HEMATOLOGY NOTES

INTRODUCTION Thrombophlebitis
 Thrombo (blood clot) + phleb (veins) + it is
(inflammation)
Common Prefixes and Suffixes from Greek and Latin  Definition: Inflammation of the vein
Prefix Meaning Anisocytosis
 a/an (decrease or absent) + iso (equal) + cyt
Cyt Cell
(cells) + osis (abnormal)
Hemo- / hemato- Pertaining to blood
 Definition: Increase in the lack of uniformity
a- / an- Without, absent decrease
of the cells in SIZE.
dys- Abnormal, Difficult, Bad
Poikilocytosis
eryhtro- red
 Definition: Increase in the variation of cells in
Leuk(o)- White
SHAPE.
Hypo- ; hyper- ; iso- Under/ decreased;
Dysmyelopoiesis
Above; Equal: same
 Definition: abnormal production in bone
Aniso- Unequal, Dissimilar
marrow contents
Poikilo- Varied; irregular
Leukocytopenia
Macro- ; mega- Large/ long; Giant
 Definition: Decrease in the white blood cells
Micro Small
Monocytopenia
Poly- Many
 Definition: Decrease in monocytes
Pan- All; overall
Leukocytophilia
Meta- After, Next; change
 Definition: Affinity to increase in white blood
Phleb- Vein
cells
Myel(o)- From BM; spinal chord
Schis- Split
Thromb(o)- Clot; thrombus BLOOD AND ITS CHARACTERISTICS
Ferr- Iron BLOOD:
Scler- Hard
-cyte Cell  A nutritive fluid that circulates in the vascular
-emia Blood system.
-penia Decreased, deficiency  Comprises 8% of our body.
-lysis Destruction, dissolving  Major body fluid
-oma Swelling; tumor  Fluids other than blood comprises 92% of the
Opathy Disease body.
-osis Abnormal Increase;  Blood is further divided into two-- the plasma
disease (55%) and the formed elements (45%).
-itis Inflammation
-phil(ic) Attracted to, Affinity for FUNCTIONS OF BLOOD:
-plasia/ -plastic Cell production or repair
1. Transport of:
-poiesis Cell production,
o Gases to facilitate gas exchange (O2 and
formation and
CO2)
development
-poietin Stimulates production o Hormones and other endocrine
secretions that regulate ell functions.
o Products of digestion to tissues where
Sample application: they will be metabolized or assimilated
Panmyelosis o Waste products of catabolism to the
 Pan (all) + myel(o) [BM} + osis (abnormal excretory organs
increase)  Transport products of metabolism:
 Definition: Abnormal increase in bone
marrow (cells or bone marrow elements).
 o transport products absorb in the
intestine and GIT
o nutrients from dietary origin
o synthesized product like cholesterol
(liver), lipoproteins
transport of products where they are
needed as nutrition
 Respiratory Function:
o transport of gasses, mainly oxygen, from
the lungs to the tissues to cell allowing
cell to breathe
o allows cellular respiration because
oxygen is vital for cellular survival
o Transport is from the lungs where the
partial pressure of oxygen is high.
Pulmonary capillaries are rich in oxygen
having a partial pressure of oxygen
equivalent to 100 mmHg.
o High partial pressure of oxygen indicates
high affinity of hemoglobin to oxygen. So
when hemoglobin reaches the lungs it
easily binds with the oxygen
o Tissues has a low oxygen level having a
partial pressure of 20 mmHg. Thus, the
affinity of oxygen in hemoglobin is low
because of the partial pressure and
acidic environment. Oxygen in the
hemoglobin will be easily released and
once it is released, it will attach to CO2.
o Carbon dioxide will be released via
exhalation
Lungs (100mmhg)
Tissues: 20mmHg
2. Regulation of the function of another organ
o Erythropoietin is a hormone that is
important in regulating red cell
production
o Hematopoiesis is a byproduct of bone
marrow
o Erythropoiesis is regulated by growth
hormone called erythropoietin which is
produced by the kidney
o Kidney: area of production,
o Bone Marrow: site of action
o Erythropoietin reaches its site of
destination by blood circulation in the
bone marrow
o Waste products needs to be excreted in
the body
o Waste products of metabolism
accumulates in the blood like urea,
creatinine, and excessive salts
o When the blood circulates in the
kidneys, some of the products of
metabolism are filtered in the kidney
and excreted in the urine
o Other waste product like carbon dioxide,
carbon monoxide are brought in the
lungs and eliminated via exhalation.
o Excessive salts and water excreted via
urination or sweating
o Failure of blood circulation will result to
waste toxicity.
3. Buffering action/function
o Blood assists in the preservation of an
almost neutral reaction in the tissues
o Helps maintain normal water balance
and fluid distribution.
 o It also regulates ionic balance like
chloride, sodium, hydrogen, potassium.
Blood will selectively excrete too many
ions and retains what the body needs
o It also eliminates excessive acids and
retains important alkali
o HgB itself is a buffering system together
with enzymes and protein
o The most important buffer that
maintains pH is HCO3:H2CO3
(Bicarbonate-Carbonic acid Buffering
system) which are maintained at a ratio
of 20 (HCO3): 1 (H2CO3)
o To maintain the bicarbonate, it needs to
be conserve in a form of Sodium
Bicarbonate (Na2HCO3) because it can
be easily loss via urination.
o Renal circulation helps in eliminating
and retention of alkali and acid
o Blood helps regulate ionic balance, acid-
base balance, and water balance in the
body
Note:
Main buffering system is the HCO3: H2CO3
maintained by sodium.
4. Maintenance of constant body temperature
o Circulation of blood minimizes variations
in local temperature.
o Blood also functions in regulating body
temperature
o During high temperature, sweating is
compensatory action to eliminate
excessive heat in the body.
o During low temperature, constriction
through shivering increases body
temperature
5. Defense
o Phagocytosis
o Blood contains proteolytic enzymes
(enzymes that help breakdown food, use
for healing of skin, muscles and joints)
and antibodies that help destroy foreign
bodies.
Phagocytosis
By definition:
It is a process wherein a cell binds to the item it wants
to engulf on the cell surface and draws the item
inward while engulfing around it. It is also used for
immunity to destroy foreign substances in blood.
Recognition and Attachment
Phagocyte receptors recognize and bind to certain
foreign molecular patterns and opsonins such as
antibodies and complement components.
Ingestion
Pseudopodia are extended around the foreign particle
and enclose it within a “phagosome” (engulfment).
The phagosome is pulled toward the center of the cell
by polymerization of actin and myosin and by
microtubules.
Killing and Digestion
 Oxygen Dependent
Respiratory burst through the activation of NADPH
oxidase. H2O2 and hypochlorite are produced.
 Oxygen Independent
The pH within the phagosome becomes alkaline and
then neutral, the pH at which digestive enzymes work.
Primary and secondary lysosomes (granules) fuse to
the phagosome and empty hydrolytic enzymes and
other bactericidal molecules into the phagosome.
 Formation of Neutrophil Extracellular Traps
Nuclear and organelle membranes dissolve, and
activated cytoplasmic enzymes attach to DNA.
The cytoplasmic membrane ruptures, and DNA with
attached enzymes is expelled so that the bacteria are
digested in the external environment.
t-cells cytokines
B-cells – IgA, IgE, major producer of plasma cells
Reference: Rodak’s Hematology Clinical principles and Applications
(6th Edition)
CHARACTERISTICS OF BLOOD
Physical Characteristics of Whole Blood
 Color: Normally red as imparted by hemoglobin
 Viscosity: 3-5x more viscious than water
 Circulates in liquid state
  Coagulates between 5-10 minutes after removal
from the body
 pH: 7.35-7.45
o Normal pH is maintained by buffers in
the body primarily the Sodium
Bicarbonate.
o 6.8-7.8: pH compatible for survival.
o Gastric juices (very acidic)
o Pancreatic Juice (very alkaline)
 Sp. Gravity (whole blood): 1.048-1.066
o Serum: 1.026-1.031
o RBC: 1.092-1.095
 Volume: 5-6L (7-8% of total body weight)
o Males: 76 ml/kg body wt.
o Females: 68 ml/kg body wt.
Note:
 The bigger you are, the more blood volume
you have.
 Females have lower blood volume in relation
to their weight.
 Blood volume is affected in the volume intake
and volume of the body.
Note:
Hemoglobin gives color to the blood. The color of the
blood depends on what color or type of the
hemoglobin present in the blood.
Note: What is the color of blood that contains of
oxyhemoglobin? Deoxyhemoglobin? Sulhemoglobin?
Carboxyhemoglobin? Methemoglobin?
Oxyhemoglin Bright red or Scarlet red
-Arterial circulation
Deoxyhemoglobin Dark red
-Venous circulation
-Reduced Hgb containing
oxygen and CO2
Sulhemoglobin Green
-formed by an irreversible
oxidation of Hgb by drugs
or exposure to sulfur
chemicals in industrial or
environmental setting. It
is formed by the addition
of a sulfur atom to the
pyrrole ring of heme and
has a greenish pigment.
Carboxyhemoglobin Cherry Red
-combination of CO and
heme iron.
-CO poisoning
Methemoglobin Chocolate brown
-formed by the reversible
oxidation of heme iron to
the ferric state (Fe3+).
Reference: Rodak’s Hematology Clinical principles and Applications
(6th Edition)
Note:
Both Oxyhemoglobin and Deoxyhemoglobin are
normal type of hemoglobin.
Capillary blood
 Mixture of both arterial and venous blood
 Color: Pale or light red because it is a mixture
of arterial and venous blood with some tissues
coming from skin.
Note: In vivo, what maintains the blood in the liquid
state?
Heparin is the natural anticoagulant which maintains
the blood in liquid state (in vivo). However, the
amount of heparin is minimal that cannot maintain the
blood in a liquid state outside the body.
NORMAL COMPOSITION OF BLOOD
1. Liquid Portion: Plasma or Serum
 55% of the total blood volume
 91-92% by weight is water
 6-7% by weight includes plasma proteins
and other substances (2%) like vitamins,
carbohydrates, etc.
Plasma proteins
Albumin: 4% (helps maintain osmotic pressure)
 Indicator of nutrition
 Functions in transport of substances that
includes bilirubin (transport in the liver for
conjugation)
  Helps keep fluid in bloodstream so it doesn’t leak  Precursor of fibrin. The one who
to other tissues, also carries hormones, vitamins converts fibrin clot to prevent
and enzymes. hemorrhage
 Has the ability to serve in osmotic and oncotic as
 Represents small portion of plasma
well as colloidal pressure.
protein
 Very important role in clot formation
because of its ability to convert into
fibrin clot
 Fibrin or coagulation factor 1
 Fibrinogen is the precursor of fibrin
 Fribrinogen will be converted by
thrombin which came from
prothrombin (Factor II)
Note:
Prothrombin is important in coagulation.
 Most important in maintaining oncotic pressure
 Oncotic pressure directed against the capillary PLASMA SERUM
wall pushing the fluid in the capillary wall Liquid portion of an Liquid portion of an
 Hydrostatic pressure is the force created by the unclotted blood Clotted blood
circulating fluid in the circulation which is Remains Fibroblast Contains no fibrinogen
responsible in pushing fluid away of the Growth Factor (FGF) factors
circulation. Strong hydrostatic pressure will lead intact
to extravasation but it is counter acted by plasma Contains less platelet Contains more platelet
protein specifically albumin by creating osmotic derivatives derivatives
or oncotic pressure
Slightly turbid or hazy Appears clearer yet
 Decrease in albumin results to decrease in due to the added darker in color because
osmotic pressure. chemical anticoagulant of the non-addition of
Globulin: 2.7% (needed for liver function, blood clotting chemical anticoagulant
and infection)
Note:
 Alpha globulin
Fibrinogen group: factors I, IV, VIII, XIII are
o Alpha 1 globulin (Alpha 1 anti-tripsin) consumable in the process of clotting.
Alpha 2 globulins (Alpha 2
macroglobulins)
o Alpha fetoprotein 2. Solid Portion: 45% of the total blood volume
o Function in the transport of enzymes,
lipids and others  RBC (erythrocytes, erythroplastids,
normocytes)
 Beta globulins o Function mainly in transport
o Allow transport of oxygen from lungs to
o Also responsible for transport
different organs and tissues to allow
o Transferrin is a beta globulin responsible
cellular respiration
in the transport of iron
o Blood is a liquid medium that circulates
in the body through pumping of heart,
 Gamma globulin
and it will enter the artery to the
o production of antibodies such as
capillary then in tissue level exchanging
immunoglobulin
carbon dioxide. It will then enter the
Fibrinogen: 0.3% (helps in blood clot for normal vein and back to the heart
blood clotting)
 o Some part of the circulating blood will
proceed to the lungs to get oxygen and
eliminate carbon dioxide

 WBC (leukocytes; leukoplastids)

o Include neutrophil, lymphocyte,
monocyte, eosinophils, and basophils
(arranged from most abundant to least
abundant)
o Function in body’s defense
o Defense mechanism involves
phagocytosis
o Phagocytosis “engulfment”- the WBCs Note:
will engulf an invading organism Hemoconia (BLOOD DUST): small refractive colorless
o Not all WBCs are active phagocytes, the particles in the blood that are probably granules from
more active phagocytes are the blood cells or minute fat globules and it has no clinical
significance.
neutrophil and monocyte
o Most active phagocytes is the
Neutrophil and also the first to reach the Function
area of infection or inflammation RBC Filled with hemoglobin which transports
o Eosinophils are less efficient phagocytes oxygen into the lungs and to the different
o Lymphocytes are not phagocytes. T- organs and carbon dioxide
cells are responsible in producing WBC Cell types dedicated to protect their host
regulatory cytokines like interleukins from infection and injury.
that will regulate the function of other -has 5 population (neutrophils,
cells while B-lymphocytes are leukocytes, monocytes, eosinophils,
responsible of antibody production (IgA, basophils)
IgE)
Platelet Also known as thrombocytes, which
o B-lymphocytes will be transformed into
maintain blood vessel integrity by
plasma cells which is the major producer
initiating vessel wall repairs.
of antibody -clot formation or hemostasis
-to form platelet plug to prevent bleeding
 Platelets (Thrombocytes; thromboplastids) Reference: Rodak’s Hematology Clinical principles and Applications
o Also known as thrombocytes (6th Edition)
o Cells that are primarily involve in clot
Reference Range
formation
RBC 3.8-4.1 M/µL or 3.8-4.1 x10^12/L
o To maintain hemostasis (refers to the
WBC 4.5-11 x10^9/L or 4,500-11,000/µL
stoppage of bleeding), by forming plug
Platelets 150-450 x 10^9/L or 150,000-450,000/µL
that will cover the injured blood vessels
Least-greatest:
o Thrombocytes: Cells for clotting RBC > Platelets> WBC
o Blood vessels react by vasoconstriction
limiting blood flow to the injured site
o After platelets will react by adhering in 3. Gaseous Portion:
the injured site forming platelet plug but o Portion where exchange between O2
platelet plug is unstable plug that blood and CO2 for normal respiration occurs.
pressure or flow may easily remove the
platelet plug
o To stabilize the platelet plug, fibrin
(fibrinogen or factor I) is needed.
 bone marrow, whereas T cell lymphopoiesis
occurs in the thymus.

Note:
SYNCRONOUS
MATURATION
-Also called normoblastic
maturation.
- The nuclear and
cytoplasmic maturation
of cell should go in the
same phase to be called
synchronous maturation.
Note: - The result of
Excessive loss of water in cases of LBM, vomiting and synchronous maturation
the like, causes dehydration and can affect the plasma is the development of
concentration. normal cells called
normocyte.
Note:
Formed elements is the formed cells called Hemocyte
includes <1% platelets, <1% leukocytes and >99%
erythrocytes. In centrifugation, hemocytes like
platelet and leukocytes settles above the RBC in the
buffy coat layer.
ASYNCRONOUS
MATURATION
-Asynchronous
maturation will result in
abnormal cell. Either the
nuclear or the
cytoplasmic maturation is
abnormal
- Most common type of
asynchronous maturation
that occurs is when the
nuclear maturation is
retarded compared to
cytoplasmic maturation.
-In the presence of defect
of DNA, nucleus will not
mature. Nucleus will slow
down maturation and as
an effect it leads to
asynchronous
maturation.

Note:
-Nucleus controls all the activity of the cell (also
Most abundant form element is the RBC that on
known as control center), it also control cell division
centrifugation, the red bottom layer is called packed
called mitosis
RBC (purely RBC).
-Nucleus cannot dictate cell division unless it is mature
-As the cell matures cell size decreases because every
CHAPTER 2: HEMATOPOIESIS time it undergo division, it split until it reaches its
TERMINOLOGIES: maturation phase.
-Since in asynchronous maturation, the nucleus is
 Dyspoiesis unable to mature normally, the cell will either not
- Abnormal formation of blood cells. divide or suffer from prolonged mitotic division.
 Erythropoiesis - Megaloblastic Maturation: prolonged or undivided
- Development of mature red blood cells from cell due to defect in nucleus.
erythropoietic stem cells. -The main reason why nuclear development becomes
retarded is because of the deficiency in DNA
- Production of red blood cell
synthesis which is commonly due to Vitamin B12
 Granulopoiesis
deficiency and folate acid deficiency
- Production of neutrophils, eosinophils, and
basophils.
 Monocytopoiesis HEMATOPOIEOSIS
- monocyte production
 Myeloid cells
 Refers to the formation and development of
- Granulocytic and monocytic cells
blood cells.
 Lymphopoiesis
 It is a continuous regulated process of blood cell
- Process in which lymphocytes (B cells, T cells
production that includes cell renewal,
and NK cells) develop from progenitor cells.
proliferation, differentiation, and maturation.
B cell lymphopoiesis is completed in the
 These processes result in the formation, Death of the Cell: Hematopoiesis include not only the
development and specialization of all of the production of new cell but also the death of the cell.
functional blood cells that are released from the
bone marrow to the circulation.
Note:
 During embryonic and fetal development,
 RBC Life Span: 120 days
hematopoiesis occurs in the yolk sac then in the
 1% of RBC reaches their 120 days life span
liver (also in the spleen and thymus). In normal
daily.
adults, hematopoiesis occurs mostly in the bone
 Bone Marrow is capable of replacing the 1%
marrow and peripheral lymphatic tissues. RBC destruction
Note:  Approximately 35,000/ µL of platelets
reached their maximum life span of 9-11 days
Proliferation is the cell production (mitosis) when they
but being replaced by bone marrow by
proliferate, they increase in number through mitosis.
producing at least 35,000 platelets daily.
Differentiation is the transformation of a stem cell
(which is a Pluripotent cell) into a specific type of Note:
blood cell or the process of stem cell to become a
Daily Turnover:
WBC, RBC or Platelet.
The number of a cell that is being destroyed daily is
being replaced by the Bone Marrow daily.
Unitarian: all blood cell came from a single parent cell
or stem cell which is the Pluripotential Stem Cell
(PPSC) that has the potential to become many kind of PHASES OF HEMATOPOIESIS
cells like WBC, any of the 5 types of WBC, RBC or
platelet.
A. INTRAUTERINE PHASE:
Morphogenesis refers to the study of morphological - Pre natal hematopoiesis
changes as cell undergoes maturation and division.
Example: an immature RBC is nucleated but as it 1. MESOBLASTIC OR MEGALOBLASTIC PHASE
undergo maturation, nucleus disappear and undergo - Chief site: yolk sac
pyknosis. Until such time, the normocyte is non- - Primitive RBC (“megaloblast of Ehrlich)
nucleated. In other words, it is the morphological
first develop within the blood island
changes that occur during blood maturation and
followed by leucopoiesis and
development (e.g. sizes, nuclear changes, etc).
megakaryopoiesis and it is produced
Functional maturation: while cells are immature, they extracellularly
perform a different function. Cell has a more defined - Embryonal hemoglobins are
function in the body as it matures, like B-cell which is synthesized during this phase
considered as an immature cell. B-cell matures into 2. HEPATIC PHASE
plasma cell and once it is transformed into plasma cell, - This phase starts on 3rd month of fetal
it is more active in the production of antibody. T cell life.
from the bone marrow goes to the Thymus to undergo - Chief site: Liver (aside from the liver as
maturation to become a functional mature T-cell the chief site of blood production,
Spleen and Thymus can also help in the
Note: production)
Destruction and production of cells are BALANCED. - Fetal Hemoglobin (HbF): synthesized
Our normal count are maintained by relatively during this phase
constant number of cells unless when we are exposed 3. MYELOID / MEDULLARY PHASE
to condition that will result to excessive or decreased - Chief site: Bone Marrow
production or excessive destruction. In this case, - This starts on the 5th month of fetal life.
number of cells are being imbalanced. It increases during the last trimester and
remains the chief site at birth.
 - Production of Adult Hemoglobin (HbA) hematopoiesis on the 5th month until birth that occurs
starts during this phase. in the medullary region of the BM. After birth, the
most dominant hemoglobin is still HbF.
MESOBLASTIC HEPATIC MEDULLARY
-19-20th day -5 -7th week
th
-5th month
-Mesoderm → -Peak: 3rd – 4th -Red Marrrow: Specific Cell Production
“blood idlands” month chief site RBC 19th -20th day
-“Hemohistoblast” -Liver: -All fetal Granulopoiesis 2nd month
→→ (Extravascular) skeletal Megakaryopoiesis 2nd month
-Primitive RBC: -Spleen & structure → Lymphopoiesis 4th month
“Megaloblast of Kidney= B-cell birth Monopoiesis 5th month
Ehrlich” -Thymus = T-cell
-WBCs & -Lymph nodes
Megakaryocytes
-Primitive
endothelial cells
-8-12 weeks active
Hb: Embryonal Hb: Fetal HgB Hb: Adult Hgb
HgB (Grower I, II & or HbA
Portland)

Note:
Hemohistioblast is a cell (like stem cell) that gives rise
to blood cells and tissue cells of an embryo.

RBCs are the first to be formed from hemohistioblast.

The hemohistioblast gives rise to the Primitive RBC or
B. EXTRAUTERINE PHASE:
“Megaloblast or Ehrlich”.
1. MYELOID / MEDULLARY PHASE
- First 3 weeks postpartum, the bone
Primitive RBCs are different from the RBC in adult life.
These are produced extravascularly and mature marrow becomes the only normal site of
through fragmentation. blood cell production.
- Red marrow is present in all fetal
skeletal structures, until the age of 2-3
years.
- 4th year: rate of BM growth exceeds
Note: need for blood cells resulting to the
Thymus is the very first well developed organ in replacement of active marrow space by
human body and is producing T-cell even during fetal
areas of fatty reserves marrow space by
life.
areas of fatty reserves.
Lymph nodes are responsible in producing T&B cells. LOCATIONS OF RED MARRROW
Children Skull, clavicle, ribs,
Once the blood islands become inactive on the 12th or vertebra, pelvis and long
beyond 12th week, the RBC formed in the liver will bones
synthesize a different kind of HgB called Fetal HgB or -occurs in flat and long
HbF. bones.
18 years and older Skull, clavicle, ribs,
Note: vertebra, pelvis & at the
proximal ends of long
On the 4th week, the bone marrow is already involve in
bones
blood production but it is not yet the main site on
hematopoiesis. BM becomes the chief site of
 2. EXTRAMEDULLARY HEMATOPOIESIS
- (Liver and Spleen): occurs normally and
in certain disease states when the BM is
unable to produce sufficient numbers of
hematopoietic cells.
- Normal Extramedullary Hematopoiesis
refers to the involvement of lymphoid
organs—organs that produce
lymphocytes like B cells / Bursa and T-
cells or T-lymphocytes produced in
Thymus.
- Thymus: primary lymphoid organs
- Spleen and Lymph Nodes: Secondary
lymphoid organs
- Spleen: when activated is involved in the
production of lymphocytes.
- During abnormal condition: liver retains
its capacity in hematopoiesis. Both the
liver and the spleen have the residual
capacity for hematopoiesis. When the
body needs blood and the bone marrow
cannot supply, the liver and spleen can
help but in short duration.
MEDULLARY EXTRAMEDULLARY
Red Marrow:
2-3 yrs old: all skeletal Thymus and Spleen
structure :Retrogression”
Adults:
Bone marrow Liver and spleen
Note:
In 2-3 years, the marrow expansion, the child’s growth
is but equal for the need for blood. Since the growth
for bone and the requirement for blood by the child is
equal, then the bones remains to be filled with the red
marrow. But starting on the 4th year and older, the
growth becomes faster than the need for blood. In
that case, the spaces of the bone marrow becomes
infiltrated with fats called Retrogression.
Retrogression is the replacement of red marrow by
marrow fats that happens in the 4th year of life.
MARROW CELLULARITY
- Hematopoietic cells compared with fat.
- Red Marrow: Hematopoietic cells
- Yellow Marrow: yellow/white marrow or fat
- 50:50 ratio of hematopoietic cells and fat.
Note:
Hypercellular /Hyperplastic BM: contains more red
than fats that is seen in leukemia
Hypocellular/ Hypoplastic: ↓red, ↑ fats
Aplastic Anemia: low blood cells characterized by
hypoplastic BM that is incapable of producing normal
blood cells because it is dominated with marrow fats.
Note: Is the bone marrow fat still capable of the
hematopoiesis? When?
Bone marrow fat is not hematopoietically active. It is
not capable of producing blood cells because it only
contains fat but it can be reactivated to become red
and help in the production of cells but in small period
of time.
THEORIES ON THE ORIGIN OF BLOOD CELLS
1. MONOPHYLETIC/ UNITARIAN THEORY
- States that blood cells from one stem
cell which is totipotent, gives rise to any
series of cell types.
- Hemocytoblast: gives rise to all blood
cells.
2. POLYPHYLETIC THEORY
- States that there are 2-3 cell origins.
There is a separate and distinct stem cell
compartment.
Sub-theories:
a. DUALISTIC- Lymphoblast and myeloblast
b. TRIALISTIC- Reticuloendothelial cells give rises to
monocytes.
3. COMPLETE THEORY- believes that there is a
separate stem cell for each cell series.
o Proginitor cells are commited cells
meaning it is already identified what cell
they will become
Note:
CFU-GM is a proginitor from CFU-GEMM that will give
rise to CFU-G and CFU-M later on become neutrophil
and monocyte respectively.

STEM CELLS PEODUCED BY THE BONE MARROW: Neutrophil and monocyte shares the same proginitor
 HEMOHISTOCLAST: one single fixed multipotent cell called CFU-GM
stem cell that gives rise to tissue and to blood
cells. CFU-Eo is the proginitor commited to become
Eosinophil
 CFU-S/ CFU-LM or PLURIPOTENT STEM CELL
(PPSC): CFU-Baso is the proginitor for Basophil
o CFU means colony forming unit.
o Colony forming unit (Stem) or Colony CFU-MegE is the proginitor commited to become
either Megakaryocte (CFU-Mega) or Erythrocyte (BFU-
Forming Unit (Lymphoid/ Myeloid)
E first then CFU-E then erythrocyte)
o Present in small numbers (constant) in
the BM The megakaryocyte and erythrocyte share the same
o Not morphologically identifiable proginitor
o Has the ability to reproduce and
differentiate any blood cells (repopulate While on the other hand, stem cell CFU-L will give rise
the BM after injury) to two proginitor cells (Pre-T/NK cell and Pre-B cell)
o Colony forming stem cells.
o 1% BM contains this stem cell Pre-T/Nk cells are committed to become T-cell or
o Can later on be the stem cell for Natural Killer cell
lymphoid or myeloid.
o One of the two daughter cell produced Pre-B cell is committed to become a B-lymphocyte
will differentiate but one will remain as
Myeloid Cell Line: Cell line of CFU-GEMM
stem cell
Lymphoid Cell Line: CFU-L cell line
o All myeloid stem cell except lymphocyte
is under Colony forming Unit Monocyte The two classifications of leukemia is the myeloid and
o Colony forming Unit Stem or PPSC is the lymphoid leukemia depending on the cell line which is
main parent of all blood cells can be damage
influence by the growth factor and can
transform into Colony forming myeloid
or CFU-GEMM (Granulocyte,
Erythrocytes, Monocyte,
Megakaryocyte) or Colony Forming Unit
Lymphoid (CFU-L) that gives rise to B-cell
and T-cell
o What will arise from CFU-GEMM and
CFU-L are the proginitor cells
o CFU-GEMM and CFU-L are uncommitted
meaning they can transform to become
any of the blood cells
Note:
Unitarian theory is correct that all cell came from one
cell called Pluripotent hematopoietic stem cell

Multipotent Stem Cells: these are Pluripotent

Hematopoietic Stem cell, CFU-GEMM and CFU-L. Since
they are multipotent stem cell they are all
uncommitted

Proginitor cells are all committed.

Morphologically recognizable precursor are the ones

that will just mature to become the next stage and
came from bone marrow.

Monocyte will still mature to become macrophage in

the tissue.

B-cell is morphologically mature but functionally

immature. It will mature to become plasma cell.

GROWTH FACTORS:

 Proteins that regulate the production of blood

cells. These include cytokines and hormones.
 Can be in the form of proteins or glyproteins
 They are regulatory factors that regulate the
production and destruction of blood.
 Can be in the form of cytokines
 Interleukin-3 (IL-3): a multipotent which means
it is capable of regulating the growth of more
than one cell line together with GM-CSF (colony
stimulating factor) which is not exclusive in
granulocyte and monocyte. It can also affect the
function of other mature cells.
 Erythropoietin (EPO): main growth factor for
erythropoiesis and Thrombopoietin (TPO) for
thrombopoiesis. Both are produced by kidneys
and liver.
 EPO mainly produced by the kidneys but small
amount produced by the liver.
 TPO mainly produced by the liver.

 Kit Ligand (KL)

- “stem cell factor” or “steel factor”
- Stimulates myeloid, erythroid and
lymphoid progenitors.
 Fit-3 Ligand (FL)
- primitive progenitor cells.
- Tyrosine like ligand because it acts on
the receptor of tyrosine kinase
- Tyrosine kinase are the one that receives
signals from the outside and
- interpret the signals to promote the
growth of progenitor cell
- Leukeimia is a disease caused by a
mutation in tyrosine kinase gene ANTIGEN MARKERS
- IL3 and GM-CSF are both multipotent These are cell surface proteins that used in the
cells recognition of lineage/differentiation

Antigen markers
CD34 Stem cell marker (lymphoid and
myeloid precursor)
CD33 Pan myeloid cells
CD31 Pan myeloid
CD11c, CD114 Monocytes
CD71 Erythroid
CD2,CD3 Lymphoid, pan T cells
CD4 Helper/ inducer T cells
CD8 Suppressor/ cytotoxic T cells
CD10, CD19, CD20 Lymphoid, pan B cells
CD16, CD56 NK cells

Release of blood cells from the bone marrow to the

circulation:

 Release of RBCs is promoted by erythropoietin,

hypoxia, cell’s stage of maturation (reticulocyte)
and the pressure exerted by the intramarrow
frowth of cells.
 WBCs. Leave the bone marrow through
chemotaxis while platelets are released to the
circulation through platelet shedding.
 RECOGNITION OF LINEAGE/MARKER
- Identifiers of the type of cell
- They can be in the form of surface
markers or internal protein
- It is called markers because their
presence is unique in that cells
- The rules of markers are very important
in Immunophenotyping
- Marker in the surface or inside the cell
will react to specific antibody
- Marker behaves like an antigen that will
react to corresponding antibody
- Cell of Differentiation is important in cell
recognition
CHANGES DURING NORMAL BLOOD CELL
MATURATION:
 Cell size: decrease
 Nuclear/ Cytoplasm (N/C) ratio: decreases
 Cytoplasm:
 Basophilia: gradually decreases
 Amount/size: increases
 In some cells, some cytoplasmic
constituents are produced.
 Nucleus:
 Size: decreases
 Nuclear chromatin: becomes more
course and clumped
 Number of nucleoli: decreases
 Staining: from reddish to bluish-purple
RULES IN BLOOD CELL MATURATION
Cytoplasmic Changes
 Basophilia
o The more the immature the cell is the
more basophilic the cytoplasm is
o The cytoplasm is rich in RNA
o Acidic or Basic refers to description of
cellular component
o Acidophilic and Basophilic is the staining
property
o Principle of staining stated that acidic is
attracted to basophilic dye.
o Acidic cellualr component love to take
basophilic dye
o The more the immature the cell is the
more RNA it has in the cytoplasm
o As it matures the amount of RNA
decreases because it is less
metabolically active
o Exception: Plasmacyte (comes from B-
cell which is immature in terms of
function. It needs to be activated by
antigen to transform into plasma cell,
plasma cell is active in antibody
production thus contains more RNA and
remain basophilic in nature)
 Granulation
o Blast cells are aggranular and only
applies to WBC and platelet
o Exception: RBC (RBC are aggranular)
o Aggranular WBC (Monocyte and
Lymphocyte). Not literally aggranular,
granules are so tiny that it can’t be seen
properly under microscope
o Granular WBC (Neutrophil, Eosinophil
and Basophil)
 Nuclear changes
o Size
 The size of nucleus and
cytoplasm is exactly opposite
 As the nuclear size decreases
the cytoplasmic amount
increases
 A more mature cell shows a
more abundant cytoplasm
 A more immature cell shows a
bigger nucleus
 Mature Red Blood Cells are non-
nucleated
  In monocytes and granulocytes, RBC Maturation Series ASCP Maturation series
while the cell is immature the (by CAP)
nucleus is round as the cell Pronormoblast Rubriblast
matures the nucleus becomes
indented Basophilic Normoblast Prorubricyte
 Nuclear Chromatin
o Immature cells contains fine and Polychromatophilic Rubricyte
delicate chromatine pattern and the
Orthochromic Metarubricyte
distribution is homogenous, as the cell
Reticulocyte Reticulocyte
matures and in preparation fro mitosis
some of nuclear chromatin aggregate
Erythrocyte Erythrocyte
and they are also more course and
clump
 Number of nucleoli
o It decreases as cell matures
 Staining
o Less basophilic in immature and
becomes more basophilic as it matures
 Cell size
o As the cell matures it becomes smaller
o Exception: Megakaryocyte (source of
platelet), the more the megakaryocyte
the more the platelet present
 Nuclear Cytoplasm Ratio
o Immature has an N/C ratio of 8:1
o Maturation it becomes 6:1
o Further maturation it becomes 4:1
o As the cell matures the N/C ratio
decreases
o The decrease in N/C ratio is parallel to
the size of nucleus

ERYTHROPOIESIS
- RBC maturation series
- Process of RBC production and maturation
- 3-5 days for complete erythropoiesis to occur
from pronormoblast (1st stage) to reticulocyte
(5th stage) and the mature RBC (6th stage)
- Production in bone marrow is from 1st to 5th
stage
- Reticulocyte is released in the circulation
becoming mature RBC
- RBC starts first as burst forming unit (BFU-E)
before becoming CFU-E

GROWTH FACTORS FOR ERYTHROPOIESIS

- The main growth factor is erythropoietin
- IL-3
- SCF
- GM-CSF
 STAGES OF ERYTHROPOIESIS

ERYTHROPOIESIS influence in erythropoiesis. Stem cell factor (CSF)

is also very important since its mode of action or
- Main site: Bone Marrow
its target cell is the main stem cell, without it
- Normally takes 3-5 days for the production of
stimulating the stem cell, the stem cell will not
reticulocyte from pronormoblast.
differentiate into progenitors. If CSF is not
- If we are to include the differentiation from the
present, the growth factors are useless because
stem cells to progenitor cells (CFU-E, BFU-E to
they have nothing to stimulate further without
erythrocyte), progenitor production until mature
the differentiation of the main stem cell.
RBCs in circulation, it takes around 18-21 days
- From CFU-E, will arise the first recognizable
that is shorten by the erythropoietin.
stage that will take 6 maturation stages.
- From the stem cell (PPSC), it will differentiate
into common myeloid stem cell (CFU-GEMM)
 ERYTHROPOIETIN
which will now differentiate into another
 Major growth factor that stimulates
progenitor which this time is more committed to
erythropoiesis
become wither Megakaryocyte lineage or
erythrocyte lineage. If it will further differentiate
 IL-3
into a more committed for erythrocyte, it
becomes BFU-E before it will become CFU-E. Note: How many maturation are there in
erythropoiesis?
There are 6 stages in erythropoiesis starting from
GROWTH FACTORS Pronormoblast-erythrocyte

- Influence the maturation and differentiation

primarily the erythropoietin and IL-3. Though it Note: In which stage does HgB occur?
is mentioned that also CSF-GM and GM-CSF has As early as pronormoblast, the cell is starting to take
up the nutrients it needs like iron (taken up from the
environment or from the storage in the BM. The
developing Red cell will take the available iron in the
BM from the ferritin), for it to synthesize heme. It is
also starting to synthesize its own globin but has not
completely make up the HgB. Hemoglin, if present, will
take up the acidic dye (eosin stain) that will result to
the pinkish color of the RBC.
In the Basophilic Normoblast (prorubricyte), evidence
of HgB synthesis is already observed. However, the
amount of HgB is in smaller amount than the other
components of cytoplasm. Since the cell is
metabolically active but still has a lot of mRNA in its
cytoplasm which will obscure the observation of HgB.
Thus, the appearance og HgB here is not obvious.
However, as it reaches Polychromatophilic
Normoblast/Rubricyte (3rd stage), the HgB is evident
now in the red cell as it gives the murky color of the
cytoplasm.
Shift cell/ Shift reticulocyte/ stressed cell or
reticulocyte: 1 day in BM; 2 days in circulation; has
many remnants of RNA; lesser function but still can
function in oxygen transport but not as efficient as the
mature RBC.
The immature RBC that is directly released from the
bone marrow will first enter the spleen and the spleen
will remove the remaining inclusions inside the cell as
well as polish the red cell membrane so that when the
cell is released by the spleen, it will now have a
biconcave shape. In this case, the mature RBC with a
biconcave shape can circulate well in the body.

HgB synthesis started in Basophilic normoblast

(Prorubricyte) but the amount is not enough to alter
the color because it contains more acidic components
in the cytoplasm. But the stage where HgB is evident
is in the Polychromatophilic Normoblast (Rubricyte).

Note: Do erythrocytes mature in the bone marrow?

No, RBCs doesn’t mature in the bone marrow.
Reticulocyte matures in the BM for 1-2 days. BM will
then releases reticulocytes in the circulation and to
further mature into mature RBC for another at least 1
day.
The 1-2 days span for the cell in the bone marrow can
be shortened in cases of anemia or when there is a
severe need of blood in the circulation. EPO will
accelerate the release of immature cells in the
circulation. If it accelerates and will stay in the BM for
just 1 day, the immature cell will stay in the circulation
as a reticulocyte for 2 days before it matures into RBC.
But if the reticulocyte stayed in BM for 0.5 days and
there is a need for it to be released in the circulation,
the retic will stay in the circulation for 2.5 days in order
to mature as RBC. The 3 days span of being a
reticulocyte should be maintained by staying in the
BM for 1-2 days and in the circulation for 1 day in
normal conditions. For cases where span in the BM as
reticulocytes is shortened, further maturation in the
circulation is extended.
Normal Retic: 2 days in BM; 1 day in circulation;
decreased amount of remnant RNA.
STAGES OF ERYTHRIPOIESIS (P.B.P.O.R.E)
1. Pronormoblast Proerythroblast (Rubriblast)
- First of the recognizable stages
- Chromatin structures: fine and delicate and no
accumulation of chromatin.
- Nucleoli: decreases
- Division: once or more than once
2. Basophilic Normoblast (Prorubricyte)
- There is a dark staining of the nucleus because of
the heterochromatin or that is the
condenstation of the chromatin.
- Division: once or more than once or twice
3. Polychromatophilic Normoblast (Rubricyte)
- Has a so called “murky cytoplasm” caused by the
appearance of HgB. RBCs are the ones that
synthesize HgB. If there is no erythropoiesis,
there is no HgB produced because HgB occurs
inside the RBC.
- It shows many shades of colors
- Division: last to undergo division and last to
undergo mitosis. As it divides and gives rise to
the next stage (orthochromic normoblast), the
orthochromic is no longer capable of division
4. Orthochromic Normoblast/ O. Erythroblast/
Metarubricyte
- Ortho: means “true”.
- Last nucleated stage and is incapable of mitosis.
- Nucleus: Pyknotic (shrunken, dehydrated which
is why it is darker in color). The nucleus is about
to be dissolved or removed and it is unfunctional
and is about to be extruded. Once it is extruded,
the stage will become a reticulocyte
- Pyknosis: nuclear dissolution.
5. Reticulocyte/ Polychromatophilic Erythrocyte
or Normocyte/ Diffusely Basophilic Erythrocyte
- First non-nucleated stage.
- Also called “Polychromasia” meaning different
shades of color particularly bluish, greyish due to
the mixture taken up by HgB and RNA inside the
cell.
- Why is it called “Reticulocye”? In the laboratory,
the ideal method to demonstrate reticulocyte is
supravital staining (“vital” means life) wherein
the cells are first stained while they are in their
living state and fixing is not required in supravital
staining method unlike in usual preparation for
blood smear.
- Supravital stains commonly used the laboratory
are Briliiant cresyl blue (BCB) and New
Methylene Blue (NMB).
- Supravital stain Principle: RNA is precipitated
for it to turn into granules thus separated from
the HgB and sometimes the granules follow into
a reticulum network pattern thus the name
“Reticulocyte”.
- Reticulocytosis: increase in reticulocytes (in
supravital stain)
- Polychromatophilia: increase in
polychromatophilic cells (Wright’s stain).
- Both Reticulocytosis and Polychromatophilia are
the same.
- The name “reticulocyte” was taken up from the
appearance of the cell when stained in supravital
stain because the cell is seen with granules
following a pattern called “reticulum network”.
But for the other name “Polychromatophilic
Erythocyte” was taken by its appearance when
stained with Wright’s Geimsa stain.
- It stays in the bone marrow as reticulocyte for at
least 1 day and is released into the circulation to
mature for another 1 day into a Red Blood Cell.
- Fully hemoglobinized but still contains remnants
of mRNA and many ribosomal RNA.
- Last stage where HgB synthesis occurs and is
completed in this stage.
- How come that it can still synthesize hgB despite
of the absence of nucleus that contains the
genetic material? In order for the cell to
synthesize protein like HgB, it needs a genetic
material (found in nucleus) that will serve as a
template for protein synthesis. In DNA
replication and RNA transcription, DNA is copied
in the form of mRNA. DNA may not be present
but remnants of mRNA (that came from DNA
when transcribed into mRNA) and ribosomal
RNA is abundant in the cytoplasm. The genetic
code of Hgb synthesis is still present in mRNA.
Thus, HgB synthesis still continues during this
stage.
- Wright’s Stain: mostly used in staining blood
cells. Has two stain composition which is the
Methylene blue (basic dye) and eosin (acidic
dye). RNA in the cell will take up methylene blue
and will appear bluish while HgB will take up the
eosin which will appear pinkish. Since HgB and
mRNA were mixed up inside the cell, the blue
color taken up by the RNA and the pink color
taken up by the hemoglobin from the
- eosin will be mixed in cytoplasm and bluish color
tend to be more dominant resulting to a
polychromatic appearance.
6. Mature Erythocyte
- Absence of nucleus, organelles, mitochondria
and RNA.
- Biconcave disk shape.
STAGE DIAMETER CYTOPLASM NUCLEUS NUCLEOLI N/C
(um) RATIO
Pronormoblas/ 14-20 -deeply basophilic With fine chromatin Usually 1-2 8:1
Proerythroblast/ cytoplasm
Rubriblast -Non-granular
Basophilic Normoblast/ 12-17 -intensely basophilic With slightly coarse Usually NOT 6:1
B. Erythroblast/ cytoplasm chromatin visible
Prorubricyte
Polychromatophilic 10-15 -blue-grey to pink- 4:1
Normoblast/ P. grey cytoplasm
Erythroblast/ Rubricyte
Orthochromic 7-12 -Pink cytoplasm Small pyknotic 1:2
Normoblast/ O. -Mitotically inactive nucleus
Erythroblast/
Metarubricyte
Reticulocyte/ 7-10 -Pink to slightly Non-nucleated
Polychromatophilic pinkish grey
Erythrocyte/ Diffusely cytoplasm
Basophilic Erythrocyte -Contains fine
basophilic remnants
of RNA
Mature Erythocyte  Non-nucleated
 Normal life span in the circulation: 120 days

megaloblastic cell. Because of the nuclear

abnormality, the DNA of the cell is incomplete since
the nucleus is not mature resulting to its incapability
to dictate a division.

Note: On the other hand, if a cell undergo 6 divisions instead

There are 4-5 cell divisions in one erythopoietic cycle
where pronormoblast as the first stage can divide
once or more than once and produces two daughter
cells that are also pronormoblast and divide again until
it matures to become basophilic erythroblast. If a cell
lineage undergo a total of 4 divisions, there will be at
least 16 RBCs produced but if it undergo 5 divisions,
there will be as much as 32 RBCs being produced. In
divisions, not all cells will be able to mature as
erythrocytes because some of them are being
destroyed during the process of maturation resulting
to a decreased number of erythrocytes produced.
As the cell divides, the cell size decreases thus, as the
cell matures, the cell size decreases.
Note: Asynchronous Maturation
In asynchronous maturation, nucleus and cytoplasm
do not mature at the same time. If the problem in the
asynch maturation is the nuclear maturation, the
intermitotic phase (PMAT) is extended or prolonged
resulting to an increase in size or will turn into a
of stopping on the 5th division, it will result to a
microcytosis or an abnormal decrease in size.
Note: RBC lifespan
Mature RBC will stay for 120 days in the circulation
performing its function. It can be shortened in
abnormal conditions but it will not exceed beyond 120
days. As it reaches 120 days, the RBC will lose it
function, crenate, its enzymes loses its function
making it metabolically inactive, speculated and
appears as a panic cell. When the old or senescent RBC
enters the spleen, Spleenic culling will occur wherein
the spleen involves removal of blood cells that are
abnormal, old, infected and blood cells with attached
antibodies.
Splenic Pitting: removal of the red cell inclusion by the
spleen.
 FACTORS AFFECTING RBC PRODUCTION
 Growth factor/ Cytokines
o Erythropoietin
-major growth factor produced by the
peritubular cells of the kidney.
-hypoxia: triggers the kidney to produce
EPO.
o IL-3
o CSF-GM
o SCF: most important growth factor
 Estrogen (female hormone): inhibit EPO
production
 Testosterone: It enhances erythropoiesis. This is
why Hgb and RBC are both higher in males than
in females. Neutrophil Granulopoiesis

 Other hormones: Pituitary & Thyroid Hormones
(T3 & T4)
 Vitamins and Minerals
o Folic acid, Vit B12: important in the
synthesis of DNA. Deficiency results to
Folate deficiency anemia and Vit B12
Anemia or Megaloblastic Anemia.
o Co, Mn, Zn, Vitamin C, E, B6, Thiamine,
Riboflavin, Pantothenic acid: important
in HgB Synthesis.
o Iron, Copper: important in HgB
Synthesis
 Proteins: globin in HgB is the protein needed in
RBC production.
- 14 days: blast stage → release of mature
granulocytes in the peripheral blood (PB).
- CFU-GM → CFU-G → Neutrophil
STAGES OF GRANULOPOIESIS
1. Myeloblast
- The usual rules in cell maturation applies
wherein the more immature the cell is, the larger
its nucleus.
- Mitotically active
2. Promyelocyte
- Azurophilic or Primary Granules starts to appear
- Mitotically active

GRANULOPOIESIS
- Includes Neutrophils, Eosinophils and Basophils.
- Share the same maturation but varies in the
morphologic changes that will occur in them.
- Progenitor Cells are:
o CFU-G: produces Neutrophils
o CFU-Eo: produces eosinophils
o CFU-Baso: produces basophils
3. Myelocyte
- The number of Azurophilic granules starts to
decrease
- Secondary granules or Specific granules are
starting to appear.
- Early and late myelocyte: continuous production
of secondary granules. Starting in the Late
Myelocyte, tertiary granules starts to occur but
will continue to appear in the metamyelocyte
stage.
- Mitotically active and this is the stage where it
undergo the last stage of mitosis.
- As the secondary granules starting to appear
from the golgi complex (which appear as an
unstained portion), the secondary granules
appear reddish tinge resulting now as if a sun
coming out called the Dawn of Neutrophilia. It
is in the Myelocyte stage where the Dawn of
Neutrophilia is observed.
 - Stage that normally appear in the circulation
4. Metamyelocyte with the amount of at least 0-5%. Since bands
- Also referred to as the Juvenile cell are still an immature cell, if it appears in the
- Nucleus starts to undergo indentation or kidney circulation with an amount of more than 5%, it
bean or peanut shaped nucleus. will now indicate Acute Leukemia
- The first stage which is incapable of mitosis. 6. Mature Granulocyte
Instead, it will undergo maturation with more
production of secondary granules. Mature Otherwise known as
- Small amounts of secondary granules are Neutrophil Polymorphonuclear
present in this stage but there is a further Neutrophil (PMN) or
production of tertiary granules Segmenter or Seg
- Basophil: dark blue to black granules Mature
- Neutrophil: pink to lilac granules Basophil
- Eosinophil: orange-red granules
Mature
5. Band/ Stab Cell/ Staff Cell
Eosinophil
- Still an immature cell.
- Nucleus undergoes elongation into the shape of
a band
- The width is one half or less than one half of the
diameter of the nucleus
- It often has the S-shaped, C-shaped, U-shaped

STAGE DIAMTER CYTOPLASM NUCLEUS NUCLEOLI N/C

(um) RATIO
Myeloblast 15-20 -basophilic & scanty non-granular -round or slightly oval 2-5 4:1
-Fine chromatin
Promyelocyte 15-21 -Basophilic & abundant -oval to round 2-3 3:1 - 2:1
-Primary granules start to appear -slightly coarser
chromatin pattern
Myelocyte 12-18 -moderate amount -oval to round none
-with few specific granules -coarse chromatin
-Secondary granules starts to
appear
Metamyelocyte 10-15 -Moderate to abundant -Indented/ kidney- none
-with specific granules bean shaped
-coarse & clumped
chromatin
Band 9-15 -specific granules -elongated or band none
shaped
-coarse & clumped
chromatin
Mature Granulocytes
Neutrophil Eosinophil Basophil
NEUTROPHIL PRECURSOR VALUES
Myeloblast 0-3% / 1-2%
Promyelocyte 1-5 %/ 2-7%
Myelocyte 6-7%
Metamyelocyte 3-20% / 15-30%
Band 9-32% / 12-18%
STAGES OF GRANULOCYTE MATURATION
 NEUTROPHIL (Polymorphonuclear
neutrophil/PMN/ Seg/Segmenter)
o Multi-segmented, normally, can be 3-4
lobes and in an older or more mature
cells they can be 5 lobes
o Abundance of 2 lobes is an indication of
abnormality known as hypolobulation.
Greater than 5 is hyperlobulation
o Cytoplasm: tiny giving grainy
appearance (pink/rose-pink violet
granules)
o Primary granules: referred to as the
Azurophilic. Presence of primary
granules in the promyelocyte stage is
not accurate for identifying which WBC
it contains
o Secondary granules: specific granules,
thus it can identify what type of
granulocyte it is by just identifying its
granules. Secondary granules will persist
until the maturation of the cell
o Eosinophil granules: red-orange in color
and larger than that of the neutrophil.
o Basophil: has few and large granules and
has a dark blue color
o Segmented neutrophils: might be
confused with the band cells but what
separate the two is the presence of
filaments in the segmented neutrophils
o Band cells: connected by a broad
connections not filaments
o Multisegmented: containing 2-5 lobes.
o Lifespan: 9-10 days
o Lifespan in the tissues depend on their
environment.
KINETICS OF NEUTROPHILS
 9-10 days
 Approximately 7 hrs. in the blood then proceed
in the tissues
(emigration(diapedesis)/transmigration)
 Life span in tissues depends on the condition. If
they migrate to the tissue without responding to
any chemical they can stay for a few hours but if
it is a response to a certain condition, their life
span lengthen up to 9-10 days because the
chemicals produced by the infected cells will
tend to increase the life span and preventing
apoptosis.
 The process of neutrophils leaving the
circulation is called Diapedesis (squeezing out)
or immigration/transmigration.
 Immigration: involves redistributing itself by
going near the vascular lining and roll and cling.
 Adhesion: is promoted by the adhesion proteins
in the endothelial cells like integrins, P selectins.
Inflammation of blood vessels make tissue
spaces wider which enable the cells to leave
 Neutrophils: capable of random and directional
motility. After random motility, they can stay in
the tissues or they can migrate to the site of
chemical attractant (directional motility). Source
of chemoreactant can be the injured tissues
 Neutrophils: first to arrive in the site of infection
 Migration: irreversible, once they arrive in the
tissue, they can no longer go back to the
circulation
 Once the cell performs its function, they die
becoming a puss cell. This is called “end-stage
cell”
 Lymphocytes: not end stage cells since they can
function again
 Neutrophils: attracted to chemoreactants
because they have receptors to it particulary the
CXCR1 and CXCR2
NOTES: How does retic released in the circulation?
- Primary factor involve is the erythropoietin which is
triggered by the hypoxia
- EPO increases mitosis increasing the number of RBC
production. It accelerate the maturation and the
release to the circulation
- First, the EPO widens the width of the marrow
sinosoids, the connection of blood vessels and
sinosoids will be widen for the entry of retic
 - EPO decreases the expression of this adhesive
receptors by the erythrocytes.
- Retic has a receptor to adhesion molecules in the
blood vessels and bone marrow. EPO decreases the
adhesion of the receptor to the retic for it to be
released
- Next factor that affects the release is the level of
maturation (cell deformability because it does not
contain nucleus)
- Another factor that has effect on the release in is the
marrow pressure exerted by the intramarrow growth
of cells. The increasing volume of RBC creates a
pressure, pushing cell in the circulation
- Blood vessels going in and out of the bone marrow,
and through this blood cells ,these cells will enter into
the circulation.
NOTES: WHITE BLOOD CELL RELEASE
- Chemotaxis meaning directional
- They leave the marrow when they are
attracted by a chemical in the tissue which are called
Chemotaxis substance (e.g. cortisol)
- Another factor that affect the release is the
number of circulating WBC. Important to note that not
all mature WBC are released to the circulation some
are stored in the storage pool and only released in the
situation where the body needs more WBC.
NOTES: PLATELETS
- Megakaryocytes are the largest cell in the
bone marrow
- For it to be release in the circulation it needs
to be extruded or protruded.
- Platelet shedding is a mechanism by which
platelet will deform into fragments for it able to be
released in the circulation
- No known chemoreactant triggers platelet
shedding.
Distribution:
BONE MARROW (M.M.S)
1. Mitotic pool (2-3 days)
o Stem cells, myeloblast, promyeloblast,
myelocyte
o Capable of reproduction and
proliferation
2. Maturation pool (5-7 days)
o Metamyelocyte, bands
3. Storage pool (11 days)
o Band , mature granulocytes
o Readily release of mature granulocytes
o Not all of them leave the B.M but will be
phagocytize
CIRCULATION
1. Marginating pool
o (1/2): Adhere all the endothelial lining
that can be formed into marginating
pool.
2. Circulating pool
o The other half will join the circulating
pool (1/2)
o 4.5-11.5 u/L of RBC count represent only
the circulating pool
o Once the marginal pool was detached,
WBC count will increase which lead to
physical or emotional stress. Reason: the
person suffers hormonal imbalance and
the detachment of granulocyte.
MAIN FUNCTION OF NEUTROPHILS:
 Defense against infections thru phagocytosis
and killing
 Destruction of foreign organisms (not Antigen
specific)
 Do not conform prolonged immunity
1. PHAGOCYTOSIS- have receptors for Fc (IgG) and
C3 (complement) have same function in
recognition of attached RBC.
PHAGOCYTOSIS
Step 1: from the circulation neutrophils move towards
chemical attractants/ first step is RECOGNITION and
moves towards the antigen
Step 2. Engulfment happens by protruding its cytoplasm
creating a vacuole called phagosome (contains the
phagocytized organism).
Step 3: Muramidase/ Lysozyme
Step 4: Killing via:
 Oxygen Dependent:
o After phagosome enters, it will fuse with
the granules of the neutrophils which is
called phagolysosomes (vacuole),
granules of neutrophil degranulate
meaning they release their contents.
Some of their contents are bactericidal
like muramidase (lysozyme)
o Does not chemically react with oxygen
o respiratory burst H2O2 and
(NaCIO/bleach) are produced
 Oxygen independent:
o degranulation
o Lysosomal degranulation together with
the presence of oxygen a chemical
reaction takes place, wherein the
oxygen will cause the release of potent
radicals which are bactericidal.
Step 5: After the killing the dead organisms will
metabolize and become waste and will become
NOTE: PAST ASSIGNMENT
exocytosis by the neutrophils. After fulfilling its function
RELEASE OF CELLS FROM BM
neutrophils also die
RBC
NOTES:
- Job syndrome (normal random, abnormal
directional)
- Lazy leukocyte syndrome (abnormal both
random and directional)
WBC
- Chronic granulocytic disease (abnormal
oxygen dependent)
PLATELETS
2. NEUTROPHIL EXTRACELLULAR TRAPS (NET)
o Form traps
o Neutrophils: once they perform their
function, will eventually die. When they
die, their components like the
cytoplasmic and nuclear components
will start to disintegrate. As the nuclear
component or neutrophils disintegrates,
the nucleoproteins from the nucleus
/nuclear material and will form into
thread like filaments or structure that
will serve as the trap.
o Some of the strands of neutrophils
contain contents like myeloperoxidase
that once the antigen is trapped, they
can still kill or destroy.
3. CAPABILITY TO SECTRETE
GRANULES:
 Primary (Azurophilic) granules:
o P.E.M.C: Peroxidase (MPO), Elastase,
Muramidase, Cathepsin.
o Starts to appear on promyelocyte stage
 Secondary granules:
o L.L.Col.B: Lysozyme, Lactoferrin,
Collagenase, (NO peroxidase in the
granules but neutrophil itself still have
peroxidase so it still react positive in
stains) β2-microglobulin
 Tertiary granules:
o ALP (late myelocyte-metamayelocyte)
 Secretory granules: responsible in secreting
other protein like the CD markers, adhesion
molecules that will promote the adhesion of
neutrophil on cell surfaces or vascular tissues
- Erythropoietin (hypoxia)
- Widens the width of marrow sinusoids
- decrease expression of adhesive receptors
- Level maturation of cell (cell deformability)
- Pressure exerted by the intramarrow growth
of cells.
- Move thru chemotaxis (e.g. cortisone)
- Diapedesis (squeeze themselves out)
- Number of circulation WBC
- Cytoplasmic shedding
MATURE GRANULOCYTE: EOSINOPHIL
 The other name for eosinophil is Acidophil
 Acidophil and eosinophil are both taken from
the staining property of the cell which takes the
acidic dye
 Methylene Blue: Basic dye
 Eosin: for acidic dye
 Eosinophil takes acidic dye or eosin thus name
eosinophil
 Reddish: Color of the cytoplasm
 Granules of eosin: takes up the eosin giving a
red-orange color
 Diameter: 9-15 microns
 Bilobed nucleus
 Distinguished by the red-orange refractile
granule of their cytoplasm
 Granules of Eosinophil is larger than the granules
of neutrophil
 Easiest to identify due to its red-orange refractile
granules, the color also serves as the pH meter
of the cell during staining
 pH of the stains for blood smear (Giemsa stain):
ranges from 6.4-6.8 pH. Lower than stated will
result to pale coloration;
 Higher pH: dark color
 Growth hormones such as IL-3, GM-CSF, IL-5
regulate eosinophil production. The role of IL-5
is very critical in the maturation as well as when
the cell is about to effect its function.
 Kinetics (distribution and migration) is similar
with neutrophil
 It joins the neutrophil in the storage pool (area
where the reserved WBC stays and will be
triggered in the condition where the body needs
it)
 Once in the circulation it stays for only 18 hours
then proceed to the tissue for 6 days
 More eosinophil will migrate (diapedesis) to the
tissues when there is an increase adrenal
corticoids production.
 Adrenocorticotropic hormone (ACTH):
stimulate adrenal glands to produce more
adrenal corticosteroid
 The effect of increase adrenal corticosteroid is to
send more blood eosinophils to the tissue
 ACTH injection will lead to decrease eosinophil
(EOSINOPENIA) in blood because eosinophils
will migrate to the tissue
 Hyperadrenalism (increase adrenal function)
will also lead to eosinopenia
 Even when there is no hormone triggering
eosinophil migration it will still migrate to the
tissue once it reaches its half-life (18 hours) in
the circulation. It will migrate specifically in the
skin, nasal membrane, lungs and GIT.
 Low number present in the blood because
mostly are found in the tissues
 During eosinophil development in the
promyelocyte stage, eosinophilic promyelocyte
has its primary granules
 Found in the primary granules are the proteins
that later on formed as Charcot-Leyden crystals.
These crystals contain lysophospholipase
coming from the cytoplasm of the eosinophils
 During eosinophil disintegration, the
components of the primary granules including
lysophospholipase will form into crystals called
Charcot-Leyden crystals
 In cases of eosinophilia cause by eosinophilic
hypersensitivity reaction, the eosinophil will
disintegrate forming crystals
 Charcot-Leyden crystals: found in body fluids
and other body specimens like in asthma
(sputum sample)
 Parasitic infection from helminths causes
crystallization of eosinophils in feces
 Joint infection cystalization can be found in the
synovial fluid
 Charcot-Leyden crystals: an indication that
there was an increase in eosinophils
 Eosinophils also contains potent oxidant as well
as cationic proteins found in granules and major
basic proteins. These contents have anti-
helminthic properties, specifically in the larval
stage of the ascaris, ancylostomiasis or
hookworm
 Loeffler’s syndrome is a type of sensitivity
reaction wherein one of the manifestations is
eosinophilia
 Eosinophil increases because of its anti-
helminthic property
NOTE:
 Eosinophil color during staining can determine
if the pH of the stain is within the range or not
 Eosinophil color is a good indicator whether or
not the stain is within the range
 Tissue eosinophil is 100x greater compared to
blood eosinophils
 Red orange granules in the eosinophils are the
secondary granules
BASOPHILS (Baso)
 Named after its staining property, it takes up
basic dye
 With few, large, dark purple to blue-black
granules
 Not normally found in tissues
 Its size is larger than that of the neutrophil and  Immediate precursor of macrophage is
eosinophil. Around 10-16 microns monocyte
 Least of the WBC, 0-1% presence in the
differential count  Mast cells are usually found in the tissue
 Growth factors involved are the IL-3, IL-5, CSF- basophils are not normally found in the tissue
GM but remain in the circulations
 Nucleus: round, indented, band or bilobed. It has
a smudged chromatin
 The appearance of nucleus is barely observed
because of the too large and too dark granules
over lapping within
 Prominent in basophil is the granulation not the
appearance
 Some of the granules may disappear during
staining (washing to be specific) because they
are water soluble
 Mast cells and Basophils are different in terms of
origin and production although may appear
similar in terms of morphology and function
 Basophils and Mast Cells are granulated with
large nucleus and often indented or bilobed.
They both have granules which contains heparin
and histamine and involved in hypersensitivity.
However, they are different and do not belong in
the same cell line
 Basophil is a true leukocyte; mast cell is not a
leukocyte though it came from bone marrow it
has a different progenitor cell

BASOPHILS
 Basophils also mediate inflammatory response
and immediate hypersensitivity reaction
 With IgE receptors so basophil can attach to IgE.
In comparison Neutrophils and monocyte has an
fc portion for IgG and C3-protein
 Granules: heparin (natural anti-coagulant),
serotonin (for vasoconstriction), histamine (for
vasodilation), eosinophil chemotactic factor A
(ECF-A)
 Basophils are involved in innate and adoptive
immunity 2
 Basophils are the least studied blood cells
because it is very few
 While basophils mediate hypersensitivity
reaction, eosinophils dampen hypersensitivity
reaction
 Basophil and Mast cells contains histamine which
is one of the signs and symptoms of allergy while
eosinophils contain histaminase that oxidizes
histamine. So eosinophils in some way can
inactivate substances produced by the basophils
EOSINOPHILS
 Eosinophil, aside from being involved in the attack
of helminth larvae they also inactivate substances
produced by basophils and mast cells and prevent
basophil and mast cell degranulation (production
and secretion of their granules)
 Eosinophils are able to dampen inflammatory and
hypersensitivity responses
 Eosinophil is attracted to chemoattractant like
Eosinophil Chemotactic factor A (ECF-A)
 In the presence of ECF-A, eosinophils will be
attracted so eosinophil number will increase
 Presence of ECF-A indicates eosinophilia
 ECF-A is secreted by basophils thus, basophils can
cause eosinophilia
 EOSINOPHIL: Hallmark of hypersensitivity reaction
because of the release of ECF-A during
degranulation of basophil and mast cells in
response to allergy

BOTH EOSINOPHIL AND BASOPHIL

  Basophils and Eosinophils are involved in anti-helminthic property. To increase eosinophils in response to
helminths, basophils are needed to degranulate to release ECF-A increasing eosinophils number.
 Basophils and eosinophils can function in phagocytosis but they are less efficient
MAST CELLS/ MASTOCYTE
 Different from basophils, though it also
originates from the bone marrow but has a
different progenitor which the Common Mast
Cell Progenitor
 Progenitor of mast cells will also enter the
circulation but does not stay in the circulation
unlike basophils
 Mast cells will migrate to the tissue and mature
as a functional mast cell
 While basophils are true leukocytes, mast cells
are connective tissue cells of mesenchymal
origin
 Almost similar morphology with basophils
except it is a bit larger in size
 Basophil and mast cells share common functions
like in hypersensitivity and inflammatory
reactions
 Basophils are initiators of hypersensitivity
reaction while mast cells are effectors of
hypersensitivity
 Granules: more numerous compared to
basophils because they are less water soluble
 Its granules contain proteolytic enzymes,
serotonin, histamine and heparin
IMMEDIATE HYPERSENSITIVITY
 During the first exposure to allergy the person
will not manifest signs and symptoms. What will
happen is that the B-cells will be activated and
be transformed into plasma cells (production of
antibody)
 Plasma cells will then start producing IgE
(produced in reaction to allergy) against the
allergen. The production of IgE will not yet
initiate the production of IgE but IgE will attach
to basophils and mast cells
 The second time the person has an encounter to
the same allergen which causes the production
of IgE, the person will manifest signs and
symptoms
 The primed (attached) IgE and mast
cells/basophils will degranulate releasing
chemical contents such as histamine and
cytokines
 The release of these chemicals causes the
symptoms of the allergy
MONOPOIESIS
- Monocyte maturation
- There are only three stages of monopoiesis
namely the monoblast, promonocyte and
monocyte
- Monocyte transform into macrophage once it
reaches the tissues
Stages
1. Monoblast
- Monoblast came from CFU-GEMMCFU-GM
CFU-M
- Large immature cell with deeply basophilic
cytoplasm and non-granular
- Has a 1-2 nuclei (mitotical active cell)
- Myeloblast and monoblast has the same
characteristic with large nucleus, agranular
cytoplasm and basophilic cytoplasm.
Differentiation between the two based on
morphology is unreliable because they look like
the same
- CD-marker through immunologic studies helps
in identification. Alternative used for the
differentiation is through the use of
cytochemical stains
- Myeloperoxidase: stain marker for primitive
myelocytic cells.
- Specific Esterase: Alpha-naphtel chloroesterase
- Granules of monocytic cells contain esterase
enzyme but of different isoenzyme. Isoenzyme
contains are positive in Alpha-naphtel acetate
esterase and Alpha-naphtel butyrate esterase
- Substrate for the myeloblast is the chloro
acetate while in the monoblast is either the
acetate and butyrate
 Monocyte (Mononuclear phagocyte) Composition and
Myeloperoxidase Sudan Specific Content
Black B Esterase - Granules of the monocyte appears ground glass
(azurophilic granules) and contains enzymes
Myeloblast + + + such as Acid Phosphatase, B-glucoronidase,
Monoblast -/ faint + -/faint+ + lysozyme(anti-bacterial), lipase, peroxidase
(smaller amount)
- Unlike granulocytes, monocytes don’t have a
2. Promonocyte storage pool in the bone marrow and as they
- Has a blue-grey cytoplasm mature they are released in the circulation
- Nucleus is slightly reduced - Life span has similarity with granulocyte from 12-
- N/C ration: 3:1 to 2:1 70 hours and from the circulation they will
- Actively dividing cell and can divide until 3 more migrate to the tissues
times - Migration toward the tissues is not influenced by
any hormones
3. Monocyte - Monocyte once leave the circulation and migrate
- Relative in size, can be between 14-20 um to the tissues is called macrophage
- Nucleus of monocyte is either round, kidney- - Thus, monocyte is the immediate precursor of
shaped; with light lobulation; folded; with fine macrophage
lacy chromatin; has an indentation Type of macrophages
- Cytoplasm: abundant, slate-grey and ground
glass
- Ground glass appearance is due to the presence
of several azurophilc granules

Monocyte Lymphocyte
largest cell in the smallest of the 5 WBCs
circulation
either deeply indented nucleus are often round
giving a horse shoe or or may have a shallow
kidney-bean shaped or indentation
slightly lobulated giving a
brain-like lobulation
has a scanty to abundant
cytoplasm and appears
sky blue but not as
abundant as the
monocyte’s cytoplasm
and colored blue-grey or
slate-grey MONOCYTE AND MACROPHAGE FUNCTION
- They make-up the Reticulo-Endothelial System
Chromatin pattern: laced-Chromatin pattern: or the Mononuclear Phagocyte System
like coarse chromatin pattern - They have a receptor for the Fc portion of the IgG
or chuncky or black and C3. They use these receptors to identify if
NOTE: The most important criteria in differentiating the cell is coated with IgG and able to destroy
monocyte and lymphocyte are the chromatin pattern this antibody coated organism
- Has the capability to remove damaged and old
red cells using their Fc portion and C3
- Macrophages in the spleen conducts spleening
culling and spitting
- Active phagocytes. Large antigen that beyond
the capacity of neutrophil to ingest or
phagocytize is ingested by the monocytes
 - Help regulate Cellular and Humoral Activity in
relation to their adoptive immunity function. T
cell is the direct effector of the Cellular response
while the B cell for the humoral
- Monocyte and the Marcophages exert a
regulatory effect to both cellular and humoral
activity by handling and processing the antigen.
First, ingestion or phagocytosis then, kill and
then handle and process the antigen and present
it to lymphocyte
- Monocyte has in their surface antigen presenting
area which they use to present antigen to the
lymphocyte and lymphocyte will produce
lymphokines (IL3) and activate macrophages
- Lymphokines(Interleukins) function includes:
activation of other resting macrophages,
activation of other lymphocytes as well as
amplification of immune respose
- Monocytes are not directly involve in cellular nor
humoral activity but they have a regulatory
effect. They are the ones sending
signal/presenting to lymphocytes to activate
other cells
- Monocyte and macrophages participate in iron
metabolism. They serve as the storage site for
iron (Ferritin, major stage form of iron)
- Macrophage are surrounded by developing RBC
trying to get iron in the storage cell. The
phenomenon by which RBC surrounds
macrophage/monocyte to get iron from the
storage site is called Suckling
phenomenon/nursing phenomenon
- Can kill malignant or tumor cells
- Can kill virally infective cells because of the
ability to secret interferon (important protein
against virus)
- Transcobalamin II: secreted by the monocyte is
important in the transport of vitamin B12
- Monocytes are also able to produce
plasminogen, the precursor of plasmin (function
in clot lysis)
LYMPHOPOIESIS
- Proliferation, differentiation and formation of
lymphocytes
- Lymphocytes are produced in two areas. Primary
lymphoid organs and secondary
lymphoid/peripheral lymphoid organs
Primary Lymphoid Organ
- Not-antigen dependent/antigen independent,
meaning there production is continuous even
without exposure to antigen the bone marrow
continuously produces B-cells, the thymus
continuously produce T-cells
- Primarily produced in Bone Marrow (B-Cells) and
Thymus (T-cells)
- All blood cells are produced in the bone marrow.
T-cells are produced in the bone marrow and
only migrate to the Thymus to mature
- Both B-cells and T-cells are capable of producing
NK cells
- B-lymphocytes represent 10-20% of the total
population
- T-lymphocytes are the majority in the population
in general representing 60-80%
- Two major population are the B-cells and T-cells
minor population is the NK cells
Secondary Lymphoid Tissues/Peripheral Lymphoid
Tissues
- Antigen dependent, start of production only
after antigen stimulation. So, the production is
not continuous.
- Mixture of T-cells and B-cells as well as NK cells
- Organ that are involved are the Spleen, lymph
nodes (waldeyer’s ring) and MALT (peyers
patches)
Stages of Lymphocyte Maturation
- There are 3 stages of maturation namely;
lymphoblast, prolymphocyte and lymphocytes
- T-cells, B-cells and NK cells are morphologically
indistinguishable the only way to distinguish
these cells are through their CD markers through
immunologic probes or CD marker testing
- Nucleus: deep purple with deeply packed
chromatin (coarse and clump); round or oval, at
times can have a shallow indentation
- Cytoplasm: sparse to abundant, pale to bright
sky blue
- Compared to other WBCs that during maturation
only undergo morphological changes,
lymphocytes, on the other hand, both undergo
morphologic and gene rearrangement
Phases
- Pre-Thymic
o Refers to the stage wherein the
precursors of T-cells are still in the bone
marrow
- Thymic Phase
o Refers to the stage wherein the T-cells
precursor already migrate to the thymus
- Post Thymic
 o Stage wherein the T-cells already leave
the tissue going to the other secondary
lymphoid tissues and the circulation
B-lymphocyte Maturation
- Progenitor comes from the bone marrow and
stays in the bone marrow until it matures into
lymphocytes
- It undergoes three stages of maturation (Pro-B
cells, Pre-B cells, Immature B cells) before it
become mature B-cells
- During the early stages (immature stage), it
undergoes gene re-arrangement while still
undergoing morphological changes
- They try to re-arrange their Immunoglobulin
gene so that each B-cells will have a unique
antigen receptor
- Mature B-cells which are not yet exposed to
antigen, leaves the bone marrow and proceed to
other secondary lymphoid organs like in the
lymph nodes. They stay as a resident in the
lymph nodes
- It is in the secondary lymphoid tissues that the B-
cells are exposed to the antigen and transform
to a more functional mature cell (plasma cell)
- B-cell when activated in the secondary lymphoid
by an antigen will divide into a memory cell (has
the ability to code specific antigen that once
exposed the body can easily recognize) and
effector cells which are the plasma cells
T-Lymphocyte Maturation
- Also passes through three stages (Pro-T cells,
Pre-B cells and immature T cells), before it
becomes a mature T-cells
- They also undergo gene re-arrangement but
here it re-arrange the antigen receptor gene, so
that each T-cells will also have a unique T-cell
receptor
- While not yet exposed to antigen, it will also
migrate to secondary lymphoid tissues where it
can be exposed into antigen. Upon exposure it
will also differentiate into a memory cell or a
effector cell
- The occurrence of different population
according to sizes (small 8-10 um, medium 10-12
um, and large 12-16 um) is accounted on their
transformation. Resting B-cells and T-cells
appear smaller, once stimulated by an antigen
they enlarge their size
- Life span: Long-lived (4-10 years) account 78-
89% of the population; Short-lived (3-4 days)
account 11-12% of the population
Lymphocytes vs other WBCs
- Unlike other WBCs lymphocytes are
heterogenous include T-lymphocytes (T-helper
and T-suppressor), B-Lymphocytes, NK cells
- Lymphocytes are not an obligate cell, meaning
they don’t die after performing the function
- Migrate into the peripheral blood and back into
the lymphatic tissue, meaning that their
migration is reversible
- There are production of lymphocyte outside the
Bone marrow or also called extramyeloid
hematopoiesis involving thymus, spleen and
lymph nodes
Function of B-cells
- Main effectors of humoral immunity
- Humoral immunity is related to antibody
productions
- Effective once transformed into plasma cells
Function of T-cells
- Main effector of cell-mediated immunity (can
directly attact antigen) including delayed
hypersensitivity reaction
- Graft rejection
- Defense against intracellular organism and
neoplasm
T-cells Subsets
- Helper T cells (CD4)- help activate B cells to
secrete antibodies and macrophages to destroy
ingested microbes, but they also help activate
cytotoxic T cells to kill infected target cells.
- Supressor T-cells (CD8)- A type of immune cell
that blocks the actions of some other types of
lymphocytes, to keep the immune system from
becoming over-active
- Cytotoxic Cell (CD8)- CD8 effector lymphocytes
are capable of killing target cells by secreting
granules containing granzyme and perforin or by
activating apoptotic pathways in the target cell.
These cells are sometimes referred to as
cytotoxic T lymphocytes
- Delayed Type Hypersensitivity Effector cells
(CD4)- a major mechanism of defense against
various intracellular pathogens, including
mycobacteria, fungi, and certain parasites, and it
occurs in transplant rejection and tumor
immunity.
Non-A Non-B population
- NK cells- effector lymphocytes of the innate
immune system that control several types of
 tumors and microbial infections by limiting their
spread and subsequent tissue damage.
- Killer Cells- enzymes that can kill tumor cells or
cells infected with a virus
Plasma Cells
- Transformed B-cells
- Oval/egg shape
- Cytoplasm: deeply basophilic because of being
metabolically active having more RNA
- Eccentric nucleus; hof (unstained area,
perinuclear area);clear area or the golgi
- Chromatin: condense coarse-blocks- “tortoise
shell”; radial or wheel-like pattern
MEGAKARYOPOIESIS/ THROMBOPOIESIS
- Refers to the proliferation, maturation and
production of megakaryocytes until the
shedding into the platelets
- May also referred to as thrombopoiesis because
the end product are the thrombocytes
- Production occurs in the bone marrow and the
progenitor cell is the CFU-Meg
- Before it become a CFU-Meg it passes through
being a BFU-Meg
- BFU-Meg is the first progenitor committed to
become megakaryocyte (note: CFU-MegE is a
committed progenitor but will either develop to
become both megakaryocyte and erythrocyte)
- Important growth factor that promotes the
development of stem cell to become a
progenitor cell is the thrombopoietin which
behave in the same manner as the
erythropoietin
- The development of progenitor CFU-MegE to
become either megakaryocyte or erythrocyte is
dependent to what specific hormone stimulated
the development (either erythropoietin or
thrombopoietin)
- The first two progenitor such as BFU-Meg and
CFU-Meg are capable of cell division or mitosis
- Light Density Colony Forming Unit Meg (LD CFU-
Meg) is no longer capable of mitosis. Instead
only its nucleus will divide. It loses its capacity to
divide but its nucleus is still capable of division
and this process is called endomitosis. This
process is unique to megakaryopoiesis
- Endomitosis is a process where there is a nuclear
duplication but no cytoplasmic duplication and
cell division
- Nuclear division in this stage increases its
chromosomal number
- This is an exemption to the general rule of the
cells when it comes to size that when the cell
mature the cell size decreases
- As a result of continues nucleus division it
become multilobulated, has a large cytoplasm
and a large cell size
- Thrombopoietin and IL-11 influence
megakaryopoiesis
- LD CFU-Meg will transform to become MK-1
(Megakaryoblast), then MK2
(promegakaryocyte), then the MK3 or immature
megakaryocyte (granular megakaryocyte) and
the mature megakaryocyte is the same as MK4
or metamegakaryocyte
- Due to endomitosis the nucleus of this cell will
appear as divided but the division will not
actually result to separation, they remain
connected and so the cell will appear as instead
multinucleated, it will appear as multi-lobulated
- The number of nucleus will increase until such
time it will reach the minimum amount of
nucleus which is 4
- Any stage having less than 4 nucleus will not be
able to produce platelets
- Platelet is produced via mechanism called
platelet shedding. First is the formation of
proplatelet process wherein the megakaryocyte
will protrude it pseudopod that will then be cut
into individual platelet thus called platelet
shedding
STAGES OF MEGAKARYOPOIESIS
Stages:
1. Megakaryoblast
- Same as the MK1
- Has only 1 nucleus that on division (endomitosis)
it become two (promegakaryocyte)
- Starting to synthesize and produce its structures.
One important structure in this stage is the
present of DMS (Demarcating Membrane
System). DMS refers to a demarcation line that
invaginates the cytoplasm of the cell which later
on divide the platelet and serve as the future
membrane structure of the platelets
- Cytoplasmic granules start to produce in this
stage but not yet visible but as it matures it
develop more
- No platelets
2. Promegakaryocyte
- Has two nuclei
 - Few cytoplasmic granules. The granules present
in this stage is called the alpha-granules (storage
granules) and the dense granules
- Platelets are not visible yet
- Present DMS
- MK2
3. Immature Megakaryocyte
- MK3
- Two or more nucleus but should not increase up
to 4
- DMS is starting to disappear or already disappear
in this stage because the cytoplasm starts to sub-
divide
- Numerous granules
- Platelet not yet visible
4. Metamegakaryocyte
- Mature megakaryocyte
- 4 or more nucleus
- MK-4
- Granules start to aggregate and form into
platelets
- When these aggregated granules are separated
it becomes the platelets
- Platelets are visible
- The more endomitosis occurs, the more nuclear
lobulation happens and the more the platelet
produce
Platelet Shedding
- First, once the metamegakaryocyte is already
mature, it extends its cytoplasm into a long
pseudopod that will thin-out at the base until
they are ready to being fragmented into an
individual platelet. This process or formation is
called the proplatelets
- The naked nucleus (with exhausted cytoplasm)
will undergo apoptosis
- In summary, the maturation time from the
development of progenitor until becoming a
platelet takes about 5 days
- Life span: 8-11 days
- Megakaryocytes are diploid
- The minimum number of platelets capable of cell
division is 4
- Some of the platelets newly produced may
appear reticulated meaning they are newly
released from the bone marrow and contains
more RNA
- Newly produced platelets from the bone marrow
is more efficient in coagulation and hemostatic
function and more active
- As they near to reach their life-span they become
smaller
- The newly released platelets are larger in size
compared to those already stayed in the
circulation
Platelets
- Products of megakaryopoiesis
- Otherwise called thromboplastins or
thrombocytes
- Technically not a cell because they don’t contain
nucleus rather, they are cytoplasmic fragments
(they don’t represent a complete cytoplasm)
- When newly released platelets appear to be
connected with each other it is called platelets
ribbons
- Size: 2-4 um in diameter, tiny around 1/8 the size
of erythrocytes
- Light blue to purple, very granular (Romanowsky
stain)
- Life span: 8-11 days
- Daily turnover: 35x 10^9/L (+/- 4.3)
- Distribution in the circulation (60%) and in the
spleen (30-40%)
- Platelet in the spleen is not permanent resident
but rather they interchange with those in the
systemic circulation and those in the circulation
can also go in the spleen
NOTE:
- When performing the platelet count, only
counted are the platelets in the circulation
- In cases of Splenectomy or the removal of
spleen, the platelets that should be in the
spleen will go in the circulation. Removal of
the spleen will therefor cause thrombocytosis
- In splenomegaly, or enlargement of spleen
instead of storing only 1/3 of the platelets it
will take more platelets and result to
thrombocytopenia
NOTE:
Function of the Platelets
- Function in hemostasis (blood stoppage)
- Capable of forming hemostatic plugs. They
are capable of adhering forming platelet
aggregates or plug that will seal damage
vessels
- Maintenance of the integrity of the blood
vessels
- Form hemostatic plugs to stop the loss
 Stage Nucl Cytopla Cytopla Thrombo 1. INFORMED CONSENT
eus smic smic cytes - Implies voluntary permission for medical
Tags Granule Visible procedure, test or medication will be given.
s
- Activities to be done should be explained to the
MK-I 1 Present Absent No
patient.
MK-II 2 Present Few No
2. EXPRESSED CONSENT
MK-III 2 or Usually Numer No
mor absent ous - May be given verbally or in writing
e 3. IMPLIED CONSENT
Metamegakar 4 or Absent Aggreg Visible - Does not require a verbal expression of consent
yocyte mor ated nor written consent. Actions can imply consent
e (e.g. the patient holds out an arm after being told
Platelets/ Thrombocytes a blood specimen is to be collected.)
 2-4um in diameter 4. HIV CONSENT
 Light blue to purple and very granular - Specify exactly what type of information must be
 Life span: 8-11 days given to inform the clients properly.
 Daily turnover: 35 x 10^9/L (+/- 4.3) - Has a pre and post counselling.
 Maturation: about 5 days o Pre-counseling: the patient should sign a
 Distribution: in the circulation (60%) and in consent
the spleen (30-40%) o Post Counseling
5. CONSENT FOR MINORS
- Parents or guardian consent is required (health
care professional who do not obtain it are liable
for assault and battery).
6. REFUSAL OF CONSENT
- An individual has a constitutional right to refuse
a medical procedure such as venipuncture.

SOURCES OF BLOOD SPECIMEN

BLOOD SPECIMEN COLLECTION
PHLEBOTOMIST
- Referred to someone who draws blood whether
through vein or skin puncture
CHARACTERISTICS OF A PHLEBOTOMIST:
 Phlebotomist must have a good interpersonal
skills
o Ability to interact with the patient
o Never lie to the patient, tell them that
the procedure might be painful but
tolerable.
 Maintain a professional attitude
o Must act and look professional
 Ensure patient confidentiality
o The profession dealt with different
people in different social classes.
Maintain confidence even though some
of the patients are a bit intimidating.
1. CAPILLARY
- An area where vein and artery meet
- Blood present is a mixture of venous and arterial
blood.
- Surrounding the capillaries are the tissue and
tissue fluids so the presence of tissue fluids in the
specimen can be observed.
- Pale in appearance because it is a mixture of
arterial and venous blood and tissue fluid.
2. ARTERIES
- Contains the blood pumped by the heart. Thus,
the blood is under pressure.
- Arteries has a thicker and more rubbery walls as
compared to venous vein.
- Bleeding stays longer in the artery compared to
the vein.
- Described as a bright red in color.
- Advantage: More uniform and consistent in
composition.

TYPES OF CONSENT
NOTE: person and patients with mastectomy, venous
 The use of tourniquet is not needed in arterial puncture is limited.
blood collection because veins can be easily
palpitated. NOTE:
 Need not to push the plunger the pressure of Skin puncture is only performed in POCT only.
the blood will allow it to flow in the syringe
and plunger will be automatically be pulled.
SKIN PUNCTURE DEVICES
 Since there is an increase pressure,
 Sterile Lancets
phlebotomist must assure that the bleeding
already stops before leaving the patient. - Standard length of the blood lancet should be
1.75mm.
- >1.7mm: not recommended
3. VEINS  Needles (Clover’s, hagedom0
- Blood obtain is dark-red in color.  Blades (Barb Parker)
- Venous blood contains most of the metabolic  Tenderfoot
products. Thus, the composition will be affected  Temderlett
by the metabolism of the tissue where it drains
from. SITES FOR SKIN PUNCTURE
1. FINGER
MICROSAMPLE - For adult, the third and fourth of non-dominant
- Known as the collection from the capillaries hand. Either the ring or the middle finger.
- Any sample that is less than 1mL. - Puncture should be on the area accessible to the
SKIN PUNCTURE thubk or facing the thumb.
- Puncture area should be “off center” nerve
- Also called Capillary/ Arteriolar/ Peripheral endings usually at the center of the finger so it is
Puncture. more painful to puncture in that area.
- A good skin puncture can yield 0.5mL of blood - Avoid heavily callous area
specimen - Depth of puncture should not be deeper than 3.1
- Can be best substitute for arterial blood gas. Site mm.
should be pre-warmed at 39-40°C for 3-5
minutes to arterialize the site.

GENRAL SKIN PUNCTURE ARE PERFORMED IN THE

FOLLOWING PATIENTS:

1. INFANTS
- Performing phlebotomy in infants can cause
phlebotomy-induces anemia. Thus, skin
NOTE: PROCEDURE FOR FINGER PRICK
puncture sis preferred.
a. Select the proper site of puncture
2. YOUNG CHILDREN
b. Disinfect with 70% of isopropyl alcohol
- 1-3 years old. c. Allow it to air dry.
- Skin puncture is performed, if a small amount of d. Puncture across the grain of the skin. Do not
blood needed in the test. prick parallel.
3. ADULTS WITH DIFFICULT VEINS
- Normally, venous puncture is performed but in
cases of obesity (difficult to palpate), geriatric
patients (fragile vein and loose skin), patients
who are undergoing chemotherapy as well as
those who are undergoing dialysis, good vein in
these patients should not be used without the
permission of the nurse in charge). Totally burnt
 2. HEEL
- For infants less than 1 year old.
- Exact location is the Medical or Lateral plantar
surface of the heel.
- Puncture should not be deeper than 2.0mm
- Puncture in the heel (center area) is not
recommended because the flesh is too thin.
Thus, bone might be punctured.
3. EARLOBE
- Flesh portion of the ear
- Less painful puncture size
- More vascularized so enough blood sample can
be gathered.
- Blood collection is done in this site normally
when testing for abnormal histiocytes.
- Also ideal in Arterialized Blood Samples. Foe ABG
test, especially when arteries of the patient is not
available.
- Advantage for ABG: can be easily pre-warmed
just by flicking.
NOTE:
 Pre-warm the puncture site at 42°C to
arterialize the site.
 Pre-warming can be through the use of pre-
warmed towel or hot pack.
ERRORS IN SKIN PUNCTURE
1. SAMPLE ERROR
- Since it is a mixture of both arterial and venous
sample, the composition is different.
- It has a lower Hgb, Hct, RBC and Platelet count
than a venous blood but has a higher WBC for
about 1000/µL.
2. EXCESSIVE MASSAGE
- With good puncture, a phlebotomist can obtain
0.5mL of blood without milking or squeezing.
- Milking; increases tissue fluid component in the
sample.
3. EXCESSIVE CRYING
- Excessive crying increases circulating WBC from
marginal pool
- False increase WBC count.
DISADVANTAGE
 Test cannot be repeated due to small volume of
blood
 Cannot be sent to National Reference
Laboratory.
SITES TO BE AVOIDED IN SKIN PUNCTURE
 INFANT:
o Central arc area of the heel and finger
o Inflamed and pallor areas (will result to
a false increase of neutrophil).
o Cold and cyanotic areas
o Congested and edematous areas
o Scarred and heavily calloused area
NOTE:
Puncture should not be more than 2mm depth
because vessels of infants are usually at 1.6mm deep.
 ADULT:
o Thumb, index finger, 5th finger
o Finger on the sire of mastectomy
o Edematous or previous puncture site.
ARTERIAL PUNCTURE
- Arterial Blood Gas Analysis (includes pO2, CO2,
pH and electrolytes) is the most common test
performed using arterial blood sample.
- Since puncturing arterial sites are bit
complicated, it is not used for routine analysis,
blood banking, chemistry and hematology.
- Sites:
o Radial: most preferred.
o Femoral: Last resort
o Scalp
o Umbilical Artery
- Performed without tourniquet since pressure is
already high. Application of tourniquet will alter
the blood gas composition of the sample.
 NOTE:
 Bleeding in this site clots longer that is why
pressure should always be applies.
 Femoral is the last resort because of the
tendency of the prolonged bleeding that may
occur.
RADIAL ARTERY
- Gauge: 23-25.
- Modified Allen Test should be performed.
- 45-60°: Angle of the needle
- Radial site has two arteries:
o Radial Artery: puncture site; cann
o Ulnar Artery: serves as the producer of
oxygen while radial cannot produce
oxygen due to the puncture. To ensure
that the ulnar artery can produce
enough oxygen, Modified Allen Test
should be performed.
BRACHIAL ARTERY
- Gauge: between 18-20
- Angle: 45-60°
NOTE:
 Scalp artery:
- can be used for children
 Among newborns whose umbilical artery can
still be drained it can still be used for blood
collection. It is the best site for newborns for
blood gas analysis
 The most appropriate anticoagulant for ABG
is the heparin (0.05 mL of heparin per 1 mL of
blood
 Heparinized blood should be placed in ice
water or coolant after collection to prevent
the consumption of oxygen by the WBC
 The best alternative if the arterial sites are not
available is the Arterialized Capillary Blood.
Acceptable for blood gas such as pH and pCO2
but not for pO2
MACROSAMPLING
- Can be done using open system, evacuated tube
system and winged infusion set 9can be done
wither using open or closed system).
MACROSAMPLING METHOD
1. EVACUATED TUBE SYSTEM
- Preferred method
- Minimize specimen contamination and exposure
to blood
2. SYRINGE OR OPEN SYSTEM
- Discouraged by the CLSI due to safety and
specimen quality issue
- May be used in small, fragile or damage vein
3. WINGED INFUSION SET (BUTTERFLY)
- Can be used with the evacuated tube system or
syringe
- Often used to draw blood from infant or children
- From hand vein and other difficult draw situation
MATERIAL FOR BLOOD COLLECTION
1. TOURNIQUET
- Release the blood when blood begins to flow
- Increases the venous filling and thus, it will be
thinner and easier to puncture
- Applied to obstruct the circulation and increase
the venous filling
- Should not be applied longer than 1 minute
- Applied to distend the vein- makes vein larger
and easier to find, and it stretches the wall of the
vein
2. NEEDLES
- With color coded hub indicating the gauge
- Gauge 21: considered standard for routine
venipuncture
- Gauge 21-23: (23 for children; 21,23,25 for
butterfly winged infusion set)
- Gauge 25: for collection of blood form the scalp,
or other small vein
- Gauge 16: for blood donation
- Length: 1 or 1.45 inches (G21-23); ½ to ¾ inch in
butterfly

3. EVACUATED TUBE SYSTEM
- Basic component: multisample needle, tube
holder, evacuated tube (with premeasured
vacuum that draws the indicated volume of
blood; Tube stopper are color coded to identify
the type of additives or the absence of additive
or special tube property
- Additives can be in a form of anticoagulant,
procoagulant, antiglycolytic agent and
polyanethol sulfonate
4. SOLUTION FOR SKIN ANTISEPSIS
- Routine collection: 70% isopropyl alcohol.
Slowly penetrate the bacteria. More than 70%
will harden the bacterial wall preventing the
penetration to the genome, thus the bacteria
will not be destroyed
- Test for legal blood alcohol level uses non-
alcohol-based antiseptic. Use instead
benzalkonium Chloride or Zephiran
- Blood Culture: Two steps (30-60 seconds scrub
should be followed) with 70% isopropyl alcohol
and then 1-10% povidone-iodine, or tincture of
iodine, or chlorhexidine, or another isopropyl
alcohol preparation.
- One step: Chlorhexidine gluconate/isopropyl
alcohol mixture or povidone 70% ethyl alcohol
mixture
- Patients like infants or adults with sensitivity to
iodine or alcohol should be given chlorhexidine
gluconate instead
SITES FOR BLOOD COLLECTION
- We are not supposed to extract blood to young
children especially to those new born, only
trained individuals can extract blood to new born
babies.
- To those who are trained they get to the
following sites:
 NEW BORN UP TO 18 MONTHS
o Superior longitudinal sinus vein
o External jugular vein
o Temporal vein
o Application of tourniquet is applied if the
vein is not prominent but not in all
instances, in collection of ABG do not
use tourniquet.
 18 MONTHS FOR 3 YEARS OF AGE
o Femoral vein
o Long saphenous vein
o Popliteal vein (we are not allowed to
take blood at this site)
 FOR ADULTS:
- The most common site is the Antecubital
- Fossa (bend of the elbow) the antecubital vein is
the vein found on the fossa. There are three
major veins on this fossa.
o MEDIAN CUBITAL VEIN:
 Cubital is formed between
basilica and cephalic.
 It is the most prominent and
well-anchored vein.
 It is the first priority vein
 If not accessible proceed to
cephalic vein.
o CEPHALIC VEIN:
 Located outwards of the body or
at the lateral side.
 Most prominent vein among
obese individuals.
o BASILIC VEIN:
 The one near at the body or the
one located towards the body.
(B for breast; located near the
breast)
  The collection at basilic is
discouraged, unless it is the only
site.
 If both arms of the antecubital
are not accessible, proceed to
the cephalic of the same arm but
if both arms of the cephalic is
not accessible then proceed to
the basilic vein.
 Basilic vein is the last resort of
the upper extremity.
 The reasons why the collection
of blood is discourage in this site
because:
1. It is very movable compared
to cephalic, cephalic is relatively
anchored.
Well achored-Cubital
Relatively anchored-Cephalic
Less anchored- Basilic
2. There are other structures
beneath or beside it.
 H PATTERN
- The veins formed as it letter “H”
- The most preferred is the MEDIAN CUBITAL VEIN
- If median is not accessible then go to the
CEPHALIC VEIN
- If the cephalic is not accessible then go to the last
resort which is the BASILIC VEIN
 M PATTERN
- The veins formed as it letter “M”
- In M pattern the median vein is still
- The most preferred vein, but it should not
collected below.
- 2nd priority is the cephalic and last is the basilica
vein.
H PATTERN M PATTERN
There is a vein that Cephalic and basilic will
dissects or separate the merge to form a Median
three major veins. vein.
Median vein is just like Median vein is from
the median cubital vein cephalic & basilic vein.

NOTE:
NOTE:
Cubital: well anchored
If both arms of the antecubital fossa are not
Cephalic: Relatively anchored
accessible, we can try the lower arm which is the
Basilic: Less anchored
superficial veins of the wrist or the dorsal hand.
 Superficial Veins of the Dorsal Hand
Difference between how it feels to palpate:
-We can get at the wrist vein but not in the beneath of
Vein- It is more rubbery in consistency
the wrist, it is connected to the cephalic however it is
Artery- It is also rubbery but it is harder as compared
movable.
to the vein and it has a pulse or it pulsate.
- We can also collect at the dorsal parts of the hand,
Nerve- It is not rubbery.
there are also major veins here like the one connected
in the basilic and cephalic.
Beside the Basilic are the following:
- The veins in the dorsal hand is still connected in the
basilic and cephalic vein, so collection at this site is still
 Brachial artery
considered as venipuncture/venous puncture.
- The effect of puncturing the artery is that, the
- We need also to locate and follow the geography of
bleeding might not stop, because the blood pressure
the patient’s vein.
is higher.
 Median nerve
- If you accidentally hit the nerve it will cause an
intense pain to the patient.

GEOGRPAHY OF THE VEIN

- We have to identify first which pattern does the
patients hand organizational of the vessel
presents.
- There are two pattern, the H and M pattern.
NOTE:
If all vessels at the upper extremity are not accessible,
we can go to the lower extremities, but it should be
the last resort.
 Superficial Veins of The Foot
-The last resort for blood collection is from the foot
veins after the arm veins have been determine
unsuitable. Always check with the hospital policy
before with this type of sampling is carried out.
- There are some patients
contraindicated with this site, like those who have
1. Diabetes Mellitus- one of the signs of DM is pro-
longed unhealing of the wounds, they have abnormal
circulation that is why there is pro-longed unhealing
and the gravity was pushed outwards.
2. Thrombotic disorder
-it refers to clot formation , clot formation is normal
if there is physiologic response but patients suffering
from abnormal thrombosis are those prone to suffers
clots, unneeded clots, & abnormal clots.
-If you are to perform venipuncture in this site to those
person who suffers thrombosis, the clot will
disseminate unlike to those who do not have
thrombosis where clot is not disseminated or you can
perform venipuncture in one site.
Vasoaclution- when the blood circulation is block by
the clot that will lead to failure of circulation.
3. Hemoglobinopathies- like those suffering from hb S
disease, wherein the sickle cell will obstruct the
circulation.
REMINDER:
When performing a venous puncture, the proper way
of handling the syringe/holder is:
• The tip of your thumb with the tips of your fingers.
• DO NOT grasp the entire assembly, otherwise you
will not see the tube if it is being filled
When you attempt to insert the needle, although we
are allowed to hold the skin to anchor the vein, it is
wrong to anchor the vessel using for finger like this:
• Putting your finger like this can cause accidents,
because there is the possibility of puncturing yourself
once the patient will move.
The correct way to anchor the vein is to pull the skin
bellow using your thumb and insert the needle bevel
up and slide on your tube.
• In sliding also of your tube make sure that it is your
thumb pushing it, while your index finger holding on
the sleeve of the evacuated tube of the holder.
• The force should not come only on one side rather
the force will meet at the center.
• When you withdraw the tube, do not just pull the
tube with your thumb and index finger because of the
possibility to withdraw the entire needle.
• You should pull the tube while pushing the
evacuated tube holder with your index finger.
• When it is already filled make sure to mix/ invert
immediately the Evacuated tube with anticoagulant to
prevent microclot formation.
• Apply gauze not a cotton and discard the needle
safely.
SITUATIONS IN THE PHLEBOTOMY
VADs-Vascular Access Devices
GENERAL RULE:
We are not supposed to extract blood on this site,
unless this are the only sites available. It should be
done with the person who has special training on this
procedure.
 ARTERIAL VASCULAR SHUNT/
FISTULA
- Fistula- means connection, it
is connection implanted surgically
to connect the arterial and veins. The
purpose of this shunt is to give access
to dialysis.
- We are not able to collect in this site
because we are not allowed to apply
the tourniquet on the same arm
with the fistula, because the shunt will
be damaged.
 The two metabolic products formed are the (1) Urea
 ARTERIAL LINE from protein metabolism and (2) Creatine from
- It is a tubing connected to an artery, like in the muscle metabolism. This are importantly measured to
radial artery. Its purpose is in monitoring assess renal dysfunction.
blood pressure and serial blood gas analysis.
- The collection of ABG can be done in this site.
- The indwelling lines are catheters connected to SITUATION ACTION
veins usually in the lower part, is connected into Sclerosed veins, scars, Select another site.
a diagram which has an opening at which drugs burns Skin in scars and burns is
can be infused and collection of blood can also thick and vascularity is
be done. not normal as well as the
- To prevent the clotting of the tubing line, it is blood circulation
connected in a vessel that is usually flushed with Tattooed arm Especially on new or
heparin/ saline. recent tattoos, the ink
- The heparin does have anticoagulant that will alter the results and
adheres on the tubing, so expect that during the the site is prone to
collection, the blood will mix with heparin/saline infection.
in a lock called the hep-lock or heparin lock/ Edema It swollen therefore veins
saline lock. are hard to palpate and is
- When collecting on this site, you need to discard contaminated with fluid.
the first 5 mL, which contained more Hematoma -Hematoma is a swelling
heparin/saline. Then collect the next volume. due to the presence of
blood clot.
 IV LINE; PREVIOUS IV SITE -Not allowed to draw on
- In the previous site, the collection is this site. Instead, draw in
not advisable in 24-48 hours from the removal of another arm or below the
the IV line because the circulation of the same hematoma.
arm still has the fluid infused in it. The fluid is still - Minimize venipuncture.
present and can alter the results. Hold pressure until
bleeding has stopped.
NOTE: What if the IV is ongoing? Streptokinase/ Tissue Minimize venipuncture.
Do not extract from it, but if it’s the only site, the same plasminogen activator Hold pressure until
arm can be used but use a different vein or lower than (tPA) bleeding has stopped.
the IV site. Streptokinase have pro-
- In general, do not extract blood from this site. longed bleeding time and
-When the extraction is done on this site, the blood clots are longer.
tourniquet should not be used due to the other lines They have more plasmin
connected to the vessels. production.
-The application of pressure caused by the tourniquet The clot will be dissolved
may damage the line of the catheter. prematurely.
-blood collection in sites where IV line is connected, Mastectomy Draw from opposite arm.
the IV fluid should be stopped for at least 2 minutes -Lymph nodes were
which is done by the nurse. removed from the breast,
- Discard the first 5 mL therefore the lymphatic
- Since it is mixed with IV fluid, we need to indicate the vessels become
container/tube that it is collected from the IV Line. obstructed leading now
What will increase in the sample? to lymphostasis wherein
It depends on what fluid is infused. the lymphatic fluid
- In NSS, Na and Cl will increase in patient values. accumulates because of
the obstructive lymphatic
- Regardless of what fluid was infused, Urea and flow leading now to
creatine will decrease because of the dilution. edema. This site is more
prone to infection
 because of the absence of blood will not
lymph nodes. enter the bevel.
Needle is not If hematoma
completely in the haven’t
SITUATION CAUSES REMEDY vein or has not occurred, push
No blood seen Needle may not Remove the reached the vein the needle until
or too little be placed at the tube and re- which will result you reach the
blood flow into center of the insert the to hematoma. vein and blood
the tube stopper causing correct angle. flow occurs.
blockage Underfilling Premature Reintroduce
Needle bevel Insert the the tubes removal of tube tube for
may be flushed needle bevel collection until
against the wall up, rotate the vacuum is
of the vein needle ¼ completely
causing blockage clockwise just exhausted.
to release the Blood stops -The vein may In close system,
bevel of the flowing have been remove the
needle. halfway during collapsed evacuated tube
Tourniquet Release the blood -Too much to regain same
applied too tight tourniquet collection pressure like lumen of the
or too long application. using a large vessel and
Note: needle against a comes back to
application small vein being dilated.
should not be -too much
more than 2 pushing of
minutes. plunger will
Tube may have Use a new tube. cause excessive
been pressure against
prematurely vein.
punctured or -The pressure of
may have sucking will cause
previously been the vein to
opened resulting collapse/
to lose it vacuum constrict.
because of Needle may have Repeat
venting air. In this been venipuncture at
case, blood repositioned or different site
cannot enter into out of the vein when
the tube. during hematoma
Needle has If hematoma venipuncture occurs. Slightly
transfixed the haven’t withdraw and
vein (going occurred, feel the vein.
through the back slightly If patient Avoid head
wall of the vein). withdraw the collapsed injury
The blood will needle until the If the patient Immediately
move out in blood enters faints stop the
nearby tissues the bevel. procedure.
(extravasation),
and it will clot
there resulting to ORDER OF DRAW FOR SKIN PUNCTURE
hematoma. In a) Tube for blood gas analysis
this case, the b) Slides, unless made from specimen in the EDTA
microcollection tube
 c) EDTA microcollection tube - Thrombin comes from the protein
d) Other microcollection tubes with anticoagulants called prothrombin (factor 2), which is converted by
e) Serum microcollection. prothrombinase complex (Activated Factor Xa,
Activated Factor Va, phospholipids and calcium)

Second Point of Conversion

- When prothrombinase complex is formed,
prothrombin is able to be converted into thrombin
- Thrombin then convert fibrinogen into fibrin.
- Note: The first mechanism of action of the
anticoagulant is either they inhibit/remove calcium.
Removal of calcium will prevent the prothrombinase
complex to be formed
- Note: Anticoagulants that initiate the removal of
calcium are the EDTA, Sodium Citrate, and oxalate.
EDTA removes calcium by chelation, while Na citrate
combines with calcium and inhibit it. Another is the
oxalate, which acts by combining to calcium.
- Note: Second mechanism is by inhibiting thrombin.
SPECIMEN PREPARATION
Anticoagulant such as heparin inhibit thrombin.
- In hematology, the following samples are used:
- Note: All anticoagulants inhibit calcium except
1) WHOLE BLOOD heparin (thrombin inhibitor). Heparin acts with
- Most common type of blood used in hematology antithrombin III in inhibiting thrombin.
- Prepared using anticoagulant so that the entire
blood components can be examined
2) PLASMA EVACUATED TUBES USED IN SERUM REPARATION
- anticoagulants
3) SERUM
TUBES ADDITIVES SECTION
- Liquid portion of clotted blood
Red (glass) None Chemistry and
- Serum can be prepared by allow it to clot and
-takes 60 Serology
separate the serum or via defibrination minutes for
- Defibrination: Removal of fibrin component of complete
the sample to get the serum. Done in an clotting
Erlenmeyer flask where whole blood is placed Red (plastic/
Silica Chemistry
and sterile glass beads are added. Afterwards, hemogard) (procoagulant) and
swirl the flask until the beads’ sound are no -This additive serology
longer audible provides more
surface for the
NOTE: CLOTTING PROCESS platelet and
coagulation
factors to be
activated
-Requires 5
minutes
Yellow/gray Thrombin STAT chemistry
orange (common
First Point of Conversion procoagulant)
- Conversion of fibrinogen (factor 1) into fibrin -Clotting time
- What converts fibrinogen to fibrin is the thrombin takes only
(factor 2a). Thrombin 5mins
through enzymatic cleavage, enzymatically converts Red/gray and -Clot activator Chemistry
fibrinogen to fibrin (clot) gold (often silica)
 - separator gel
(complete
clotting is 30
minutes)
NOTE:
-Procoagulants promote clotting by either providing
more space for the platelet to be activated or shorten
the coagulation mechanism causing the conversion of
fibrinogen into fibrin
-After centrifugation the gel separator can be found in
the middle of serum and pack RBC. Gel separator is
inert material that has a thixotrophic activity.
Thixotrophic meaning has the ability to change from
thick to thinner gel
 Most Commonly Used Anticoagulant is EDTA
(K2, K3, Na)
- Most commonly used anticoagulant in
hematology
- Preferred anticoagulant in hematology for
complete blood count and platelet counts
- Advantage in platelet count: EDTA prevents
platelet from aggregating together and platelet
adhesion
- Disadvantage in platelet count: It often result to
platelet satellitism
- EDTA is also ideal in the preparation of blood
smear if the preparation is done after 2-3 hours
of collection only, although blood smear can be
prepared using freshly collected blood. Beyond
2-3 hours morphological changes/distortion can
be observed:
o WBC: vacuolation of cytoplasm; more
homogenous nuclei; irregular or poorly
defined cytoplasmic borders and
irregularly shaped nuclei
o Platelet: increases in size then
disintegrate
NOTE:
The purpose of smear is to examine the WBC for
differential count
- Prolonged storage of RBC in EDTA will result to:
o First, they will crenate, after 6 hours RBC
will start to swell thus, the volume is also
increased resulting to increase
Hematocrit (packed cell volume), mean
cell volume (mcv), osmotic fragility test
(oft)
o Erythrocyte sedimentation rate (which
undergo rouleaux formation/pile of
count appearance, for the first 10
minutes) will be slowed down, decreases
and will not undergo in a rouleaux
formation due to swollen cells. Rouleaux
formation allows the cell to settle faster
o Corpuscular Hemoglobin Concentration
(MCHC) will also decrease. Formula is:
HgB / Hct
o Prevention: Refrigerate at 4°C, the cell
will be valid for 24 hours to use in the
test such as Hct, MCV, OFT, ESR, MCHC.
Beyond 24 hours only cell count can be
performed.
- Hemoglobin remains stable for several days in
EDTA even RBC already lysed and shrink
- Platelet satellitism: one disadvantage of EDTA.
The platelet surrounding the neutrophil. Platelet
surrounding the neutrophil can still be
recognized in smear but if automated reading is
done, the system will not be able to count those
surrounding the neutrophil thus resulting to
falsely decrease platelet count
- Platelet satellitism occurs only if the patient
develop antibody against EDTA. IgG coated
platelets (formed as antibody against EDTA) will
attach to the fc portion of neutrophil forming
platelet satellites. Remedy is the usage of
Sodium citrate tube instead of using EDTA tube.
However, because the ratio of citrate additives
to blood is 1:9 it is more diluted. So whatever,
result of platelet count gathered in Sodium
citrate it should be multiplied to the dilution
factor which is 1.1
- EDTA is not recommended in Coagulation test
o EDTA interferes in the interaction
between the thrombin and fibrinogen
o It does not preserve well the factor V
(labile factor) which is important
coagulation factor unlike in Citrate
o Citrate more preferred for coagulation
testing because it preserves labile
factors such as factor V and VIII
- Ratio: 1.5mg of EDTA: dL of blood
- The minimum amount of blood that should be
placed in the EDTA tube is atleast haft-full. Less
than required will result to excessive
anticoagulation. Excessive anticoagulation will
result to:
o EDTA is hypertonic anticoagulant if the
blood volume is not balanced
o RBC shrinkage, will decreases
hematocrit (measure of volume), MCV,
ESR (whether swollen RBC or shrinkage
it will still prevent the rouleaux of the
erythrocyte)
o Increases MCHC (because of decrease
hematocrit)
o WBC: degenerative changes
o Platelets: swell and break, thus will
result as falsely increase.
- Commercial name includes the versine (Na) and
sequestrene (K2 and K3)
- K2 and K3 are more commonly used that the Na
because it is more soluble when added to the
blood
- K2 is found in powdered formed while the K3 is
added in the tube in liquid form
- Lavander stopper (for hematology) is the most
commonly used EDTA stopper
- Pink stopper, has a spray-dried EDTA and
commonly used in blood banking
- White stopper, has EDTA and gel separator
additives for molecular diagnostic
- Tan (plastic) stopper, contains k2 EDTA for lead
testing
 Sodium Citrate Anticoagulant
- Second most used anticoagulant
- It works as anticoagulant by inhibiting calcium
- Two formulations of Sodium Citrate: 0.105 M
(glass tube)/ 0. 109 M (pastic tube), same as the
3.2% concentration and other 0.129 M or the
3.9%
- Light blue stopper contains the 3.2%, use in
coagulation testing because it preserves well the
labile coagulation factors (Factor 5 and 8)
- In 3.2% the ration of anticoagulant to blood is
1(sodium citrate) :9 (blood)
- Black stopper contains 3.9% of the sodium
citrate and use for ESR particularly in original
Westergren procedure. The ratio is 1 (sodium
citrate) : 4 (blood sample)
- Coagulation test where Sodium Citrate is used
include prothrombin time (pt) and Activated
Partial Prothrombin time (APTT), particularly the
3.2% sodium citrate.
 Heparin Anticoagulant
- Also called the Mucoitin polysulfuric acid
- Best anticoagulant, because it exist in the body
as a natural anticoagulant and best in preventing
hemolysis
- Does not cause much distortion thus best in
preventing hemolysis
- Can be used in vivo, actually used in intravenous
anticoagulant therapy
- Only anticoagulant that does not act on calcium
but rather it inhibits the thrombin. Thus, it
functions as antithrombin
- Ratio: 0.1-0.2 mg/ mL of Whole blood
- In hematology heparin is used in the following
procedures:
o Osmotic fragility test (OFT): Determines
the ability of the RBC to absorb
hypotonic solution without lyse.
Increase osmotic fragility will result to
hemolysis, thus the positive result is
hemolysis. Therefore, it is required that
the sample used for this test is hemolyze
free sample. Defibrinated blood can also
be used in OFT test. Best sample to be
used is heparinized blood and then the
defibrinated blood. Best anticoagulant
for OFT is the heparin
o Methemoglobin assay
- Heparin cannot be used in the following
hematology procedures.
o It cannot be used in coagulation studies
because it inhibits thrombin. Citrate on
the otherhand, which inhibit calcium can
still be used in the coagulation study
because the complex (XaVa-platelet
phospholipidcalcium) that will convert
prothrombin into thrombin can still be
reversed. Meaning upon addition of
calcium reagent calcium coagulation will
still occur. Heparin inhibition to
thrombin is irreversible
o It cannot be used for smear:
 EDTA for smear preparation is
recommended
  Heparin shows bluish fermentation will not be prevented, then it will
background upon application of result to increase ethanol
Romanowsky stain. The pH of - Fluoride disadvantage in chemistry: Can only be
heparin is not compatible with used for glucose. It should not be used in
the Romanowsky stain potassium measurement neither for urea
 Heparin also causes the WBC measurement because this anticoagulant
and platelets to clump resulting inhibits urease
to falsely decrease WBC - Test for both Blood glucose and Blood urea
- Light green and green/black contain lithium nitrogen examination should be in Lithium
heparin and gel separator and used for chemistry iodoacetate stopper
tests
- Green contains either Sodium or Lithium Heparin  YELLOW STOPER
also used in chemistry - Contains sodiumpolyanethol sulfonate to allow
- Tan (glass) stopper contains heparin and used for the culture of bacteria because SPS inhibits the
lead testing antibiotics if present in the blood circulation. It
- Royal Blue stopper contains Sodium and also inhibits antibody, and complement protein
Na2EDTA, used in trace element and toxicology from destroying the bacteria
testing - Used for culture test
- Function to increase the bacteria yield in the
OTHER ADDITIVES
blood
 GRAY STOPPER
- Contains either oxalate or antiglycolytic agent,
 YELLOW STOPER WITH BLACK TYPE
they are not anticoagulant. Additives include
- Contains acid citrate, dextrose.
Sodium fluoride (enolase) or Lithium
- Good anticoagulant for RBC. It also acts a
iodoacetate and is used for serum glucose,
support medium
ethanol determination. Glucose if not preserved
- Since it contains acid citrate then it is an
will decrease 10% every hour.
anticoagulant and since it contains dextrose it is
- Gray stopper containing Sodium fluoride still
a nutrient for RBC
produces serum as its liquid component because
- Blood Bag in blood donation contains ACD
sodium fluoride is not anticoagulant
- Primarily used in transfusion, HLA (human
- Plasma Preparation in fluoride containing tubes
leukocyte antigen) typing
should be through the addition of oxalate to
sodium fluoride.
 OXALATES
- Sodium fluoride combines with magnesium
- Anticoagulant that is less commonly used in
forming magnesium fluoride, thus inhibiting
hematology
magnesium. Enzyme enolase which is needed in
- Potassium Oxalate (K2C2O4) or Paul-Hellers
glycolysis is magnesium dependent thereby
oxalate has the disadvantage of causing red cells
inhibiting enolase in the glycolytic reaction will
to shrink
result to preservation of glucose. Glucose in
- Ammonium Oxalate (NH4C2O4 has the
fluoride can be preserved up to 3 days
disadvantage of causing swelling
- Lithium Iodoacetate preserve glucose by
- Double Oxalate or the mixture of both potassium
inhibiting glyceraldehyde-3-phosphate (G3P).
and ammonium oxalate are used to neutralize
G3P is an intermediate product in glycolysis,
the shrinkage and swelling effect. Ratio is 2 parts
inhibiting it will discontinue the process of
of Potassium oxalate for every 3 parts of
glycolysis. It can preserve glucose only for 24
Ammonium oxalate
hours
- Oxalate is not ideal for blood smear preparation
- Fluorides and Acetates are also used in ethanol
because of the possibility of cell distortion
testing. Ethanol is a product of carbohydrate
fermentation, if glycolysis is not inhibited
  THROMBIN AND SOYBEAN TRYPSIN INHIBITOR
- Used in hematology, particularly in coagulation
procedure
- Used in the measurement of Fibrin degradation
products (FDP)

ORDER OF DRAW
- Order of draw is needed to prevent
contamination and carry-over of additives and
sample
- By following the order of draw though the carry
over will not be prevented, but the effect will be
OLDER ORDER OF DRAW
minimized
- Separate order for both closed and open system
EVACUATED TUBES AND SYRINGE (CLSI UPDATED)
FOR EVACUATED TUBE SYSTEM (OLDER)
1. Blood Culture Tubes (yellow stopper)
1. Culture tubes
o Minimizes bacterial contamination
2. Plain/Non-Additive Tubes
2. Sodium Citrate Tube (usually the Light blue)
3. Citrate
3. Serum Tubes (either the red, red with black, yellow,
4. Heparin
yellow with gray)
5. EDTA
o To prevent the content of blood culture and
sodium citrate to interfere in heparin and EDTA 6. Oxalates/Fluoride
tube serum tube should be drawn first
FOR SYRINGE (OLDER)
4. Heparin Tubes, with or without gel (light green and
1. Culture Tubes
green)
2. Citrate
o Causes the least interference amongst the
remaining tube 3. EDTA
5. EDTA (purple) 4. Heparin
o Causes more interference 5. Fluoride
6. Antiglycolytic tubes (gray) 6. Plain

COMPLICATIONS IN PHLEBOTOMY
NOTE:
 Immediate Local Complication
Pneumonic: Stop, Light Red, Stay Put,
Green Light, Go o Ecchymosis
S- Sterile o Localized Hemoconcentration or venous stasis
o Circulatory failure
L- Light Blue o Syncope or Fainting
R- Red
S- Serum separator tube  Late Local Complication
P- Plasma separator tube or PST o Thrombosis
G- Green o Thrombophlebitis
L- Lavander o Allergies (aptients can be allergic to
G- gray
antiseptics, adhesive glue in bandages,
and latex. Use alternate antiseptic if
 required; paper tape placed over folded
gauze or self-adhesive bandage material
can be used in place of adhesive
bandages).

 Delayed General Complication (transmission of

blood-borne infections)

HEMATOLOGY LECTURE MIDTERM NOTES

ENUMERATION OF BLOOD CELLS

- Also called hemocytometry, because it entails have to dilute to disperse the blood cell. Diluting
the use of hemocytometer as guiding for cell pipets are used in preparation of dilution.
count.

3 METHODS TYPES OF DILUTING PIPETS

AUTOMATIC PIPETS:
 TURBIDIMETRIC
- Simple as observation.  TRENNER
- The more cell there are, the more turbid the - Is an automatic pipet because it can be filled
sample is. with blood via capillary action.
 MICROSCOPIC - It is similar with the usual RBC pipet. However,
- Manual method, wherein we prepare the the RBC pipet is attached with a sucking tube to
sample we dilute it and count them under the allow the entry of blood into it.
microscope. - It also has a red bulb with a single graduation
 AUTOMATED on top of the bulb which it 101.
- Most routine cell-counting procedures in the
laboratory in the hematology laboratory today.
However, performance of manual method is
still necessary when counts exceed the linearity
of an instrument or when an instrument is non-
functional and there is no backup instrument,
or in an extreme situation when testing is done  UNOPETTE
in the field. - A plastic disposable pipet that already contains
HEMOCYTOMETRY into it the diluent inside the reservoir of the
container.
- Numerical evaluation of formed elements of - There is a unopette for RBC counting, WBC
blood cells in a known volume of blood. counting as well as for platelet counting.
- Estimation of the number of blood cells in a
known volume of blood.

NOTE:
RBC: several millions in µL
WBC: several thousands in µL
Platelet: several hundred thousand in µL.

If we simply focus the blood sample in a microscope,

it’s very impossible to count them because they are
too much overlapping one another therefore we
 WBC PIPET THOMA PIPET RBC PIPET
White Color of bead Red
Smaller Size Larger
NOTE: UNOPETTE Larger Bore smaller
Components: 10 / 11 Volume 100 / 101
 Plastic reservoir: Inside it is the diluting fluid. .5, 1.0, 11 Calibrations .5, 1.0, 11
 Diluent: 1:10-1:100 Dilutions 1:100-1:1000
o RBC: NSS diluent
o WBC: Acetic acid diluent
o Platelet: Ammonium oxalate diluent 𝐕𝐨𝐥. 𝐨𝐟 𝐭𝐡𝐞 𝐛𝐮𝐥𝐛 𝐓𝐕−𝟏
DF= 𝐕𝐨𝐥.𝐨𝐟 𝐭𝐡𝐞 𝐛𝐥𝐨𝐨𝐝 or DF= 𝐕𝐨𝐥.𝐨𝐟 𝐭𝐡𝐞 𝐛𝐥𝐨𝐨𝐝
 Thin plastic at the bottom that seals the fluid
in it.
 Has a pipet to contain the blood that is filled
via capillary action WBC PIPET VOL. IN THE RBC PIPET
 Overflow chamber DILUTION BULB DILUTION
 Pipet shield: to protect the pipet from 100 .1 1000
breaking 20 .5 200
10 1 100
Procedure:
- Fill the pipet with blood and puncture the
diaphragm using the tip of the pipet shield. SOLUTIONS:
Transfer the blood into the reservoir WHITE BLOOD CELLS
𝐓𝐕−𝟏
containing the diluent, mix and attach the DF=
𝐕𝐨𝐥.𝐨𝐟 𝐭𝐡𝐞 𝐛𝐥𝐨𝐨𝐝
overflow chamber into the diaphragm.
𝟏𝟏−𝟏
DF=
𝟎.𝟓
DF= 20
𝐓𝐕−𝟏
DF=
𝐕𝐨𝐥.𝐨𝐟 𝐭𝐡𝐞 𝐛𝐥𝐨𝐨𝐝

𝟏𝟏−𝟏
DF=
𝟎.𝟏
DF= 100
𝐓𝐕−𝟏
DF=
𝐕𝐨𝐥.𝐨𝐟 𝐭𝐡𝐞 𝐛𝐥𝐨𝐨𝐝

𝟏𝟏−𝟏
DF=
𝟏
NON-AUTOMATIC PIPETS: DF= 10
RED BLOOD CELLS
DILUTING PIPETS DF=
𝐓𝐕−𝟏
𝐕𝐨𝐥.𝐨𝐟 𝐭𝐡𝐞 𝐛𝐥𝐨𝐨𝐝
 THOMA PIPETS
𝟏𝟎𝟏−𝟏
- Commonly used in the manual procedure DF=
𝟎.𝟓
- It is a non-automatic pipet due to the need to DF= 200
suck the blood and the diluting fluid. 𝐓𝐕−𝟏
DF=
𝐕𝐨𝐥.𝐨𝐟 𝐭𝐡𝐞 𝐛𝐥𝐨𝐨𝐝

𝟏𝟎𝟏−𝟏
DF=
𝟎.𝟏
DF= 1000
 𝐓𝐕−𝟏 NOTE:
DF=
𝐕𝐨𝐥.𝐨𝐟 𝐭𝐡𝐞 𝐛𝐥𝐨𝐨𝐝
 RBC pipet can also be used for WBC, Platelet
𝟏𝟎𝟏−𝟏 count, Sperm cell count, CSF, etc.
DF=
𝟏  Use of pipets depend on the dilution to be
DF= 100 used.

NOTE: NOTE: DILUTION/ PIPETS TO BE USED

Blood will first suck unto the calibration of 0.5. Then
the next we suck into the pipet is the diluent. As we
suck in the diluent, blood including the diluting fluid
flows into the bulb. As we fill the volume up to the
last calibration, the stem of the Thoma pipet will now
only contain the diluting fluid. There is no blood
present in the stem because it is already displaced by
the diluting fluid. Dilution occurs only in the bulb. No
dilution will take place in the stem.
When computing the diluting factor, we are only
after the volume in the bulb. We don’t consider the
volume of 1 that remains in the stem. This is the
reason why we subtract 1 from the total volume.
When using Thoma pipet (WBC/RBC), the dilution
factor should be TV-1 divided by the volume of blood.
Represented in a formula of:
𝐓𝐕−𝟏
DF= 𝐕𝐨𝐥.𝐨𝐟 𝐭𝐡𝐞 𝐛𝐥𝐨𝐨𝐝
When do we prepare 1:10, 1:20, 1:100, and 1:200
dilution?
- In the usual procedure, in WBC we use the
1:20 dilution and in RBC we usually use the
1:200 dilution. However, if the cell count is
too high or too low, we have to adjust the
dilution. If the patient is high in WBC (like in
Leukemia cases), even if we follow the usual
procedure of 1:20, the blood cells are still
difficult to count due to its increase amount
of cells. In this case, we need to increase the
dilution. Same is true to patients who has low
in blood cell count (Anemic). The less cell
counted, the less precise the count is. If we
follow the usual 1:20 solution, the cells are
too few to count therefore we have to
increase the amount of blood to decrease the
dilution.

1:20 dilution: usual dilution in WBC pipet BOARD QUESTION: If the anticipated count is
1:200 dilution: usual dilution in RBC pipet 32,000/µL or 𝟑𝟐. 𝟎 × 𝟏𝟎𝟗 /L, what dilution are we
0.5: usual blood volume going to follow or prepare?
ANSWER: 1:100 dilution and 1:200 dilution
Dilution Range: highest and lowest dilution that a
pipet can contain.
 Rule in dilution: The more blood you use, the DILUTING FLUIDS
less diluted it is. While, the less blood you  Cheap & economical
add, the more diluted it is.
- Example: NSS
 0.1: the least volume of blood that we can
 Easy to secure and prepare and stable
add which has the highest dilution.
- If the diluting fluid evaporates, it becomes
 1: the maximum amount of blood that we
can add which has the lowest dilution concentrated causing red cells to crenate. It
should be stable.
 With preservative action
Anticipated Recommended Pipet to be - It should not distort the blood cells (prevent
WBC count Dilution used swelling).
(___ x 𝟏𝟎𝟗 / L)  With high specific gravity
0.1-3.0 1:10 WBC - To let cells settle faster and prevent them from
3.1- 30.0 1:20 WBC moving for easier counting.
> 30.0 1:100 RBC - Too high can cause crenation.
≥100.0 1:200 RBC
 With buffer action
- Capable of maintaining the shape of the cells
 Non-allergic and non-corrosive
 WHITE BLOOD CELLS
 - reagent should be hypotonic fluid  Described as similar to improved Neubauer but
- Intact blood cells will have difficulty in counting. it has a depth of 0.2 mm
RBCs can obscure WBC count. HYPOTONIC  Has two ruled areas
SOLUTION will hemolyze RBCs but it will not  Measurement of 4x4 mm
cause any changes in WBCs leaving them  Divided into 16 equal parts
present in the counting chamber and will be
easier to count. NOTE:

 RED BLOOD CELLS

- reagent should be isotonic fluid
- RBCs are the most fragile cells that is why there
is a need to maintain its morphology to be able
to count them.
- It will be easier to count because they are more
plenty than WBCs.

HEMOCYTOMETER Area= L x W
Area= 4 x 4
- Will guide us in counting. Area= 16 mm2
- Improved Neubauer: Most commonly used
- Thick slide with ruled platforms where the Volume (1 square)= L x W x D
counting is done. Volume (1 square)= 4 x 4 x .2
- Has a special cover slip. Volume (1 square)= 3.2mm3
- DEPTH: Space between the cover slip and ruled
TV= volume x no. of squares
chamber.
TV= 3.2 mm3 x 2
- Depth: 0.1 or 0.2 depending on the device used.
TV= 6.4 mm3

2. SPEIRS-LEVY
 Depth: 0.2 mm
 Ruled area/ counting area: 4
 Volume (1 square): 2.0 mm3
 Total volume: 8 mm3
 Has 10 equal squares arranged in 2
rows
 Has 4 ruled areas
 Dimension: 5x2
 Used in counting fewer number of cells
(Eosinophil & Basophil)

NOTE:

3 COMMONLY USED ACCORDING TO RULINGS

1. FUCHS-ROSENTHAL
 Depth: 0.2mm
 Ruled area: 2 squares
 Volume (1 square): 3.2 mm3 Vol= L x W x D
 Total volume: 6.4 mm3 Vol= 5 x 2 x 0.2
 Vol= 2 mm3 Vol= 1 x 1 x .1 = 0.1 mm3
Total vol. of 4 WBC square: 0.1 x 4 = 0.4 mm3
TV= volume x no. of squares RBC count
TV= 2 mm3 x 4 - use the central squares
TV= 8mm3 - each RBC squares has 25 intermediate
NOTE: ADVANTAGE OF FUCHS-ROSENTHAL AND squares and divided into 16.
SPEIRS-LEVY
 Used in counting fewer number of blood RBC squares
cells - only use the 5 squares (4 small corner
 Absolute Eosinophil count squares and the central square).
 Absolute Basophil count

3. IMPROVED NEUBAUER
 Depth: 0.1 mm
 Rules squares/ counting area: 2
 Volume/sq: 0.9 m mm3
 Total Vol= 1.8 mm3
 Dimension: 3 x 3 mm2
 Area: 9 equal squares  0.004: volume of 1 small RBC square
 Divided into 9 equal squares Solution:
 Not all squares are used in counting Vol= L x W x D
 Mostly used in routine manual cell Vol= 0.2 x 0.2 x 0.1
Vol= 0.004 mm3
counting.
 Similar to Fuchs-Rosenthal but differ in
Total Volume= Vol x No. of squares
depth and square Total Volume= 0.004 x 5
 Contains less than the other two Total Volume= 0.02 mm3

NOTE:
SUMMARY OF THE COMMONLY USED
HEMOCYTOMETER
TYPE DEPTH DIMENSION VOLUME
Fuchs- 0.2mm 2 ruled V= 3.2
Rosenthal areas: 4x4 mm3
mm TV=
6.4mm3
Vol (1 square)= L x W x D
-each ruled
Vol (1 square)= 3 x 3 x .1
area is
Vol (1 square)= 0.9 mm3
divided into
16 large
TV= volume x no. of squares
squares
TV= 0.9 mm3 x 2
-each large
TV= 1.8 mm3
square is
NOTE: further
WBC count divided into
- only use the 4 large corner squares 16 small
- each WBC squares is divided into 16 squares
intermediate squares Speirs- 0.2mm 4 ruled V= 2.0
Levy areas: mm3
WBC squares: 2x5mm TV= 8 mm3
Vol= L x W x D
 After diluting discard the first 2 to 3 drops of blood.
-each ruled
area is
divided into
10 equal
squares
arranged in
2 rows
Neubauer 0.1mm 2 ruled V= 0.9
areas: mm3
3x5mm TV= 1.8
mm3 When loading the chamber, do not touch the cover
-each side is slip in order to prevent the smudging of the sample.
divided into Settle for 2-10 mins.
9 equal WBC – Low Power Objective
squares RBC – High power objective
Platelets – High power objective
DILUTING FLUIDS
When counting, start at the first square on the top
 FORMOL- CITRATE (DACIE’S)
most and the left most.
- Most ideal diluting fluid of RBC
- Formalin; 3% Sodium citrate
 Preserves the cell.

 TURK’S SOLUTION
- GI. Hac; 1% Aq. Gentian violet
- Adv: it will make the nucleus of the WBC’s more
prominent.

 1-3% ACETIC ACID

- Most ideal diluting fluid of WBC Inverted “L” rule: all cell touching the L-shape will be
counted. The right most and the left most will not be
counted.

NOTE: Eshridge:
For 2 lines: consider the inner line as boundary line .
If the outside is touched, it will not be considered.
For 3 lines: consider the middle line as boundary line.
If the last line is touched do not apply the rule.
 CELL COUNTING SOLUTION 𝟏 𝒄𝒖𝒎𝒎
VCF=
𝟎.𝟎𝟐 𝒄𝒖𝒎𝒎
VCF= 50 𝒎𝒎𝟑
FORMULAS:
Cell count/ cumm= cells counted x DFx VCF Cell count/ cumm= cells counted x DFx VCF
Cell count/ cumm =480 x 200 x 50
𝒕𝒗−𝟏
DF = Cell count/ cumm= 4,800,000/mm3
𝒃𝒍𝒐𝒐𝒅

𝟏 𝒄𝒖𝒎𝒎 “For every 1 RBC that we count accurately or

VCF= inaccurately, it will be multiplied by the factor which
𝒕𝒐𝒕𝒂𝒍 𝒗𝒐𝒍𝒖𝒎𝒆
is 10,000”
Note:
1 cumm/ 1 𝒎𝒎𝟑 is constant.
NOTE:
 The only acceptable RBC count is the
WBC SQUARES automated RBC count.
WBC squares VOL= L x W x D = 1 x 1 x .1 = 0.1  Manual RBC count is considered as
WBC squares VOL= 0.1 x 4 unreliable, because RBC are too small to
WBC square TV= 0.4 all 4 squares count, and they are too plenty.
 Most precise manually perform is the
𝟏 𝒄𝒖𝒎𝒎 hematocrit count.
VCF = 2.5 all 4 squares
𝟎.𝟒 𝒄𝒖𝒎𝒎

NOTE:
𝒏𝒐.𝒄𝒆𝒍𝒍𝒔 𝒙 𝑫𝑭 𝒙 𝟏 𝒄𝒎𝒎
RBC SQUARES  Cell/cumm3=
𝒕𝒐𝒕𝒂𝒍 𝒗𝒐𝒍𝒖𝒎𝒆
RBC squares VOL = L x W x D = 0.2 x 0.2 x .1 = .004
RBC squares TV= 0.004 x 5 = 0.02 𝒏𝒐.𝒄𝒆𝒍𝒍𝒔 𝒙 𝑫𝑭
𝟏 𝒄𝒖𝒎𝒎  Cell/cumm3=
VCF= = 50 all 5 squares 𝒕𝒐𝒕𝒂𝒍 𝒗𝒐𝒍𝒖𝒎𝒆
𝟎.𝟎𝟐 𝒄𝒖𝒎𝒎
𝒏𝒐.𝒄𝒆𝒍𝒍𝒔 𝒙 𝑫𝑭
 Cell/cumm3=
𝒂𝒓𝒆𝒂(𝒎𝒎)𝒙 𝒅𝒆𝒑𝒕𝒉 (𝟎.𝟏𝒎𝒎)

WBC EXAMPLE: 𝒏𝒐.𝒄𝒆𝒍𝒍𝒔 𝒙 𝑫𝑭 𝒙 𝟏𝟎

# of cells counted = 107  (Rodak’s) Cell/cumm3=
𝒂𝒓𝒆𝒂(𝒎𝒎)

𝟏𝟏−𝟏
DF = = 𝟐𝟎 Accuracy is the nearest of the value to the true value.
.𝟓 The less squares you count, the less precise it is.
𝟏 𝒄𝒖𝒎𝒎
VCF=
𝟎.𝟒 𝒄𝒖𝒎𝒎
VCF= 2.5 𝒎𝒎𝟑 CORRECTED WBC COUNT
5 or more NRBC/100 WBC
Cell count/ cumm= cells counted x DFx VCF 𝑈𝑛𝑐𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡
Cell count/ cumm =107 x 20 x 2.5 Corrected WBC count= 𝑁𝑅𝐵𝐶 𝑥 100
100+# 𝑜𝑓 𝑊𝐵𝐶𝑠
Cell count/ cumm= 5350/cumm 100

NOTE:
“For every 1 WBC erroneous counted it will cause an The effect of hypotonic solution to RBC is to
equivalent of 50 cells error.” hemolyze it so that the WBC will still remain.
RBC EXAMPLE: However only the mature and non-nucleated RBC are
# of cells counted = 480 fragile to hypotonic soln.
𝟏𝟎𝟏−𝟏
DF = = 𝟐𝟎𝟎 The 5 nucleated RBC will be found in stained blood
.𝟓
smear.
5 or more NRBC need to recompute because it will
lead to false increase.

Steninger: more than 5 will not be included

Rodak’s & Turgeon: 5 or more

EXAMPLE:
𝟓𝟑𝟓𝟎
𝒙 𝟏𝟎𝟎 = 𝟒𝟗𝟎𝟖/uL
𝟏𝟎𝟎+𝟗

Correct the value by subtracting the 4,905 to 5,350. REPORTING

5,350 – 4,905= 445

The 445 is caused by nucleated RBC Old Cells/cumm New / SI unit _ x 10x/L
WBC X 103/mm3 WBC X 109/L
NOTE: Where do we count platelet in counting
RBC X 106/mm3 RBC X 1012/L
chamber?
We count it in 5 RBC squares or in 10 RBC squares. Plt X 103/mm3 Plt X 109/L

NOTE: VIEWING CELLS UNDER THE MICROSCOPE

LPO- it is able to view the whole big square
HPO- it is only able to view the small 1 square
You will know that you are in the WBC squares if you
will see either diagonal lines or vertical lines.
EXAMPLES: Old cell/ cumm
WBC: 5,350/mm3 = 5.35 x 103/mm3
(we moved the decimal into 3 decimal places)
RBC: 4,100,000 = 4.1 x 106/mm3

EXAMPLES: New/ SI unit _ x 10x/L

WBC: 5,350/mm3 = 5.35 x 109/L

Note:
For the decimal point: move the decimal point to the
left 3 times.
For the exponent: there are 6 zeros in 1M uL,
therefore, add 3+6=9
You will know that you are in the RBC squares if what
RBC: 4,100,000 = 4.1 X 1012/L
lies around it are all squares then the upper part are
Plt: 245,000/uL = 245 x 109/L
vertical lines.

If you are in the first square of the RBC, what lies on REFERENCE RANGES:
its left side are all horizontal lines and the upper part RBC count: (male) 4.5-5.9 x 1012/L
are all vertical lines. (female) 4.1-5.1 x 1012/L
WBC count: 4.4-11.3 x 109/L
NOTE: Platelet count: 150-450 x 109/L
The identification of NRBC is not in hemocytometer
counting. Instead, NRBCs can be seen in stained
blood smear (diff count).
 ENUMERATION OF BLOOD CELLS
Units used
 Relative count: % (in relation to)
o It is only expressed in relation to how
many cells we counted off.
EXAMPLE:
𝟓𝟎
Reticulocyte= 𝒙 𝟏𝟎𝟎 = 𝟓% out of 1000 RBC
𝟏𝟎𝟎𝟎
there are 5% reticulocytes.
 Absolute Count: _/vol
o It gives us the exact number of cells per
unit volume of blood.
o Vol can be /L, /uL,/cumm
EXAMPLE:
5,500/uL = 5.5 x 109/L
ERRORS IN MANUAL HEMOCYTOMETRY
 Technical/Human Errors
- Errors was introduced by the Medical
Technologist.
- Human error is preventable
 Equipment/Standard Errors
- Errors was introduced by the equipment
like microscope
- preventable
NOTE:
 NBS-certified pipets & hemocytometers
 WBC pipet = (+ / - 3 %) ACCEPTABLE
 RBC pipet = (+ / - 5 %) ACCEPTABLE
 Inherent/Field Error
o Poisson’s law of distribution – cells are
settled at random
- It is an error of what you are looking into
- Theoretical: 10 mins
- Practice: once the cells stop to move then
start to count.
- Check also the distribution of cells
NOTE: GOOD DISTRIBUTION
WBC: no >15 between the highest and the lowest
numbers of WBCs.
RBC: no >20 difference for the RBC count
>2 SD= Recharge
 The difference of two chamber should not be
>2 SD, if it exceeds in 2 then recharge.
= or <2 SD = compute for average count
 Example:
Square 1: 120
Square 2: 116
120-116= 4, is within 2 SD, therefore, get the
average.
 According to Rodak’s, the difference of two
platforms should be <10%.
HEMOGLOBIN
- Main component of RBCs that carries oxygen
from the lungs to the body tissues and return
carbon dioxide from the tissues back to the
lungs. Measurements of the hemoglobin from
the venous or capillary blood aids in the
detection of a variety of conditions that alter the
normal hemoglobin concentration of the blood
(e.g. anemia or polycythemia).
METHODS OF HEMOGLOBIN MEASUREMENT
1. GASOMETRIC METHOD (VAN SLYKE OXYGEN
CAPACITY)
- Based on the oxygen carrying capacity of the
hemoglobin.
- Indirect method
2. CHEMICAL METHOD
- Chemical method is based on the presence of
Iron.
- Fe2+: component of the hgb.
- Indirect method
3. GRAVIMETRIC METHOD
- Specific gravity of blood is affected by the hgb.
The major composition of blood which is
responsible in giving its weight is hemoglobin.
- Indirect method
4. COLORIMETRIC METHOD
- Based on hemoglobin itself.
- Direct Method
 General Basis of
Methods measurement
Following the Gravimetric Method
Gasometric Oxygen Indirect
Method Method Oxyhemoglobin Normal Can be
Chemical Iron (Fe2+) Deoxyhemoglobin Normal (can measured
Method still bind (only the O2)
Gravimetric Specific gravity with O2)
Method Carboxyhemoglobin Abnormal Not
Colorimetric Hemoglobin Direct Sulfhemoglobin (cannot bind measured
Method with O2) because it
cannot bind
with O2
1. GASOMETRIC METHOD (VAN SLYKE OXYGEN Expected that the values are
CAPACITY) LOWER because it only
 VAN SLYKE APPARATUS measured the active and
 Only the oxygen is being measured normal hemoglobin.
2. CHEMICAL METHOD
 PRINCIPLE:
A. WONG METHOD
o A given sample of blood can be
 Iron is liberated by H2SO4 and
equilibrated with O2 under standard
potassium persulfate.
conditions of temperature and pressure.
 Hemoglobin: contains heme &
o It measures directly the amount of
globin.
oxygen present in a blood sample. After
 Heme: contains FeH
measuring the oxygen, the O2
 Globin: protein
concentration can be converted into
 Separation of heme by globin by
Hgb.
treating the sample with sulfuric
 EQUATION: 1 g Hgb = 1.34 ml O2
acid & potassium persulfate,
o HUFNER FACTOR: every gram of hgb can
the globin will precipitate by
actually bind to 1.39 ml of oxygen. Once
tungstic acid.
the oxygen is already known, we can
 PROTEINS: precipitated by tungstic acid
convert it in hemoglobin.
 Iron → Ferric thiocyanate
 LIMITATION: It cannot measure hemoglobin that
 Ferric thiocyanate is the end
cannot bind with oxygen. Measures active
product and it will be measured
hemoglobin only.
spectrophotometry.
 Beer’s law: The intensity of the
NOTE:
color of a solution. The end
 Oxyhemoglobin: Normal hemoglobin with
product is directly proportional
oxygen
to the solution of the substance.
 Deoxyhemoglobin: hemoglobin with less
oxygen  Iron is being measured
 Carboxyhemoglobin: hemoglobin with  Equation: 1 gm hb = 3.47 mg
carbon dioxide content. iron
 Methemoglobin: hemoglobin with Fe  Fe →Hgb
 Sulfhemoglobin: hemoglobin containing A. ASSENDELFT METHOD
sulfur.
3. GRAVIMETRIC METHOD
Carboxyhemoglobin, Sulfhemoglobin, and  Based on specific gravity
Methemoglobin are considered abnormal  Uses a standard CuSO4 solution (in 40 tubes)
hemoglobin and are not capable of binding with with specific gravity of 1.035 to 1.075 at interval
oxygen. As a result, these types of hemoglobin are not of .001
capable for oxygen transport. Thus, it cannot be  A drop of patient’s blood is placed into each
measured in Gasometric method. tube. The drop of blood becomes coated with
 copper proteinate and for about 12 seconds, the  Consists of different colors and it is
blood drop may either: arranged in rotating disc.
o Sink (if heavier/denser): the blood sp.  Each color has corresponding
Gravity is greater than the solution concentration.
present in the tube.  Inaccurate: gives as much as 30%
o Float (if lighter/less dense), the blood error.
has the sp. Gravity lighter than the  The automated version is
solution. acceptable.
o Remain (if it has same density and the
CuSO4 solution) in the suspension.

NOTE: NORMAL VALUES

FEMALE 1.053 = 12.5 g/dL hb
MALE 1.055 = 13.5 g/dL hb

NOTE:
 Gravimetric is no longer performed in 3. ACID HEMATIN
routine clinical laboratory, but it remains to
be the acceptable method in the mass blood
donation.
 Only one reagent having a specific gravity of
1.053 is needed in mass blood donation.

4. COLORIMETRIC METHOD
A. DIRECT VISUAL COLORIMETRIC (TDAA)

1. TALLQUIST METHOD
 Patient’s undiluted blood is
absorbed unto an absorbent pad
and the color is compared with a
lithographed color scale
representing values from 10-100%.
 Inaccurate: gives as much as 50%
error.
2. DARE’S HEMOGLOBINOMETER
 Blood is drawn by capillary action
between 2 glass plates and the color
of blood is matched with a rotating
disc of red tinted glass with varying
thickness and color.
 PRINCIPLE: hemoglobin is converted
into alkali hematin upon addition of
an alkali. Abnormal hb are converted
to alkali hematin and are thus
measured.
 Hb+acid →Acid hematin
 The reagent (acid) is 0.1N HCl
 Hb+alkaline → alkali hematin
 The intensity of the brown color
depends on the amount of
hemoglobin.
o More Hgb: darker the color
of brown.
o Less Hgb: lighter the color of
brown.
 The end product which is the brown
color which will be diluted with
distilled H2O until the brown color
matches the color of the comparator
black. (g/L,g/uL)
  DISADVANTAGE: does not measure into an oxygenated form. To do that
inactive forms procedure, we mix:
o 0.02 mL whole blood is
reacted with 5 mL of 0.07N
NH4OH / 0.1% Na2CO3 then
mixed for proper
oxygenation.
o Oxyhemoglobin→Absorba
nce is read at 540 nm
against reagent blank.
NOTE: o Does not measure other
derivatives: methgb,
carboxyhgb, sulfhgb.
o The relevance of this is in
diagnosis of acute
hemolysis.
o Methods:
Allow the solution to stand
for 3-5 mins.  Harboe – 415 nm
Methods: Sahli-Hellige; Sahli-adams; Sahli-hayden (Soret band: 400-
hayden-hauusser; Newcomer; Osgood-haskin. 430 nm)
 Naumann-hb
4. ALKALI HEMATIN catalyzes the rapid
 PRINCIPLE: hemoglobin is converted oxidation of
into alkali hematin upon addition of benzidine by H2O2;
an alkali. Abnormal hb are converted More sensitive but
to alkali hematin and are thus less accurate
measured. o Cyanmethemoglobin
 Produces a more stable pigment but method: Most accurate
is not ideal for infants and children
 Wherein the major hgb of infants &  Reagent: Drabkin’s Composition
children is HgbF, one major
characteristic of fetal hgb is, it is NOTE: DRABKIN’S REAGENT (N.P.P.S.D)
resistant to acid & alkali.
Non-ionized - Detergent reagent: enhances
 METHODS: Wu; Klegg & King detergent lysis
 Will .1 N sodium hydroxide (sterox - When blood is mixed with a
[Harleco] or solution of potassium cyanide
B. PHOTOELECTRIC COLORIMETRIC Triton) and potassium ferricyanide,
the erythrocytes are lysed
1. OXYHEMOGLOBIN METHOD thereby producing
 Oxyhgb- oxygen containing Hgb hemoglobin.
 What is measured in this method is - The hgb is needed to be
hemolyzed for it to react to
the entire hemoglobin with the
another reagent.
oxygen.
Potassium - HbFe+2 → HbFe+3
 There is instances that instead of Ferricyanide Methemoglobin
oxygen, carbon dioxide is attached (K3Fe[CN]6) - Potassium ferricyanide
in hemoglobin. The best thing to do oxidizes hgb into
in that case is to allow the methemoglobin that
hemoglobin to oxygenate first, so combines with potassium
that all the Hgb will be converted cyanide
 - Methemoglobinemia:
abnormal increase hgb
Potassium - In combination of potassium
Cyanide ferricyanide and potassium
(KCN) cyanide it will produce
hemiglobincyanide
(cyanmethemoglobin) whose
absorbance is measured at
540 nm against the reagent
blank.
Sodium - Gives off cyanide to combine
Bicarbonate with:
 Methemoglobin to
make the end product
Cyanmethemoglobin
 Hemiglobin (Hi) to
make the end product
Hemiglobincyanide
(HiCN)
Dihydrogen - Uses sodium carbonate as the
Potassium buffered solution
Phosphate - Incubation time is 10 mins
KH2PO4 - In modified Drabkin’s reagent,
(NaCO3- the sodium carbonate has
ORIG been replaced by KH2. The
Drabkin’s) effect of KH2 will accelerate
the reaction, shortening now
the incubation from 10 mins
to 3 mins.
- Advantage: All type of
hemoglobin is transformed to
cyanmethmoglobin (except
sulfhemoglobin)
2. CYANMETHEMOGLOBIN METHOD
NOTE: HEMOGLOBIN (gm/dL)
𝑨𝒖
𝑩𝒆𝒆𝒓′ 𝒔 𝒍𝒂𝒘 𝒇𝒐𝒓𝒎𝒖𝒍𝒂: 𝑪𝒖 𝒙 𝑪𝒔
𝑨𝒔
𝑨𝒃𝒔𝒐𝒓𝒃𝒂𝒏𝒄𝒆 𝒐𝒇 𝒕𝒆𝒔𝒕
𝒙 𝑺𝒕𝒂𝒏𝒅𝒂𝒓𝒅 𝒄𝒐𝒏𝒄.
𝑨𝒃𝒔𝒐𝒓𝒃𝒂𝒏𝒄𝒆 𝒐𝒇 𝑺𝒕𝒂𝒏𝒅𝒂𝒓𝒅
𝑨𝒃𝒔𝒐𝒓𝒃𝒂𝒏𝒄𝒆 𝒐𝒇 𝒕𝒆𝒔𝒕
𝒙 𝟏𝟓𝒈𝒎/𝒅𝒍
𝑨𝒃𝒔𝒐𝒓𝒃𝒂𝒏𝒄𝒆 𝒐𝒇 𝑺𝒕𝒂𝒏𝒅𝒂𝒓𝒅
Example:
Au = 0.575
As = 0.524
Hgb = 16.5 g/dL
PRINCIPLE OF ABSORBANCE: The darker the color of the
end product, the more light will be absorbed. Any light
that is not absorbed by the solution will be transmuted.
CHEMICAL REACTION:
- The more hgb that reacts with the reagent, the
more amount of the product is produced. The
more end product, the darker the color.
- The darker the color, more hemoglobin.
- Color = conc. Of the unknown
PROCEDURE:
a. 1:251 blood-reagent
b. 20 µl of blood + Drabkin’s reagent
c. 3 minutes at room temperature
d. Absorbance at 540 nm against reagent blank
e. Determine concentration
f. Standard curve
g. Calculate (Beer’s Law)
PREPARATION OF STANDARD CURVE:
a. Will use standard reagent of hgb 15 g / dl
b. Prepare dilutions to achieve certain conc.
c. Read abs/ at room temperature of each dilutions
@ 540 nm
d. Plot absorbance at room temperature against its
concentration.
 PHYSIOLOGIC ERRORS
- Physiologic error is in the blood.
 Turbidity
- Will cause more light to be absorbed.
o Lipemic blood: high lipids, milky,
chylous. Fats cannot be separated
through centrifugation
 Remedy: Use patient blank
o High leuokocyte count: High WBC count
50,000/uL will cause turbidity of the
sample. This can be separated through
centrifugation.
NOTE: REFERENCE VALUES
 Remedy: Centrifuge and use
Male 140-175 g/dL
supernatant.
Female 123-153 g/dL
o Abnormal hemoglobin: Hgb S and Hgb C
At birth 150-200 g/dL
will become resistant to lysis, it will not
release the hgb and it cannot react with
NOTE:
potassium ferricyanide and the other
10 dL in 1L:
reagent.
To convert g/L to g/dL: divide by 10
 Remedy: dilute sample 1:1
distilled water (Hypotonic)
NOTE: Why is there a higher amount of hemoglobin at
o Easily precipitated proteins:
birth?
 0.1 gm of K2CO3/L orig.
At birth, both flat and long bones are active in
Drabkin’s
hematopoiesis. Whenever there is an RBC production,
hemoglobin production also occurs.  Multiple myeloma
 Produces bence jones protein
and large amount of IgM and it
SOURCES OF ERRORS: CYANMETHEMOGLOBIN will affect the plasma cells when
- Considered as reference procedure for both mix in drabkins reagent, the
manual & automated. solution will become turbid.
- Most accurate method because it is stable.  Waldenstrom
- It measures all hemoglobin except macroglobulinemia
sulfhemoglobin.
- Sulfhemoglobin is an abnormal hemoglobin QUALITY CONTROL OF DRABKIN REAGENT
formed when a sulfide radical reacts with hgb,  Use fresh reagent
the addition is irreversible.  Reagent: pale yellow with a pH of 7.0 – 7.4
- Once the color of the reagent changed into dark
TECHNICAL/ HUMAN ERRORS or pale yellow, it is an indication that the reagent
 Inaccuracy of the pipet is deteriorated.
- Dilution is inaccurate on pipets with cracks on its  Abs= 0 @ 540 nm
tip.  Storage: amber bottle at room temperature
 Pipetting error - Exposure to light can oxidize the reagent, and
 Use of unmatched cuvette some analytes in the blood.
- Should be clean & free from any finger prints.  Standard: 15 g/dL
 Deteriorated reagent  Stable in a polyethylene bottle at 2 – 8°C.
- Cyanmethemoglobin uses 2 reagents (Drabkins
and standard reagent)
- The standard reagent is not added to the sample.
 Oxidation of the reagent
 CLINICAL SIGNIFICANCE production which will increase now the
INCREASED HEMOGLOBIN: hemoglobin level.
- Decreased in oxygen is compensated in
 Polycythemia increase in RBC by the increase of
- Hgb increases in blood cells due to abnormal hemoglobin.
increase of blood production.
 Dehydration (burns, diarrhea)
QUANTITATION OF FETAL HEMOGLOBIN (HbF)
- Burns & diarrhea has massive loose bowel, which
- Adults have 2% HgbF. Higher than 2% is
contains more water and also plasma water.
- Loose of plasma water will result to cell considered abnormal.
concentration and will lead to
hemoconcentration. NOTE: Why are we going to measure the
percentage of HgbF in total Hgb?
DECREASED HEMOGLOBIN: To separate the hgbF from adult Hgb.

 All types of Anemia

- Anemia is a group of condition, and all are A. ALKALI DENATURATION
characterized in decreased hemoglobin.  PRINCIPLE: When added in alkali, all
 Leukemia hemoglobin except HbF will suffer from
- Disease of the bone marrow, wherein there is an denaturation of alkali reagent.
excessive production of leukemic cells and the  The methods of measuring or
normal cell production will decrease. quantitating hgbF are all based on
- Decrease RBC count leads to a decreased characteristics of hgbF being resistant to
hemoglobin. both acid and alkali treatment.
- Because of the excess production of leukemic  Using the alkali the A1 and A2 will
cells, the patient develops anemia, this is one of denature then the F will still remain.
the reasons why the hgb will decrease. a. BETKE METHOD
b. SINGER METHOD
NOTE: HEMOGLOBIN
 The purpose of this method is to
 Slightly decreases after 50 years of age
determine the percentage of F
 Hemoglobin is lower if lying down
in the blood.
- The interstitial fluid will shift inside the
 (+) control: drop of blood from
circulation resulting now in hemodilution, the
blood cells will be diluted in more plasma fetal umbilical blood.
fluid.  PROCEDURE:
 Higher in the morning and lower in the  RBC hemolysate +
evening Drabkin’s NaOH +
- Circadian effect, there are some tests that (NH4)2SO4→ ppt
affect the time of collection. denatured HbA will be
 Increased in smokers removed by filtration.
- It is increased especially in chronic smokers;  Filtrate(HbF)-measured
they have a higher value of abnormal hgb spectrophotometrically
(carboxyhemoglobin).  __ % HbF
- 10% of the hemoglobin of the chronic smokers
is the carboxy hgb. REFERENCE VALUE:
 Increased among males  Mod by Betke: 0.2% - 1.0%
 Increased in high altitude - (Less than 1 %)
- The higher the altitude, the atmospheric  National Committee for Clinical Laboratory
oxygen is lower resulting now to hypoxia. Standards (NCCLS): HbF quantitation (2-40%)
Hypoxia triggers the erythropoietin. As a  RID: <2% (most preferred method)
result, there is an increase in the RBC  Column Chromatography: >40%
 High Performance Liquid Chromatography
 - most acceptable method but not a routine  CLINICAL SIGNIFICANCE:
test in clinical laboratory  Hereditary Persistence of Fetal
 Elevated HbF: some hemoglobinopathies Hemoglobin (HPFS)
 B-thalassemias: Hereditary Persistence of -even distribution of hbF among
Fetal Hemoglobin. red cells
 Thalassemia and
Test: patient sample + KOH Hemoglobinopathy
-heterogenous distribution of
B. ACID ELUTION METHOD (Modification of the HbF among cells.
Kleihauer-Betke by Shepard)
HEMOGLOBIN ELECTROPHORESIS
 To assess whether the distribution of
A. CELLULOSE ACETATE ELECTROPHORESIS (pH
HbF in all red cell is the same. (homo or
8.4-8.6)
hetero distribution)
 Method of separating substances or
 For detection of fetal red cells in
molecules on the basis of their net
maternal circulation
charge.
 EXAMPLE: Bloody Amniotic fluid- fetal
 Movement of molecules on the
red ceels appears red. And if those red
electromagnetic field
cells are A then its maternal red cell.
 LIMITATION: cannot be easily identified.
 Useful in diagnosis of Hemolytic Disease
of Fetus & the New born.
 PROCEDURE:
 Prepare blood smear
 Air-dried blood film
 Elution: citric acid-phosphate
buffer (pH 3.3)
 Stain-Ehrlich acid Hematoxylin
 Counterstain-Erytrosin:
Standard test for quantitating  Anion: negatively charge (-)
fetomaternal hemorrhage  Cation: positively charge (+)
 INTERPRETATION:  Electrode:
 Maternal red cells: appears as  Cathode: negatively charged (-)
ghost cells  Anode: positively charged (+)
 Fetal red cells: appears as rose
pink in color

 Proteins are bipolar, it means they have

both cathode and anode electron
“AMPOTHERIC”
 Therefore, Hgb is neutral
 But if the protein is placed in a medium
In adults: almost all red cells
(cellulose acetate), it will have an altered
appear ghost cells
electrical charge. It will have net
negative charge.
NOTE:
The importance of cellulose acetate is in separation of
four major and significant hemoglobin. Which are
hgbA, hgbF, hgbS (ABNORMAL,most common hgb
variant. Related in sickling RBC), HgbC (third most
common Hgb variant.
Both of the abnormal hgb is associated in
crystallization of the hemoglobin.
Since the movement is towards the anode, the
mixture of hemoglobin need to place in an area nearer
the cathode to observe how far do they go and how
fast do they go.
 The more negatively charged ions, the faster
it is to migrate to the anode.
 The movement of the hemoglobin is
dependent on net negative charge.
 The Amino acid component of globin gives the
negative charge of the hemoglobin.
NOTE: How do we know if the specific hemoglobin is
S or F?
We placed them in control, which will help us to
identify.
B. CITRATE AGAR ELECTROPHORESIS (pH 6.0-6.2)
 Separates hgb that migrate together on
cellulose acetate.
 SEPARATION OF HgBs:
a) interactions among Hb variants,
b) Interaction of agar and citrate
buffer ions;
c) Altered electrical charge of the
various HgbS at acid pH
- They are not all positive or
negative charge, their charge is
altered.
 PROCEDURE:
 Place the specimen exactly at
the center.
 Once the current is applied
some goes to cathode and some
are going to the anode
 We cannot compare the
mobility of one another because
they have a different pattern of
migration.
 RELEVANCE: S and C migrates alone
NOTE:
Cellulose acetate electrophoresis
Slowest to fastest arrangement
CSFA
Mnemonics:
Crawl Slow Fast Accelerate
Citrate Agar electrophoresis
Cathode: F A Anode: S C
Anode: CS Cathode: AF
NOTE: CHARACTERISTIC OF HbF
 Resistant to alkali denaturation and
acid elution
 Slower on electrophoresis than HbA
 Has high affinity with O2
 Has a decreased affinity with 2,3 DPG
or Diphosphoglycerate
-- 2,3 DPG is a molecule that
affects affinity of hgb to oxygen.
HEMATOCRIT (HCT) MEASUREMENT
HEMATOCRIT (HCT)
- Otherwise known as the Packed Cell Volume
(PCV). In some, they define it separately that Hct
is the methodology of doing it and Packed Cell
Volume is the test that we are to report. But this
time, we can interchange. We may be referring
 to the method or to the test itself. Either Hct or
PCV is used.
- Ratio of the volume of erythrocytes to that of the
whole volume.
- Refers to the proportion of whole blood that
consists of red blood cells.
- Expressed as a percentage (%) of the total blood
volume.
- Hct provides clinicians with an estimate of the
body’s red cell volume and thus, the blood’s
oxygen-carrying capacity.
- Hct determination is useful in screening for
diagnosing, or monitoring a number of
conditions and diseases that affects the
proportion of blood (e.g. Anemia).
- Unit/ Reported:
o Percentage (%)
o Decimal fraction (L/L)
REFERENCE VALUES:
Male 0.42-0.50 / 41.5-50%
Female 0.36-0.45 / 35.9-44.8%
NOTE:
Buffy coat contains WBCs and platelets. We can also
make use of the buffy coat layer to estimate for the
WBC of a patient. For every buffy coat layer, there are
approximately 5,000 -10,000 WBC.
NOTE:
If we are to prepare purely buffy coat to see mainly
the WBCs and platelets, then do not use whole blood
in preparing the blood smear. Rather, use the buffy
coat only and we can make use of the capillary tube to
collect the buffy coat to be smeared.

FORMULA: METHODS OF HEMATOCRIT (HCT) MEASUREMENT

1. INIDRECT HCT
𝐏𝐂𝐕 𝐏𝐂𝐕 - One of the automated principles.
HCT= or HCT= 𝒙 𝟏𝟎𝟎
𝐓𝐕 𝐓𝐕 - Automated Hct can be measured through
CONDUCTIVITY or in INDIRECT WAY.
Whereas:
- The Hematology Analyzer/ Hemoanalyzer will
TV= the total volume of the blood
not centrifuge the blood sample. It will only
PCV= volume of the RBC
EXAMPLE: CALCULATE.
𝐏𝐂𝐕 - Derived from the values of RBC count and Mean
HCT=
𝐓𝐕 Cell Volume (MCV). Depending on the Hct value,
𝟒𝟖 you can divide it by 10.
HCT=
𝟏𝟎𝟎
FORMULA:
𝐌𝐂𝐕 × 𝐑𝐁𝐂 𝐜𝐨𝐮𝐧𝐭
HCT= 0.48 or 48% HCT=
𝟏𝟎
NOTE:
There is no need to indicate
the L/L unit for decimal
2. DIRECT (MANUAL/ SPUN HCT)
fraction.
- Requires centrifugation
- Hematocrit Centrifuge/ Hemofuge: a special
centrifuge used in this method.
- Can be performed using:
OTHER USES OF HCT o Micro Method: small volume of blood
o Macro Method: e.g. method by
 Used in the calculation of absolute indices/
Wintrobe.
constants or Red Cell Indices (MCV, MCHC)
 In Rough Quality Control calculations
 WINTROBE METHOD- DOUBLE OXALATE
o 1 Hct point= 0.34 gm Hb/ 100mL
- Macro method
o 1 Hct point= 107,000 RBC/cumm of
- Makes use of the Wintrobe’s tube which has two
whole blood
graduation (1 in left, 1 in right) wherein we read
o Buffy coat= WBC count
the Hct using the right side graduation.
 The buffy coat portion can be used in the
preparation of buffy coat smears.
 - Double Oxalate: anticoagulant of choice when SUMMARY:
using Wintrobe Method. MICROHEMATOCRIT MACROHEMATOCRIT
METHOD METHOD
 HADEN’S METHOD: 1.1% Na Oxalate Microhematocrit Tube Wintrobe Tube
 VAN ALLEN: 1.6% Na Oxalate  Lenth: 70 or  Length: 11cm or
 SANFORD MAGATH METHOD: 1.3% Na Oxalate 75mm 110 mm
 BRAY’S METHODS: Heparin  Bore: 1mm  Diameter: 3mm
 Blood column: at  Blood column:
 ADAMS MICROMETHOD: makes use of the
least 5cm 100mm or 10cm
hematocrit centrifuge.
 Plus/Seal: clay  Graduations:
and paraffin Left: 0-10; ESR
 Centrifugation Right:10-0; Hct
(RCF): 10,000-  Centrifugation
15,000 g for 5 (RCF): 3,000 rpm
minutes for 30 minutes

EQUIPMENT NEEDED IN PERFORMING MICROHCT

NOTE: LAYERS FORMED IN SPUN HECT
RBCs: the densest or has the highest
Sp. Gravity of all the cells that is why
they settle at the most bottom upon
centrifugation.
Buffy Coat: second layer; consists of
WBCs and platelets. WBCs lies at the
bottom of the buffy coat due to
higher sp. Gravity (denser) while
platelets lie right above the WBCs.
Plasma: liquid portion that is
separated
Note: If we are to arrange these cells, mature/non-
nucleated RBCs lie at the most bottom part. If there
are immature RBCs, they lie just above the mature
RBCs. The more nucleated the cell is, the less dense it
is.
NOTE: WAYS OF REPORTING
Cell count= cells/ cumm
= no. of cells/ µL
= no. of cell x 𝟏𝟎𝟗 /L (WBC)
= no. of cells x𝟏𝟎𝟏𝟐 /L (RBC)
METHOD
1. HEMATOCRIT READER
2. SEALANT/ CLAY SEALANT
3. PARAFFIN SEAL
4. BLOOD COLLECTION MATERIALS
5. MICROHEMATOCRIT CENTRIFUGE/ HEMOFUGE
6. CAPILLARY TUBES
- Internal diameter: 1mm
- Length: 70 or 75 mm / 70cm or 7.5 cm
- Types:
o Heparinized: red band; used when
directly collecting blood from skin
puncture.
o Non-heparinized: blue band; used when
the blood is already in an EDTA tube or
mixed in an EDTA coagulant in order to
prevent shrinkage of the RBCs and
decreased volume of Hct and MCV
NOTE: PROCEDURE (MICROHCT)
a) Tubes are filled with blood on the opposite
side of the band. Fill the capillary tube with
blood in ¾ full. When it is in ¾ full already, the
tube approximately contains 0.5mL or 50µL.

Hgb= gm % ; gm/L ; gm/dL

Hct= % or L/L b) When using heparinize, invert it upside down
in order for the blood and the heparin to be
mixed.
 c) Let the blood move downward until the edge
of the tube to start sealing. Do not seal if blood
is placed on the other side of the band to
prevent spillage or displacement of the blood.
Start sealing with clay first which is the one in
direct contact with the blood because it is
denser and will totally seal or prevent the f) Measure total volume, height of packed cell
blood from leaking. volume excluding the buffy coat. Inclusion of
d) To hold the clay in place, follow it up with the buffy coat will result to an erroneously
paraffin so that the clay will not be washed out increased Hct reading.
because during centrifugation, the force will
𝐏𝐂𝐕 𝐏𝐂𝐕
be going outwards. The clay will be washed off HCT= or HCT= 𝒙 𝟏𝟎𝟎
𝐓𝐕 𝐓𝐕
if it is too soft. The length of the seal
(clay+paraffin) should be at least 4-6mm.
NOTE:
 Foot rule: can be used to measure the PCV
 Microhematocrit Reader: made up of two
rotating plates

e) After sealing, balance the centrifuge and

centrifuge the capillary tube at 10,000-15,000
g for 5 minutes.

How to use:
Note: To prevent the blood from spilling yet allows the a. Set capillary tube into the groove of the sliding
red cells to settle, the sealed area should be pointing curve
outwards. Once the machine rotates, the centrifugal b. Set bottom of blood cell in tubes into 0% line.
force is to push the blood outwards. c. Move cursor and set upper end of blood
plasma in tubes onto 100% line
d. Move scale and set red line onto upper end of
blood layer and read the measure value.

MACROHEMATOCRIT
MACRO METHOD (WINTROBE)

 Length: 11cm or 110 mm

 Blood column: 100mm or 10cm
 Graduations:
Note: Appearance after centrifugation. o Left: 0-10; ESR
o Right: 10-0; Hct
 Diameter: 3mm
 Centrifugation (RCF): 3,000 rpm for 30 minutes
FORMULA:
Vol % Hct=
𝑯𝒕 𝒐𝒇 𝑷𝑪𝑽
× 𝟏𝟎𝟎
𝑯𝒕 𝒐𝒇 𝒘𝒉𝒐𝒍𝒆 𝒃𝒍𝒐𝒐𝒅
REFERENCE RANGES:
Male 0.42-0.50 / 41.5- 50.4 %
Female 0.36-0.45 / 35.9- 44.6 %
At birth 0.45-0.60
NOTE: Macrohematocrit
 Unlike for the micro method wherein the
centrifugation is only for 5 minutes, in macro
method, the more blood you use, the longer
is the centrifugation time yet lower centrifugal
force.
 Manual calculation is done in Macro method
unlike in micromethod where hematocrit
reader is used for measuring.
 It is more accurate to read in millimeters (mm)
because of the small graduations.
NOTE: HCT are expected to be ELEVATED in the
following conditions:
POLYCYTHEMIA VERA
- One condition wherein Hgb is elevated
because in this condition, the RBC count is
high due to an increase production or too
much production of RBC. In fact, all blood cells
(WBCs, platelets, RBCS) are increased in this
condition.
- The measure of this RBC volume is the Hct.
Therefore, Hct is also elevated. The higher
RBC, the higher PCV.
HEMOCONCENTRTION
- Increase in Hct is not due to an increase in cell
prodution but rather the decrease of plasma
concentration resulting to
hemoconcentration which can also be due to
dehydration.
DEHYDRATION
NOTE
Hematocrit (PCV) is one of the values or tests that is
greatly affected when blood specimen collection is
wrong particularly the prolonged application or tight
application of tourniquet. Because of this, the fluid
inside the plasma will be pushed outwards but the cell
will remain in the circulation. However, some of the
plasma fluid will move outside resulting now to
hemoconcentration.
Change in patient’s position from supine to upright or
sitting position can cause hemoconcentration. The
plasma volume of the patient will move outside the
interstitial space resulting to hemoconcentration.
In this situation, the effect on PCV is not great. Around
8% will be the erroneous effect in the PCV.
NOTE: HCT are expected to DECREASE in the
following conditions:
ANEMIA
- Group of conditions characterized by
decreased in RBC count, Hgb, and Hct.
Decrease in RBC is the one that leads to the
decrease in the Hgb concentration and Hct.
- Total volume is not changed. However, the
PCV has decreased because of the decrease in
production.
HYDRATION
- Hydration will lead to diluted PCV resulting to
a deceased Hct.
- The effect of hydration is hemodilution. By
increasing the fluid inside the circulation (Shift
of extravascular fluid) will cause dilution of the
suspended RBCs resulting now to a decrease
in the measured PCV.
- When a patient is given an IV fluid, this too can
result to hemodilution.
- Other cause is the change in patient position
that if a patient is in a lying position, the fluid
outside the blood vessel will move inside the
blood vessel resulting to hemodilution.
INTERPRETATION:
From left to right:
1st tube: decreased plasma volume due of
dehydration.
2nd tube: increased in RBC and decreased in plasma
volume because of the abnormal production of RBC;
“Polycythemia Vera”
3rd tube: normal
4th tube: Normal; despite the total reduction in blood
volume, the reduction is proportional. This type of
condition is somewhat an unreliable condition or is
unreliable to evaluate and interpret the Hct of this
condition. This happened during acute blood loss. In
acute blood loss, there is an increased loss of blood
but in proportion resulting now to normal Hct.
Compensatory: blood cell undergo vasoconstriction
so that at least the amount of blood being lost is
limited. But the effect of this is the decrease in
circulation because the blood is somewhat obstructed
resulting to a decrease in oxygenation. To expand the
blood vessel, the blood volume should also expand
and the blood volume will come from the bone
marrow but it is too long for the bone marrow to
compensate blood loss. To immediately compensate
blood loss is the shift of fluid from the interstitial
tissues but the effects would be hemodilation.
Note: taking the Hct of a patient immediately after an
acute blood loss is one of the sources of errors.
5th tube: decreased RBC, increased plasma volume
because of the hemoconcentration.
6th tube: normal vol of RBC, but increased in plasma
volume, because of the hydration.
HEMATOCRIT SOURCES OF ERRORS
TECHNICAL ERRORS/ HUMAN ERRORS
 Excess anticoagulant
- Will cause the RBC to shrink and shrinkage will
lead to decrease in Hct.
 Prolonged-standing of blood sample prior to
test
- Will cause RBCs to crenate and swell. When a cell
is swollen especially at the 6th hour of letting the
sample to stand, it will result to an elevated Hct.
- EDTA: most common anticoagulant used but it is
most preferred to use immediately within the
first 2-3hrs from collection.
- Remedy: gently invert the sample 60x to ensure
adequate distribution of the blood cells.
PHYSIOLOGIC ERRORS
 Insufficient mixing of blood
- Will cause either a decrease or increase in Hct
depending on which blood component collected.
- In prolonged standing, the red cells will lie at the
bottom of the tube. The upper portion of a
prolonged standing blood will result to
decreased Hct. But if you will aspirate blood at
the bottom, it will then result to an elevated Hct.
 Improper sealing of tube
- Some of the components will be lost.
- Improper sealing will cause the RBCs to escape
from the tube during centrifugation.
 Inadequate centrifugation/ allowing the tubes
to stand too long after centrifugation
- 5 minutes: required time for microhematocrit
centrifuge
- 30 minutes: required time for macrohematocrit
- Inadequate centrifugation will cause the cells to
disperse resulting to an increase of height
formed. Allowing the cells to stand after
centrifugation for too long will lead to
erroneously increased.
- Protocol of the laboratory:
o >55% Hct value:
-will cause polycythemia vera condition.
To prevent misdiagnosis, before
reporting very high Hct, we have to
make sure that we followed the
procedure correctly.
-Protocol: centrifuge it again.
o If in 2nd centrifugation is still be greater
than 55%, the result is ready to be
reported.
o Re-centrifugation is required if the tube
is not read 10 mins after initial
centrifugation because letting it stand
for 10 mins will cause the RBC to
disperse again. Thus, erroneously
elevated
 Reading errors (Parallax)
- Parallax Error: error in reading volumes due to
the different position of the eyes or the volume
being read.
- It is more accurate to read the volume in eye
level.
 Trapped plasma (poikiolocytes & anisocytes)
 - It refers to the volume of plasma, trapped in
between red blood cells.
- Spun hematocrit: higher compared to
automated hematocrit. It is higher for about 1-
3%, but it will be greater than 3% in the presence
of poikilocytes and anisocytes. Thus, it will lead
to higher hematocrit reading.
- In the presence of abnormal size or shape of
cells, the more spaces would be left in between
that will be filled with plasma, that will trap
plasma.
 Values taken immediately after acute blood
loss
- It will show a proportional decrease in PCV and
the plasma. It will result to normal hematocrit
count, but after few minutes shift of fluid from
the extravascular into intravascular occurs then
that is the true hematocrit of the patient. The
values taken immediately are not reliable,
because there is no change in the result.
- True reflection of acute blood loss will be
observed after 48 hours.
 Dehydration
- The hematocrit will appear elevated not because
of true abnormal increase in production, but
only because of an alteration of the body fluid of
the patient.
 Stasis
- Slowing down/stoppage
- Example: Prolonged application of tourniquet,
the circulation was slow down in the arm
because of the prolonged application of the
tourniquet.
RULE OF THREE
- To check the accuracy of the RBC, Hgb, and Hct
values generated by an automated analyzer or
by manual methods.
- The rule of three applies only to RBCs that are
NORMAL in size and in hemoglobin content.
FORMULA:
Hb (g/dL)= RBC × 3
 Range: +/- 1.5
Hct (%)= Hb × 3
 Range +/- 3
EXAMPLE:
Hb (g/dL)= RBC × 3
Hb (g/dL)= 4.7 × 3
Hb (g/dL)= 14.1 g/dL
 Range: +/- 1.5
 Range: 12.6 – 15.6 g/dL
Hct (%)= Hb × 3
Hct (%)= 14.7 × 3
Hct (%)= 44.1 g/dL
 Range: +/- 3
 Range: 41.1 – 47.1 g/dL
INTERPRETATION:
- If the values coincide, and the results obtained from
the blood smear evaluation appears to exhibit normal
RBCs or appears to be normocytic and normochromic,
the results obtained are ready to be reported. The
results should not be reported if the acquired values
match with each other yet the blood smear shows
abnormal RBCs. Possible causes of a falsely low
hemoglobin concentration or a falsely high hematocrit
should also be looked into.
- If the values do not coincide and the RBC appears to
be abnormal, the results obtained are ready to be
reported. On the other hand, the values acquired are
not reliable when the readings do not agree with each
other yet tend to have normal cells visible in the blood
film.
If the values do not correlate, more research should be
done to find the source of the problem. When a
discrepancy is found, a control specimen should be
examined. In this case, random error may have
happened if appropriate results for control are
achieved. When inexplicable inconsistencies are
discovered, the specimens tested before and after the
specimen in question should be analyzed to see if their
results are consistent with the rule.
RED CELL INDICES/ ABSOLUTE INDICES
- Used to define the size and hemoglobin content
of the red blood cell.
 o The normal appearance of RBC is salmon
pink, the central pale area does not
contain hgb.
o Normal RBC with normal Hgb: the size
of the central palor should be 1/3 the
diameter of the RBC
o Hypochromic: If the central palor is
greater than 1/3, it means that the rbc
lacks hgb.
o We can describe the shape and the hgb
content of the rbc through the
examination of smear, but not always
accurate.
 MEAN CORPUSCULAR VOLUME (MCV)
- Used in classifying types of Anemia.
- With the computation of MCV alone, we can
already morphologically classify anemia as to
whether the anemia is Normocytic, Microcytic, FORMULA:
or Macrocytic.
- The single best index aid in morphologic
classification of anemia.
- For the estimation of the average SIZE of
the RBC
- Size and volume are directly proportional.
Since it is directly proportional, the value of
the MCV is also used to estimate the
average size of RBC.
- Not dependable when RBC vary markedly in
size. The relationship of value and size
becomes unreliable and undepandable.
 Dimorphism: dual morphology
- Ex. Mixture of normal and macrocytic cells
 Reticulocytosis
- Is a condition where reticulocyte volume is
higher, are immature cells and they are
larger than the normal cells and we can see
again the dimorphism.
𝑯𝒄𝒕(𝑳/𝑳 ) 𝑯𝒄𝒕(%)
MCV= × 𝟏𝟎𝟎 𝒐𝒓 × 𝟏𝟎
𝑹𝑩𝑪 (𝟏𝟎𝟏𝟐/𝑳) 𝑹𝑩𝑪 (𝟏𝟎𝟏𝟐/𝑳)

REFERENCE RANGE:
 MEAN CORPUSCULAR HEMOGLOBIN (MCH)
- It is not very well important. 80-100 fL (1fL=10-15L)-normocytic
<80 fL – Microcytic
 MEAN CORPUSCULAR HGB CONCENTRATION
>100 fL- Macrocytic
(MCHC)
- To make the classification more complete
together with the hgb concentration EXAMPLE:
 RED CELL DISTRIBUTION WIDTH (RDW) 1. Hct= 0.44 L/L ; RBC ct= 3.8 x 1012/L
- The use of MCV & MCHC in the morphologic 𝑯𝒄𝒕 (𝑳/𝒍)𝑿 𝟏𝟎𝟎𝟎
classification of anemia applies only when 𝑹𝑩𝑪(𝟏𝟎𝟏𝟐/ 𝑳)
𝟎. 𝟒𝟒 𝑿 𝟏𝟎𝟎𝟎
the red cells are normal in size or when they = = 𝟏𝟏𝟓. 𝟕𝟗 𝒇𝑳 𝑴𝒂𝒄𝒓𝒐𝒄𝒚𝒕𝒊𝒄
𝟑. 𝟖
belong only in one population.
- If the cells are diverse or varied in size, the 2. Hct= 41% ; RBC ct= 4.6 x 1012/L
MCV is not reliable. What is more reliable is 𝑯𝒄𝒕(%)𝒙𝟏𝟎
the RDW or index of anisocytosis.
𝑹𝑩𝑪(𝟏𝟎𝟏𝟐/ 𝑳)
OTHER INDICES 𝟒𝟏𝒙𝟏𝟎
= 𝟖𝟗. 𝟏𝟑 𝒇𝑳 𝑵𝒐𝒓𝒎𝒐𝒄𝒚𝒕𝒊𝒄
 Color index (CI) 𝟒. 𝟔
 Volume index (VI) 3. Hct=0.37 L/L ; RBC ct= 5.1x 𝟏𝟎𝟏𝟐/ 𝑳
𝑯𝒄𝒕 (𝑳/𝒍)𝑿 𝟏𝟎𝟎𝟎
 Saturation index (SI)
𝑹𝑩𝑪(𝟏𝟎𝟏𝟐/ 𝑳)
 Mean corpuscular Diameter 𝟎. 𝟑𝟕𝑿 𝟏𝟎𝟎𝟎
 Mean 𝟓. 𝟏
= 𝟕𝟐. 𝟓𝟓𝒇𝑳 𝒎𝒊𝒄𝒓𝒐𝒄𝒚𝒕𝒊𝒄

MEAN CORPUSCULAR VOLUME (MCV)

- Refers to the average VOLUME of the MEAN CORPUSCULAR HEMOGLOBIN (MCH)
individual red cells in a given blood sample - The average weight of Hgb per RBC
- Hematocrit packed cell volume test - Always correlates with the MCV and MCHC
 - Directly proportional to the size of the RBC &
the concentration of hgb in the cell. 3. Hb= 137 g/L ; Hct= 0.47
- Does not always give us an accurate 𝟏𝟑𝟕𝒙 𝟏𝟎𝟎
= 291.48 g/dL Hypochromic
information, because it always follows the 𝟒𝟕
value of the MCV.
- Whenever the value of MCV and MCHC is
low then the value of MCH is also low. MCV & MCHC
- We do not interpret the MCH. There are 3 general classifications of anemia according
to morphology:
FORMULA:
𝑯𝒈𝒃 (𝒈/𝒅𝑳) 𝑯𝒈𝒃 (𝒈/𝒅𝑳) 1. Normocytic & Normochromic RBC Anemia
MCH= 𝒐𝒓 x 10
𝑹𝑩𝑪 (𝟏𝟎𝟏𝟐/ 𝑳) 𝑹𝑩𝑪 (𝟏𝟎𝟏𝟐/ 𝑳) - Normal size and hemoglobin
- Main Cause: lack of production
- Example: Aplastic Anemia
REFERENCE RANGE: 2. Microcytic & Hypochromic anemia
27-33 pg (1pg= 𝟏𝟎−𝟏𝟐 ) - Example: Iron Deficiency Anemia
- A patient with IDA does not always show
<27 pg (microcytic) microcytic hypochromic red blood cells.
>33 pg (macrocytic) There is a transition wherein when we
develop anemia, we don’t immediately
have the IDA. Rather, we start to develop it.
NOTE:
As the IDA develops further, the cells
“Normocytic but Hypochromic”
change from normocytic, normochromic
It is possible that we can see an abnormal cell with a
into microcytic hypochromic
normal size but it lacks hgb.
3. Macrocytic & Normochromic Anemias
- Example: B12 deficiency anemia & folate
MEAN CORPUSCULAR HEMOGLOBIN deficiency anemia, it describes as
CONCENTRATION (MCHC) megaloblastic.
- Refers to average Hgb concentration of red
cells in a given volume of blood. NOTE:
Some books, there are 4 general classification
FORMULA: wherein the 4th one is, the cells are normocytic but
hypochromic.
𝑯𝒈𝒃 (𝒈/𝒅𝑳) 𝑯𝒈𝒃 (𝒈/𝒅𝑳)
MCHC= 𝒐𝒓 x 100
𝑯𝒄𝒕 (𝑳/𝑳) 𝑯𝒄𝒕 (%)
RED CELL DISTRIBUTION WIDTH (RDW)
- Represents the ratio of the standard
REFERENCE RANGE: deviation to the MCV (taken with the width
32-36 g/dL or 320-360 g/L of the histogram)
 32-36 g/dL- Normochromic - Use RDW: If there is variation in sizes and
 <32 g/dL – Hypochromic shape.
 >36 g/dL- Spherocytic - MCV and MCHC: not applicable when there
is a variation in shapes and sizes.
- We don’t compute it manually; the machine
EXAMPLE:
will derive it from the printed histogram or
1. Hb= 13.6 g/dL ; Hct= 0.42
𝟏𝟑.𝟔 𝒙 𝟏𝟎𝟎
generated histogram.
=32.38 g/dL Normocytic - Index of relative ANISOCYTOSIS (variation in
𝟒𝟐
size)
2. Hb= 14.2 g/dL ; Hct= 38% - →POIKILOCYTOSIS (variation in shape)
𝟏𝟒.𝟐𝒙 𝟏𝟎𝟎
=37.37 g/dL Spherocytic
𝟑𝟖
REFERENCE RANGE: FORMULA:
11.6 – 14.6% % 𝑯𝒈𝒃 𝒈𝒊𝒗𝒆𝒏 𝑯𝒈𝒃𝒙 𝟏𝟎𝟎
C.I. = %Hb=
 Values within – in uniform, same population % 𝑹𝑩𝑪 𝑺𝒕𝒅.𝒉𝒈𝒃 (𝟏𝟔)
 Values below – are not significant
 Values above- cells are significant 𝑹𝑩𝑪 𝒄𝒕.𝒙 𝟏𝟎𝟎
%RBC=
𝑺𝒕𝒅.𝑹𝑩𝑪 𝒄𝒕.(𝟓.𝟎)

HISTOGRAM
- Graphical representation on how cell
REFERENCE VALUE:
distributed, or if the cells are well
0.9-1.1
distributed.

NOTE:
Higher than the normal range will lead to Pernicious
Anemia (macrocytic/megaloblastic anemia B12
deficiency)
 Hgb= 14.0 g/dL ; RBC ct = 4.8x1012/L

 VOLUME INDEX (VI)

Y-axis: depicts cells distribution/frequencies
X-axis: depicts the cell volume. Average size of a red cells as compared with the
- If cells belong to the same volume, the average size of a normal red blood cell.
histogram will show a peak that is too high FORMULA:
which is the normal curve “bell-shaped % 𝑯𝒄𝒕 𝒈𝒊𝒗𝒆𝒏 𝑯𝒄𝒕𝒙 𝟏𝟎𝟎
curve” a.) V.I.= %Hct=
% 𝑹𝑩𝑪 𝑺𝒕𝒅.𝒉𝒄𝒕(𝟒𝟕)
- when the peak is high, the width becomes
narrow and the measurement of the width
is the RDW
REFERENCE VALUE:
0.9-1.1
Increased in infants
Decreased in older children
 Hct = 44% ; RBC ct= 4.8x109/L

-
 SATURATION INDEX (S.I.)

Average amount of Hgb per unit volume of RBC in

relation to normal.

- if the cells are varried FORMULA:

𝑪.𝑰. % 𝑯𝒈𝒃
- if the peak is low the cells will be broad and S.I.= OR
𝑽.𝑰. % 𝑯𝒄𝒕
broad width will show a high RDW.

OTHER BLOOD INDICES

 COLOR INDEX (CI) REFERENCE VALUE:
- Average amount of hgb in each RBC 0.80-1.20
compared with the average amount in a
normal RBC.
- Has a similarity with MCH
  MEAN CORPUSCULAR DIAMETER (MCD) WBC DIFFERENTIAL COUNT
- The average diameter of RBC in microns. - Involves both Quantitative and qualitative
- Price Jones method (Direct micrometry) study of the types of WBCs
- Normal Range: 6-9 microns - It represents the percentage distribution of
- Normal diameter 7-8 um normocyte the different WBCs.

Review on WBC morphology

- The thinner the smear the larger the cell

will appear.
- Not accurate MORPHOLOGY
NEUTROPHIL 9-15 um, has 3-4 lobes
MEAN CORPUSCULAR THICKNESS (MCAT) with granules pink/lilac.
LYMPHOCYTE smallest of the WBC’S,
has round nucleus &
shallow indentation,
scanty or few amount of
cytoplasm and color sky
blue
MONOCYTE largest cell, deeply
indented nucleus “brain
like”, has abundant
FORMULA: amount of cytoplasm
𝑴𝑪𝑽 contains tinny reddish
MCAT = 𝑷𝒊 (𝑴𝑪𝑫)𝟐 granules giving now the
−−−−−
𝟐 ground glass
appearance, and the
color in contrast to the
color of lymphocytes
REFERENCE VALUE: cytoplasm is slate-gray
1.7-3.5 microns or bluish gray.
EOSINOPHIL Has red orange
granules, bilobed
nucleus sometimes
spherical.
BASOPHIL has also spherical &
bilobed nucleus but the
appearance of the
cytoplasm is not very
clear because the
nucleus is covered often
by the large and very
dark granules
sometimes appear blue-
black.
 NOTE:
If we see an immature cell like band or
metamyelocyte, it should be count differently or
separately.

GLASS SLIDE-COVERSLIP METHOD (BEACOM)

It also includes quantitative and qualitative study of
thrombocytes as well as age and morphologic
abnormalities of erythrocytes.
4 GENERAL STEPS:
- Place 1 drop of blood near one end of a glass
slide. Place coverslip.
- As soon as the blood spreads, push forward
the coverslip along the surface of the slide.
- This method is uncommonly performed.

1. Preparation of blood smear

2. Staining of the smear
3. Counting of the cells
4. Reporting

COVERSLIP METHOD (EHRLICH) TWO-GLASS SLIDE METHOD (WEDGE SLIDE/ PUSH

- BM preparations
- NOT for routine Peripheral Blood Smear
- ADV: superior leukocyte distribution
- DISADV: It is more technically difficult
PROCEDURE:
SMEAR)
- Most convenient and most commonly used.
- One of the glass slides serves as the spreader
and the other one is the stationary slides
where the smear will be imprinted.

a. Prepare 2 clean glass slides

b. On the surface of the glass slide place 1 drop of
blood at the center of coverslip held between
thumb & forefinger
c. Invert another coverslip rotated 45o
d. As soon as the blood spreads, pull apart the 2
coverslips in a firm and steady motion. PROCEDURE:
e. Never touch the coverslip with fingers
a. Prepare a smear, place a drop of blood not too
large & not too small to one end of the slides.
b. The size of the blood is 2-4 mm
c. The distance from the end of the slides to
another end should be atleast 1 cm (not less than
1 cm)
 d. Because if we draw the blood backwards with
less than 1 cm it will drawing until at the very end
of the slide.
e. When we positoned it we maintained an angle
between 30-45° (Henry), 25-40° (Steninger)
f. The more viscous the blood sample is, the lower
should be the angle. While the less viscous like
sample from anemic patient the higher should
be the angle.
g. The blood should not be in front of the spreader
slide, do not push the sample, and pull it behind
the spreader slide.
h. And wait the blood drop to spread evenly at the
edge.
i. Push or pull the slides depending on the position
of your hand, with the rapid and smooth motion
no hesitation and no stopping.
DISADVANTAGE:
 Tendency of larger cells to settle at the slide
edges & feathered ends
 ADV: easier to find anbonrmal cells
 Poor distribution of nucleated cells
 Greater trauma to cells, we might see broken cell
or smudged cells. Sumdged cells or also called
squash cells they are actually lymphocytes that
are fragile, commonly seen in chronic
lymphocytic leukemia.
SPREADER SLIDE:
- Must be narrower than the stationary slide,
the use of another slide will prevent the
production or preparation of the smear that
is narrower.
- The immature and larger cells are often
located at the two sides of the slides, while
the smaller cells are at the center.
- If the smear is as white as the slide that will
occupy entirely the width of the slides then
it will not be able to focus the two sides’ part.
- Spreading edge: clean, smooth, polished &
thin
o Presence of dirt or clotted blood
may result to gritty appearance.
- With beveled edge.
AUTOMATED SMEAR
MINIPREP/ HEMAPREP
- Differential count PBS & Hct are often
performed manually to verified those results
generated to automated machine.
- Utilize the wedge procedure
- A precision blood spreader prepares dual
smears simultaneously at a constant angle
and speed.
- But the spreader here is not another slide, it
is already part of the device.
- It is semi-automated only.
- ADV: it allows us to prepare two slides
simultaneously at a constant single speed.
HEMASPINNER:
- Glass slides are placed on a platform inside
the instrument
- It is more automated
- Instrument spins causing the slide to be
covered with a monolayer of cells beam of
sensor light to detect blood distribution.
- Uses beam of sensor light to detect the
filling.
- The type of smear that we produced here is
called spun smear.
 ADVANTAGES DISADVANTAGES WET THICK SMEAR:
 Even distribution  Longer
- On a glass slide, place a large drop of blood
 Consistency of preparation
together with NSS to emulsify and cover it
preparation  Smears cannot be
with coverslip. Examine it just like hanging
 Large done outside the
examination area lab drop preparation.
 Fewer broken  No specific - It is used to observe the motility of the
cells location for parasite.
 We can do the abnormal cells
DRY THICK SMEAR
examination in  Centrifugal force
any part of the may cause - It is prepare in diagnosis of Trypanosmomes
smear. distortions. together with thin smear. “thick & thin
 Can examine the smear”
entire area - The thick & thin smear can prepare on the
same slide or on separate slide.
- For thick smear, place a large drop of blood
at the center of the slide and spread it into
the size of 25 centavo coin and let it dry.
After drying immerse it in water or add drop
of distilled water. (Need to remove Hgb)
then stain it with wright or giemsa stain.
NOTE:
Purpose: screening of parasitic infection or
Whole blood is used in coverslip smear, wedge smear
blood parasites.
and Spun smear.
- The thin smear is prepare with the same
manner it is prepare with the wedge smear.
BUFFY COAT SMEAR But after drying we should not immerse it
- It is used in wedge method water instead we immediately fix in
- For finding reactive /immature or abnormal methanol. After fixing dry it and stain.
cells that are present in small numbers - Purpose: specie identification
- Nucleate RBC is located just beneath of the - Methanol: the best fixative in smear.
buffy coat.
o Megaloblastic nucleated RBC or
hypersegmented neutrophils
- For easier location of bacteria & parasites
- Buffy coat smear is considered a concentrate
smear for WBCs, thus it recommended when
the WBC count is <1x109/L
- Only around 3-5 WBC may be seen in
emmersion field, but in buffy coat smear we
see a lot. CHARACTERISTICS OF A PROPERLY PREPARED
WEDGE SMEAR
THICK BLOOD SMEAR
 Length: must occupy at least ½ to 2/3 of the slide
- Uses whole blood
- For blood parasites examination  Should be free of waves, holes, and ridges
o Malaria; CA; Plasmodium  Should have smooth and even appearance
o Filaria  Should be narrower than the stationary slide
o Trypanosomes  Must have a gradual transition from thick to thin
o Spirochetes  Thin enough to allow proper fixation
- We may prepare the thick smear as wet  Should terminate in a feathery tail (the end
thick smear. should be like tongue or finger shape)
  Should contain at least 10 LPF in which 50% of
the RBCs do not overlap each other; the
remainder may overlap in doubles or triples and
the central pallor should be clear.
IMPROPERLY PREPARED BLOOD SMEARS
A- Doesn’t have uneven distribution
B- Has waves ridges, due to some hesitation
C- Bullet shape & short
D- Short
E- Failure to wait the blood sample to spread
evenly.
F- Presence of holes is caused by net over
foils, gritty appearance.
G- Uneven spreading, blood sample is not
place at the center.
H- There is a good smear at the sides, but the
center appears to be dry because of the
delay smearing.
OIL OJECTIVE 1000x
CAUSES OF THICK SMEAR
 Too large drop of blood
- Greater than to 2-3mm will result to thick
smear
 Too fast spread
- The spreading should be rapid and smooth,
but not too rapid.
 Too high angle
- Required angel is 25-45° greater than this
will cause thick smear.
 High hematocrit
- High viscosity is difficult to spread.
- Remedy: add NSS to the blood sample
prior to smearing to lessen the viscosity.
- Lower the angle.
- The lower the angle, the thinner the smear.
NOTE: EFFECTS OF THICK SMEAR
 Excess plasma causes nucleated cells to shrink
 RBCs form rouleaux
CAUSES OF THIN SMEAR
 Too small drop of blood
- Less than 2mm is inadequate
 Too slow spread
- The slower we spread, the more pressure we
apply on the spreader.
 Too low angle
- Lower than 25o is inadequate will cause the
blood smear very long & very thin.
 Low hematocrit
- Have lower PCV with higher plasma volume
and lesser viscosity.
NOTE:
 Increased number of smudged cells
 RBCs become artificially spheroid with
distorted shape
 More nucleated cell on the edges.
 CAUSES OF GRITTY APPEARANCE
- Accumulation of nucleated cells (large
number of leukocytes; use of heparin)
o Heparin causes the blood to clump,
causes a dark background.
- Too slow spreading/delay in spreading
o Too slow in spreading will cause the
blood to dry.
- Using only a part of the drop of blood
- Rough edge or dirty spreader slide.
ARTIFACTS OF BLOOD SMEAR PREPARATION
Blood Sample That Can Be Used in Smear Preparation
1. Fresh capillary blood sample
2. EDTA anticoagulated blood sample, it has more
volume therefore we can prepare more smear,
we may also delay the preparation (the delay
should not be longer than 2-3 hours).
EDTA
1. Monocyte vacuolation
2. Reactive lymphocytes→ vacuolated cytoplasm
(“swiss cheese”) w/ convoluted nuclei
3. RBC speculation
- Become echinocytic, the borders are no
longer smooth as it has already spicules.
4. Dohle bodies may disappear
5. Basophilic granules
- Clinical sig. of basophilic granules it indicates
abnormal RNA metabolism. But in artifact it
is just an artifact without clinical significance
at all it is because of the delay in preparation
of blood smear.
6. Drying artifacts:
 Humid environment
 Severe anemia
 Water contamination of fixative
- Red cell artifacts
 RBC= “moth-eaten” appearance
- Hairy appearance of lymphocytes
- Shrinkage of normal lymphocytes.
METHODS OF DRYING BLOOD SMEAR
 Air drying (fan)
 Use of low flame
 Use of oven
 Do NOT blow on the slide
- When blowing air unto the blood smear, we
also introducing moisture from our breath,
moisture will cause an artifact “echinocytic”
ROMANOWSKY STAINS:Dmitri Leonidovich
POLYCHROMATIC STAINS
- Produce multiple colors
COMPOSITION:
 METHYLENE BLUE (+/BASIC STAIN)=DNA
- Tetramethyl thionine; trimethyl thionine
(Azure)
 EOSIN B/Y (-/ACIDIC STAIN)= BASIC HGB
- Ionization; no ionization, no staining will
occur.
- In examination of malarial blood smears
ideal pH 7.2
 FIXATIVE: Methanol
- wash water must be neutral, tap water is
highly discourage because it has some
contaminants
EXAMPLE OF ROMANOWSKY STAINS:
 Wright- the most commonly use
 Giemsa- For examination of malaria parasites,
the most commonly use is the giemsa stain or
not na leishman stain.
 Modified Wright-Giemsa
 Leishman
 May-Grunwald
 Jenner
  MacNeal
WRIGHT’S STAIN: PREPARATION
- Wright’s powder 1g
- Acetone-free Methanol 500 mL
- 1 Week at 37°C then RT/Ref temp.
o (+) 1 gm Giemsa powder/ 500 stain
shortens the incubation.
WRIGHT-GIEMSA STAIN: PREPARATION
- Wright powder 9g
- Giemsa powder 1g
- Acetone-free methanol 2,910 ml
- Age for 30 days
o We have to ripen for oxidation/
polychroming/ripening/aging.
o Without ripening there is no effect
on the blood cells.
- Fix then apply stains as well as the buffer,
and we wash off the excessive stains we dry
then prepare for examination.
METHODS OF STAINING
1. RACK METHOD
- Makes use of a staining rack, we place the
blood smears with fixatives then we drain-
off the fixatives, flood the blood smears with
stains like the eosin & methylene blue and
then the buffer. Once the buffer is applied
we can observe a greenish metallic sheen on
the surface of the stain. Which indicates the
mixing of the buffer to the stain.
- After staining, we will discard or drain off the
stains.
2. DIP METHOD
- It is more practical, we are going to prepare
pulping jars or vessels we fill this with the
staining reagent with (1) fixative; (2) Eosin
and (3) methylene blue. Deep now the slides
on the solutions.
3. AUTOMATED MATHODS
- Follows the principle of dip method, it’s just
that it is performed by the machine.
- In manual method, we manually handle the
slides, manually dip the slides unto the
staining reagents.
- In automated method, same dipping will
occur. The slides here are suspended but it is
done by the machines. The machines has the
carrousel to rotate the suspended smears, it
also has an arm it moves up & down to
immerse the smears as well as to agitate the
smears.
CELLS/ CELLULAR COMPONENT
1. BASOPHILIC
- It takes up the basic dye, the nature of this
component is Acidic in nature.
- Contains more acid like DNA, because it
takes up more of the basic stain. (MB)
2. ACIDOPHILIC/ EOSINOPHILIC
- Takes up the acidic dye, it has higher affinity
to acidic stain (Eosin). Example of this
component is the Hgb, it is more basic as to
its nature does it will take up the acidic dye.
(pinkish/reddish)
3. NEUTROPHILIC
- Cellular component that react to both & give
both characteristics of acidic & basic color
they are referred to as Neutrophilic which
has neutral reaction.
- Example: Neutrophil
CRITERIA FOR A GOOD STAINS SMEAR
MACROSCOPIC: the blood smear should be pink to
reddish-brown.
MICROSCOPIC:
 Erythrocytes: appear salmon pink
 Platelets: purple-blue to lilac cytoplasm with
red-purple granules
 PMN: Nuclei = purple-blue; cytoplasm= pinkish
tan; granules= pink to lilac
 Lymphocytes: light blue cytoplasm
 Monocytes: faint blue-gray cytoplasm with tiny
red-purple granules
 Eosinophils: red-orange granules
NOTE:
The color of these granules serves as the pH
meter of the stain/buffer.
 Basophils: dark purple granules
 CAUSES OF RED STAIN
 Too acidic buffer or stain
- Lower than 6.4-6.8 will lead to reddish stain
 Excess buffer
 Insufficient staining time; excessive washing
- Will result to under stained smear.
 Very thin smear
- Fewer cells are deposited on the blood
smear.
 Contaminants in wash water
 Exposure of buffer or stain to avid fumes
 Old stain
 Mounting the coverslip before drying.
CAUSES OF BLUE STAIN
 Too alkaline buffer or stain or was water
- pH >6.8
 Too little buffer; excessive staining time
- Too short application of buffer, excessive
staining will result to over staining of blood
smear.
 Short drying period; inadequate washing
 Thick smear; old smear
- The thicker the smear, the darker it will
appear.
- Old smear related to proteins, in an old
smear the plasma proteins will start to dry
causing now the dark appearance
 Protein abnormality; low Hct
- Related to proteins
 Heparinized blood sample
- Disadv: WBC= gritty appearance
- Disadv: pH of this anticoagulant is
incompatible to the stain. Thus, in
Romanowksy stain, it gives dark blue or dark
background.
 Very high leukocyte count with many blasts
- More blast cells (immature cells)
MANUAL DIFFERENTIAL COUNT
- WBCs are count in consecutive fields as we move
the slide. The methods we will mention are
based on how we move the slide.
BATTLEMENT METHOD
- Also described as “Serpentine” because it moves
like a serpent starting from one end and moves
towards the other end of the slide.
- Most commonly used method.
- Examination are done in the body of the blood
smear where the cell are not too much
overlapping and we move it from side to side.
- Similar to the cross-sectional method but certain
patterns are being followed in battlement
method where we start on the side edge of the
smear and move the field three times towards
the center. As we move the field, we count the
WBCs consecutively while classifying them
according to type. After moving towards the
center, move two fields vertically and back to the
side edge two times.
- Purpose and advantage: to compensate on the
inconsistency distribution of cells since most of
the large cells as well as nucleated cells are
located at the edge while the smaller cells (e.g.
lymphocytes) are located at the body or at the
center of the smear. Smears are examined in
such a way that we have the chance to observe
the edges and the center of the smear.
LONGITUDINAL STRIP METHOD
- Cells are again count consecutively as we move
the fields from end to end and go back from the
same end to the other end until we are able to
complete the differential count of 100 WBCs.
- Longitudinal Strip Method is ideal only when the
smear is too thin enough. With the wedge smear,
longitudinal method is not possible because in
wedge smear, it is impossible to perform a
differential count on the thickest part (head)
because the cells are too much overlapping and
are distorted. Examination cannot also be done
in the feathery tail. Examination are only limited
to the body of the smear. Thus, we cannot do
this method but it can be done if the smear is a
spun smear.
- SPUN SMEAR: prepared with the use of a
hemaspinner wherein the entire slide is
deposited with a single layer (monolayer) of
blood cells. Examination can be done on which
 ever part of the smear due to the consistency in
the preparation.
STEP METHOD
- Fields are moved in a patterned movement
following a step pattern. We always have to
pass from the edge or the side and then the
center to compensate the distribution of cells.
PROCEDURE
1. Check slide identification
2. Perform patient specimen orientation.
- As medtechs perform differential count, must
orient themselves with the patient’s results to
know what to expect on the blood smear. (E.g.
When the patient’s CBC results shows ↓Hgb,
expect for the RBCs to appear to have a large
central pale area.)
- Used to detect inconsistencies
3. LPO- scan to review blood film.
- LPO>HPO>OIO
o Check feather edge for fibrin (possible
if the blood is not properly mixed with
the anticoagulant which will negate the
examination). Fibrin formation can
result to a decreased cell count because
some cells will be trapped in the fibrin
clot. As a result, examination becomes
inaccurate.
o Check film edges for excessive
leukocytes. It is but normal to find
more WBCs in edges compared to the
center of the smear. Especially the
larger ones like neutrophils and
monocytes. While lymphocytes are
located in the body or center of the
smear.
o Edges should contain <2-3 times more
WBCs than in the body of the film.
o Verify the acceptable number of
leukocytes
o Verify stain quality (If the RBCs appear
salmon pink, if platelets appear purple
or bluish, if other cells are well stained)
o Examine RBC distribution patterns and
shapes
4. Shift to HPO and perform an HPO scan to
determine examination area. Body of the smear
is where the examination is done. We will select
which part of the body the examination should
will be done. Criteria are:
o At least 50% of the RBCs should not be
overlapping and at the same time their
central pale area (central palor) should
also be prominent.
5. Oil Immersion Examination
o Perform WBC differential count
o Estimate Platelets (8-20 platelets/ oif)
o Estimate WBC
o PUPROSE OF
ESTIMATION (manual
or automated): Though
estimated platelets
and WBCs are not
being reported, we
only estimate or use
the estimated values
to validate the results
or correlate with the
directly performed
palatelet and WBC
count.
NOTE:
In routine differential count, we count a total of 100
WBCs as we classify them according to their types.
But there are conditions that will excuse us from
counting 100, we can count less than 100 (E.g. 50) or
there are conditions that will require us to count
more than 100.
NOTE: How do we verify the acceptable number of aggregates of platelets, then the estimates will not be
WBCs? valid. Considering the number of platelets
We correlate it with the WBC count of the patient. If aggregated, it will exceed 8-20 platelets / oif and
the WBC count is too low, counting 100 WBCs will be clumped platelets cannot be counted. We count the
very time consuming. In this case, we can count for a total number of platelets in 10 fields and getting its
less than 100 WBCs (E.g. 50). On the other hand, if average by dividing it into 10 (representing the 10
the WBC count is very high, we can count a total of fields) then multiply it by a factor or 20,000
200 WBCs. The more cells there are, the WBCs should
also be counted. 𝒕𝒐𝒕𝒂𝒍 𝒏𝒖𝒎𝒃𝒆𝒓 𝒐𝒇 𝒑𝒍𝒂𝒕𝒆𝒍𝒆𝒕𝒔
𝑷𝒍𝒕 𝒄𝒐𝒖𝒏𝒕/µ𝑳 =
𝟏𝟎
× 𝟐𝟎, 𝟎𝟎𝟎
 50 cells: WBC count is < 𝟏 × 𝟏𝟎𝟗 /L or >1000
/µL
Example:
 200 cells: WBC count is > 𝟒𝟎 × 𝟏𝟎𝟗 /L or 𝐭𝐨𝐭𝐚𝐥 𝐧𝐮𝐦𝐛𝐞𝐫 𝐨𝐟 𝐩𝐥𝐚𝐭𝐞𝐥𝐞𝐭𝐬
>40,000 /µL 𝐏𝐥𝐭 𝐜𝐨𝐮𝐧𝐭/µ𝐋 = × 𝟐𝟎, 𝟎𝟎𝟎
𝟏𝟎
 More WBC should be counted: >100,000 /µL 𝟏𝟖𝟎
𝐏𝐥𝐭 𝐜𝐨𝐮𝐧𝐭/µ𝐋 = × 𝟐𝟎, 𝟎𝟎𝟎
In either case, whether we count 50 or 200 cells, the 𝟏𝟎
basis of reporting should still be of a 100 total. Platelet count/ µL= 360,000/ µL
NOTE: How to estimate WBCs?
If the count is less than 100, we multiply the count by Get the average of the number of WBCs observed in
a factor to make it a total of 100. 10 fields. We will now multiply it by the usual factor
Example: of 2,500 or 2,000 (if HPO) or 3,000 (if LPO).
WBC count= 50 Note: estimated counts are not being reported.
Neutrophils= 25
𝒕𝒐𝒕𝒂𝒍 𝒏𝒖𝒎𝒃𝒆𝒓 𝒐𝒇 𝑾𝑩𝑪𝒔
25 x 2 = 50% 𝑬𝒔𝒕𝒊𝒎𝒂𝒕𝒆𝒅 𝑾𝑩𝑪/µ𝑳 = × 𝟐, 𝟓𝟎𝟎
𝟏𝟎
Leukocytes= 20
20 x 2 = 40%
Monocytes= 5
5 x 2 = 10% WBC DIFFERENTIAL COUNTING
Total converted cells= 100

If we counted more than 100, we have to report it

based on 200 totality. In this case, we divide the cells
in a factor to convert it to a total of 100.
Example:
WBC count= 200
Neutrophils= 140
140 ÷ 2 = 70%
Leukocytes= 40
40 ÷ 2 = 20%
Monocytes= 10
20 ÷ 2 = 10%
Total converted cells= 100

NOTE: What is the purpose of counting 200 cells yet

reporting only 100 cells? MORPHOLOGY
To improve precision or the reproducibility of the test
NEUTROPHIL  3-4 lobes of nucleus (can be
or result.
more than 4)
NOTE: How to estimate platelets?
 Rose pink or pink to lilac
After locating a good area of the smear, observe first
granules
the number of platelets per field. For the estimate to
BANDS  Less mature cells but
be valid, There has to be at least 8-20 platelets/ oil
normally appear in the
immersion field (oif) wherein each of this oif, there
circulation
are approximately 200 RBCs. Therefore, if we see
  Appears with an elongated - Keep counting and registering by pressing
nucleus with C, U or S- buttons until the total of 100 is reached.
shape. - Record the register counts and set back to zero
 Not separated by filaments before using it again to another count.
LYMPHOCYTES  Smallest WBC
 Dark nucleus and scanty
cytoplasm but some may
also show abundant
cytoplasm.
 Round nucleus, but some
will also show a slight
indentation on its nucleus.
 Cytoplasm: pale blue or sky
blue
MONOCYTES  Largest of the WBCs
 Nucleus: lighter color with
deep indentation giving a
kidney bean-shape
appearance
 Cytoplasm: greyish-blue;
PROCEDURE OF DIFFERENTIAL COUNTING
abundant amount
EOSINOPHILS  Granules: orange-red color
 Most of them are bilobed
or three lobulation
BASOPILS  Granules: blue-black or dark
blue large granules

 TALLY COUNTER
- Cannot be used in differential counter because
it will not show the types of WBCs.
- Used for total cell counting or total
enumeration.
- It only counts the number of WBCs regardless
of its type.
a. After identifying the correct area, move the
slide either using the battlement method or
longitudinal strip. As we move the field, we also
count the WBCs we encounter consecutively.
b. Register the number of cells on the differential
counter according to its type.
c. Keep moving the slide until we are able to
complete the required number of cells which is
100.

 DIFFERENTIAL COUNTER
- Counts and identify the cells separately
- We also count if we see immature cells and
count them separately because they have
different clinical significance from that of the
mature cells.
OTHER CRITERIA
 50 cells: WBC count is < 1 × 109 /L or >1000 /µL
 200 cells: WBC count is > 40 × 109 /L or >40,000
/µL
 We count a total of 100 cells at first but when
the following listed below are observed, then
there is a need for us to add more 100 cells.
Additional 100 cells are counted when:
o >10% Eosinophils
o >2% Basophils
 o 11% Monocytes
o Lymphocytes > Neutrophils because
normally, Neutrophils have greater
number than the lymphocytes.
NOTE: Exception to the rule
o It is valid if the lymphocytic observation
(increase number of lymphocytes) is
observed on the blood samples of
children because children will normally
show more lymphocytes than
neutrophils.
METHODS OF CLASSIFYING NEUTROPHILS
1. ARNETH’S CLASSIFICATION
- Classification based on age and the age is
dependent on the number of lobes or
segments.
- It separates the young ones from the more
mature ones based on their lobulation.
- The less lobes there are, the more immature
the cell is. The more segments or lobes present,
the more mature the cell is.
- Neutrophils with no indentation or lobulation
but only a round nucleus
CLASS NO. OF LOBES % o Infection, appendicitis and acute
DISTRIBUTION
I No indentation; 1 round or 5% (blast
indented nucleus cells)
II 2 nuclear lobes/ divisions 35%
III 3 nuclear divisions 41%
IV 4 nuclear divisions 17%
V 5 or more 2%
Note:
Class I and II are immature.
Class III represents the mature forms and is the most
abundant.
Class IV and V are still mature but represents the
older forms. The older the neutrophil is, the more
segments are formed.
2. SCHILLING’S CLASSIFICATION
- It still separates the neutrophils according to
age but the age this time will depend on the
cytoplasmic granulation.
- The less granules there are, the less immature
the cells is. The more granules there are, the
more mature the cell is.
- Makes use of the Schilling’s Hemogram to tally
the cells.
o Left: 1-4; younger forms
o Right: mature forms; mature
eosinophils, lymphocytes, basophils and
monocytes.
- Left Shift: If more cells are registered on the left
side, it indicates an increase in the younger
forms.
- Right Shift: If more cells are registered on the
right side, it indicates an increase in the mature
cells.
- Normal blood sample: shows a Right Shift
because in a normal blood sample, the most
abundant are the mature neutrophils followed
by the mature lymphocytes.
NOTE: SCHILLING’S HEMOGRAM
SHIFT TO THE LEFT/ LEFT SHIFT
- Increase in younger forms
 Regenerative:
o ↑Young forms with accompanying ↑
WBC count
sepsis
 Degenerative:
o ↑ young forms with Normal or
↓WBC count
o Typhoid Fever and TB
SHIFT TO THE RIGHT/ RIGHT SHIFT
- Increase in mature forms
 Pernicious anemia or megaloblastic anemia
-Both RBCs and Neutrophils will appear
abnormal. Neutrophils appears large and are
hypersegmented which indicates the mature
or older neutrophils.
3. FILAMENTOUS AND NON-FILAMENTOUS
- Based on the presence of filaments that
connects the lobes or the segments of the
nucleus.
o Filamentous: mature form; nuclear
lobes are separated with filaments
 o Non-filamentous: an immature form; if
the lobes are not separated by
NOTE:
filaments but only by thinned out
portions of the nucleus; Band cells. Reference Values from Brown

REPORTING
1. RELATIVE COUNT
- Percent or relative distribution of cell Reference values by Rodak’s and Henry
- To be reported as percentage or decimal
- Total WBC count is never a relative count

2. ABSOLUTE COUNT
- Actual number of cell per unit volume of blood
- Differential can be reported either as an
absolute or relative count
- Can be done through direct count NOTE: SAMPLE COMPUTATION:
- To get the absolute count: Relative count x total Result in patient A shows a relative monocytosis.
WBC count

SAMPLE COMPUTATION:
WBC count: 10.0 × 𝟏𝟎𝟗 /L
Diff Count: Relative Absolute
Count (%) count
Neutrophil 62% 6,2000/ µL
Lymphocyte 26% 2,600/ µL
Monocyte 8% 800/ µL
Eosinophil 4% 400/ µL

Patient B shows leukocytosis with absolute

Relative Absolute neutrophilia, absolute monocytosis and absolute
Count Count eosinophilia
PMN 35-71% 1500-7400/uL
Lymphocyte 24-44% 1000-4400/uL
Monocyte 1-10% 100-1000/uL
Band/Stab 0-6% 0-700/uL
Eosinophil 0-4% 0-400/uL
Basophil 0-2% 0-200/uL
 CLINICAL SIGNIFICANCE
PHYSIOLOGICAL VARIATION
- Circulating cells are volatile
o Easily leave the circulation like for
instances where neutrophil respond to
chemotaxin cause by infection
- Corticosteroid hormones cause lymphocyte and
eosinophil to disappear from the circulation
within 4-8 hours
o increase adrenal production producing
corticosteroid which will signal
lymphocyte and eosinophils to migrate
to tissues thus there will be
lymphocytopenia and eosinophilia
o Cushing syndrome will result to
lymphocytosis and eosinophilia
- Epinephrine (produced during stress) cause
granulocytosis within minutes
o Marginating WBC will detach and will be
in the circulation
- Neutrophilia
o Physiologic
 Increase during stress
 Response to the therapy
 Response to physical and
emotional stress
o Pathologic
 Infections
 Inflammatory response to tissue
injury
 Malignancy (neoplastic growth)
 Metabolic disorder
 Drugs, chemicals, venoms
- Neutropenia
o Inherited causes:
 Stem cell disorders (decreased
CFU-GM)
 Genetic disorder of the immune
system
 Disorders of cellular
development
 Intrinsic defects - Fanconi’s,
Kostmann’s, cyclic neutropenia,
Chédiak-Higashi
o Acquired causes:
 BM/stem cell destruction-
Radiation, cytotoxic drugs
(chemotheraphy), benzene
(destroys bone marrow)
 Severe folic & Vit. B 12
deficiency decreasing the
production
Others:
- megaloblastic anemia (severe folic & Vit. B 12
deficiency decreasing the production),
myelodysplasia, marrow failure, marrow
replacement
- Infectious - any overwhelming infection
(Infectious diseases- tularemia, typhoid,
brucellosis, hepatitis, influenza, measles,
mumps, rubella, IM, malaria)
- Drugs – cancer chemotherapy, chloramphenicol,
sulfas/other antibiotics,
phenothiazines,benzodiazepine, antithyroids,
anticonvulsants, quinine, quinidine,
indomethacin, procainamide, thiazides
INCREASE NEUTROPHIL DESTRUCTION
- Liver disease, storage diseases, LE
- Infectious - any overwhelming infection
(Infectious diseases- tularemia, typhoid,
brucellosis, hepatitis, influenza, measles,
mumps, rubella, IM, malaria)
NOTE:
if bacterial infection involves destruction, neutrophil
decreases
Note: when neutrophils had performed their function,
they are destroyed since they are an end stage cell
Others:
- Toxins - alcohol, benzene compounds
- Immune-mediated - collagen vascular disorders,
RA, AIDS
- thurst - starvation, hypersplenism
- Basophils releases Histamine and ECF-A which
will stimulate eosinophil to release histaminase
to modulate allergic reaction thus increasing
eosinophil
- Eosinophils granules have cationic protein and
major basic protein to destroy the larval stages
of helminths thus eosinophil is increased in
parasitism (helminths – round worms)
- Loefflers is a kind of allergic reaction of the lungs
wherein eosinophil is increase in the lungs
- Eosinophilia
o Allergic reaction - asthma, hay fever,
drug reaction, allergic vasculitis, serum
sickness
o Parasitism - Nematodes, Trematodes,
Cestodes (trichinosis, filariasis,
schistosomiasis)
o Hypoadrenalism – addison’s disease
  Decrease corticosteroid thus
decrease eosinophil and
lymphocytes in the tissues
 Neoplastic diseases - Hodgkin
lymphoma, CML, collagen
vascular disease, ulcerative
colitis, Ltryptophan eosinophilic
myalgia, T cell lymphomas
Others:
- Inherited, malignant or reactive
- Inflammatory - eosinophilic fasciitis
- Nonparasitic infections - systemic fungal, Scarlet
fever, chlamydial pneumoniaof infancy,
occasionally gonnorrhea and hansen’s disease
- Associated with recovery phase of most
infections during drug specific parasite
manifestation
- Skin disorder - psoriasis, eczema, pemphigus,
dermatitis, herpetiformis, erythema multiforme,
exfoliative dermatitis ; eosinophils are >200 in
the skin & appear in the linings of the skin
- Respiratory - pulmonary eosinophilic syndromes
(Löffler’s, tropical pulmonaryeosinophilia),
ChurgStrauss syndrome
- Idiopathic hypereosinophilic syndromes -
affecting heart, liver, spleen,CNS, other organs
- Others - certain drugs, hematologic and visceral
malignancies, GI inflammatory diseases,
sarcoidosis, Wiskott-Aldrich syndrome
EOSINOPENIA
o Hyperactivity of the adrenal cortex
(hyperadrenalism) as in cushing’s
syndrome
o Note: when corticosteroid is increased in
the blood it will caused the eosinophil
and lymphocytes it will proceeds
towards the tissues thus decreasing
eosinophil and lymphocyte count thus
decrease in corticosteroid & blood
eosinophil & lymphocytes
o ACTH administration – Thorn test ;
stimulates adrenal to increase
production resulting to decreased
eosinophils in circulation
o Severe infectious granulocytosis
o Labor eclampsia
o After electric shock treatment
o Glucocorticoids, prostaglandins and
epinephrine administration
BASOPHILIA
o Systemic mast cell disease, chronic
hypersensitivity states in the absence of
the specific allergen
o Hypothyroidism
o Ulcerative colitis
o Estrogen therapy;
o Long-term foreign antigen stimulation
o Myeloproliferative disorders
Others:
- Allergy - food, drugs, foreign proteins
- Chronic hemolytic anemia - especially post
splenectomy
- Infections - variola, varicella; Ulcerative colitis
- Inflammatory diseases - collagen vascular
disease, ulcerative colitis
BASOPENIA
o During acute infections
o Stress; Hyperthyroidism;
o Increased levels of glucocorticoids
o Reactive (Immune hypersensitivity
reaction)
LYMPHOCYTOSIS
o Infections - many viral, pertussis,
tuberculosis, toxoplasmosis, rickettsial,
Whooping cough, brucellosis, sometime
tuberculosis, secondary syphilis, Viral
hepatitis, IM, mumps cytomegalovirus,
Chronic inflammations - ulcerative
colitis, Crohn’s
o Hypoadrenalism and sometime
hyperthyroidism
o Ulcerative colitis and immune diseases
(serum sickness, idiopathic
thrombocytopenia purpura)
Others:
- Immune mediated - drug sensitivity, vasculitis,
graft rejection, Graves’, Sjögren’s
- Hematologic - ALL, CLL, lymphoma
- Stress - acute, transient
LYMPHOCYTOPENIA
o Immune deficiency syndromes
o Adrenal gland hypersensitivity, adrenal
corticosteroid exposure, congestive
heart failure
o Destructive - radiation, chemotherapy,
corticosteroids
o Aids destroys T lymphocytes
 o Debilitative - starvation, aplastic anemia, AUTOMATION IN HEMATOLOGY
terminal cancer, collagen vascular  WALLACE COULTER
disease, renal failure, far-advanced - First person to introduce automation in
tuberculosis hematology
o Defects of lymphatic circulation such as - 1949: Coulter Counter by Wallace Coulter
intestinal lymphagiectasia, disorders of - Mode of Action: Hgb, RBC, WBC,
intestinal mucosa, thoracic duct Differential count, Platelet, Blood Indices
drainage - Coulter Principle: electrical
impedance/resistance
Others:
- Infections - viral hepatitis, influenza, typhoid  CLINICAL CHEMISTRY
fever, TB - is the first fully automated in the
- AIDS associated - HIV cytopathic effect, laboratory & hematology is semiautomated
nutritional imbalance, drug effect
- Congenital immunodeficiency - Wiskott-Aldrich USES OF HEMATOLOGY ANALYZERS:
syndrome a. Fully automated – utilizes a bar scanner for patient
- Abnormal lymphatic circulation - intestinal specimen identification
lymphangiectasia, obstruction, thoracic duct
drainage/rupture,CHF b. Semi-automated
Process: technologist feeds the blood into the
- MONOCYTOSIS machine which has a needle/probe which will
o TB, SBE, Rickettsial diseases, syphilis, aspirate the blood for testing. Manual input of
hepatitis patients’ data is also employed.
o LE, RA, ulcerative colitis
o Many cancers, lymphomas and c. Complete automated (ex.: walk away machine)
myeloproliferative disorders Process: feeding of blood is made done by the
machine, moves sample from one area to another
& prepares blood smear & stain
Others: Result: well prepared smear & CBC results
- Infections - tuberculosis, subacute bacterial
endocarditis, syphilis, protozoan, rickettsial
Principle Used by Various Multichannel Instruments
- Recovery from neutropenia
in the Clinical Laboratory
- Hematologic - leukemias, myeloproliferative
Method Impeda Conducti Light Cytochemi
disorders, lymphomas, multiple myeloma
(Instrum nce vity Scatt stry
- Inflammatory diseases - collagen vascular
ent) er
disease, chronic ulcerative colitis, sprue,
Abbott X X X
myositis, polyarteritis, temporal arteritis
Others ABX X X X
- solid tumor, immune thrombocytopenic Siemens X X
purpura, sarcoidosis Coulter X X X
Sysmex X X X
MONOCYTOPENIA
o Monocytopenia is uncommon as an  AUTOMATION
isolated finding. - the use of laboratory instruments and
o Following administration of processing equipment to perform clinical
glucocorticoids (During therapy with laboratory assays with only minimal
prednisone, monocytes fall during the involvement of the medical technologist
first few hours after the first dose but
return to above original levels by 12  CBC- contains different parameters that will
hours) measure the different cells
o Hairy cell leukemia (HCL)
o During overwhelming infections that  HEMOCYTOMETER PRINCIPLE: used in Cell
also cause neutropenia count in manual method
 I - CELL COUNTING: 2 CLASSIC AUTOMATED
METHODS FOR COUNTING CELLS:
a. Electric/Electronic Impedance/Resistance/Gating
- Principle employed by Coulter Counter
Model S & S+
b. Optical Detection/Light Scattering
- Technicon Autoanalyzer H1, Ortho
Diagnostic, Fisher Auto-cytometer
THE BASIC COMPONENTS COMMON TO MOST
HEMATOLOGY ANALYZERS
a. HYDRAULICS
- Includes the aspirating unit, dispensers,
dilutors, mixing chambers, aperture baths,
and/or flow cells, and hemoglobinometer
b. PNEUMATICS
- Vacuum and pressures for operating values
and moving the sample through the system
c. ELECTRICAL SYSTEMS
- Electronic analyzers and computing
circuitry for processing data
1. ELECTRIC IMPEDANCE
- Involvement of resisted electricity
- Basis: Size
PROCESS:
1. Blood is diluted in an isotonic solution (NSS – not a
very good conductor of electricity)
2. Diluted blood is place in the bath containing 2
compartments ((-) inner & (+) opposing electrode)
which is connected by a small opening the aperture
(diameter: 100 µm)
RULE:
The 2-electrode located on each side of compartment
must be opposing to allow current conduction
3. Cells are allowed to pass through the aperture &
the passage of blood cell will cause an
impedance/resistance of electricity.
4. Each time the cells passes, it causes an impedance
which is converted into a voltage pulse
RULE:
The number of cells that will pass through is
equivalent to the no. of pulses that will be generated.
In short, the number of voltage pulses is directly
proportional to the cell count.
RULE:
The larger the cell, the more it will occupy the entire
aperture, thus it will cause greater impedance, thus,
the higher the height of the voltage or height of
aperture. The smaller the cell, the less electricity it
will impede, thus, the lower voltage pulse created.
RULE:
The height of the pulse is directly proportional to the
size of the cell which is directly proportional to the
volume. Therefore, the height of the voltage pulse is
directly related to cell size & volume
The number of voltage pulses is equal to the no. of
cells that pass through.
In short: Blood is diluted in an isotonic solution and
drawn through an aperture. Since cells are non-
conductive, the passage of cell through the aperture
increases the resistance (impedance) of the electrical
path between 2 electrodes that are located on each
side of the aperture.
 Changes in electrical resistance are counted as
voltage pulses.
 Number of Voltage pulses is directly proportional to
the number of cells/cell count.
 Amplitude/height/size of the pulse is directly
proportional to the volume of the cell
 Determines both cell volume & count.
2. LIGHT SCATTERING
- Source of Light: LASER
- a diluted blood specimen passes through a
steady stream (located at both sides of the
blood which do not mix w/ blood sample
but it causes blood cells to pass through in a
single pile in a narrow dynamic –
hydrodynamic focusing). As each cell passes
through the sensing zone, a beam of LASER
light is focused. Depending on the
characteristic cell in size & internal
complexity (granulated or lobulated) it
scatters light in different directions which is
measured via photodetectors & scattered
light is converted into an electrical pulse
KINDS OF SCATTERED LIGHT:
a. Side scatter/high angle scatter/ 90 degrees
angle light scatter
b. Low angle/forward scatter
Basis of Scattered Light:
a. Reflection – when light goes up
b. Diffraction & refraction – bending of light
Analysis of cells basis: size, shape & complexity
LASER – light amplified dissimulated emission of
radiation
Photodetectors – measures the amount of intensity of
the light transmitted
NOTE:
In hematology what is counted is the number of cells,
not the type of antigen or antibody involved
NOTE:
 Number of pulses is directly proportional to
cell count
 Patterns of scatter are measured at various
angles to obtain information on the cell
structure.
 Forward Low Angle Scatter:
- cell size/volume
- If cells are large, the higher the light
scattered & vice versa. The lesser the cells,
the smaller the cells generated & vice versa
RULE:
No. of cells generated, is equivalent to no. of cells &
the height & amplitude of voltage pulse is equivalent
to voltage size & volume.
 Right/High Angle Scatter:
- internal structures (ex.: cytoplasmic
granularity)
- In Internal Structure: Cytoplasmic
Granularity & Nuclear Lobularity are
measured at a high angle scatter & the
location of photodetector is at high angle.
When cells are granulated, the greater the
amount of light is reflected thus the higher
is the voltage pulse & vice versa.
 Differential Scatter
 Combination of forward & high angle
 Used in analysis of: RBC (Machine will
compare the RBC volume in relation to
Hb) & WBC (vol. vs nuclear lobularity)
CELL COUNT
Sample: EDTA
Note: check for presence of clots &
agglutinations. If there are presence of micro-
clots this will clog the machine & probes/needle
of machine
Remedy: immediately mix the blood w/
anticoagulant
Dilutions: (Manual)
Purpose: disperse the blood cells in cell counting
Thoma Pipette Dilution:
1:20 WBC 1:200 RBC
Dilutions: (automated)
a. WBC & Hb = 1:500 (usual dilution, but may vary
among manufacturers – 1:250 or 1:750)
b. RBC & Plt = 1:50,000
Reason: 1.) RBC are in millions (3.8/µL); 2.) Make
presence of WBC negligible
NOTE:
 WBC is hypotonic thus causing lysis for RBC
leaving WBC only & releasing Hb thus the
machine measures both WBC & Hb. RBC is
isotonic, WBC are intact with plt thus all will
be measured but some of WBC w/ same RBC
size will be counted as RBC & if dilution is
high WBC will be negligible except when WBC
is very high (>50,000/uL) this will cause a
significant error
 Light impedance & scattering are all for RBC,
WBC & Plt count
II-DIFFERENTIAL COUNT
 Manual – smear & microscopic examination
 Automated – histogram & cytogram
 Cytogram – Gives 5-part differential count
HISTOGRAMS
 Gives a 3 parameter Differential count w/ both
relative & absolute count
 Line graph, graphic representation of 1 cell, the
size/volume (x-axis) versus cell frequency (y-axis)
Left shift: increase small cells on the left
Right Shift: increase large cells on the right
 produced in RBC, WBC & platelet measurements
 Classify WBC into 3 only
 Derived from the histogram are blood Indices as
well as WBC differential.
RBC HISTOGRAMS
- RBC histogram: 36 – 360 fL
 It is where MCV & RDW (width of histogram) are
derived.
 Histogram appears SYMMETRICAL/Gaussian shape
when cell population is homogenous’ WIDER or
FLATTENED shape if there is more variation in the
size of the cell
 NOTE:
 Normal population: Bell shape curve w/ high
peak & is narrow indicates
 The wider the width, the flatter is the
histogram
PLATELET HISTOGRAMS
- Platelet histogram: 2 - 20 fL
- Where Mean Platelet Volume (6.5 – 12 fL)
& Platelet Distribution Width are derived
NOTE:
When values are >20 fL, it will be counted as RBC.
 Purpose of RBC & Platelet Distribution:
mean volume & distribution width
 Purpose of WBC Histogram: differential
count
 WBC Histogram Distribution: Granulocytes
(eosinophil, basophil & neutrophil),
Monocytes (also contains blast) &
lymphocyte.
II-DIFFERENTIAL COUNT
 WBC Histogram: (used in the 3-parameter
Differential count)
 Purpose: separates WBC according to their
population base on size & volume
WBC Populations on the histogram
 35 - 90 fL: Lymphocytes
 90 - 160 fL: Mononuclears/Mid cells/Mono -
(Monocytes, Promyelocytes, Myelocytes, Blasts)
 160 - 450 fL: Granulocytes - (Mature WBC, Blasts,
Metamyelocyte, Bands)
NOTE:
 Limitation: whenever there is a high value of
mid or granulocytes, manual method must be
done
 Automation has a lysing & shrinking effect
due to histomatolysin thus causing partial
monocytes shrinkage & partial granulocyte
swelling
 When Mid Cells are increase, identification
of which is increase is necessary & the same
goes with granulocyte.
SCATTERGRAM / CYTOGRAM / DOT PLOTS:
- Rule: the more dots present, the more cells
are present in the population
- Basis: Size & Complexity
Y-axis: size X-axis: internal complexity
(granularity)
 Population of cells are presented in dots
 a visual display of 2-cell characteristics such as
internal cell complexity and relative size
 It is used in the 5-part automated differential
 Atypical cells are pushed through the upper portion
of histogram while smaller cells are in the lower
portion & the more granulated cells are push
towards the right of x-axis while the less granulated
are placed towards the left
 WBCs are differentiated into 5 traditional categories
REAGENTS:
 Cytochemical stains – stains specific chemical
component of the cell (ex.: enzyme in the cell);
cytogram channels
 Peroxidase dye: PMN & other cells
 Alcian blue/Toluidine blue dye: basophil
 Lipase/Esterase dye: monocytes
 Fluorescent dyes: for specific cell population (NRBC)
III – HEMOGLOBIN :
CYANMETHEMOGLOBIN PRINCIPLE
 Golden standard, most accurate and
measures all types of hemoglobin except
for sulfhemoglobin
 Wavelength: automated: 525 nm
manual: 540 nm
 basis: automated: spectrophotometric
 chemical reaction basis:
cyanmethemoglobin principle
IV – HEMATOCRIT
2 WAYS OF MEASUREMENT:
a. DIRECT (MANUAL)
 spun hematocrit which requires spinning or
centrifugation
 common to have the trap plasma which
increases PCV
 used to calibrate the automated because it is
the most precise & accurate on RBC
measurement
b. INDIRECT METHOD (AUTOMATED)
- being calculated
- Formula derived from:
MCV = Hct/RBC, thus Hct = MCV x RBC
 Machine directly measures MCV from RBC
histogram & directly count the RBC thus only Hb
is unknown.
 Automated hematocrit will always be 1-3% (some in RBC is erroneous Hct is
2-3%) lower: erroneous causing RBC
 In manual, Hct is directly measured & direct indices to be erroneous
counting of RBC thus deriving MCV but in except for RDW
automated procedure, Hct is indirectly NRBC -  Increase WBC Corrected
derived & RBC count is measured resist WBC if >5
 When manual Hct lower than spun Hct, it is lysis in  Hypotonic solution, causes NRBC
unacceptable because in spun Hct, trapped hypotoni RBC to lyse
plasma is common. c
solution Formula for uncorrected WBC:
IV- INDICES thus it uncorrected x 100 / NRBC+100
 MCH & MCHC are calculated from measure & will be
derived values counted
as WBC
 RDW
Cold  Agglutination of RBC at cold T Pre-warm
agglutin’ causing decrease RBC count EDTA by
NOTE:
s – Ab & will be counted as 1 w/ simply
Errors in RBC & MCV count, Hct becomes erroneous
reacting high volume, increasing MCV holding
and since automated machine computes MCHC using
at cold the
MCHC = Hb/Hct, causing erroneous Hct, MCHC will
temperat  On prolonged standing sample to
also be erroneous
ure (0-10 of EDTA, it will cause disperse
C) morphological distortion thus the RBC as
INTERFERING FACTOR ON MOST HEMATOLOGY long as it
refrigerate for 24 hours to
ANALYZERS is not
retard changes
CONDITI PARAMETER AFFECTED RESOLUTI hemolyze
ON ON  Increase MCV d
WBC  False increase in RBC count Dilute  Inaccurate Hct
over 50, Reason: Isotonic diluting fluid sample & Dirt Increase cell count which
000/uL of RBC preserves the RBC, WBC rerun depends on cell size. Increase is
& Plt. Some WBC are counted due to increase impedance &
as RBC because the dilution is affecting light scattering
increased at 1:50,000 thus
 Giant  Decrease Plt 1. Examine
making WBC negligible but blood
platele
when WBC is too high, presence film &
t Platelets – volumes between 2-
of WBC will cause false increase observe
20 fL
of WBC
 platele for
t presenc
 False increase Hb e of
clumps
Reason: Hb measurement is
 Increase RBC platelet
based on spectrophotometric satellitis
procedure wherein the amount m & if
Platelet clumps – recognize as
of light absorb is directly present
larger structure which can be
proportional to the amount of
 satelliti counted as RBC recollect
light measured thus increase Perform
sm in
WBC causes sample to be spun
sodium
turbid hematocri
 Decrease Plt citrate
Rule: The more turbid, the t
however
higher the absorbance & (accuracy
Satellitism – platelets adhere blood
concentration measured is based
on a neutrophil or monocyte & becomes
on skills)
occurs on EDTA especially more
 Hematocrit diluted
when individual develops Ab on
Reason: calculated using the (1:9 or
IgG which will attach on Fc
formula: RBC x MCV & when 1.1) thus
portion of neutrophil or
 monocytes thus machine it must optical interference causing
counts it as 1 cell resulting to be increase of amount of light
decrease Plt multiplie absorb thus causing
d to 1.1 increasing Hb & other
to indices
return Hemolysi  False Decrease RBC & Hct In vitro:
the 10% s  Increase MCHC & MCH recollect
loss due In vivo:
to  Hemolysis together with no
sodium lipemia & icterisia will remedy
dilution cause optical interference but
because they will alter the performed
2. Manual color or appearance of Hb
counts sample manually
or film  Hemolysis will also alter & use a
estimate RBC patient
s to  Hemolysis causes blank
correlat erroneous Hb because if in
e the vitro hemolysis occur the
values in plasma will contain Hb
automat Old  Increase MCV due to swollen Recollect
ed plt specime RBC
(average n  Decrease Plt count due to plt
plt x swelling & disintegration
20,000)  Automated differential count
& if it because WBC morphology
correlat becomes altered
es,  Schisto  Decrease RBC  Manual
report cytes  Inaccurate HCT & indices counts
Lipemia  Increase Hb, MCH  Follow  Spun Hct
(Hb/RBC), MCHC (Hb/Hct) manufac Schistocytes – a generic term  Recalcul
turer’s indicating red cell fragments ate
Icteric – yellow or orange instructi  Microc (triangle, helmet, bite, indices
sample caused by carotene, ons ytes microspherocytes) due to
bilirubin or Vitamin A ; possible (<80 fL) hemolysis thus it will no longer
cause is indirect  Recalcul be counted as RBC because
hyperbilirubinemia ; from the ate they become smaller
word icterus or bilirubin indices Resistant  Increase WBC  Manual
Hyperbilirubinemia – yellow RBCs (Hb  Inaccurate Hb & decrease Hb,
pigment sample ; water S or C) – Hb due to RBC did not lyse adding
insoluble will be water
Chylous – turbid counted (hypoto
Lipemic – increase lipid ; also as WBC nic) to
called chylous due to increase & Hb is lyse RBC
chylomicron which are the not  Manual
lightest proteins that causes release WBC for
turbidity & even when & will affected
centrifuge it will not settle not react WBC
Lipoproteins: HDL, VLDL, w/ count
Chylomicrons Drabkins  Recalcul
ate
 Lipemic (turbid) & Icteric
indices
samples (yellowish) causes
 Fragile  Decrease WBC
WBCs  May increase Plt
(e.g.
chemoth  In chemotherapy the
erapy) chemical infused in patients
is not selective thus
destroying normal cells,
targeting WBC nuclei
resulting to fragile WBC &
this WBCs will not lyse thus it
is now counted as Plt
Manual
counts INDICATIONS OF PBS REVIEW
 Abnormal cells
 Abnormal RBC morphology
 Pancytopenia – overall decrease of blood cells
 Increase Mid cells & Granulocytes
 Presence of microorganism
 RBC/WBC/Platelet inclusions

SOURCES OF ERROR
a. CAUSES OF POSITIVE ERRORS: Erroneous Increase
 Aperture plugs – plasma containing high
amount of protein will coat the aperture & will
precipitate causing plug formation
 Extraneous electrical pulses (improperly
grounded or shielded equipment)
 Bubbles in the sample causing reflection or
scattered light due to vigorous mixing
 Improper setting of aperture current or
threshold
 Agglutinated red cells or platelets thus altering
WBC

b. CAUSES OF NEGATIVE ERRORS: Erroneous decrease

 Excessive lysing of RBCs thus no RBC will be
counted
 Improper setting of aperture current or
threshold
 Agglutination of RBCs, WBCs or platelets

QUALITY CONTROL FOR HEMATOLOGY ANALYZERS

 XB
- a quality control check in which the
averages of the MCV, MCH, MCHC are
tracked for batches of 20 patients
- Also known as the moving average

 DELTA CHECKS
- Patients test results are compared to his
previous results (WBC, Plt, MCV & Hgb)
- utilize for all clinical laboratory
- 2 Reasons of comparison: determine the
physiologic change or human error

 FLAGS
- suggest cells types/abnormalities or
performance error
- Remedy: indicate investigation before final
reporting.
 HEMATOLOGY FINALS NOTES
HEMOGLOBIN
- A respiratory pigment that allows cellular
respiration
- Red color: accounted to iron component
- A conjugate protein (heme + globin) synthesized
in the RBC
- Represents more than 1% of the total body
weight & it occupies 28% of the RBC mass.
- Main protein component: globin
- Non-protein component: heme
- Main Function: Transport oxygen from the lungs
where the oxygen tension is high, to the tissues,
where the oxygen tension is low which allows
cells & tissues to expire.
- Often describes as the “Respiratory pigment”
because it gives the red color of RBCs and allows
respiration or allows oxygen to cells.
- Function: Transport oxygen & CO2 thus allowing
exchange of O2 & CO2 in the alveolar capillaries
& tissues; transports nitrous oxide (NO)
NITROUS OXIDE (NO)
- Substance produce by endothelial cells which
forms the endothelial line
- Function: cause vasodilation; relax the smooth
muscles surrounding the blood vessels especially
the small blood vessels, thus increasing the
blood flow.
- Enhanced by: Hemoglobin
NOTE:
Vasoconstriction: thickened walls and small lumen
Vasodilation: wider lumen, thus, more blood volume
will enter and transported.
VASODILATION PROCESS
- Blood vessels in the lumen increases in diameter
or dilate causing more blood volume to enter
thus the more O2 is delivered. Contrary, when it
constricts, NO causes vessels to lower blood
volume & O2 delivered.
RELATIONSHIP OF HGB TO NO
- NO, once released into the plasma by
endothelial cells, if NO remains free in the
plasma, it will be short lived. But when bound to
hemoglobin, it will be stable & be transported
to different areas of the body (hypoxia tissues).
In tissues w/ decrease O2 (hypoxic tissue), the
body will compensate by delivering more O2
thus the vessels have to vasodilate allowing
more entry of blood & hemoglobin transports
NO to hypoxic tissue in order for NO to cause
vasodilation allowing more blood & O2 supply to
tissues thus correcting the condition. In contrast,
if tissues have increased O2, hemoglobin will
inactivate NO to restrict further vasodilation &
causing vasoconstriction limiting O2 delivery.
Thromboxane A2 (TX2) & serotonin from the
platelets also causes vasoconstriction.
NOTE:
 Hemoglobin when fully saturated: 1 g = 1.34
mL O2, approximately 600 g of 5 – 7 L is
hemoglobin which is able to carry 800 mL of
O2 coming from the lungs brought into the
tissues
 Transport of O2 is influence by affinity of
hemoglobin to O2 which is regulated
depending on action: to carry or release O2.
FACTORS THAT AFFECTS AFFINITY OF HEMOGLOBIN
TO O2
1. pO2
- RULE: increased partial pressure to the lungs
thus the affinity is high allowing Hb to be fully
saturated but if its pressure is low in the tissue,
the affinity is also low causing Hb to release O2
- In the lungs, the pO2 is high at 100 mmHg or 95
– 98% pO2 thus allowing increased affinity &
causing Hb to be fully saturated. Oxygenated Hb
will proceed towards the tissues. In the tissues,
 pO2 is low approximately 20 mmHg or <30%
thus decreases the affinity & Hb will readily
release the O2 to the tissues in exchanged for
the CO2 carried to the lungs to be excreted.
CHEMICAL REACTIONS IN CO2 & O2 EXCHANGE:
a. TISSUE LEVEL (VENOUS BLOOD)
- There is increased CO2 conc. as a product of
metabolism (glycolysis)
1. CO2 will diffuse into RBC
2. CO2 will react w/ H2O forming Carbonic Acid
which is catalyze by an intracellular enzyme:
carbonic anhydrase
3. Carbonic acid will dissociate into H & HCO3
4. H will easily replace O2 thus releasing O2 &
Hb binds w/ H
5. O2 released will proceed to the tissues
6. Binded Hb & H will form hemoglobinic acid
7. HCO3 will stay in the RBC however some
leaves the RBC in exchange of Cl (chloride
shift) which will maintain electroneutrality
8. RBC w/ reduced O2 will proceed towards the
lungs
b. LUNG LEVEL
1. Due to high pO2, O2 will readily enter RBC
thus replacing H binded w/ Hb
2. H will bind w/ HCO3 forming carbonic acid
which will dissociates in the presence of
carbonic anhydrase into H & CO2
3. CO2 will leave the RBC entering now the
pulmonary capillaries to be exhaled by the
lungs
IN SHORT:
CO2 which is greater in tissues diffuses the RBC w/
H2O forming H2CO3 which will dissociate into H &
HCO3. Some HCO3 will diffuse out to prevent
excessive accumulation in exchange w/ Cl (chloride
shift) while H will combine w/ Hb making it more
acidic. Wherein at acidic pH this will decrease the
affinity thus O2 will be easily released & will be
replaced by H. The released O2 will proceed to the
tissues thus Hb will oxygenate the tissues & what will
remain is the Hb w/ H which will proceed to the lungs.
H will be replaced by O2 & Hb will become oxygenated
& H will combine w/ HCO3 forming H2CO3 which will
diffuse into H & CO2. CO2 will be exhaled.
SUMMARY HOW CO2 IS TRANSPORTED: CO2 will be
transported more as HCO3
NOTE: 4 WAYS HOR CO2 IS TRANSPORTED:
1. Plasma – HCO3 ; HCO3 which leaves RBC &
becomes the major form of CO2 which is 70%
2. RBC – 20% HCO3
3. 5% Carbamino Hb – some CO2 which did not
participate in chemical reaction & thus
remained as CO2 & combined w/ Hb known as
carbamino Hb.
4. 5% CO2
2. pH
- RULE: the lower the pH the lower the affinity
- Also binds to other substances such as hydrogen
ions (which determine pH of the blood), CO2 &
2, 3 diphosphoglycerate (2,3 – DPG). These
substances do not bind to hemoglobin at the
oxygen – binding sites, however, binding of
these substances to hemoglobin affects the
affinity of hemoglobin to oxygen. Conversely,
binding of oxygen to hemoglobin causes changes
in the structure of the hemoglobin molecule thus
affecting its ability to bind other gases or
substances.
NOTE:
As CO2 enters the blood, it combines with H2O to form
carbonic acid (H2CO3), a relatively weak acid, which
dissociates into H+ and HCO3-. Blood acidity is
minimally affected by the released H+ ions because
blood proteins, especially hemoglobin, are effective
buffering agents. The conversion of CO2 to H2CO3 is
catalyzed by carbonic anhydrase, an enzyme presents
inside the RBCs. Because the enzyme is present only in
the RBCs, HCO3– accumulates to a much greater
extent within the red cells than in the plasma. The
capacity of blood to carry CO2 as HCO3 – is enhanced
by an ion transport system inside the RBC membrane
that simultaneously moves a HCO3- out of the cell and
in exchange for a chloride ion. The simultaneous
exchange of these 2 ions, known as the chloride shift,
permits the plasma to be used as a storage site for
bicarbonate without changing the electrical charge of
either the plasma or the red blood cell.
 HEMOGLOBIN COMPOSITION  Arrangement of all amino acids of globin
1. 1 GLOBIN chains are precise & exact no. of genes is also
- a globular protein necessary.
and is a tetramer  2 genes from the mother & father each are
which is made up of inherited thus forming 4 alpha chains.
4 polypeptide  Alpha & Beta are the genes affected by
chains which Thalassemia because of the deficiency in the
genes which will result in the synthesis of the
contains individual
chains thus deficiency in the synthesis of the
heme inserted to
entire Hb.
each thus giving a
ratio of 1 globin : 4 heme
- among each polypeptide chain, 2 are different GREEK SYMBOL NO. OF GENES
making up the tetramer of globin which is the NAME AMINO
globular protein component ACIDS
- Composed of 2 a & b (Hb A1) or 2 a & g (Hb F) ALPHA (for α 141 4
Hb A)
GLOBIN CHAINS BETA β 146 2
 Composed of 2 sets (dimer) of 2 different DELTA δ 146 2
polypeptide chains GAMMA λ 146 2
 Synthesize on specific ribosome which depends (for Hb F)
on the genetic make-up dictated by DNA which EPSILON Ԑ 146 2
will determine the type of Hb (for
 Difference in the polypeptide chain relates both to embryonic
the sequence & to the number of amino acids in Hb)
the chain ZETA (for ζ 146 2
 The spatial arrangement of each alpha-beta pair is embryonic (Steineinger)
symmetrical Hb)
 Contacts between the subunits are by means of H- 141
bonds (Rodak’s)
 Depending on the polypeptide chain component,
this will now give rise to the different types of Counterpart
proteins of A
(Henry’s)

REASON FOR DIFFERENT POLYPEPTIDE CHAINS: Follow: 146

 Differences in amino acid composition, amino
acid number and amino acid sequence which
depends on the gene. NOTE:
 RULE: whatever the gene has, it will dictate Thalassemia – caused by deficiency in the genes =
the type of protein or polypeptide chain which deficiency in chains = deficiency in Hb = anemia
will be synthesize by the ribosome Short arm of Chromosome 11 – Epsilon, Delta,
Gamma, Beta & Zeta
Short arm of Chromosome 16- Alpha
NOTES:
 All types of Hb have the same heme but the
globin portion of each varies. Hb TYPE POLYPEPTIDE (GLOBIN) CHAINS
 In embryonic life, Zeta is the counter part of a GOWER-1 2ζ 2Ԑ
which is responsible for Gower 1 & 2, Portland GOWER-2 2α 2Ԑ
& Hb F together with Epsilon. PORTLAND 2ζ 2λ
 Counter part of Epsilon: Beta, Delta & Gamma Hb A 2α 2β
Hb A2 2α 2δ
HB F 2α 2λ
GLOBIN CHAIN SYNTHESIS: completed and this will be released as a
polypeptide chain
a. Due to the dependency on the genetic
composition, synthesis starts in the nucleus B chain = 146 Arrangement: 1. Valine 2.
b. It is w/in the nucleus that the DNA which carries Histidine
genetic information is located
c. Since DNA do not leave the nucleus, it will be b. Chains of amino acid will undergo twisting
copied in the form of mRNA. forming coil or helixes. B chain has 6 helixes from
d. Each gene is stored in the form of segment and A to H. It is between the E and F helix that the
will be transcribed in the form of mRNA. heme is inserted particularly in the histidine. It is
e. mRNA will leave the nucleus and will be scanned in the heme that the oxygen is bound to.
by ribosomes (main globin synthesis) which will RULE: for every 1 polypeptide chain there is, 1
decode the mRNA which serves as a template. heme is inserted which contains the ion
f. For every set of 3 nucleotide will be interpreted
as 1 amino acid. 2. 4 HEME
g. tRNA carries specific type of amino acids which - Inorganic iron groups that assist in binding O2
will be connected to the template connected by (each iron atom can bind and then release an
polypeptide bonds. oxygen molecule)
- Synthesis in the mitochondrion of RBC
FACTORS AFFECTING HEMOGLOBIN AFFINITY FOR - Where O2 binds thus carrying 4 mol of O2
OXYGEN - Has a volume of 1.34 mL O2
- Main function: Transport O2 in exchange for
FACTOR FACTOR IS FACTOR IS CO2
INCREASED DECREASED - Common to all types of Hb:
BLOOD o 4 Protoporphyrin IX: nitrogenous
TEMPERATURE substance formed in the mitochondrion;
where the iron will be attached.
BLOOD pH increase pH Decrease pH o 4 Iron: maintained in Fe2 or else it
(BOHR EFFECT) increase affinity decrease cannot transport O2.
of Hb to O2 in affinity of Hb to
the lungs O2 in the
tissues

RBC 2,3 DPG Increased in the Decreased in

tissues thus the lungs thus
decreasing increasing
affinity of Hb to affinity of Hb to
O2 O2

CO2 (HALDANE NOTE:

EFFECT)  Mature RBC cannot synthesize Hb because it
Hb F has no nucleus and no mitochondrion.
 Not all heme synthesis occurs in the
ABNORMAL Hbs mitochondrion. What only occur in the
mitochondrion is the first and final reaction
while the intermediate reaction occurs in the
cytoplasm or cytosol
SYNTHESIS:

a. mRNA scans every coding triplet carried by tRNA

until a total of 146 B and 141 A chains is
HEME SYNTHESIS:
1st Reaction: Condensation between Succinyl CoA
from tricarboxylic cycle & glycine in the mitochondrion
1st Product: 5-aminolevulinic acid/D-ALA which will
leave the mitochondrion goes into the cytoplasm and
undergoes several changes until it becomes the
coproporphyrinogen 3 which goes back into the
mitochondrion to form in the heme.
EXPOUNDING HEME SYNTHESIS:
a. Reaction between Succinyl CoA from
tricarboxylic cycle & glycine in the
mitochondrion is catalyzed by aminolevulinic
acid synthase w/ the cofactor B6/pyridoxal
phosphate forming now the 5-aminolevulinic
acid or D-ALA
b. 5-DLA will leave the mitochondrion
proceeding to cytoplasm where it undergoes
several changes where it becomes
porphobilinogen – hydroxymethylbilane –
uroporpophyrinogen 3 –
coproporphyrinogen 3 wherein all of these
are enzymatically catalyzed
c. coproporphyrinogen 3 will enter the
mitochondrion where it is converted to
protoporphyrinogen IX which is oxidized to
protoporphyrin 9 (immediate precursor of
heme)
d. Iron from plasma will combine to form the
heme.
e. Heme will leave the mitochondrion and goes
into the plasma where it combines with
polypeptide chains wherein it binds with
some A & non-alpha such as B chain. These 2
will form dimers and combined to form
tetramers thus completing heme synthesis
RULE: globin chains combine due to their
charges thus forming a combination
NOTE:
 Beta has high affinity thus it will bind to more
A thus causing more A1
 Delta second to the highest will bind to A
forming the A2 = 3.5% of total Hb
 Gamma contains few & will bind to A forming
Hb F = 1-2%
IRON SYNTHESIS:
a. Iron is bound to its transport protein called
transferrin/siderophilin
b. Iron will be transported in ferric state/Fe3
c. RBC membrane have receptor for transferrin
which will be recognized and transferrin
enters w/ iron bound to it
d. Iron will enter the cytoplasm and reduced into
ferrous.
e. Ferrous form will combine w/ protoporphyrin
9 forming now the heme.
NOTE:
 Ferrocheletase/heme synthase enzyme –
Enzyme catalyzing the combination of iron to
protoporphyrin 9
 FERROPROTOPORPHYRIN: synonym of
combined heme
2,3 DIPHOSPHOGLYCERATE (DPG)
- Sometime/transient resident
- Molecule that regulates Hb affinity to O2 & how
Hb releases O2 to tissues thus w/out it Hb will
not release O2 & the affinity of Hb to O2 will be
very high & thus its function is to decrease the
affinity causing release of O2
- a salt produced in the Rapoport-Luebering shunt
in the RBCs
RAPOPORT-LEUBERING SHUNT
- part of glycolytic pathway/embden-Meyerhof
which is unique in RBC
EMBDEN-MEYERHOF PATHWAY
- occurs anaerobically
- 2 SHUNTS: Hexose Monophosphate & Rapoport-
Luebering
- IMPORTANCE: necessary for the formation of
2,3-DPG which is an intermediate to 1,3-
diphosphoglycerate & 3 phosphoglycerate.
BASED ON OXYGENATION OF HB, HB ASSUMES 2
FORMS WHICH ARE REVERSIBLE:
a. Oxyhemoglobin/Relaxed form - Hb saturated
w/ O2 w/ 4 moles of O2
b. Deoxyhemoglobin/Tense form
OXYGEN RELEASE INFLUENCED BY 2,3 DPG & TAKE UP
- During release of O2, O2 will not be released
simultaneously but respectively.
- Take up of O2 in the lungs will be respectively
too.
 - The entry of 1 O2 into Hb molecule will increase
the affinity causing the entry of 3 O2 & same
goes with the release of O2 however affected by
insertion of 2,3 DPG
a. When the 1st O2 is released, B chains will be
pull apart thus allowing entry of 2,3 DPG
b. 2,3 DPG maintains its attachment w/ the B
chain forming a salt bridge which lowers the
affinity of Hb to O2. The remaining O2 will
eventually be released into the tissues
c. Hb assumes a tense form or deoxygenated
form in tissues which will circulate back into
the lungs & will gain O2
d. The deoxyhemoglobin containing the 2,3
DPG in the lungs w/ increase pO2, the entry
of 1 O2 into Hb will affect & make alpha and
beta chains slightly move by 15 degrees thus
breaking the salt bridge
e. Once salt bridge is broken, 2,3 DPG will be
released causing increase affinity of Hb to O2
thus allowing entry of O2 resulting to relaxed
form or oxyhemoglobin
BREAKDOWN OF HEMOGLOBIN
GLOBIN: consists of amino acids (141 or 146) go back to
the amino acid pool
HEME: Fe & Protoporphyrin 9
IRON:
- split off & removed by its attachment to a B-1-
globulin
- reused
- stored (ferritin or hemosiderin)
 Breakdown of Hb occurred when RBC are
destroyed in the 120th day
 Red cells upon production will survive for 120
days in circulation & once it gets old, it cannot
maintain its shape & metabolic activity will be
decrease.
 Old RBC will be recognized by the macrophages
of the spleen (splenic culling)
 Spleen – filter of blood which removes abnormal
illusion, cells & old RBC
 Senescent RBC – old RBC
 Macrophages of RBC: will cull or lyse the old RBC
which will cause the release of Hb (extravascular
hemolysis)
2 WAYS OF DESTRUCTION OF RBC:
1. Extravascular – occurs outside the blood vessel,
in the macrophages of the spleen
2. Intravascular – lysis inside blood vessel even
when 120 is not reached
 Once Hb is released, it will be broken to following
components:
EXTRAVASCULAR
1. HEME AND GLOBIN SEPARATES
a. Globin will be recycled as amino acids
wherein the amino acids will separate and
will proceed to the cytoplasm or plasma
forming the amino acid pool making it as a
reservoir for protein synthesis.
b. Heme will release Fe & Protoporphyrin.
c. The iron bound to the B1 globulin attached
to transport protein will split off & be
removed to its attachment to B1 globulin.
Iron will be released & may be reused in Hb
synthesis or other protein synthesis or be
stored in the form of ferritin or hemosiderin
Ferritin – major form of storage form of iron
in the spleen or bone marrow
d. Protoporphyrin ring will break off at the
alpha methane bridge by heme oxygenase
resulting to biliverdin (green pigment) & CO
(toxic gas from endogenous source)
o During degradation of Hb, we release CO
thus the more lysis inside the body, the
more CO is generated
o In extravascular hemolysis, the amount
of CO released is normal & be easily lost
through exhalation
BREAKDOWN OF BILIVERDIN
BILIVERDIN
- precursor of bilirubin
- Comes from protoporphyrin 9
 - Formed w/in the macrophages & reduced into
bilirubin
2 TYPES OF BILIRUBIN
1. Conjugated
2. Unconjugated
- What comes out of bilirubin
- Non-polar water insoluble & when numerous in
plasma it causes jaundice
a. Unconjugated bilirubin will be released in the
plasma & for it to be normally excreted it will be
brought by albumin (transport protein) into the
liver for conjugation
b. In the liver w/ UDP-glucorynyl transferase which
will catalyze the addition of 2 glucoronic acids to
bilirubin forming now the conjugated
bilirubin/bilirubin diglucuronide which is water
soluble
c. Bilirubin diglucuronide will be stored in the bile.
COMPOSITION OF BILE: bile salts, bile acids, bile
pigments (conjugated bilirubin)
FUNCTION OF BILE: digestion of fats
d. During digestion, bile is released into the small
intestines (duodenum) via the common bile
duct.
e. Bilirubin will be acted by bacterial intestinal
enzymes converting it into urobilinogen. ½ of
urobilinogen will be reabsorb to the liver for
reprocessing while some will be filtered by the
kidneys and be excreted into the urine
f. Urine urobilinogen will give off the dark yellow
color of urine (normal)
g. The other half will be excreted into the feces
giving a color of light yellow, light brown to dark
brown due to urobilin and stercobilin coming
from urobilinogen
INTRAVASCULAR HUMAN HEMOGLOBIN
- Same w/ extravascular but w/out the
macrophages.
- Lysis occurs in the circulation
NORMAL HUMAN HEMOGLOBINS:
1. EMBRYONIC HEMOGLOBIN
- Produced in the mesoblastic stage
- First 3 months after conception
- zeta chain = analogue of a-chain
- epsilon chain = counterpart of the gamma, beta
and delta chains
3 EMBRYONIC HEMOGLIBIN:
- DIFFERENCE: Composition of globin structure
a. Hb Gower I (2 zeta & 2 epsilon)
b. Hb Gower II (2 alpha & 2 epsilon)
c. Hb Portland (2 zeta & 2 gamma)
2. Hb F (2 alpha & 2 gamma)
- Major Hb of the fetus and the newborn
- Has high affinity to O2 thus it will not easily
release the O2
- Resistant to both acid elution and alkali
denaturation
- After birth, smaller amounts of HbF are
produced
- Diseases associated: HPFH & B. Thalassemia
- Increased would lead to hypoxia
- In fetus, anaerobic respiration occurs, for it to
conserve its O2, it contains Hb F, but for new
born, aerobic respiration, Hb F is still increased
thus decreased O2, this decrease is
compensated by the increased Hb F
AFTER BIRTH, SMALLER AMOUNTS OF HbF ARE
PRODUCED:
o 6 months = <8%
o After age 2 years = <2%
o Adults = < 1-2%
GLOBIN SYNTHESIS:
- Hb F, Gower 1 & 2 & Portland are Hb that are
synthesized separately & produced separately
- Primitive RBC are the very first blood cell
produced during the mesoblastic stage which
will oxygenate the embryo’s tissue. This
primitive RBC contains the embryonic Hb
a. Zeta & Epsilon are synthesized in the
mesoblastic stage forming the Gower 1
Note: synthesis of zeta & epsilon is high at the
start but declines thereafter, thus decreasing
embryonic Hb.
b. After few days, alpha chains are synthesized
wherein a small amount of it combines w/
epsilon forming the Gower 2
c. A small amount of Gamma is synthesized &
combines w/ zeta forming the Portland
d. No synthesis of Epsilon and Zeta would lead to
dissolution of embryonic hb wherein it only
 persists for at least 3 months. As a  Methemoglobin is not a compound but only
counterpart, the A & Gamma increases. an Hb containing an oxidized Fe (Ferric)
e. Combination of these 2 forms the Hb F during  Sulfhemoglobin is a Hb w/ sulfide radical
the hepatic stage  Oxy & DeoxyHb are normal Hb formed during
f. Synthesis of A chain persist until after birth the normal transport of gases. These are
while gamma starts to decrease after birth & normal both in presence & function.
as a counterpart B chain gradually increases.  Carboxy, Met & SulfHb are abnormal in
g. Combination of A & B chain forms the Hb A1. function but normal in presence. These are
h. Delta chain, does not peak & combination of present in small amount in circulation.
this to A chain will form the Hb A2  Affinity of carboxyhemoglobin to Hb is >200x
thus instead that O2 binds,
carboxyhemoglobin binds w/ Hb thus it has an
abnormal function & is present in very minute
3. Hb A1 (2A & 2B): Major normal adult hemoglobin
concentration.
(97%)
 Everyday 3.5% of Hb is converted into
4. Hb A2 (2A & 2D): methemoglobin but since RBC have reducing
o Accounts for 1.5 % - 3.5 % of normal capacity, it is reduced back into a normal Hb.
adult Hb Deficiency in reducing capacity of RBC, RBC
o Increased in some – B Thalassemia’s, can no longer reverse the reaction thus
hyperthyroidism and in some cases of increase in methemoglobin causing
megaloblastic anemia methemoglobinemia.
 Affinity of Hb to O2 is dependent in the state
HEMOGLOBIN MOLECULAR STAGE OF LIFE of Fe – ferrous
STRUCTURE  Oxidation reduction is reversible, meaning it is
oxidized but it is also reduced.
Gower 1 2Z&2E EMBRYONIC
 Sulfhemoglobins presence & function is both
Gower 2 2A&2E EMBRYONIC abnormal thus it must not be present in
circulation. It is the only Hb that is not
Portland 2Z&2G EMBRYONIC measured by cyanmethemoglobin procedure.
 Conversion of Hb to Sulf is irreversible.
Fetal (F) 2A&G FETAL

A1 2A&2B ADULT
FORMS OF HEMOGLOBIN
A2 2A&2D ADULT NORMAL

1. OXYHEMOGLOBIN (HbO2)
- Circulates in the arterial circulation
HEMOGLOBIN COMPOUNDS - Formed when the Hb passes through the
OXYHEMOGLOBIN O2 + Hb Hb F, A1 & pulmonary capillaries of the lungs
- The O2 is loosely bound and unstable for easier
A2
release into the tissues
DEOXYHEMOGLOBIN CO2 + Hb Hb F, A1 & - Gives a scarlet red/bright red color
A2
NOTE:
CARBOCYHEMOGLOBIN CO + Hb Hb A2 Basilic is not appropriate for blood collection due to
possibility that brachial artery might be hit
METHEMOGLOBIN Ferric Hb Hb A1 & A2

SULFHEMOGLOBIN S + hb Hb A1 & A2 2. DEOXYHEMOGLOBIN

- Reduced form however increased CO2
- Hemoglobin with CO2 (carbaminohemoglobin)
NOTE: wherein CO2 binds to the free amino group of
the Hb to form carbaminohemoglobin
 - Gives a dark red color due to presence in venous
circulation
- Formed when Hb passes through the tissues
ABNORMAL
1. CARBOXYHEMOGLOBIN (HbCO2)
- Hemoglobin with CO (affinity of CO to Hb is 200-
210x greater than the affinity of O2 thus it binds
more in Hb)
- Normally present in body due to endogenous CO
source (<1%) but have an abnormal function
because it cannot bind with O2
- From the breakdown of protoporphyrin into
biliverdin & CO which is the endogenous source
which is only minimal (<1%). At the same time,
for people who are exposed to normal
atmospheric O2, the half-life of CO is only 4
hours & 50% is lost by exhalation & this is not
enough to cause CO poisoning
- Chief Sources of CO (exogenous): automobile
exhaust, industrial wastes, tobacco smoking
(10% of Hb of people expose to exogenous
source)
- Imparts a cherry red in color in blood & skin
- Absorption wavelength: 576 nm
CRITICAL VALUE:
5g /100 ml which may cause irreversible tissue
changes since COHb cannot bind w/ O2, thus it cannot
transport O2 thus it cannot oxygenate the tissues &
lungs. There are body tissues that are very dependent
on O2 (brain cells). When brain cells are depleted w/
O2 this will result to cell damage & brain cell damage
is irreversible. If values are >50 – 70% this may lead to
asphyxiation which is experienced by a person being
strangled, it is a condition wherein the patient is
depleted w/ O2 thus the patient will lose
consciousness & may lead to death, however
treatment may be employed w/ hyperbaric O2
Treatment: administration of hyperbaric O2 – involves
MEDICAL IMPORTANCE:
high amount of conc. O2 w/ pressure in a pressurize
room in a pressurize tube. Although the affinity of
COHb is greater than O2, w/ higher amount of
pressurize O2, the O2 exposure can displace CO from
attachment to Hb thus bringing the CO to its normal
Hb range
MANAGEMENT OF CYANIDE POISONING
NOTE: RAPID TESTS FOR HbCO
a. NaOH test
- Composition: 40% NaOh + EDTA – blood
- Process: warm gently
- Result: HbCO: red color HbO2: Black-Brown
b. Dilution test (addition of water)
- Compostion: 1 mL blood + 50 mL water
- Result:
o HbCO: cherry red – pink or bluish red
o HbO2: Yellowish Red
c. Tannic Acid test
- Composition: 1% Tannic acid which will tend
to acid digest the blood resulting to black-
brown color
- Result:
o HbCO: red precipitate
o HbO2: Black-brown discoloration
2. METHEMOGLOBIN/ HEMIGLOBIN (Hi)
- Iron (Fe++) is oxidized into its Fe+++ form
- Not a compound but an oxidized Hb
- Absorption wavelength: 630 – 635 nm
- Screening tests:
o Aeration: blood retains its color
o Addition of 1% MB: blood turns red
- Have an abnormal function but normally present
in blood w/ conc. of 1.5% because every day
almost 0.5 – 3% Hb is converted to hemoglobin
that does not accumulate even if it is oxidized it
is reduced back to normal Hb as oxidation
reduction is reversible. But if amount of
oxidation is >1.5-3.5% g/dL (10%) it will cause
cellular damage causing cyanosis wherein the
patient will suffer from bluish discoloration of
the skin as well as difficulty of breathing due to
depletion of O2.
- If present in blood at high conc. >1.5-3.5% g/dL
(10%) this will result to a chocolate brown color
- With strong affinity w/ cyanide forming
hemoglobincyanide (HiCN) which may take
place inside the body. It is important in the
treatment of cyanide poisoning. Free cyanide
in the circulation is very toxic.
- NO2 is given which will cause the oxidation.
NO2 will convert Hb into hemiglobin causing
oxidation of the patients Hb thus once
 hemiglobin is formed, it can bind to the
produced cyanide forming into HiCN. The free
poisonous cyanide is converted into less
poisonous cyanide.
 Maintenance of Hemoglobin in the Normal
Reduced state (Fe++): Reducing properties of
RBC
PRODUCTION OF:
 Reduced Diphosphopyridine dinucleotide
(DPNH) in the presence of methemoglobin
reductase (diaphorase)
- Reaction in Embden-Meyerhof Pathway that
Generates NADH:
o NAD is reduced into NADH which has a
reducing potential thus deficiency of it
will not make it to reduce the ferric into
ferrous.
 Reduced Triphosphopyridine nucleotide (TPNH)
in the presence of Glucose-6-PO4
dehydrogenase
- produced in Embden-Meyerhof Pathway in the
Hexose Monophosphate shunt
- Process: from phosphorylation of glucose to
glucose 6 phosphate it enters the hexose
monophosphate shunt & converted into 6-P
gluconate catalyze by G-6-PD with a coenzyme
NADP wherein in this reaction it is reduced which
is similar to TPNH. With the reduced NADP it can
caused the production or reduction of more
glutathione which is also a reducing potential.
The major reducing capacity of RBC is found in
the hexose monophosphate shunt because in
this shunt, it can be able to produce 1.) NADPH
& 2.) GSH
 Glutathione and its associated enzymes
o Glutathione is the major anti-oxidant
since it has a reducing property w/
NADPH
o Reduced glutathione is important in
protecting the Hb from being oxidized
NOTE:
Reduced NAD can bring back methemoglobin into
normal Hb, ferric to ferrous
OTHER REDUCING AGENTS:
a. METHYLENE BLUE
- Often given to patients suffering from
methemoglobinemia
- It will cause the reaction of glucose 6-P to be
faster wherein the H of NADH will be
transferred to the Hb thus reducing Hb
however the transfer of H is slow wherein
methylene blue will enhance this reaction
b. ASCORBIC ACID: a potent reducing agent &
also an anti-oxidant
METHEMOLOBINEMIA
- increase in the methemoglobin conc. in blood
CONDITIONS OF METHEMOGLOBINEMIA
a. Inherited (enzyme deficiency): commonly
attributed to NADH-Methemoglobin reductase
deficiency/Diaphorase deficiency which is
important in reducing ferric to ferrous
b. Inherited M (methemoglobin)
- results of various amino acid substitutions in the
globin chain that directly affect the heme group
- due to increase methemoglobin, RBC cannot
keep up thus it cannot reduced back to
hemoglobin
METHEMOGLOBIN VARIANTS
- Different structural abnormality but are not
protected from being oxidized meaning they are
easily oxidized
1. M-saskatoon
2. M-boston
3. M-iwate
4. M-hydePark
5. M-milwaekee
c. Acquired:
- effects of chemical or therapeutic agents (aniline
dyes in food, nitrate & nitrite-rich water and
foodstuffs ; anti-malarial drugs & sulfonamides)
- Therapy: Ascorbic acid & Methylthionimium Cl
NOTE:
 Methylene blue is not a reducing agent but
only accelerates the reaction.
 Carboxyhemoglobin and methemoglobin are
present in small quantities in the serum
however its function is abnormal.
SCREENING TEST FOR METHEMOGLOBIN 123 – 153  Higher in the morning & lower in
Composition: 1% Methylene Blue g/L the evening
Result: red or pink color of blood
AT BIRTH:  Lower if lying down

3. SULFHEMOGLOBIN (SHb) 150 – 200  Increased in smokers

- Formed when organic sulfides combine with Hb
- Observed in patients taking in oxidant drugs
(phenacetin & acetanilid, sulfonamides)
- Once Hb is converted into sulfhemoglobin it
remains as it is thus it is Irreversible for 120 days
however it can combine w/ CO forming
carboxysulfhemoglobin
- It is the irreversible formation which prevents it
to be measured by drabkins reagent thus it
cannot be converted in cyanmethemoglobin
- NOT normally present in the blood thus its
presence at 0.5 g/100 mL is a critical level
causing damage to tissues
- Imparts a greenish color
- Unstable Hb that can easily precipitate into a
Heinz bodies, an inclusion bodies, which when
present to RBC, they tend to attach themselves
on RBC membrane making RBC membrane rigid
thus cells w/ Heinz bodies are prone to hemolysis
leading to hemolytic anemia. Even normal Hb
can form few Heinz bodies when denatured
- Absorption wavelength: 600 - 620 nm
g/L
 Increased in high altitude
because the atmosphere is very
thin thus O2 is thin
 Estrogen tends to inhibit
erythropoietin while androgen
will tend to stimulate more
OTHER FORMS OF HEMOGLOBIN
a. CYANMETHEMOGLOBIN
- Feriicyanide + Fe++ of Hb
- An in vitro test that may also occur inside the
body (in vivo)
- Its presence can also be induced in vivo (inside
the body) especially during cyanide poisoning.
- The most stable among the Hb pigments thus
when performing cyanmethemoglobin
procedure the Hb result can be delayed because
the color produced does not fade
- Absorption wavelength: 540 nm

RFERENCE INCREASED Hb DECREASED Hb b. GLYCOSYLATED HEMOGLOBINS

VALUES - Also called “Glycated Hemoglobin” or simple
FOR  Polycythemi  All “Glycohemoglobin”.
HEMOGLO a - increase anemia - Described as “fast hemoglobins” because they
BIN RBC migrate fast in electrophoresis.
 Leukemi
production - Irreversibly glycosylated at 1 or both N-terminal
a
valines (or lysine) of the β-chains
 Dehydration
 Oligochr - Refers to the addition of a glucose molecule to
– due to
omia the hemoglobin molecule. Such addition is non-
hemoconcen
enzymatically catalyzed. The only basis of when
tration w/
the glucose binds in hemoglobin is the level of
decrease
plasma glucose that when the levels of plasma
plasma
glucose is elevated (like in hyperglycemia, DM
volume due
Type I and II), the excessive glucose in the plasma
to burns &
can bind with the hemoglobin called glycation or
diarrhea
glycosylation which is irreversible wherein once
MALE: PHYSIOLOGIC VARIATIONS the glucose is added to the Hgb molecule, this
Hgb will remain to be glycated so that the RBC,
140 – 175  After 50 years of age – slight once its Hgb is glycosylated, it will remain
g/L decrease glycolyated during the entire life span of the
FEMALE: RBC. This will now lead to the formation of:
o Hb A1a
 o Hb A1b days. It is important index in diabetes control,
o Hb A1c: the FBS & RBS are more accurate as an index of
 Major fraction which serves as metabolic control for the past 2-4 months,
an index of the metabolic meaning the glycosylated Hb is the same for the
control over that last 2-4 next 4 months.
months. This is more preferred
OXYGEN DISSOCIATION CURVE
in monitoring glycemic control.
Oxyhemoglobin
 Elevated 2-3 folds in patients
dissociation curve
with Diabetes Mellitus (DM).
(ODC), is a curve that
 Preferred than FBS and RBS.
plots the proportion of
NOTE: HBA1c hemoglobin in its
saturated form on the
 Patients with DM are being monitored
Y axis against the
for their glycemic control and if their
prevailing oxygen
medications can control the increase
tension on the X axis. It
of their glucose. The monitoring
is slightly S shape.
should be a form of review on their
Left shift indicates increase O2 saturation
conditions for the past 2-4 months.
Right shift indicates that O2 is released in the tissues.
 The present HbA1c levels of the
This relates oxygen saturation and partial pressure of
patient reflects on his/her HbA1c
O2 in the blood (PO2), and is determined by
levels for the past 2-4 months.
hemoglobin affinity for oxygen wherein the higher the
 HbA1c is just for monitoring and not
affinity, the curved shift towards the left & vice versa.
for diagnosis for DM.
Affinity is the main factor that influences the shifting.

- Necessary for diabetic individual

FACTOR SHIFT CAUSED BY
- Fast Hb
- Hb w/ attached glucose which occurs when FACTOR FACTOR
plasma glucose is high. Glycation is not INCREASE DECREASE
enzymatically catalyzed. When glucose attached
to Hb, the only factor which will promote this is pH Left shift due to Right shift due
the increased in plasma glucose or increase affinity to decrease pH –
hyperglycemia. – alkalosis acidosis

NOTE: 2,3 DPH/BPG Right shift due do Left shift due to

 To which part of the Hgb will the glucose decrease affinity increase affinity
attach? due to increase due to decrease
o Glucose attaches at 1 or both N- DPG DPG
terminal valines (or lysine) or the β-
CO2 (haldane Right shift due to Right shift due
chains.
effect), H+ & increase of these to decrease of
 Only HbA1 are glycosylated Hgb because the
glycosylation occurs in the β-chain. Therefore, Cl- - all factors thus salt these factors
only Hgb has the β-chain. maintains the bridge is thus salt bridge
salt bridge stabilized thus is not stabilized
decreasing the thus increasing
2 REACTIONS ARE INVOLVED: affinity the affinity
1. REVERSIBLE Blood (Body) Right shift due to Left shift due to
2. AMADORI: irreversible meaning the glucose Temperature increase decreased
attached to the terminal valine or lysine can no Temperature temperature
longer be detached. & once the Hb is Rule: the
glycosylated it will remain glycosylated until 120 higher the
temperature,
the faster is DISORDERS RELATED WITH ABNORMAL
the HEMOGLOBINS
metabolism 1. THALASSEMIA
- Synthesis defect
Hb F Left shift due to Right shift due - Decreased or non-existent production of one or
increase Hb F to decreased Hb more globin chain type
-Has innate
thus it will not F thus it will - For both α & β, there is a deletion in the gene
increased
easily release O2 easily release resulting to incomplete synthesis of A & B chain.
affinity which
O2 - Diverse group of genetic disorders characterized
is needed by
fetus in an by a primary, quantitative reduction in globin
anaerobic chain synthesis for hemoglobin.
environment CLASSIFICATIONS:
in the uterus
thus if A. ALPHA THALASSEMIA
increased in - Indication of Deletion:
adults, this 1. Silent carrier - (_A/AA) (AA/_A) = ¼ thus no
may lead to signs & symptoms of anemia
deoxygenatio 2. Thalassemia trait/A-Thal minor – (_ _/AA)
n. (_A/A_) = 2/4 w/ mild signs & symptoms
3. Hb H disease – (_ _ /A _) = ¾ thus the
Abnormal Hbs production of Alpha chain becomes too
(depends on insufficient & B chain will be used in excess
characteristic resulting to Hb having all B chains (4 B)
of 4. Bart’s Hydrops fetalis – (_ _/ _ _) = 4/4 thus
abnormality) no synthesis of A chain, as early as fetal life,
Hb F which consist of 2 A & 2 G, there would
be no Hb F synthesis & what will be
SEVERAL ABNORMALITIES OF HEMOGLOBIN synthesize is the G chain (4 G).
1. HEME IS ABNORMAL
- Iron is deficient thus affecting Hb synthesis B. BETA THALASSEMIA
leading to IDA - Indication of Deletion: 0 superscript
- Deficient Protoporphyrin IX because of enzyme 1. Beta Thalassemia Major/Cooley’s anemia –
defect along the synthesis leading to excess iron B0/B
& iron will not be utilized in the absence of 2. Beta Thalassemia Intermedia – B+/B+,
protoporphyrin 9. Unused iron will precipitate present but cannot effect the synthesis of B
into siderotic granules leading to sideroblastic chain because of partial suppression.
anemia 3. Beta Thalassemia Minor/Cooley’s trait -
B0/B0, the patient is devoid of Hb A1
NOTE:
because the patient cannot synthesize B
Sideroblastic Anemia – problem in the utilization of
chain & what is elevated is Hb F.
iron due to deficiency in protoporphyrin 9

2. HEMOGLOBINOPATHIES
2. GLOBIN IS ABNORMAL - Disorder in structure
- Ex.: Hb A1 – 2 A w/ 4 genes & 141 amino acids & - Results from the alteration of the DNA genetic
2 B chains w/ 2 genes & 146 amino acids code for the chains
- 2 FACTORS: - Conditions characterized by qualitative
o Alpha Thalassemia structural abnormalities of the globin
o B Thalassemia polypeptide chains that result from alteration of
 the DNA genetic code for those chains. Structural Hb S (SICKING HB)
abnormalities may involve amino acid. - The most well-known hb variant characterized
by substitution of glutamic acid by tRNA to valine
STRUCTURAL DEFECTS ON A OR B CHAIN RESULTING TO
on the 6th amino acid position of the β-chain.
Hb VARIANT:
Glutamic: CAG Hb S: β 6(A3) Glu → val
Valine: GUG
1. SUBSTITUTION
- Ex.: Hb Hb S: β 6(A3) Glu → val
- Common to black population
- The effect of alteration from glutamic acid to
2. DELETION
valine is a negative charge wherein glutamic is
- some amino acid is deleted
highly negatively charge compared to valine thus
- reason of thalassemia
leading to loss of negative charges & Hb
Ex.: Hb Gun Hill: (β91 to β 95) → 0 becomes less negative
- In electrophoresis in cellulose acetate, S
3. FUSION migrates slowly than A & F due to substitution of
- Hb Lepore-Baltimore: (G(1-50) B(86-146)) – caused by amino acid 6
cross over during mitosis resulting to fusion - Inheritance:
o Homozygous (SS) – sickle cell anemia
4. ELONGATION/ ADDITION o Heterozygous (AS) – sickle cell trait
5. Hb CONSTANT SPRING (a+31c)
EFFECTS OF HB S:
REASON FOR DELETION & ELONGATION: mutation in  Blacks who are suffering from this are
genetic code protected from being infected from
developing severe falciparum malaria
REASON OF HEMOGLOBINOPATHIES: mutation of gene (Resistance). Hb C, E, B-Thalassemia & G-6PD
NOMENCLATURE deficiency also confer protection in terms of
resistance but not immunity meaning these
1. COMMON NAME
patients may still be infected but may not
a. Morphology- sickling Hb (HbS)
proceed to chronic stage.
b. Place where they were discovered- (Hb
o Falciparum are intracellular species &
Boston) when present in RBC & when RBC is
2. SCIENTIFIC NAME sickle the parasite inside also dies
- Gives structural defect of Hb does no complication arise
- Example: Hb Hb S: β 6(A3) Glu → val o Falciparum metabolizes Hb, when Hb
B: affected polypeptide chain crystalizes it forms into a crystal &
6: sequential amino acid number(s) affected falciparum may no longer metabolize
(primary structure) the Hb
A3: Helix number involved (secondary structure: o RBC w/ falciparum will be
A-H); not necessary as long as primary structure phagocytosis before it causes severity
is indicated
Glu: nature of the abnormality (substitution, HOMOZYGOUS (SS) SICKLE CELL ANEMIA
deletion, addition or globin chain fusion) - Severe anemia because the patient does not
PRIMARY STRUCTURE synthesize the normal Hb A

- sequence or arrangement of amino acid NOTE:

On electrophoresis:
NOTE: o Hb S – 80 -90%
Hemoglobinopathies commonly affects the B chain. o Hb F – 10 -20 %
o Hb A2 – Normal

- Increased RDW
 - Retarded growth & sexual maturation & patient
suffers sickle cell crises
- Main cause of Sickle Cell Crises: sickling
- Normally when Hb S is fully oxygenated, Hb S is
fully soluble
- But when Hb S is w/ reduced O2, Hb S becomes
insoluble wherein it polymerizes (change in
molecular arrangement) & forms into tactoid
crystals/fluid crystals (elongated & thin, pointed
at both ends) leading to sickling & rigid cell
membrane thus it is no longer adjustable leading
to vassoocclusion thus it cannot easily pass
through the small circulations
- Sickling is reversible but repeated sickling leads
to permanent sickling due to permanent damage
to RBC membrane
- Causes vasso-obstruction
- Vasoocclusive crises occurs commonly on small
circulations like in the fingers & toes thus hand-
foot syndrome / dactylitis (inflamed or swollen,
cyanotic due to absence of circulation) occurs
- Vassoocclusion also occurs in bone & joint
causing pain
- Splenic circulation is also minute, since spleen
also serves as a filter of blood thus its circulation
is too small, that only deformable red cell may
pass through. Due to increase activity of spleen
& vassoocclusion, spleen also enlarges
(splenomegaly) causing sequestration of more
blood leading to hypovolemic shock. When
spleen is enlarged & bone marrow is affected
thus both are abnormal in function leading to
decrease blood production to the marrow
(aplastic crises) thus the immune system is
defective & the patient becomes prone to
infection (infectious crises) as a result of all of
this organ damage occur (marrow lungs)
- The no. 1 cause of death is infection & in children
the most common is S. pneumoniae.
NOTE:
SICKLE CELL TRAIT (AS)
• Predominant Hb is A, only 30-45% is Hb S
• Patient usually have no symptoms & sickling is
uncommon to occur unless in cases of extreme tissue
hypoxia because S is minimal in concentration
NOTE:
Hb S – Thalassemia:
o Hb S-A
o Hb S-B – more severe
NOTE:
OTHER SICKLING HBS:
o Abnormality: Hb S: β 6(A3) Glu → val + unique B-
substitution
o Hb C-Harlem, C-Ziguinchor, S-Travis
HEMOGLOBIN C
-
Composition: B 6 (A3) Glu → Lys
- A beta structural defect, where glutamic acid is
substituted w/ lysine
HOMOZYGOUS: Hb C DISEASE (CC)
- N/N in size & shape anemia with numerous
target cells (bull’s eye)
- Hb CC crystals formed when deoxygenated &
dehydrated & the crystals are hexagonal or rod-
shaped crystals or bar of gold shape & blunt ends
& does not deform the cell meaning it retains its
shape but becomes rigid
HETEROZYGOUS: Hb C TRAIT (AC)
- RBCs – slightly hypochromic
- Less abnormalities
HEMOGLOBIN SC DISEASE
- Signs & symptoms are milder than SS but more
severe than sickle cell
- Electrophoresis: C = S = 50 – 50%
- Cytoplasm appears folded like a pocketbook cells
(RBC w/ cytoplasm folded)
- Hb SC easily crystalize forming a fingerlike
projection w/ more than 1 protrusions where
one is long & protrude away. It also in the form
of hand in gloves crystals or pointing – finger or
Washington monument crystal
OTHER ABNORMAL HEMOGLOBINS
a. Hb METHEMOGLOBIN
- M-Saskatoon, Boston, Iwate, HydePark,
Milwaukee = all have different amino acid
substitution but the effect is the same & not
protected from oxidation
- Methemoglobinemia w/ congenital cyanosis due
to overwhelming oxidation even at baby stage &
the blood appears chocolate brown
- Easily precipitate & forms into Heinz bodies
-
b. HbS W/ ABNORMAL AFFINITY TO OXYGEN
1. Hb w/ Increase Affinity
- Example: Hb Chesapeake (a 93 Arg → Leu) – affects
affinity of Hb to O2 & O2 will shift to left

2. Hb with Decrease Affinity
- Example: Hb Kansas (B 120 Asp → Thr) – shift to the
right
NOTE:
Abnormal Hb are connected by weak bonds thus they
easily precipitate & flocculate
TEST FOR UNSTABLE HB
1. ISOPROPANOL PRECIPITATION TEST
- When heated, 17% Isopropanol at 37 C normal
Hb do not precipitate but few will do & unstable
Hb will easily precipitate.
- Purpose of Isopropanol: weaken the bonds
2. HEAT DENATURATION/INSTABILITY TEST
- At 50 C for 3 hours, normal Hb will not flocculate
but unstable Hb will
3. HEINZ BODY STAINING
- If ppt & flocculation happens in vivo forming
Heinz bodies which are not easily seen in normal
wrights blood smear
- Supravital staining is employed (brilliant cresyl
blue & crystal violet)
NOTE:
RED BLOOD CELLS
CHARACTERISTIC OF A NORMAL RBC:
 Shape: Biconcave disk
o Purpose:
 Promotes gas exchange
between CO2 & O2
 For RBC to be flexible &
deformable in order to squeeze
through the microcirculations or
else it will be prone to lysis.
 Diameter: 7 – 8 um
o <7: microcyte
o >8: macrocyte
 Thickness (MCAT): 1.5 – 2.5 um
 Average volume (MCV): 90 fL
 Average Surface Area: 160 um2
 Shape & Deformity: Flexible & deformable
o Responsibility of: shape, integrity of cell
membrane & biconcavity.
 Mature: anucleated w/ a dense outer rim
 Central pallor: 1/3 of its diameter, it is pale due
to lack of Hb
o >1/3: RBC is hypochromic
 Size: similar to small lymphocyte
o Rule: Slight variation in size & shape is
acceptable but increase in variation will
be abnormal
 Cytoplasm: uniformly pink without inclusions
 Average lifespan: 120 days
o <120 days: RBC undergoes pre-mature
hemolysis
 Chromium 51 (Cr51): used to measure the
effective survival of RBCs in vitro
o Half survival time range: 25-32 days
o <25 days: RBC undergoes hemolysis
o Process: Collect blood from the patient.
Radioactive Cr is added on the blood.
Chromated RBC are infuse back
intravenously. The disappearance of
chromated RBC will be measured by
performing blood cell count in a gamma
ray for every 1 – 2 days for 10 – 14 days.
Cr is eluted from circulation 1% a day, its
disappearance is shorter
o Purpose: label RBC in order to be
differentiated from unlabeled RBC
 Half – life: no. of days wherein at least 50% of
radioactivity of Cr or gamma ray disappear
from circulation
 Eliptocytes are usually utilize for thinner &
deformed cells due to acquired condition
while ovalocytes are egg shape & not thin,
which are often seen in hereditary type.
RBC MEMBRANE
- underlie the lipid layer & regulate shape &
deformability because if the membrane is rigid,
the cells cannot reshape itself & not flexible
- surrounds the cellular cytoplasm or internal
environment
- selective transport membrane: separates the
internal environment to external
o does not easily allow diffusion of
substances
o It selectively allows the entry of water &
electrolytes, inlet & exit
- consist of a phospholipid bilayer
 RBC MEMBRANE COMPOSITION:
1. 50% PROTEINS
a. INTEGRAL PROTEINS
- crosses phospholipid bilayer
- high amount of sialic acid for imparting negative
charge to maintain zeta potential
NOTE:
Zeta potential
- RBC repel each other due to negative cloud
surrounding them caused by sialic acid. When
zeta potential becomes altered this result to
rouleau formation.
- Component: Glycophorin A & component a
(band 3)
- Function: serves as membrane protein
mechanism in order to allow passage of
molecules either inward or outward
b. PERIPHERAL PROTEINS
- Component (cytoskeleton): Spectrin & Actin
- found facing the inner or cytoplasmic
environment or facing outwards found on the
outer or inner surface
- adds up to the adherence of lipids
MEMBRANE ENZYME SYSTEM FOUND OF THE SURFACE
OF RBC:
- Form the Active Transport System
a. Na/K ATPase
- Regulates entry of Na & K in & out of the cell in
order to maintain Na as a major extracellular
cation while K is the major intracellular cation
thus when it is impaired there would be
excessive inlet & outlet of cations. If excessive
cation entered, this will result to hypotonicity of
RBC causing now the entry of water, swelling &
lysis of RBC
b. Ca/Mg ATPase
2. 40% LIPIDS
a. PHOSPOLIPIDS
- Serves as a liquid sealer, because it is liquid
stabilized by proteins where they are connected
- Arrangement serves as liquid sealer wherein
there is 2 phospholipid layers containing a
phosphate head which is polar thus it reacts with
liquid environment with hydrocarbon tail which
is hydrophobic meaning it does not interact with
watery environment. The polar heads are
exposed to the watery environment while the
hydrocarbon tail is facing inwards because if
outwards it does not interact with watery
environment.
KINDS:
1. EXTERNAL SURFACE: phosphatidyl choline,
glycolipids & sphingomyelin
2. INTERNAL SURFACE: Cephalin,
phosphatidylinositol & phosphatidylserine
b. CHOLESTEROL
- Amount depends upon the concentration of
plasma cholesterol, bile acids & activity of the
enzyme LCAT
- Maintains regulation of membrane fluidity &
permeability to electrolytes. It also maintains the
surface area volume of RBC
- Water content of RBC depends mainly on its
ionic gradient wherein the greater the ionic
gradient, RBC becomes hypertonic thus water
enters RBC & too much entry of water causes
swelling of RBC & eventually burst & decrease
surface area volume
AMOUNT DEPENDS UPON THE CONCENTRATION OF:
1. PLASMA CHOLESTEROL: if plasma cholesterol
is high, patient has hypercholesterolemia
(liver diseases) macrocytes & target cells are
present, because of loading of some
cholesterol into the cell membrane causing
the cell to widen
2. BILE ACIDS – for solubilization
3. Activity of the enzyme LCAT
3. 10% CARBOHYDRATES
- Some serve as red cell antigens (e.g. ABH antigen
on surface of RBC)
- Often in combination w/ lipids (glycolipid) or
protein (glycoprotein)
- Ex.: Glycophorin A, B , C/D
ABH – blood antigen ABO – blood
group Ab
NOTE:
 A-spectrin, Ankyrin, Band 3, B-Spectrin,
Protein 4.2 & 4.1 & Glycoprotein are
 important in maintaining the shape & size of
RBC & abnormality of these will lead to
deformation of cells.
 Defective A-spectrin, cell will be spherocytes
or pyropoikilocytes
METABOLIC ACTIVITIES IN THE RBCS
- RBC is metabolically active when it is maturing
because when it becomes mature it does not
have nucleus & mitochodria
RULE:
The more immature the RBC, the more metabolically
active it is which is related to the amount of the RNA
which indicates the cellular immaturity & metabolic
activity.
1. EMBDEN MEYERHOF PATHWAY/GLYCOLYTIC
PATHWAY
- Since RBC are already devoid of mitochondria,
glycolytic pathway goes anaerobic.
- Main Function of Anaerobic Glycolysis:
production of the cells energy which is ATP
- Process: for every glucose metabolize, it will
produce 2 ATPs for the glyceraldehyde-3-
phosphate & for 2 ATPs for Dihydroxyacetone
Phosphate thus a total of 4 but the net energy
utilized is a net of 2
- Major source of red cell’s ATP (90-95%)
2. HEXOSE MONOPHOSPHATE SHUNT/PENTOSE
PHOSPHATE SHUNT
- Arises from Embden Meyerhof Pathway but it
undergoes oxidative glycolysis providing 5 –
10% ATP
- Contains the major anti-oxidant of cells
- Main function: generates NADPH and reduced
glutathione (GSH) in the presence of Glucose 6-
PO4 dehydrogenase (G6PD). GSH protects the
hemoglobin from oxidation by peroxides.
- Process: If G6P converts into 6-
phosphogluconate, it produces NADPH & the
enzyme used is G6PD & the substrate is NADP
which is reduced into NADPH which may
become H donor wherein it transfers the H to
ferric iron & ferric becomes ferrous. It also
maintains the glutathione in the reduced form
which also serves as a reducing agent wherein it
detoxifies the toxic radicals (H2O2 – strong
oxidizing agent causing oxidation of lipids & Hb
& this is detoxify into H2O & O2). Glutathione
also brings back the ferric form of iron into
ferrous.
3. RAPOPORT-LUEBERING SHUNT
- Generates 2,3-DPG /2,3-BPG which regulates
the affinity of Hb to O2 by lowering the affinity
promoting release of O2 causing a shift to the
right
4. METHEMOGLOBIN REDUCTASE PATHWAY
- Reduction of methemoglobin by NADPH is more
efficient in the presence of methemoglobin
reductase (cytochrome b5 reductase) which
serves as an intermediate electron carrier. It also
prevents oxidation of heme by reducing ferric to
ferrous with the use of NAD.
CONNECTIONS OF THE 4 PATHWAYS
- EMBDEN MEYERHOF PATHWAY is an anaerobic
& goes as anaerobic glycolytic pathway, mainly
for the production of 90-95% of RBC ATPs. The
HEXOSE MONOPHOSPHATE SHUNT is important
for the production of NADPH & GSH for the
maintenance of Hb iron in the reduced form
because it can reverse the ferric into ferrous.
GSH can also detoxify H2O2 converting it into
H2O & O2. RAPOPORT-LUEBERING SHUNT is
necessary for production of 2,3 DPG which
maintains affinity of Hb to O2.
METHEMOGLOBIN REDUCTASE PATHWAY
produces NADH which serves as reducing
property & may also donate its hydrogen to
cause reduction of ferric into ferrous.
 BREAKDOWN OF RBCs - Process: Hemoglobin is released into the plasma
- As RBC ages, there is a decrease in its enzymes and can be filtered by the kidneys. Plasma
causing slow reaction, thus causing a decrease in haptoglobin and hemopexin salvage the
ATP production & metabolic activity decreases & released hemoglobin so that its iron is not lost in
everything also decreases causing now the urine
distortion thus if more ions enter the RBC, more - Ex.: when RBC is not deformable they undergo
water will enter causing now swelling and size intravascular hemolysis (spherocytes)
also decrease and an increase in density.
- This natural deterioration leads to its
MECHANISM TO CONSERVE THE HB:
phagocytosis. When cells are deformed it will be
1. HAPTOGLOBIN
recognized by splenic macrophages as a damage
- synthesize by the liver & released into the
or senescent RBC wherein spleen filters blood to
plasma
removed old & damage cells (splenicullin), this
- Function: combine w/ hemoglobin preventing
process happens extravascularly. Approximately
Hb from being filtered by the kidneys &
1% of RBC leave the circulation each day & are
hemoglobin-haptoglobin will be brought into the
broken down by mononuclear phagocytic
liver for metabolism. Hb will undergo breakdown
system
by separating heme & globin. Globin will be
recycled as amino acids, & goes back to amino
a. 90% EXTRAVASCULAR (MACROPHAGE-
acid pool. Heme is recycled as iron is
MEDIATED)
immediately utilized or stored as ferritin but
- destruction of senescent red cells by splenic
haptoglobin cannot repeat the function, it will
macrophages
carry Hb to the liver but it cannot repeat its
- release of Hb occurs inside the macrophages &
function, it cannot go back. Thus, in increase
unconjugated Hb is released in the circulation
hemolysis haptoglobin is also depleted.
- Process: Hemoglobin undergoes degradation
- In short: haptoglobin binds w/ free Hb thus
within the macrophage where iron is stored as
forming hemoglobin-haptoglobin & delivered to
ferritin, amino acids of globin are returned to
the liver
the metabolic amino acid pool and
protoporphyrin is converted to bilirubin which
2. ALBUMIN
is released into the plasma and excreted by the
- Process: Hb in plasma becomes oxidized into
liver in bile.
methemoglobin which combines with albumin
forming methemealbumin, this will be brought
b. 10% INTRAVASCULAR DESTRUCTION
into the liver for processing.
(MECHANICAL HEMOLYSIS)
- In short: albumin bind to methemoglobin
- may be caused by the turbulent environment in
forming methemealbumin, & increases in
the circulation
hemolysis due to increase formation
- lysis occurs in the circulation thus release of Hb
is directly in the plasma & increase in
intravascular hemolysis can cause the RBC to
appear pink. Hb is a low molecular weight
protein that can be easily filtered by the kidneys
& be excreted into the urine causing
hemoglobinuria making urine red or pink &
when Hb stayed in room temp. for prolonged
time it will be oxidized into methemoglobin.
When Hb is excreted, it will lose the components
(60% Fe, Protoporphyrin 9, globin & amino acids)
but the body has a compensatory mechanism to
conserve the Hb.
 LABORATORY EVALUATION OF RBC
 RBC count
 Hemoglobin
 Hematocrit
 RBC Indices
 Peripheral Blood Examination
o Blood Smear for differential counting &
morphology examination of RBC (PBS)
 Reticulocyte measurement
 Osmotic Fragility test
 Erythrocyte Sedimentation Rate

CRITERIA FOR PATHOLOGIC RBC

 Size (7-8 um)
 Variation in size (80-100 fL)
 Area of Central Pallor (1/3 & clear)
3. HEMOPEXIN
 Cytoplasm (clear w/out inclusion)
- Process: Hemopexin combines w/ heme. When
heme is separated from methemoglobin it can
be still conserve by binding of hemopexin to
heme forming the hemopexin complex & will be
brought to the liver
- In short: heme bind with hemopexin forming
heme-hemopexin complex

NOTE:
Whether intravascular or extravascular, iron is
recycled.

ANISOCYTOSIS
 increase in variation in size thus increasing RDW a measure of variation in size
 Refers to variation in color after staining which is related to the hemoglobin content of the cell, since it is the
hemoglobin that takes up the stain wherein the appearance of the cell after staining is directly proportional to
the hemoglobin content
CELL TYPE MORPHOLOGIC APPEARANCE DEFECT OR CHANGE ASSOCIATED CONDITIONS
NORMOCYTE Biconcave disc  Normal
 Not all cells are normal there is a classification
known as normocytic anemia which are normal
in size (MCV: 80-100) but there is anemia which
is related to the decrease in production
MICROCYTE  Smaller RBCs  Main cause: Any defect in heme or globin that
 Diameter: <7 µm results in IMPAIRED HB SYNTHESIS thus cells
 MCV= <80 fL undergo extra division & the cells hardly
reached its optimum MCHC
Note: It is efficient to use MCV  Develops from:
because diameter depends on the o ineffective iron utilization, absorption,
smear prepared. or release
o decreased or defective globin synthesis
 Diseases caused:

MACROCYTE  Larger than 10 µm
diameter due to failure of
mitosis
 Diameter: >9 µm
 MCV= >100 fL
o IRON DEFICIENCY ANEMIA: affects
heme
o SIDEROBLASTIC ANEMIA (heme) – iron
precipitate due to protoporphyrin 9
deficiency leading to siderotic granules
& its occurrence in RBC appears as
sideroblast
o THALASSEMIA (globin)
o ANEMIA OF CHRONIC
INFLAMMATION/DISEASE:
inflammation activates macrophages &
T lymphocytes causing increased
production of cytokines that will inhibit
normal iron metabolism thus affecting
heme synthesis
o LEAD POISONING – lead inhibits
enzymes (heme synthase) in heme
synthesis
CAUSES:
a. ACCELERATED ERYTHROPOIESIS – fast RBC
prod. Until faster release of reticulocyte in
circulation as a compensation to blood loss or
lysis. If patient suffers from blood loss or lysis,
blood cell count decreases causing hypoxia &
the kidneys will produce increase
erythropoietin causing acceleration of
erythropoiesis thus the bone marrow will
release even immature reticulocytes
(stress/shift reticulocytes) thus it is larger in
size
b. DEFECTIVE DNA SYNTHESIS – affects nuclear
maturation resulting to nuclear arrest which is
prolonged mitosis due to immaturity of nucleus
which is the central control of the cell due to
deficiency in DNA synthesis thus it undergoes
slow maturation & mitosis leading to large
cells; failure of division
c. IN CONDITIONS WHERE MEMBRANE
CHOLESTEROL AND LECITHIN LEVELS ARE
INCREASED – cell membrane is loaded w/
cholesterol thus increasing cell membrane &
the cell due to hypercholesterolemia this is due
to disturbance in the lipid conc. of plasma &
may be seen in obstructive liver disease
wherein liver is the main organ that
metabolizes fats and where VLDL came from.
Obstructive liver disease leads to incomplete
metabolism of fats thus patients suffer from
hypercholesterolemia.
 HEMOLYTIC ANEMIA & ACUTE BLOOD LOSS –
bone marrow compensates for the blood loss
 or lysis by producing immature RBC thus high
reticulocyte count
 MEGALOBLASTIC PROCESS – affected by
impaired DNA synthesis because cells fail to
divide or suffer from long mitosis resulting to
oval macrocytes
 CHEMOTHERAPY – the chemical infused to
destroy the malignant cells often target the
DNA of cell thus affecting DNA
 LIVER DISEASE - increase cholesterol lipids
causing loading to phospholipid bilayer
 HYPOTHYROIDISM – thyroid is necessary for
metabolism, T3 & T4, for regulating other
hormone
 POST-SPLENECTOMY – not only macrocytes
persist in circulation but also all cell
abnormalities. The spleen filters 5 – 10% of
blood allowing inclusions & abnormal cells to
be removed thus removing the spleen will lead
to no filtration of blood causing abnormality in
circulation.
 ALCOHOLISM
DINMORPHIC RBCs  Presence of 2 distinct populations of red cells that may differ in size, shape or
hemoglobin content thus it may be a mixture of hypochromic cell & normochromic
cell or a microcytic cell or macro
 SEEN IN:
o ANEMIA AFTER TRANSFUSION – after transfusion, on the examination of
patient’s blood smear, there is 2 population of cell, the patient RBC &
transfused RBC, the cells are pale (hypochromic) & microcytic, mixed w/ the
transfused blood (normocytic & normochromic)
o IRON DEFICIENCY DURING THERAPY – cells are microcytic & hypochromic
o IRON DEFICIENCY & VIT. B12 – Iron stored in bone marrow, but storage of
iron is not proportional where the other parts are proportional while the
others are not, thus the developing cells near w/ iron develops as normocytes
while cells w/ developing iron develops as microcytes thus it is a mixture of
microcytic & normochromic cells
o COMBINED VIT. B12 /FOLATE – macrocytosis, 2 most common causes of
megaloblastic anemia DEFICIENCY AN IN SIDEROBLASTIC ANEMIA –
microcytosis, normocytic cell ; a group of anemias that show different
morphology

ANISOCYTOSIS & POIKILOCYTOSIS MACROCYTOSIS GRADING (no

GRADING variation)
Percentage of RBC differ in size or shape from normal RBCs.
Normal 5% 1+ (slight) 25%
Slight 5-10%
1+ 10-25% 2+ to 3+ (moderate) 25-50%
2+ 25=50%
3+ 50-75% 4+ (marked) >50%
4+ >75%
Used if cells vary in size and shape
ANISOCHROMASIA
 variation in the hemoglobin concentration
 absence of uniformity of color
 directly relates to Hb
 Color of cells when stained depends on the Hb
CELL TYPE MORPHOLOGIC APPEARANCE DEFECT OR CHANGEASSOCIATED CONDITIONS
NORMOCHROMIC Homogenous with central Normal
pallor (1/3)
Measure: MCHC
HYPOCHROMIC  RBCs show central DEFECT IN HB SYNTHESIS (HEME/GLOBIN) DUE TO:
pallor (exceeds 1/3 of  decrease in heme synthesis
the diameter of the red  defect in globin chain synthesis
cell)  Iron Deficiency Anemia (IDA)
 Thalassemia
ANULOCYTE/ GHOST  Thin & poorly
CELLS hemoglobinized cells RULE: Not all hypochromic cells are microcytic but all
- very hypochromic (anulocyte) microcytic cell are hypochromic
cells
ANISOCHROMASIA & POIKILOCYTES
SPHEROCYTIC Cells that lack central pallor  Defects in the cytoskeletal proteins
and with reduced diameter  Hereditary and acquired spherocytosis
TARGET CELLS  Central pale area is
filled with hemoglobin
resulting to bulls eye &
further surrounded by
thin rim of Hb
 On side view, there is
protrusion on the
center
POLYCHROMASIA PROBLEM: difference in RNA & Hb among RNA & Hb
 Refers to the display of different shades of color which is Diffused bluish-gray tint of
RBCs & is not directly related to Hb content
 Already fully hemoglobinized but the display of different colors is caused by the
differences in the affinity of the cellular component (Hb & rRNA) wherein Hb takes
up the acidic dye & the rRNA takes up the basic dye
 Exemplified by the fifth stage of erythropoiesis: reticulocytes which is the first non-
nucleated stage but it has remnants of RNA & is fully hemoglobinized

RULE: The more immature, the more RNA it has thus the more chromatophilic it is.
 Not directly related to Hb content but on the RNA content
 The reddish or orange color caused by eosin of Hb & the bluish color stained by
methylene blue of rRNA will be mixed in the cell & the colors produced is mixed
resulting to diffuse coloration of cells
 Increase polychromatic cells indicates immature cell (reticulocyte) which is
synonymous to reticulocyte

APPEARANCE: diffuse coloration, no central pale area

RETICULOCYTE IDENTIFICATION:
 Supravital stains (new methylene blue & brilliant cresyl blue) – appropriate staining
of living reticulocyte & the cells will appear as larger cells w/ granules (RNA).
  Supravital stains precipitate the RNA thus they appear as dark blue precipitate
against pale blue cytoplasm. The granules appear in a network or reticulum thus
called reticulocytes

HYPOCHROMIA GRADING POLYCHROMASIA GRADING

1+ Area of central pallor is ½ of cell diameter Slight 1%
2+ Area of pallor id 2/3 of cell diameter 1+ 3%
3+ Are of pallor is three-quarters 2+ 5%
4+ Thin rim of hgb 3+ 10%
4+ >11%

1+ (slight) Central pallor: 1/3 ro 2/3 of cell’s diameter 1+ 1-3 polychromatophilic cells/ field
2+ to 3+ More than 2/3 2+ 3-5 PcC/f
(moderate)
4+ (marked) Thin rim on the periphery 3+ >5 PcC/f

VARIATION IN SHAPE OF RBC

POIKILOCYTOSIS- increase variation in shape ; named after their shape
PROTEINS – maintain shape & size of the cell, preventing it from collapsing
LIPIDS – main fluid barrier

 Abnormality in proteins, lipids & carbohydrates affects the shape of the cell
CELL TYPE MORPHOLOGIC DEFECT OR CHANGE DEFECT OR CHANGE ASSOCIATED
APPEARANCE ASSOCIATED CONDITIONS
CONDITIONS
OVALOCYTES  Range from egg- HEREDITARY OVALOCYTES:
(ELLIPTOCYTES) shape, slightly oval ELLIPTOCYTOSIS: (25-  Myelodysplastic syndrome
to sausage, rod, or 90%)  Thalassemic syndrome
pencil forms  decrease in  Megaloblastic syndrome –
 Despite of their skeletal megaloblastic
shape they function membrane ELLIPTOCTE:
normally depending CHON band 4.1  Hereditary elliptocytosis
on their hemoglobin  increase heat  Idiopathic myelofibrosis
content, flexibility & sensitivity of  Small numbers may be
deformability spectrin acquired in:
 Some appear o iron deficiency,
normochromic or ACQUIRED: 10% o megaloblastic
hypochromic  No problem or anemia,
meaning they are deficiency o myelophthisic
abnormal in shape affecting the anemia,
but normal Hb protein o thalassemias &
content thus membrane but sickle cell anemia
function normally the cell
 Some are became
normocytic but distorted
elongated sized but because of
the MCV is the different
same. Some are oval condition
& macrocytic
(macroovalocyte)
which is a significant
 observation related
to megaloblastic
anemia
 Hb appears to be
concentrated at the
two ends of the cell,
leaving a normal
central pallor area.

NOTE: Membrane
abnormality for both is the
same
NOTES:
Peripheral proteins can be found on the surface or inner surface underlying the phospholipid layer, these proteins are
connected to the transmembrane proteins & phospholipid bilayer which adds on the integrity thus deficiency in
spectrins, protein 4.1, 4.2 will alter the membrane thus deformed.
SPHEROCYTE  sphere & not  indicates a TWO MOST COMMON CAUSES:
biconcave hemolytic  HEREDITARY
 Smaller in diameter process SPHEROCYTOSIS/INTRINSI
than normal RBC & (hemolysis C MEMBRANE DEFECT –
w/ smaller volume results from a protein or spectrin
because it is fully membrane abnormality, deficiency in
distended due to abnormality) ankyrin or protein band 3.
increase hb thus it cannot Spectrin dimer is
 On side view it has a enter onto the connected to the integral
small biconcavity microcirculatio protein, ankyrin underlies
but very small not n & as they the phospholipid bilayer,
enough to make enter they these add to the integrity
them flexible or immediately of phospholipid bilayer
deformable burst or lyse thus a deficiency in these
 With concentrated protein will make the
hb content phospholipid bilayer,
 No visible or weaker, & some of portion
decrease central of phospholipid will be
pallor easily detached into the
form of microvesicles.
IN SHORT:
A deficiency in spectrin, ankyrin &
protein band 3 leads uncoupling of
phospholipid layer & some of lipids
will be easily detached into the
form of microvesicles. Repeated
detachment leads to a smaller cell
having no central pallor,
spherocyte.

OTHER EXPLANATION:
Weak membrane causing weak
dissociation of deficiency in
cytoskeletal protein

 AUTOIMMUNE
HEMOLYTIC ANEMIA: Ab
 to the red cell will
attached to the red cell w/
their FC portion exposed
which will be recognized
by macrophages that have
Fc receptors on their
receptors & they pit off.
Macrophages pull off Ab
attached on RBC but as it
pulls off, it removes
portion of RBC. The
remaining fragment of RBC
will reseal to form a
smaller cell w/ a filled
volume. Repeated pitting
may occur resulting to
microspherocyte.

SPLENIC CULLING – removal of old

& damage red cell
PITTING – process of removing
inclusions & Ab attached to RBC

OTHER MECHANISMS (LOSS OF

MEMBRANE):
 NORMAL AGING PROCESS:
lose enzymatic properties
& will lack ATP caused by
decreased in enzymatic
activity of enzymes involve
in Embden Meyerhof
pathway resulting to lack
of ability to maintain their
shape & size. Na/K ATPase
needs ATP for activity thus
when cell lacks ATP, this
can no longer be
maintained & transport of
Na/K is compromised & as
an effect the cell can be
crenate or distorted.
 STORAGE PHENOMENON
– after transfusion of
stored blood, patient
blood smear shows lots of
spherocytes but no
abnormality in the
membrane but the cells
undergo change in their
shape due to storage
lesion. During the storage
of blood in blood bag, it
will be consumed, the cell
 will lack glucose to
produce ATP & the main
source is glycolysis, but as
the cell lack glucose this
will lead to lack of ATP
production.
 SEVERE BURNS:
Since phospholipid is a fat,
it is easily dissolve by heat
thus will fragment &
vesicle out of phospholipid
bilayer resulting to
spherocyte & repeated
fragmentation leads to
microspherocyte.
BURR CELL  Microscopically burr  Depletion of BURR CELL
& echinocyte ATP 1. Occur in situations that
appear similar.  Exposure to cause a change in tonicity
ECHINOCYTE hypertonic (hypertonic) of the
 Sea urchin solution intravascular fluid plasma
 Crenation occurs in seen in dehydration &
vitro azotemia related to uremia
 An artefact &
insignificant which 2. ASSOCIATED W/ RENAL
may be seen on INSUFFICIENCY - wherein
thinner part of the kidneys important in
cell removing metabolic waste
 With evenly in the blood, it also filters
distributed, blood but do not remove
uniformly sized 10- abnormally shaped cells &
30 blunt spicules or inclusions. It functions to
bumps remove metabolic
products, dissolve
RULE: Both reactions are chemicals & substances
reversible (salts, acids, & excessive
water). In renal
BURR CELL insufficiency the kidneys
 Very clinically cannot function, resulting
significant which to non-removal of
indicates changed in metabolic products such as
the plasma tonicity the non-protein
 Microscopically the nitrogenous substances,
appearance of the blood urea nitrogen &
spicules is the same creatinine, these
w/ echinocytes thus substances builds up in the
relate in conditions. blood altering the tonicity
 Crenation occurs in thus cells will crenate
vivo while still in the
 Reversible in intravascular fluid.
formation, when
patient underwent RULE: The no. of burr cell indicates
dialysis thus NPN is the severity of renal damage.
cleared out from the
 plasma maintaining ECHONOCYTES
the normal tonicity 1. Several hours old
of plasma anticoagulant blood stored
 With irregularly at room temperature
sized & unevenly causing an artefactual
spaced 10-30 blunt effect due to prolonged
spicules storage at prolonged
temperature
2. Stored blood causes
crenation of cell due to
depletion of ATP &
biochemical activities

RULE: Both reactions are reversible

ACANTHOCYTES  Irreversible  May be caused  ASSOCIATED W/ END
formation by changes in STAGE LIVER DISEASE –
 No longer a disc the ratio of liver is main organ which
shaped, with few (3- plasma lipids. metabolizes & stores fats
12) irregularly Since cell such as cholesterol &
spaced, pointed membrane is lipoproteins but when it is
spicules/thornlike made up of 2 diseased it will not be able
projections of layers of to metabolize the different
various lengths and phospholipids phospholipids &
widths + cholesterol cholesterol resulting to
 Indicates that maintain abnormal ratio in plasma.
permanent the When there is
membrane damage, permeability of hypercholesterolemia due
some are long & membrane to to liver disease this may
short thus it the passage of lead to abnormal ratio of
indicates that the ions. plasma lipids leading to
cell can no longer  Changes in distortion of RBC into
regain its normal cholesterol, acanthocyte.
shape lecithin &
 sphingomyelin NOTE: The conc. of cholesterol
depends on the conc. of plasma
cholesterol, activity of LCAT & bile
acids.

 ALCOHOL INTOXICATION –
affects liver leading to
hepatic cirrhosis, the
alcohol has a direct effect
on RBC
 ALCOHOLIC CIRRHOSIS
 PK DEFICIENCY – pyruvate
kinase is involved in EMP,
it catalyzes the conversion
of phosphoenolpyruvate
to pyruvate wherein ATP is
produced thus deficiency
in PK leads to deficiency in
ATP which is important in
 maintaining cell
membrane
 CONGENITAL
ABETALIPOPROTEINEMIA
– absence of
betalipoprotein which is
related to the inability of
the liver to metabolize it.
LDL is necessary but must
not be in excess thus the
absence of betalipoprotein
leads to red cell distortion
 VITAMIN E DEFICIENCY –
Vit. E is needed for
preventing peroxidase
oxidation of the cell
membrane
POST-SPLENECTOMY – removal of
spleen, all abnormalities will be
seen in the circulation
STOMATOCYTES  Normal sized cell Membrane defect that COMMON CAUSES: HEREDITARY
(MOUTH CELLS) with an elongated results in high cellular  HEREDITARY
HYDROCYTE or a slit like mouth sodium and low STOMATOCYTOSIS – main
or stoma of central potassium content problem in the transport
pallor of Na/K. Due to defect in
 cup-shaped on SEM the transport of Na/K,
 fully hydrated cell there is high cellular Na
due to defect in the thus water enters into the
transport of Na/K cell & the cell becomes
ATPase hydrated & low K
 swollen cell due to  RH NULL DISEASE – second
increase water major blood group (rhesus
content blood group), the Ag in this
blood group are the capital
D indication of positive or
negative & also C & E. Rh
null means no D, C & E Rh
Ag located on the cell
membrane. Rh Ag are
integral part of cell
membrane for the
transport of molecules
thus absence affects the
maintenance of RBC thus
transport of Na/K is
altered leading to
stomatocytosis

ACQUIRED CASES: MOST

COMMON CAUSE:
 Obstructive liver disease
 Alcohol excess or
intoxication
 LESS COMMON:
 myelodysplastic
syndromes
 hydroxyurea therapy –
therapy for leukemia
 Hereditary stomatocytosis
TARGET CELLS  Has a bulls eye, w/ a  Increase in HB DEFICIENCY:
(CODOCYTES)/ button or cholesterol &  Hemoglobinopathies
PLATYCTES/LEPTOCYT concentrate Hb thus phospholipids  Thalassemia
E a dark center  Excess surface  Hemoglobin C Disease or
followed by clear Hb area to volume Trait
area & followed by ratio  Iron deficiency anemia
thin rim of Hb  Any hemoglobin
 May have or have abnormality
not clinical
significance (Hb OTHERS:
deficiency)  Liver diseases – affects
 Large cell w/ small level of plasma cholesterol
Hb as compared to  LCAT deficiency –
its volume or size hypercholesterolemia,
causing the some of cholesterol will
redistribution of Hb load causing the cell to be
into a button of Hb larger in size & Hb content
at the center (Iron  Post-splenectomy
deficiency anemia,
thalassemia,
hemoglobinopathies
)
 With a central area
or hb surrounded by
a colorless ring and
a peripheral ring of
hb
 bell-shaped (codon)
or tall hat shaped on
SEM
 resembles a tall
Mexican hat cell on
SEM
 non-specific finding,
seen on several
findings & are
artIfactual in nature:

1. Drying Artefact –
slow drying due to
humid environment
causing Hb to
crystallize at the
center & dissolve
forming a
concentrated Hb at
 the center resulting
to a target cell
2. Decrease Hb

PLATICYTES - On front view,

it appears as a flat cell
LEPTOCYTE – remains
connected w/ the peripheral
Hb but clinical significance is
the same
NOTES:
ACANTHOCYTE: abetalipoproteinemia TEARDROP CELL: myelofibrosis
BURR CELL: renal disorder TARGET CELL: non-specific

POIKILOCYTES SECONDARY TO TRAUMA

EXCESSIVE PHYSICAL TRAUMA IN THE CVS DUE TO:
1. Presence of fibrin strands causing physical trauma to the red cells passing through leading to fragmentation
2. Blood vessels that are abnormally too small for RBC to circulate

TWO PATHWAYS OF FRAGMENTATION ARE RECOGNIZED

a. Alteration of normal fluid circulation (DIC, TTP, MAHA)
b. Intrinsic defects of the red cell (spherocytes, antibody altered red cells, inclusions
CELL TYPE MORPHOLOGIC DEFECT/S OR CHANGE/S
APPEARANCE ASSOCIATED CONDITIONS
SCHISTOCYTES  Very significant FRAGMENTATION DUE TO:
(SCHIZOCYTES) finding indicating a 1. Exposure of cells to
hemolytic anemia abnormal conditions like
heat (affects RBC in
General term for fragments circulation) or mechanical
of RBCs varying in shapes: trauma
 Helmet cells 2. Contact with fibrin
 Triangular cells strands or damaged
 Keratocytes/Horn endothelium – fibrin
cell deposits & fibrin strands
present slicing of RBC
during their passage. If
the current of blood flow
is strong this will push the
cell to the fibrin strand
causing now
fragmentation seen in
microangiopatic anemia &
disseminated
intravascular coagulation
3. Intrinsic defects of RBCs –
RBC has the problem
 Spherocytes (no more
adjustment in shape
& volume), Ab altered
red cells (recognized
by macrophages &
hemolyze them), Red
ASSOCIATED
CONDITIONS
Hall mark of Hemolytic
anemias
CAUSES:
PROSTHETIC HEART
VALVE – damaged on
artificial heart valve will
exposed artificial
components such as
rubber, causing damage
to RBC & RBC suffer from
physical trauma leading
to hemolysis
MICROANGIOPATHIC
HEMOLYTIC ANEMIA &
DISSEMINATED
INTRAVASCULAR
COAGULATION
SIMILARITIES:
coagulation mechanism
is activated causing now
the formation of blood
clots due to fibrin
threads deposited on the
circulation causing now
obstruction & limit the
normal circulation

KERATOCYTES (HORN KERATOCYTE – pair of
CELL), BITE CELLS, spicule surrounded by a
DEGMACYTES gap
BITE CELL – bite
appearance
 Erythrocytes with a
pair of spicules or
horns surrounding
a gap in the cell
outline
cells containing
inclusions (pitting of
cell causing formation
of bite cell), etc.
BITE CELL: Results from
removal of a Heinz body from
a cell by splenic macrophages
(splenic pitting) or when an
erythrocyte is caught on a
fibrin strand or during pitting.
 When a cell has an
inclusion it will pit off
or pull off by
macrophages thus
removing a part of
RBC & the cells will
assume a bite cell
1. Heinz inclusion on a cell
will pass through a limited
basement membrane
leading to physical
trauma. If clots are
bigger the more
obstruction which will be
difficult for RBC to pass
thru.
DIFFERENCE:
MAHA – abnormal
formation & deposition
of blood clots occurs in
blood vessels
DIC – affects all vessels
since it is disseminated
CLOSTRIDIAL
INFECTIONS – toxin
produced damages RBC
& activate coagulation to
cause abnormal clot
formation
THROMBOTIC
THROMBOCYTOPENIC
PURPURA & HEMOLYTIC
UREMIA SYNDROME
SIMILARITIES: platelet
aggregates causes
obstruction of circulation
due to abnormal
activation of platelets
wherein when platelets
are aggregated they form
together to form a
thrombus
Hemolytic anemias
G6PD deficiency
 2. Rigid portion will be left &
SEMILUNAR BODIES  Half-moon / crescent
cells
 Large pale pink
staining ghost of the
RBC
 Complete round w/ a
visible crescent shape
of Hb or complete
crescent
TEARDROP CELLS  Pear-shaped or
(DACROCYTES) teardrop-shaped
w/ a single
elongated point
 One end is
rounded the other
end is pointed
resulting to a pear,
drop or rocket
shape
 Hall mark of
myelofibrosis
if able to pass through it
will become a teardrop
cell but if removed RBC
will reseal & adjust the
volume & shape
 Frequently observed Overt Hemolysis
in malaria which is a
hemolytic disorder &
in other conditions
causing overt
hemolysis.
 As RBC is hemolyze it
will release its Hb
content & only a small
portion will be left
that is remain
attached on the
membrane indicating
an overt hemolysis
 Caused by excessive MOST COMMON:
squeezing & results to
fragmentation during MYELOFIBROSIS W/
splenic passage MYELOID METAPLASIA –
 Vessels constricted bone marrow is
causing RBC to infiltrated by fibrous
squeeze itself which materials, thus there is
causes too much overgrowth of fibrous
squeezing resulting to cells thus if bone marrow
permanent damage & is unable to produce
may no longer revert blood cells resulting to
to its shape thus metaplasia & spleen is
loosing capacity to involve in the production
regain normal shape of RBC which may suffer
also of fibrosis causing a
limitation of its
circulation. Blood vessels
in the spleen are
normally small or minute
but in myelofibrosis the
more they become
limited thus increase
microcirculation causing
teardrop cell which is the
hall mark of
myelofibrosis.
HYPERSPLENISM –
spleen enlarge causing
circulation to be limited
PRESENCE OF INCLUSION
BODIES – makes RBC

MICROSPHEROCYTES &  Are red cell
PYROPOIKILOCYTE
 MCV 60 fL
 Smaller than
 Also lack central
OTHER APPEARANCES RELATED W/ HEMOLYSIS:
Results from repeated
fragments fragmentation of RBC & from
thermal damage to cell
membrane
spherocytes
KNIZOCYTES – appear like
pinched bottle having
rigid like Heinz bodies
which commonly
attached on the
membrane of RBC
making the membrane
rigid & when it passes
through the splenic
circulation which is too
limited, it has to squeeze
itself leading to its:
1. Detached & cell
becomes a bite cell
2. Pulled & be pointed
because it is the last to
exceed the membrane
causing a tear drop shape
MYELOPHTHISIC
ANEMIA – bone marrow
is infiltrated w/ abnormal
cell like in leukemia thus
circulation becomes
limited
UNCOMMON:
Pernicious anemia
B thalassemia
MICROSPHEROCYTE:
Severe burns – thermal
injury on RBC membrane
wherein fats are easily
damage causing the
membrane to vesicle, if
detachment is mild it will
result to spherocyte but
if there is continuous
fragmentation, it will
become the largest
fragment, the
microspherocyte
PYROPOIKILOCYTE:
Hereditary
pyropoikilocytosis –
there is a membrane
damage of red cell due to
sensitivity to heat. A
normal RBC fragments at
49 C but cells with HPP
will fragment at low T
(45-46)
PYKNOCYTES – distorted
or irregularly contracted
BLISTER CELL – with thinned portion due to cell caught constriction on one of its part red cells, seen in
on fibrin strands wherein it might have encountered indicating that it might blister hemolytic anemia
along the circulation such the portion of the membrane or bud off wherein the bud
is pull apart. may detached & become
defragment. The remaining
fragment, the larger one may
become a spherocyte or the
smaller being
microspherocyte, seen in
hemolytic anemia normally in
infants but w/ lesser number

POIKILOCYTES & CRYSTALS SECONDARY TO ABNORMAL HEMOGLOBIN CONTENT

CELL TYPE MORPHOLOGIC DEFECT/ CHANGES ASSOCIATED CONDITIONS
APPEARANCE
DREPANOCYTES/SICKLE  Most insoluble of  Polymerization of Sickle cell anemia
CELLS the Hb variants hemoglobin Indicates Sickling Hb
 Fully soluble Hb  Sickling is
when exposed to reversible when
normal O2 but exposed to normal
when O2 but repeated
deoxygenated it sickling causes
becomes insoluble permanent
& crystallizes at damage on the
low O2 tension. membrane &
The crystals are assumes
thin & elongated
with pointed ends
altering the shape
of the cell having a
curved, straight L,
V, & S shape

HB CRYSTAL HB CC CRYSTAL
 With one or more  Hexagonal
fingerlike, blunt-pointed  Both ends are blunt &
projections that protrude rectangular
from the cell membrane  Parallel sides
leaving a pale area at the  Formed within the membrane (RBC) but does not
opposite end deform RBC causing a normal shape but it makes
 Has more than one protrusion & the longest one RBC rigid thus the RBC is not deformable &
protrude away flexible thus prone to hemolysis
 Washington monument, Pointing finger, Hand  Bar of gold
glove  Crystalizes when Hb is O2 or dehydrated
 INCLUSIONS: ABNORMAL HEMOGLOBIN PRECIPITATION
REASON:
1. Precipitation of abnormal component in the cell
2. Disturbance or abnormality in the developmental or maturation of RBC (developmental organelles)
INCLUSION MORPHOLOGIC DEFECT/S OR CHANGE/S ASSOCIATED CONDITIONS
APPEARANCE
HEINZ BODIES  Large (1 to 3 Results from  G-6-PDH DEFICIENCY
um), single or precipitation of ANEMIA – most common
multiple small, hemoglobin that has disorder associated w/ Heinz
round, oval or been denatured body. G-6-PDH is an involve
serrated, in hexose monophosphate
purplish shunt. When G6P is
inclusions on RBC converted into 6PG catalyze
periphery by G6PD & the cofactor is
(distorts the NADP which becomes
membrane) NADPH a reducing property
depending on of RBC which in turn
the oxidation maintains GSH into reduced
form, which serves as the
STAINS (SUPRAVITAL major antioxidant property
STAINS): Crystal violet & of RBC which can breakdown
Brilliant Cresyl Blue H2O2 into H2O & O2
preventing oxidation of Hb.
The NADPH formed will
reduced the ferric back to
ferrous thus protecting RBC
to oxidation. Deficiency will
result to deficiency in NADPH
& GSH
 RED CELL INJURY DUE TO
CHEMICAL INSULT OR
UNDER OXIDANT STRESS
(sulfonamides) – causes
accumulation of oxidized Hb
 HEMOGLOBINOPATHIES
AND THALASSEMIA MAJOR
 UNSTABLE HEMOGLOBIN
SYNDROMES
 ZURICH & KOLN HB –
abnormal Hb prone to
precipitation & oxidation,
unstable Hb
 POST-SPLENECTOMY
HB H INCLUSIONS  Small greenish- Precipitate of Hb H, an Seen in Hb H disease
blue inclusion abnormal Hb which has 4 Alpha Thalassemia
bodies polypeptide chains as B
 Seen in occurs when A chain is
unstained cells not completely
 May also be seen synthesize
w/ supravital
stain
 Golf ball w/ pits
when numerous
in RBC
HOWELL-JOLLY BODY  Small, coarse  Nuclear CAUSES:
round granules remnants  SPLENECTOMY
 deep blue or red-  with Feulgen &  FUNCTIONAL
purple, appears Methyl Green HYPOSPLENIA - spleen
singly & is off the Pyronin stains w/ decreased function
center but not at (stains for DNA) thus unable to remove
the periphery  seen when there inclusion
 DNA containing is increase
inclusion karyorrhexis DISORDERS ASSOCIATED W/
meaning it is a (fragmentation KARYORHEXIS:
nuclear fragment of nucleus) •ACCELERATED OR ABNORMAL
 Very tiny ERYTHROPOIESIS
inclusion w/ <1  hemolytic anemias – too
um much RBC are
hemolyzed thus bone
marrow compensate by
accelerating RBC
production thus nuclear
dissolution is also fast &
more nuclear fragments
will be seen
 megaloblastic anemia –
caused by abnormal
nuclear dissolution (Vit.
B12 & Folate deficiency)
PAPPENHEIMER BODIES  small (2-3 um)  Unused Iron  SIDEROBLASTIC
SIDEROTIC GRANULES faint blue deposits ANEMIA – red cell
coccoid bodies precipitate & cannot make use of the
that aggregate in become siderotic iron due to deficiency in
small clusters at granules protoporphyrin 9 thus
the periphery of  Sometimes may iron precipitate &
the cell. appear as Ringed becomes siderotic
 Smaller than H-J sideroblast granules
bodies, deep  THALASSEMIA – affects
blue & are more globin
likely to be  HEMOGLOBINOPATHIES
multiple - affects globin
 Iron containing  ALCOHOLISM – affects
granules positive enzymes in heme
when stained w/ synthesis
Perls Prussian  POST-SPLENECTOMY
 blue & named as  CONDITIONS LEADING
siderotic TO
granules & if HEMOCHROMATOSIS or
positive when HEMOSIDEROSIS (iron)
stained w/ – both increase iron or
wrights/giemsa it iron overload
is named as
pappenheimer
bodies
BASOPHILIC STIPPLING  Dark blue Aggregates of ribosomes DEFECTIVE OR ACCELERATED
granules, and mitochondrial HEME SYNTHESIS
multiple, uniform remnants thus acidic in  Lead or other heavy
and evenly component because they metal poisoning –
distributed as took up the basic dye affects hb synthesis
fine dots or large due to rRNA  Thalassemia - globin
granules  Hemoglobinopathies -
 Often confused globin
w/  Megaloblastic anemia -
pappenheimer rarely
bodies (+
Prussian Blue) PYRAMIDINE-5-NUCLEOTIDASE
but basophilic DEFICIENCY
stippling do not Explanation:
stain w/ Prussian When RNA degrades, they
blue thus it is not release their purine &
seen due to lack pyrimidine bases. For the
of iron but if complete degredation of
present in wright pyramidine bases, it needs
& Prussian then Pyramidine-5-nucleotidase thus
it is siderotic deficiency of Pyramidine-5-
granules nucleotidase, RNA degradation
will also be defective or
APPEARANCE: incomplete
COARSE: granules are
much more outlined and
easily distinguished &
larger
PUNCTATE: coalescing
into smaller forms
DIFFUSE: Granules are
fine blue dusting
CABOT RINGS Reddish-violet thread- Remnants of Dyserythropoiesis
like that assumes a loop, microtubules of mitotic megaloblastic anemias
or an incomplete ring or spindle & can reshape Homozygous
a figure of 8 into a loop or a fragment Thalassemias
of the nuclear  Lead poisoning
membrane  Other severe anemia
 Post splenectomy
STRUCTURES NOT SEEN IN WRIGHTS: STRUCTURES NOT SEEN IN WRIGHTS:
1. Granules of reticulocyte 1. Polychromatophilic cell
2. Siderotic granules 2. Pappenheimer bodies
3. Heinz bodies
 ABNORMAL RED CELL DISTRIBUTION
AGGLUTINATION Clumping of RBCs Presence of antibodies on  Cold agglutinin
Indication of Ag-Ab the RBC surface produced  Autoimmune
reaction by patient (autoAb) or from Hemolytic Anemia
different person (isoAb)

ROULEAUX  Linear alignment of Increased concentration of  HYPERPROTEINEMIA’S

RBCs appearing as globulin – plasma protein
stack of coins component is altered
 RBC due to resulting to alteration of
destruction of zeta the negative charges
potential surrounding the RBC
 Side by side flat which is seen in
surfaces Multiple myeloma &
 biconcave surfaces Waldenstrom’s
in apposition macroglobulinemia
appearing as a which are plasma cell
stack of coins or discrasias which are
flat plates leukemias affecting
plasma cell causing
increase proliferation of
plasma cells

 MULTIPLE MYELOMA –
most of the affected
plasma cell will
produce excessive IgM

 WALDENSTROM’S

PROTOZOAN INCLUSIONS
 RBC is normal but infected by hematozoic parasite
a. PLASMODIUM SPECIES
• Seen in different stages of smear mostly ringed forms

1) MALARIAL INCLUSION
• Obligate intraerythrocytic parasites
• w/ hepatic stage (liver) where they rapture the liver & proceed to
rupture the RBC & metabolize the Hb but not completely & leave behind a
pigment (hematin or hemozoin pigment)

PIGMENTS INSIDE THE INFECTED RBC:

a. P. vivax - Schuffners dots
b. P. falcifarum – Maurer’s dots
c. P. malariae – ziemann’s dots
d. P. ovale – james dots

2) BABESIA PARASITE
• Parasite of cattle’s causing red fever on cattle’s
• Discovered by babes & may also affect men
• w/ ring forms in RBC
 ARTIFACTS
a. FIXATION b. HEAT ARTEFACTS
• When fixative is contaminated w/ water which EFFECTS:
must be water free methanol 1. Red cell bud off into vesicles which might be reported
EFFECTS: microspherocytes
1. Refractile rings in red cells 2. WBC disintegrate
2. Mouth eaten cells – caused by blowing leading to 3. Proteins coagulate producing small weak basophilic
spicules particles which are similar to platelets

WHITE BLOOD CELLS

CLASSIFICATION OF WBC
a. Granulocytes (neutrophil, eosinophil, basophil) & Non-granulocytes (lymphocytes & monocytes)
 Non-granulocytes have granules however too small to be seen
 Cytoplasm of monocytes appear like a ground glass due to the tiny granules present

b. Polymorphonuclears (those w/ segmented or lobulated nuclei – neutrophil, eosinophil & basophil) &
Mononuclears (unsegmented nucleus – monocytes & lymphocytes)
c. Phagocytes (non -specific function – neutrophil (most active phagocyte), monocyte & macrophages,
eosinophil & basophil (both are less efficient phagocytes) & Immunocytes (involve in production of Ab – B
cells)

Formation: Bone marrow & lymphoid tissues (thymus (T-cells), spleen & lymph nodes)
General Function: Defense
NON-MALIGNANT/REACTIVE CHANGES OF WBCS
CYTOPLASMIC MORPHOLOGIC DEFECT OR CHANGE ASSOCIATED CONDITION/S
CHANGES APPEARANCE
TOXIC  Large purple to Altered primary  SEVERE INFECTION – a reaction of the
GRANULATION black azurophilic granules infection
granules especially EXPLANATION: during infection since
in the neutrophils  Neutrophils have neutrophil is involve in this mechanism, it
which contains primary granules undergoes several changes:
primary granules. but when it is 1. EXEMPLIFY & ENHANCE ENZYME
 Larger than 2- altered, it PRODUCTION as they need to
degree granules & appears as a toxic destroy the invading organism by
stain dark blue-black granulation which phagocytosis. The phagocytized
are larger than material must be destroyed thus the
the usual primary cell produces lots of enzymes to
granules perform respiratory burst & must
also alter its granulation by
increasing the function of its
granules. Enzymes such as the
myeloperoxidase is contained in the
primary granules. As the cell
increase its function w/ the
granules, the granules also function
to increase their content resulting to
alteration & toxic granulation.
2. SHIFT TO THE LEFT – even immature
WBC will be released as a
 compensation for the severe
infection
3. INCREASED PHAGOCYTIC ACTIVITY
– in infection, the neutrophils have
to phagocytized the invading
organism & the phagocytized cell
will be contained inside the vacuoles
& it will appear as many toxic
vacuolation & granulations. The
cytoplasm also appears as Dohle
bodies as larger inclusion.
 CHEMICAL POISONING
 TOXIC STATES

 Toxic granulation is a normal reaction in

these conditions
DOHLE  Blue, round, or Aggregates of free INFECTION – due to increase activity of the
BODIES/AMATO mostly elongated ribosomes cell
bodies
 Represents rRNA CELLS INCREASE METABOLIC ACTIVITY AS A
 Not permanently RESPONSE IN:
seen in cytoplasm  Severe infections, burns,
except for may- Chemotherapy, surgery
hegglin anomaly  If these conditions disappear,
 Single or multiple Amato bodies will also
arranged in parallel disappear
rows
MAY-HEGGLIN ANOMALY – an inherited
condition, thus appearance of amato is
permanent

 Basophilic stippling: If RNA

precipitates & seen as granules in
RBC
 Amato bodies: if RNA precipitates &
seen as granules seen inside of WBC
 RNA is important in metabolism, as
the cell increases its production of
enzyme, this will increase metabolic
activity thus increased RNA function.
CYTOPLASMIC  Many presence Represents end stage SEPTICEMIA, SEVERE INFECTIONS, TOXIC
VACUOLATION indicate phagocytosis of phagocytosis STATES
particularly the end
stage  During phagocytosis the phagocytize
 Presence of many material will be contained in a vacuole
toxic vacuoles thus increasing the vacuole inside the
containing the neutrophil. Once in the vacuole the
phagocytized phagosome will fused w/ lysosome
material w/ the forming the phagolysosome. Once
contents of lysosome destroyed the debris will be contained in
or debris of the a vacuole
phagocytized
material
  Large empty areas
within the cell
ATYPICAL/  Cells show foamy & Seen in INFECTIOUS
REACTIVE abundant cytoplasm MONONUCLEOSIS/KISSING DISEASE –
LYMPHOCYTE/  ≥40% associated w/ caused by Epstein barr virus
DOWNEY CELLS infectious
mononucleosis

INHERITED DISORDERS
CYTOPLASMIC CHANGES MORPHOLOGIC APPEARANCE ASSOCIATED CONDITION/S
ALDER-REILLY  Large, coarse blue – black granules Associated w/
GRANULATION in all WBCs MUCOPOLYSACCHARIDOSIS/G
 Abnormal polysaccharide granules ARGOYLISM – abnormalities in
which are not completely mucopolysaccharide
metabolize due to an enzyme metabolism
deficiency (congenital disorder) which leads to non-  Hurler’s
metabolism of mucopolysaccharide & prevents the  Hunters
formation of secondary granules

MAY-HEGGLIN  Resembles Dohle bodies but are permanently LEUCOPENIA, VARIABLE

ANOMALY present THROMBOCYTOPENIA, GIANT
 Presence of gray – blue spindle shaped inclusions in PLATELETS
the cytoplasm of Neutrophils and Monocytes

FUNCTIONS OF NEUTROPHIL:
1. DIAPEDESIS – squeezing out through the circulation via the endothelial lining & proceeds via:
Random motility – no particular direction
Direct motility/Chemotaxis – w/ particular direction
2. PHAGOCYTOSIS – killing of phagocytized agent
INHERITED ABNORMAL GRANULOCYTE FUNCTION
CYTOPLASMIC DESCRIPTION
CHANGES
JOB’S SYNDROME  Impaired directional motility
 Random movement of phagocytes is normal thus neutrophil may still move
elsewhere but directional motility is impaired because cells respond slowly to
chemotactic agents thus do not directly proceed to the chemical attractant.
EFFECT: it gives more time for the bacteria to proliferate or cross the infection. If bacteria
are dislodged in the tissue, & immediately phagocytized thus it will not be able to cause
infection but due to slow movement of cells, the bacteria will be able to lodge in the site of
infection causing infection.
LAZY LEUKOCYTE  Both random and directional movement of the cells are defective thus neutrophil
SYNDROME will only stay but the organism will approach the neutrophil thus phagocytosing it
  Cells fail to respond to inflammatory stimuli but have normal phagocytic and
bactericidal activity.

NON-MALIGNANT: DEFECTIVE KILLING OF MICROORGANISMS

CYTOPLASMIC DESCRIPTION
CHANGES
CHEDIAK – HIGASHI 
Characterized by large, blue to grayish round granules or inclusions in Neutrophils
ANOMALY that vary in size and color
 Stain positive with peroxidase, Sudan black B (SBB) and Acid phosphatase (ACP)
stains.
 Represents the fused primary granules. Primary granules contain the usual
composition such as peroxidase, sudan black b & ACP but these are abnormally
package, meaning they are normal in contents but abnormally package, thus
appears as chediak-higashi anomaly.
IN SHORT: granules are normal in content but abnormally packaged thus it cannot perform
its normal function
CHRONIC  Phagocytes ingest all but cannot kill catalase (+) organism because of lack of
GRANULOMATOUS appropriate respiratory burst
DISEASE  Neutrophils have normal direct & random motility & phagocytosis

RESPIRATORY BURST – reaction inside the neutrophil needed for the killing of organism.

In respiratory burst the neutrophil is able to produce potent oxygen radicals like H2O2 &
O2 for killing organisms. In chronic respiratory disease, the respiratory burst function of
neutrophil is decreased or inappropriate thus inadequate production of H2O2 & O2 which
are important bactericidal agent.

Catalase + Organisms: Staph aureus – produce enzyme catalase which will breakdown
H2O2 into H2O & O2

OTHER APPEARANCES
TART CELL  A monocyte (phagocyte) that engulf a lymphocyte (engulf material)
 Formation is no known significance

FERRATA CELL  a stimulated or atypical lymphocyte with denser and more opaque cytoplasm
 associated with SBE
REIDER CELL  a lymphocyte with notched, lobulated, segmented, clover – leaf like nucleus
 associated with Chronic Lymphocytic Leukemia
L.E. CELL (LUPUS  Neutrophil engulf the lysed nucleus of another neutrophil containing a large purple
ERYTHEMATOSUS) homogenous round inclusion (phagocytized cell) with nucleus wrapped around the
phagocytized material which occupies the entire cytoplasm of neutrophil thus
neutrophils nucleus is pushed around the side appearing as a wrapping itself into the
phagocytized cell
 For it to occur there has to be a neutrophil, ANA or L.E. factor & nucleus of another
neutrophil
 Seen in SLE & other autoimmune disorders because it is related to the presence of
anti-nuclear Ab (ANA)
HAIRY CELL  A lymphocyte affected by hairy cell leukemia
  with abundant hair-like cytoplasmic projections

SEZARY CELL  Round lymph cell with nucleus that is grooved or convoluted
 Seen in Sezary syndrome which is an advanced case of mycosis fungoides. Both are T
cell disorder but they represent different stage of the disorder:
Mycosis fungoides – only skin is affected by T cells ; initial stage
Sezary Syndrome – when affected T cell migrate deeper into the dermal layer, lymph
nodes & visceral organs
 Mononuclear cell w/ a brain-like nucleus or cerebriform nucleus & a narrow rim of
cytoplasm
FLAME CELL  plasma cell with red to pink cytoplasm that appears like a flickering candle
 Associated with increase in IgA associated w/ multiple myeloma which is a malignancy
of plasma cell which is responsible for the production of Ig.

GRAPE CELL/ MOTT  plasma cell that contains small colorless (or blue or pink) vacuoles or globules
CELL/ MORULA CELL  large protein globules giving the appearance of grapes
 blue or pink protein that are excessively produced in multiple myeloma

RUSSEL BODIES  accumulation of IgG found in the cytoplasm & also seen in plasma cell

DUTCHER BODIES  intracellular crystalline structures of abnormal IgG

 inclusions of nucleus

NON-MALIGNANT/REACTIVE CHANGES
UCLEAR CHANGES MORPHOLOGIC APPEARANCE DEFECT OR CHANGE ASSOCIATED
CONDITION/S
HYPOLOBULATION 
Neutrophils have single or Decreased segmentation PELGER-HUET ANOMALY:
bilobed nuclei TRUE – inherited
 The 2 lobes appear as a PSEUDO – acquired
peanut or dumbbell shape or
a pince-nez spectacles (pair of
eye glasses)
HYPERSEGMENTATION  Neutrophils show more than 4 Abnormal DNA synthesis Megaloblastic anemia
lobes  Cells are abnormally
 Abnormal in size & lobulation large
& large as a macropolycyte
2 TYPES:
Normal size of neutrophil: 9-15 a. B 12 deficiency
um anemia
b. Folate deficiency
anemia
 OTHER NUCLEAR AND CYTOPLASMIC CHANGES
PYKNOTIC CELL  shrunken and dehydrated nucleus of cells that are about to die

PYKNOSIS – cell dissolution

SMUDGE CELL  Nuclear remnant of lymphocyte, seen after smear preparation

 Thumbprint appearance
 Associated w/ Chronic Lymphocytic Leukemia wherein cells are fragile thus they are easily
smudge.

BASKET CELL  Nuclear remnants of granulocytic cell

 Seen in Chronic Granulocytic Anemia
 Basket appearance

BARR BODY  Drumstick like body protrudes away from some of segments of neutrophils & attached on
one of the lobes of neutrophil nucleus
 Indicate an extra X chromosome & seen in females

AUER RODS  Pink or red rod-shaped structures

 Fused primary granules seen in immature neutrophils
 If seen in bundles this are called faggot cells
 Seen in Acute Myeloid Leukemia

INHERITED DISORDERS OF MONOCYTES & MACROPHAGES

Monocytes & macrophages are also involved in phagocytosis & also dispose of. They help in metabolism of lipids &
carbohydrates where they need enzymes & deficiency of these enzymes leads incomplete metabolism thus
incomplete disposal. Non-metabolized substances are contained in the cytoplasm causing macrophage overload.
DISORDER DESCRIPTION ENZYME DEFICIENCIES
MUCOPOLYSACCHARIDOSES  Deficiencies of specific enzymes involved I Hurler’s (most common)
/GARGOYLISM in the degradation of IV Morquio – Ullrich
mucopolysaccharides II Hunter’s
 Non-metabolized mucopolysaccharide will V Scheie
be contained in the cytoplasm of the cell III Sanfilippo
as granules VI Maroteaux – Lamy
 Alder-Reilly granules represents the non-
metabolized mucopolysaccharide. It is
associated w/ the enzyme deficiencies.
LIPIDOSES (abnormal lipid metabolism)
DISORDER DESCRIPTION & ENZYME DEFICIENT
GAUCHER’S DISEASE (MOST  lack of  - glucosidase resulting inability to metabolized glucocerebroside
COMMON) which will accumulate of in the spleen, liver and bone marrow

Gaucher cell – cytoplasm is distended with many glucocerebroside; sponge-like

appearance, crinkled or crumpled tissue paper
NIEMANN – PICK DISEASE
SEA –BLUE HISTROCYTES
 deficiency of sphingomyelinase resulting to accumulation of sphingomyelin
and cholesterol in the macrophages
 Foamy appearance
 w/ non-metabolized lipid-rich granules that stain blue green with polychrome
stains
 accumulation in the spleen and BM of histocytes giving an appearance of a
blue sea
 s/s: hepatomegaly, thrombocytopenia

FABRY’S DISEASE  lack of a-galactosidase resulting to accumulated glycolipid trihexosyl ceramide

but will not give an unusual appearance of macrophage
WOLMAN DISEASE  deficiency of acid esterase resulting to accumulation of TG and cholesterol
TANGIER’S DISEASE  Patient is unable to produce HDL (good cholesterol) which responsible for the
metabolism of the cholesterol by bringing it into the liver to prevent
hypercholesterolemia, as a result, cholesterol esters accumulate

TESTS FOR NEUTROPHIL FUNCTION: EVALUATION OF RESPIRATORY BURST

NTR (NITROBLUE  screening test for neutrophil function (ability of neutrophils to attack bacteria)
TETRAZOLIUM  test for the ability of normal neutrophil to reduce normal yellow water-soluble dye
REDUCTION) TEST into an insoluble blue formazan
 abnormal neutrophils are unable to reduce the yellow water-soluble dye into blue
formazan
CHEMILUMINESCENCE  Based on the ability of the cell for emission of low-level light pulses by
stimulated cells.
 Normal neutrophils when not stimulated or in resting state do not emit light
but when stimulated they emit a low level of light.
 Abnormal neutrophils w/ impaired respiratory burst cannot emit light whether
stimulated or resting
BOYDEN MICROPORE  Utilizes a micropore filter where neutrophils are counted. This is placed on an
FILTER abrasion to determine the ability of neutrophils to migrate
 Tests migration of neutrophil (fastest to migrate) in response to a chemotactic
factor.
REBUCK SKIN WINDOW  Requires performance of superficial abrasion on the skin to evaluate the
speed, type and number of phagocytes that respond to a skin abrasion.
 On top of the abrasion is the slide. After, perform a count on the slide &
observe.
 PLATELET
 Cytoplasmic fragments of megakaryocyte
 Small packages of cytoplasm that are nipped off from
the cytoplasm of megakaryocyte
 Are complex and metabolically active. They function
in hemostasis by interacting with their environment
to initiate and control hemostasis
GENERAL CHARACTERISTICS OF THROMBOCYTES
Composition: 60% proteins, 30% lipids, 8% CHO,
minerals, water, nucleotide, glycogen & glucose (main
ATP source)
Shape & Origin: Thin disc cytoplasmic fragment
Diameter: 2-4 um
Volume: 5 – 7 fL
Reference Platelet Count: 150,000 – 450, 000/ uL
Daily Turnover: 35 x 109/L (+/- 4.3) & then destroyed by
the spleen & produced by the bone marrow
2 pools: 1/3 spleen & 2/3 circulation
Lifespan: 8-11 days in vivo
Function: Hemostasis or stoppage of bleeding by
forming into plugs for the maintenance of vascular
integrity & continuity & blood coagulation
HEMOSTASIS
During vessel injury the blood contained in the
vasculature may leave the circulation (extravasation).
The blood vessels will constrict to limit the entry of
blood into the vessel causing limited blood loss. The
constriction of vessels is aided by thromboxane A2 &
serotonin from the platelets. Platelets immediately
form into plugs to prevent further bleeding. The plug is
an initial plug, w/ the current of flowing plug, the plug
may be removed or dislodge thus bleeding continues.
When secondary hemostatic mechanism is activated
when fibrin is form of fibrinogen, the fibrin thread will
be measured around the platelet plug, stabilizing now
the platelet plug thus further enhancing the hemostatic
effect of platelets.
NOTE:
In the 4 stages of megakaryopoiesis, only the
metamegakaryocyte (MK 4) with a 4 nucleus or more,
will be able to shed off as platelet.
 THROMBOPOIETIN – growth factor for
megakaryopoiesis produced mainly by the
liver & some in kidneys
 ENDOMITOSIS – division of nuclear
components but no cellular division thus as
cell mature it increases in size
 PLATELET BITS/STRING/RIBBON – hydrolytic enzymes
interconnected platelets indicating newly
released form BM which may be increased in
number in cases of hemolytic anemia &
hemorrhagic anemia
PLATELET STRUCTURE: FUNCTIONAL PLATELET
ZONES
A. PERIPHERAL ZONE: outermost zone
 Glycocalyx bears the different glycoproteins (Gp
1a, 1b, 1c, 2a, 2b, 3, 4, 5) which are not present
in other cell membrane. These glycoproteins
serves as surface for the adherence of other
coagulation factor (CF 1, 5, 7, 11, 12, 13)
Ex.: fibrinogen converted into fibrin on the
surface of platelets
Platelet can only aggregate to another
platelet if it has the important glycoprotein
for aggregation
NOTE:
SUB-MEMBRANOUS AREA: Underlies the plasma
membrane, this contains the communication w/ the
external environment of cell via receives message
from the outside
B. SOL-GEL ZONE:
 Consists of a stable gel that regulates
arrangement of internal organelles.
 Contains communication of organelles &
maintains location of the different organelles
 Where important protein serving as
cytoskeleton of the cell is located
MICROTUBULES (tubulin) & MICROFILAMENTS
(actin & myosin) – provide the cytoskeleton thus
maintaining the discoid shape of platelets
 When actin & myosin interacts, they form
into actomyosin where they form into
thrombosthenin
NOTE:
THROMBOSTHENIN – important contractile acting
force, provides contractile force after activation of
platelets.
C. ORGANELLE ZONE:
 Where the different organelles, alpha granules
(most abundant), dense bodies, mitochondria,
lysosomes and peroxisomes are located.
 Alpha & dense granules are for storage of
important contents while mitochondria for
ATP production, lysosomes containing
 ALPHA GRANULES CONTENT ROLE IN SUBSTANCE SOURCE
a. High molecular weight kininogen – cofactor in HEMOSTASIS
coagulation system Promote High Molecular Alpha granules
b. Factor 5 – cofactor for coagulation system coagulation Weight
c. Fibrinogen – same as coagulation factor 1, when Kininogen
acted by thrombin it is converted into fibrin thus it Fibrinogen;
is the precursor of fibrin Factor V von
d. Von Willebrand Factor – for platelet adhesion Willebrand
e. Thrombospondin – platelet function Factor
f. Platelet Factor 4 – platelet function Promote ADP; Calcium Dense bodies
g. Beta thromboglobulin – blood vessel repair aggregation Platelet factor 4 Alpha granules
h. Platelet Derived Growth Factor – blood vessel repair Thrombospondin Alpha granules
 When a vessel is injured, it will undergo healing Promote Serotonin Dense bodies
where smooth muscles & fibrous tissues must vasoconstriction Thromboxane A2 Membrane
regenerate. Beta thromboglobulin & PDGF both precursors phospholipids
stimulate smooth muscle repair & chemotactic Promote Platelet derived Alpha granules
to fibroblast vascular repair growth factor
i. Plasminogen – when acted on by its activator, Beta
converts into plasmin ; important for fibrinolysis thromboglobulin
Fibrinolysis – dissolution of the clot by plasminogen Other system Plasminogen Alpha granules
affected Alpha 2-
j. A-2 antiplasmin – Inhibits plasminogen to prevent antiplasmin C1
excessive clot dissolution esterase
k. C1 esterase inhibitor – found in complement system inhibitor

DENSE GRANULES: CAMAS

a. ADP – for platelet aggregation & adhesion
b. ATP – for platelet aggregation & adhesion
c. Calcium – for platelet aggregation & adhesion
d. Magnesium – for platelet aggregation & adhesion
e. Serotonin – for vasoconstriction

PLASMA COAGULATION FACTORS – roman numerals

PLATELET FACTORS – ARABIC NUMBERS
PLATELET FACTORS
a. PF 3 – platelet phospholipids found on surface of
platelet
b. PF 4 – inhibitor of heparin thus anti-heparin factor
MEMBRANE SYSTEM
 2 membrane systems
a. OPEN CANALICULAR SYSTEM – delivery routes thus
it is where nutrients enter & waste products leave
the cell
b. DENSE TUBULAR SYSTEM – remnant of endoplasmic
reticulum ; site of arachidonic acid synthesis and
functions as a calcium sequestering pump
MICROSCOPY FOR PLATELET COUNT
 light & phase contrast microscopy
PLATELET ESTIMATION
IMPORTANCE: for correlation for performed platelet
count
FORMULA: (total no. of platelets per field) / 10 x 20, 000
REPORTING: qualitative/descriptive reporting
Ex.: (70 / 10) x 20,000 = 140, 000 ; slight decreased
 Correlate w/ the chart
TYPE OF BLOOD SAMPLE PERFORMED
EDTA
 because it prevents platelets from adhering &
aggregating together thus appear separated
under the microscope but if examined via fresh
blood, platelet appears clump or aggregated
  DISADVATAGE OF EDTA: platelet sateletism for
patients who develop Ab against EDTA
DIRECT PLATELET COUNT REPORTING: 150 – 450 x 109/L
PROPERTIES AND FUNCTIONS OF PLATELETS
1. ADHESION: platelet to non-platelet
 Adherence to injured blood vessel
 Ability of platelets to attach/stick to non-
platelet surface. This allows platelets to adhere
to damaged endothelium.
 requires plasma von willebrand factor and gp
ib-ix
NOTE: MODE OF ACTION
When activated or discoid, they change their
morphology where their cytoplasm will have
expansions for adherence. When an injury occurs,
some of the connective tissue & smooth muscles
components enters the circulation & be exposed to
the circulation such as the collagen. Collagen is
outside the blood vessel which maintains the integrity
of blood vessels, but during injury, some of the
collagen are exposed to the plasma & platelets. The
platelets adhere on the exposed collagen via the GP
1B9 complex but GP 1B9 complex will not adhere
directly to the collagen but needs a receptor the, von
willebrand factor thus allowing adhesion to occur.
2. PLATELET RELEASE REACTION
 Alpha & dense granules release substances that
will contribute to platelet aggregation and
activation of the coagulation system
Ex.: Serotonin for vasoconstriction – when
vessels are injured vessels constrict
Thromboxane A2 form the cytoplasm of the
membrane phospholipids
ADP from the dense granules promotes
aggregation for efficient plug strength
3. PLATELET AGGREGATION: platelet to platelet
 Platelets stick to one another platelet to form
an initial platelet plug.
 Induced by stimuli such as ADP, thrombin, TxA2,
collagen & epinephrine.
 Requires Gp IIb-IIIa and plasma
fibrinogen/FACTOR 1.
 FACTOR 1 serves as the adhesive glycan or
glue of the 2 platelets together
4. VASOCONSTRICTION & CLOT FORMATION
QUALITATIVE PLATELET DISORDERS
ADHESION DEFECTS:
1. BERNARD-SOULIER: DEFICIENCY/DEFECT IN GP IB-IX
 Characterized by large platelets,
thrombocytopenioa, prolonged and Bleeding
Time.
 Platelets show normal aggregation with
epinephrine, ADP and collagen but not with
ristocetin.
2. VON WILLEBRAND’S DISEASE
Aggregation test: same with Bernard-Soulier
PLATELET AGGREGATION STUDY TEST
 Platelets + agonist (aggregating agents) =
stimulate aggregation
RESULT: BOTH AGGREGATES TO: ADP, collagen &
epinephrine
BOTH DO NOT AGGREGATES TO: Ristocetin
AGGREGATION DEFECTS
1. GLANZMANN’S THROMBASTHENIA:
DEFICIENCY/DEFECT IN GP IIB-IIIA
 Platelets show normal aggregation with
ristocetin but not with epinephrine, ADP nor
collagen.
2. AFIBRINOGENEMIA (severe/absent),
HYPOFIBRINOGENEMIA (decrease) OR
DYSFIBRINOGENEMIA (defective)
RELEASE DISORDER/GRANULE DEFECTS/STORAGE
GRANULE DEFECT
1. Gray platelet syndrome – deficiency in Alpha
granules where platelets appear large pale or gray
or pale blue wherein platelet must be blue or purple
2. Wiskott-aldrich syndrome – deficiency in dense
granules that appears as smallest platelets
3. Chediak-higashi – deficiency in dense granules
4. Hermansky-pudlak syndrome – deficiency in dense
granules
QUANTITATIVE PLATELET DISORDERS
THROMBOCYTOPENIA: (<150, 000)
1. Decreased production – BM is ineffective in
producing platelets due to:
a. Megakaryocyte Hypoproliferation
b. Ineffective thrombopoiesis
 c. Marrow replacement/myelopthesis – BM space
is replaced by abnormal cells (cancer, leukemic
or myeloma cells)
2. Dilutional – related to massive blood transfusion
which is the transfusion of >1.5 L blood per 24 hr.
The available circulating platelet of the patient will
be diluted by transfused blood. If transfused blood
is not fresh do not have viable platelets.
 Outside the body platelets lifespan is only 3
days
3. Increased destruction (in vivo)
a. Immune – presence of Ab mostly IgG such as in
immune thrombocytopenic purpura
ITP – caused by autoimmune Ab destroying the
platelets
b. non-immune – no Ab implicated such as in
abnormal activation of platelets (thrombotic
thrombocytopenic purpura)
TTP – platelets are consumed as they are
converted into platelet aggregates or thrombus
leading to decreased in platelet count
4. Splenic sequestration – in cases of splenomegaly,
platelets will be sequestered more in spleen than in
circulation thus decreased platelet count
5. Acquired
a. Uremia – increase in NPN thus damaging cells
b. Paraproteinemias - increase in abnormal
protein
THROMBOCYTOSIS/THROMBOCYTHEMIA
1. Primary Thrombocytosis – excessive production is
the main defect
 Myeloproliferative diseases – disorder of the
bone marrow that results to excessive
production of blood cells such as in cases of
polycythemia vera which is excessive
production of all blood cells ; uncontrolled
production caused by the permanent damage of
bone marrow
 Essential Thrombocytosis – excessive
production of abnormal platelet
2. Secondary/Reactive thrombocytosis – reaction to
another condition ; irreversible
 hemolytic anemias
 hemorrhagic blood loss anemia
BOTH: body will compensate for the loss of
blood by causing splenic mobilization wherein
the 1/3 in the spleen will move into the
circulation as a compensation thus transiently
increasing platelets but returns to normal after
a few hours
 splenectomy
 SPECIAL HEMATOLOGY PROCEDURES
TEST FOR SICKLING HEMOGLOBINS
 SICKLE CELLS are distorted cells appearing as
thin, dense and elongated cells with both ends
pointed. SICKLING is caused by the
PRECIPITATION of an ATYPICAL HEMOGLOBIN
(commonly hemoglobin S) which DISTORTS the
RED BLOOD CELLS into a SICKLE or CRESCENT
SHAPE. HEMOGLOBIN S is fully soluble when
oxygenated but becomes insoluble when the
oxygen level is decreased. It first POLYMERIZES
then forms into CRYSTALS which cause the red
cell to become RIGID.
SCREENING TESTS
 PRINCIPLE: HbS forms an insoluble tactoid
crystals when oxygen supply to the red cell is
decreased. When Hb S is deoxygenized outside
the cell into the plasma it will cause the solution
to be TURBID.
 Positive result:
o Sickle RBC
o Turbidity
 Degree of sicking or turbidity depends on the
concentrstion of HbS in the red cell is decreased.
HB S in the RBC
1. SEALED WHOLE BLOOD METHOD (SCRIVER &
WAUGH)
 PRINCIPLE: RBC take on a SICKLE-LIKE SHAPE
when oxygen supply to the red cell is decreased.
HbS forms INSOLUBLE TACTOID CRYSTALS when
exposed to LOW OXYGEN TENSION.
 PROCESS: Perform IN VIVO. Using a RUBBER
BAND, the rubber band is TIED at the BASE of the
FINGER or at the BALL of the finger intended to
be prick & maintain it for at least 5-10 minutes
to cause obstruction in order to obstruct the
circulation thus decreasing the oxygen & Hb S
will form into crystals. Prick the deoxygenated
finger & drop into the center of the slide & cover
w/ cover slip. Seal the sides w/ petroleum jelly or
paraffin to prevent entry of atmospheric O2
which contains abundant O2 which may cause
the sickled cell to revert to normal shape.
Incubate for 1 hour at room temperature &
examine microscopically if still none, incubate
for 2 hours until 3rd hour. Perform interval
microscopic examination.
 POSITIVE RESULT: 1. Sickle RBC (Hb S) but does
determine if heterozygous AS or homozygous SS
 REPORTING: positive or negative
NOTE:
 Degree of sickling depends on the
concentration of the Hb S in the RBC
 Readings are made at an hourly interval for 2-
3 hours
 POSITIVE RESULT is diagnostic but does not
distinguish hb s trait & hb s anemia
2. SODIUM METABISULFATE METHODS (DALAND
& CASTLE)
 REAGENT: 2% Sodium metabisulfide or sodium
bisulfate which are reducing agent which bind &
remove O2 from the blood
 PRINCIPLE: a drop of blood is mixed w/ a drop
of 2% sodium metabisulfite (a reducing agent)
on a slide, & the mixture is sealed under a
coverslip. The hemoglobin inside the RBCs
becomes deoxygenated causing polymerization
& the resultant sickle cell formation
 PROCESS: add a drops of blood & the reagent
into the center of the slide. Cover the slide w/
coverslip & seal the sides. Observe
microscopically.
 POSITIVE RESULT: sickled RBC deoxygenated by
reducing agent
3. DITHIONITE SOLUBILITY TUBE TEST
 REAGENT: sodium hydrosulfite (dithionite)
removes O2
 SPECIMEN: whole blood w/ EDTA heparin or
sodium citrate in order to prevent clotting
 PRINCIPLE: Red cells are lysed by saponin
allowing Hb to escape. Sodium dithionine binds
& removes oxygen from the test environment.
Hb S polymerizes in the deoxygenated state &
forms a precipitate in a high-molarity
phosphate buffer solution. The tactoid refract
or deflect light & make the solution turbid.
 PROCESS: anticoagulated whole blood + sodium
dithionite. Saponin lyses the RBC releasing now
Hb. Hb binds w/ sodium dithionite which
removes O2 from solution causing Hb to
crystalizes but polymerization happens outside
 the RBC thus it will not cause sickling but
turbidity of solution & is determined by
refraction & deflection of the solution in the
presence of light. Observe specimen for
turbidity by holding the tube 2.5 cm in front of a
newsprint or a card reader w/ thin black line
 RULE: if the solution is observed in the solution
then the solution is not turbid thus negative
result but if lines are no longer visible then it is
positive for tactoid crystals & sickling.
 POSITIVE RESULT: turbidity
NOTE:
 Turbidity indicates presence of sickling Hb
regardless of any genotype
 Screening method of choice
4. HEMOCARD Hb A & S
 PRINCIPLE: Hb A & S contains monoclonal Ab
(IgG), which specifically bind to the amino acids
at or near the 6th position of the B-chain of the
Hb S & A
NOTE:
 Hb S monoclonal Ab will react w/ the B-chain
of Hb S but not w/ the Hb A
 Hb A monoclonal Ab will react w/ the B-chain
of the Hb A but not w/ Hb S
 Abs are adsorbed to suspended metal sol
particles giving the reagent raspberry-like
color
NOTE:
 After electrophoresis, densitometry is
performed to determine the density of the
bands created by the migrating proteins
converted into concentration.
 In cellulose Acetate, at an alkaline pH, Hbs are
negatively charge thus they migrate towards
the anode. Hb S together w/ D & G upon
migration appears as a band of protein thus
densitometry is performed to determine the
concentration of Hb S.
 On Citrate Agar, at an acidic pH, Hb S migrates
towards the anode thus citrate agar is more
confirmatory.
RETICULOCYTE COUNT AND ITS ASSOCIATED
CORRECTIONS
RETICULOCYTES
 Are young erythrocytes that are in a discrete,
penultimate phase of maturation where the
nucleus has been removed, however, some of
the extranuclear RNA remains in the cytoplasm.
 The term “reticulocyte” is derived from the fact
that the cell contains a small network of
basophilic materials called reticulum which is
demonstrable only by supravital stain.
Reticulocytes is the 5th stage of erythropoiesis
having no nucleus as well as contains remnants
of RNA. The presence of RNA is used to
determine presence of reticulocyte which is
stained w/ supravital stain which will precipitate
the RNA into granulofilamentous particles.
 In the peripheral blood using Romanowsky stain,
they are called as polychromatophilic
erythrocytes. Reticulocyte count and its
associated corrections can be used to assess
bone marrow erythrophoietic activity. Wherein
BM is the main organ that produces
erythrocytes.
 PRINCIPLE: The ribosomal RNA is stained
supravitally. Any non-nucleated RBC that
contains 2 or more blue-stained
particles/granulofilamentous materials, is
counted as reticulocyte.
 PROCEDURE: In a test tube, add an equal
number of blood with supravital stain (new
methylene blue or brilliant cresyl blue or janus
green for granulofilamentous staining). Incubate
for 10-20 minutes inside of an incubator to
enhance the staining because staining of living
cell is longer because they resist staining due to
their intact membrane. After incubation, remix
the sample because the reticulocyte has a low
specific gravity thus they tend to float in the
solution. After, transfer a drop into a slide &
prepare smear.
 METHODS OF STAINING: Wet method and Dry
Method (schilling’s method)
METHOD OF COUNTING:
A. LIGHT MICROSCOPE: DIRECT COUNT
 Reticulocytes are enumerated among 1000
RBCs in areas where RBCs are close but not
 overlapping and reticulocytes appear to be well
stained.
 Formula:
RETIC % = (No. of Retics / 1000 RBCs observed) x 100
B. MILLER DISC METHOD
 Miller Disc is a small disc, connected to one of
the ocular
 The calibrated Miller disc appears in the field
with 2 squares: a large square and a small square
inside the large square which is 1 /9 the size of
the larger square which can be located anywhere
 Reticulocytes are counted in the large square
while RBCs are counted in the small square in
successive fields on the film until a total of 500
RBCs have been counted
 RULE: include in the count the reticulocyte
present in small squares
FORMULA:
𝒕𝒐𝒕𝒂𝒍 𝒏𝒐. 𝒐𝒇 𝒓𝒆𝒕𝒊𝒄𝒔 𝒊𝒏 𝒕𝒉𝒆 𝒍𝒂𝒓𝒈𝒆 𝒔𝒒𝒖𝒂𝒓𝒆
Retic (%)= × 𝟏𝟎𝟎
𝒕𝒐𝒕𝒂𝒍 𝒏𝒐.𝒐𝒇 𝑹𝑩𝑪𝒔 𝒊𝒏 𝒕𝒉𝒆 𝒔𝒎𝒂𝒍𝒍 𝒔𝒒𝒖𝒂𝒓𝒆 ×𝟗
REFERENCE VALUE:
ADULTS: 0.5 - 1.5 %
NEWBORN: 2.5 - 6.5 %
NOTE:
 Counterstain (wrights or giemsa) is not
advisable because it may destroy the image
 Reticulocyte have lower specific gravity
 Increased glucose inhibits staining thus
granules may not be seen when observed.
INCLUSIONS CONFUSED WITH RETICULOCYTES
 Pappenheimer bodies – iron containing
granules ; stain w/ Romanowsky or Prussian
blue stain (siderotic granules)
 Howell-Jolly bodies – appears singly ; stain
smear w/ Romanowsky to differentiate
 Heinz bodies – appears pitted golf ball ; stain
w/ supravital stain
 Hb H – appears at the periphery attached to
the membrane of RBC ; stain w/ supravital
stain
 Artifact – if stained smear is stored longer it
may create into precipitate which may
deposit in the surface of RBC & be mistaken
as the granules thus filter the stain & move
the fine adjustment. Artefacts when you are
out of focus appear as refractile, while
granulofilamentous particles will disappear.
ASSOCIATED CORRECTION
ABSOLUTE RETICULOCYTE COUNT
 Actual no. of reticulocytes in 1 L of whole blood
o ARC = Retic% / 100 x RBC count =
(_x1012/L) x 1000
o EXAMPLE: (1.5% / 100) x 4 000 000 = 60,
000
o REFERENCE VALUE: 25, 000 – 75, 000
CORRECTED RETIC COUNT / RETICULOCYTE INDEX
 This corrects the reticulocyte count to a normal
Hct to allow correction for the degree of patient
anemia. The percentage of reticulocytes may
appear increased because of early release into
the circulation or because of a decrease in the
no. of mature RBC in the circulation
o CRC: Retic % x (Hct (L/L) / 0.45 L/L)
0.45 L/L = normal mean Hct
o REFERENCE VALUE: 1% (depends on the
degree of anemia)
RETICULOCYTE PRODUCTION INDEX/SHIFT
CORRECTION (RPI)
 More appropriate test for determining efficiency
of BM compensation for anemia
 General indicator of the rate of erythrocyte
production increase above normal in anemias
meaning the previous production rate increases
for compensation for blood loss
 Index calculated to correct for the presence of
shift reticulocytes that otherwise may falsely
elevate the visual reticulocyte count.
 Done if shift cells/stress reticulocyte are present
& computing it
 RULE: the more severe the anemia, the higher
the production must be to normalize the
condition
o RPI = Corrected Reticulocyte count /
Maturation time (days)
Maturation Time: depends on the
patient’s hct
o REFERENCE VALUE: 1 maturation time if
hct is 0.45
 RETICULOCYTE MATURATION
HCT TIME (DAYS)
40-45% 1.0
35-39% 1.5
25-34% 2.0
15-24% 2.5
<15% 3.0
NOTE:
 Reticulocyte mature in circulation after 1 day
but in the presence of anemia the bone
marrow has to release even the immature
reticulocyte. Immature reticulocyte stay
longer in the circulation
 RULE: the shorter the immature reticulocyte
spent in the bone marrow, the longer they are
in the circulation
 RULE: the lower the Hct, the longer the
maturation time because of the severity of
anemia
CLINICAL SIGNIFICANCE OF RETICULOCYTES
METHODS:
 Increased in hemolytic & hemorrhagic anemias
OSMOTIC FRAGILITY TEST (OFT)
 OFT measures the ability of the RBCs to take in
fluid without lysing. As the cells take in water
(hypotonic solution), they become bloated or
swollen. If the cell is normal or biconcave disk, it
can take in more water w/out lysis because the
biconcavity can be distended w/out lysing but
the cell is already a spherocyte, its volume is
occupied thus as it takes more water, it will
immediately hemolyzed. It Reflects RBC shape
and size (surface area to-volume ratio). This is
used to detect spherocytosis, because
spherocytes rupture in saline concentrations
near the normal level. It may also detect target
cells, which, owing to their reduced hemoglobin
content, are able to withstand osmotic stress
and rupture only at very dilute saline
concentrations.
 IMPORTANCE: Diagnosis of Hereditary
Spherocytosis (HS) and other hemolytic anemias
associated with spherocytes.
CLINICAL SIGNIFICANCE OF OFT
 ABILITY OF CELL W/OUT LYSING IS AFFECTED
BY: shape & size of the RBC, which in turns
depends on the volume, surface area wherein a
hypochromic cell has a wider area than
spherocytic cell and functional state of the cell
membrane.
 SPECIMEN: Heparinized blood or defibrinated
blood because other anticoagulants alter the pH
of the blood
o Blood sample should be obtained with
minimum trauma and stasis thus upon
collection of the blood there must be no
hemolysis
o Ensure uniform size of blood drops
NORMAL RESULT:
a. Incomplete hemolysis: 0.45 % saline, solution
w/ pinkish tinge & an intact button of RBC at
the bottom upon centrifugation
b. Complete hemolysis: 0.35 % saline, red
solution w/out an intact button at the bottom
Note: Spherocytes show hemolysis as early as 0.60%
saline indicating prone to lysis or fragility
1. SANFORD METHOD
 Whole blood is pipetted to each of a series of
hypotonic saline solutions of graduated
concentration.
 Visual observation
 (+) result: Hemolysis
2. DACIE’S METHOD
 Uses hypotonic saline buffered with PO4 at ph
7.4 Hemolysis is determined
spectrophotometrically at 540 nm
 If the degree of spherocytosis is mild the degree
of hemolysis is not detected at first thus incubate
for hours to determine presence of hemolysis
 (+) result: Hemolysis
NOTE:
 INCUBATED OFT: sterile incubation at 37C for
18 – 24 hrs to detect mild forms of hereditary
spherocytosis.
a. INCREASED OFT (cells are fragile): Hereditary
spherocytosis; red cells with abnormal
membrane; severe G-6-PDH deficiency;
Pyruvate kinase deficiency; hemolytic anemias
 characterized by abnormal membrane or
enzyme deficiency
b. DECREASED OFT (cells are resistant to lysis due
to enough surface area to volume ratio thus
these are hypochromic cells & target cell): Sickle
cell anemia, severe Iron deficiency anemia,
thalassemia
o Sickling is reversible, if a sickle cell
intakes water it reverses back to its
normal shape, thus ability of sickle cell
to react in a hypotonic solution will
depend on the degree of sickling
ERYTHROCYTE SEDIMENTATION RATE
 Measures the degree or rate of settling of
erythrocytes at the bottom of the tube in
plasma in an anticoagulated whole-blood during
a specified period of time (1 hour)
IMPORTANCE:
1. Non-specific measure of inflammation
o The main factor affecting the rate of
settling is the presence of varied plasma
protein such as acute phase proteins
produced during inflammation
2. It is a good index for the presence of a hidden but
active disease like in the case of inflammation &
tuberculosis
3. A hematology test to indicate inflammatory
response to tissue injury however it does not
determine the severity of inflammation
4. Follow the progress of certain disease but not
identify the severity of the condition
5. It measures the suspension ability of the RBCs
6. It measures the abnormal concentration of
fibrinogen and globulin
STAGES OF ESR:
1. LAG PHASE/INITIAL ROULEAUX
 occurs during the first 10 minutes & the settling
is slower but after the first 10 minutes where
they form in rouleau, RBC sediment faster
 ROULEAU – cells in apposition to one another w/
their flat surface
2. DECANTATION PHASE/ PERIOD
RAPID/CONSTANT SETTLING
 occurs within 40 minutes.
3. FINAL SETTLING/PACKING
 occurs during the last 10 minutes settling slower
at the bottom of the tube
FACTORS AFFECTING SETTLING OF RBC
1. ERYTHROCYTE FACTORS
 RBC SIZE OR MASS: directly proportional to ESR
or rate of settling
 RULE: the larger, the faster is the settling & the
smaller RBC is, the slower they settle
 NUMBER OF RBC: inversely proportional
o In anemia where there are only few
cells present thus more space to settle.
In anemia, lesser cells, faster settling. In
polycythemia vera, cells are crowded
thus settling is slower.
 PRESENCE OF ANISOCYTES & POIKILOCYTES:
slower settling due to no rouleaux formation
wherein these cells are abnormal in shape &
size
2. PLASMA FACTORS
 PLASMA VISCOSITY: inversely proportional to
ESR meaning the more viscous the plasma is, the
slower is the rate of settling
o The most important factor which affects
the rate of settling is the plasma
composition
 PLASMA COMPOSITION: increase fibrinogen, α-
1 globulin, α-2 globulin: increase ESR thus
altering the zeta potential of cells causing
formation rouleaux
o increase albumin & lecitihin: decreases
ESR due to rouleaux formation inhibition
3. MECHANICAL/ TECHNICAL FACTORS
 POSITION OF THE TUBE: should be exactly
straight vertical because a slight tilting of the
tube 3 degree causes 30% error thus faster
settling
 INCUBATION ENVIRONMENT: should be free
from any movement or vibration at room
temperature however fluctuation caused
vibration causes a significant error because
vibration causes more cells settle at the bottom
OF
 INCUBATION TEMPERATURE: should be at room
temperature
 LENGTH AND DIAMETER OF THE TUBE: the
longer & wider the tube, the faster is the ESR due
 to increase pressure & wider space for cells to ESR: WESTERGREN METHOD
settle down AGE MALES FEMALES
 ANTICOAGULANT: should not be over- <50 y/o 0-15 mm/hr 0-20 mm/hr
anticoagulated (EDTA or citrate) of causes slower >50 y/0 0-20 mm/hr 0-30 mm/hr
ESR because cells are shrunken which may not >85 y/o 0-30 mm/hr 0-42 mm/hr
form into rouleau Children: 0-10 mm/hr
 TIME OF SETTING UP: first 2 hours or 6th hour CLINICAL SIGNIFICANCE OF ESR
from collection because beyond that cells will a. INCREASED ESR IN DUE TO INFLAMMATION OF
swell thus no rouleaux formation THE TISSUES
 Inflammations; acute & chronic infections,
ESR METHODS tuberculosis, multiple myeloma
1. WINTROBE & LANDSBERG METHOD  Rheumatic fever, rheumatoid arthritis,
(WINTROBE TUBE) myocardial infarction, nephrosis
 WINTROBE TUBE: glass tube w/ thick walls  SBE, Waldenstrom’s macroglobulinemia,
where one end is open & the other is close & hepatitis, menstruation, pregnancy
feeding of blood is done manually, w/ 2
graduation 10 on top & 0 at the bottom (left) b. DECREASED OR NORMAL ESR IN
for microhematocrit reading while for ESR it is 0  Polycythemia due to many blood cells,
on top & 10 on the bottom (right). Internal spherocytosis, conditions associated with Hb S
diameter is 3 mm & total height is 11 cm but & Hb C disease
column of blood is 10 cm  TO DETERMINE DECREASED ESR: if cells do not
 MATERIAL: wintrobe tube, Pasteur pipet w/ settle at all
thin needle pipet
 ANTICOAGULANT: Ammonium-Potassium OTHER METHODS OF ESR
Oxalate or wintrobe mixture or double oxalate 1. GRAPHIC-CUTLER METHOD: uses 3.8% sodium
citrate and Cutler tube
2. WESTERGREN METHOD (WESTERGREN TUBE) 2. LINZENMEIER METHOD: uses 3.8% sodium
 WESTERGREN TUBE: plastic or glass tube, both citrate and Linzenmeier tube
ends are open. Length is 200 while the blood 3. MICRO-LANDAU METHOD: uses 5 % sodium
column is 200 mm. The internal diameter is 2.65 citrate
(+- .15) 4. SMITH MICRO METHOD: uses 5 % sodium citrate
 MATERIALS: Westergren tube w/ stopper, 5. CRISTA OR HELLIGE-VOLLMER METHOD
commercial tube w/ anticoagulant, Westergren 6. ROARKE-ERNSTENE METHOD: uses heparin
rack 7. BRAY’S METHOD: 1.3% sodium oxalate
 ANTICOGULANT: Original Westergren = 3.8% NEW ESR METHOD
sodium citrate 1. Automated Erythrocyte Sedimentation Rate
 Modified Method = 2 ml of EDTA- a. Ves-Matic system – based on optoelectronic
anticoagulated blood with 0.5 ml NSS or 3.8% sensor wherein it measures the change in
sodium citra as diluents in order to adjust hct opacity of a column of blood as
 REFERENCE: adjust hct 0.35 sedimentation occurs
 PROCESS: Westergren tube is pushed into a b. Sedimat 15 – based on infrared
bottle of blood, the blood will move up via measurement wherein tubes are slightly
capillary action making sure that the blood is inclined thus the normal values are also
properly mixed via inversion & incubate for 1 adjusted
hour in ESR rack by letting it stand on a c. ESR STAT PLUS system - based on how fast
Westergren rack cells settle upon centrifugation for pediatric
 REPORTING: mm/hr or mm patients because it requires smaller samples
o More accurate if hct is adjusted to 0.35
to limit effect of anemia on RBC
 ZETA SEDIMENTATION RATIO (ZSR)
- Uses zetafuge which applies controlled
centrifugation of blood, producing alternating
compaction and dispersion of erythrocytes to
measure how closely the erythrocytes approach
one another under a specific standardized
gravitational force
- Also uses small amount of blood due to a special
capillary tube, 75 mm in length with an internal
diameter of 2 mm
- After centrifugation, Zetacrit% is determined at
the “knee” of curve to determine zeta
sedimentation ratio
FORMULA:
𝒉𝒄𝒕 %
ZSR (%) = × 𝟏𝟎𝟎
𝒁𝒆𝒕𝒂𝒄𝒓𝒊𝒕 %
REFERENCE VALUE:
(ZSR): 41 – 54%
ZETA SEDIMENTATION RATIO
51-54% Borderline normal
55-59% Mildly elevated
60-64% Moderately elevated
>65% Markedly elevated
ADVANTAGES OF ZSR
 It requires a smaller amount of blood
 It is not affected by anemia and is faster than
other methods
 Reference range is the same for both sexes
 Has only 1 reference value
 Faster due to involvement of centrifugation
ABSOLUTE EOSINOPHIL and BASOPHIL COUNTS
 DILUTION FOR EOSINOPHIL & BASOPHIL: 1:10
to increase observation because the higher the
dilution the fewer the cells seen
 DILUTING FLUID FOR EOSINOPHIL GRANULES:
o Phloxine – stains eosinophil red
o Propylene glycol – lyses RBC
o Heparin – inhibits leukocyte clumping
o Na Carbonate – enhances eosinophil
granule staining & hemolyze other WBC
o ALTERNATIVE STAINS: Pilot’s solution &
Randolph’s stain
NOTE:
ABSOLUTE EOSINOPHIL COUNT:
 FORMULA: Absolute count x WBC count
WBC COUNT: 9000/ uL
DIFFERENTIAL COUNT:
 NETROPHIL: 0.60
 LEUKOCYTE: 0.30
 MONOCYTE: 0.08
 EOSINOPHIL: 0.02
NOTE:
 There are some cells that needs to be counted
directly such as the eosinophil & basophil
 We rarely see eosinophil & very rare for
basophil thus performing count directly &
manually is necessary
THORN TEST (Test for adrenocortical functions)
- Does not determine bone marrow function but
adrenal cortical function
- Perform direct absolute eosinophil count w/
fasting sample w/ ACTH injection. After,
introduce ACTH in order to stimulate the adrenal
gland to produce corticosteroid hormones which
will cause the circulation to leave the circulation
& proceed towards the tissues. After 4 hrs., draw
second specimen for absolute eosinophil count.
After 4-8 hours ACTH injection, when the adrenal
gland reacts it will produce corticosteroid
hormones causing now eosinophil to proceed to
the tissues thus giving a lower count. If the
adrenal gland function is normal it will be able to
cause increase production of adrenal hormones.
- NORMAL: 2nd count should at least be 50%
lower than initial count
o If 1st & 2nd count is the same meaning
adrenal gland is not functioning causing
hypoadrenalism
ABSOLUTE BASOPHIL COUNT
- uses Cooper and Cruickshank stain (neutral red)
- COUNTING CHAMBER: Speirs – Levy or Fuchs-
Rosenthal
- DILUTION: 1:10
LUPUS ERYTHEMATOSUS (L.E.) CELL PREPARATION
- An LE cell is a neutrophil or macrophage that has
phagocytized (engulfed) the denatured nuclear
material of another cell that has been lysed by
anti-nuclear Ab. Its formation is characterized by
 the production of a number of autoantibodies.
The most important of these are the antinuclear
antibodies which occur in the serum of patients
with some autoimmune disorders including
systemic lupus erythematosus (SLE).
Demonstration of LE cells therefore suggests the
presence of these antinuclear antibodies also
termed as the LE factor
TO PERFORM LE:
 The LE factor or antinuclear antibodies causes
nuclear lysis (lysed nucleus becomes a
homogenous amorphous mass) and this material
is then phagocytized by a neutrophil.
 Extruded nuclei is acted by antinuclear
antibodies
 The phagocytic neutrophil which has
phagocytized the lysed nucleus is now called an
L.E. cell. (seen on a buffy coat smear)
 Rosette formation a flower like formation which
has an extruded nucleus surrounded by smaller
neutrophils is NOT considered positive
PROCEDURE:
a. Collect blood w/out an anticoagulant, 10-15 mL
WB & allowed to completely clot for 1 hr at 37 C
or 2 hr at RT
b. Discard the serum to demonstrate the LE cell by
obtaining the buffy coat layer
c. Get the blood clot & macerate to extrude some
of nucleus thus if antinuclear antibodies is
present it will act on the extruded nucleus thus
lysing the nucleus
d. Macerated blood will be transferred into 3-4
wintrobe tube & centrifuge at 2550 rpm for 30
mins.
e. After centrifugation, there is a higher volume of
buffy coat
f. Remove serum to obtain buffy coat & perform
buffy coat smear
g. Stain w/ wirghts & giemsa & observed for LE cell
which is a neutrophil that has engulf a large mass
h. Phagocytic neutrophil appears to wrap into the
lysed nucleus
OTHER ANTI-NUCLEAR ANTIBODY TEST
a. FLUORESCENT TEST (has replaced the LE test)
- more accurate because it identify what kind of
nuclear Ab is produced by patient
- serological test
- Uses fluorescein-conjugated antihuman IgG
BONE MARROW STUDY
COLLECTION OF SPECIMEN
 ADULTS: posterior iliac crest (posterior
preferred or anterior) – more common because
the tissue is more thinner at the hips & sternum
 CHILDREN: tibia
PROCESS: the aspirating needle must be able to reach
the medulla
 Examination of BM requires examination of
blood film which must be collected first because
bone marrow collection requires boring
because the outer layer of bone is calcified thus
it is very painful & stressful for the patient
which causes falsely increase WBC
1. TREPHINE BIOPSY/CORE
- Utilizes the jamshidi needle for obtaining the
core to get a rounded portion of the BM tissue
thus it will not disturb the nearby tissue
- This is taken before aspiration biopsy to avoid
any disruption of marrow architecture.
- Requires small sample of BM
- Imprint biopsy is prepared by touching the
specimen on a slide.
- FIXATIVE: 5% Zenker fluid
2. ASPIRATION
- This follows immediately trephine biopsy
- Disturb the architectural pattern of cell
- 1-3 mL of marrow in a 30-35 mL syringe w/ EDTa
to prevent cellular distortion & processed within
1hr.
- NEEDLE: university of Illinois sternal needle
BONE MARROW SMEAR PREPARATION:
1. WEDGE OR COVERSLIP METHOD – 2 coverslip
method giving excellent distribution of blood
cells
2. PARTICLE SMEAR/SQUASH METHOD – on slide
place the core collected & cover w/ another slide
& squash
3. BUFFY COAT (CONCENTRATE) SMEAR – collect
in a wintrobe tube & prepare buffy coat smear
same w/ wedge smear & centrifuged for 8-10
mins. at 2800 rpm this will compensates for
 hypocellular marrow and allows for examination o HYPOCELLULAR/HYPOPLASIA:
of large numbers of nucleated cells without incomplete development in one or more
interference from fat or RBCs. lines thus it contains more fats thus less
4. HISTOLOGIC SECTION (CELL BLOCK) blood production like in aplastic anemia
o FIXATIVE: 10% formalin, Zenker glacial
MARROW DIFFERENTIAL
acetic acid, or
- include all nucleated hematopoietic cells EXCEPT
o B5 FIXATIVE STAINS: Romanowsky; iron
megakaryocytes & macrophages
stain; H and E
- at least 500 cells are counted preferably 1000
NORMAL MARROW CELLS cells, 500/slide (2 slides)

 Hematopoietic cells – stain w/ romanowsky Guidelines for adult bone marrow differential in
stain concentrated smears, 1000 cell counts
 Lymphoblasts CELL TYPE RANGE (%)
 Monocytic cells Eryhroblast 18-24
 Myelocytic cell Myeloblasts, Type I 0-1
 Erythrocytic precursors Myeloblasts, Type II 0-2
Promyelocytes 1-4
 Macrophages/ Histiocyte or osteoclast
Neutrophils & Precursors 53-63
o with storage iron – stain w/ iron stain
Monocytes 0-2
(Prussian blue stain)
Eosinophi;s & Precursors 1-3
o with lipids (Gaucher cells)– monocyte or
Basophils & Precursors 0-1
macrophage w/ cytoplasm appearing a
Lymphocytes 8-12
crinkled tissue paper because of Plasma Cells 0-2
leucocerebrocytes)
 Mast Cells / Tissue Basophil
 Osteoblasts – immature bone cells, waterbug or MYELOID ERYTHROID RATIO
comet appearance - ratio between the granulocytic precursors &
 Osteoclasts/macrophages – for bone marrow erythroid precursors but only the nucleated
resorption precursors
- on smear, count all myeloid cells & erythroid
precursor which has 3 pronormoblast &
basophilic normoblast & 4 orthochromic
normoblast & for granulocyte only the
neutrophil, eosinophil & basophil
o M:E RATIO = 2 granulocyte:1 RBC – 4:1
o AVERAGE: 3:1

SMEAR PREPARATION FOR MALARIA

MARROW CELLULARITY
- Percentage of marrow space occupied by
hematopoietic cells compared w/ fat (yellow or
red marrow)
 MARROW FAT: hematopoietic cell = 1 : 2 (adults)
 MYELOID: Erythroid (M:E) ratio = 2 : 1 - 4 : 1
 RED MARROW CELLULARITY
o HYPERCELLULAR/HYPERPLASIA:
increased in one or more cell lines w/
few fats thus increase marrow
production like in polycythemia vera
EXAMINATION: SPECIMEN
- EDTA-anticoagulated blood or fresh capillary
blood collected before the initiation of
treatment.
- At least two thick and two thin peripheral blood
films should be made.
a. THIN BLOOD SMEAR
- For morphologic examination and species
identification and determination of Percent
arasitemia
- PREPARATION: same as wedge method
 o A drop of blood w/ 2-3 mm & 1 cm away
from the end & smear via wedge method
o First fixed in methanol then add distilled
water to let RBC to hemolyze to
removed Hb then stained w/ pH of 7.2
pH due to hemozoin pH & examine
o Cells are still intact & not overlapping
thus observing morphology
b. THICK BLOOD SMEAR
- For screening purposes because cells are
distorted
- PREPARATION: place 3 small drops or large drop
of blood close together near one end of the slide.
Spread in a 25 centavo coin & dry. With one
corner of a clean slide, stir the blood for about
30 seconds to mix the three drops over an area
approximately 1 to 2 cm in diameter
o First dehemoglobinized; air-dried then
stained
c. STAINS FOR MALARIAL BLOOD SMEAR
- Wright’s; Giemsa
CYTOCHEMICAL STAINS
CYTOCHEMISTRY
- Defined as the microscopic study and
identification of chemical constituents within
individual cells. It is useful in the identification of
malignant cell types on the basis of cytoplasmic
or nuclear chemistry; cellular constituents that
are present in abnormal form or amount; lack of
cellular constituent; and cells exhibiting
functional abnormalities
LEUKEMIA
- refers to group of disorders
- In the category of cell line, leukemia can be
differentiated to either Myeloid or Lymphoid
Leukemia
- In the basis of Blast cell
o Pre-dominance of lymphoblast is called
Acute Lymphocytic Leukemia
o Pre-dominance of mature with few
lymphoblast is called Chronic
Lymphocytic Leukemia
o Acute leukemia refers to a type of
leukemia with poorer prognosis with an
increase predominance of blast cells or
immature cells
o The decrease of blast cells and increase
of mature cells is called chronic
leukemia
o Predominance of myeloblast is
associated with Acute Myeloid
Leukemia
o Predominance of mature myelocyte is
associated with Chronic Myeloid
Leukemia
- Blast cells are not easily identified because of
their common characteristics (larger in size, large
nucleus, smaller and deeply basophilic
cytoplasm), cytochemical stains are used to
differentiate in the absence of CD marker
- Acute Myeloid Leukemia can be further classified
knowing it is a stem cell
- French American British System classified
leukemia based on the morphology of the cells
after staining with Romanowski stain and their
reaction to cytochemical stain
- FAB further classified ALL as ALL1, ALL2, ALL3.
While AML is classified into seven categories
(AML1-AML7)
- In AML:
o AML1-AML3 predominance cell is
Myeloblast, promyeolocyte myelocyte
(granulocytic series)
o AML4 and AML5 uses alpha naphthyl
Chloroacetate esterase
o AML4 stain positive in both Specific and
non- specific esterase because it
contains both granulocytic and
monocytic cells
o AML5- is the acute monocytic leukemia
o AML6 is called Erythroid Leukemia,
leukemic cells are precursors of
erythrocyte. Positive in Periodic acid
Schiff
o AML7 leukemia of megakaryocytes
called acute megakaryoblastic leukemia
- One example for Chronic Lymphocytic Leukemia
is the Hairy Cell Leukemia which affects B-cells.
Hairy cells contain ACP.
o ACP isoenzyme is present in all blood
cells and can be inhibited by tartaric
acid except the isoenzyme number 5
 which is the one present in hairy cell
leukemia
- In Chronic Myeloid Leukemia the predominant
cells are the mature myeloid cells especially the
granulocytes. CML is also called Chronic
Granulocytic Cells. Granulocytes can range from
50,00-300,000
- Having a very high WBC count can also be
observed in Leukemoid Reaction which is due to
underlying condition
- Both leukemoid reaction and chronic myeloid
leukemia are characterized by a very high WBC
count. However, Leukemoid Reaction is
reversible that when the underlying condition is
resolved the WBC will return to normal count.
CLM is a permanent condition. To differentiate
Leukemoid reaction from CML, alkaline
phosphatase is used.

ENZYMATIC STAINS
Peroxidase/Myeloperoxi - The stain itself does not contain the peroxidase enzyme. The peroxidase enzyme
dase (MPO) is inside the cell, the composition of the stain is the substrate for peroxidase
enzyme
- Myeloperoxidase is a normal constituent of the primary granules of the
myelocytic cells which is observe as early as promyeolocyte
- Stain marker for primary granules & auer rods (fused primary granules)
- Stain positive in AML but negative in ALL
- Differentiates AML from ALL
- Stain marker for immature myeloid cells
- Stain positive in granulocyte but not in lymphocytes
- Positive Activity: reddish-brown deposits (in cytoplasm of granulocytes and
monocytes)
- Note: Myeloperoxidase enzyme deteriorates; Stain should be done only on fresh
specimens
Alpha-Naphthyl Acetate - Use to detect the granulocyte of monocytic origin
Esterase & Alpha- - Non specific because it can be seen in other cells
Naphthyl Butyrate - The stain does not contain esterase but rather substrate for the enzyme
Esterase (Non-Specific - If the cell contains the enzyme it will catalyze the hydrolysis of the butyrate or
Esterases/NSE) acetate in the stain
- Unfixed sample can be used so long in the dark for as long as 2 weeks
- Marker for cells of Monocytic origin
- Other cells (+) : Megakaryocytes, Platelets, Histiocytes, Plasmacytes, some T-
lymphocytes
- Positive Activity: red-brown/dark red
Leukocyte/Neutrophil - Stains NEUTROPHILS (the only leukocytes that contain this activity)
Alkaline Phosphatase - Neutrophils contain various amount of ALP
(LAP/NAP) - Principle: Differential count involving neutrophils
- Differentiates Leukemoid reaction (LR) from Chronic myeloid leukemia (CML)
- Reference Value: 30 –185 LAP score
- Increase LAP score is observed in the following:
o during the last trimester of pregnancy - Hodgkin disease
o Polycythemia vera - Multiple myeloma
 o Aplastic anemia - Obstructive jaundice
- KAPLOW’s Scoring (Count 100 cells and grade them as follows)
o 0 = no staining
o 1+ = faint & diffuse staining
o 2+ = pale moderate amount of blue staining
o 3+ = strong blue precipitate
o 4+ = deep blue or brilliant
- To compute multiply the counted neutrophils by their grade
- Normal to High indicates Leukemoid reaction
- Below than normal value to zero indicates CML
Tartaric Acid Resistant - For the diagnosis of Hairy cell leukemia (HCL)
Acid Phosphatase (TRAP) - General Principle: ACP is Detected in almost all blood cells but when treated with
tartaric acid it is inhibited except isoenzyme number 5
- Labile if unpreserved
- Sample should first be fixed and be stored at -20 C and can be used atleast 2
weeks
- Other Tartrate Resistant cells: Sezary cells; Histiocyte; T-cell of acute lymphocytic
leukemia
- Activity is indicated by purple to dark red granules in cytoplasm
Cyanide-Resistant - For identification of Eosinophilic components
Peroxidase - Positive Activity: brown

Naphthol ASD - Marker for mature & immature Neutrophil and mast cells
Chloroacetate Esterase - Substrate is the chloroacetate
(Specific Esterase) - Use to identify immature or primitive granulocyte ALM1-ALM3
- Positive Activity: bright red granules in cytoplasm
- This enzyme is stable and may last for months
Terminal - Stains DNA polymerase immunoperoxidase
Deoxyribonucleotidyl - Marker for immature Lymphoid cell
Transferase (TdT) - Positive in almost 90% cases of ALL
- Also useful in the detection of the “lymphoblastic transformation” of chronic
myeloid leukemia (CML)
- Detects blastic transformation of CML
o CML can transform into an acute leukemia
o Acute leukemia more than or equal to 30% blast cells
o CML can transform either AML or ALL. If the blast cells presence stain
positive in MPO stain and SBB it is AML while if it stain positive in TdT it
means the it transforms into ALL
Acid Phosphatase - Present in all hematopoietic cells and found in lysosomes
- Activity is indicated by purple to red granules
- Unstable, cannot be stored

NON-ENZYMATIC STAINS
Prussian Blue stain - Stains siderotic granules (for the diagnosis of sideroblastic anemia)
- Used to detect hemosiderin in urine
- Use to identify iron in the ferric state (Fe+3)
Periodic Acid Schiff (PAS) - Stain reacts w/ aldehyde groups in glycogen, mucoprotein, glycoproteins, and
high molecular weight carbohydrates
- Does not detect stains instead it combines with the aldehyde group of glycogen
- Positive to all blood cells except normal erythroblast or pronormoblast
 - Positive erythroblast indicates that it is affected by AML6
- Positive in erythroblasts in Di Guglielmo disease / Erythroleukemia
- L1 and L2 also stain positive in PAS
- The positivity of L1 to Pas is described to be chunky or block-like positivity
- L2 is not that large as L1
- Negative Activity: bright fuchsia pink (Pattern of staining varies with each cell
type)
- Also differentiates Acute Lymphoblastic leukemias
- Peroxidase of eosinophil is resistance to cyanide.
- Cyanide resistance peroxidase is used to identify peroxidase of eosinophils
Sudan Black B (SBB) - Marker for phospholipids and lipids
- Has the same staining reaction as myeloperoxidase
- Gives the same information as Peroxidase in the interpretation
- Positive in AML
- Advantage over myeloperoxidase is that it is more stable, because
myeloperoxidase detects enzyme which is somewhat labile and may disappear in
prolong storage. Sample stained in SBB can give reliable result for months
- Differentiates AML from ALL
- (+) Result: Dark purple-black granules
- Notes:
 Can be done on stored specimens.
 More sensitive than chloroacetate in the demonstration of mature &
immature neutrophils and mast cells
Toluidine Blue O - A metachromatic stain
- Binds with mucopolysaccharides in blood cells
- For the recognition of basophils and mast cells
- (+) Result: Reddish-Violet
 HEMATOLOGY 2
GLOSSARY
HEMOSTASIS
- Stoppage of bleeding
- To maintain normal fluidity of blood in vivo.
There must be a balance in hemostasis. If not
needed, the clot should not be formed, but when
needed, the clot should be formed.
- Imbalance in hemostasis system will lead to
excessive bleeding/hemorrhage or excessive
clotting or abnormal thrombosis.
- Bleeding can be external or internal, severe or
mild which is indicated by red purple or blue
black which occur in the vessels particularly in
the capillaries thus blood deposit on the skin and
causes discoloration of the skin
PETECHIAE
- purplish red, pinpoint hemorrhagic spots in the
skin caused by loss of capillary ability to
withstand normal blood pressure and trauma
- diameter of <3mm
PURPURA
- produced by hemorrhage of blood into small
areas of skin, mucous membranes, and other
tissues
- Red-purple-brownish yellow
- diameter of >3 mm but <1 cm
- larger than petechiae or a combination of
petechiae
- simultaneous rupture of capillaries
ECCHYMOSIS/ BRUISE
- a form of purpura in which blood escapes into
large areas of skin or mucous membranes, but
not into deep tissue
- Black/blue – greenish brown or yellow
- diameter of >3 cm
- due increase rupture of capillaries but not on the
visceral organs but directly on the skin
NOTE:
All of these are caused by the breaks of small vessels
particularly the capillaries.
CAPILLARIES
- smallest blood vessels w/ the thinnest lining or
walls thus easily ruptured which may be caused
by increase in blood pressure/hypertension
leading to discoloration of the skin depending on
the amount of blood that deposited on the skin
NOTE:
Discoloration of the skin depends on the volume of
the blood.
COLOR TRANSITION
- bluish black if blood volume is greater or bluish
reddish/purplish to greenish to dark brown or
greenish brown to yellow
NOTE:
 Changes in the color of skin is associated with
the degradation of Hb wherein Hb is degraded
into globin & heme wherein heme is further
broken down into iron & protoporphyrin &
protoporphyrin is oxidized into biliverdin
(green) & further converted into bilirubin
(yellow)
 Petechiae, purpura & ecchymosis are
indication of primary hemostasis
POSSIBLE CAUSES OF RUPTURE OF CAPILLARIES:
1. Increase in blood pressure or hydrostatic
pressure
2. Pressure exerted outside
DISORDERS AFFECTING THE INTEGRITY OF CAPILLARIES:
1. Platelet abnormalities
2. Blood vessel wall abnormalities
NOTE:
 Even during normal situations, there are gaps
formed in between the endothelial lining
which is made up of connected endothelial
cells
 Whenever gaps formed, platelet functions to
aggregate to plug the gaps in order to
maintain continuity of blood vessel thus
limiting blood loss. Upon aggregation,
platelets are consumed leading to increase
appearance of petechiae or platelet may not
 HEMATOLOGY 2
properly perform its function due to platelet
abnormalities
 Petechiae and Purpura may occur without
platelet & blood vessel wall abnormalities
EPITAXIS- nosebleed
HEMARUTHROSIS
- leakage of blood into a joint cavity
- bleeding in the joints resulting to clot formation
causing obstruction in the movement causing
damage to the affected joint leading to crippling
which occur in severe hemocoagulation
deficiencies such as hemophilia
HEMATEMESIS
- vomiting of blood
- content of esophagus or stomach which is acidic
(HCl)
- brown color blood due to reaction of blood to
HCl
- pH of <7.35
HEMOPTYSIS
- expectoration of blood secondary to
hemorrhage in the larynx, trachea, bronchi, or
lungs
- coughs out blood
- red color w/ pH of 7.35 – 7.45
SPUTUM- product of the goblet cells of the lungs
HEMATOMA
- a swelling or tumor in the tissues or a body cavity
that contains clotted blood
- clotted blood with swelling
TRANSFIXATION- through & through of needle causing
hematoma
THROMBUS/CLOT
- In vivo blood clot causing vascular occlusion and
tissue ischemia
- composed of mostly platelets occurring in the
arteries causing arterial thrombosis or fibrin & a
small amount of platelet occurring in vein
causing deep vein thrombosis wherein both
causes obstruction.
DEEP VEIN THROMBOSIS
- vaso-obstruction causing the amount of blood
flow to be limited & the tissue will suffer from
ischemia due to decrease O2
EMBOLUS/EMBOLISM
- some parts of the clot detach & there is increase
in thrombus causing increase in pressure causing
the lumen to be smaller & the heart
compensates causing increase blood output &
pressure leading to rupture & detachment of clot
& the detach clot may obstruct smaller vessel
THROMBOSIS- formation, presence of a clot in a blood
vessel
 2 TYPES:
o PHYSIOLOGIC – if activation of
coagulation system to form a thrombus
is normal
o PATHOLOGIC – inadvertent formation of
blood clot
NOTE:
CAUSES OF PATHOLOGIC THROMBOSIS:
 TTP
 Cholesterol Plaque formation seen in DM
which thickens causing the vessel to be
sclerotic resulting to less capacity of the vessel
to dilate & constrict thus tissue reaction occur
causing obstruction leading to slower
circulation & this increases platelet activation
& will form into thrombus
 Varicose veins / superficial thrombosis
 Deep veins thrombosis
 Arterial thrombosis
HEMATURIA
- presence of intact red cells in the urine or simply
blood in urine
- red with intact RBC thus presence of turbidity or
smoky red
HEMOGLOBINURIA- presence of hb in the urine with
clear red color.
 HEMATOLOGY 2
METHEMOGLOBINURIA – methemoglobin in urine or
oxidized Hb in urine due to storage at room temperature
giving a color of chocolate brown urine
MENORRHAGIA – excessive menstrual bleeding
MELENA
- Stool containing dark red or black blood due to
oxidized Hb.
- Bleeding in the upper GIT such as in stomach or
upper part of small intestine.
HEMATOCHEZIA
- The passage of blood in feces.
- Red color bleeding in lower GIT such as in rectum
or colon.
OTHER CAUSES OF RED COLOR IN STOOL
 Tomato, red beets, dragon fruit, diet
 NOTE:
o the passage of blood in feces
o red color bleeding in lower GIT such as in
rectum or colon
HEMOSTASIS IS A COMPLEX MECHANISM THAT:
1. Retains the blood within the vascular system
during periods of injury.
- vessels constrict/ vasoconstriction/vasospasm
thus limiting blood passage leading to limited
blood loss
2. Localizes the reaction involved to the site of
injury.
- Platelet plug formation by first adhering of
platelet & will release their content & will
aggregate causing plug formation thus stoppage
of bleeding initially.
3. Repairs and re-establishes blood flow through
the injured vessels
- Fibrin clot formation by converting fibrinogen
into fibrin causing fibrin clot formation which will
stabilized the plug formation by forming a
meshwork.
- Promoted by thrombospondin & platelet derived
factor for smooth muscle & connected tissue
muscle repair thus dissolving fibrin.
2 STAGES OF HEMOSTASIS
a. Primary – consist of vasoconstriction & plug
formation
 Components: blood vessels (endothelial lining),
platelets
b. Secondary – consist of fibrin clot formation
 Component: Coagulation factors
(Fibrinogen/CF1 converted by thrombin from
prothrombin wherein the conversion of
prothrombin to thrombin is caused by CF10a, 5a,
Ca & PL to fibrin), Inhibitors (deactivates further
action) & Fibrinolytic proteins
PLASMIN/FIBRONOLYSIN – dissolves/lysis clot
FIBRINOLYSIS – process of dissolution of clot
PROCOAGULANTS- Promoters of coagulation
 Components: clotting factor for fibrin,
phospholipids, platelets for plug formation
ANTICOAGULANTS- Prevents excessive formation of
platelet plug
 Components: Natural inhibitors (ATIII, proteins-
C & S), Fibrinolysis
RESULT OF BOTH:
a. Thrombosis – increase procoagulant, decrease
anticoagulant
b. Hemorrhage – decrease procoagulant, increase
anticoagulant
CAPILLARY FRAGILITY TEST
- This test measures the ability of small capillaries
to retain blood when subjected to increased
hydrostatic pressure and anoxia.
- It is a non-specific evaluation to measure
capillary weakness and deficiencies in platelet
number and function meaning when there is
abnormal platelet number, there is abnormal
CFT this is due to thrombocytopenia or when
there is abnormal plt count but normal plt
function thus abnormal CFT
- Decreased capillary resistance causes the
capillaries to rupture which leads to bleeding
and formation of petechiae.
 HEMATOLOGY 2
- Requires exposure to increase hydrostatic
pressure & anoxia by applying a positive
pressure via BP apparatus or Tourniquet using
Rumpel-Leede Tourniquet Test or Negative
pressure from the outside using suction cup
- Measures fragility or stability of capillaries as
well as a non-specific evaluation of platelet
number & function
RESULT: petechiae
RUMPEL-LEEDE TOURNIQUET TEST
1. Examine the forearm, hand, and fingers to make
certain that no petechiae are present.
2. With a blood pressure cuff, apply 100 mmHg
pressure to upper arm. To those who do not
have a blood pressure cuff, use a tourniquet or
rubber/cloth strip instead. Apply the tourniquet
not too tight, not too loose to employ just
enough pressure.
3. Maintain pressure for 5 minutes.
4. Release cuff and wait for 5 – 10 minutes before
making a final reading.
5. Examine the forearm, hands and fingers for
petechiae.
NOTE:
Disregard any petechiae within ½ inch of the
blood pressure cuff (tourniquet) because this
may be due to pinching of the skin by the cuff.
6. Count the number of petechiae and roughly
interpret as follows:
1+ A few petechial on the anterior part of the
forearm
2+ Many petechial on the anterior part of the
forearm
3+ Multiple petechial over the whole arm and back
of the hand
4+ Confluent petechial on the arm and back of the
hand
HEMOSTASIS
VASCULAR SPASM
- very first reaction to injury
- function of blood vessels
NOTE:
 Vascular spasm & platelet plug formation are
under the primary hemostasis while the clot
formation is under the secondary hemostasis
 The primary hemostatic response towards
injury is immediate but since the platelet plug
is unstable the effect is short term
 Secondary hemostatic response is more
complex but the response is quite delayed but
once the clot is formed & is stabilized it will be
efficient in effect which is long term
 In primary hemostasis, the platelets clump
together & in secondary hemostasis, fibrin
monomers are formed & connected together
or polymerized thus forming a meshwork
around the platelet aggregate thus stabilizing
the platelet aggregate
 In the presence of meshwork, some of the
blood cell in the circulation (RBC) are trapped
in the fibrin meshwork & add up to the bulk &
the larger the size of the clot, the more
efficient it is as a plug
THREE HEMOSTATIC COMPONENTS
EXTRAVASCULAR - Play a part in hemostasis by
COMPONENTS providing back pressure on the
injured vessel causing some to
flow outside & the tissues will
absorbed & the blood causing
swelling which will exert a back
pressure preventing the further
release or extravasation of the
blood
DEPENDS ON:
a) BULK or amount of surrounding
tissue.
b) TYPE of tissue (skeletal muscles
will be absorbed more as
compared to lose connective
tissues).
c) TONE of the surrounding tissue
(tissues that lack tonicity will not
 HEMATOLOGY 2

absorbed that much such as PRIMARY HEMOSTASIS SECONDARY

tissues of elderly) HEMOSTASIS
VASCULAR - Involves the blood vessels INVOLVES: INVOLVES: activation of a
COMPONENTS Vasoconstriction, Platelet series of plasma proteins
DEPENDS ON: plug, Platelet adhesion, in the coagulation system
a) SIZE of the blood vessels release reaction & until fibrin clot formation
b) AMOUNT of smooth muscle aggregation (final product)
within their wall ACTIVATED BY: ACTIVATED BY: large
c) INTEGRITY of the endothelial desquamation and small injuries to blood vessels
cell lining injuries to blood vessels and surrounding tissues
Procoagulant substances Tissue factor exposed on
ENDOTHELIAL LINING are exposed or released cell membranes or
- composed of endothelial cells by damage or activated circulation
INTRAVASCULAR - Includes cells, plasma proteins & endothelial cells in order
COMPONENTS fibrinolytic agents such as the to activate the secondary Ex.:
plasminogen, plasmin & tissues hemostasis 1. Collagen exposed to
plasminogen activator and the surrounding tissue,
inhibitor causes platelets to
Platelets and biochemical in the aggregate & adhere &
plasma. activate CF 12. COLLAGEN
is the chief component of
 Clots are actually the the connective tissues.
plasma not the RBC 2. Tissue Factor
 There are inhibitors for introduced to the
coagulation and circulation because of
fibrinolysis large injury to the tissues,
 PECAM & ICAM contains it will activate factor 7
adhesion molecules causing the in vivo
 Platelets are also coagulation
involved in 2nd COMPONENTS: vascular COMPONENTS: platelet
hemostasis by exposing intima & platelets & coagulation system
their surface for the Rapid, short-lived Delayed, long term
clotting factors to be response response
activated & react Ends with platelet plug Naturally occurring
formation inhibitors in blood will
Primary Hemostatic block activated
ARTERIES VEINS CAPILLARY Disorders: Blood vessel & coagulation factors so
SIZE Large Smallest; platelet disorder that widespread
easily coagulation does not
rupture occur.
LININGS 3 layers of 3 layers of Endothelium FIBRINOLYSIS – slow breakdown & removal of fibrin
endothelial endothelial (inner) and clot as healing of the injured vessel occurs
lining, lining, Epithelial
smooth smooth cells
muscle and muscle and SPECIAL FUNCTIONS OF VASCULAR ENDOTHELIUM
connective connective
1. MECHANICAL ABILITY
tissues tissues
WALL Thicker Thinnest a. Vasodilation
o Prostacyclin – induces
vasodilation ; produce by
endothelial cell

b. Vasoconstriction
- 2 Substances that Promotes
Vasoconstriction:
o Serotonin/5 hydroxytriptamine
– from dense granules
o Thromboxane A2
NOTE:
Both serotonin & thromboxane A2 activates the
surrounding tissues for it to constrict causing now
limited amount of blood to enter
2. SYNTHESIS
- Aids in anticoagulation & procoagulation
 ANTICOAGULANT: during intact skin ; by smooth
surface allowing blood cells to circulate
smoothly
o Prostacyclin & nitrous oxide – secreted
by intact endothelial cell; induces
vasodilation & inhibit platelet
aggregation
o Heparan sulfate – Increases the activity
of antithrombin 3 (major inhibitor of
thrombin
o Tissue Factor Pathway Inhibitor – major
inhibitor of tissue factor/extrinsic
pathway which is initiated by factor 12
o Anti-thrombin 3 – from the liver; major
inhibitor of the entire coagulation
system
o Thrombomodulin – Inhibitor of Factor
2a; binds w/ thrombin thus inhibiting
thrombin & cause deactivation of
protein C which is the cofactor of protein
S. Protein C comes from the liver
produced as an inactive protein which is
inactivated by thrombin
thrombomodulin. Protein C becomes an
inhibitor. Protein C, thrombomodulin &
thrombin combines to form a
trimolecular complex & with endothelial
cell as a receptor, Protein C becomes
activated
HEMATOLOGY 2
 PROCOAGULANT: during vascular damage
o Serotonin & thromboxane A2 –
vasoconstriction
o Von Willebrand Factor – from alpha
granules which is involve in platelet
adhesion & in the endothelial cell they
are stored in the weibel palate
o ADAMTS-13 – cleaves ultra large von
Willebrand factor which may adhere
even in the absence of damage which
may lead to TTP
o P-selectin – produce by other WBC &
produce by endothelial cell ; enhances
adhesion to damage
o Collagen & Tissue Factor exposed –
enters the circulation during damage &
be exposed wherein collagen will cause
platelets to adhere & aggregate &
activate coagulation factors such as
Factor 7 & 7a causing clotting factors to
occur for tissue factor
o Fibrinolysis – produces tissue
plasminogen activator which converts
plasminogen into plasmin however
excessive TPA causes excessive plasmin
causing excessive fibrinolysis thus
PLASMINOGEN ACTIVATOR INHIBITOR is
activated to inhibit activation of
plasminogen in order to decrease the
synthesis of plasmin & THROMBIN
ACTIVATABLE FIBRINOLYSIS INHIBITOR
inactivates fibrinolysis
&
 HEMATOLOGY 2

PRIMARY HEMOSTASIS: Components, Functions, ADHESION •P-selectin; Procoagulant

Disorders MOLECULES Intracellular
BLOOD VESSELS (Vascular Intima) Adhesion
Molecules
ANTITHROMBOTIC, FIBRINOLYTIC AND COAGULANT •Platelet
SUBSTANCES RELEASED FROM OR FOUND ON THE endothelial
SURFACE OF ENDOTHELIAL CELLS cell adhesion
molecules
SUBSTANCE ACTION HEMOSTATIC
(PECAMs)
ROLE
•Help cells
PROSTACYCLIN •Inhibits Anticoagulan stick to each
(PGI2) platelet t and to their
activation surroundings
•Stimulates
vasodilation
HEPARAN SULFATE Coats the Anticoagulan NOTE:
endothelial t  Vascular disorders affect hemostasis
cell surface  Bleeding occurs if the connection of the
and weakly endothelial lining is loose.
enhances  Connective tissues are disseminated in every
activity of part of the body. Thus, all tissues of the body
antithrombin with defective connective tissue will also be
-3 affected.
THROMBOMODULI •Endothelial Anticoagulan
N (ENDOTHELIAL surface t
PROTEIN C receptor for Fibrinolytic BLEEDING DISORDER CAUSED BY VASCULAR
RECEPTOR) thrombin DEFECTS
(binds & HEREDITARY CONNECTIVE TISSUE DEFECTS
inactivates
thrombin) 1. EHLERS-DANLOS SYNDROME
•Enhances - Sex-linked ; caused by a defect in the gene
anticoagulant responsible for peptidase enzyme production
and which converts procollagen to collagen (chief
fibrinolytic component of the connective tissues of blood
action of vessels)
protein C - Characterized by: Hyper-extensive joints and
TISSUE FACTOR Controls Anticoagulan hyperplastic skin
PATHWAY Activation of t - LABORATORY FINDINGS: Normal Coagulation
INHIBITOR the extrinsic Test & Platelet function studies
pathway
TISSUE Converts Fibrinolytic
2. MARFAN SYNDROME
PLASMINOGEN plasminogen
- Defect in fibrillin
ACTIVATOR to plasmin
ADENOSINE Stimulates Reduces - Characterized by: Elongated upper & lower
vasodilation blood flow digits.
rate
VON WILLEBRAND Required for Procoagulant 3. PSEUDOXANTHOMA ELASTICUM
FACTOR (VWF) platelet - Autosomal recessive ; defect in elastic tissue
WEIBEL-PALADE adhesion to - Characterized by: calcified & formed into
BODIES site of injury plaques thus affecting the ability of the vessel to
 HEMATOLOGY 2
constrict & dilate and structurally abnormal
Elastic fibers
- Yellowish plaque, observed on skin where the
skin is abnormally loose (neck)
ACQUIRED CONNECTIVE TISSUE DEFECT
1. VITAMIN C DEFICIENCY (SCURVY/SCORBUTUS)
- Vitamin C deficiency common among children
thus prone to petechial formation
- IMPORTANCE OF VITAMIN C: for the formation
of the intact structure of the vascular basement
membrane & in synthesis of collagen for
hydroxylation of Vitamin C’s proline & lysine
- Characterized by: gingival bleeding; hemorrhage
into subcutaneous tissues and muscles; large
hemorrhagic areas may develop just below the
eyes; splinter-like hemorrhage may also appear
in the fingernail beds
- LABORATORY FINDINGS: CFT = usually positive
2. SENILE PURPURA
- Related with normal aging process;
discoloration of the skin which may not
disappear & will remain as age spot because
even the function of the macrophages which
supposed to remove it has also declined
- LABORATORY FINDINGS: CFT = positive ;
Bleeding time = normal or only slightly prolonged
HEREDITARY ALTERATION OF VESSEL WALL
STRUCTURE
1. HEREDITARY HEMORRHAGIC TELAGIECTASIA
(RENDU-OSLER-WEBER SYNDROME)
- Common among Scandinavian population ;
discoloration of the skin where the skin is thin ;
blood vessels are abnormally thin thus they
dilate & will be seen as spots
- Autosomal dominant trait characterized by thin-
walled, focally disorganized and dilated blood
vessels with a discontinuous endothelium
appearing as red pinpoint lesions most
commonly on the face, lips, tongue, mucous
membranes of the mouth and nose, ears,
conjunctiva, palms and soles
- LABORATORY FINDINGS: bleeding time, platelet
function tests, capillary fragility test and
coagulation test are all normal
2. CONGENITAL HEMAGIOMATA (KASABACH-
MERRITT SYNDROME)
- Disorder associated with tumors composed of
many large vessels ; benign & can be surgically
remove
- Formation of fibrin clots, platelets consumption
and red cell destruction secondary to vascular
obstruction occur at the site of tumor in the
mucous membrane
ACQUIRED ALTERATIONS OF VESSEL WALL
STRUCTURE
1. DIABETES MELLITUS
- Deposition of glycosylated proteins leading to
thickening of capillary basement membrane
plaques affecting often the capillaries of the
renal glomeruli and retina
2. AMYLOIDOSIS
- Excessive amyloid or cholesterol deposit in
small vessels thus forming plaques causing now
inflammatory process leading to cellular
inflammation and activation of cellular
inflammatory process leading to injury & the
normal vessel flow will contain thrombus
formation
- Causes vasoobstruction thus less oxygenation &
may also lead to rupture resulting to stroke &
other condition
ENDOTHELIAL DAMAGE
1. AUTOIMMUNE VASCULAR PURPURA
- Problem with antibodies
A. DRUG INDUCED PURPURA
- Common drugs that induce purpura:
sulfonamides & iodides quinine, procaine,
penicillin, aspirin, sedatives, coumarin
- Endothelial cell might no longer be recognized as
self-cells & the immune system will induce Ab
against the endothelial cell & iodide or
sulfonamide
- May be due to development of antibodies to
vessel walls components, development of
immune complexes and change in vessel wall
permeability
 HEMATOLOGY 2

B. ALLERGIC PURPURA/ANAPHYLACTOID NOTE:

PURPURA
- Associated with certain food and drugs, cold
temperature, insect bites and vaccinations
- HENOCHS PURPURA – associated with
abdominal pain secondary to GIT hemorrhaging
- SCHONLEINS PURPURA – associated with joint,
especially in the knees, ankles and wrist
 Only metamegakaryocyte has the ability to
produce platelets
 The more nuclear lobes, the more platelets it
will produce
4 FUNCTIONAL ZONES OF PLATELETS

2. INFECTIOUS PURPURA a. PERIPHERAL ZONE

- Associated with bacterial ; viral, rickettsial & - Contains glycocalyx which contains
protozoal infections or substance it produces glycoproteins (4, 6, 9)
- Contains glycoprotein 1b/9 for platelet adhesion
PLATELETS & GP 2b/3a for platelet aggregation
CHARACTERISTICS:

- Cytoplasmic fragments from megakaryocytes b. ZOL GEL ZONE

- Contains gel components that maintains
Diameter 2-4 um & 1/8 the diameter of different organelles at their position & allows
RBC them to communicate with one another
Site of production Bone marrow - Contains the cytoskeleton of the cell which
2 Pools 1/3 in spleen maintains the size & shape of platelet for it to not
2/3 in circulation
collapse which is made up of microtubules and
Growth Factor Thrombopoietin
microfilaments, the actin & myosin forms the
Manner of Division Endomitosis – increase
contractile protein, thrombosthenin.
nuclear component while
cytoplasmic component is
absent c. ORGANELLE ZONE
Rule As it matures, the cell size - Contains the dense granules, alpha granules
increases (most abundant), mitochondria & lysosomes
Mean Platelet 8-10 fL
Volume (MPV) d. MEMBRANE SYSTEM
Reference Platelet 150,000-450,000/ uL o Open Canalicular System – Membrane
count that allows communication to the
Daily Turnover 35 X 109/L (+/- 4.3) outside environment & route of entry &
Average production 2,000 – 4,000/megakaryocyte exit of substances
Lifespan 8-11 days o Dense Tubular System – for calcium
Function Maintenance of vascular pump & arachidonic acid production
integrity and blood
coagulation

DEMARCATING MEMBRANE SYSTEM

- Demarcation lines in cytoplasm ; where platelet

fragmentation occurs
- Appears as early as megakaryoblast & prominent
in promegakaryocyte & absent in megakaryocyte
& metamegakaryocyte.
 HEMATOLOGY 2

PLATELET FACTORS

PF1 Plasma coagulation factor V

PF2 A globulin that inhibits antithrombin III;
increases platelet aggregation and
accelerates interaction of thrombin and
fibrinogen (fibrinoplastic platelet factor)
PF3 A lipoprotein (platelet phospholipid) found
in platelet granules & membrane and
required in 2 steps of the coagulation
process ; functions for coagulation factors to
assemble in order to cause fibrin formation
; component of prothrombinase together
w/ CF10, 5a, Ca for conversion of
prothrombin into thrombin for secondary
hemostasis ; assembly molecule
PF4 A glycoprotein stored in the alpha granules
and is extruded during the platelet release
reaction; aids in ADP-induced platelet
aggregation and inhibits effect of heparin

NOTE:
 In heparin therapy, patients might
develop autoantibodies against PF4
in Heparin Induce
Thrombocytopenia disease
 Heparin Induce Thrombocytopenia
– Acquired condition caused by
autoimmune disorder caused by
heparin therapy
PF5 Platelet fibrinogen
PF6 A plasma inhibitor associated with platelets
PF7 Cothromboplastin
PF8 Antithromboplastin factor
PF9 Accelator globulin stabilizing factor
PF10 Serotonin found in the dense granules
 HEMATOLOGY 2

ROLE IN SUBSTANCE SOURCE PRINCIPAL FUNCTION/COMMENTS

HOMEOSTASIS
Promote HMWK (high molecular Alpha Granules  Contact Activation of intrinsic coagulation
coagulation weight kininogen) pathway
 Cofactor of 12a in converting prokalycrine into
kalycrine
Fibrinogen  Converted to fibrin for clot formation
Factor V  Cofactor to Factor 10 to convert prothrombin
into thrombin in fibrin clot formation
Von Willebrand Factor  Assists platelet adhesion to subendothelium
to provide coagulation surface
Promote ADP (strong) Dense bodies
Aggregation Calcium  Promote platelet aggregation
Platelet factor 4 Alpha granules  Calcium is involved in coagulation
Thrombospondin  Thrombospondin & platelet factor 4 is for
platelet function
Promote Serotonin Dense bodies
Vasoconstriction Thromboaxane A2 Membrane  Promotes vasoconstriction at injury site
precursors (inhibited phospholipids  Thromboxane A2 is a very strong aggregator
by aspirin therapy) of platelet
Promote Platelet derived Alpha granules  Promotes smooth muscle growth
vascular repair growth factor  For vessel repair
Beta-thromboglobulin  Chemotactic for fibroblasts
 For vessel repair
Other systems Plasminogen (from Alpha granules  Precursor to plasmin, which induces clot lysis
affected liver converted into
plasmin by TPA &
inhibited by A2-
antiplasmin)
A2 – antiplasmin  Plasmin inhibitor, inhibits clot lysis
C1 esterase inhibitor  Complement system inhibitor

PHYSICAL PROPERTIES AND FUNCTION OF - Von Willebrand Factor is necessary for areas
PLATELETS where circulation is with high shear (arteries &
1. ADHESION (platelet-to-injury) arterioles) such that if the platelets will simply
- Platelets during injury first cling & roll on the adhere, It will be easily dislodge but in veins &
damage endothelium & adhere on damage capillaries, since the pressure is not that strong,
endothelium the platelets can directly adhere on the exposed
- Sticking of platelets to a non-platelet structure; surface such as GP 6
platelets adhere only on detached or injured - Reversible.
endothelium
- Requires the GP Ib/IX in complex with V for 2. PLATELET RELEASE REACTION
adhesion which serves as a receptor for Von - Platelet undergoes shape or morphological
Willebrand Factor which is readily available in change. Alpha & dense granules release
plasma substances that will contribute to platelet
- Plasma Von Willebrand Factor is used for aggregation and activation of the coagulation
platelet attachment to the exposed system.
subendothelial matrix such as the collagen
 HEMATOLOGY 2
- Releases ADP from dense for aggregation,
serotonin from dense for vasoconstriction &
thromboxane A2 from membrane phospholipid
for vasoconstriction & aggregation
- Irreversible
3. PLATELET AGGREGATION (platelet-to-platelet)
- Requires Gp IIb-IIIa as a receptor for fibrinogen
and plasma fibrinogen/Factor 1 which serves as
glue or adhesive for platelet to platelet adhesion
- Viscous metamorphosis
- INITIAL AGGREGATION: reversible
- SECONDARY AGGREGATION: irreversible
- AGGREGATING AGENTS: ADP, Thrombin,
Collagen, Thromboxane A2, Epinephrine,
Arachidonic Acid, Ristocetin, Thrombospondin,
Reptilase (from reptile)
OTHER FUCNTIONS:
4. VASOCONSTRICTION
- Enhanced by serotonin released from platelets &
thromboxane A2 from membrane phospholipids
- Reaction in primary hemostasis
5. CLOT FORMATION
- Platelet factor 3 or the assembly molecule is
needed for the formation of active plasma
thromboplastin
- Reaction in secondary reaction
QUALITATIVE PLATELETS (Platelet-vessel wall
interaction)
A. ADHESION DEFECTS:
1. BERNARD-SOULIER SYNDROME
- Features:
o Deficiency in Gp 1b/IX thus an intrinsic
defect
o Platelets are abnormally low & abnormal
morphology
o Abnormally large platelet/giant platelet
w/ thrombocytopenia & prolonged
bleeding time
o AGGREGATION STUDIES: platelet
 Abnormal: Ristocetin
 Normal: ADP, collagen &
epinephrine
2. VON WILLEBRAND DISEASE
- Most common coagulopathy
- Autosomal inheritance & function at
chromosome 12 synthesized by the endothelial
cell & platelets; stable.
- Autosomal inheritance in Chromosome 12;
deficiency in plasma VIIIc: VWF.
- Characterized by prolonged bleeding time &
same aggregation test result with Bernard-
Soulier; prolonged APTT due to deficiency or
decrease in CF 8c
- FACTOR 8C/ANTIHEMOPHILIC FACTOR –
cofactor in the intrinsic pathway
- ACTIVATED PARTIAL THROMBOPLASTIN TIME –
test for intrinsic pathway
- Multimeric molecule w/ a receptor for GP
Ib/IX/V complex & collagen which is used to
bind w/ platelets causing platelet adhesion
- Contains Ag epitope & has a receptor for & a
protein carrier for FACTOR
8C/ANTIHAEMOPHILIC 8C which is a sex-linked
inheritance in the X chromosome synthesized by
the liver & is labile thus it is prone to proteolysis
thus causing secondary deficiency causing
prolongation of APTT but becomes relatively
stable w/ Von Willebrand Factor.
ANTIHEMOPHILIC FACTORS:
 A – Factor VIII (2 components: 8:VWF for
adhesion & 8C for coagulation intrinsic pathway)
 B – Factor IX
 C – Factor XI
LABORATORY: PLATELET COUNT AND MORPHOLOGY:
generally NORMAL
COMMON SIGN: mucocutaneous bleeding
BLEEDING TIME (severe) & APTT TEST (prolonged) –
screening test
DIAGNOSIS: STANDARD VMD TEST PANEL TESTS:
1. Quantitative VWF test (VWF Ag assay) – to
determine if VWF is quantitative or qualitative.
2. VWF activity test/ VWF RCo assay – qualitative
test to determine ability of vWF to bind to
platelets.
 HEMATOLOGY 2

3. Factor VIII activity assay – to determine the

extent of VWF disease which may lead to
NOTE:
secondary deficiency of Factor 8
ACQUIRED VWF DEFICIENCY:
TREATMENT: - hypothyroidism, autoimmune,
lymphoproliperative & myeloproliferative,
 Cryoprecipitate – contains the complete disorders; benign monoclonal gammopathies;
component of factor VIII & VWF wherein this Wilms tumor ; intestinal angiodysplasoa;
control bleeding by supplying the deficient congenital heart disease ; pesticide exposure;
Factor VIII & VWF but it is no longer & hemolytic uremic syndrome
recommended because it does not undergo viral
inactivation wherein the specimen source is the
B. AGGREGATION DEFECTS (Platelet-platelet
blood donor.
interaction)
 Desmopressin acetate (1-desamino-8-D
arginine vasopressin/ DDAVP) – standard
1. GLANSMANN’S THROMBASTHENIA
treatment which cause release of VWF & Factor
- Features:
8 from endogenous sources in order to prevent
o Deficiency/ Gp IIb-IIIa; Inherited as an
bleeding.
autosomal recessive trait
 A plasma-derived factor VIII/VWF concentrate
o Bleeding time is prolonged, platelets are
CLASSIFICATIONS OF VON WILLEBRAND DISEASE morphologically normal but isolated w/
one another
TYPES DESCRIPTION o AGGREGATION STUDIES: Platelets
1  Partial quantitative deficiency of von aggregate normally with Ristocetin but
Willebrand factor (vWF) not with Epinephrine, ADP, Collagen
 Most common to occur about 75 -80% ;
a quantitative disorder
2. (CONGENITAL) AFRIBRINOGENEMIA OR
2  Qualitative (Function) deficiency of vWF
HYPOFIBRINOGENEMIA
 Level of vWF is normal but function is
o AFIBRINOGENEMIA: Acquired/Inherited
abnormal
total absence or severe deficiency in
2A  Decreased platelet-dependent vWF
fibrinogen.
function with selective deficiency of high-
molecular-weight multimers (the vWF is o HYPOFIBRINOGENEMIA:
more susceptible to proteolysis by Acquired/Inherited slight decrease in
ADAMTS-13 which is secreted by fibrinogen.
endothelial cells which destroys low o DYSFIBRINOGENEMIA: Inherited lack of
molecular weight vWF) fibrinogenemia
2B  Increased affinity for platelet
glycoprotein lb/IX/V DISORDERS OF PLATELET SECRETION & SIGNAL
TRANSDUCTION DEFICIENCY OF GRANULES
2M  Decreased platelet receptor binding
(STORAGE POOL DISORDERS)
(glycoprotein lb/IX/V)
ALPHA GRANULES DEFICIENCY
2N  (Normandy Variant) Impaired factor VIII
binding site leading to deficiency to APTT 1. GRAY PLATELET SYNDROME
thus APTT is prolonged - Deficiency of alpha granules
 Autosomal Hemophilia (royal disease) - Platelets aggregate abnormally & are abnormally
3  VWF is absent or nearly absent from large
plasma - Characterized by larger platelets colored gray to
 Rarest vWF & severe or complete blue-gray, and thrombocytopenia
deficiency
- LABORATORY: Prolonged Bleeding time;
 Autosomal Recessive
decreased aggregation with all agents.
 HEMATOLOGY 2
2. QUEBEC PLATELET DISORDER
- Autosomal dominant disorder characterized by
abnormal proteolysis (destruction of proteins) &
deficiency of alpha granules
- Abnormal increase in urokinase-type
Plasminogen Activator
- Platelet count is normal or decrease
- Characterized by delayed bleeding when
exposed to trauma (12-24 hrs from the time of
exposure) from mucocutaneous bleeding.
DENSE GRANULES DEFICIENCIES
1. WISKOTT-ALDRICH SYNDROME
- (Sex-linked) deficiency of dense granules, with a
defect in cytoskeletal assembly.
- Characterized by predominance of small
platelets
- TRIAD OF SYMPTOMS: thrombocytopenia,
recurrent infection & eczema
2. HERMANSKY-PUDLAK SYNDROME
(AUTOSOMAL)
- TRIAD OF SYMPTOM: oculocutaneous albinism,
accumulation of ceroid-like pigment in
macrophages & bleeding tendencies
3. CHEDIAK-HIGASHI (AUTOSOMAL)
- TRIAD OF SYMPTOM: albinism (no pigment),
recurrent infection (immune deficiency) & giant
lysosomes
4. THROMBOCYTOPENIA WITH ABSENT RADII
SYNDROME (TAR)
- Reduced radial bone or totally absent ;
appearing like a t-rex syndrome
- Structural defect in beta granules with
NOTE:
corresponding abnormal aggregation responses
and thrombocytopenia
- Characterized by severe neonatal
thrombocytopenia and congenital absence or
extreme hypoplasia of the radial bones (most
pronounced skeletal abnormality), cardiac and
other skeletal abnormalities
- Abnormal aggregation
- Observed as early as birth
DISORDERS OF PLATELET PROCOAGULANT
ACTIVITIES
- In resting platelets, phosphatidylserine (PS) &
Phosphatidylethanolamine (PE) are located
predominantly on the inner leaflet while
phosphatidylcholine has the opposite
distribution & this arrangement is maintained
by Aminophospholipid Translocase Enzyme.
During platelet activation, it undergoes a
morphological change such that its cytoplasm
will be extruded with extensions & the
distribution of phospholipids will be scrambled
wherein phosphatidyl serine &
phosphatidylethanolamine will be exposed &
acted by the enzyme, phosphatidyl
scramblase/PF 3 & coagulation factors will be
activated & be assembled
1. SCOTT SYNDROME
- Surface expression of phosphatidylserine is
decreased
- Disorder of calcium-induced membrane
phospholipid scrambling that when a platelet is
activated it can assume a resting phase of
platelet & may also be activated wherein the
exposure of phosphatidyl serine is deficient thus
scrambling occurs & affects the secondary
hemostasis
2. STORMORKEN SYNDROME
- Platelets are always in an activated state without
prior activation
- Defect in aminophospholipid translocase
- Phosphatidyl choline is always on the outside
causing abnormal platelet activation
 Phosphatidyl Choline – major component of
the outer layer
 Phosphatidyl serine &
Phosphatidylethanolamine – internal later
 HEMATOLOGY 2
ACQUIRED DEFECTS OF PLATELET FUNCTION
1. DRUG-RELATED
 ASPIRIN – permanent cyclooxygenase inhibition
which is needed in the synthesis of thromboxane
A2 thus affecting platelet aggregation
- Phospholipid A2 will convert the membrane
phospholipid of platelets into cyclooxygenase
- Prevents conversion of arachidonic acid into
prostaglandin by cyclooxygenase leading to
deficiency in thromboxane A2
 NSAIDs and other COX-2 inhibitors (naproxen
and ibuprofen)
 Other Antiplatelet agents: TICLOPIDINE AND
CLOPIDROGEL – blood thinning drugs making
blood viscous ; affects fibrinogen & Gp2b/3a
 Dextran – coating of platelets thus platelet
Causes:
cannot adhere & aggregate
2. PARAPROTEINEMIAS ((MULTIPLE MYELOMA
AND WALDENSTROM MACROGLOBULINEMIA)
- Dysproteneimias – Excessive plasma proteins to
lead to hyper-viscosity syndrome making the
circulation slow leading to thrombosis & the
pressure is increase
- Disorder of the bone marrow thus production of
intrinsically abnormal platelets
- Increase in bens jones protein &
immunoglobulins wherein for MM it is increase
IgG while for WM it is increase IgM
3. RENAL DISEASE
- Uremia: increase in non-protein nitrogenous
substances ; leads to decreased thromboxane
synthesis causing platelet destruction ;
Decrease: adhesion, platelet release, and
aggregation ; Increase: non-protein nitrogenous
substances
QUANTITATIVE PLATELET DISORDERS
- Platelets function is normal but platelets are
few or increased in number
SIGNIFICANT PLATELET LEVELS (platelet count and
clinical manifestation):
 150 – 450 x 109/L – REFERENCE PLATELET
COUNT
o <150, 000: thrombocytopenia
o >450, 000: thrombocytosis
 < 100,000/ uL – ABNORMALLY LOW; significant
decrease ; bleeding is not apparent
 30,000 – 50,000/ uL – BLEEDING IS POSSIBLE
especially when exposed to trauma or bleeding
secondary to surgical procedures
 <30,000 – SPONTANEOUS BLEEDING, meaning
even without trauma or surgical procedure,
bleeding may occur in the internal organs
 <5,000/ uL – SEVERE SPONTANEOUS BLEEDING,
may cause intracranial bleeding and bleeding in
visceral organ due to inability of platelets to
maintain the continuity of blood vessels ;
considered as a life-threatening condition
THROMBOCYTOPENIA
1. DECREASED PRODUCTION
- Bone marrow defect, where platelet production
remains abnormal
 MEGAKARYOCYTE HYPO-PROLIFERATION in:
o APLASTIC ANEMIA – affects the bone
marrow; bone marrow is infiltrated with
fats which are haematopoietically
inactive; all blood cells are decrease.
o MAY-HEGGLIN – inherited condition;
abnormally large megakaryocyte but are
decrease in number; WBC w/ dohle
bodies.
o TOXIC CONDITIONS – when the toxic
agents destroy megakaryocyte
 INFECTIVE THROMBOPOIESIS – Infective
thrombopoiesis due to decrease in
thrombopoietin which is mainly produced by the
liver and a few in kidneys ; associated among
chronic alcoholics among ethanol abused thus
damaging platelet production
 NEONATAL THROMBOCYTOPENIA – among
newborns who are exposed to infection inside
the uterus ; associated with infections with
toxoplasma, rubella, cytomegalovirus, and
herpes and HIV (TORCH); and in-utero exposure
to certain drugs (particularly chlorothiazide
diuretics and the oral hypoglycemic
tolbutamide)
 HEMATOLOGY 2
 MARROW REPLACEMENT/MYELOPTHISIS –
replacement of entire bone marrow with
abnormal cells such as cancerous cells
2. BLOOD TRANSFUSION
- Transfused platelets will add on the patients
platelets improving now the patients platelet
count
- Occurs after transfusion of platelet-containing
blood products (recipient’s plasma is found to
contain alloantibodies to antigens on the
platelets of the transfused blood product)
- Normal lifespan of platelets in blood banks is
only 5 days
 DILUTIONAL LOSS – blood product transfused
does not contain enough viable platelets thus it’s
effect is to dilute only the patients’ blood and
improve the blood volume ; Massive transfusion
where patient receives 4 or more units of blood
for 24 hours may also lead to dilution of patient’s
platelets ; transfusion of intravenous fluids and
pure plasma
 POST TRANSFUSION PURPURA – occurs a week
after ; related to antibody if the patient is already
been sensitized or received blood component
before causing now antibody production against
the platelets ; severe type of thrombocytopenia
3. INCREASES DESTRUCTION OR CONSUMPTION
a. NON-IMMUNE
- premature activation of platelets in secondary
aggregation causing platelets to be consumed
- associated with activation and consumption
1. THROMBOTIC THROMBOCYTOPENIC
PURPURA (TTP): caused by a deficiency of a
ADAMTS-13 ; common in young adults ;
persistence of ultra large VWF which may
bind prematurely to platelets causing
platelets to aggregate which is irreversible &
the clot may detach & may cause
vasooclussion; platelets are consumed when
formed into thrombus ; deficiency in
disintegrin and metalloproteinase with a
thrombospondin type 1 motif, member 13
(ADAMTS-13)
2. DISSEMINATED INTRAVASCULAR
COAGULATION (DIC) – patient produces a
substance that will activate both coagulation
and fibrinolysis leading to imbalance in
coagulation as clotting occurs there is a
corresponding fibrinolysis, when clot occurs
some coagulation factors are consumed
such as the CF 1, 5,8 and 13 and platelets ;
occurs elsewhere the body ; involves the
activation of both coagulation and
fibrinolysis due to liberation of
thromboplastic substance by damaged or
abnormal cells
3. HEMOLYTIC URENIC SYNDROME (HUS) –
associated with diarrheal or non-diarrheal
a. DIARRHEAL among children after
children has been infected with GIT
problem with any organism that can
produce a shiga-like organism the
verotoxin (E. coli H7:0157, Strep.
Pneumoniae, shigella) where it will it
bind to glomerular capillaries on the
endothelial cells causing them to be
activated and become thrombotic
promoting thrombosis; often occurs in
the kidneys ; It is hemolytic when
platelets become thrombus they cause
lysis ; urine appears as pinkish ;
Associated with mild febrile illnesses,
certain immunizations, and
gastrointestinal disturbances (E.coli
O157:H7 or other Shiga/Vero toxin-
producing bacteria)
b. NON-DIARRHEAL among children
associated with vaccination
4. PLATELET LOSS ON ARTIFICIAL SURFACE – in
cases of artificial heart valve, though it is
match, when platelets are in contact to
them, platelets are destroyed
5. INFECTIONS (eg. Dengue) – leads to platelet
consumption, because dengue infects some
of the macrophages and monocytes which
produces cytokines & the effect make the
endothelial lining hyper permeable and
platelets will plug the area until platelet
count is consumed
 HEMATOLOGY 2
b. IMMUNE DESTRUCTION
- Associated with antibody (IgG)
1. IDIOPATHIC/IMMUNE
THROMBOCYTOPENIC PURPURA (TTP) –
cause is unknown for ITP; common among
young children ; due to presence of anti-
platelet antibodies of the IgG type
associated with previous infection; most
common in young females <15 y/o ; if occurs
in children, the prognosis improves but in
adults it is life threatening
2. DRUG-INDUCED: HEPARIN-INDUCED
THROMBOCYTOPENIA (HIT)- patient
develops IgG antibody specific for heparin-
platelet factor 4 complexes; A. common side
effect of unfractionated heparin
administration
4. SPLENIC SEQUESTRATION
- Seen in hypersplenism and splenomegaly where
there is enlargement of spleen thus sequestering
>1/3 of platelets & keeping 50-90% of platelets,
leaving 10% of platelets in the circulation thus
leading to thrombocytopenia.
5. UREMIA
- Increase of NPN in the blood due to inability of
the kidney to remove these substances due to
renal insufficiency; also causes platelet damage.
THROMBOCYTOSIS/ THROMBOCYTHEMIA
- Increases platelet count (>450,000/uL)
1. PRIMARY THROMBOCYTOSIS
- main defect ; abnormal production ; seen in
myeloproliferative disorders where there is
abnormal proliferation of blood
a. POLYCYTHEMIA VERA – chronic
myeloproliferative disorder ; all blood cells
are increase
b. ESSENTIAL THROMBOCYTOSIS – increase
platelets >1, 000, 000/uL w/ abnormal
function
c. CHRONIC GRANULOCYTIC LEUKEMIA –
leads to abnormal marrow cell production
2. SECONDARY/ REACTIVE THROMBOCYTOSIS
- due to underlying condition ; Seen in splenic
mobilization and in hemolytic anemias
a. SPLENIC MOBILIZATION – occurs in
hemorrhagic conditions ; due to acute
hemorrhage, platelets moves into systemic
circulation causing transient increase in the
platelet circulation ; response to stress such
as in acute blood loss anemias ; platelets
formed and mature from the spleen
b. RAPID REGENERATION – involves the bone
marrow but production is normal ; during
hemolytic anemia where there is a need to
loss of blood cells thus the bone marrow will
immediately try to compensate and increase
production of blood cells ; platelets newly
released from the bone marrow indicated by
reticulated platelets in circulation in order to
respond to underlying condition
LABORATORY EVALUATION OF PRIMARY
HEMEOSTASIS
- Evaluates platelet and blood vessels.
A. DIRECT METHOD OF PLATELET COUNT
- Can be performed directly or indirectly and can
also be done manually or with the use of
automated machines.
- Manual Procedures for platelet count can be
done with the use of Light Microscopy Principle
or the Phase-Contrast Microscopy Principle.
- EDTA: Anticoagulant of choice whether
automated or manual procedure. It prevents
platelets from adhering and as well as
aggregating together.
- When aggregation and adhesion occurs, this will
result to a false decrease in platelet count.
However, even if the blood is EDTA
anticoagulated, platelet count should be
performed as early as possible. Preferable within
the first 3-5 hours from collection.
o 5 Hours: if blood is left in a room
temperature (20°C). Otherwise, on
prolonged standing, platelets will start
to swell and break into smaller
fragment resulting to a false increase in
platelet count.
 HEMATOLOGY 2
o 24 hours at 4°C: cell count (Platelet, RBC
count, WBC count) are still valid if
performed within the first 24 hours.
AUTOMATED PRINCIPLE:
- Counted within the volume range of 2-20 fL. All
cellular factors that falls within this value are
considered by the machine as platelets.
a. Electric impedance – platelets are counted in
triplicate (3x)
b. Light scattering
DILUENT:
1. LIGHT MICROSCOPY METHOD (Tocantin
SOURCES OF ERRORS:
Method)
- Manual counting and requires additional stain
- Principle: Hemocytometry
- Counting chamber: improved Neubauer
 Rees & Ecker Diluent: contains formalin as a
fixative which preserves platelets and RBCs. It
also contains Sodium Citrate as an additional
anticoagulant and a supravital stain which is the
Brillian Cresyl Blue (BCB).
2. PHASE – CONTRAST MICROSCOPY METHOD
- Identify cells based on their index of refraction.
- Reference method for manual platelet counting.
- Platelets are counted in 5R squares & appear as
refractile or glistening bodies
- Counting Chamber: Spencer-Bright line (S-B)
1475
 Brecker Cronkite Method
o 1% NH4 Oxalate diluent: no fixative and
stains. It removes RBCs from the
background wherein RBCs are
hemolyzed and platelets will remain in
the field.
PROCEDURE FOR ALL:
PHYSIOLOGIC VARIATION:
a. First dispense 2-3 drops of the mixture before
loading the counting chamber
b. Dispense the mixture on both side of the
counting chamber
c. Place the loaded counting chamber on the wet
house counting chamber which contains a
moistened cotton/filter paper w/ distilled water
to provide moisture and prevent evaporation for
15 minutes to allow the cells to settle
d. After 15 minutes, place on the microscope &
start counting on the 5 small square or 25 central
square or 10 small squares
e. Compute:
FORMULA:
𝑇𝑉−1
DF=
𝐵𝑙𝑜𝑜𝑑 𝑣𝑜𝑙𝑢𝑚𝑒
1 𝑚𝑚3
VCF=
𝑛𝑜.𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 ×𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒𝑠
Plt. Count= no. of platelet counted × 𝑉𝐶𝐹 × DF
1. PLATELET CLUMPS – occurs if blood is not
immediately mix w/ anticoagulant during
collection causing false decrease platelet count.
2. PLATELET SATELLITISM
- Surrounding of platelets in a neutrophil.
- Common among individuals who have develop
Ab (IgG) against EDTA.
- Platelets attaches on WBC, band cells &
monocytes due to cell receptors on their cell
surface.
- Causes false decrease platelet count.
- Remedy: collect using Na citrate & perform
manual platelet count. However, platelet must
be multiplied by 1.1 to correct the dillutional
effect of Na citrate to blood.
3. EXCESS EDTA (1/2 full)
- Platelets will swell & fragmentize causing false
increase in platelet count.
- Fill EDTA half full (2.5 mL)
4. 3 HOURS AT RT – platelet will swell &
disintegrate causing false decrease platelet
count thus, refrigeration of the sample at 4°C for
24 hours.
1. Decreased at birth
2. Increased after the 1st week of birth until adult
normal values.
3. Decreased during menstruation.
 HEMATOLOGY 2

B. INDIRECT METHOD release cuff & wait for 5-10 minutes before
reading.
1. FONO’S METHOD - The forearm, hands, and fingers are then
- DILUENT: 14% of MgSO4 – prevents platelets examined for petechiae 5 inches away from
from adhering & aggregating. blood pressure cuff.
- If there is a need to re-perform, perform on the
PLATELET ESTIMATION
other arm. It can only be repeated on the same
- The average number of platelets observed in 10 arm after a week.
fields & divided by 10 & multiplied by 20,000 in - NORMAL: 0 to occasional petechiae.
a blood smear anticoagulated with EDTA thus - INTERPRETATION: +, ++, +++, ++++
appearing clear & separated at the neck region
counted via cross sectional (side by side) or b. HESS/SUCTION TEST (Negative pressure)
battlefield method (serpentine) - A suction cup (2cm diameter) is placed in close
- In capillary puncture platelet appears as clump. contact with the skin at midpoint of upper arm
Thus, resulting to a false decrease of platelet for 1 min (pressure of 200-250 torr). An area
count. within a circle of 1 cm diameter is observed for
- Platelet shows 8-20 platelets per oil immersion petechiae 5 minutes after removal of suction
field of 200 RBC & 2-3 platelet clumps only and cup.
reported as follows: - CLINICAL SIGNIFICANCE: Positive in
thrombocytopenia, hypofibrinogenemia, and in
0-49,000 Marked decreased vascular purpura.
50,000- 99,000 Moderate decreased
100,000-149,000 Slight decreased TESTS FOR PLATELET FUNCTION & VASCULAR
150,000-199,000 Low normal FUNCTION
200,000-400,000 Normal 1. BLEEDING TIME
401,000-599,000 Slight increase - Measures the ability of the small blood vessels
600,000-800,000 Moderate increase to control bleeding after injury.
Above 800,000 Marked increased - Time it takes for a standard wound to stop
bleeding at a standard pressure (40mmHg)
- Standardized both incision & pressure applied
PLATELET & VASCULAR FUNCTION
- FACTORS AFFECTING BT:
1. TOURNIQUET TEST/CAPILLARY FRAGILITY TEST
a. Number of platelets and the ability of
- Tests the ability of small capillaries to retain
platelets to form plugs.
blood when subjected to increased hydrostatic
b. Thickness and vascularity of the skin.
pressure and anoxia.
c. Ability of the blood vessels to constrict.
- Affected by capillary integrity & platelet function
d. Severe coagulation factor deficiency causing
and number.
prolonged bleeding time.
- Becomes normal in presence of
- PROLONGED BT:
thrombocytopenia, hypofibrinogenemia &
o When platelet count is lower than 30-
vascular purpura
50,000/uL, or when platelets are
- Requires application of pressure which is applied
dysfunctional in VWD.
as a positive or negative pressure.
o After ingestion of aspirin/aspirin-
containing compounds, anti-
a. RUMPLE-LEEDE METHOD (positive pressure)
inflammatory, anticoagulants, and
- PRINCIPLE: an inflated blood pressure cuff on
some antibiotics.
the upper arm is used to apply pressure (100
mmHg) to the capillaries for 5 minutes. After,

2. PLATELET ADHESIVENESS/RETENTION TEST
- Reference Values: 26-60%
a. GLASS BEAD METHOD/SALZMAN METHOD
- Collect 2 whole blood samples, 1 using EDTA and
the other using glass bead collecting system.
Perform platelet counts separately. (higher in
EDTA)
- COMPUTATION:
𝑝𝑙𝑡 𝑤𝑖𝑡ℎ𝑜𝑢𝑡 𝑔𝑙𝑎𝑠𝑠 𝑏𝑒𝑎𝑑𝑠−𝑝𝑙𝑡 𝑤𝑖𝑡ℎ 𝑔𝑙𝑎𝑠𝑠 𝑏𝑒𝑎𝑑𝑠
%= × 100
𝑝𝑙𝑎𝑡𝑒𝑙𝑒𝑡 𝑤𝑖𝑡ℎ𝑜𝑢𝑡 𝑔𝑙𝑎𝑠𝑠 𝑏𝑒𝑎𝑑𝑠
b. BORSHGERVINCT METHOD (in vivo test)
- Perform separate platelet count on venous
blood and capillary blood samples
- Formula:
𝑝𝑙𝑡 𝑐𝑜𝑢𝑛𝑡 𝑣𝑒𝑛𝑜𝑢𝑠 𝑏𝑙𝑜𝑜𝑑−𝑝𝑙𝑡 𝑐𝑜𝑢𝑛𝑡 𝑐𝑎𝑝𝑖𝑙𝑙𝑎𝑟𝑦 𝑏𝑙𝑜𝑜𝑑
%=
𝑝𝑙𝑎𝑡𝑒𝑙𝑒𝑡 𝑐𝑜𝑢𝑛𝑡 𝑖𝑛 𝑣𝑒𝑛𝑜𝑢𝑠 𝑏𝑙𝑜𝑜𝑑
3. PLATELET AGGREGATION TEST
- Aggregating agents; thrombin, arachidonic acid,
ADP, collagen, epinephrine and ristocetin.
- Currently, this test is considered the gold
standard for evaluation of aspirin resistance.
- PRINCIPLE: Aggregating agents added to a
stirred suspension of PRP induce a shape change
and aggregation of platelets. As a result, the PRP
changes from a turbid suspension to tone that
transmits more light (clear) as the aggregates
are formed. The aggregometer records changes
in optical density in the form of a graph.
READING OF AGGREGATION RESPONSE:
1. PLATELET-RICH PLASMA AGGREGOMETRY: light
transmittance aggregometer
2. WHOLE-BLOOD PLATELET AGGREGOMETRY:
electrical impedance
3. OPTICAL LUMI-AGGREGOMETER: for
simultaneous measurement of platelet
aggregation and the secretion of ATP.
HEMATOLOGY 2
OTHER TESTS
CLOT RETRACTION TIME
- PRINCIPLE: When blood coagulation is complete,
clot normally undergoes retraction where clot
becomes denser and serum is expressed.
Normally, clot retraction begins within 30
minutes after the blood has clotted &
completed within 24 hours.
- Normal clot retraction requires normal number
of functioning platelets. Ca, ATP, fibrinogen,
normal interaction of the platelets with
fibrinogen.
- Thrombosthenin; ratio of the plasma volume
and red cell mass; activity of a retraction-
promoting principle in serum & nature of the
surface on which CRT is being measured.
CLINICAL SIGNIFICANCE:
× 100
- CRT is poor when platelet count is less than
100,000/uL
- in dysfibrinogenemia or hypofibrinogenemia,
paraprotinemias
a. HIRSCHBOECK METHOD (Castor oil method)
- Qualitative: test for the presence or absence of
retraction
- Formation of dimpling/droplet like serum on the
surface of blood drop.
- Normal values equals to 15-45 minutes.
b. STEFANINI METHOD (test tube)
- Normal: clot retraction begins within 1 hour,
complete within 18 to 24 hours.
c. MAC FARLANE METHOD
- Provides quantitative estimate of the degree of
retraction.
- Normal values equals to 44 - 67%.
𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑒𝑟𝑢𝑚
- %CRT= 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 × 100
𝑏𝑙𝑜𝑜𝑑
 HEMATOLOGY 2
SECONDARY HEMOSTASIS: COMPONENTS,
PATHWAYS, TESTS; DISORDERS
SECONDARY HEMOSTASIS
- involves coagulation factor deficiency
(hemophilia A/B/C)
- Depends on availability and functionality of
different plasma coagulation factors.
PLASMA FACTORS
- These are soluble plasma coagulation proteins
that have crucial functions in the formation of a
clot. These proteins circulate as inactive
zymogens (proenzymes) that become activated
during the process of coagulation and, in turn,
react with other factors in the system to
ultimately generate thrombin which converts
fibrinogen to a localized fibrin clot.
CLASSIFICATION OF COAGULATION FACTORS:
 Substrate: are the substance upon which
enzymes act.
 Cofactors: accelerate the activities of the
enzymes that are involved in the cascade. (TF,V,
VIII, HMWK)
 Enzymes: serine proteases and transaminase
NOMENCLATURE:
- Except for Prekallikrein (PK) and HMWK which
are referred to by their name only, each factor
was assigned a Roman numeral number in order
of its discovery. The use of “a” that follows as
Roman numeral denotes the activated form. A
“f” refers to fragmented factor (e.g. XIIf)
GENERAL CHARACTERISTICS: (SUMMARY)
 All are synthesized by the Liver except factors
VWF, III, and IV
 Deficiency in any factor may produce abnormal
hemostats; except of factors XII, PK and HMWK
 Labile factors: V and VIII (short shelf life)
 Factors prematurely activated by refrigerator
temperature.
QUESTIONS TO REMEMBER:
 Composition of Prothrombinase: Xa-Va-Ca++-
PL
 Composition of the intrinsic tenase: IXa-VIIIa-
Ca++-PL
 Factors present in fresh plasma: all are
present: no deterioration
 Factors present in aged serum: II, VII, IX, X
(prothrombin); XI, XII, HMWK, PK (contact)
 Clotting → fresh serum: (-) I, II, V, VIII, XIII
 Storage: plasma: (-) V and VIII (labile factors)
 Adsorption → absent in plasma(-): II, VII, IX,
X
 Present in adsorbed plasma: I, V, VIII, XIII
(fibrinogen): XI, XII, HMWK, PK (contact
group)
 Most abundant: fibrinogen and prothrombin
→ less likely to be deficient.
 Factor II → 80% convert to thrombin and only
20% remain in circulation
 Factors in the Common Pathway: I, II, V, X
 Factors tested by both PT and APTT: I, V, II, X
 Fibrinogen Factors: I, V, VIII, XIII
 Coagulation factor deficiencies not related to
bleeding: XII, PK, HMWK.
GENERAL RULE:
All coagulation factors are produced by the LIVER.
EXCEPT factors: VWF, III, and IV
VON WILLEBRAND FACTOR (VWF)
- Produced by endothelial cells or endothelial
lining (Weibel palate bodies), platelets and
megakaryocytes (alpha granules).
FACTOR III: THROMBOPLASTIN
- Abundant: produced by all tissues in the body
except blood cells.
- When abnormally produced can trigger DIC
patients release thromboplastin-like substance
that will activate both coagulation and
fibrinolysis.
- Crude extract from the tissue
- Component: tissue factor and phospholipid
- COMPLETE: Tissue factor and phospholipid
 HEMATOLOGY 2
- PARTIAL/INCOMPLETE: phospholipid
component only
- PTT: reagent used is complete thromboplastin
- APTT: reagent used is phospholipid from partial
thromboplastin
FACTOR IV (CALCIUM)
- Abundant in the plasma in ionized form.
- Can interact in coagulation mechanism.
- Sources: diet, bone, reabsorbed in the filtrate
ZYMOGENS: II, VII, IX, X, XI, XII, AND PK
 Zymogens – proenzymes: inactive enzymes; no
enzymatic function unless activated.
 II (Prothrombin): inactivated → IIa (thrombin) →
fibrinogen → fibrin; and activate other factors.
 VII (stable factor: no fxn) → VIIa → activate
factor X and IX
 PK → kallikrein → enzymatic ability: serine
proteases
SERINE PROTEASES
- When zymogen is activated and functions as
serine proteases → which when activated,
functions to activate the next enzyme or factor
in the pathway.
- e.g. XIIa → activate XI → XIa → IX → IXa → X →
Xa → prothrombin → thrombin
NOTE:
EXCEPT: I and XIII
- Factor I (fibrinogen): never activated (Note: never
write it as Ia) → write it as fibrin or fibrin monomer
(activated)
e.g. thrombin once formed will activate fibrinogen to
become fibrin (wrong) → use the word cleaves
- Last to occur/be activated: cleavage of fibrinogen to
fibrin.
FIBRINOGEN: ultimate substrate in the coagulation
mechanism (acted by thrombin).
Cofactors: V, VIII, III, HMWK
FACTOR XIII: FIBRIN STABILIZING FACTOR/LAKI-
LORAND FACTOR
- (activated by thrombin) → transaminase or
transglutaminase enzyme → create covalent
bonds between and among fibrin monomers →
stabilization (cross-linkages) → fibrin as
insoluble clot
- Fibrin polymer: first clot formed (soluble and
unstable) unless factor XIII acted on it to stabilize
it.
o The one stabilizes the unstable and
soluble clot by forming covalent linkages
or cross-linking the fibrin monomers
together.
NOTE:
EXCEPT: Co-factors: V, VIII (need to be activated); III,
HMWK (need not to be activated)
Co-factors: increase activity of another coagulation
factor
Activated by thrombin: V → Va and VIII → VIIIa →
function as cofactor.
Va cofactor of Xa → Prothrombinase (Xa-Va-Ca++- PL)
Xa → directly converts prothrombin to thrombin;
need Va to increase its function
VIIIa cofactor of IXa → Intrinsic tenase (IXa-VIIIa-Ca++-
PL) needed to activate factor X to Xa
VIIIa: increases the activity of IXa in activating X
Factor III (Tissue factor) – cofactor of VIII- when
combined to factor III, will be able to generate X and
Xa
Extrinsic tenase: TF-VIIa → activate Factor X
HMWK: cofactor of XIIa; enhances XIIa activity
Function of XIIa: 1.) XIIa → PK → Kal; 2) activate XI
→ XIa
NOTE:
XII, PK, and HMWK deficiency – NO HEMOSTATIC
(BLEEDING) ABNORMALITY (IN VIVO STATEMENT)
IN VITRO: Prolonged APTT
In some, PK and XII deficiency  thrombosis
(abnormal clot formation)
NOTE:
FACTOR I (FIBRINOGEN)
- First to be discovered and most abundant
- Fibrinogen deficiency is unlikely to occur.
- PLASMA PROTEIN (absent in serum due to
consumption → fibrin) → NOT a serum
protein)
LABILE FACTORS: V and VIII (AHF-A)
- Immediately disappear in stored plasma at
room or ref temperature (in vitro).
 HEMATOLOGY 2

- Factor V (labile factor/proaccelerin) – most

labile.
- Factor VIII: labile in vitro and in vivo if it is not
being complexed with VWF.
- APTT (intrinsic pathway test): prolonged in
factor VIII deficiency and liver diseases.

FACTOR VII (STABLE FACTOR)

- IN VITRO: most stable → present in plasma
even if it is stored for several hours.
- IN VIVO: has shortest circulating life (3 hours)
and it is the first to disappear in the
circulation.
- In liver disease: first to be deficient
- FXN: in extrinsic pathway, it is the only
coagulation factor involved in extrinsic
pathway
- Prothrombin Time Test (extrinsic): prolonged
in VII deficiency and liver diseases.
- Prothrombin time: test that is more sensitive
in evaluating liver diseases, vitamin K
deficiency or antagonist.

PLASMA COAGULATION FACTORS (13)

- 12 coagulation factors assigned with Roman

numeral number (RNN) and named according to
order of their discovery.
- Two assigned with RNN but often referred by
their name: Factor III (Tissue factor or Tissue
thromboplastin) and IV (Ionized calcium).
- Factor VI: assigned to a discovered coagulation
factor (removed)  same as activated factor V
(Va): did not move/rearranged assignment of
coagulation number.

COAGULATION FACTOR NOT ASSIGNED WITH ROMAN

NUMERAL NUMBER:

1. HMWK (Kinin system)

2. PK (Kallikrein system)
- Belong and functions to other system.
- Also have a role in coagulation (minor).
3. Platelet factor 3: Platelet phospholipids →
exposed when platelets are activated:
phosphatidyl serine (major) and phosphatidyl
ethanolamine → assembly molecule →
formation of enzyme complex.
 HEMATOLOGY 2

NUMERAL NO. PREFERRED NAME & FUNCTION BIOLOGIC HALF- MEAN

SYNONYM/S LIFE; REMARKS CONCENTRATION
I Fibrinogen Thrombin 2-4 days 200-400 mg/dl
substrate
II Prothrombin Serine protease 3-4 days 10 mg/dl
 Prethrombin
III Tissue Factor Cofactor Lipoprotein found in None
 Tissue Thromboplastin most of the body
tissues; insoluble
IV Calcium Mineral Ionized state 8-10 mg/dl
V Proaccelerin Cofactor 36 hrs 1 mg/dl
 Labile factor Most unstable &
 Accelerator globulin deteriorates
(AcG) Rapidly at RT
VII Proconvertin Serine protease 3-6 hrs; disappears 0.05 mg/dl
 Stable factor rapidly from the
 Serum Prothrombin blood when
Conversion Accelerator production is halted
 Autoprothrombin I (e.g. coumarin)
VIII:C Antihemophilic factor (AHF) Cofactor 10-14 hrs 0.01 mg/dl
 Antihemophilic globulin Bound to vWF
(AHG) (Factor VIII complex)
 Antihemophilic factor A
(AHF A)
 Platelet cofactor I
IX Plasma thromboplastin Serine protease 18-24 hrs 0.3 mg/dl
component (PTC)
 Christmas factor
 Antihemophilic factor B
(AHF B)
 Platelet cofactor 2
X Stuart-Prower factor Serine protease 40-60 hrs 1 mg/dl
 Stuart factor May be stored up to
 Prower factor 2 mons at 40C
 Autoprothrombin III
XI Plasma thromboplastin Serine protease 40-70 hrs 0.5 mg/dl
antecedent (PTA) Circulates in
 Antihemophilic factor X complex with
(AHF C) HMWK
XII Hageman factor Serine protease 50-70 hrs 3 mg/dl
 Glass factor Heat stable; can be
 Contact factor stored at 40C for 3
mons
XIII Fibrin stabilizing factor (FSF) Function? 11-14 days 2 mg/dl
 Laki-Lorand factor
 Fibrinase; Fibrinoligase
 Plasma
transglutaminase
Prekallikrein (PK) Serine Protease 35 hrs 35-50 mcg/ml
 HEMATOLOGY 2

 Fletcher Factor
High Molecular Weight Kininogen (HMWK/HK) cofactor 9-10 hrs 5 mg/dl
 Fitzgerald factor; Contact activation
cofactor
 William factor; Flaujeac factor
Other Procoagulants:
VWF von Willebrand factor Factor VIII carrier; 24 hrs 1 mg/dl
Functions in platelet
adhesion
Platelet factor 3 Phospholipids- Assembly molecule Released by
primarily the platelets
Phosphatidylserine,
PF3

NOMENCLATURE FOR FACTOR VIII OF THE Test: Bleeding time test

INTERNATIONAL COMMITTEE ON THROMBOSIS AND Portion of molecule responsible for
HEMOSTASIS binding to endothelium and supporting
- In circulation, FACTOR VIII circulates in complex normal platelet adhesion and function.
with vWF. Tested by bleeding time
VIIIC: Ag Test: Immunologic monoclonal Ab
Antigenic property of procoagulant
2 MAJOR COMPOSITIONS portion as measured by immunologic
monoclonal antibody techniques
a. High Molecular Weight VWF (autosomal/
vWF: Ag Formerly known: VIIIR: Ag
chromosome 12)
Test: Laurell rocket or
- stable and serve as protein carrier for VIII and immunoradiometric factor assay
low molecular weight VIII:C (sex-linked in X Factor VIII-related antigen, which is a
chromosome) property of the large vWF portion of the
- labile if not carried by vWF  in plasma circulate molecules and measured by immunologic
in complex techniques of Laurell rocket or
b. Low Molecular Weight VIIIC (sex-linked in X immunoradiometric assay
chromosome) The antigen portion of the protein
- labile if not carried by vWF)  in plasma portion of vWF; now vWF:Ag
circulate in complex VIIIR:RCo Ristocetin cofactor activity
Allows platelets to interact or aggregate
VIII/vWF VIII:C and VWF with ristocetin
The molecule as it circulates in the plasma Ristocetin cofactor activity, which is
Composed of VIII:C and VIII:vWF portions factor VIII-related activity required for
non-covalently bound aggregation of human platelets with
VIII:C Function as a cofactor in intrinsic ristocetin in in vitro aggregation studies.
pathway coagulation (antibiotic no longer used
Test: Activated Partial Thromboplastin therapeutically)
Time (APTT/ PTT)
Portion of molecule acting in intrinsic
system as cofactor to IXa (with calcium) in NOTE:
the conversion of factor X to Xa. Tested by FACTOR DEFICIENCY- Leads to bleeding
partial thromboplastin time XII, PK & HMWK Deficiency- No bleeding abnormality
The pro-coagulant portion
VIII: vWF Function in primary hemostasis:
adhesion (platelet function)
 HEMATOLOGY 2

THE 3 GROUPS OF COAGULATION FACTORS (12)

- Based on their common characteristics or
properties

1. FIBRINOGEN GROUP
- Factors: I, V, VIII, XIII
- They are large molecules
- Act as Substrate for Plasmin
- Consumed during the process of coagulation
- They are susceptible to denaturation (most
labile).
- Increase in concentration during an
inflammatory response.
- Increased in women using contraceptive pills
and in pregnancy

2. PROTHROMBIN GROUP (VIT.K DEPENDENT

GROUP)
- Factors II,VII,IX,X
- They are dependent on vitamin K for their
synthesis in the liver
- Oral anticoagulants: cause a functional decrease
in these factors by producing abnormal proteins
that cannot bind calcium
- They are not consumed during coagulation
- They are stable compounds that are well
preserved in stored plasma.
- They require calcium as a cofactor for binding to
phospholipids surfaces.
- Are readily adsorbed from the plasma by BaSO4,
or Al(OH)3.
- They are decreased in patients with problems in
gastrointestinal absorption

3. CONTACT GROUP
- Factors: XI,XII, Prekallikrein, HMWK
- They are fairly stable and not consumed during
coagulation.
- They require contact with negatively charged
surface for activation.
- They are closely related to fibrinolytic, kinin, and
complement systems.
 HEMATOLOGY 2

Characteristics FIBRINOGEN GROUP PROTHROMBIN GROUP CONTACT GROUP

Members I, V, VIII, XIII II, VII, IX, X XI, XII, HMWK, PK
Consumed during CONSUMED NOT CONSUMED NOT CONSUMED
Coagulation/ Exception: Factor II-
clotting partially consumed

Factors in serum ABSENT in Serum PRESENT PRESENT

PRESENT in Plasma
Adsorbed by NOT ABSORBED ADSORBED NOT
BaSO4/ Al(OH)3

Factors present in PRESENT ABSENT PRESENT

BaSO4 adsorbed
plasma
Vit. K dependent NOT REQUIRED
Require Ca++ for NOT
binding to PL
surface
HIGHEST Molecular Weight
MOST LABILE
(V and VIII)
Absent in stored plasma: V
and VIII
Substrate to plasmin (main
CHON for fibrinolysis)
Thrombin cleaves factor I, V,
VIII, XIII
Found in platelets:
I and V: alpha granules
XIII – general cytoplasm of
the platelets (not stored in
granules)
EXCEPT: VIII:C  plasma
bound to VWF (protein
carrier)
Increase in inflammation
(APP) and pregnancy; in
women using contraceptive
(V and VIII)
DEPENDENT NOT REQUIRED
LEVELS NOT AFFECTED
(For functional ability of the
factors)  binding on surface
of platelet or any phospholipid
REQUIRES NOT
Needs to be carboxylated in
order to interact with Ca++ in
the presence of vit. K
STABLE STABLE
Vitamin ADEK fat soluble
vitamins  should be
properly digested bile for
fat solubilisation PRESENT IN ALL SAMPLES
If there’s an obstructive
jaundice  bile can’t be
drained in duodenum
Undigested fats and vit K.
will not be reabsorbed Closely related to fibrinolytic,
kinin and complement systems
Decrease GIT absorption
problems: (e.g. Obstructive 1. XII  XIIa = coagulation
jaundice, excessive bacterial 2. XIIa  PK Kal  Kininogen
overgrowth) = Kinins
3. XIIa-Kal: plasminogen
Affected by oral activator  plasmin =
anticoagulants: immediate fibrinolysis
action: coumarin, 4. Plasmin  complement
coumadin, warfarin - inhibit activation
synthesis of vit. K; vit. K
antagonist
 HEMATOLOGY 2

NOTE: THEORIES ON COAGULATION

ADSORPTION: process of removal of different
coagulation factor; adherence of smaller molecule to 1. MORAWITZ/CLASSICAL THEORY (1905)
larger molecule or surface. - Believes that there are 2 stages coagulation:
BaSO4/ Al(OH)3: large molecule unto which small a. Thrombin formation: by the combination of
molecules adhere prothrombin plus calcium ions plus tissue
Prothrombin Group: needs Vit. K for them to interact thrombokinase.
with Ca++ → bind with platelet PL
Excessive fibrinolysis: plasmin destroys intact factor V
and VIII

b. Fibrin formation: the formed thrombin acts

on the fibrinogen in the plasma.

2. HOWELL’S THEORY (1910)

- This theory believed that there are 5
coagulation factors involved: (Fibrinogen,
FACTORS II, VII, IX, X: Terminal AA (Glutamic acid): Calcium, Prothrombin, Anti-prothrombin,
requires additional –COOH (carboxyl radicals)  Thromboplastin)
gamma carboxylation (addition of another –COOH)
 to make it an anion (net negative charge)
interact with cation (Ca++) adhere or assembly on
the surface of platelet phospholipid (negatively
charge).
Gamma carboxylation: Vitamin K is required as a
cofactor for Factor IX to gain a negative charge for it
to attach with calcium.

Sequence of reactions:
NOTE:
1. Clotting (Serum): (-) I, V. VIII, XIII a. Prothrombin is inactivated by anti-
2. Adsorption: (-) II, VII, IX, X prothrombin/ heparin
3. Storage Age: (-) V and VIII b. When prothrombin needs to be activated
(vessel injury) → Thromboplastin neutralizes
anti-prothrombin and releases prothrombin
COAGUALATION MECHANISM c. Prothrombin → (Ca++ and thromboplastin) 
- Coagulation is a complex series of cascading Thrombin
reactions involving development of enzymes d. Thrombin + Fibrinogen → Fibrin
from their precursor. As one enzyme formed, it
converts the next zymogen to its activated 3. MODERN THEORIES: “WATERFALL” &
enzyme. This process continues until a fibrin “CASCADE” (1964)
meshwork clot has formed. Fibrin forms a - By Breckenridge and Ratnoff
meshwork in and around the primary hemostatic - 3 distinct phases:
platelet plug-like spider web, producing a stable
physical barrier.
 HEMATOLOGY 2

 Activation of factor X via the intrinsic or extrinsic

pathway (start of COMMON PATHWAY).
 Conversion of prothrombin to thrombin
 Conversion of fibrinogen to fibrin
 Advocated the phases of coagulation

PHASES OF COAGULATION: Coagulation is divided

into four phases
1. Contact Activation Phase (XII – IX)
- Starts with the activation XII (comes in contact
with negative surface), then activation of
prekallikrein to kallikrein; and factor XI.
2. Activation of Factor X (via intrinsic Pathway or
via Extrinsic Pathway)
- Formation of Thrombin: Conversion of
Prothrombin to Thrombin
3. Formation of Fibrin
COAGULATION CASCADE
IN VITRO COAGULATION
1. INTRINSIC SYSTEM
- All necessary factors are IN the blood
(circulation) VIII, IX, XI, XII, HMWK, PK
- All components are found in the circulating
blood. To be activated, the blood must have
direct contact with a foreign object such as a
damaged blood vessel or glass. This results to
absorption of XII of the negatively-charged
collagen exposed by vessel wall damage.

Sequence of reactions:

a. Factor XII is activated by the foreign material

into XIIa;
b. XIIa reacts weakly with XI, prekallikrein (also by
XIIf), and plasminogen, thus converting them
into their forms
 HEMATOLOGY 2
- Formed kallikrein feeds back to XII and causes
enzymatic cleavage producing more XIIa.
c. XIa in the presence of Ca activates IX
Note: IX can also be activated by VIIa-Ca-TF complex:
(Current concept of Coagualation)
d. IXa in the presence of Ca, platelet phospholipids,
and VIIIa activates X.
NOTE:
APTT: deficiency in any factors  PROLONGED APTT
Activators: Exposed collagen (major component of
connective tissue that surrounds and support BV)
and chemical  XII (contact factor)  XIIa 
perform several functions:
XII (glass factor/contact factor)  XIIa
1. Coagulation cascade: 1. (CONTACT ACTIVATION
PHASE: Activate XI  IXa); (IXa-VIIIa –Ca++-PL:
Intrinsic tenase); convert PK to Kal
2. Initiate fibrinolysis: XIIa  plasminogen to
plasmin
e.g. Which of the pathway occurs when the blood is
placed on a tube or glass slide and allowed to clot?
INTRINSIC PATHWAY  coagulation XII (glass factor)
 XIIa
2. EXTRINSIC SYSTEM/ TISSUE FACTOR PATHWAY
- All factors needed are in the tissue NOT in the
circulation. Produce by all tissues but not by
blood cells. ONLY FACTOR VII is involved.
- Does not occur outside the body unless you add
a reagent
- Tissue Factor/Tissue thromboplastin
introduced in the circulation when there’s
vessel/tissue injury.
- In classical concept: TF activates VII  VIIa and
combines with VIIa
- In current concept: VIIa (inert material) is
already available in the circulation in small
amount (1-2%)  combine with TF/Factor III
(cofactor)
 EXTRINSIC TENASE: TF + VIIa = TF/VIIa 
activates X in common pathway
 Tissue factor is necessary for the
activation of this system. All cells with
the possible exception of those in the
blood, contain tissue factor. When
injury occurs, the tissue factor is
released and acts as a cofactor in
initiating coagulation. The released
tissue factor bind to VII and activates it
to VIIa. Then, VIIa, together with
calcium, activates X to Xa of the
common pathway.
 VII  prothrombin time: deficiency of
VII  prolonged PT
3. COMMON PATHWAY
- Where both intrinsic and extrinsic pathway
usually meet or merge. The application is in in-
vitro coagulation. Superseded by the current
concept of the coagulation  REVISED
COAGULATION MECHANISM (In vitro:
laboratory tests)
- Begins with activation of factor X via the
Intrinsic Pathway (IXa-VIIIa-Ca-PL) or via the
Extrinsic Pathway (VIIa-III/TF) to form
Prothrombinase/Xa-Va-Ca-PL. This
prothombinase complex converts prothrombin
to thrombin which enzymatically cleaves
fibrinogen into fibrin.
- Thrombin activates XIII  XIIIa
(transaminase/transglutaminase)
- 4 FACTORS INVOLVED: X, V, II, I  tested by
PT/APTT.
 HEMATOLOGY 2
NOTE:
In performing a laboratory test on coagulation, follow
and interpret the test using the CLASSICAL CONCEPT
OF COAGULATION
Routine Screening Test for coagulation factor
deficiency: COAGULATION TESTS: measure how long
does the plasma take to form a clot; end result 
clot formation
Intrinsic: APTT
Extrinsic: PTT (Prothrombin Time)
PT: prolonged; APTT: Normal  Extrinsic factor
deficiency (VII)
PT: Normal APTT: prolonged  Intrinsic factor
deficiency (XII, XI, IX, VIII, HMWK, PK)
Both prolonged  Coagulation factor deficiency of
the common pathway (X, V, II, I)
In APTT/PTT (in vitro testing): XIII is not included 
not detected
XIII not needed for the formation of clot  stabilize
the clot
NOTE:
COAGULATION ROLES OF THROMBIN: activates V,
VIII, XIII, cleaves fibrinogen + activates XI.
FIBRIN FORMATION
FIBRINOGEN STRUCTURE:
- D-terminal domains and a central-E domain
- 3 types of polypeptide chain (pairs):
o + Fibrinopeptides (2A)
o Alpha (2B) beta
o Gamma (no fibrinopeptide)
- Fibrinopeptide: removed by thrombin
- Fibrinogen: soluble protein; not seen in plasma,
but once fibrinopeptide A and B is removed 
insoluble (clot is seen).
 alpha, beta and gamma chains terminates in D-
domain and meet at the center coil E-domain
 exposing alpha and beta fibrinopeptides 
thrombin (proteolytic enzyme) cleaves
fibrinopeptides A and B  removed  fibrin
monomer (insoluble)  several formed 
polymerization: end to end and side to side
connection  fibrin polymer (clot) soluble and
unstable clot  fibrin cross-linking by factor
XIII  covalent linkages in D-terminal 
stabilized fibrin
STEPS IN CONVERTING FIBRINOGEN TO FIBRIN
1. Thrombin cleaves the alpha and beta chains
only (releasing fibrinopeptides A and B) 
soluble fibrin monomer/unstable gel.
2. Fibrin monomers aggregate spontaneously end
to end, side to side to form soluble fibrin
polymers thus vulnerable to enzyme plasmin
(also soluble in 5M urea).
3. Factor XIII: XIIIa + Ca crosslinks adjacent fibrin
monomers by forming covalent bonds to form
stabilized fibrin.
OTHER RELATED SYSTEMS:
1. Kinin System: this system is involved in
chemotaxis, pain sensation, and mediate
inflammatory responses, increased vascular
permeability, and vasoconstriction and induce
smooth muscle contraction.
2. Fibrinolytic system: The Kallikrein, XIIa, &
HMWK act as plasminogen activator.
3. Complement System: this mediate immune and
allergic responses.
 HEMATOLOGY 2
CLASSICAL (IN VITRO)
- Does not explain how physiologic hemostasis
occur in vivo.
- Interdependent pathways
IN VIVO CONCEPT OF COAGULATION/ CURRENT
CONCEPT/ REVISED CONCEPT OF COAGULATION
- Current Concept of the Coagulation Cascade (In
vivo coagulation)
- INITIATING step: exposure of TF and its reaction
with VII.
2 FACTORS ACTIVATED BY TF/VIIa: X and XI 
production of thrombin
a. INITIATION: Extrinsic tenase  3%- 5% lIa
- Small effect of activation of IX and X + Tissue
Factor Pathway Inhibitor (naturally occurring
inhibitor) inhibits VIIa and Xa  less thrombin
production.
b. PROPAGATION (platelets)  95%
lIa/Thrombin:
- Self-perpetuating /autocatalytic.
- When complexes are increased in phospholipids,
the more thrombin production.
NOTE:
The key initiating step is the exposure of TF to the
circulation and reaction of TF with factor VII. The TF-
factor/VIIa complex can enzymatically activate
factors X and IX. The initial activation of factor X to Xa
may be important in getting the coagulation cascade
started, however, tissue factor pathway inhibitor
(TFPI) rapidly inactivates the TF-VIIa-Xa complex. The
major action of the TF-VIIa complex in vivo is the
activation of factor IX to IXa, which then activates
factor X to Xa. Consequently, prothrombin is
converted to thrombin, then fibrinogen is converted
to fibrin. Factors Xa and IIa have positive feedback
activity on earlier steps of the cascade.
NOTE:
FACTOR XII: In vivo coagulation; their role is not
necessary; can be by passed; XI can be activated by
thrombin; deficiency will not cause bleeding
conditions.
Key initiating step: cause to start the reaction:
activation of XII and VII (more important to occur);
VIIa/TF  series of reaction
Xa and IIa (positive feedback activity: once formed go
back to their initial reaction and proceed to cause
more activation)
 Xa  VII  VIIa (2 chain) more effective in
function
 VIIa (single chain) produce by other factors or in
plasma; less efficient.
 Thrombin: once formed, it can cause increase in
production.
THROMBIN FEEDBACK MECHANISM
ACTIVATION OF COAGULATION
1. Autocatalytic or self-perpetuating: many more
thrombin from small amount present
2. LOW level of thrombin activates V and VIII
3. Activates XIII  XIIIa; calcium dependent
4. Induces platelet aggregation; agonist
5. Also activates factor XI; current concept
6. Procoagulant: Activates TAFI (thrombin
activatable fibrinolysis inhibitor) – inhibits
fibrinolysis; produced by endothelial cell
INHIBITOR TO COAGULATION
1. Increased thrombin: destroys Va and VIIIa  prevents
clotting/continuous clotting Disseminated coagulation
2. Activate Protein C (w/ Ca++ and thrombomodulin) 
inhibitor of coagulation (CHON S)  plasmin generation
 inactivation of Va Vi and VIIIa  VIIIi
3. Promote plasmin generation (fibrinolysis)
 Controls excessive coagulation
 Increase concentration of thrombin (HIGH level);
destroys V and VII; activated protein C
 Protein C and S increase plasminogen activation
 HEMATOLOGY 2
QUESTIONS TO REMEMBER:
1. What may activate II: PROTHROMBINASE: II  (Xa-
Va-Ca-PL)  IIa
2. What may activate XII: in vivo: collagen or any
other negative surface or chemicals; in vitro: glass
contact
3. What may activate X: X  (IXa-VIIIa-Ca-PL)  Xa
4. What may activate V and VIII: THROMBIN (IIa): V
(thrombin)  Va ; VIII  (thrombin)  VIIIa
5. What may activate XI: XI  (XIIa, IIa)  XIa for
propagation
6. What may activate IX: Extrinsic tenase and XIa: IX
 (VIIa/TF) or (XIa)  IXa
7. What activation reactions can be by-passed: XII 
XIIa (even without, coagulation can still occur)
FIBRINOLYSIS
- Last phase of Coagulation
- Re-establishment of blood flow → lysis of clot.
- It is defined as the dissolution of clot or lysis of
clot by plasmin or fibrinolysin.
- Plasmin: not normally present in plasma and it is
derived from plasminogen, which is produced by
the liver.
- Plasminogen: normally present in plasma;
precursor synthesized by liver; it will convert into
plasmin by plasminogen activators (3 activation
phases).
EXCESSIVE/ UNREGULATED FIBRINOLYSIS:
- May lead to disorders: inability to clot/excessive
clotting.
REGULATE FIBRINOLYSIS: REGULATORY PROTEINS
 PLASMINOGEN ACTIVATOR INHIBITOR (PAI)
- It inhibits the activators of plasminogen to
prevent excessive production of plasmin.
 ALPHA-2-ANTIPLASMIN
- Inhibit plasmin; prevent excessive function of
plasmin
FIBRIN DEGRADATION PRODUCTS (FDP) OR FIBRIN
SPLIT PRODUCTS (FSP)
- released after the fibrin clot is broken down
ACTIVATION OF PLASMINOGEN → PLASMIN
WAYS OR PHASES: PLASMINOGEN ACTIVATORS
1. INTRINSIC ACTIVATION: XIIa AND KALLIKREIN
- by XIIa-Kallikrein-HMWK
- Contact factors are closely related to the
fibrinolytic system: → XIIa-Kal → plasminogen
 plasmin
 HEMATOLOGY 2
2. EXTRINSIC ACTIVATION: TPA (tissue type)
- Tissue Plasminogen Activator(TPA): produced by
endothelial cells
3. EXOGENOUS ACTIVATION
- introduced into patient: Urokinase and
Streptokinase Therapeutic and fibrinolytic agent:
prevent occurrence of abnormal clot
o Urokinase: present in the urine
o Streptokinase: exogenous; comes from
strep organisms
INHIBITORS
 Plasminogen: Histidine-rich glycoprotein
 Activators (TPA): Thrombin Activatable
Fibrinolysis Inhibitor(TAFI) & Plasminogen
Activator Inhibitor (PAI)
 Plasmin: plasmin inhibitor (alpha-2-antiplasmin;
alpha-2-macroglobulin)
FIBRINOGEN LYSIS
FIBRINOGEN
- Made up of three polypeptide chains: alpha,
beta, and gamma.
ALPHA CHAIN
- first site that plasmin will be acted on:
1. Plasmin will cleave alpha chains  Fragment X
(D-E-D)
- Fragment X: first fragment to be formed;
largest.
2. Fragment X will be acted on by plasmin
asymmetrically (one side) to separate one of
the D’s  Fragment Y (E-D combination)
3. Plasmin will act on Fragment Y and separate E
from the D.
FIBRINOGEN DEGRADATION PRODUCTS (FDP)
 X and Y: early degradation products
 D and E: late degradation products
NOTE:
Fibrinogen Degradation Product = XYDE
FIBRINOLYSIS
FIBRIN
- Fibrin monomers (connect end-to-end (D-D
connection) and side-by-side (D-E-D connection)
 fibrin polymerization XIIa (stabilizes clot;
crosslinks by forming covalent linkages between
D-D).
FIBRIN DEGRADATION PRODUCTS X, Y, D-D DIMER, E
- Acted on by Plasmin
D-DIMER/ D-D PRODUCT
- indication (specific) of fibrin lysis (clot formed)
- main difference between the products
YY COMPLEX/ YY FRAGMENT: E-D-D-E
DXD COMPLEX: D-D-E-D-D
 HEMATOLOGY 2

DIMER: clot Fibrinolysis is activated within 24-48 hours.

MONOMER (FIBRINOGEN): no clot formed COAGULATION MECHANISM

Coagulation and Fibrinolysis should have balance
Excessive coagulation → thrombosis
Excessive fibrinolysis → bleeding

NOTE:
 Fibrin Degradation Product: X Y D (D=D; D-
dimer) E
 In normal coagulation/hemostasis/fibrinolysis
→ X, Y, D (D-D Dimer), E will be cleared by the
liver but not in excessive fibrinolysis and liver
diseases; or if increase production of FDP → NATURALLY OCCURRING INHIBITORS OF
bleeding. COAGULATION AND FIBRINOLYSIS
SERPIN FAMILY (Serine Protease Inhibitors)

EFFECTS OF FDP: INHIBIT OR INTERFERE IN NORMAL SERINE PROTEASES

COAGULATION - Those when activated there function is to
 X and Y: with anticoagulant effects activate another enzyme in the cascade.
 Y and D: inhibit fibrin polymerization - e.g. VII → VIIa → IXa
 Fragment E: inhibits thrombin
NAME FUNCTION
NOTE: Tissue factor pathway With Xa, binds TF:VIIa
Primary Hemostasis: rapid in response; platelet plug: inhbitor
short lived and unstable Thrombomodulin Endothelial cell surface
Secondary Hemostasis: slower response; fibrin clot: receptor from thrombin
long live and stable Protein C Serine protease
Protein S Cofactor
SUMMARY OF STEPS IN HEMOSTASIS Antithrombin Serpin
1. Vessel injury Heparin Cofactor II Serpin
2. Vasospasm (TxA2; serotonin) Z-dependent protease Serpin
3. Platelet plug → PRIMARY HEMOSTASIS inhibitor
4. Insoluble fibrin clot
Alpha 1 -protease Serpin
5. Clot retraction
inhibitor (alpha1 –
6. Clot dissolution or fibrinolysis
antitrypsin)
Alpha2-macroglobulin Serpin
Clot formation (in vivo)= 6 minutes
Clot formation (in vitro) = Red tube (15-20 minutes →
1 hour completely clotted)
 HEMATOLOGY 2

INHIBITOR COAGULATION FIBRINOLYSIS NOTES

Component(s) Component(s)
inhibited inhibited
Antithrombin III Thrombin, VIIa, IXa, Plasmin  First inhibitor discovered and tested
Xa, XIa and XIIa and  Main inhibitor of the entire coagulation
Kallikrein mechanism; major inhibitor of thrombin
 Enhanced by a co-factor Heparin
 It is a serpin.

THROMBIN FUNCTIONS:
1. Induces platelet aggregation
2. Activates factors XIII, V, VIII, XI
HEPARIN: does not remove Ca++

ROLE of HEPARIN: Cofactor of AT-III  inhibit

thrombin; serves as a bridge. Heparin when
bound to AT-III causes a morphological change to
be more specific to the size of thrombin.
Thrombomodulin Thrombin  It binds to thrombin and inhibits
thrombin (functions to activate protein
C).
 Produced by endothelial lining and it is a
membrane bound.
 Thrombin when bound to
thrombomodulin can’t activate factor VIII
and V instead it will activate protein C to
become Activated Protein C (APC).
 Trimolecular complex: Thrombomodulin
+ thrombin + Protein C + (receptor:
EPCR)  APC.
Thrombin INHIBIT
Activatable FIBRINOLYSIS
Fibrinolysis
Inhibitor(TAFI)

Plasminogen
Activator Inhibitor
(PAI)
Tissue Factor Xa & TF:VIIa  Kunitz-type serine protease inhibitor
Pathway Inhibitor complex that has a double inhibitory effect
(TFPI)  Major inhibitor of extrinsic pathway
3 Kunitz type:
Lipoprotein Kunitz 1: binds with VIIa; inactivating therefore
Associated VIIa-TF complex
Coagulation Kunitz 2: binds with Xa; inactivating Xa
Inhibitor (LACI) Kunitz 3: unknown fxn

Extrinsic Pathway X (intrinsic tenase: IXa-VIIIa-Ca-PL) or (extrinsic

Inhibitor (EPI) tenase: TF-VIIa) Inhibited by TFPI  Xa Inhibited
 HEMATOLOGY 2

by TFPI  NO: CAN’T HAPPEN: II 

(prothrombinase)  IIa

Alpha-2 Thrombin & Plasmin  Inhibits plasmin when alpha-2

macroglobulin Kallikrein antiplasmin is depleted.
C’1 inactivator/ XIIa, XIa,& Plasmin  Inhibitor in the complement pathway
C’1 esterase Kallikrein
inhibitor
Protein C and S Va and VIIIa Enhances plasmin  Cofactor: Protein S
system formation   Vit. K dependent inhibitor
ENHANCES  Inactivates inhibitors of plasminogen
FIBRINOLYSIS activators
 Produce as zymogen by the liver
 Activated by thrombin
 APC will inactivate VIIIa and Va  VIIIi
and Vi
Protein S: exists in 2 forms in plasma
Bound to C4bBP(complement pathway;
inflammation): 60%
Free Protein S: 40%  can ONLY serve as
cofactor
In inflammation (excessive coagulation): 85-90%
bound and 10-15% is free: can’t inactivate VIII
and V.
Alpha-1- XIa Plasmin  Inhibits plasmin only when a-2
antitryspin/ A-1 antiplasmin and a-2 macroglobulin are
protease inhibitor depleted.
Alpha-2- Plasmin  Major inhibitor of plasmin
antiplasmin  First to inhibit plasmin
Z-dependent Xa, XIa  Enhanced by its co-factor protein Z
protease inhibitor (CHON –Z) additional protein that is vit. K
(ZPI) – dependent like II, VII, IX, X
 Serpin – inhibits serine protease

Protein C inhibitor Thrombin, APC, TPA, urokinase  Anticoagulant

thrombin-  Pro-coagulant
thrombomodulin  Fibrinolytic inhibitor
complex

QUESTIONS ASKED:
1. Factors in the intrinsic pathway: VII, IX, XI, XII, HK, PK (deficiency in any prolongs APTT)
2. What are the cofactors: TF-VIIa; Va-Xa; VIIIa-IXa; HK-XIIa
3. The only transaminase/transglutaminase among the coagulation factor: XIII
4. What stabilizes fibrin: XIIIa
5. Vitamin K-dependent: II, VII, IX, X, C, S, Z
V- (labile factor) Pro-accelarin / acceleratorgobulin
VII- (stable factor) Pro-convertin / plasma conversion
IX- Plasma thromboplastin component = anti hemolytic factor B
XI- Plasma thromboplastin antecedent = anti hemolytic factor C
 HEMATOLOGY 2

Coagulation Group Pathway Test - One reagent in APTT/PT is calcium, therefore the
factor involved prolonged excessive citrate in blood sample will bind with
If factor is the reagent calcium  prolonged test result;
deficient calcium is inactivated by excessive citrate.
(PT/APPT/
BOTH) 2. Clots in specimen
Fletcher Contact Intrinsic APTT - Clotting happens due to consumption
factor (PK) group
coagulation factors (I, V, VII, XIII).
Accelerator Fibrinogen Common Both
- REJECT SAMPLE even the clots are micro clots
Globulin (V) group
- In coagulation studies, presence of even micro
Antihemophili Fibrinogen Intrinsic APTT
c factor A clots would cause the rejection of sample.
(VIII:C)
Proconvertin Prothrom Extrinsic PTT 3. Hemolysis and Traumatic phlebotomy
(VII) bin - Tissue Factor introduction
Plasma Contact Intrinsic APTT - Hemolyzed RBC: exerts thromboplastin-like
thromboplasti activity
n antecedent - Excessive probing  damage tissue 
(XI) thromboplastin: TF:VIIa  coagulation
Plasma Prothrom Intrinsic APTT mechanism.
thromboplasti bin
n component; 4. Icterus; Lipemia
Christmas - Affects spectrophotometry: more light
factor (IX)
absorbed  concentrated.
Stuart-Prower Prothrom Common BOTH
factor (X) bin COLLECTION OF SAMPLE
1. NEEDLE GAUGE
 Adult (<25ml): G20 - 21; 1- 1.25 in.
LABORATORY EVALUATION OF COAGULATION AND
FIBRINOLYSIS  Adult (>25 mL): G19; 1 or 1.25 in.
- All coagulation testing critically depends on the  G23: Child or adult with friable, or hardened
quality of the specimen. Minimum tissue veins.
trauma and the avoidance of hemolysis are  G20, 21, or 23: Syringe with winged-needle set
essential. It is therefore important to adhere to o The smaller the bore, the slower the
principles of proper specimen collection, blood will flow into the needle
handling and preparation. o The larger the bore, the easier will blood
will flow  prevent HEMOLYSIS.
GENERAL INSTRUCTION FOR COAGULATION TESTS o For G23(smaller): Avoid hemolysis by
- Time is important: Coagulation test like in XIII gently pulling of the plunger/ gentle
assay; any factors that may affect time should be aspiration
avoided. 2. SYRINGE
- Plastic syringe or silicone-coated
SOURCES OF ERRORS AFFECTING COAGULATION TESTS: - AVOID using GLASS in all procedure: use plastic
1. Blood volume is inadequate causing excess test tube, pipettes and syringes
anticoagulant - Glass will activate XII (glass factor)
- Recommended ratio: 1:9 using Na citrate
- In routine hematology: half full is accepted 3. ANTICOAGULANT
- 3.2% sodium citrate
- In coagulation studies: tubes should be at least
90% full.
 HEMATOLOGY 2
- If patient’s Hct is more than or equal to 55%,
adjust the volume of citrate since excess citrate
will bind with reagent calcium.
- ADJUSTING THE VOLUME OF CITRATE:
o C= (0.00185) (V)(100-H)
o Where:
 V = blood volume in mL
 H = patient’s Hct
 C= volume (mL) of anticoagulant
needed.
4. TEMPERATURE
- Most of the tests are performed at 37°C
- Perform: STAT
- But if there is delay  Freeze the PPP only; DO
NOT FREEZE WHOLE BLOOD/ BLOOD WITH RBC.
- Storage:
o PT (extrinsic pathway: VII (stable
factor)): uncentrifuge (with Na citrate)
and well capped at room temperature
for 24 hours
o Never refrigerate samples for PT: cold
temp  cold activation: VII and XI
o APTT/PTT (intrinsic pathway factors:
VIII, IX, XI, XII, HK, PK): uncentrifuge for
4 hours at 4°C.
o Factor VIII (labile at room and ref. temp.)
note also V (labile factor; common
pathway).
o Never leave at RT: VIII will deteriorate
o Test to monitor unfractionated heparin
therapy is APTT: heparin+antiheparin
factor (PF4)
o Unfractionated heparin (PTT) 
separate/centrifuge in 1 hour  Platelet
poor plasma (PPP) to prevent the action
of PF4 against the heparin.
o PPP: at 20°C for 2 weeks; at -70°C for 6
months.
SAMPLE PREPARATION
- Sample Used:
o Whole Blood
o Plasma
 Anticoagulant used: 3.2%
Sodium Citrate
 Aspirate always using plastic
pipette.
 Platelets are found in the buffy
coat layer.
NOTE:
Platelet Poor Plasma (PPP) preparation (<10,000/uL)
 Centrifugation: 2000 xg for 10 minutes;
Rodak’s: 1500 g for 15 minutes
 Collect only the upper 3⁄4 of the plasma
layer.
 Test immediately because exposure to air
changes the pH due to loss of carbon dioxide
(↑pH).
 Store plasma at 4°C but not to exceed 2
hours or rapid freezing to -20°C.
Platelet Rich Plasma (PRP) Preparation (200,000 –
300,000/uL)
 Centrifugation: 60 – 100 xg for 10 minutes at
RT; Rodak’s 50 g for 30 mins
 Separate upper portion of the sample
NOTE:
Coagulation factors are in plasma.
2 concentration of sodium citrate:
3.2% SODIUM CITRATE (recommended by NCLSI)
 V and VII (preserve well)
 Less affected by polycythemia vera
 Normal Ratio: 1 (citrate): 9 (blood)
In px with high hct: adjust volume of citrate to prevent
excessive citrate which will bind to reagent calcium.
3.8% SODIUM CITRATE
 easily affected by high hct samples or polycytemia
vera.
DO NOT use EDTA (labile factors are not well preserve,
inhibit thrombin-fibrinogen interaction, affects calcium)
and HEPARIN (inhibits thrombin).
 HEMATOLOGY 2
NOTE:
ROUTINE SCREENING TEST (PT/APPT): Light blue first
SPECIFIC FACTOR ASSAY (like VIII, VII assay): use a
prime/dummy tube, red top, light blue
FOR OPEN SYSTEM: Two-syringe method: after
collecting blood using the first syringe, detach
syringe without withdrawing the needle and replace
with another syringe. The blood in the first syringe
will be discarded and the blood in second syringe is
the one be used.
RULE:
1st: DISCARD
2nd: USE
NOTE:
What factors will deteriorate at room temperature? V
and VIII
Refrigerator temperature affects what factors? VII
and XI
PT plasma samples capped, are stable for 24 hours at
what temperature? Room temperature
APTT samples are stable for 4 hours if stored at 4°C.
COAGULATION TESTS
- All based on the time it takes for the blood
samples to form into a clot whether plasma or
whole blood.
- Accuracy in measuring time is necessary because
little excess may be reported as falsely
prolonged instead of normal.
TEST FOR INTRINSIC AND COMMON PATHWAYS
FACTORS TO DETECT: VII, IX, XI, XII, HMWK, PK
CONTACT FACTORS: I, II, V, X
TISSUE FACTOR: where activation starts in vivo.
1. COAGULATION TIME (Whole Blood)
- All of the components needed for clotting are
present in the blood.
- When blood is placed in a glass test tube or glass
slide, the blood will be activated.
- Factor XII is activated first by contact with glass
slide (PK to Kalikrein; XIIa activate XI to XIa; Xia
activates IX to IXa).
- Activation starts with contact activation phase
- Measures the time required for whole blood to
clot after it has been removed from the body
- Affected by amount of blood used.
A. SLIDE METHOD
 REFERENCE VALUE: 2-4 minutes
 REQUIREMENT: glass slide method
B. CAPILLARY METHOD (Date and Laidlaws
Method)
 REFERENCE VALUE: 2-4 minutes
 REQUIREMENT: Non-heparinized
capillary tube (blue ring) or else no
clotting will occur.
PROCEDURE FOR BOTH (SLIDE AND CAPILLARY
METHOD):
a. Fill the non-anticoagulated capillary tube & let it
stand for 2 minutes
- NOTE: do not wipe off the first drop of blood but
include it.
b. Start time as soon as blood starts to appear from
the site of puncture
c. For the slide method place 2-3 drops of blood in
a slide while for capillary tube, fill it with whole
blood
d. Wait for 2 minutes initial time before checking
for fibrin clot formation
e. CHECKING:
o SLIDE METHOD: check by using the same
lancet from collection by pricking into
the blood sample and raising the blood
 HEMATOLOGY 2
sample until a thin fibrin thread is
observed as thin as the hair & record the
time when the thin fibrin thread is
observed as the clotting time
o CAPILLARY METHOD: Break the glass
tube on one side without pulling them
apart and slowly pull them apart and
observed for a thin fibrin thread
connection, connecting both fragments.
f. If no fibrin thread is observed wait for another
30 seconds and check again and if it is not yet
visible again continue waiting for another 30
seconds and check again.
NOTE:
BOTH NORMAL VALUE: 2-4 minutes at RT
AFFECTED BY: amount of blood used
Micromethod requires fewer blood sample while for
macromethod, the longer is the clotting time.
C. LEE AND WHITE METHOD (Macromethod)
o REFERENCE VALUE: 7-15 minutes
o DISADVANTAGE: Longer clotting
time due to usage of larger blood
volume.
PROCEDURE (LEE AND WHITE METHOD):
a. Place 1 mL of each into 3 tubes with equal size
and dimension (12 x 75)
b. Label the tube as 1, 2 and 3
c. Extract 4 mL of whole blood
d. Start time as soon as blood appears at the
puncture site.
e. Place 1 mL of blood in each tube starting from
the 3rd tube up to the first
f. Discard the last remaining 1 mL
g. Stoppered the three tubes and incubate at 37°C
in a water bath for 5 minutes undisturbed
h. After 5 minutes, check for clot formation by
tilting the tubes at 45° angle
i. observed first the tube 1 up to tube 3
j. If blood flows to the tube, it is not yet fully
clotted thus incubate further for 30 sec.
k. After 30 secs, check tube 1 again and when
tube 1 does not spill its content upon total
inversion, clotting time is complete and record
- NOTE: do not report clotting time in tube 1
l. After 30 secs. Check for clotting for tube 2 and
same procedure will be employed as with tube
1
m. Once clotting is complete in tube 2, wait for
another 30 secs. Before reading tube 3
n. Once 30 secs is reach, check for clotting with
tube employing the same procedure with tube
and report the clotting time
NOTE:
TIME DIFFERENCE BETWEEN TUBES: 30 SECONDS
REPORTING: from time the blood starts to appear until
complete clotting time in tube 3.
REFERENCE VALUE: 7- 15 MINUTES
2. ACTIVATING CLOTTING TIME (reliable)
- PRINCIPLE: Whole blood contains all the
components necessary to produce a clot when
put into a glass tube. By adding an activator (e.g.
Diatomite) and keeping blood at constant 37°C.
- The test becomes more reliable and rapid.
- REFERENCE VALUE: 75-120 seconds
- ACTIVATOR: Diatomite – shortens clotting time
and enhances the coagulation factors
NOTE:
PLASMA CLOTTING TIME
- Coagulation factors are in the plasma
- Plasma is the one which clots
- Plasma clotting is translucent
1. No clot
2. Clotted
3. Clotted
4. Clotted
3. PLASMA RECALCIFICATION TIME
- Plasma was prepared using Na Citrate thus Ca is
removed.
- Plasma is calcium free thus add calcium (reagent)
- Upon addition of calcium take the time it takes
for plasma to clot
 HEMATOLOGY 2
- Reference value depends on the plasma used
(platelet-rich plasma or platelet poor plasma)
- No longer performed
- PRINCIPLE: except for Ca, normal PRP/PPP
contains all the components necessary for
generating fibrin.
- REFERENCE VALUE:
o PRP: 100-150 seconds
o PPP: 130-240 seconds
4. ACTIVATED PARTIAL THROMBOPLASTIN TIME
(APTT)
- Routine screening test
- Thromboplastin/Tissue Factor is involved in the
extrinsic pathway which activates VII to VIIa
and binds to it
- Test for Heparin therapy
- REAGENT: partial thromboplastin: cephalin
(phospholipid only) & 0.025 M Ca (reaction
initiation
- SAMPLE: Platelet Poor Plasma (PPP) (Hct <55%)
to avoid inappropriate with citrate and plasma
volume.
- ACTIVATORS: Kaolin, Celite, Ellagic acid
- PURPOSE: for determination of deficient in
intrinsic pathway
- PROCEDURE: Starts with the activation of FXII
upon contact with a negative surface. In vivo, it
may be collagen while for in vitro it is the glass
test tube. Coagulation cascade will not proceed
if intrinsic tenase and prothrombinase will not
be formed thus no fibrin formation. Both
intrinsic tenase and prothrombinase requires
Ca & phospholipid. The source of phospholipid
source is the platelets but since the sample is
platelet poor plasma, there would be no source
of phospholipid. While, Ca is removed by
citrate. Thus, cephalin and Ca is added in order
to observe clotting. Upon addition of cephalin
and Ca, observed the time it takes to form fibrin
clot
- Most useful procedure for routine screening of
coagulation disorders in the intrinsic system, for
detecting the presence of circulating anti-
coagulant and for monitoring heparin therapy.
Also measures factors present in the common
pathway, except for platelet and factor XIII.
- PRINCIPLE: Expect for Ca and Platelet
phospholipid (PPL), PPP contains all the
coagulation factors needed for the generation
of intrinsic prothrombinase. When Ca is added
with “incomplete” thromboplastin (serves as
platelet substitute), intrinsic prothrombinase is
generated.
- Difference:
o APTT: w/ activator
o PTT: w/out activator
NOTE: APTT
FACTORS THAT MAY PROLONG THE TEST:
A. Any factor deficiency (Intrinsic (VIII, IX, XI, XII,
HMWK, PK) and common pathway (I, II, V, X))
B. Presence of inhibitor – Factors VIII-I and FIX-I
C. Heparin (immediately process the sample and
prepare the plasma within 1 hour to prevent PF4 from
inhibiting the heparin)
D. <60 mg/dL fibrinogen – Factor 1 belongs to
common pathway.
E. Presence of increased Fibrin Split Products (FSP) –
all clot-based test will be prolonged in the presence of
FSP.
NORMAL VALUE: 25 – 35 SECONDS
NOTE:
 All of clot-based test cannot measure Factor
XIII deficiency & FXIII will be normal because it
is not needed for formation of clot.
 When thrombin acts on XIII, it will now
stabilize the clot.
 XIII is involved in common pathway.
TEST FOR EXTRINSIC AND COMMON PATHWAYS
EXTRINSIC: VII
COMMON PATHWAY: I, II, V, X
1. PROTHROMBIN TIME (PT) / QUICK’S TEST
- Starts with the action of VII.
- Dominant pathway that occurs in vivo, once
thromboplastin is introduced in the circulation,
it immediately combines with VIIa already
present in circulation in vivo but in vitro, VII
must be activated by thromboplastin. In vitro,
there is no source of thromboplastin when
 HEMATOLOGY 2

placed in a test tube. The reagent  Oral anticoagulants (warfarin, coumarin,

thromboplastin will provide the thromboplastin
which comes from tissues. Complete
thromboplastin is needed because it has to
supply the other missing factors such as
phospholipid and thromboplastin. The
composition of complete thromboplastin is
tissue factor and phospholipid. The tissue factor
in the reagent will activate VII and the
phospholipid in the reagent will supply the
missing phospholipid. Since citrate is utilized, Ca
is also removed thus 0.025 M CaCl2 is utilized
as a substitute. Upon addition of
Thromboplastin and CaCl2, record the time it
takes for plasma to form into clots
- Useful screening procedure for the extrinsic
coagulation mechanism including the common
pathway, also monitors oral anticoagulant
therapy with coumadin.
NOTE: PT
 PRINCIPLE: when tissue extract or
thromboplastin is added to PPP along with Ca,
it reacts with factor VII leading to subsequent
activation of factor X.
 NORMAL VALUE: 10 – 12 SECONDS
 THROMBOPLASTIN SOURCE: rabbit brain
extract or similar
 SPECIMEN: Citrated PPP to prevent excessive
citrate in plasma
 REAGENTS: Complete Thromboplastin (TF &
phospholipid) and 0.025 M CaCL2
 PURPOSE: in monitoring extrinsic and
common pathway deficiency, more sensitive
for monitoring oral anticoagulant therapy,
vitamin K deficiency and liver diseases
coumadin).
SOURCES OF ERRORS:
 Prolonged in the presence of heparin
contamination; can be shortened in traumatic
venipuncture.
REPORTING:
 INR to monitor effect of therapy and dose of
oral anticoagulant
ADDITIONAL REMINDER:
 Prolonged PT causes VII to disappear
immediately since it has a shortest lifespan in
vivo.
INTERNATIONAL NORMALIZED RATIO (INR)
- A standardized way of reporting PT in
monitoring oral anticoagulant therapy,
calculated as a ratio of the patient’s PT to a
control PT standardized for the potency of the
thromboplastin reagent developed by the
World Health Organization (WHO).
- Reference Values for INR:
o 2.0-3.0
 In prevention and treatment of
venous thrombosis
 Treatment of pulmonary
embolism
 Prevention of stroke in MI
 Peripheral arterial disease
 Prevention of systemic
embolism in atrial fibrillation
 Cardiac value replacement
(tissue valves).

o 2.5-3.5
NOTE:  In prevention of recurrent MI
PROLONGED PT IS SEEN IN:  Reduction of mortality in MI
 Any factor deficiency under the extrinsic (VII,  Mechanical prosthetic heart
I, II, V, X) and common pathway. valve (high risk).
 Fibrinogen concentration <100mg/dl
 Dysfibrinogenemia – abnormal fibrin FORMULA:
𝑝𝑎𝑡𝑖𝑒𝑛𝑡 𝑃𝑇
structure affecting PT. INR= [ ]𝐼𝑆𝐼
𝑃𝑇 (𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑚𝑒𝑎𝑛)
 Vitamin K deficiency (II, VII, IX, X) and certain
liver disease (affecting all factors except, VWF,
Ca & TF synthesis) thus prolong PT and APTT
 HEMATOLOGY 2

SURFACE ACTIVATION – initiates intrinsic pathway Perform

when factor XII is in contact with any surface; mixing
monitored by APTT. studies

TISSUE THROMBOPLASTIN
2. STYPVEN TIME/ RUSSEL VIPER VENOM TEST
- for extrinsic and monitored by PT - Uses a venom (from (Viperarusselli/Daboia
INTRINSIC PATHWAY – PTT russelli) with a “thromboplastin like” action
- Principle: Addition of the venom bypasses the
VII – PT activation of VII and directly activates factor X
INTRINSIC PATHWAY AND FACTOR VII – common - Reagent: Russel Viper Venom 
pathway; APTT & PT Thromboplastin-like (“VIIa”): VIIa like activity 
immediately activate X.
- Activation of VII is bypassed in the reaction
because reagent have VIIa-like activity
- Initial utilization: Differentiate X and VII
deficiency
o X deficiency: ST: Prolonged
o VII deficiency: ST: Normal
- Clinical utility now: for identification or detect
the presence of Lupus anticoagulant (LA) or
Anti-phospholipid Antibody (APA) – antibody
against phospholipid; inhibits PF3 and
phospholipid (interferes normal coagulation)
prolonged test.
- Screening test performed using diluted plasma:
Dilute Russel Viper Venom Test (DRVVT) or
Dilute Stypven Time (DST) for Lupus
Anticoagulant: factor activity INCREASES with
increasing dilutions of patient plasma used in the
assay (Corresponding dilutions of LA/APA).
- Performed in combination with APTT: ST and
APTT  identify LA/APA.
TEST INTRINSIC EXTRINSIC COMMON - Interpretation:
PT- None Prolonged None o CF deficiency (dilution) : more
prolonged Factor VII prolonged
APTT- deficiency o LA-causing (diluted): improved in the
normal test/ shorten
APTT- Prolonged None None  Rationale: while the plasma is
prolonged VIII, IX, XI, being diluted, the LA is also
PT- Normal XII, HMWK, diluted  increase CF activity
PK - Circulating anticoagulant: abnormal
deficiency
anticoagulants that inhibit certain coagulation
Perform
proteins.
additional
testing - Specific inhibitor: inhibitor/anticoagulant that
BOTH PT & None None Prolonged inhibits specific coagulation protein like factor
APTT- I, II, V, X VIII or IX
prolonged
 HEMATOLOGY 2
- Non-specific inhibitor/ non-specific circulating
anticoagulant: inhibitor/anticoagulant that
inhibits no specific coagulation protein like LA.
INDIRECT TESTS:
FOR FIBRINOGEN DEFICIENCY ONLY
THROMBIN TIME (FIBRINOGEN DEFICIENCY TEST)
- Modification of Thrombin Clotting Time (TCT) by
Clauss.
- Principle: Addition of thrombin bypasses all
coagulation pathways except the polymerization
of Fibrinogen.
- Specimen: citrated PPP
- Reagent: standardized thrombin solution and
calcium.
o Fibrinogen --- Standard Thrombin + Ca++
 Fibrin
- Will not detect deficiency in the CF VII, V, X, VIII,
IX, XI, XII, HK, PK  reactions bypassed
- Cannot determine deficiency or availability of
thrombin.
- DETECT FIBRINOGEN DEFICIENCY ONLY
- Reactions involved conversion of fibrinogen to
fibrin.
- Measures the availability of adequate and
functional fibrinogen
- Used to monitor heparin therapy and is sensitive
to presence of increased levels of FSP.
- Presence of FSP: XYDE; inhibits fibrin
polymerization and inhibits conversion of
fibrinogen to fibrin by thrombin  inhibits
clotting/prevent clot formation  increased/
prolonged TCT (more sensitive).
- Monitors Heparin Therapy
o Heparin: anti-thrombin; only
anticoagulant (in vivo and in vitro) that
does not remove Ca++; functions to
inhibit thrombin by increasing the
activity of AT-III.
o APTT and TT: monitors heparin therapy
- Reference value: 17-25 secs. (may depend on
the thrombin concentration)
- Prolonged Thrombin time:
o Fibrinogen level is below 75-100 mg/dL;
functional disorder of fibrinogen
o Presence of Heparin, fibrin degradation
products (FDP) and thrombolytic agents
o Normally prolonged in the newborn and
in multiple myeloma.
APTT: intravenous anticoagulant
PT: oral anticoagulant  INR
TT: fibrinogen deficiency and heparin therapy
TT together with APTT: Heparin therapy
DST together with APTT: LA
REPTILASE TIME
- Uses the reagent reptilase enzyme (collected
from Bothropsatrox) which has a thrombin-like
effect.
- Functionality same as thrombin time: IDENTIFY
PRESENCE, AVAILABILITY AND FUNCTIONALITY
OF FIBRINOGEN.
- REAGENT: Reptilase (“IIa” – like): toxic and
potent: very unsafe to handle (should not be in
contact in open skin  blood will clot)
- Fibrinogen --- Reptilase  Fibrin ; immediate
clotting will occur
- PRINCIPLE: Reptilase bypasses all coagulation
pathways except the polymerization of
fibrinogen.
- FIBRINOGEN: 3 polypeptide chains (alpha, beta
and gamma) ends with D-terminals and a central
E domain where alpha, beta and gamma
polypeptide chains meet and coil extended at E
central domain is fibrinopeptides A and B.
- If thrombin cleaves fibrinogen: thrombin
cleaves both fibrinopeptides A and B.
- If reptilase cleaves fibrinogen: reptilase cleaves
only fibrinopeptides A.
- Effect are similar: Fibrinogen  fibrin monomer
 polymerization
 HEMATOLOGY 2

- Reptilase cleaves I  monomers polymerize F XIII Deficiency (soluble Dissolved Not

(end-to-end)  formation of clot clot) dissolved
o Thrombin: end to end and side to side Excessive fibrinolysis Dissolved Dissolved
connection (excessive plasmin)
o Reptilase: end to end only
- Prolonged reptilase time is observed in OTHER TEST:
Fibrinogen deficiency and other functional
disorder of fibrinogen; presence of FSP/FDP 1. BETHESDA ASSAY: determine and measure the
- REFERENCE TIME: 18-20 secs titer of specific factor inhibitor of VIII.
- More sensitive to fibrinogen deficiency 2. ECARIN CLOTTING TIME

NOTE: HISTORICAL TESTS:

 All coagulation assay is affected by the Basis of coagulation tests
presence of FSP  prolonged PROTHROMBIN CONSUMPTION TEST
 BOTH (Fibrinogen Assay) are used to identify
- Deficiency in factors V, VIII, IX, X, XII or platelets
availability and functionality of fibrinogen and
will slow the rate of prothrombin conversion
both sensitive to fibrinogen deficiency or
dysfunctional  prolonged test leaving significant amount of prothrombin in the
serum.
- Prothrombin Time (PT) is performed on SERUM
THROMBIN REPTILASE - Deficiency in some factors will SLOW the rate of
TIME (standard TIME prothrombin conversion leaving significant
thrombin Rgt) (thrombin-like amount (60%) of prothrombin in serum
rgt.) o Slow pathway: Prothrombin (60%) 
Heparin – both Prolonged Normal or
Thrombin (40%)
APTT and TT Slightly
- Can’t identify which coagulation factor is
Prolonged
deficient  needs mixing studies.
(overdosed)
(+) FSP – all Prolonged Prolonged - GENERAL RULE: Only consumable factor are
clotting test FIBRINOGEN GROUP (I, V, VIII and XIII) and
Decreased Prolonged Prolonged factor II.
Fibrinogen - During clotting (serum): II (20%; residual
prothrombin)  IIa (Thrombin: 80%)
- SERUM: product of clotting of blood
DUKERT TEST (5M UREA SOLUBILITY TEST)

- Test for Factor XIII

- PRINCIPLE: clot formed in normal plasma is
insoluble in 5M urea during a 24-hr incubation.
- If factor XIII is deficient in the patient’s plasma,
the clot is dissolved in less than 24 hours by the
urea.

1% monochloroacetic acid Incubate for 24 hours

at RT
Clot + 5M Clot+
THROMBOPLASTIN GENERATION TEST (TGT)
Urea NaCl
(NSS) - By Biggs & Douglas
Normal (insoluble clot) Not Not - Uses BaSO4/Al(OH)3- adsorbed plasma or fresh
dissolved dissolved serum
 HEMATOLOGY 2
- Basis of mixing studies AUTOMATED INSTRUMENTS:
- No longer performed
- Mixing agent + Patient’s plasma  clotting
- Mixing agents: BaSO4/Al (OH)3 – Adsorbed
plasma; Fresh serum; stored serum.
HICKS-PITNEY TEST
- Modification of Thromboplastin Generation
Time (TGT) and requires re-calcification of
diluted plasma.
AUTOMATION IN COAGULATION
- Detects formation of FIBRIN
FIBRINOMETER
- A semi-automated mechanical instrument that RESULT AND INTERPRETATION:
detects fibrin strand formation using a wire loop
or hook.
- Only machine in hematology that uses electro-
mechanical (manual) principle.
- Fibrinometer has wells where samples are
placed, and has a wire loop or needle which dips
in the blood sample in the wells and keeps
dipping in 0.5 seconds interval (up and down)
until fibrin thread or clot formation is observed.
When fibrin threads from the sample connects
with the wire loop, stop the time and record the
clotting time
- Semi-automated.
- Identify fibrin formation FACTOR ASSAYS:
PHOTO-OPTICAL
- Depends on the increase in light scattering
associated with the conversion of soluble
fibrinogen molecules to the insoluble
polymerized fibrin clot.
- Machines measures: increase in light scattering
- Identify fibrin formation from fibrinogen
- In plasma, fibrinogen is dissolved thus it is not
able to scatter the light into different direction
- Fibrinogen  Fibrin (insoluble) fibrin particles
will block and scatter the light in different
direction.
SEMI-AUTOMATED INSTRUMENTS:
 Electra 750 and 750A, Fibrin timer series; and FP
910 coagulation analyzer
 Ortho Koagulab 16S and 40A
 Coag-A-Mate X2 and XCm
 MLA Electra 700 and 800
MIXING STUDY OR SUBSTITUTION STUDIES
- Performed when PTT & PT are prolonged to
determine whether it is caused by a nonspecific
inhibitor, specific factor inhibitor, or coagulation
factor deficiency, and to determine the deficient
coagulation factor.
- Procedure: Mix equal parts of patient’s plasma
plus Fresh normal plasma then repeat the
prolonged test.
 Presence of a pathological circulating
anticoagulant: if test is not corrected (remains
prolonged) when mixed with mixing normal
plasma.
 Factor deficiency: if repeated test is corrected
when mixed with normal plasma. The next thing
to do is to identify the deficient factor by
performing additional mixing study or factor
assay.
 Identification of deficient factor: mix equal parts
of patient’s plasma plus an appropriate mixing
sample.
1. Fibrinogen assay: Clauss modification of
Thrombin time.
 SINGLE FACTOR ASSAY:
o Quantification of remaining deficient
factor; determine the factor level in
relation to
o Applicable only in A and B: sex linked
o Not for: C (autosomal)
o Do dilution: 1:10; 1:20; 1:40; 1:80  CT
interval (Reference Curve).
Hemophilia: Factor Level vs. Severity
Severity Factor Level Manifestation
Severe <1% Spontaneous
bleeding; bleeding
with minor surgery or
trauma
 HEMATOLOGY 2

Moderate 1-5% Spontaneous bleeding Factor Deficiency Mixed Reagent Result

uncommon; may (Patient’s
bleed with surgery or sample)
trauma. VII Factor-VII No correction
Mild 5-20% No spontaneous Deficient Plasma
bleeding; may bleed
with major trauma or
surgery 4. Factor XIII assay: Chromogenic assay
Example: - Transglutaminase/Fibrinase/Transaminase
APTT: prolonged PT: Normal reaction  Release of ammonia
APTT: not corrected by Factor XI- deficient plasma but is - Directly proportional: More ammonia release:
corrected by Factor IX-deficient plasma more XIII deficiency; Less ammonia release: less
 What type of hemophilia? XIII deficiency
o Hemophilia C - (Glu DH Rxn): NADPH consumption is measured
by decrease of A at 340 nm
2. Single factor assays using PTT - Absorbance is inversely proportional to factor
- For factors VIII (A-most common), IX (B- second XIII activity.
common), and XI (C-rarest) deficiencies - Inversely proportional: High absorbance:
(hemophilias). Increase XIII deficiency; Lower absorbance:
- Uses a factor-depleted platelet-poor plasma (like Normal or Increase
factor VIII-depleted PPP which provides normal - Factor XIII: Assay cannot be detected by PT and
activity of all procoagulant except factor VIII). APTT
- Normal plasma  normal result
e.g. Px plasma + factor deficient plasma NOTE:
Prolonged Test (PT, APTT/Both coagulation test) 
APTT reagent
CONSIDER/POSSIBLE REASONS: px is suffering in
PT reagent
coagulation factor deficiency or abnormal component
FACTOR MIXED REAGENT RESULT (inhibitor/circulating anticoagulant) that keeps
DEFICIENCY inhibiting the factors  perform mixing study
(Patient’s Reagent: 1:1 mixture
- Equal parts: Patient’s plasma + correction
Sample)
reagent or mixing reagent
VIII Factor-VIII Deficient No correction
PRINCIPLE:
Plasma - If patient is deficient and we add a mixing
Factor-IX Deficient Corrected reagent that contains all necessary
Plasma components  corrected: factor deficiency.
IX Factor-VIII Deficient Corrected - If patient is deficient and we add a mixing
Plasma reagent that contains all necessary
Factor-IX Deficient No correction components but still not corrected: presence
Plasma of abnormal component.

e.g. repeat prolonged test but: (1:1) Px plasma+

3. Single factor assays using PT normal fresh plasma(contains all coagulation factor)
- For factors II, V, VII, and X deficiencies
 HEMATOLOGY 2

PROLONGED TEST
INTERPRETATION NORMAL FRESH PLASMA
Factor Deficiency Normal: Corrected test
Inhibitor; C Remains Prolonged: not
anticoagulant corrected test

NOTE:
After Coagulation test (prolonged)  perform mixing
study with Fresh Normal Plasma (FNP): identify
whether it is coagulation deficiency or presence of
abnormal component  then identify which factor is
deficient or abnormal component
PROLONGED TEST: Deficiency  continue mixing
studies
PT: VII
APTT: VIII, IX, XI, XII, HK, PK
PT and APTT: I, II, V, X

MIXING STUDIES
MIXING/CORRECTION REAGENT CF PRESENT CF ABSENT
FRESH NORMAL PLASMA ALL NONE
ADSORBED PLASMA I,V, VIII, XIII, XI, XII, HK,PK II, VII, IX, X
AGED PLASMA I, XIII, II, VII, IX, X, XI,XIII, HK, PK V and VIII
FRESH SERUM II, VII, IX,X,XI,XII,HK,PK I, V, VIII, XIII
AGED SERUM VII,IX,X,XI,XII,HK,PK I, V, VIII,XIII, II (residual II
deteriorates)
ADSORBED SERUM XI, XII, HK, PK I, V, VIII, XIII, II, VII, IX, X
FACTOR DEFICIENT PLASMA Deficient of particular CF; removed through immunoadsorption
Commercially available plasma (named)
e.g. Factor VIII deficient plasma  deficient of VIII

3 WAYS COAGULATION FACTORS ARE REMOVED: EXAMPLES

PROBLEM 1: PT: Prolonged & APTT: Prolonged
1. Clotting/Coagulation: Consumable factors (I, V,
XIII) and partially II. 1. Establish whether it is coagulation deficiency or
2. Adsorption: Adsorbable factors presence of abnormal component
3. Storage/Aging: Labile factors (V and VIII) Perform test with FNP  if corrected: CF
- Aged Serum: residual prothrombin deficiency
 HEMATOLOGY 2

2. Identify which pathway is affected: Common  Elimination:

pathway. Adsorbed plasma (VIII and XI present) (IX absent)
3. Coagulation Factor Deficiency (CFD) (I, II, V, X)  Fresh serum (IX and XI present) (VIII absent)
Mixing study.
ANSWER: Factor VIII deficiency
4. CFD: Pxt Plasma + Mixing Rgt.  Repeat PT and
APTT.

PROBLEM 2: COMMON PATHWAY PROBLEM 3: PT and APTT – prolonged

 Coagulation Factor Deficiency: I, II, V, X  Both are corrected by fresh serum but not with
1. Pxt Plasma + Mixing Rgt: Fresh Serum (I, V stored serum. Identify factor/s deficient.
absent; II, X present)  Repeat PT and Aptt.  Common pathway deficiency: I, II,V, X  factor II
Deficient: deficiency
I = not corrected
II = corrected A. VIII B. I C. V D. II E. X
V = not corrected  Elimination:
X = corrected o Fresh serum (II and X present) (I, V
2. Pxt Plasma + Mixing Rgt: Aged Plasma (V absent)
absent; I, II, X present)  Repeat PT and APTT o Stored serum (X present) (I, V, II absent)
Deficient:
I = corrected NOTE:
II = corrected WAYS OF ANALYZATION
V = not corrected: prolonged  ELIMINATION
X = corrected  WRITING
 RECORDING
PROBLEM 3: PT and APTT are prolonged and corrected
by addition adsorbed plasma. Which coagulation is/are
deficient? PROBLEM 4: PT and APTT – prolonged

 Pxt Plasma + Mixing Rgt: Adsorbed Plasma ( II  Both are corrected with fresh serum but not with
and X absent; I and V present)  Repeat PT and adsorbed plasma.
APTT o Fresh serum: II, VII, IX, X, XI, XII, HK, PK
 Deficient: (present)
I = corrected o Adsorbed plasma: II, VII, IX, X (absent)
II = not corrected  What may be mixed to identify the factor
V = corrected deficient?
X = not corrected o Stored serum: II and X

ANSWER: Factor I and V deficiency PROBLEM 5: PT and APTT – prolonged

PROBLEM 4: PT: Normal APTT: Prolonged  COMMON PATHWAY: I, II, V, X

 Corrected (it contains the CF) by adsorbed
plasma but NOT(absent) by fresh serum Factor
VIII deficiency
 Factor deficiency in INTRINSIC PATHWAY: VIII, IX,
XI (common deficiencies to occur); (XII, PK, HK –
deficiency will not cause bleeding disorder but
may cause prolongation of the test and are very
rare to occur)
 Both are NOT corrected with fresh serum but
corrected with adsorbed plasma.
o Fresh serum: I, V,VIII,XIII (absent)
o Adsorbed plasma: I,V, VIII, XIII (present)
 What may be mixed to identify the factor
deficient?
o Aged Plasma: I
 HEMATOLOGY 2

PROBLEM 6: PT and APTT – prolonged

 COMMON PATHWAY: I, II, V, X

 Both are NOT corrected with fresh serum but
corrected with adsorbed plasma and aged
plasma.

FACTOR PROLONGED TEST/S CORRECTED BY NOT CORRECTED BY

DEFICIENCY
I PT and APTT  Adsorbed plasma  Fresh Serum
 Aged Plasma  Aged Serum
 Adsorbed Serum
II PT and APTT  Aged plasma  Adsorbed plasma
 Fresh serum  Aged serum
 Adsorbed serum
V PT and APTT Adsorbed plasma  Aged plasma
 Fresh serum
 Aged serum
 Adsorbed serum
VII PT  Aged plasma  Adsorbed plasma
 Fresh serum  Adsorbed serum
 Aged serum
VIII APTT Adsorbed plasma  Aged plasma
 Fresh serum
 Aged serum
 Adsorbed serum
IX APTT  Aged plasma  Adsorbed plasma
 Fresh serum  Adsorbed serum
 Aged serum
X PT and APTT  Aged plasma  Adsorbed plasma
Stypven Time  Fresh serum  Adsorbed serum
 Aged serum
XI APTT ALL NONE
Presence: APTT and Stypven Time NONE ALL
AntiPL-
Antibody
NOTE: Factor XIII Assay cannot be detected by PT and APTT
 HEMATOLOGY 2

MIDTERMS - The effect of coagulation is consumption of

DISORDERS OF FIBRINOLYSIS coagulation factors (I, V, VIII and XIII) and
PLASMIN platelets. The effects of activation of fibrinolysis
is production of FSP. Main result: continuous
- Main protein involved that dissolves the clot
bleeding
and may cause excessive fibrinolysis.
- Regulatory proteins: alpha 2 antiplasmin: main
- Comes from plasminogen in the presence of
inhibitor of plasmin; and other inhibitors are
plasminogen activators like tissue plasminogen
consumed because of too much production of
activator and activated factor XII (XIIa) when
plasminogen activators.
produced can dissolve fibrin and if excess
fibrinogen. TESTS FOR FIBRINOLYSIS
EUGLOBULIN CLOT LYSIS TIME
FUNCTIONS OF PLASMIN:
- Principle: Plasma euglobulins are precipitated
 Plasmin  fibrinogen  Fragment X, Y and
with 1% HAC. Precipitates are redissolved and
Fragment D, E
clotted with thrombin. The clot is incubated, and
 Cross-linked fibrin  X-oligomers and D-dimer
the time for complete lysis at 37°C is measured.
NOTE: - Procedure: Precipitation of plasma euglobin
In both degradation and split products of fibrin or with 1% Acetic acid  re-dissolved ppt  add
fibrinogen are produced. thrombin: fibrinogen immediately converted
into fibrin  plasminogen present in fraction
now present in fibrin will be slowly converted
1. PRIMARY FIBRINOLYSIS (FIBRINOGEN LYSIS)
into plasmin by activators of plasminogen 
- This is due to the release of excessive activators
fibrin clot with plasmin  check the
of plasminogen resulting to the conversion of
ability/activity of plasmin to dissolve the clot 
plasminogen to plasmin in the absence of fibrin
at 37°C  CLOT LYSIS TIME
formation
- TEST FOR PRIMARY FIBRINOLYSIS
- Fibrinolytic system: activated in the absence of
- Detects excess production of plasmin
clot formation.
- Separation through PRECIPITATION: inhibitors
- Main problem: Increase activators of
left in the supernatant
plasminogen that leads to increase production of
- Plasma Euglobulin Fraction contains
plasmin.
Plasminogen, Activators of plasminogen and
- Since the function of plasmin is not specific to
fibrinogen.
the clot, it also destroys intact fibrinogen
- Normal clot lysis time: within 2-4 hours.
together with factors V and VIII.
- Clot lysis in less than 2 hours is indicative of
- Effect of plasmin on plasminogen: will cause the
abnormal fibrinolytic activity
release of FSP which interferes in normal
- Increase fibrinolysis is seen in: circulatory
clotting; unable to clot  BLEEDING.
collapse; adrenalin injections; sudden death;
- To inhibit the role of plasmin: treatment
pulmonary surgery; pyrogen reaction; obstetric
involves introduction of anti-fibrinolytic
complications
drugs/antiplasmin.
TESTS FOR SECONDARY FIBRINOLYSIS
2. SECONDARY FIBRINOLYSIS (FIBRIN LYSIS) - Detects fibrin monomers
- This clot dissolution results to increase FSP/FDP
ETHANOL GELATION TEST (BREEN AND TULLIS) AND
that interfere with coagulation and platelet
PROTAMINE SULFATE TEST
function.
- In DIC where there is a simultaneous occurrence - Principle: 50% ethanol (and Protamine sulfate)
of both coagulation and fibrinolysis. will cause the soluble fibrin monomers
 HEMATOLOGY 2

complexes to dissociate resulting in the product came from primary or secondary

polymerization of the monomers and fibrinolysis.
subsequent gel like clot formation or - POSITIVE: presence of FSP  agglutination.
paracoagulum (paracoagulation)
PRIMARY SECONDARY
- Both tests are designed to detect the presence
FIBRINOLYSIS FIBRINOLYSIS
of fibrin monomers in the plasma. A screening
Platelet Normal Decreased
procedure utilized as an aid in the diagnosis of
count  Platelet  Platelets
DIC does not are
- Normally, there is NO gel formation. Because undergo consumed
there should be no fibrin monomers. aggregati during clot
- Presence of paracoagulation: indicates on thus is formation
presence of fibrin monomers and secondary not
fibrinolysis. consume
- Differentiate Primary and Secondary Fibrinolysis: d
o Negative: Primary fibrinolysis RBC Normal RBC
o Positive: Secondary fibrinolysis morpholog  No clot fragmentation:
y formatio Schistocytes
D-DIMER TEST/ D-D ASSAY n thus  Caused by
there is trauma
- Specific to SECONDARY FIBRINOLYSIS only no
- Measures fibrin formation (presence of D-dimer trauma
indicates: fibrin clot and fibrin lysis) caused in
- D-D fragment arises from the degradation of RBCs in
cross-linked fibrin. Its presence is specific of the
fibrin formation. circulatio
- The test uses latex beads with monoclonal Ab n
that will react only with D-dimer (Turbidimetry PT and Prolonged Prolonged
or Nephelometry) APTT  Excessive  Consumpti
- Normal: <200ng (D-dimer)/ (blood) mL : action of on of I,V,
<200ng/mL. plasmin VIII and XIII
on I, V during clot
- Increased results may be seen in DIC,
and VIII formation
thrombosis, phlebitis or other condition

characterized by fibrin clot formation. excessive
THROMBO-WELLCOTEST (TEST FOR FSP) fibrinolysi
s
- PROCEDURE: Whole blood + thrombin (for FDP’s or POSITIVE POSITIVE
complete clotting of whole blood)  serum + FSP’s
soya bean enzyme (as inhibitors)  diluted D-dimer NEGATIVE POSITIVE
serum + latex particles (coated with anti-FSP)  Euglobin POSITIVE NEGATIVE
presence of FSP  agglutination Clot Lysis
- Whole blood is added to thrombin to ensure Protamine NEGATIVE POSITIVE
complete clotting and soya bean enzyme Sulfate(SO
4) Test
inhibitor after incubation, the patient’s serum is
Ethanol NEGATIVE POSITIVE
diluted and mixed with latex particles that have
Gelation
been coated with anti-fibrin split products.
- Determines presence of split or degradation
products but will not identify whether this split
 HEMATOLOGY 2

THROMBOTIC DISORDERS
THROMBOSIS: platelet or fibrin clots

PHYSIOLOGIC THROMBOSIS: normal response due to

injury.

PATHOLOGIC THROMBOSIS

- Abnormal that may lead to obstruction of

circulation.
- Can be acquired or inherited.
- Risk factors:
o Acquired conditions are related with life
events such as age, smoking and other
diseases.
o Inherited conditions are related with
congenital deficiencies in the regulatory
proteins (inhibitors) and defective
factors.

TYPES OF PATHOLOGIC THROMBOSIS

a. ARTERIAL THROMBOSIS
- Composed of many platelets with small amounts
of fibrin, RE and white cells – “white clot”.
- CAUSES: Hypertension, Hyperviscosity,
qualitative platelet abnormalities and
atherosclerosis.

b. VENOUS THROMBOSIS
- Few platelets and blood cells, and MANY FIBRIN
- Composed of large amounts of fibrin and red
cells.
- CAUSES: Abnormalities: associated with slow
blood flow (hyper viscosity of blood), activation
of coagulation system, platelet dysfunction,
leukocyte activation: produces a lot of adhesive
molecules, anatomical abnormalities of blood
vessel wall, impairment of the fibrinolytic
system, and deficiency of physiologic inhibitors
- RISK FACTORS: Life events: age, smoking, DM,
hypercholesterolemia.
 HEMATOLOGY 2

INHERITED THROMBOTIC NOTES ASSOCIATED ACQUIRED

DISORDERS CONDITIONS
Anti-thrombin (AT) Deficiency Recurrent venous thrombosis DIC, liver disease, nephrotic
AT-III: main inhibitor of thrombin syndrome, oral contraceptives and
(continuous activation of V, VIII, XI, pregnancy
XIII)
Heparin Cofactor II Deficiency Not associated with thrombosis
Regulatory Proteins important in inactivation of V and VIII
Protein C Deficiency Thrombopoietin Vitamin K deficiency, liver disease,
malnutrition, DIC and warfarin
therapy
Protein S Deficiency Vitamin K deficiency, liver disease
and DIC
Activated Protein C Resistance Factor V Leiden is not inactivated  excessive clot formation
(Factor V Leiden) Mutated factor V  Resistant to inactivation of activated Protein C  Va remains
to be activated
Prothrombin Mutations Increase in the concentration of plasma prothrombin
Other Inherited Thrombotic Disorders:
 Elevated activity levels of Factor VIII are associated with venous thrombosis embolism
 Factor XII deficiency: XIIa serves as plasminogen activator  deficiency: decrease production of plasmin and
persistence of clot in the circulation
 Dysfribrinogenemia
 Hyperhomocysteinemia
 Tissue Factor Pathway Inhibitor Deficiency: TFPI main inhibitor of tissue factor pathway
THROMBOTIC THROMBOCYTOPENIC PURPURA (TTP)
 Deficiency of ADAMTS-13 (metalloproteinase enzyme important for the cleavage of ultra large vWF)
 activation of platelets  platelet plug  obstruction

HEMOLYTIC UREMIC SYNDROME

 Associated with diarrhea
 Caused by verotoxin or shigatoxin producing bacteria: E.coli 0157:H7 or Shigella strain
 This toxin will cause damage to glomerular capillaries and activation of platelets  hemolytic anemia;
fragmentation of RBC
COMMON SIGNS: TTP and HUS are fever, petechiae, neurologic signs (more often occur in TTP) and renal diseases
(more often occur in HUS); this happens when platelet aggregates is present in small circulations.
 HEMATOLOGY 2

ACQUIRED THROMBOTIC DISORDERS - It inhibits coagulation by combining with

1. LUPUS ANTICOAGULANT / ANTIPHOSPHOLIPID
SYNDROME
- Lupus anticoagulant does not inhibit in vivo
coagulation but may cause prolonged in vitro
tests.
2. HEPARIN-INDUCED THROMBOCYTOPENIA
- Development of antibodies against heparin-
platelet factor 4 complex.
- This immune complex causes platelet activation,
platelet microparticles, thrombocytopenia, and
hypercoagulable state.
NOTE:
ANTICOAGULANT THERAPY
Prevention of thrombosis: can be anticoagulant
drugs, antiplatelet or thrombolytic drugs
ANTIPLATELET DRUGS – prevent activation and
aggregation and are most effective in the treatment of
the arterial diseases; platelets are more often
associated.
1. ASPIRIN – MOST COMMONLY USED: Inhibits
Thromboxane A2 synthesis by irreversibly actetylating
the enzyme cyclooxygenase; acetylsalicylic acid;
irreversibly affects platelet function by inhibiting the
cyclooxygenase (COX) enzyme and thereby the
formation of thromboxane A2 (TXA2).
2. OTHER ANTIPLATELET DRUGS: dipyridamole,
thienopyridines, ticlopidine and clopidogrel.
ANTICOAGULANT DRUGS
- Inhibit thrombin and fibrin formation.
- Prevention of venous thrombosis.
antithrombin-III in inhibiting thrombin and Xa.
2. ORAL ANTICOAGULANTS: COUMADIN,
WARFARIN, DICUMAROL
- These inhibit vitamin K-dependent factors and
other vitamin K-dependent proteins
- They alter hepatic synthesis resulting to inability
of the liver to carboxylate the glutamyl residue
of the factor leading to their functional
deficiency. These factors / proteins formed are
referred to as PIVKA/des – gamma-carboxy
proteins.
- Coumadin (warfarin): crosses the placenta and
is present in human milk.
- Therapy is monitored by PT / international
normalized ratio (INR).
- When overdosed, high levels vitamin K reverse
the action.
- For long term management.
THROMBOLYTIC DRUGS
- Used to break down fibrin clots to restore
vascular function and to prevent loss of tissues
and organs.
- Also used in acute arterial thrombosis for
immediate thrombolysis.
SERVE AS PLASMINOGEN ACTIVATORS
1. UROKINASE – this is not a fibrin specific; it is used in
the treatment of venous thromboembolism, MI and
thrombolysis of clotted catheters.
2. STREPTOKINASE – not fibrin specific.

1. INTRAVENOUS ANTICOAGULANT: HEPARIN

- The anticoagulant activity of heparin is enhanced
by binding to AT.
- Heparin-AT complex inactivates thrombin and
Factor Xa.
- Heparin dosage is monitored by APTT &
activated clotting time.
- When overdosed, its action is reversed by
protamine sulfate.
- Effect is immediate because it enters directly
into the vessel.
 HEMATOLOGY 2

RECAP: PRIMARY AND SECONDARY HEMOSTASIS

PATTERNS OF CLINICAL BLEEDING IN DISORDERS OF HEMOSTASIS

PRIMARY HEMOSTASIS SECONDARY HEMOSTASIS
Characteristics platelet/vascular problem coagulation factor problems or presence of
inhibitors and systemic disease
Onset Spontaneous bleeding, immediate after trauma Delayed after trauma
Sites Skin, mucous membranes Deep tissues
Form Petechiae, ecchymosis Hematomas (bluing of skin with tumor/swelling
 clot within tissues
Mucous Common (nasal, oral, gastrointestinal, Less common
membrane genitourinary)
Other sites Rare in deep tissues Joint, muscle, central nervous system,
retroperitoneal spaces
Clinical examples Quantitative Platelet Defects: Factor deficiency, liver disease, acquired
 Thrombocytopenia inhibitors
Qualitative Platelet Defects: (adhesion,
aggregation or release reaction, vascular defects)
 platelet defects, vWD, scurvy

DISORDER OF COAGULATION, FIBRINOLYSIS AND THROMBOSIS

INHERITED DISORDERS OF COAGULATION
COAGULATION FACTORS DEFICIENCY

All CF are produced by the liver except PF3, CF IV(Ca++) and vWF
FACTOR INHERITED COAGULOPATHIES ACQUIRED NOTES
INHERITANCE COAGULOPATHY COAGULOPATHY
PATTERN
PROTHROMBIN GROUP
II Autosomal Prothrombin Liver disease Least to occur
recessive deficiency Vitamin K deficiency
VII Autosomal F VII deficiency (II,VII,IX,X)  functional Most profound
recessive deficiency; no gamma disorder/deficiency;
carboxylation shortest circulating life 
first to disappear.
Oral anticoagulant PT: immediately prolonged;
therapy: inhibits vit. K  more sensitive to liver
functional deficiency diseases and vit. K.
X Autosomal F X deficiency Bile duct obstruction (gall
recessive stone)  incomplete
IX Sex -linked digestion and absorption Hemophilia B; Christmas
of vit. K  functional Disease
deficiency
FIBRINOGEN GROUP
I Autosomal Afibrinogenemia Severe liver disease Rarest to occur
recessive DIC
 HEMATOLOGY 2

Autosomal Dysfibrinogenemia Fibrinolysis Consumable factors during

dominant Normal levels of I but clotting
has abnormal Sensitive to high levels of
structure plasmin
V Autosomal Owren’s disease
recessive Labile Factor
Deficiency
Parahemophilia
XIII Autosomal F XIII deficiency
recessive
VIII Sex linked Inflammatory liver
(hepatic) diseases
 factor VIII increases
CONTACT GROUP
XI Autosomal Most common among
recessive contact factors: Hemophilia
C but least likely to occur in
hemophilia
XII Autosomal F XII deficiency; 2nd most common; not
recessive Hageman deficiency related to bleeding but
related to
thrombosis/abnormal clot
formation
PK Autosomal Fletcher trait
recessive
HK Autosomal Fitzgerald trait
recessive

HEMOPHILIAS
HEMOPHILIA

- Love of hemorrhage
- Inherited disorders characterized by a deficiency of the anti-hemophilic factors.

HEMOPHILIA A B C
FACTOR DEFICIENT VIII (VIII:C) IX XI
OTHER NAME Royal disease and Classic Christmas disease Rosenthal syndrome
hemophilia
INHERITANCE Sex-linked Sex-linked Autosomal
OCCURENCE 1 in 5,000-10,000 males 1 in 25,000 -30,000 in males Ashkenazi Jews
REGARDED AS Most severe congenital 2nd most severe congenital Least and rarest to occur
bleeding anomaly bleeding anomaly same S/s
with A but milder
PROLONGED TEST APTT APTT APTT
TREATMENT Commercial Factor VIII Factor IX concentrate Fresh frozen plasma (FFP) or
concentrate (prevent (prevent bleeding) whole blood: Replenish
Does not correct bleeding) Recombinant F-IX ( in deficient factor
hemophilias but only Recombinant F-VIII (in developed countries: No single blood component
ARREST BLEEDING or developed countries: standard standard treatment and
treatment and management) management)
 HEMATOLOGY 2

PREVENT POSSIBLE DDAVP (1-desamino-B-

BLEEDING arginine-vasopressin)

Contraindication: ASPIRIN,
ANALGESICS (anti-platelets)

Notes: Royal disease: first observed in Named after Stephen Rosenthal: means “rose-
royal family of England (Queen Christmas; case published valley”: German compound
Victoria: Carrier) (December 22, 2011) in The
New England Journal of
Medicine

1. HEMOPHILIA A/CLASSIC HEMOPHILIA with major trauma or

- A sex-linked disorder characterized by a surgery
deficiency of Factor VIII:C
- Most common hemophilia
- Clinical signs and symptoms depend on NOTE:
remaining factor VIII:C level  Generally applies to both Factor VIII and factor
IX deficiencies; may not apply to deficiency of
- Therapy:
other factors.
o Commercial Factor VIII concentrate
 To determine the remaining factor level 
o DDAVP (1-desamino-B-arginine-
Factor Assay (+ Factor VIII –deficient plasma
vasopressin) or + Factor IX-deficient plasma)
 Bleeding manifestation: Hematoma,
2. HEMOPHILIA B (CHRISTMAS DISEASE) hemarthrosis  severe hemophilias
- Sex-linked disorder characterized by a deficiency
of Factor IX
- Second most common hemophilia with clinical
signs and symptoms similar to hemophilia A HOW IS SEX-LINKED HEMOPHILIA (A AND B)
- Therapy: Factor IX concentrate TRANSMITTED?
- Applies PUNNET SQUARE
SEVERITY OF HEMOPHILA A AND B - 50:50 Rule of inheritance of genes including
the sex chromosomes( XX:Female; XY: Male)
- Severity of bleeding depends on factor level For females:
which classify hemophilias as severe, moderate, XXH: Only one chromosome is affected  CARRIER
or mild; applicable only to sex-linked hemophilia:  Carrier (does not manifest s/s of hemophilias
A and B but can transmit abnormal gene to either son
or daughter)
CLASSIFICATION FACTOR COMMON XHXH: When both X chromosome have the abnormal
OF BLEEDING LEVEL MANIFESTATION gene  HEMOPHILIAC
Severe <1% Spontaneous bleeding;  Happens only if father is hemophiliac and
bleeding with minor mother is either hemophiliac or carrier
surgery or trauma XHO: One X chromosome is inactivated; Female is
Moderate 1–5% Spontaneous bleeding suffering from Turner’s syndrome  HEMOPHILIAC
uncommon; may bleed
with surgery or trauma For males:
Mild 5- 20% No spontaneous XHY: Once X is affected  HEMOPHILIAC (one’s that
bleeding; may bleed have the disease and shows s/s)
 HEMATOLOGY 2
 HEMOPHILIAC (one’s that have the disease
and shows s/s)
IMAGE 1: Union of Carrier Mother and Normal Father
will produce sons: (50% normal and 50%
haemophiliac) and daughters: (50% normal and 50%
carrier)
IMAGE 2: Union of Normal Mother and Hemophiliac
Father will produce sons: (100% normal) and
daughters: (100% carriers)
NOTE:
HOW TO DETECT HEMOPHILIA
 Signs and symptoms
 Presence of FAMILY HISTORY of bleeding
among male relatives in the mother’s side
 Laboratory tests: APTT and Clotting Factor
Assays
EARLY CLUES FOR THE DETECTION OF HEMOPHILIA
 Prolonged bleeding after cutting the umbilical
cord
 Hematomas and joint swelling in babies
beginning to creep, crawl and walk
 Limited range of motion of certain joint
 Refusal to use limb (young child)
 Prolonged bleeding after circumcision
 Prolonged bleeding after tooth extraction
 Swelling and hematomas after blood
extraction or IM injection
 Muscle tightness
3. HEMOPHILIA C (ROSENTHAL SYNDROME)
- An autosomal recessive disorder characterized
by a deficiency of Factor XI
- Has an increased incidence among Ashkenazi
Jews
- Bleeding is mild unless stressed by trauma or
surgery
- Considered as silent type
- Not associated with bleeding disorder or just
mild bleeding disorder: explain by current
coagulation pathway
- Therapy: Fresh frozen plasma or whole blood
4. PREKALLIKREIN ; HMWK ; FACTOR XII
DEFICIENCY
- Autosomal recessive disorders with no
associated bleeding tendencies.
- Patients are more vulnerable to excessive
clotting (thrombosis).
ACQUIRED DISORDERS OF COAGULATION
- No genetic abnormality or anomaly has been
associated with these condition. Gene is
something that underwent mutation due to
some mutagens. Main causes are the other
underlying conditions or diseases.
1. INHIBITORS
A. NON-SPECIFIC FACTOR INHIBITORS
- Directed not against coagulation factor but
other molecules and proteins involved in
coagulation.
- Example: Lupus Anticoagulant (LA)
NOTE:
LUPUS ANTICOAGULANT (LA)/ ANTIPHOSPHOLIPID
ANTIBODIES (APA)/ ANTICARDIOLIPIN ANTIBODIES
 A non-specific inhibitor directed against
platelet phospholipid and phospholipid-
protein complexes.
 Its presence will prolong
 Directed to epitopes of proteins bound to
phospholipids  THROMBOSIS
 HEMATOLOGY 2
 Prolonged: APTT (effect is less common) and
dilute Rusell Viper Venom Test (dRVVT)
o The combination of these two tests
serves as a screening test for the
presence of Lupus Anticoagulant.
 Immuno assays (ELISA): for confirmatory test
as well as for titer.
 Not at risk from suffering bleeding conditions.
Instead, they are more at risk to suffer from
THROMBOSIS.
B. SPECIFIC FACTOR INHIBITORS
o These are IgG autoantibodies
directed against specific
coagulation factors (e.g. Factor
VIII:C, IX, X and Factor XI inhibitors)
NOTE:
FACTOR VIII:C INHIBITOR
 Most likely to occur
 Commonly developed from patients suffering
from B-cell malignancies, autoimmune
diseases (Systemic Lupus Erythematosus), and
elderly.
 Production may also be induced in patients
who have received the Factor VIII
concentrate.
 Present bleeding conditions similar with
Hemophilia A (Acquired hemophilia).
 Prolonged APTT and will serve as a screening
test to detect VIII:C Inhibitor.
 Bethesda Assay-titer: determine the titer of
antibody.
 High-dose Factor VIII: given to prevent from
suffering bleeding conditions.
 Immunosuppressant drugs (Cytoxan,
Rituximab): given for longer management of
the patient. It is used to inhibit or prevent the
immune system from continuous production
of antibody against factor VIII.
2. VITAMIN K DEFICIENCY
- More on functional deficiency and less on
synthesis deficiency
- Will result to functional decrease of Factor II, VII,
IX, and X, protein C, S and Z
- Causes of Vitamin K Deficiency:
 Decrease intake of vit. K.: Malnutrition;
source of Vit. K is diet (green leafy
vegetables)
 Use of oral anticoagulant (warfarin,
coumarin, dicoumarol and other
derivatives) or presence of Vitamin K
antagonist: inhibits vitamin K 
inhibiting normal synthesis of vit. K
dependent coagulation factors 
referred as PROTEINS INDUCED BY
VITAMIN K ANTAGONIST (PIVKA) 
abnormal in function
 Decreased normal flora in the GIT
(bacteroides fragilis, E. coli); prolonged
use of strong antibiotics: broad
spectrum antibiotics  damage normal
flora  growth of abnormal flora
 Decreased intestinal absorption intake
 Intestinal infection and Biliary duct
obstruction (obstructive jaundice).
 Vitamin K is fat soluble vitamin it
requires bile for its digestion thus
obstruction along the normal flow of bile
which can be caused by the presence of
tumor or gallstone that constricts this
bile duct and cause obstruction to bile
flow  inadequate digestion and
absorption.
NOTE:
 Infants: given vitamin K at birth to
improve the baby’s synthesis of vitamin
K dependent factors; intestine is sterile.
Bacteria (from maternal skin) grows
only when the baby start to breastfeed.
 Importance of Vitamin K: gamma
carboxylation of II, VII, IX, X; the addition
of this carboxyl radicals to their terminal
glutamic acid will cause them to gain a
negative charge thus will allow them to
interact with Calcium which is positively
charged and on bind to platelet
phospholipid which is negatively
charged.
 HEMATOLOGY 2
3. LIVER DISEASE
- Both clearance and synthesis function will be
affected  Bleeding disorders.
- Leads to synthesis deficiencies (FVII deficiency is
the most profound; first to decrease due to its
short circulating lifespan)
- Decrease clearance activity of activated
components including plasminogen activators
- Increase in FSP due to increase fibrinolysis
- DECREASES: VII (first), PK, I
 Factor VII: most profound to decrease.
When the liver is unable to produce
coagulation factors, Factor VII is the first
to decrease due to its short circulating
life span.
 PK: one of the first to decrease
 Factor I (Fibrinogen): last to decrease
- INCREASE: VIII; if the liver disease is
inflammatory in nature, Factor VIII increases.
NOTE:
Liver functions in the synthesis of all coagulation
factors except calcium and tissue factor and in
clearance, it removes activated coagulation
components to prevent further activation and
excessive coagulation. It also removes
plasminogen activators to prevent excessive
fibrinolysis or excessive production of plasmin.
It removes products of fibrinolysis: Fibrin split
products.
4. MASSIVE TRANSFUSION
- Replacement of more than 1.5 L blood volume
or more than 3 units of blood bags in 24 hours
resulting to dilution of coagulation factor;
DILUTIONAL COAGULOPATHY (in transfusion of
pRBC and NSS) and increased introduction of
anticoagulants (each unit contains 63 mL of
anticoagulant; 10% or more enough to interfere
to normal coagulation).
5. DISSEMINATED INTRAVASCULAR
COAGULATION (DIC)
- Disseminated: clot formation occurs elsewhere
in the body
- Often described as CONSUMPTION
COAGULOPATHY
- CAN OCCUR IN ANYONE, NO AGE GROUP NOR
GENDER PREFERENCE.
- Occurs as complication of another disorder 
causes release of thromboplastic substance 
initiates both Coagulation and Fibrinolysis: both
system simultaneously occurs and may either
predominate the other.
 Result from the liberation of thromboplastic
substance that activates coagulation
 Release of thromboplastic-like substance may
be secondary to trauma, sepsis or toxin that
tissues suffering from this may cause the release
of inflammatory substances that may then exerts
thromboplastic-like activity
 Complication of childbirth obstetrics as
placental tissue are separated, they release
tissue fluids that can exert thromboplastin-like
activity, so the substance will activate both
coagulation and fibrinolysis.
 Plasminogen is also activated through the
contact phase (TPa)
NOTE:
Thus, coagulation and fibrinolysis occur
simultaneously and either may dominate, resulting to:
 Consumption of coagulation factors (I,V, VIII
and XIII) and platelets as thrombi are formed
and deposited locally and widely in the
circulation
o As COAGULATION is activated,
platelet aggregates  they are
consumed resulting to
thrombocytopenia and bleeding.
The platelet aggregate will also
obstruct normal circulation 
thrombosis  vasoocclusion
because of thrombus within the
circulation. Because of the clot
deposited in the tissues, the
 HEMATOLOGY 2

normal flow of oxygen and blood Hematom II, VIII, IX Umbilical X, XIII
will be obstructed resulting to as cord
ischemia or decrease in tissue bleeding
oxygenation  tissues die Mucosal II, VIII, IX, XI Miscarriag I, XIII
(necrosis): kidneys, lungs, liver bleeding e
and brain. Hemarthr VII, IX, X Thrombosi Abnormal
 RBC fragmentation  SCHISTOCYTES osis s fibrinogens;
o Clot deposits cause trauma to red Postsurgic I, II, V, VII, LA;
cells; some are caught or slice/cut al VIII, IX, X, inhibitor
off by fibrin strands  bleeding XI, XIII deficiencies
fragmentation or lysis  Intracrani VII, VIII, IX, Asymptom FXII, PK, HK
hemolysis lead to presence of RBC al XIII atic
fragments called schistocytes on bleeding -They do
circulation and blood smear. not play a
 Increased in FDP and D-dimer major role
o FIBRINOLYSIS ACTIVATION: in in vivo
production of fibrin coagulatio
split/degradation products: XYDE n
 interferes in normal platelet NOTE:
function as well as in the Factors VIII and IX is the most severe coagulation
polymerization of fibrin factor deficiency.
monomers  affects normal
coagulation  more bleeding will
occur. CYTOCHEMICAL STAINS
CYTOCHEMISTRY

NOTE: DIC - Microscopic study and identification of chemical

LAB FINDINGS: all laboratory test will appear constituents within individual cells. It is useful in
abnormal like platelet count (decrease: the identification of malignant cell types on the
thrombocytopenia) and prolonged bleeding time and basis of cytoplasmic or nuclear chemistry;
clotting time(PT, APTT, TT, ST) cellular constituents that are present in
abnormal form or amount; lack of cellular
TREATMENT: underlying cause of DIC should be constituent; and cells exhibiting functional
considered abnormalities.
 Plasma and platelet transfusions – restore
consumed BRIEF INTRODUCTION TO LEUKEMIA
 Heparin –prevent further activation of
clotting mechanism - Leukemia refers to group of disorders
- In the category of cell line, leukemia can be
differentiated to either Myeloid or Lymphoid
Leukemia
CLINICAL MANIFESTATIONS OF COAGULATION - In the basis of Blast cell
FACTOR DEFICIENCIES o Acute Lymphocytic Leukemia: Pre-
dominance of lymphoblast.
TYPE OF COAGULATI TYPE OF COAGULATI o Chronic Lymphocytic Leukemia:
BLEEDING ON FACTOR BLEEDING ON FACTOR Pre-dominance of mature with few
DEFICIENCY DEFICIENCY lymphoblast.
Easy II, VIII, IX Delayed I, XIII o Acute leukemia: refers to a type of
bruising wound leukemia with poorer prognosis with
healing
 HEMATOLOGY 2
an increase predominance of blast
cells or immature cells
o Chronic Leukemia: the decrease of
blast cells and increase of mature
cells.
o Predominance of myeloblast is
associated with Acute Myeloid
Leukemia
o Predominance of mature myelocyte
is associated with Chronic Myeloid
Leukemia
- Blast cells are not easily identified because of
their common characteristics (larger in size, large
nucleus, smaller and deeply basophilic
cytoplasm), cytochemical stains are used to
differentiate in the absence of CD marker
- Acute Myeloid Leukemia can be further
classified knowing it is a stem cell.
- French American British System classified
leukemia based on the morphology of the cells
after staining with Romanowski stain and their
reaction to cytochemical stain.
- FAB further classified ALL as ALL1, ALL2, ALL3.
While AML is classified into seven categories
(AML1-AML7).
- In AML
o AML1-AML3 predominance cell is
Myeloblast, promyeolocyte myelocyte
(granulocytic series)
o AML4 and AML5 uses alpha naphthyl
Chloroacetate esterase
o AML4 stain positive in both Specific and
non- specific esterase because it
contains both granulocytic and
monocytic cells
o AML5- is the acute monocytic leukemia
o AML6 is called Erythroid Leukemia,
leukemic cells are precursors of
erythrocyte. Positive in Periodic Acid
Schiff.
o AML7 leukemia of megakaryocytes
called acute megakaryoblastic leukemia.
- One example for Chronic Lymphocytic Leukemia
is the Hairy Cell Leukemia which affects B-cells.
Hairy cells contain ACP.
o ACP isoenzyme is present in all blood
cells and can be inhibited by tartaric
acid except the isoenzyme number 5
which is the one present in hairy cell
leukemia.
- In Chronic Myeloid Leukemia the predominant
cells are the mature myeloid cells especially the
granulocytes. CML is also called Chronic
Granulocytic Cells. Granulocytes can range from
50,00-300,000
- Having a very high WBC count can also be
observed in Leukemoid Reaction which is due to
underlying condition.
- Both leukemoid reaction and chronic myeloid
leukemia are characterized by a very high WBC
count. However, Leukemoid Reaction is
reversible that when the underlying condition is
resolved, the WBC will return to normal count.
CLM is a permanent condition. To differentiate
Leukemoid reaction from CML, alkaline
phosphatase is used.
ENZYMATIC STAINS
PEROXIDASE/MYELOPEROXIDASE (MPO)
- The stain itself does not contain the peroxidase
enzyme. The peroxidase enzyme is inside the
cell, the composition of the stain is the substrate
for peroxidase enzyme
- Myeloperoxidase is a normal constituent of the
primary granules of the myelocytic cells which is
observed as early as promyeolocyte.
- Stain marker for primary granules & auer rods
(fused primary granules)
- Stain positive in AML but negative in ALL
- Differentiates AML from ALL
- Stain marker for immature myeloid cells
- Stain positive in granulocyte but not in
lymphocytes
- Positive Activity: reddish-brown deposits (in
cytoplasm of granulocytes and monocytes)
- Note: Myeloperoxidase enzyme deteriorates;
Stain should be done only on fresh specimens
ALPHA-NAPHTHYL ACETATE ESTERASE & ALPHA-
NAPHTHYL BUTYRATE ESTERASE (NON-SPECIFIC
ESTERASES/NSE)
- Used to detect the granulocyte of monocytic
origin
- Nonspecific because it can be seen in other cells
 HEMATOLOGY 2
- The stain does not contain esterase but rather
substrate for the enzyme
- If the cell contains the enzyme it will catalyze the
hydrolysis of the butyrate or acetate in the stain
- Unfixed sample can be used so long in the dark
for as long as 2 weeks
- Marker for cells of Monocytic origin
- Other cells (+): Megakaryocytes, Platelets,
Histiocytes, Plasmacytes, some T-lymphocytes
- Positive Activity: red-brown/dark red
LEUKOCYTE/NEUTROPHIL ALKALINE PHOSPHATASE
(LAP/NAP)
- Stains NEUTROPHILS (the only leukocytes that
contain this activity)
- Neutrophils contain various amount of ALP
- Principle: Differential count involving
neutrophils
- Differentiates Leukemoid reaction (LR) from
Chronic myeloid leukemia (CML)
- Reference Value: 30 –185 LAP score
- Increase LAP score is observed in the following:
o during the last trimester of pregnancy -
Hodgkin disease
o Polycythemia vera - Multiple myeloma
o Aplastic anemia - Obstructive jaundice
- To compute, multiply the counted neutrophils by
their grade.
- Normal to High: Leukemoid reaction
- Below than normal value to zero: CML
TARTARIC ACID RESISTANT ACID PHOSPHATASE (TRAP)
- For the diagnosis of Hairy cell leukemia (HCL)
- General Principle: ACP is Detected in almost all
blood cells but when treated with tartaric acid it
is inhibited except isoenzyme number 5
- Labile if unpreserved
- Sample should first be fixed and be stored at -
20°C and can be used atleast 2 weeks
- Other Tartrate Resistant cells: Sezary cells;
Histiocyte; T-cell of acute lymphocytic leukemia
- Activity is indicated by purple to dark red
granules in cytoplasm
CYANIDE-RESISTANT PEROXIDASE
- For identification of Eosinophilic components
- Positive Activity: brown
NAPHTHOL ASD CHLOROACETATE ESTERASE (SPECIFIC
ESTERASE)
 Marker for mature & immature Neutrophil and
mast cells.
 Substrate is the chloroacetate
 Use to identify immature or primitive
granulocyte ALM1-ALM3
 Positive Activity: bright red granules in
cytoplasm.
 This enzyme is stable and may last for months.
TERMINAL DEOXYRIBONUCLEOTIDYL TRANSFERASE
(TDT)
 Stains DNA polymerase immunoperoxidase
 Marker for immature Lymphoid cell
 Positive in almost 90% cases of ALL
 Also useful in the detection of the
“lymphoblastic transformation” of chronic
myeloid leukemia (CML)
 Detects blastic transformation of CML
o CML can transform into an acute
leukemia
o Acute leukemia more than or equal to
30% blast cells
o CML can transform either AML or ALL.
 AML: (+) MPO stain and SBB
 ALL: (+) TdT, it means that it
transforms into ALL.
ACID PHOSPHATASE
 Present in all hematopoietic cells and found in
lysosomes
 Activity is indicated by purple to red granules
 Unstable, cannot be stored.
NON-ENZYMATIC STAINS
PRUSSIAN BLUE STAIN
 Stains siderotic granules (for the diagnosis of
sideroblastic anemia).
 Used to detect hemosiderin in urine
 Used to identify iron in the ferric state (Fe+3)
 HEMATOLOGY 2
PERIODIC ACID SCHIFF (PAS)
 Stain reacts w/ aldehyde groups in glycogen,
mucoprotein, glycoproteins, and high molecular
weight carbohydrates.
 Does not detect stains instead it combines with
the aldehyde group of glycogen.
 Positive to all blood cells except normal
erythroblast or pronormoblast
 Positive erythroblast indicates that it is affected
by AML6.
 Positive in erythroblasts in Di Guglielmo disease
/ Erythroleukemia.
 L1 and L2 also stain positive in PAS
 The positivity of L1 to Pas is described to be
chunky or block-like positivity
 L2 is not that large as L1
 Negative Activity: bright fuchsia pink (Pattern of
staining varies with each cell type)
 Also differentiates Acute Lymphoblastic
Leukemia.
 Peroxidase of eosinophil is resistance to cyanide
 Cyanide resistance peroxidase is used to identify
peroxidase of eosinophils
SUDAN BLACK B (SBB)
 Marker for phospholipids and lipids
 Has the same staining reaction as
myeloperoxidase
 Gives the same information as Peroxidase in the
interpretation.
 Positive in AML.
 Advantage over myeloperoxidase is that it is
more stable, because myeloperoxidase detects
enzyme which is somewhat labile and may
disappear in prolonged storage. Sample stained
in SBB can give reliable result for months.
 Differentiates AML from ALL
 (+) Result: Dark purple-black granules
NOTE:
 Can be done on stored specimens.
 More sensitive than chloroacetate in the
demonstration of mature & immature
neutrophils and mast cells.
TOLUIDINE BLUE O
 A metachromatic stain
 Binds with mucopolysaccharides in blood cells
 For the recognition of basophils and mast cells
 (+) Result: Reddish-Violet
ADDITIONAL NOTES:
Primary fibrinolysis/fibrinogenolysis: Increase
activators of plasminogen  increased plasmin: not
normally present
Secondary fibrinolysis: Release of thromboplastin-like
substance  activates both coagulation and
fibrinolysis  increased plasmin.
Naturally occurring inhibitors: naturally present in the
circulation and their purpose is for normal regulation
of coagulation mechanism. They maintain
homeostasis. Examples: AT-III; TFPI, Protein C, Protein
S, alpha-2 macroglobulin, alpha-2 anti-plasmin, etc.
Abnormally, we could also develop inhibitors or
autoantibodies. Under ACQUIRED COAGULATION
DISORDER; not normally present in the system or
circulation but we produce them when our system is
abnormally activated.
INHIBITORS: autoantibodies that cause prolongation
of certain test because they interfere in normal
coagulation.
2 CATEGORIES:
1. SPECIFIC
 inhibitor directed against specific coagulation
factor like FVIII, FIX or FVII inhibitor FACTOR
VIII: C INHIBITOR
 Most common and severe coagulation factor
inhibitor in VIII:C portion not the complex
 Inhibit Factor of VIII: C only: does not have an
effect on vWF.
 Prevents VIII:C in participating as cofactor in
the formation of intrinsic tenase.
 Normally present in elderly with B-cell
anomalies
 Patients are high risk for suffering bleeding
 Prolonged: APTT
 Bethesda assay – titer: antibody
measurement
 Acute management  high-dose F VIII  not
all will be inhibit  coagulation proceeds (for
short term only).
 HEMATOLOGY 2
 Long term management  MYELODYSPLASTIC, MYELOPROLIFERATIVE and
Immunosuppression  patient is given
cytosan  inhibit production of all antibodies:
should be well-planned.
2. NON-SPECIFIC
 directed against factor or protein involved in
coagulation but is not specific to a certain
coagulation factors; targets anything.
LUPUS ANTICOAGULANT/APA/ ANTICARDIOLIPIN
ANTIBODY
 Inhibits phospholipids and proteins bound to
phospholipids but it has no anticoagulant
effect in vivo  does not lead to bleeding.
 Directed to epitopes of proteins bound to
phospholipids  Thrombosis; abnormal
clotting
 Seen in immunocompromised patients:
higher in patients suffering from SLE, HIV, and
other autoimmune disorders that affects B-
cells production
 Effect is different in vivo and in vitro
 In vitro: has anticoagulant effect and causes
prolongation of clotting time tests like APTT
and dRVVT; less likely in PT
 Immunoassay: for diagnosis  ELISA
LYMPHOPROLIFERATIVE DISEASES
MYELODYSPLASTIC SYNDROME
 myelo – bone marrow; dys – abnormality; plastic
– development  abnormality in development
of cells arising from bone marrow.
MYELOPROLIFERATIVE DISEASE
 Abnormal proliferation of cells in the bone
marrow.
MYELODYSPLASTIC-MYELOPROLIFERATIVE
 Abnormal proliferation and development of cells
in the bone marrow.
NOTE:
NORMAL LIFE: cell production = cell destruction 
NORMAL cell count
Main parent cell: PPSC  Stem cell: LSC (T and B
lymphocytes and non-A non-B population) and MSC
(RBCs, Platelets, Monocytes and granulocytes)
UNILINEAGE DYSPLASIA: only one cell line is affected
MULTILINEAGE DYSPLASIA: one or more cell line is
affected
 HEMATOLOGY 2

MYELODYSPLASTIC MYELOPROLIFERATIVE MYELODYSPLASTIC-

Myelodysplastic Syndrome (MDS) MYELOPROLIFERATIVE
or Dysmyelopoetic Syndrome
(DMS)
abnormality in development of abnormal proliferation of cells in the bone abnormal proliferation and
bone marrow cells marrow development of cells in the bone
marrow
MAIN ABNORMALITY: MAIN ABNORMALITY: Uncontrolled Can’t classify as MDS or MPD alone
Hypercellular maturation  cells proliferation of one or more of the BM  MDS/MPD  with variable
are abnormally large of cells Dysplasia might be present (little) increases in cells as well as
myelo-cell line (RBCs and WBCs but not common cytopenia and morphologic
but NOT LYMPHOCYTES) dysplasia
Abnormal morphology: RBC and Abnormal number of cell: RBC and WBC Show mixed characteristics of MDS
WBC and MPD.
MAIN DIFFERENCE: Many MAIN DIFFERENCE: Little or no dysplasia
dysplasia
Apoptosis (programmed cell Proliferation outspaces apoptosis
death) predominates  easily
perish/destroyed
BLOOD PICTURE: Cytopenia  BLOOD PICTURE: Overproliferation 
decrease in blood cells increase in blood cells
Pre-leukemia DISORDERS: DISORDERS:
• Not leukemia but can 1. Leukemia: genetic involvement; 1.Chronic Myelomonocytic
progress to leukemia  complicated classification Leukemia (CMML)
px are at risk of suffering A. ACUTE LEUKEMIA 2. Atypical Chronic Myeloid
leukemia (AML) B. CHRONIC LEUKEMIA Leukemia (aCML)
• Common in elderly 3. Juvenile Myelo-Monocytic
2. Chronic Myeloproliferative Disorders
patient: Age 50 and above Leukemia (JMML)
• Acquired condition 4. MDS/MPD-U: unclassified
CLINICAL COURSE/PROGNOSIS:
survival
Shorter than CMPD longer than
Acute Leukemia  AML: short
clinical course
DISORDERS: Refractory 
unresponsiveness
1. Refractory Cytopenia Unilineage
Dysplasia (RCUD)
Refractory Anemia (RA): only red
cells are affected
Refractory Neutropenia (RN): only
neutrophils are affected
Refractory Thrombocytopenia
(RT): only thrombocytes or
platelets are affected
2. Refractory Anemia with Ringed
Sideroblast (RARS): immature
RBCs with siderotic granules
 HEMATOLOGY 2

3. Refractory Anemia with

Multilineage
Dysplasia (RAMD)
Refractory Anemia with Excess
blast (RAEB)
4. 5q- SYNDROME OR
ISOLATED 5q DELETION
5. MDS-U: unclassified

TWO SYSTEMS EMPLOYED IN CLASSIFICATION OF LEUKEMIAS AND MYELOID DISORDERS

The FAB Classification The WHO Classification

French-American British System
Group of scientist and doctors
Basis: Morphologic appearance 
examination of blood smear stained with
Wright’s or Giemsa; cellular level  not
accurate and precise
FIRST BASIS
RA Refractory anemia
RARS Refractory anemia with ringed sideroblasts
RAEB Refractory anemia with excess blasts
CMML Chronic Myelomonocytic Leukemia
RAEBIT Refractory anemia with excess blasts in
transformation
World Health Organization
Basis includes more criteria: morphology,
chromosomal study (genetic abnormality) and
immunologic probe (CD markers); more
genetic level of disease
CURRENT BASIS
Refractory anemia RCUD
Refractory anemia with ringed sideroblasts
Refractory cytopenia with multilineage dysplasia
Refractory anemia with excess blast
Type I
Type II: characteristics are similar with RAEBIT
Myelodysplastic syndrome unclassifiable
5(q) chromosome abnormality

MYELODYSPLASTIC SYNDROME (MDS)

 MDS are group of disorders that result from clonal abnormalities of hematopoietic pluripotential stem cells. These
are characterized by hypercellular maturation of the erythroid cells, granulocytes and megakaryocytes.
 They are often described as “Pre-Leukemias” FEATURES
 Occur primarily in persons over age 50
 Characterized by peripheral Cytopenias
 Also characterized by presence of dysplasia of the myeloid cell lines
 Clinical course is shorter than CMPD and longer than acute leukemias.
 HEMATOLOGY 2

MAJOR CLASSIFICATION OF MDS/DMS (WHO Classification, 2008)

MDS PERIPHERAL BLOOD FINDINGS BONE MARROW NOTES

FINDINGS
Refractory Cytopenia Unicytopenia Unilineage dysplasia: Refractory:
with Unilineage Dysplasia No or rare blasts (<1% blast) only one cell is affected; Unresponsive to either
(RCUD) >10% of cells in one treatment or stimulation.
• Refractory anemia (RA): myeloid lineage Dysplastic: should be
RBCs (most common) more than or equal to
• Refractory neutropenia 10% of cells.
(RN) Main abnormality:
•Refractory hypercellular maturation
thrombocytopenia: Prognosis: >5 years; good
Platelets prognosis Risk of
acquiring
leukemia  AML (6%) 
Refractory Anemia Dyserythropoiesis: BM: <5% blasts AML: poor prognosis
megaloblastoid maturation (RA): none or <15%
(ovalocytes); ringed sideroblasts
macroovalocytes ( more than 8
micra)
PB: <1% blasts; reticulocytopenia
 increased apoptosis

Refractory anemia with Features are similar with RA. •Erythroid dysplasia only Reasons for the presence
ring sideroblasts (RARS) Dyserythropoiesis: •15% or more ringed of ringed sideroblast:
megaloblastoid maturation sideroblasts (ONLY Abnormal development
(ovalocytes); DIFFERENCE with RA) of cells and impaired RBC
macroovalocytes ( more than 8 •<5% blasts hemoglobinization: Iron
micra) ppt  siderotic granules
PB: <1% blasts; reticulocytopenia Stain to confirm presence Heme synthesis:
 increased apoptosis of iron: mitochondria; near
Anemia, no blast PRUSSIAN BLUE nucleus: iron not utilized
 siderotic granules
around nucleus forming
a ring
Prognosis: >5 years
 AML (2 years)

Refractory Cytopenia Cytopenia(s) Dysplasia in >10% of cells Multilineage: more than

With Multilineage PB: more than or equals to 10% in two or more myeloid one lineage in myelo cell
Dysplasia (RMCD) dysplasia in more than one cell lineages (neutrophil &/or line is affected; any
lines: (> 2 cell line) erythroid precursors combination
No or rare blasts (<1%) &/or Prognosis: 33 months 
<1 x 109/L monocytes; megakaryocytes <5% AML (11%)
monocytopenia is observed blasts in marrow + (less
No Auer rods than 15%) or - ringed
 HEMATOLOGY 2

sideroblasts; No Auer
rods

Refractory Anemia With Cytopenia(s) Unilineage or PROGNOSIS: less than

Excess Blasts 1 (RAEB-1) <5% blasts multilineage dysplasia 2 years Incidence to
<1 x 109/L monocytes Hypercellularity progressing as AML
NO AUER RODS dyspoiesis: 5-9% blasts (25%)
without Auer rods

Refractory Anemia With Cytopenia(s) Unilineage or Incidence to progressing

Excess Blasts 2 (RAEB-2) 5-19% blasts multilineage dysplasia as AML (33%)
<1 x 109/L monocytes Hypercellularity
+ or – ; with or without: Auer rods dyspoiesis: 10-19% blasts
(fused primary granules) with Auer rods

Myelodysplastic Cytopenia(s) •Unequivocal dysplasia

Syndrome Unclassified <1% blasts in <10% of cells in one or
(MDS-U) more myeloid cell lines
PB: Rare blast when accompanied by a
cytogenic abnormality
considered as
presumptive evidence for
a diagnosis of MDS
•<5% blasts
MDS Associated With Anemia •Normal to increased 5q: long arm of
Isolated Del (5q) Usually normal or increased megakaryocytes with chromosome; there’s
Also known as: platelet count increased hypolobulated deletion of gene 
5-q Syndrome or Isolated No or rare blasts (<1%) nuclei affecting megakaryocytes
5q Deletion •<5% blasts p-arm: short arm
•No Auer rods Prognosis is good
 HEMATOLOGY 2
MYELODYSPLASTIC/MYELOPROLIFERATIVE
DISORDERS
These are disorders that show features of both
myelodysplastic and myeloproliferative disorders. They
are characterized by variable increases in cells as well as
cytopenia and morphologic dysplasia.
TYPES:
1. CHRONIC MYELOMONOCYTIC LEUKEMIA
(CMML)
 Predominant feature is persistent monocytosis
of >1 X 109/L for more than 3 months duration;
with <20% blasts (monoblasts) and few
promonocytes in the peripheral blood and bone
marrow with dysplasia in one or more myeloid
cell line.
 Cytochemical stain: (+) with alpha naphthyl
acetate and alpha naphthyl butyrate esterase
stains  nonspecific esterase stains
 Used to be under MDS in FAB classification.
 In WHO, it is under MDS-MPD  used currently
 Chronic means long/persistent or prolonged;
more than 3 months
NOTE:
TYPES:
 CMLL-1: <5% blasts and promonocytes in the
PB, <10% in the BM.
 CMML-2: 5-19% blasts and promonocytes in
the PB, or 10-19% in the BM  more severe
o Whenever the blast is increased, the
disease is more severe and the
poorer the prognosis
ACUTE LEUKEMIA: poor prognosis; ↑ blasts
CHRONIC LEUKEMIA: good prognosis; ↓ blasts
ESTERASE STAINS:
 SPECIFIC: for immature granulocytes like
myeloblast  alpha naphthyl chloroacetate
esterase (combined substrate)
 NON-SPECIFIC: for monocytic cells  alpha
naphthyl acetate and alpha naphthyl
butyrate esterase stains
2. ATYPICAL CHRONIC MYELOID LEUKEMIA, BCR-
ABL1 NEGATIVE
 Characterized by leukocytosis with prominent
dysgranulopoiesis  (mature and immature
granulocytes)
3. JUVENILE MYELOMONOCYTIC LEUKEMIA
(JMML)
 This is a clonal disorder of predominantly
granulocytic and monocytic lineages.
 MDS/MPD: no Ph1 chromosome
 Juvenile - immature or young patients
 Previously referred to as Ph1 negative CML or
Monosomy 7.
o Normal chromosome: 23 pairs  22
pairs autosomes and 1 pair of sex
chromosomes; but in monosomy 7, no
pair.
 Ph : Philadelphia chromosomes; abnormal
1
translocated chromosome seen in patient
suffering CML ( leukemia and stem cell disorder
wherein most patients but not all have Ph1 )
o Ph1 (+): mostly adults; when Ph1 is
present, it will fall under CML (MPD).
o Ph1 (-): infants or newborns and young
children.
4. MYELODYSPLASTIC/MYELOPROLIFERATIVE
DISEASE, UNCLASSIFIABLE
 These include those cases that meet the criteria
for MDS/MPN but do not fit into one of the
specified subcategories.
 HEMATOLOGY 2

MYELOPROLIFERATIVE DISORDERS (MPD) ENVIRONMENTAL:

LEUKEMIAS
 Group of disorders: Leukemia is a disease of
the blood-forming tissues, predominantly the
bone marrow. It is a malignant neoplasm
characterized by disorderly, purposeless, and
uncontrolled proliferation of one or more of the
hematopoietic cells in the bone marrow.
HEMATOPOIETIC CELLS
 Medullary: bone marrow: disorder  leukemia
(cancer of bone marrow of the blood forming
cells)
 Extra medullary: thymus, lymph nodes 
lymphoma
ETIOLOGY: genetic mutation and/or oncogene activation
3. CHEMICAL AGENTS: carcinogen or leukomogens
 Through inhalation, ingestion or simply exposure
4. IONIZING RADIATION
 Strong dosage
5. VIRUSES: EBV and HTLV (I and II Retrovirus)
 Because of their nature (viral replication)
 EBV and HTLV (I and II Retrovirus): Burkitt
lymphoma  ALL –L3 (leukemic phase)
 Bacteria: can’t cause leukemia: reproduce or
divide by binary fission
 Virus: enters host cell  dissolve its capsid to
release its viral genes or genetic component then
combine with host’s cell gene  form a new
gene  new viral elements  replicate inside
cell using their own and host’s gene  mutation.

 Genetic mutation: cancerous gene  over 6. IMMUNOLOGICAL DEFECTS

proliferation  Patient’s immune system is suppressed 
 Oncogene activation: a gene when activated genetic mutations/oncogene activation  Prone
converts normal gene/cell into cancerous one  to suffer cancer: *Kaposis sarcoma: HIV 
Onco means cancer Breakdown of the immunosurveillance that
normally keeps neoplastic growths in check.
IMPLICATED/ASSOCIATED CONDITIONS: Leukemia  Especially seen in lymphocytic leukemia and
occur as a combination of genetic and environmental lymphoma
factors. 7. NEOPLASIA
GENETIC:

1. HEREDITY/ GENETIC
 Familial incidence: cancer that runs in the
family.
2. CHROMOSOMAL ABNORMALITY/CONGENITAL
FACTORS
 Ph1 chromosome (CML)
 Trisomy 21 (Down syndrome)  predisposed
to suffer AML
 Translocation of a part of Chromosome 8 -14 
or written as t (8:14)  Burkitt lymphoma (ALL-
L3)
o Leukemia and lymphoma are two
different terms but lymphoma can
progress to leukemia when the
cancerous cells in the lymph nodes
infiltrate the bone marrow  leukemic
phase.
 HEMATOLOGY 2

NOTE:
CLINICAL FEATURES: Cancerous cell clone will
grow rapidly and the expense of normal
hematopoietic cells
Leukemic cells (LC) are cancer cells that grows
rapidly or replicate easily thus spread rapidly; found
in bone marrow  space is limited by hard bone 
LC grow rapidly  occupy the bone marrow at the
expense of the normal ones  overcrowd and
infiltrated and take up the nutrients fast
(competition) for normal cells  decreased normal
hematopoietic cells and increased abnormal cells or
LC.

CONSEQUENCES MANIFESTATIONS TREATMENT FOR LEUKEMIA

Decrease RBC production
Decrease normal WBC production
Decrease platelet production
1.Systemic Symptoms 1. Chemotherapy: destroy all cells
- Decreased RBC: including normal  monitor CBC
 Anemia: Fatigue, headaches, 2. Radiotherapy: radiation destroys
dizziness, paleness and others also abnormal cell.
seen in leukemia with anemia 3. Bone Marrow Transplant
- Decreased WBC: multitude of infection  - Graft vs. Host (GVHD)
fever, increase sweating 4.Differentiation Treatment:
- Decreased Platelets: Bleeding (more manipulation of growth or
important to address) and Clotting replication of cancerous cell into a
problems less or non-cancerous one  turn
into mature cell
2.Hypermetabolism - All Trans Retinoic Acid (ATRA)
5. Supportive Management:
Transfusion of blood for anemia and
Antibiotics for infections
 HEMATOLOGY 2
NOTE:
2 MAJOR CLASSIFICATION OF LEUKEMIA BASED ON
CELL LINE:
1. Myeloid/ Non-Lymphoid/ Myeloblastic/
Myelocytic/ Myelogenous Leukemia (AML or ANLL)
2. Lymphoid/ Lymphocytic/ Lymphoblastic/
Lymphogenous Leukemia
BASED ON THE NUMBER OF BLAST CELLS:
 Upper: immature/blasts  increased in blast
cells  ACUTE LEUKEMIA (AML or ALL)
 Acute: more blast (30 (FAB) or 20 (WHO) more
percent)
 Lower: Mature cells increased  CHRONIC
LEUKEMIA (CML or CLL)
 Chronic: less blast (20 (WHO) or 30 (FAB) less
percent); more mature cells
e.g. Lymphocytic/Myeloid cell line
Acute/Chronic  number of blast or prognosis/clinical
course
NUMBER OF BLAST (> or < 20/30):
 Acute: more
 Chronic: less
PROGNOSIS:
 Acute: Days to months (poor; short life span)
 Chronic: 1-2 years or more (good, longer life
span)
4 MAJOR TYPES OF LEUKEMIA
1. Acute Myeloid Leukemia (AML)
 increased in blast: myeloblast (>30%); stem
cell disorder; elderly and newborns
2. Chronic Myeloid Leukemia (CML)
 decreased blast(<30%); increase mature cell:
myelocytes
3. Acute Lymphoblastic Leukemia (ALL)
 increased in blast: lymphoblast (>30%);
common in children
4. Chronic Lymphoblastic Leukemia (CLL)
 decreased blast(<30%); increase mature cell:
lymphocytes
 HEMATOLOGY 2

CLASSIFICATIONS OF LEUKEMIAS
No. of WBC (PB)
(15 x 109/L)
CHARACTERIZATION:

 LEUKEMIC: based on the number of WBC in

peripheral blood (PB)  if WBC count is higher
or more than 15x 109/L
o In leukemia, WBC/RBC count is high
however these cells are abnormal in
function because it produced by
abnormal bone marrow.
 ALEUKEMIC: leukemia if WBC count less than
15x 109/L
o Absence of leukemic or blast cells in the
circulation, leukemic cells are confined
ONLY in bone marrow
 SUBLEUKEMIC: leukemia if WBC count less than
15x 109/L found in the bone marrow and in the
circulation.

There are presence of blast and leukemic cells  both in bone marrow and peripheral blood

CRITERIA CLASSIFICATION; CHARACTERISTICS

I. Cell line 1. Myeloid/Myelocytic/Myelogenous Leukemia/Non-Lymphocytic Leukemia
2. Lymphoid/Lymphocytic/Lymphogenous Leukemia/Lymphoblastic
II. % of Blasts in 1. ACUTE LEUKEMIA:
the Peripheral  With increased blast cells in the bone marrow and peripheral blood
blood and  FAB critertia: >30% bone marrow blasts
bone marrow  WHO criteria: >20% bone marrow blasts
 Prognosis: Rapidly progressive, with a shorter prognosis of days to 6 month
2. CHRONIC LEUKEMIA
 Less than 10% blasts in PB
 With better and longer prognosis
3. SUB-ACUTE LEUKEMIA
 10-30% blast in the PB plus other symptoms
 Prognosis: at least 2-6 months
III. Number of 1. LEUKEMIC LEUKEMIA: WBC count is more than 15000/uL
WBCs in the 2. SUB-LEUKEMIC LEUKEMIA: WBC count is less than 15,000 uL, and
Peripheral  Presence of immature or abnormal cells in the PB
blood 3. ALEUKEMIC LEUKEMIA: WBC count is less than 15,000/Ul
 No immature nor abnormal cells in the PB
TWO SYSTEMS FOR CLASSIFYING LEUKEMIA
French-American British 1976; group of scientist
(FAB) Criteria: Blood smear with Romanowsky stain (Wright’s or Giemsa)  observe
Classification morphology (M1-M7; L1-L3) Cytochemical staining  identify cell line affected
>30% Acute Leukemia
World Health Improvements in 1997, 2001, 2008, 2014  classification is subject to changes
 HEMATOLOGY 2

Organization (WHO) Criteria: Cellular morphology, cytochemical stains, immunologic probes of cell
classification markers (CD markers), cytogenetic or chromosomal abnormalities and clinical
syndrome
Current basis: Standard system of classifying leukemia >20%  Acute Leukemia

NOTE: ACUTE LEUKEMIA  Key myeloid antigens: Myeloperoxidase,

 Acute Myelocytic Leukemia (AML or ANLL)
 AML is a stem cell disorder with
predominance of Blast Cells (>20%) in the
blood or marrow and may resemble acute
infection at presentation. It affects all ages,
but increases with older age (>60 years). This
is also the most common form of acute
leukemia during the first few months of life.
 Stem cell disorder/leukemia  affect all cell
line that arise from it CFU- GEMM
 Ionizing Radiation, Leukomogens;
Congenital/Genetic factors (Down’s
syndrome); Viruses, Neoplasia (cancer that
transform into different or poorer type of
cancer; transformation of one type of
malignancy into another form of malignancy)
Clinical and Laboratory features of AML
 Affects all ages, but increases with older age
 Two peaks: newborns or babies (almost
always AML) and elderly (>60 years)  In
children, ALL is more common
 May resemble acute infection at
presentation.
 Requires 20% blasts in blood or marrow for
diagnosis.
CD13, CD33, CD117, CD14/CD64
 Signs and symptoms: Anemia, infection;
bleeding (decrease in plts.); coagulation
mechanism abnormalities (disturbance in CF)
(AML –M3)
 FAB: Classified into seven; requires 30% or
more blast in the myeloid series in the
marrow or circulation.
 WHO: requires 20% or more blast in the
myeloid series in the marrow or circulation
 Key myeloid antigens: Myeloperoxidase,
CD13, CD33, CD117, CD14/CD64
 Classification made by morphology,
cytogenetics, flow cytometry and
cytochemistry  Recurrent
cytogenetic abnormalities that characterize
defined subtypes

AML
(M1, M2, M3: MPO (+); SBB (+); aNCAE(+)); TdT
( -)
Myeloid M4 and M5: aNAE and aNBE (+)
M6: PAS (+)
Leukemia
CML
LAP (+)
Leukemia
ALL
MPO (-); SBB(-); Alpha NCAE( -); (TdT (+) L1-L3)
Lymphocytic
Leukemia L1 (intense) and L2: PAS (+)

CLL
 HEMATOLOGY 2

French-American-British (FAB) Classification of the Acute Myeloid Leukemias (stem cell disorder)
CRITERIA: MORPHOLOGY AND REACTION TO CYTOCHEMICAL STAINS
M0 (AML with minimal differentiation/ Undifferentiated leukemia)
 Cells are not well differentiated
 Cytochemical staining: Negative with SBB and peroxidase  stains used to identify cells in myeloid series
(granulocytes).
MYELOCYTIC or GRANULOCYTIC SERIES
M1 (Acute Myeloblastic L. without Maturation) Myelocytic origin
No maturation: >30% myeloblast
Auer rods present (fused primary granules with pencil or
rod like shaped; spindle-shaped, red-purple)

Primary granules: promyelocyte

Secondary granules: myelocyte
M2 (Acute Myeloblastic L. with Maturation) Myelocyctic origin
With maturation: >30% myeloblast with >10%
granulocytic component (promye- to neutrophil) 
mature forms
Auer rods present
Similar to WHO: t (8:21)

M3 (Acute Promyelocytic Leukemia) With heavy granulation or Hypergranulation 

Also called Hypergranular Promyelocytic Leukemia abundant promyelocyte: primary granules of
granulocytes
Many Auer rods in bundles called “faggot cells”

WHO: t (15:17)

Associated with DIC: normal tissue (damage)  release

of thromboplastic substances  Secondary fibrinolysis:
ACTIVATION of:
Coagulation  Fibrinolysis promyelocyte lysis 
lysosomal contents  activate coagulation and fibrinolytic
cascade
MONOCYTIC SERIES [(+) with a-naphthyl acetate esterase & a-naphthyl butyrate e.]
M4 (Acute Myelomonocytic Leukemia) Myelo represents granulocytic cells
Also called “Naegeli” monocytic Leukemia Monocytic represents monocytic cells
>20% of PB WBCs are monocytes or monocytic
precursors the rest are mixture of the granulocytic cells
Predominance of both myelocytic and monocytic cells at
80:20 ratio WHO: inv (16)

M5 (Acute Monocytic Leukemia) >80% of BM elements are monocytic series; mono-, pro-
Also known as Schilling’s Leukemia and monocytes

WHO: t (9:11)
 HEMATOLOGY 2

TYPES:
M5a: poorly differentiated monocytic Leukemia;
predominant cell is promonocyte (increased in BLASTS;
>80% are mostly monoblasts)
M5b: well differentiated; more of promonocytes and
monocytes (more MATURE)
ERYHTRPID SERIES
M6 (Pure Erythroid Leukemia) With neoplastic Myeloblasts & Erythroblasts
Cancerous cells in BM represents: >50% are erythroid cells
Other names: in all stages of maturation
Erythroleukemia
Erythremic Myelosis Cytochemical Stains (+) w/ Periodic Acid Schiff (PAS)
DiGuglielmo Disease PAS: stain for aldehyde; stains glycogen in the cells;
combined with aldehyde portion of the glycogen therefore
it is positive in ALL BLOOD CELLS except the normal
erythroblast or precursor or immature RBCs Normally,
erythroblast should be negative in PAS however in AML-
M6; erythroblast are abnormal  Positive in PAS.
MEGAKARYOCYTIC SERIES
M7 (Acute Megakaryocytic Leukemia) Predominantly Megakaryoblasts (>30%) and
micromegakaryoblast Cytochemical stain: Negative (-) w/
SBB and peroxidase

NOTE:
 HEMATOLOGY 2

World Health Organization (WHO) CLASSIFICATION: AML

In order to classify the cell under AML they should be

positive with the following: Key Myeloid Antigens:
CD13, CD33, CD117, and CD14/CD64

Primitive Myeloblasts: (+) Myeloid associated antigens:

MPO (peroxidase enzyme in primary granules), CD13,
CD33, CD117

More mature myeblasts: produces enzymes  (+)

Cytochemical stains: MPO (peroxidase), SBB (lipids),
CAE (enzymes; esterase)

WHO Classification - Main Categories of AML;

current basis
1. AML with recurrent cytogenetic abnormalities
 Translocation (t) and inversion (inv)
 M3

2. AML with multilineage dysplasia

 There is a combination of cell line affected

3. AML, Therapy related: has mutagenic effects 

cause mutation
 Alkylating agents: chlorambucil,
cyclophosphamide, thiotepa, and busulfan
 Exposure to radiation
 Appears 5 years after exposure to both
alkylating agents and/or radiation: MDS  AML

4. AML, Not otherwise specific/classified (sub-

classified by morphology and
immunophenotype)
 FAB: M1-M7 (except M3) retained as sub-
classification

With stars: AML with recurrent cytogenetic

abnormalities (translocation and inversion) 
Myeloid associated Ag: MPO, CD13, CD33, CD117.
Second table: AML, Not otherwise specific/classified;
retained FAB
M3: under AML with recurrent cytogenetic abnormalities
 APL with t (15:17).
 HEMATOLOGY 2

KEY FEATURES OF THE MAJOR ACUTE MYELOID LEUKEMIAS (AML)

Category Cell Morphology Cell surface markers Cytogenetics Prognosis

AML with t (8;21) >20% blasts, >10% CD 13, 33, 117, t(8;21)(q22;q22) More favorable
maturing granulocytes, CD19, CD34 AML1/ETO
Auer rods, dysplasia
AML with inv (16) Blasts with both CD 13,33,14,4,64 inv(16)(p13;q22) or More favorable
monocytic & t(16;16)(p13;q22)
neutrophilic
differentiation,
increased
eosinophils/immature
eosinophils
APL with t (15;17) Promyelocytes with CD 13,33 t(15;17)(q22;q12) More favorable if
azurophilic granules, CD2, +CD117 PML/RARa Variants responsive to ATRA
Auer rods all involve 17q12
AML with (t9;11) Monoblasts and CD 33,65,4, HLA-DR t(9;11)(p22;q23), Intermediate
promonocytes MLLT3-MLL
predominate
AML, therapy Multilineage dysplasia, CD 13, 33, 34, +CD 11q23 Less favorable;
related RS, increased basophils 56, 57 abnormality Median survival < 3
seen with years
topoisomerase
II
inhibitor-associated
AML
AML with t(6;9) Any morphology may be CD 13, 33, 38, DEK-NUP214; t(6;9) Less favorable
seen but HLADR, CD117 (p23;q35)
AML with inv(3) or myelomonocytic is most CD13, CD33, CD38, Inv(3)(121; q26.2) or Less favorable
t(3;3) common Any HLA-DR, CD117 t(3;3) (q21;q26.2)
morphology may be RPN1-EV11
seen except APL
AML with t(1;22) Megakaryoblastic CD41, CD61, square} t(1;22)(p13;q13) Less favorable
morphology CD13, CD33 RBM15-MKL1
with small and large
megakaryocytes
AML with FLT3 Any Any t(6;9)(p23;q34), Less favorable
mutation/duplication t(15;17)(q22;q12) or
normal
AML with NPM1 Myelomonocytic and CD13,CD33, CD34 is NPM1 mutation, More favorable in
mutation monocytic features negative cytoplasmic the absence of FLT3
expression
AML with CEBPA Variable, generally CD13, CD33, CD65, CEBPA mutation More favorable
mutation similar to less CD11b, CD15
differentiated AMLs in
FAB scheme (M1,2)
 HEMATOLOGY 2

CYTOCHEMICAL STAINS: Cytochemical Reactions in Acute Leukemia

CYTOCHEMICAL CELLULAR ELEMENT BLASTS IDENTIFIED NOTES

REACTIONS STAINED
ENZYMATIC STAINS
 Enzymatic staining: the stain is not the enzyme. (e.g. CAE stain  enzyme is what we want to detect which is
inside in the cell) Purpose/Composition of stain: serve as a substrate to cause a reaction
Myeloperoxidase (MPO) Neutrophil primary Myeloblasts strong positive Differentiates ALL from
granules and auer rods Monoblast faint positive ANLL
For AML identification
(+) Activity: reddish brown
deposits (in cytoplasm of
granulocytes and
monocytes)

MPO enzyme deteriorates;

stain should be done only
on fresh specimens
Luekocyte Alkaline Present in tertiary granules Identify CML (leukemia; uncontrolled proliferation) from
Phosphatase of neutrophils leukemoid reaction (LR): transient
Reference Value: 30-185 LAP score

CML: WBC: >50,000/uL  LAP score: <0

LR: WBC: >50,000/uL  LAP score: Normal to High

Smear with LAP stain  observe microscopically  count

100 neutrophils  Grade

Kaplow’s LAP SCORE:

0 = no staining
+ = faint and diffuse staining
++ = pale and moderate amount of blue staining
+++ = strong blue ppt
++++ = deep blue or brilliant

Computation: Multiply the number of cell

by their grade e.g.
0 = 30  0
+ = 50  50
++ = 20  40
= 90 LAP score

Increased LAP Score:

(Controls: During the last trimester of pregnancy),
Polycythemia vera, Aplastic anemia, Hodgkin disease,
multiple myeloma, obstructive jaundice
Specific esterase Cellular enzyme Myeloblasts strong positive For AML identification
 HEMATOLOGY 2

alpha-Naphthyl Marker for mature and

chloroacetate esterase immature neutrophil and
mast cells
(+) Activity: bright red
granules in cytoplasm
This enzyme is stable and
may last for months
Nonspecific esterase Cellular enzyme Monoblast strong positive For M4 and M5
alpha-naphthyl Acetate Marker for cells of
Esterase and alpha- monocytic origin
Naphthyl Butyrate Other cells: (+)
Esterase Megakaryocytes,
Platelets, Histiocytes,
Plasmacytes, some T-
lymphocytes
(+) Activity: red-brown/
dark red
Terminal deoxynucleotidyl Intranuclear enzyme Lymphoblast positive Present in the nucleus of
transferase (TdT) immature lymphocytes
primarily lymphoblast
Stain for ALL
Important in identifying
blastic transformation of
CML
e.g. CML  TdT  ALL CML
 MPO  AML
Tartaric Acid Resistant Acid For hairy cell leukemia(HCL) Other tartrate resistant Activity is indicated by
Phosphatase (TRAP) cells: sezary cells, purple to dark-red granules
histiocytes, T cell of acute in cytoplasm
lymphocytic leukemia
ACP: 5 isoenzymes
1-4: inhibited by tartaric
acid  NEGATIVE
5: resistant to tartaric acid;
inside lymphocytes of HCL
 positive to TRAP
Cyanide- Resistant For identification of Eosinophils (+) Activity: brown
Peroxidase Eosinophilic components
Acid Phosphatase Present in all hematopoietic Activity is indicated by
cells and found in purple to red granules
lysosomes Unstable and cannot be
stored
NON-ENZYMATIC STAINS
Periodic acid-Schiff (PAS) Glycogen Variable, coarse, block-like It stains glycogen,
positivity often seen in mucoprotein,
lymphoblast and glycoproteins, and high
pronormoblasts, molecular weight
myeloblasts usually carbohydrates by
negative although faint combining with the
 HEMATOLOGY 2

diffuse reaction may aldehyde portion of the

occasionally be seen glycogen.

Positive in erythroblasts in
Di Guglielmo disease/
Erythroleukemia

(-) Activity: bright

fuschia pink (pattern
of staining varies with
each cell type)

Principle: positive in all

blood cells except normal
erythroblast but the
abnormal erythroblast in
M6 is positive in PAS.
For AML- M6 Identification

Gives a faint reaction with

lymphocytes in ALL;
Differentiates
ALL-L1 from ALL-L2 of ALL
 Both positive
L1: chunky or block-like
positivity of granules
Sudan Black B (SBB) Lipids and phospholipids Myeloblasts strong positive Differentiates ALL from
Monoblast faint positive ANLL
For AML identification
Gives the same
information as peroxidase
in the interpretation (+)
Result: dark purple-black
granules
Can be done on stored
specimens More sensitive
than chloroacetate in the
demonstration of mature
and immature neutrophils
and mast cells
Perl’s Prussian Blue Stains siderotic granules Use to detect hemosiderin
( for diagnosis of in urine
sideroblastic anemia)
Toluidine Blue O Binds with For recognition of basophils A metachromatic stain:
mucopolysaccharides in and mast cells gives different colors
blood cells (+) Result: Reddish Violet
 HEMATOLOGY 2
ACUTE LYMPHOCYTIC LEUKEMIA
 All is characterized by an uncontrolled growth of
abnormal lymphoid cells. It is primarily a disease
of childhood (young children) and adolescence,
accounting 15% of childhood cancers and up to
75% of childhood leukemia.
 Peak: 2-5 years of age
 Clinical Manifestations: The same with other
acute leukemias but onset (s/s) is more sudden.
o In general, leukemia is characterized by
anemia (decreased RBC), recurrent
infection and bleeding disorders
 Immunocompromised patients:
2 MAJOR CLASSIFICATIONS OF LYMPHOCYTES:
o Humoral Immunity: B cell ALL: signs
include lymphadenopathy.
Splenomegaly and hepatomegaly.
Eventual infiltration of malignant cells
into the meninges (lymphoblasts appear
in the CSF), testes, or ovaries.
o Cell-mediated Immunity: T cell ALL:
there may be a large mass in the
mediastinum (near ribs) leading to
compromised/abnormality of regional
anatomic structures. More common in
teenage males.
 Prognosis: depends on age at the time of
diagnosis, lymphoblast load/tumor burden,
immunophenotype, and genetic abnormalities.
o The younger (common) the age the
better prognosis. In adults  worse
prognosis
o Immunophenotype: type of ALL: (L1, L2,
L3).
 L3: most severe and have worst
prognosis.
 L1: better prognosis among all
the types.
o T-ALL: bad prognosis
o Worse outcome if the count of
lymphoblast is too high (greater than 20
to 30x109/L)  poor prognosis:
hepatosplenomegaly, and
lymphadenopathy all are associated
with worse outcome (uncommon in
AML)
o Secondary lymphoid organs: spleen and
lymph nodes  needs antigenic
stimulation.
o Primary lymphoid organs: bone marrow
and thymus.
 Common causes of death are infection (1st) and
bleeding (2nd).
 In children, the common causes of infections are
S. aureus, P. aeruginosa, C. albicans, H. influenza,
and P. mirabilis  parasites are not that
pathogenic but causes severe outcome
especially in immunocompromised individuals.
1. B cells: humoral immunity  produces
antibodies
2. T cells: cell-mediated immunity
NOTE:
Unlike neutrophils, it involves on defense but the
activity is PHAGOCYTOSIS.
TOTAL NORMAL WBC COUNT: 4,500 -11,000/ 4.5-
11.0x109/L Peripheral blood lymphoblast counts
greater than 20 to 30 x 109/L
Laboratory Findings:
 WBC count vary from low (<5.0 x 109/uL) to
high (>100 X 109/L)  poor prognosis; most
are lymphocyte.
 Most abundant WBC: Neutrophil (60-80%); 
lymphocyte (20-40%);  monocyte 
eosinophils  basophils
 In ALL, predominant cell is immature
lymphoblasts.
 BM is hypercellular/hyperplastic (red marrow;
has more hematopoietic cells uncontrolled
production of lymphocytes) and heavily
infiltrated with lymphoid cells.
NOTE:
ALL- most are lymphoblasts indicated by immature
amount of basophilic cytoplasm
AML – most are myeloblasts
Hard to differentiate: Immature cells: larger in size,
large nucleus with basophilic cytoplasm  importance
of cytochemical staining and CD markers
differentiation.
 HEMATOLOGY 2

mature
cells are
seen in this
type; negative
in CD34
T-ALL CD2, CD3, CD4, CD5, Rare and more
CD7, CD8, TdT common on
teenage
males.

WHO: Immunophenotyping

- Regardless of the classification: from CFU-L 

lymphoid antigens: CD34, TdT (nucleus of
lymphoblasts).
Further classification of B-ALL: Based on recurrent genetic
ALL IMMUNOPHENOTYP NOTES abnormalities: 7 types T-ALL not further classified
SUBTYPE E
Early CD34, CD19,
5% (less NOTE:
(pro/pre- cytoplasmic CD22,
common in CALLA: monoclonal COMMON ACUTE
pre) B-ALL TdT LYMPHOBLASTIC LEUKEMIA ANTIGEN: present on
children);
the leukemia cells of 70% of all patients; not present
11% (most
on normal peripheral lymphocytes but not specific
common in
for leukemia because it is also present on normal BM
adults)
cells that are positive for TdT and HLA-DR antigen;
Intermediat CD34, CD19, CD10, Common B-
only aids in identification of type II: intermediate B-
e (common) cytoplasmic CD22, ALL;
ALL
B-ALL TdT Common ALL FAB Classification of ALL: based on difference in
antigen morphology and cytochemical staining
(CALLA) are
Bases of FAB: Occurrence of individual cytologic
found  CD10
features (cell size; chromatin; nuclear shape, nucleoli,
has
degree of basophilia in the cytoplasm & presence of
relationship
cytoplasmic vacuolation); Degree of heterogeneity in
with CALLA
distribution among the leukemic cell population of
Pre B-ALL -CD34, CD19, 15%
some or all of those cytologic features
cytoplasmic CD22, (child);
Hand-mirror cells: lymphoblast that has
cytoplasmic m, 10%
(adults) contracted cytoplasm
TdT (variable)
 OIF= RBC  Neutrophils  Lymphocytes 
commo Monocytes  Eosinophils  Basophils
n in
children
less in
adults;
most
 HEMATOLOGY 2

Criteria L1 L2 L3
(Burkitt type)
Cell size Small, homogenous Large, heterogeneous (large Large; homogenous
and small)
Nucleus Regular with occasional Irregular; clefting/indentation Regular, oval to round
clefting; Rare with occasional is common
clefting
Nucleoli Rare/small, inconspicuous Present (often large) 1-3 (vesicular)
Chromatin 1-3 (vesicular) Variable Finely stippled
Cytoplasm Scanty Moderate Moderately; vacuolation of the
cytoplasm
Basophilia Moderate Variable Intense
Incidence 15 y/o and below Older than 15 y/o Rarest to occur; Burkitt lymphoma
(ALL in t(8;14)
general is
more
common in
young
children (2-
5 y/o)
Most common and has the best Rarest and has the worst or
prognosis poorest prognosis;

NOTE: ALL resulting to gene fusion (BCR-ABL gene). Ph1 is

First stage of life: Infants and newborns: AML
Young Children: ALL
Adults: CML
Older adults: CLL
Late/Last stage of life: Elderly: AML
CHRONIC MYELOPROLIFERATIVE DISORDERS
(CMPD)
- CMPDs are a group of acquired, malignant
disorders that develop from the proliferation of
an abnormal pluripotential stem cell.
1. CHRONIC MYELOGENOUS LEUKEMIA
(CML/CGL)
 CML is a stem cell disorder commonly affecting
adult. Some patients show a chromosomal
abnormality called the Ph1 chromosome
(formed by the translocation of long arm of
Chromosome 22 to long arm of Chromosome 9
present in around 90% of CML adults, while
infants and children <2 years of age are often
Ph1 (-).
 Also known as Chronic Granulocytic Leukemia
(CGL): most abundant cells is granulocytes
(neutrophils)  Disease of adults
 Philadelphia chromosome: Ph1 or Ph’  BCR-
ABL gene  CML; truncated Chromosome 22.
o Chromosome 9 is longer chromosome;
chromosome 22 is shorter  both have
p arm (short) and q arm (long)
o Chromosome 9 (ABL gene is detached;
became longer) reciprocal translocation
 Chromosome 22 (part is truncated in
BCR; but not detached; became shorter
due to truncation)  truncated
Chromosome 22 (Ph’): where new gene
is formed: BCR-ABL gene  cause
chronic leukemia  more oncogenic
effect.
 HEMATOLOGY 2
o Truncated chromosome 22: Ph1 has
BCR-ABL gene expressed as mRNA (has
genetic code); template for CHON
synthesis  DNA transcription 
production of p210 protein: influence
 chronic phase of leukemia 
excessive production of mature cells
(granulocytic cells)
NOTE:
Truncated chromosome 22: Ph1 has BCR-ABL gene 
expressed as mRNA (has genetic code); template for
CHON synthesis  DNA transcription  production of
p210 protein: influence  chronic phase of leukemia
 excessive production of mature cells (granulocytic
cells)
Imatinib mesylate: drug for CML which targets p210
protein  produced as an effect of newly produced
BCR-ABL gene  Dominated by granulocytes 
neutrophils
Clinical Features: Splenomegaly; Hepatomegaly; Bone
pain
LAB FINDINGS:
•WBC count: 50-600 X 109/L; ↓/0 LAP score
•Basophilia; Eosinophilia; Monocytosis
•Bone marrow: hypercellular with predominant
granulocytic cells (different stages of maturation);
more red marrow  more hematopoietic cells)
• ↓/0 LAP score:
o Differentiated with leukemoid reaction (LR):
transient; cells with normal function 
normal to high LAP score.
o CML: bone marrow defect; cells with
abnormality  0 to low LAP score
o Neutrophils (Tertiary granules): with highest
and only cell with LAP activity  LAP is also
called Neutrophil Alkaline Phosphatase (NAP)
• Increased granulocytes: Basophilia; Eosinophilia;
and Monocytosis
o PPSC  CFU-GEMM  CFU GM  CFU-G
(granulocyte) and CFU-M (monocyte)
• RBC and Plt. count decreased: Anemia and bleeding
disorder
• M:E = 10:1 or higher
o M:E– relationship between nucleated
granulocytic cells and precursor versus
precursors of erythrocytes
o In bone marrow, granulocytic cells is higher in
number than erythrocytes
o Normal ratio: 2-4:1
 Every 1 RBC there is 2-4 granulocytic
precursors
o In ALL, hypercullar BM but M:E ratio is not
affected or increased because what increases
are lymphoid cells
PROGNOSIS: Not all CML patients have Ph’
• Ph1 (+) patients have better prognosis than Ph1 (-)
• Ph1 (+) : mostly Adults  CML; prognosis is better.
• Ph1 (-) : Infants and <2 y/o  JMML or Monosomy 7
under MSD/MPD
o If you have abnormal chromosome the better
is your prognosis.
o Effect of presence of Ph1 in CML  better
prognosis
o If you have abnormal chromosome the better
is your prognosis
• High risk of transforming from chronic to blast 
“blast transformation” or “acute exacerbation blast
crisis” is indicated by an increase of >20% blasts in the
PB or BM.
o If myeloblast fails to mature  acute
leukemia; >20% blasts  can change in cell
line due to release of abnormal chemicals
and substances like cytokines that will
influence the differentiation of cell.
CML/CGL  ALL: TdT (+) AML: MPO (+)
 HEMATOLOGY 2

Acute Leukemia Chronic Leukemia NOTE:

>20% are blasts/ <20% blast; more mature  Later stage  BM infiltrated with fibrous
immature cells  cells tissues  resembles and become
abnormal indistinguishable with BM myelofibrosis.
Fail to mature  More mature cells o Hallmark mark: teardrop cells in
prone to destruction myelofibrosis and later stage of
of blasts cell primary polycythemia vera.
Unlikely to become If blasts are destroyed  Hyperviscosity syndrome  increased cells
chronic  Can’t revert before maturing  can but few plasma volume  flow slower than
back to chronic progress to Acute Leukemia usual  increased pressure (to compensate)
leukemia o The slower flow  thrombotic
Poor prognosis Better prognosis condition.
NOTE:
Management: Repeated phlebotomy
2. POLYCYTHEMIA VERA  Prevent hyperviscosity of blood
 This is characterized by pancytosis/panmyelosis  Removes blood: Blood removed can’t be
due to clonal proliferation with an increase in all transfused  abnormal blood
cellular BM elements, specifically the red cell  Every blood volume removed; iron (60% in hb)
mass (RCM). is loss  possible develop Iron Deficiency
 Laboratory: Anemia (IDA).
o Generally lower EPO level
o High RBC count (>10M)
NORMAL POLYCYTHEMIA VERA
▪ Normal RBC count: 3.8-5.2
HCT = PCV x 100 2: Relative polycythemia
Millions TV vera
▪ Increased in all RBC 1: 50/100 x 100= 50% Decreased in plasma
measurement volume  changing the
o High Hct (>55% in males; >47% in total volume
females) 50/75 x100 = 66.67% 
o High Hb: >18.5 g/dL (M); >16.5 g/dL (F) increased hct
o High LAP, WBC count: 40-50,000/uL In here, bone marrow is
o High Plt count (>1000 x 109/L); more not defective but the hct
than 1 million is increased due to
reaction with underlying
▪ Normal plt. count: 150-
condition
450,000/uL
o High Basophils

WHO CRITERIA FOR THE DIAGNOSIS OF PV 3: True or Primary

polycythemia vera
 Elevated hb> 18.5 (M), 16.5 (F) g/dL, or other Plasma volume is the
evidence of increased Red cell volume. same however the PCV is
 Presence of JAK2 V617F or similar mutation such increased.
as AK2 exon 12 mutation. 75/100 x 100 = 75% 
increased hct
o Janus kinase gene  mutated gene 
dictates abnormal
Increased PCV  due to
production/proliferation and mutation increased production in
of cells. the bone marrow
(defective BM)
 HEMATOLOGY 2

presence of
abnormal
hemoglobin

Inappropriate
response
Associated with some
kidney diseases

3. ANGIOGENIC MYELOID METAPLASIA/

IDIOPATHIC MYELOFIBROSIS/ MYELOFIBROSIS
WITH MYELOID METAPLASIA/ PRIMARY
MYELOFIBROSIS
- AMM is characterized by fibrosis,
megakaryocytic and granulocytic hyperplasia in
the bone marrow (marrow fibroblast).
- This is caused by an increase in defective
platelets as a result of dysplastic
megakaryocytopoiesis. These dysplastic cells
Polycythemia/Erythrocytosis: Characterized by an are prematurely destroyed resulting in the
INCREASED in HEMATOCRIT which may be due to: release of their a-granules that contain platelet-
1. A true increase in Red Cell Mass: PV/Primary derived growth factor (PDGF) which stimulates
polycythemia and secondary polycythemia fibrosis.
2. Just a decreased in plasma volume (relative o Fibrosis (marrow fibroblast)  caused
polycythemia) by dysplastic megakaryopoiesis 
DIFFERENTIATION abnormal megakaryocytes and platelets
Primary Secondary Relative  easily destroyed and lysed 
Polycythemia Polycythemia Polycythe released the contents of their granules
With bone No bone marrow mia
(alpha  PDGF (normally, promote
marrow defect defect No bone
vessel repair; abnormally, cause fibrous
marrow
defect tissue proliferation  scar formation
This is the Appropriate/compens Associated and dense)
myeloprolifera atory with a - Blood film shows predominance of teardrop
tive disease  Characterized decrease cells. Marrow fibrosis often result to “dry tap”
Absolute type by an increase in the during bone marrow aspiration.
Belongs to in the RCM plasma - Bone marrow failure to produce blood 
CMPD &Hct due to volume increased in fibrous tissue and depleted blood
some with no cells; what will produce are liver and spleen that
identifiable increase in has residual function for hematopoiesis
cause RCM & however there would also be fibrosis in these
 High in RCM is EPO organs especially in the spleen.
secondary to
- With increased blood cells and fibrous tissues
high in EPO
- EXTRAMEDULLARY FIBROSIS: fibrosis in the
 Hypoxia: can
be seen in liver and spleen
high altitude, - Middle aged to older people; rare to occur in
high O2 children
affinity and - Bone marrow: 2 normal tissues
 HEMATOLOGY 2

o Yellow: Marrow fats - Spontaneous aggregation even in the absence

o Red: Hematopoietic marrow of vessel injury  Megakaryocyte proliferation
- Blood film shows predominance of teardrop with large and mature morphology; no or little
cells. granulocyte or erythroid proliferation.
o PBS: teardrop cells  hallmark finding; - Platelet aggregation studies show platelets have
indicates the disease decreased/abnormal aggregation with
o Formation of teardrop cell can be epinephrine.
medullary (BM) and extramedullary - In vivo aggregation  platelets tend to obstruct
(SPLEEN) circulation
o Due to narrow circulation in the fibrotic o Two types of thrombosis:
BM and spleen  RBCs squeeze ▪ Arterial: involves platelets 
themselves  deform in order to pass arterial vasoocclusion by
through or enter the limited platelets aggregates 
circulation severe damage in cell decreased circulation and
membrane  retained the shape  oxygenation  called ischemia
TEARDROP CELL ▪ Venous: involves fibrin
- During bone marrow aspiration/core Biopsy 
University of Illinois needle targeting marrow o Arterial or venous thrombosis: 
sinusoids where bone marrow sample is Transient Ischemic Attacks (e.g.
aspirated. priapism (penis); digital ischemia
o Marrow fibrosis often result to “dry (peripheral tissues (digits: toes and
tap” during bone marrow aspiration. finger))
o DRY TAP: difficulty in aspirating that is ▪ More on arterial  decreased
very painful to the patient oxygenation
o Bone marrow should be spongy but due
▪ Toe ischemia: bluish coloration
to fibrous tissues infiltrated it becomes
on the toes
scarred/hard  hard aspiration.
▪ Priapism is a prolonged
erection of the penis. The
4. ESSENTIAL/PRIMARY THROMBOCYTOSIS
persistent erection continues
- ET characterized by thrombocytosis with
hours beyond or isn't caused by
spontaneous aggregation of abnormal platelets.
sexual stimulation. Priapism is
Platelet aggregation studies show platelets have
usually painful. Although
decreased aggregation with epinephrine.
priapism is an uncommon
- 2008 WHO criteria: platelet count of
condition overall, it occurs
>450x109/L. Megakaryocyte proliferation with
commonly in certain groups,
large and mature morphology; no or little
such as people who have sickle
granulocyte or erythroid proliferation; does not
cell anemia.
meet WHO criteria for CML, PV, IMF, MDS, or
▪ Priapus: minor fertility god in
other myeloid neoplasm; demonstration of
Greek mythology, who was also
JAK2 (V817F) mutation or other clonal marker,
the protector of livestock, fruit
No evidence of reactive thrombocytosis.
plants, and male genitals. He
- PRIMARY: main defect is production  bone
was depicted as having an
marrow defect  cancer of BM  excessively
oversized and permanent
produce thrombocytes
erection.
- SECONDARY: in reaction, also known as
reactive thrombocytosis
 HEMATOLOGY 2

NORMAL SMEAR ABNORMAL SMEAR (Essential thrombocytosis)

Platelet count: 8-20/OIF that

contains 200 RBCs.

ET/ PRIMARY THROMBOCYTOSIS POLYCYTHEMIA VERA

Increased platelets (ONLY) with NO increased in RCM
and other blood cells
JK mutation gene
PRIMARY THROMBOCYTOSIS
Platelet count often exceeds 1M/uL or >1000 x109/L
Not reactive: Increased in the production by defective
bone marrow  JAK2(V617F) mutation
With permanent damage, unless treated with bone
marrow transplant
Increased in all blood cells with prominent increased RCM
JK mutation gene
SECONDARY/REACTIVE THROMBOCYTOSIS
Platelet count is high more than 450,000/uL but rarely
exceeds 1M/uL or >1000 x 109/L
Platelet pool: 1/3 spleen and 2/3 circulation
Reactive: When 1/3 platelet in the spleen is mobilized into
the circulation  reactive thrombocytosis (transient)

Abnormally produced due to defective marrow  Normally produced (from spleen; just mobilized) 
Abnormal platelet function normal platelet function
 HEMATOLOGY 2
CHRONIC LYMPHOPROLIFERATIVE DISORDERS
1. CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)
 CLL is characterized by abnormal proliferation
of lymphocyte that are unresponsive to
antigenic stimuli. CLL is less likely to undergo
blastic transformation but CLL patients are
predisposed to develop autoimmune hemolytic
anemia (AIHA)
o Less likely to undergo blastic
transformation
▪ does not become ALL or AML
o Dormant B-cells: unresponsive to Ag
stimulation and treatment
▪ B cells are stimulated by
antigens  transform into
plasma cells  produce
antibodies.
▪ Decreased IgG production: IgG
(2nd; produce at chronic phase,
but once produce it continues
 confers long term or lifetime
protection but in here, it is
decreased thus patients prone
in reinfection).
▪ Affected B-cells does not
produce normal IgG but
produces abnormal ones that is
directed against the patient’s
RBC leading to AIHA.
▪ Warm Autoimmune Hemolytic
Anemia (WAIHA)  IgG –
related: warm type
▪ Recurrent skin infection:
Herpes zoster or Shingles 
pruritus
▪ Chickenpox: initially appears as
papules on the skin and when
reoccurs it is now called herpes
zoster
▪ IgM (1st; peaks immediately
but declines rapidly  does not
confers lifetime protection; cold
type AIHA).
 PROGNOSIS: 3-4 years
NOTE:
CLINICAL SIGNS AND SYMPTOMS INCLUDE:
 Enlarged lymph nodes (lymphadenopathy),
spleen (splenomegaly), and liver
(hepatomegaly)  CLL will more likely show
these S/s over CML.
 Pruritus due to recurrent skin infection (often
with Herpes zoster).
LAB FINDINGS:
Lymphocyte count: 10-150 X 109/L  very high and
mature
 More on lymphocytes: thin rim cytoplasm;
morphologically normal but is fragile 
during smearing  smudge cell  CLL
▪ Differentiate smudge cells from
basket cell  nuclear remnants of
granulocytes; seen in chronic
granulocytic leukemia (CGL/CML)
▪ Smudge cells: formed IN-VITRO 
caused by manner of smearing; does
not seen in wet preparation.
 Lymphoblasts: larger and basophilic
cytoplasm
Many smudge cells on the blood smear
Hypogammaglobulinemia.
TREATMENT/MANAGEMENT:
1. Leukapheresis
 Apheresis: separation technique for
blood components  Whole blood:
PRBC, Plasma (PPP and Fresh plasma,
cryoprecipitate and cryosupernate),
Platelets (plateletpheresis), and WBC
through leukapheresis  separates
into Granulocytes and/or
lymphocytes
2. Regular injection with gamma-globulins (IgG) 
manage infection.
2. HAIRY CELL LEUKEMIA/ LEUKEMIC
RETICULOENDOTHELIOSIS
 HCL shoes presence of small lymphocytes with
many fine cytoplasmic extensions.
o It affects B-cells with presence of ACP 5
o Microscopically, cells have cytoplasmic
hair-like protrusions
 Prognosis is quite good (benign), and can be
longer than 10 years.
o Benign and has a good prognosis
 HEMATOLOGY 2
 Both CLL and HCL  more common in older
adults (>50 y/o) and more on males
 Pancytopenia  unique finding and important
feature of HCL.
NOTE:
 Clinical sign and symptoms: splenomegaly is
most consistent (splenectomy is often
considered).
 Laboratory diagnosis: Cytochemical stain 
TRAP (+)
o Isoenzyme 5 of ACP  unique
because it is resistant and not
inhibited by Tartaric acid  when
employed with ACP stain  ACP 1-4
(negative); ACP 5 (positive)
3. PRO-LYMPHOCYTIC LEUKEMIA
 CLL is characterized by an increase in
prolymphocytes, with almost total replacement
of the bone marrow. This has a poorer
prognosis than HCL and CLL.
 RARE BUT MOST SEVERE
 PROGNOSIS: poor (<1 year)  life expectancy
if left untreated.
NOTE:
LAB FINDINGS:
 HIGH LYMPHOCYTES WITH PREDOMINANCE
OF PROLYMPHOCYTES  less mature
o STAGES of lymphocyte maturation:
LYMPHOBLAST  PROLYMPHOCYTE
 LYMPHOCYTE
OTHER LYMPHOCYTE and PLASMA CELL NEOPLASMS
DIFFERENT STAGES OF A SINGLE NEOPLASTIC
DISORDER
 Both affects T-cells
o T-helper cell (lymphatic tissue)  skin:
MYCOSIS FUNGOIDES
o SKIN: affected T-cells will cause damage
in the epidermal layer  parakeratosis
and pautrier’s microabscess. Skin
become scale that causes eczematoidal
or fungal lesions.
o Histological finding: accumulation of
lymphocytes called  Pautrier’s
microabscess in epidermal biopsy
 SEZARY SYNDROME: Flow through lymphatic
circulation and reach lymph
o Blood smear: Sezary cells  lymphoid
cells with convoluted or irregular
nucleus
NOTE:
MYCOSIS FUNGOIDES
 This affects the mature T cells. This is an
early stage of Sezary syndrome and is
characterized by pruritus, eczematoidal
psoriasis form non-specific exfoliative
dermatitis. Diagnostic evaluation of the skin
reveals Pautrier’smicroabscesses (clusters of
lymphocytes forming on the skin)
accompanied by parakeratosis.
 EARLIER STAGE
SEZARY SYNDROME
 This stage manifests infiltration of the lymph
nodes and viscera, characterized by band-like
infiltrates of lymphocytes with cerebriform
nuclei called Sezary cells.
 ADVANCE STAGE
PLASMA CELL DYSCRASIAS
Paraproteinemia: MM and WM  malignancies
involving plasma cells.
 Excessive proliferation of plasma cells even in
the absence of antigenic stimulation 
excessive production of abnormal
immunoglobulins.
1. MULTIPLE MYELOMA
 This is the most common clonal proliferative
malignancy of plasma cells.
 Monoclonal gammopathies  abnormal cells
produce a single type of immunoglobulins.
 Of the cases, approximately 50% produce only
IgG; 20% only IgA; and 15% only the L-chain
o Plasma cells: 50% - only IgG; or 20% -
only IgA and/ or 15% - only L-chain 
interfere in platelet function
 Marrow infiltration often leads to bone pain.
o Myeloma cells: cancerous and leukemic
plasma cells that cause increased
 HEMATOLOGY 2
production of antibodies  tumor
formation in bone marrow  lytic bone
lesions  bone pain
NOTE:
Clinical sign and symptoms:
 Lytic bone lesions
 Hyperviscosity: due to abnormal proteins in
the plasma
o Causes hypertension and
Cardiovascular Disease (CVD)
 Bleeding disorders
o Platelets and endothelial cells are
coated with the abnormal proteins
 preventing the expression of
glycoproteins and other receptors 
abnormal adhesion and aggregation
Laboratory findings reveal:
o Chemistry: Bence Jones Protein (BJP)
o Myeloma cells may produce BJP 
part of immunoglobulin but
represent only the L-chain  Low
MW  easily passes through
glomerulus  seen in urine 
urinary screening
o Urine screening: unique solubility
characteristic  when heated at
certain temperature it can
precipitate and be dissolved
 Not heated or <40°C = clear/
soluble
 40-60°C = urine becomes
turbid due to precipitation
of protein; BJP  insoluble
 100°C = clear; BJP  soluble
o Urinalysis: many hyaline casts and other
casts and positive BJP
o Hematology:
o Elevated ESR: Rouleaux formation
 Because of the abnormal
proteins produced, zeta
potential of the RBC is
altered  rouleaux
formation (normally in vivo
RBC does not form into
rouleaux  repel each other
through zeta potential; RBC
has sialic acid in their
phospholipid layer that
imparts a negative charge)
ESR: 3 stages
 Initial Rouleaux: first 10 mins
 rouleaux  becomes
heavier
 Rapid Settling: succeeding 40
mins  larger sediment will
settle faster
 Final Settling: last 10 mins
o Hypercalcemia: due to abnormal
metabolism of calcium  bone
lesions and pain
BLOOD FILM FINDINGS
 Flame cells: plasma cell with red to pink
cytoplasm associated with increase in IgA
o Plasma cells with cytoplasm filled
with IgA
o Appear like a flickering candle
 Dutcher bodies: intranuclear crystalline
structures of abnormal IgG.
o Contains precipitated IgG in the
nucleus of the plasma cells
 Grape cell/Mott cell, Morula cell: plasma cell
that contains small colorless or blue/pink
protein vacuoles that appear like grapes
o Plasma cells whose cytoplasm are
filled with several protein globules 
russel bodies: are globules seen in
the cytoplasm containing proteins
and IgG  made the cell look like a
bundle of grapes
o Russel bodies: accumulations of IgG

 Rouleaux formation
2. WALDENSTROM MACROGLOBULINEMIA
 This is the second most common type of plasma
cell dyscrasia.
 Almost similar with MM
 Myeloma plasma cells are more involve in the
production of IgM.
o Malignant plasma cells show increase
production of IgM (>3 g/dL)
o IgM: pentamer; big
macroglobulinemia
 Also called Lymphoplasmacytic lymphoma 
can affect lymphocytes that are not transformed
into plasma cells.
 Hyperviscosity: due to macroglobulins
NOTE:
LAB FINDINGS:
o Increased IgM (>3 g/dL)
o Rouleaux formation  Increased ESR
o BJP can be produced
DIFFERENCE BETWEEN WM AND MM
1. WM: predominantly IgM
MM: predominantly IgG/IgA/L-chain
2. MM: affects B-cells and plasma cells precursors
WM: only plasma cells are affected
RARE CONDITIONS:
3. PLASMA CELL LEUKEMIA
 >2x109/L plasma cell count
4. HEAVY CHAIN DISEASE (HCD)
 This shows an abnormal synthesis of heavy
chains (Gamma HCD; Alpha HCD)
HEMATOLOGY 2
LYMPHOMAS
- Lymphomas are cancers of the lymph nodes
characterized by uninhibited growth of cellular
elements normally found in lymphatic tissues
resulting to lymph node enlargement.
o Lymph nodes are well-organized
because they are involved in the
immunity.
- Proliferative disease affecting some of the blood
forming cells and tissues outside the bone
marrow  lymph tissues: lymph nodes and
vessels
- May affect either B-cells or T –cells
1. NON-HODGKIN LYMPHOMA (NHL)
 A more common type than Hodgkin lymphoma.
It is a B-cell lymphoma characterized by painless
lymph node enlargement (cervical nodes are
 most often involved)
 Cervical nodes  GIT lymph nodes 
enlargement.
 Represent 60% of lymphoma cases
 CAUSES: congenital immunodeficiency diseases
and viral agents
o Since it is a cancer, mechanism isn’t
clearly defined.
 CLASSIFICATION:
o Low grade: enlargement is slow; can
take years for the enlargement of lymph
nodes to be diagnosed as one
o Intermediate grade: Rapid enlargement
o High grade: More rapid enlargement
2. BURKITT HODGKIN LYMPHOMA
 This is a B-cell neoplasm associated with EBV
and HIV infections. It is endemic among African
children (observed as jaw mass). Cytogenic
abnormality involves a translocation of C-myc
gene on chromosome regions (Ch8:14)
 One type of non-hodgkin lymphoma which can
be B-type or T-type  can affects both B-cells
and T-cells
 Lymphoma that when it infiltrates the bone
marrow  it becomes leukemic  ALL-L3
 ALL-L3 and Burkitt  related to EBV and HIV
 Problem: translocation of a gene between 8 and
14 (more common; written as t (8;14)) or 8 and
 HEMATOLOGY 2
2 or 8 and 22; the gene translocated is is C-myc
gene
o C-myc gene is a normal gene that both
regulates proliferation and apoptosis
but its translocation will cause it to be
abnormal thus it will become
overexpressed  abnormal function
 Prominent feature: rapid proliferation of
affected cell  rapid enlargement of lymph
nodes
 DIAGNOSIS: Lymph node biopsy: diffused
lymphoid proliferation (many lymphoid cells;
monocyte and macrophage: clears cellular
debris)  STARRY SKY.
NOTE:
WHO Classification:
Endemic: more common in African children 
enlarged jaw or with jaw mass or upper lymph nodes
Sporadic: more common in Unites States 
American  begins at GIT  enlargement of GIT
lymph nodes
Immunodeficiency: EBV and HIV
3. HODGKIN LYMPHOMA
 This is characterized by painless enlarged lymph
nodes usually in the neck and/or in some, the
cervical nodes that spreads in an orderly fashion.
o If enlargement is rapid (severe cases) 
usually is painful.
o Orderly fashion enlargement  it
follows lymphatic circulation
 More on B cell lymphoma
 Signs and symptoms include:
o GENERAL: fever, night sweats, or 10%
unexplained weight loss in 6 months, or
a combination of both.
 Diagnosis: lymph node biopsy  SPECIFIC
IDENTIFICATION
 Hallmark finding: REED-STERNBERG (R-S) CELL
or LACUNAR HISTIOCYTES
 Giant lymph cell that is usually 4-8x larger than
lymphocytes.
 Giant multi/binucleated: more common and
each half is identical to each other
o Nuclei  shows distinct nucleolus with
very prominent nuclear halo and distinct
nuclear envelope  giving owl-eyes
image
 When cut in half; almost identical  MIRROR
IMAGE CELL.
 When seen is pathognomonic of the disease
 CBC is not the appropriate procedure because it
is not a cancer of the bone marrow thus the
disease have a normal CBC.
 But in cases wherein the lymphoma becomes
leukemic; CBC can be included.
 LEUKEMIC PHASE OF LYMPHOMA: CBC 
Leukocytosis and lymphocytopenia are observed
in advance cases  poor prognosis.
TWO CRITERIA:
A. ANN HARBOR CLASSIFICATION OF HIGKIN
LYMPHOMA
 Region of RN (I-IV):
o Suffix A: no s/s
o Suffix B: with s/s
o Subscript E: indicates involvement of
extra lymphatic tissue
o Subscript S: indicates involvement of
the spleen.
 Stages:
o Stage I: only one lymph node region
affected
o Stage II: two or more lymph node
region affected but all located in one
part of the diaphragm either upper or
lower.
 HEMATOLOGY 2

o Stage III: two or more lymph node

region affected but are disseminated in
both parts of the diaphragm
o Stage IV: involvement of an extra
lymphatic tissue (spleen, liver or any
other organs)

B. RYE CLASSIFICATION (WHO)

 based on extent of lymphocyte infiltration and
the abundance of R-S cells
a. Lymphocyte Predominant: Abundant
Lymphocytes with normal and abnormal
forms; no fibrosis; with few R-SCs
b. Nodular Sclerosis: Moderate Lymphocytes
with nodulating collagen bands; R-SCs in
clear zones
c. Mixed Cellularity: Moderate lymphocytes in
diffuse pattern
d. Lymphocyte Depleted: few lymphocytes
with diffused fibrosis and many R-SCs

Stage Rye Histological Prognosis

Classification Features
I Lymphocyte Lymphocytes BEST
predominant and R-SCs
II Nodular Nodules of GOOD
sclerosis lymphoreticu
lar cells and
lacunar RSCs
III Mixed Mixture of FAIR
cellularity lymphocytes,
eosinophils,
plasma cells
and RSCs
IV Lymphocyte Lymphocytes POOR
depleted and RSCs
 HEMATOLOGY 2

FINALS o Proper position of patient during

phlebotomy: in comfortable position,
whether upright/sitting or lying in bed.
ERYTHROCYTE DISORDERS
o Sudden change in position:
 ERYTHROCYTIC DISORDER: affects RBC
Hemodilution: upright to lying: shift of
functions  oxygen transport as conducted by
fluid from extravascular to
its hemoglobin content thus affecting the oxygen
intravascular/circulation  decreased
delivery function of RBCs called ANEMIA.
RCM.
 ANEMIAS: are group of clinical conditions o Hemoconcentration: lying to upright:
associated with either a decrease in the number shift of fluid from intravascular to
of total red blood cells, a decrease in the
extravascular increased RCM (PV) 
concentration of hemoglobin, and/or a decrease REMEDY: wait for at least 20 minutes to
in the hematocrit (in any or all of the RCM) in
elapse for the fluid to equilibrate
relation to the individual’s age, gender and o IV fluid: hemodilution
geographic location.
o Anemia conditions can be absolute or CLINICAL SIGNS:
relative.
General signs and symptoms due to lack of oxygenation
o Decrease in RCM:
in tissues
 Hemoglobin (single lab test
important in screening anemia)  Easy fatigability and dyspnea on exertion  lack
 RBC count of oxygenation
 Hematocrit  Vertigo and faintness  drop in circulation thus
 Decrease oxygen delivery in less circulation and oxygenation in the brain
tissue preventing normal o Nervousness/faint  REMEDY: patient
cellular respiration. must lay down improve circulation in
 POLYCYTHEMIA VERA: all findings are opposite the brain
of anemia  increase in all  increase in RCM  Palpitation, rapid bounding pulse, systolic
(absolute PV) or decrease murmurs and headache o Due to lack
plasma/hemoconcentration (relative PV)  oxygenation  systolic murmur  heart
hematocrit (single lab test important in compensates
screening PV) o Brain demands large amount of oxygen
therefore the heart compensates by
TYPES:
redistributing the circulation of blood
 ABSOLUTE: True decrease in RCM  Decreased through transporting more oxygen to
in production (bone marrow defect that does not the brain causing an increase in cardiac
release a normal RBCs); increased loss output.
(hemorrhagic); increased destruction o Compensation of heart: increase cardiac
(hemolytic) output  increase pressure
o Normal plasma volume but PRBCs or o Low Blood Pressure due to decrease
PCV is decreased blood output causing increase cardiac
 RELATIVE: related to volume of plasma: plasma output causing increase pressure
expansion  hemodilution: shift of fluid from leading to headache  Pallor (lacks
extravascular to intravascular; false hemoglobin  heme: gives the normal
interpretation: decreased in cell count, hb and red pigment of blood)
hct.  low BP (lack of blood)
o Specimen collection: Very important to  Slight fever (especially when coexistent with an
consider infection) and dependent edema
 HEMATOLOGY 2
SPECIFIC CLINCAL SIGNS
JAUNDICE: yellow discoloration of the LEG ULCERS: open wounds most
skin, eyes, nail bed due to commonly seen lower extremities
accumulation of unconjugated because it is affected by thrombosis
bilirubin (persist in circulation(yellow  in sickle cell anemia  rigid cells
plasma); water insoluble and bound  hyperviscosity of blood leading to
to albumin; not excreted in the urine) thrombosis
 specific sign seen in hemolytic
anemia In normal condition: RBCs are containing CHON (myoglobin and
discocyte, flexible and deformable  cytochrome) and tissues  heme
pass through small vessels.
In sickle cell anemia, rigid cells and color of blood and flesh.
not deformable therefore they
obstruct the circulation leading to
hypervicosity of blood  thrombosis
 vasoocclusion decrease in
oxygenation  delay wound healing
FRONTAL BOSSING: seen in NEUROLOGIC DEFICIT: seen in
Thalassemias: Betathalassemia major Vitamin B12 deficiency  decrease
(more common) and DNA production leading to
hemoglobinopathies megaloblastic anemia (affects blood
cell maturation) and destruction of
Cause: Skull (flat bone) that is active in CNS tissues due to inefficient myelin
hematopoiesis. In B-thalassemia sheath  severe cases: mood swings
major: no hemoglobin(A1) which is and change in behavior 
the major hemoglobin in adults so the Megaloblastic madness
blood production tries to compensate
 red marrow expansion inside the Vit. B12: important and serve as a
flat bone producing more blood cells cofactor in many metabolic reaction:
 bone structure also expands  DNA synthesis and maintenance of
skeletal deformity myelin sheath of the brain and spinal
B-THALASSEMIA: manifest mongoloid cord or CNS tissues
features due to skeletal deformity
KOILONYCHIAS: spooning of nail bed
 seen in Iron
Deficiency Anemia
IDA  hookworm infections
Iron is very much needed by the body:
hemoglobin and other heme
contains iron that give offs normal
CYANOSIS: seen in
methemoglobinemia  increase in
abnormal hemoglobin: hemoglobin
Hemoglobin “M”s  methemoglobin:
hb that is not protected from
oxidization  easily oxidized (ferrous
to ferric form)  Hb M Boston, Hb M
Iwate, Hb M Hyde park, Hb M
Saskatoon, Hb M Milwaukee, Hb M
Osaka and Hb M Fort Ripley.
Methemoglobin is normal in blood
but in small amounts: >3.5% but if
increased it becomes abnormal
because it cannot transport oxygen
especially in distal tissue like fingers.
 HEMATOLOGY 2
COMPENSATORY MECHANISMS
PHYSIOLOGIC RESPONSE
Immediately occur
. Shift to the right of the Oxygen Dissociation Curve
Y-axis = oxygen saturation; increase affinity of hb to O2
X-axis = partial pressure of oxygen; decrease affinity of
hb to O2
2.Increase 2,3 DPG shift to right in ODC and more
oxygen will be released towards the tissue
TWO FORMS OF HEMOGLOBIN BASED ON
OXYGENATION:
1. Oxyhemoglobin – “R” relax form; No 2,3 DPG;
fully oxygenated  increased affinity to
oxygen; but once in tissue there will be
decreased in the affinity 2,3 DPG inserts in the
hb
2. Deoxyhemoglobin – “T” tense form; with 2,3
DPG that lowers the hb affinity to oxygen
release oxygen in the tissue
3.Selective redistribution of blood flow to areas of
highest oxygen demand like in the brain
4.Increased cardiac output
RETICULOCYTE
 Immature cell
 Normal count: Adults: 0.5-1.5% (average: 1%
Retics)
HEMATOLOGIC RESPONSE
Take time to occur
Erythropoiesis: 3-5 days (from pronormoblast to
reticulocyte)
 Normally: 3.5 days will be spent in BM and 1 day
in the circulation mature RBC
 Anemia: reticulocyte will be released in the
circulation less than its normal (<3 days); if the
number of days in the BM is shorten, the longer
the number of days the reticulocyte
will take in the circulation
Increase in erythropoietin (EPO)
 Increase erythroid precursors
 Accelerates rate of proliferation and maturation
 Accelerate release from the BM to the PB
Increase shift cells/stress reticulocyte
SHIFT/ STRESS CELLS/ RETICS
 Prematurely released
 More immature than reticulocyte
 Increased RNA
 HEMATOLOGY 2
o Maintains 1% retics everyday
o In anemia, prolonged in the circulation
 increased retics count; and if they are
shift (more immature more RNA)
Romanowky stain – polychromatic
MAJOR CAUSES OF ANEMIA
1. GENETIC FACTORS
- Abnormalities in the structure of some
molecules present in the RBC.
a. Hemoglobin C, S: can’t function well in
delivering oxygen leading to anemia.
b. Cell membrane: abnormal spectrin and
ankyrin due to abnormal gene  RBC easily
deformed  spherocytes/elliptocytes)
c. Enzymes: affects metabolism of RBC (like
G6PD deficiency  needed in hexose
monophosphate shunt )
2. ENVIRONMENT FACTORS
- Factors that interfere with the normal
development and functioning of RBCs.
a. Lead exposure may lead to Sideroblastic
Anemia (SDA); lead interfere in heme
synthesis affecting the utilization of iron 
iron cannot be utilized therefore precipitate
as siderotic granules
b. BENZENE – laboratory reagent that exposure
may lead to developing aplastic anemia;
toxic and damages BM thus preventing
production of blood cells
c. Failure to acquire nutrients, vitamins,
minerals (e.g. from vegetables) 
Nutritional Deficiency Anemia: Iron
deficiency (IDA), Lacks dietary vegetable and
meat (poultry product)  B12 and Folate
Deficiency
3. PATHOLOGICAL FACTORS
- infectious organisms and parasites
a. PARASITES
o In anemia, prolonged in the circulation 
increased retics count; and if they are shift
(more immature more RNA)
Supravital stain – bluish granules More granules  shift cells
o D. latum - tapeworm that lives in ileum;
interferes in the absorption of Vit. B12
anemia leading to Vit. B12 deficiency
anemia or fish tapeworm anemia; often
referred as bothricephalus pernicious
anemia
o Hookworms – Ancylostoma duodenale
and Necator americanus: blood sucking
nematodes that attaches in the mucosal
of the small intestine through their
buccal cavity. As they move to one area
to another, the area of attachment will
be left behind as chronic/minute
bleeding area and mechanical sucking it
will cause blood loss (hb loss  iron
loss especially chronic infection with
heavy worm burden  IDA)
o Malaria/Plasmodium spp. and Babesia
– live inside the RBC  ruptures and
destroy RBCs hemolysis  hemolytic
anemia
4. IATROGENIC FACTORS
- These are results of side-effects of legitimate
medical treatment/procedures that may
interfere with the normal development and
function of RBC
a. Prolonged use of antibiotics:
Chlorampenicol – destroys BM leading to
development of aplastic anemia b. Surgery
– hemorrhagic/blood loss anemia
 HEMATOLOGY 2

LABORATORY DIAGNOSIS NOTE:

- To screen for the presence of anemia  Reference Value:
performs CBC  Normocytic: 80-100 fL
1. HEMOGLOBIN (ANEMIA) AND HEMATOCROT  Microcytic: <80 fL
(PCV)  Macrocytic: >100 fL
 Single test to screen anemia: Hemoglobin  if FORMULA:
low, proceed with stated test below. 𝑯𝒄𝒕 (𝑳/𝑳) × 𝟏𝟎𝟎 𝑯𝒄𝒕 (%) × 𝟏𝟎
𝒐𝒓
𝑹𝑩𝑪 (𝟏𝟎𝟏𝟐 /𝑳) 𝑹𝑩𝑪 (𝟏𝟎𝟏𝟐 /𝑳)
NOTE:
HEMOGLOBIN
 Reference Value:  MCHC: refers to average Hb concentration of
FORMULA: red cells in a given volume of blood
𝑨 𝒐𝒇 𝒕𝒆𝒔𝒕 (𝒖𝒏𝒌𝒏𝒐𝒘𝒏)
NOTE:
𝑨 𝒐𝒇 𝒔𝒕𝒂𝒏𝒅𝒂𝒓𝒅
Reference Value:
× 𝑺𝒕𝒂𝒏𝒅𝒂𝒓𝒅 𝒄𝒐𝒏𝒄. (𝟏𝟓𝒈𝒎/𝑳)
= 𝒈/𝑳 𝒐𝒓 𝒈/𝒅𝑳 (𝒘𝒊𝒕𝒉 𝒅𝒆𝒎𝒊𝒄𝒂𝒍)  Normochromic: 320-360 g/L or 32-36% or 32-
HEMATOCRIT 26 g/dL
 Reference Value:  Hypochromic: <32%
 Male: 140-175 g/L  Spherocytic  full of Hb: >36%
 Female: 123-153 g/L FORMULA:
𝑯𝒈𝑩 (𝒈/𝒅𝑳) 𝑯𝒈𝑩 (𝒈/𝒅𝑳) × 𝟏𝟎𝟎
 At birth: 150-200 g/L 𝒐𝒓
FORMULA 𝑯𝒄𝒕 (𝑳/𝑳) 𝑯𝒄𝒕 (%)
1. INDIRECT:
𝑯𝒄𝒕 = 𝑷𝑪𝑽 × 𝑹𝑩𝑪 𝑪𝒐𝒖𝒏𝒕
3. RETICULOCYTE COUNT; RETICULOCYTE
2. DIRECT
𝑯𝒕 𝒐𝒇 𝑷𝑪𝑽 × 𝟏𝟎𝟎 PRODUCTION INDEX (RPI) OR SHIFT
= 𝑽𝒐𝒍. 𝒐𝒇 %𝑯𝒄𝒕 CORRECTION
𝑯𝒕 𝒐𝒇 𝒘𝒉𝒐𝒍𝒆 𝒃𝒍𝒐𝒐𝒅 (𝑻𝑽)
𝒐𝒓  Compensatory mechanism of BM is to increase
𝑷𝑪𝑽 its reticulocyte production  increased
= 𝑳/𝑳 (𝒅𝒆𝒄𝒊𝒎𝒂𝒍)
𝑻𝑽 reticulocyte count.
 RPI: to measure whether the increase in
production is parallel or compensatory to the
2. BLOOD INDICES: MCV, MCHC, RDW
degree of anemia
 For morphological classification of anemia
o Index calculated to correct for the
 RDW: represents the ratio of the standard
presence of shift reticulocytes that
deviation to the MCV )Width of histogram)
otherwise may falsely elevate the visual
o Index of relative anisocytosis; may also
reticulocyte count
indicate poikilocytosis
o Increased in hemolytic and hemorrhagic
REFERENCE VALUE: anemia
 11.6- 14.6% o Reference value: 1 if hct is 0.45
 >14.6% indices variation in sizes
NOTE:
RETICULOCYTE COUNT:
 MCV: refers to the average volume of PCV of the  Reference value: 0.5 -1.5%  average: 1%
individual red cells in a given blood samples.
o It is used as an estimation of the average RETICULOCYTE PRODUCTION INDEX (RPI)
size of the RBC  Reference value: 1 if hct is 0.45
𝑪𝒐𝒓𝒓𝒆𝒄𝒕𝒆𝒅 𝒓𝒆𝒕𝒊𝒄𝒖𝒍𝒐𝒄𝒚𝒕𝒆 𝒄𝒐𝒖𝒏𝒕
o No dependable when RBC vary markedly  FORMULA:
𝑴𝒂𝒕𝒖𝒓𝒂𝒕𝒊𝒐𝒏 𝒕𝒊𝒎𝒆 (𝒅𝒂𝒚𝒔)
in size.
 HEMATOLOGY 2

4. MYELOID: ERYTHORID RATIO B. ANEMIA ASSOCIATED WITH DEFECTIVE HGB

 Ratio between granulocytes and erythrocytes SYNTHESIS
precursor inside the BM  Defect in globin or heme synthesis
 Normal ratio: 3:1  1 RBC precursor: 3  Heme: IDA, SDA
Granulocytic precursors  Globin: thalassemia
 CGL/CML ratio: >10:1  M:E ratio increases
 Anemia: 3:3  1:1  M:E ratio decreases C. ANEMIA WITH VITAMIN B12 OR FOLATE
DEFICIENCY
5. OTHER SPECIAL TEST DIAGNOSTIC OF THE  Nutritional deficiency anemia: IDA, B12 or
SPECIFIC TYPE OF ANEMIA folate deficiency anemia
 AIHA: serologic test: antibody testing to
indentify the specific autoantibody D. ANEMIA ASSOCIATED WITH IMPAIRED BONE
 Pernicious anemia: Schilling’s test MARROW OR STEM CELL FUNCTION
 Aplastic anemia; bone marrow is infiltrated with
CLASSIFICATION OF ANEMIA fats thus reduced or no red marrow that is the
- Some anemia have more than 1 pathologic
active marrow producing blood cells.
mechanism (e.g. B12 deficiency  tapeworm
infection + nutritional deficiency + antibody) and
E. ANEMIAS ASSOCIATED WUTH DECREASED RED
go through more than one morphologic stage
CELL SIRVIVAL AND INCREASED RED CELL
(IDA: microcytic-hypochromic; normocytic-
DESTRUCTION
hypochromic)
 Prone to damage: hemolytic anemia
1. MORPHOLOGIC CLASSIFICATION
2. ETIOLOGIC CLASSIFICATION
F. ANEMIA SECONDARY TO BLOOD LOSS
3. PHYSIOLOGIC CLASSIFICATION
 Hemorrhagic anemia:
 The ability of the bone marrow to respond
o Chronic blood loss
physiologically by producing erythrocytes is
o Acute blood loss iii. Post-acute
measured by RPI  Ineffective
hemorrhagic anemia  IDA
erythropoiesis (RPI <2.0)
 Effective erythropoiesis (RPI >3.0)  e.g. RPI= 4
means that the rate of production has increased
4x than its usual for compensation

NOTE:
Some anemia have more than 1 pathologic
mechanism & go through more than one morphologic
stage.

ETIOLOGIC CLASSIFICATION OF ANEMIAS: CAUSE OF

ANEMIA
A. RELATIVE ANEMIA
 Increase in plasma volume  hemodilution
 RBCs are NOT reduced but is diluted with
increase plasma volume
 TRUE/ABSOLUTE ANEMIA: reduced in number
of RBC due to defective production
 HEMATOLOGY 2

MORPHOLOGIC CLASSIFICATIONS OF ANEMIA

- Usually followed
- Basis: size and hemoglobin

MCV: according to size
<80 fL = microcytic
80-100 fL = normocytic
>100 fL = macrocytic
MCHC: hemoglobin concentration RDW: a measure of the degree of
MCH is not helpful in morphological anisocytosis and poikilocytosis  used
classification of anemia because it always because MCV and MCHC cannot be rely
follow the MCHC upon when the cell population is diverse.
<32% = hypochromic
32-36% = normochromic machine gives its value from histogram
>36% = spherocytic
According to MCV and MCHC: single
population
3 CLASSIFICATION OF ANEMIA
1. Normocytic – normochromic
2. Microcytic – hypochromic
3. Macrocytic – normochromic
4. Normocytic - hypochromic  gradual
changes seen in IDA

MCV: Single red cell index that can be used to CBC  anemia  retic count  morphologic classification
morphologically classify anemia; will suffice alone 
microcytic; macrocytic; normocytic

MICROCYTIC: the most common anemias included are

those
characterized by defect in hb: heme: SDA, IDA; globin: ACI,
Thalassemia  in general thalassemia fall under
microcytic anemia

GLOBIN DEFECTS: deficiency in alpha and beta chain

synthesis: thalassemia; defect in amino acid
structure/composition: hemoglobinopathies  fall under
normocytic anemia (Hb C and S) except Hb E disease or
trait.
 HEMATOLOGY 2

MACROCYTIC: include problems in DNA synthesis 

megaloblastic anemia

NORMOCYTIC: more on destruction (hemolytic) and

decrease in production (aplastic: BM failure)

MICROCYTIC HYPOCHROMIC MACROCYTIC HYPOCHROMIC NORMOCYTIC NORMOCHROMIC

Main cause: defective hb synthesis Commonly Macrocytic Commonly Normocytic
Heme synthesis: IDA, SDA, ACI Globin  Vitamin B12 deficiency  Hypoproliferative anemia
synthesis: alpha or beta thalassemia and anemia  Folic acid  Myelophthisic anemia
Hb E disease deficiency anemia  Hemolytic anemia
 Hemoglobinopathies
Commonly Microcytic Occasionally Macrocytic  Acute blood loss anemia
 Iron deficiency anemia  Hypoproliferative anemia  Anemia of chronic disease
 Sideroblastic anemia   Refractory anemia  Renal and endocrine
Thalassemia  Liver disease diseases
 Hemolytic anemia  Paroxysmal nocturnal
Occasionally Microcytic  Acute blood loss anemia hemoglobinuria
 Anemia of chronic
disease/inflammation Occasionally Normocytic
 Hemoglobinophaties (Hb E  Early iron deficiency
disease  Refractory anemia
The cells will keep on dividing until their optimum MCHC is
reached. In normal erythropoiesis, it takes only 4-5
divisions for mitosis  Pronormoblast will divide once
producing 2 basophilic normoblast (BN) and these BN will
divide twice and the last to go division is the rubicyte  4x
division = 16 ; 5x division = 32

Every time the cell divides the cell size becomes smaller

In microcytic-hypochromic, since due to decrease hb, it

will not reach its optimum MCHC therefore it will keeps on
dividing thus decrease in cell size Microcytic: extra division
Hypochromic: decrease Hb.
 HEMATOLOGY 2
MICROCYTIC- HYPOCHROMIC ANEMIAS
 ETIOLOGY: Microcytosis is caused by one or
more extra cell division due to the depression of
the MCHC. This conditions also exhibits
hypochromia which is cause by an impairment in
the hemoglobin synthesis due to abnormalities
in either the heme or globin synthesis or both.
1. IRON DEFICIENCY ANEMIA (IDA)
 Iron Metabolism: Iron is absorbed in the ferrous
form, later oxidized in the mucosal cells to the
ferric form. Absorbed iron passed on directly to
the blood stream by the mucosal cells, but most
of it is bound to Apoferritin, which then forms
Ferritin. Iron in the bloodstream is transported
by Transferrin which delivers it to the
mononuclear phagocytic cells (MPC) of the
bone marrow and other issues. During
erythropoiesis, the MPC of the bone marrow
incorporated iron into the developing red cell.
 NORMAL BODY IRON (FE): 4,000 mg
o 3 COMPARTMENTS: iron is distributed
among these compartments and sub-
compartments.
 STORAGE: in the form of ferritin
(major form in the liver and
tissue) and hemosiderin
 TRANSPORT: protein
transporter for iron
 FUNCTIONAL: hemoglobin,
myoglobin, iron-containing
enzyme. Most of the iron is in
the hemoglobin approximately
60%; thus deficiency in iron will
affect hemoglobin resulting in
anemia.
 IRON KINETICS:
o Fe++ & Fe+++ (diet)  Fe++ (stomach)
 Fe+++ (Mucosal cells)
 What the body can absorb is
only the ferrous form; the ferric
form can be reduced into
ferrous form in the stomach by
our gastric juices (very acidic
due to HCl) to promote
absorption, when the ferrous
form of iron reaches the small
intestine (particularly in
duodenum and jejunum) they
will be absorbed by mucosal
cells and immediately cause
oxidation of ferrous thus
converting back into ferric form.
The ferric form will be released
into the plasma circulation by
the mucosal cells.
o Fe+++  bloodstream
 Fe binds with apoferritin to form
FERRITIN (major storage form of
iron  stored in tissues and
liver).
 Fe-transferrin  transported to
Mononuclear Phagocytic Cell
(MPC) going into the bone
marrow and liver.
NOTE:
 In bone marrow, during erythropoiesis, when
the developing red cell start to synthesize hb,
mononuclear cell will transport and
incorporate iron into developing RBC 
incorporation is called ROPHEOCYTOSIS
 ROPHEOCYTOSIS: During hemoglobin
synthesis in immature red cell, mononuclear
cell will incorporate the iron thus iron (Fe+++)
will be released and immediately reduced into
ferrous (Fe++) and incorporate itself into
protoporphyrin IX to form heme.
ETIOLOGY/ RELATED CAUSES: IDA
1. CHRONIC BLOOD LOSS
 Most common cause of IDA in adults.
 HEMATOLOGY 2
 Small volume but chronic
 Male: GIT bleeding  unnoticed because it
occurs inside unless it develops into other
serious complications.
 Female: prolonged menstruation, bleeding
after childbirth or cesarean
 ACUTE BLOOD LOSS will NOT lead to IDA
STAGES IN THE DEVELOPMENT OF IDA
2. INCREASE DEMAND
 especially among pregnant women (30-
75mg), adolescents and infants
 Not pregnant female: 10-20 mg thus female
needs more iron due to monthly blood loss
 Pregnant: ferrous sulfate supplement to
prevent IDA
 Adolescent: fast growing  more iron needed
by the body
 Infants: breastmilk – poor in iron  pure
breastfed infants may develop IDA called
“milk anemia of infancy”  Male has less iron
demand because they do not have monthly
menstruation/blood loss
3. MALABSORPTION
 Absorption of iron happen in small intestine
(duodenum and jejunum).
 Regardless of the amount of iron take in, the
absorption is limited to 10% of dietary iron
 Celiac disease (sprue): disorder characterized
by antibody destruction of the small intestines
 destroyed absorption sites  total
gastrectomy: decreased absorption sites
 Intestinal Parasites:
o N. americanus: 0.03 ml/day  per
worm
o A. duodenale: 0.15 – 0.25 ml/day 
per worm
o T. trichiura: 0.005 ml/day  per
worm
4. POOR DIET
5. FREQUENT BLOOD DONATION (225 gm/unit)
 450 ml: blood donated
PROMOTE ABSORPTION REDUCE ABSORPTION
Ferrous form Ferric form
Inorganic form Organic form
Acids: HCl, Vitamin C Alkali: Antacids
Pancreatic secretions
Solubilizing agents: Precipitating agents:
sugars and amino acids phosphates
Iron deficiency Iron excess
Increased erythropoiesis Decreased erythropoiesis
infection
 STAGE I: Iron depletion stage: Iron is mobilized
from stores, storage iron decreases, plasma
ferritin decreases, iron absorption increases.
o First to decrease is iron storage
compartment which is measured by
transferrin13
o Serum ferritin first decreases and
reflects the iron storage or ferritin
o Heme is consisting of iron and
protoporphyrin IX. Deficiency in iron
would lead to increase in
protoporphyrin IX and will accumulate
inside the RBC which is measured as Free
Erythrocyte Porphyrin  FEP is increase
o Since functional iron is still normal then
hemoglobin and hematocrit are normal
thus, it is characterized as normocytic
and normochromic
o Normal serum iron and TIBC
 STAGE II: Iron deficient erythropoiesis: after
iron stores are depleted, the plasma iron
concentration falls, saturation of transferrin falls
below 15% and the percentage of sideroblasts
decreases in the marrow.
o Iron storage (ferritin) and transport iron
is depleted
o Decrease in serum iron with increase
FEP
o Increase TIBC: indirect measurement of
transferrin
o Transferrin: transport two molecules of
iron simultaneously
 When transferrin is bound into
iron because iron is normal, it is
fully saturated thus it will no
longer bind with additional iron.
In the absence of iron (IDA),
transferrin remains to be free.
 HEMATOLOGY 2
When added with the reagent
iron, it will bind with iron.
 Transferrin when it is not
saturated, its binding capacity
for iron is increased measured
by TIBC.
o Functional iron is still normal thus the
anemia is normocytic – normochromic
 When the cells are in the
circulation, they are fully
hemoglobinized and in the
presence of IDA, the mature RBC
will not be affected because the
mature RBC have fully
synthesized their hemoglobin.
 IDA affects the developing RBC
in the bone marrow wherein the
bone marrow will immediately
suffer in decreased in iron
because of the storage form of
iron.
 Aside from the liver and tissues
having the storage form, there is
also in the bone marrow which
will be first depleted.
 STAGE III: Iron deficiency anemia: anemia is now
detectable. The anemia is at first normochromic
acid normocytic, gradually becomes microcytic
IRON PROFILE TESTS FOR THE DIFFERENTIAL DIAGNOSIS
and finally microcytic & hypochromic.
OF IRON DEFICIENCY ANEMIA
o Iron deficient red cells from the bone
marrow will be released and will appear
as microcytic as reflected by the low
hemoglobin (hypochromic).
o Findings: decrease hemoglobin, serum
iron, ferritin ; increase TIBC
LABORATORY FINDINGS OF IDA
- In early IDA, RBCs are N/N; in later stages, RBCs
become Microcytic-Hypochromic. Severe cases
will exhibit poikilocytosis & anisocytosis 
Reticulocyte count is low; Platelet count is
slightly elevated
- Bone marrow smear is negative for hemosiderin
granules
- Serum iron is decreased, and serum ferritin may
be reduced to zero; increase Free Erythrocyte
Protoporphyrin (FEP); increase Total Iron-
Binding Capacity (TIBC)
- General blood picture: microcytic-hypochromic
with poikilocytosis and anisocytosis and elevated
RDW.
o RDW: measure in the variation of size
and shape
- Reticulocytopenia: Even though the red marrow
tries to compensate, the red cells are
ineffectively growing therefore
reticulocytopenia.
- Slight thrombocytosis: If it is associated with
bleeding because bleeding is also one of the
causes of IDA, chronic bleeding (most common
cause of IDA) especially in adults, it may also be
characterized by slight thrombosis.
- Prussian blue staining: hemosiderin granules are
negative.
 SERUM FERRITIN
o Reflects the body’s tissue iron stores
o Decrease in iron causes increase
protoporphyrin 9. Though normally, RBC
produces more protoporphyrin than
iron, but in the absence of iron, the more
the protoporphyrin will accumulate
causing increase FEP. It is free because it
is supposed to bind with iron but since
there is no iron, it remains to be free
erythrocyte protoporphyrin. Some
measured it as ZEP because the FEP can
combine with zinc and measured as zinc
erythrocyte protoporphyrin
o Reference value: 12-300 ug/dL
 SERUM IRON
 HEMATOLOGY 2
o A sensitive indicator or iron depletion
but has a marked diurnal variation by as
much as 30% with its highest values in
the morning and lowest values late in
the day.
o Reference value: 50-160 ug/dL
o Decreased in IDA
 FREE ERYTHROCYTE PROTOPORPHYRIN (FEP)
o Normally, red cells produce slightly more
protoporphyrin than what is needed, but
when iron is deficient, protoporphyrin
build up in the erythrocytes
o Reference value: 10-99 ug/dL
o Increased in IDA; decreased in most
SDAs.
 TOTAL IRON BINDING CAPACITY (TIBC)
o Binding capacity: measure the ability of
transferrin to bind with iron. Wherein, if
tranferrin is not bound to iron, it will
bind. But if the transferrin is already
saturated with iron, it can not further
bind with other iron (in the reagent).
o Wherein in normal iron status, iron is
plenty thus transferrin carry two iron
molecules.
o In IDA, iron is depleted thus they carry
no iron thus transferring is free. When
the reagent (iron) is added, the
transferrin can bind with it because TIBC
is increase.
o Reference value: 250-400 ug/dL
o Increased in IDA
 TRANFERRIN SATURATION/ PERCENT
SATURATION OF TIBC
o Measures how transferrin is saturated;
percent saturation
o Reference value: 20-55%
o Decreased in IDA
NOTE:
Since iron is not only for hemoglobin synthesis but also
important for the tissues, enzymes, myoglobin and
other proteins, these tissues and proteins will also be
damaged due to IDA.
CLINICAL FEATURES OF IDA
 TONGUE- ATROPHY: The tongue is red because
of iron in heme present in hemoglobin in blood
and myoglobin in tissues, but since it lacks iron,
it will atrophy (become smaller).
 ANGULAR STOMATITIS: the lips will get dry and
have cracks
 CHEILITIS: the drying and inflammation of the
lips; associated with IDA
 “Webs” of tissue or partial strictures at the
junction of the esophagus and hypopharynx:
there will be cracks and wounds inside 
resulting now to difficulty in swallowing; affects
entire GIT.
 CHRONIC GASTRITIS – decreased gastric fluid
secretion which is important because it is the
gastric juice that is acidic which helps in the
absorption of iron. The acidity of gastric juices
helps reduce the ferric into ferrous in order to
absorb the dietary iron even in the ferric form.
 NUMBNESS & TINGLING SENSATION: peripheral
tissues nerves will be affected especially the
distal parts of the body (toes, fingers, and feet).
 KOILONYCHIA: distortion of the nail bed, spoon
shaped of the nails due to lack of iron in the
tissues
 PICA: unusual and compulsive appetite for a
certain kind of food or non-food item; among
children with severe IDA wherein they even have
abnormal craving for unusual food.
 SPLENOMEGALY: spleen function to filter blood
and remove a lot of abnormal cells so it will be
overworked and abnormal cells will clog in the
splenic circulation resulting enlargement of
spleen.
NOTE:
Aside from the general manifestations of anemia,
other manifestations include paresthesia, pica,
koilonychia, atrophy of epithelium of the tongue with
burning or soreness, fissures/cracks or ulcers at the
corners of the mouth, difficulty swallowing owing to
webs of tissue or partial strictures at the junction of
the esophagus & hypopharynx. Chronic IDA also
manifests gastritis and splenomegaly.
GENERAL BLOOD PICTURE OF IDA
 Microcytic-hypochromic
 HEMATOLOGY 2
o Central pallor: >1/3 diameter
o Normal RBC has the same size as the
small lymphocyte
 So roughly, even with the absence of MCHC
computation; blood picture alone can suffice
that the cells are hypochromic  central
pallor is greater than 1/3 of the diameter.
TREATMENT OF IDA
 Dependent on the cause of IDA.
 Important to consider the etiology for the
treatment of anemia.
 Since iron is primarily deficient, iron supplement
is given.
 If the main cause of IDA is malnutrition, ferrous
sulfate supplement may be given.
 If the cause is chronic blood loss, blood loss must
be supervised and give iron supplement to
monitor the progression of disease.
 If it is caused by parasite, eradicate the parasite.
NOTE: RECAP
IRON DEFICIENCY ANEMIA
 Described as microcytic: hypochromic type of
anemia
 Morphologic changes: At first it is normocytic
and as it becomes more severe, it becomes
microcytic.
IRON DISTRIBUTION: 3 POOLS/ COMPARTMENTS
a. IRON STORAGE POOL: includes ferritin
measured using serum ferritin and
hemosiderin measured via Prussian blue
staining.
b. TRANSPORT COMPARTMENT: iron bound to
transferrin measured as serum iron or
transferrin saturation or total iron binding
capacity (TIBC).
c. FUNCTIONAL IRON COMPARTMENT: iron
present in functional proteins such as
hemoglobin, myoglobin and other enzymes.
2. SIDEROBLASTIC ANEMIA (SDA)
 It is a group of anemias characterized by
abnormalities in the synthesis or enzyme
involved in synthesis of heme.
 SDA are a group of heterogeneous disorders
characterized by hypochromic red cells, which
are microcytic in the hereditary forms and often
macrocytic in the acquired forms, as a result of
defective heme synthesis, the red cells may also
be mixed with normochromic cells (dimorphic
appearance).
 Associated with defective protoporphyrin
synthesis which leads to excessive accumulation
of iron. The unused iron is deposited in the
mitochondria of normoblasts as siderotic
granules.
NOTE:
In SDA, the first enzyme (D-ALA synthase) is commonly
affected or if not the last enzyme (ferrochelatase). If
the D-ALA synthetase is affected, defective or
deficient, it will slow down the very first reaction
which is the condensation between succinyl CoA and
glycine and deficiency in this reaction would lead to
deficient D-ALA and the following reactions will
become retarded or slow down thus protoporphyrin
IX will also be deficient. When iron is transported in
the RBC, it will not be utilized because of the
deficiency in protoporphyrin IX; wherein iron is
supposed to bind with protoporphyrin IX but in the
absence of protoporphyrin IX, the iron will be
unutilized and the non-utilized iron will precipitate
inside the RBC, inside the mitochondria of RBC
becoming now the siderotic granules. When siderotic
granules are found surrounding the nucleus of RBC, it
is called the ringed-sideroblast which are immature
RBC containing siderotic granules which are non-
utilized iron.
COMPARISON:
 In IDA, iron is deficient thus increase in
protoporphyrin IX
o Negative in prussian blue staining
 HEMATOLOGY 2
 In SDA, protoporphyrin IX is deficient thus iron
will not be utilized and precipitate as siderotic
granules
o Not in all types of SDA, the
protoporphyrin IX will be decrease.
There are some types wherein
protoporphyrin IX will be increased.
o Dimorphism is observed in
sideroblastic anemia.
o Some cases, protoporphyrin IX is
increase or decrease depending on
which stage in the heme synthesis is
inhibited.
o SDA is highly positive in Prussian blue
stain since iron is not utilized
CLASSIFICATION/ TYPES OF SDA
1. INHERITED
 Caused by an abnormal gene
 Inability to inherit the gene needed by the
enzyme
 Characterized by microcytic cells
2. ACQUIRED
OTHER RELATED DISORDER:
 Exposure to agent that will inhibit the function of
the enzyme like in lead poisoning wherein lead
interferes with some of the enzymes involved in
heme synthesis.
 Characterized by macrocytic cells.
 Example: lead poisoning  lead may interfere in
the synthesis by inhibiting pyridoxal phosphate
ALA synthetase, ALA dehydrase or
ferrochelatase. When ferrochelatase or heme
synthase (which catalyzes the addition of
protoporphyrin IX with iron) is inhibited by lead
 protoporphyrin IX may no longer bind with
iron thus there would be protoporphyrin IX and
iron build up
A. HEREDITARY SIDEROBLASTIC ANEMIA
 Sex-linked recessive trait, mostly caused by
decreased in D-ALA synthase activity
 Deficiency of D-ALA synthase causes the
retardation of heme synthesis pathway
 Associated with microcytosis
B. AQUIRED SIDEROBLASTIC ANEMIA
 Due to exposure
 Associated with macrocytosis
 Acquired Idiopathic Sideroblastic Anemia
(AISA)/ Refractory Anemia with Ringed
Sideroblast (RARS)
o Mostly encountered in elderly patients
(older than 50 y/o) affecting both sexes.
Actual etiology of the disease is still
unknown but consistent findings
included decreased activity of ALA-
synthetase or an abnormal high
requirement for the cofactor pyridoxial-
5PO4 to maintain ALA synthetase RBCs
are hypochromic, despite presence of
large quantities of iron.
 Secondary Sideroblastic Anemia: Secondary to
some agents that interfere with heme synthesis
(Lead Poisoning; Alcohol, Drug-induced)
o Known etiology
 PORPHYRIAS:
o Group of disorders characterized by the
accumulation of porphyrins.
o Diseases characterized by impaired
production of heme (can be hereditary
or acquired) causing porphyrins to not
be completely changed and metabolized
resulting them to accumulate inside the
RBC or liver cells.
o The causes of porphyria can be the same
with those of SDA and the more
prominent feature is the accumulation
of porphyrins.
 Lead poisoning can also lead to
porphyrias
o Acquired types are usually associated
with enzyme deficiency. The products
from earlier stages (porphyrins) in the
pathway accumulate in the cells that
actively produce heme proteins such as
RBC and hepatocytes. These porphyrins
 HEMATOLOGY 2
can also be excreted in the urine or feces
or be deposited in body tissues.
NOTE: PORPHYRIA
MAIN FEATURE: accumulation of porphyrins
 Porphyrins accumulate first in the RBC
because RBC is involved in heme synthesis and
in the liver because it is involved in the
synthesis of myoglobin.
o During damage of RBC, when it is
hemolyze, as it reaches its 120th day,
the RBC will hemolyze and all its
contents will diffuse out into the
plasma including the porphyrins.
o When liver cells are damaged, they
also release their contents like,
porphyrins into the plasma.
Porphyrins will be elevated in the
plasma and can be filtered by the
kidneys to appear in the urine
(porphyria) where the urine appears
as port wine red. Some are excreted
in the feces.
o It is more accurate and precise to
measure porphyrins in the feces than
in urine.
o Deposit in body tissues – when
porphyrins deposit in the skin, the
skin becomes photosensitive.
Porphyrins absorbs the zorret band
coming from the sun, so when
exposed to sun, the skin of the patient
suffers from sun burn, blindness
(eyes), brain damage or psychiatric
abnormality (brain).
o Some teeth and bones especially
among the growing children wherein
some of the substances will
fluorescence. Teeth and bones are
fluorescent because of the deposited
porphyrins.
PORPHYRIA: associated with vampire stories
 A common disease affecting the royal families
in Romania and Transylvania.
 Characterized by bleeding teeth due to lack of
hemoglobin; evasion from sunlight because
they are prone to suffer sunburns and
blindness; fluorescing teeth due to deposition
of fluorescing porphyrins in the bone
 Most common clinical features:
Photosensitivity and Psychiatric or neurologic
symptom due to deposition of porphyrins
 Hematologic manifestations: Acute
intermittent porphyria (abdominal pain),
congenital erythropoietic porphyria (port
wine red urine), variate porphyria (psychosis
and photosensitivity) & erythropoietic
protoporphyria (psychosis)
HEMOCHROMATOSIS
 Another disease related to Iron metabolism:
TRUE IRON OVERLOAD.
 TO COMPARE: SDA  Iron wasn’t used in the
phase of normal iron (NORMAL: Iron; Cannot be
used/metabolized = NO IRON OVERLOAD)
 NORMAL: RATE of IRON ACQUISITION (1%) =
RATE of IRON LOSS (at least 1% of Iron daily = Red
cell destruction 1% daily)
o Rate of Loss (1%) daily is replaced by
what is absorbed (1%) daily
 HEMOCHROMATOSIS: RATE of IRON
ACQUISITION > RATE of IRON LOSS
o Iron acquisition exceeds the rate of iron
loss  iron is accumulated in the body
 Results when the body’s state of iron acquisition
exceeds the rate of iron loss. It can be acquired
or hereditary (mutations affecting the proteins
of iron metabolism). The body’s first reaction is
to store excess iron in the form of ferritin, then
in the form of hemosiderin within cells.
CAUSES OF HEMOCHROMATOSIS
 ACQUIRED: Transfusion-related
o It cannot be due to DIETARY REASONS,
excessive iron in the diet is unlikely to
cause hemochromatosis because our
absorption for Iron is LIMITED
 HEMATOLOGY 2

o Commonly caused by TRANSFUSION- o Fe++ (+O2)  Superoxide and Free

RELATED radicals – strong oxidizing agents 
 IDA: one factor leading to this PEROXIDATION of membrane lipids
condition is Frequent Blood results to:
Donation that every time we  Cell respiration - (Destroying
donate blood we lose iron. Mitochondrial Membrane) –
 Hemochromatosis: Every time Impaired ATP production = No
the recipient receives blood Cellular Respiration
(transfusion) the recipient gains  Lysosomal enzymes - (Impairing
Iron. Lysosomal Membrane) -
• Example: Patients with Beta- Rupture = Release of Hydrolytic
Thalassemia would require Enzyme
frequent donation or  Lysosome = Suicide Bag of the
transfusion of blood (PRBC/WB) Cell because once it releases its
they gain Iron thus repeated hydrolytic enzymes it can lead to
transfusion will lead to Autolysis (Cell will digest itself)
Transfusion-Related cell damage it will be destroying
Hemochromatosis. the nucleus, nuclear membrane
and in general will cause an
 INHERITED: Mutations affecting the proteins of irreversible membrane damage
iron metabolism of the cell and the organelles.
o Results to Less Metabolism: More Iron  Irreversible membrane damage
Accumulation. (Cell Membrane)
o Excessive Iron (Iron Overload): most of o In the presence of Oxygen, free ferrous
the Iron will first be stored as ferritin, but iron initiates the generation of
when overwhelmed will be stored as superoxide and other free radicals,
hemosiderin. which results in the peroxidation of the
o 2 Storage form of iron: 1st - Ferritin  membrane lipids.
when overwhelmed  2nd - o Results to damage of cell membrane,
Hemosiderin nuclear membrane, mitochrondrial and
o Both storage forms have limits and when lysosomal mnembranes. These events
both get overwhelmed, the remaining ultimately affect cellular respiration,
excessive Iron will have nowhere to be enzyme digestion, cell death, and organ
stored thus the remaining Iron (Fe++) in damage.
the presence of free oxygen (O2) will o Hemosiderin also deposits into tissues
now cause the generation of and organs leading to further organ
SUPEROXIDES and FREE RADICAL which damage:
are TOXINS and STRONG OXIDIZING
AGENT that cause oxidation of proteins,
lipids and cells  causing
PEROXIDATION of Membrane Lipids,
Cell Membrane, Mitochondrial
Membrane, Lysosomal Membrane and o SKIN AND PANCREAS: “bronze diabetes” –
other organelles, even the Nuclear bronze skin plus diabetes meaning the
Membrane leading now to impaired ATP Hemochromatosis have already affected the skin
production. and pancreas.
 HEMATOLOGY 2
 SKIN: Bronze/ golden color due to
cellular damage and accumulation of
excessive iron. The skin will absorb the
excessive Iron and it will accumulate
resulting to a BRONZE SKIN COLOR
 PANCREAS: Pancreas will be destroyed
and its production of Hormones
(regulate Carbohydrate Digestion) like
NOTE:
INSULIN, GLUCAGON etc.  with that it
BOTH IDA and SDA  heme synthesis defect
affects the Glucose Metabolism and can
Thalassemia  globin synthesis defect
lead to Diabetic Mellitus
o LIVER: Cirrhosis-induced jaundice subsequent
Liver Cancer
o HEART: Congestive Heart Failure
o Other more organs can be passively destroyed
SCREENING TEST FOR HEMOCHROMATOSIS:
 Transferrin saturation (common test performed)
and Ferritin (excessively overwhelm).
 Measurement of Transferrin Saturation
o Transferrin is fully saturated due to
excess Iron/ Iron overload ELEVATED
Transferrin Saturation
 Transferrin and Iron-binding Capacity(TIBC) will
be DECREASED
o NOT THE APPROPRIATE TEST for
HEMACHOMATOSIS
TREATMENT:
 THERAPEUTIC/REPEATED PHLEBOTOMY
o Most practical treatment to prevent
excessive Iron and prevent the
successive chain reaction
o Some patients require 2-3 times removal
of blood/phlebotomy  removal of
excessive Iron
 IRON-CHELATING DRUGS to bind/chelate excess
iron for safe excretion.
o Desferrioxamine/Desferal
 Intake of Vit. E and C
o Slow down chain of reaction but effect
will be overwhelming  inefficient;
retard.
o Can somehow help because they are a
REDUCING SUBSTANCE (especially Vit. E)
resulting to the RETARDATION of the
Iron and
o Free Oxygen Reaction
o But itself can be overwhelmed and will
only be able to SLOW DOWN the CHAIN
of REACTIONS = Inefficient
o It should be alongside the
Therapeutic/repeated phlebotomy and
Iron-chelating drugs
 Hemoglobin are usually dimers, their globin
portion has 4 polypeptide chains  2 different
types of chain (alpha and beta).
o Example: A1 (heme + globin)  4 PPC
(2 alpha and 2 beta chains)
o To synthesize the chains, it need
genes (complete number) that is
inherited from parents (one per each
parent).
 Alpha chain: needs 4 alpha
genes
 Beta chain: 2 beta gene
o Each PPC has its amino acids
 Alpha chain: made up of 141
AA  per chain
 Beta chain: 146 AA  per
chain
 HEMOGLOBINOPATHIES: condition
characterized by qualitative structural
abnormalities of the globin polypeptide chains
that result from alteration of the DNA genetic
code for those chains.
o Structural defect due to defect in
amino acids; classified as normocytic
anemia except Hb E disease
 THALASSEMIA: diverse group of genetic
disorders characterized by a primary,
quantitative reduction in globin chain
synthesis for hemoglobin
o Synthesis defect (alpha or beta) 
disorder characterized by defective or
deficient number in chains due to
deletion of some gene thus proteins
that is encoded by the gene can’t be
synthesize.
 HEMATOLOGY 2

3. THALASSEMIAS CLASSIFICATION CLINICAL HEMOGLOBI

 Thalassemias are the results of impaired MANIFESTATIO N
(deficient) globin chain synthesis. These may N
affect either the alpha or beta chain synthesis. Silent carrier Normal 3 functional
(one-gene hematologically, alpha gene
Greek No. of No. of Chromosome deletion) with few target A1= normal
Name genes Amino Location cells, elliptocyte (97%)
Acids
Alpha 4 (2 141 16 s/s:
mother; 2 asymptomatic
father)  normal
Beta 2 (1 146 11
mother; 1
father) Alpha- Mild anemia, 2 functional
Delta 2 146 11 Thalassemia trait: microcytic- alpha gene
Gamma 2 146 11 (two-gene hypochromic, remaining
Epsilon 2 146 11 deletion) elliptocytes, thus
Zeta 4 146 16 target cells synthesis of
(Rodak’s) Hb A1 is
slightly
decresed:
normal A1
levels atleast
97%.

Hb A (60%);
Hb Barts
(510%)
A. ALPHA THALASSEMIA HbH disease Microcytic, One
(Three-gene hypochromic, Hb functional
o A deficiency in the synthesis of Alpha-
deletion) H inclusions, gene
globin chains. Each individual has two
high retics remaining
sets of two alpha genes. Suppression of that
all four genes is needed to completely A1: 2 alpha & 2 is not enough
suppress alpha chain synthesis. The beta to cause
usual mechanism of suppression is by A2: 2 alpha and 2 normal
gene deletion. delta production of
o Main common cause: gene deletion or Hb F: 2 alpha and alpha
no inherited gene. 2 gamma polypeptide
o Deletion is indicated by blank: e.g. – Since alpha chain.
a/aa  one gene deletion; --/aa  two chain production
deletion; --/a  three deletion; --/--  is affected Hb H  seen
and these an pitted golf
four gene deletion.
hemoglobin ball in PBS.
needs the alpha
chain thus their
production will
also be
 HEMATOLOGY 2

retarded  RBC CLASSIFICATION OF BETA THALASSEMIA

will compensate Beta Beta Beta
by Thalassemia Thalassemi Thalassemi
increasing the Major/ a Major/ a Major/
synthesis of beta Cooley’s Cooley’s Cooley’s
chain  anemia anemia anemia
4 beta chain 0 0 + +
(B l B ) (B l B ) (B0l B)
producing Hb H
•Complete lack of •Caused by a •Sufficient
Bart’s Hydrops Stillborn, Hb Bart’s:
Beta globin partial quantities of
fetalis (four-gene hydrops tetramer Hb
production suppression of HbA1 is
deletion) (enlarged head) consisting of
•Most affected the Beta genes produced.
-Hb Bart’s has 4 gamma
patients exhibit •Symptoms •Hemoglobin
abnormal chains.
retarded growth are similar levels are
function
with mongoloid with that of slightly low,
wherein it can’t Since there is
facial features and Beta but blood
promote no
severe anemia Thalassemia smear shows
oxygenation of production of
•Marked by major, but similar
tissues. alpha chains
characteristics depend on the morphology as
due to total
changes in RBC extent of gene in Thalassemia
deletion of
morphology; suppression Major or
alpha gene,
microcytosis, • May produce Intermedia
the RBC will
hypochromia, varying •HbF is about
compensate
anisocytosis and amounts of A1 2-3%
by producing
poikilocytosis. depending on •HbA2 level is
gamma
Numerous target the degree of elevated 
chains thus
cells, Howell-Jolly suppression has delta chain
formation of
bodies and
Hb bart’s.
siderocytes. TYPES:
•HbF level is Homozygous
elevated (40-60%) (B+l B+): 2 beta
• Can synthesis A2 gene is
• Most severe of partially
all B-thalassemia suppressed
that requires Heterozygous
B. BETA THALASSEMIA frequent (B+IB): One
o Characterized by a deficiency in Beta- transfusion gene is
globin chain synthesis. Each individual partially
has one set of two beta genes. suppressed
Suppression or absence of these beta and the other
one is normal
genes results to deficient beta chain
synthesis.
o Gene deletion is indicated by 0
 HEMATOLOGY 2
NOTE:
Hereditary Persistence of Fetal Hemoglobin and B-
Thalassemia Major: highest concentration of Hb F can
be seen.
In thalassemias and other disorders characterized by
abnormal hemoglobin synthesis like IDA and SDA, it is
very common to see target cells  nonspecific finding
unlike schistocytes that is a hallmark finding in
hemolytic anemia indicating hemolysis and teardrop
cells in myelofibrosis
4. ANEMIA OF CHRONIC INFLAMMATION (ACI)
 These diseases are grouped into chronic
inflammatory diseases and malignant diseases.
It is the inflammation that may lead to anemia.
 Malignant disease/ cancer
 Chronic inflammatory disease
o Infectious TB
o Osteomyelitis
o Non-infectious RA
o SLE and Crohn’s disease
MECHANISMS OF ANEMIA OF CHRONIC DISEASES:
1. Altered Iron Metabolism  Microcytic
 Among the 4 pathophysiology, most common to
happen due to increase in acute-phase protein
and it is a direct effect of inflammation
2. Direct inhibition hematopoiesis, decreases
production of EPO. moderate destruction 
normocytic (do not involve iron)
 During acute/chronic inflammation, the
macrophages and lymphocytes are activated
thus produce cytokines of different types that
includes tumor necrosis factor-alpha (TNF-
alpha), Interferon-gamma and other
interleukins. Some of these cytokines will cause
inhibition of bone marrow function leading to
decrease hematopoiesis, inhibition of kidney
function thus decreased erythropoietin
production and some may cause direct damage
on RBCs
NOTE:
Anemias that develop in chronic diseases are caused
by the effects of the increased inflammatory
cytokines. (i.e. interleukins) produced by activated
cells during the chronic inflammation: These cytokines
may cause any of the following mechanisms:
1. ALTERED IRON METABOLISM OR IMPAIRED
FERROKINETICS
 Activated lymphocyte will produce IL-6 that
will act on the liver, it will activates the liver to
produce increase hepcidin (Positive Acute
Phase
 Protein: proteins that immediately increase
during inflammation) caused by an increase of
the following acute phase reactants that
interfere with iron metabolism:
o HEPCIDIN: this causes the
degradation of the transmembrane
protein ferroportin used by
macrophages and hepatocytes to
export iron into plasma.
 HEMATOLOGY 2
 Increase in hepcidin will result
to failure of iron to be
released in the plasma
because iron will be retain in
the cells
 Decrease iron absorption and
release  decrease plasma
iron
 FERROPORTIN: transport
protein found in the intestinal
enterocytes(cells of
duodenum), hepatocytes,
and macrophages
o LACTOFERRIN: An iron-binding
protein in the tertiary granules of
neutrophils. Its avidity for iron is
greater than that of transferrin.
During inflammation, it scavenges
available iron at the expense of
transferrin.
o FERRITIN: developing RBCs do not
have a ferritin receptor, thus, cannot
utilize iron for hemoglobin synthesis.
 Increase in the production of
apoferritin that binds with
iron to form ferritin, thus iron
will not be utilized because it
is trap in ferritin.
2. MODERATE INCREASE IN THE DESTRUCTION
OF RBC
3. DECREASED PRODUCTION OF
ERYTHOPOIETIN
 Production of inflammatory cytokines (such as
tissue necrosis factor-a and interleukin-1 from
activated macrophages and interferon-g from
activated T-cells) also impairs proliferation of
erythroid progenitor cells, diminishes their
response to erythropoietin, and decreases
production of erythropoietin by the kidney
5. HEMOGLOBIN E
 Only hemoglobinopathies wherein its
morphology is microcytic
 It a structural and synthesis defect
(hemoglobinopathy and thalassemia)
 Involves same mutation as Hb C(B6) wherein
Glu  Lysine but the point of
mutation(substitution) occurred in 26th amino
acid
o Hb C (B6 glu  lys) and Hb S(B6 glu 
val) has structural defect but Hb E has
both structural and synthesis defect
 Decrease in B-chain synthesis
 2nd most common hemoglobinopathies
MACROCYTIC ANEMIA
 MCV: >100 fL
 A macrocytic cell seems to have more
hemoglobin content compared to normocytic
but in relation to its size it is normal. 
Macrocytic  normochromic.
TWO TYPES OF MACROCYTIC ANEMIA:
 In cell maturation, the nuclear-cytoplasmic
maturation must be synchronous in order for the
cells to be normocytic when released. In
asynchronous maturation, the maturation of
nucleus and cytoplasm is not the same that can
lead to megaloblastic maturation.
1. NON-MEGALOBLASTIC
 Normal maturation therefore normal
development however when released in the
circulation they appear large
 PATHOPHYSIOLOGY: Aplastic anemia (but most
are in normocytic classification), chronic liver
disease, alcoholism and other disease or
condition that can lead to hypercholesterolemia
and reticulocytosis.
 LIVER DISEASES (chronic, obstructive jaundice),
LCAT deficiency:
o LIVER: main organ where cholesterol is
stored and metabolized thus when
damaged cholesterol will not be
completely metabolized leading to
hypercholesterolemia/ excess
cholesterol in plasma
o LCAT – enzyme involve in the
esterification of cholesterol thus when
 HEMATOLOGY 2
deficient the cholesterol(free) in the
plasma will accumulate leading now to
hypercholesterolemia
 Cholesterol in the plasma can
be in the form of free
cholesterol or cholesterol ester
 The RBC membrane consists of
phospholipid bilayer which
have some cholesterol,
proteins. CHO in the layer. The
cholesterol embedded in the
membrane depends on plasma
cholesterol that when
cholesterol plasma is increased,
there will be excessive loading
of cholesterol into the plasma
membrane thus enlarging the
size of the cell.
 RETICULOCYTOSIS: any condition resulting to
premature release of reticulocytes in the
circulation
o Reticulocyte (5th in erythropoiesis) are
immature cells seen as large cells but
they underwent maturation in the BM
o PBS: supravital stain: reticulocyte
(reticulocytosis)
o Wright’s Stain: polychromatophilic cell
(polychromatophilia)
o Shift/Stress cells: more larger
o Released as a compensatory
mechanism of bone marrow to the
following condition:
 Acute blood loss
 Hemolysis or hemolytic anemia
 Bone marrow infiltration: filled
with abnormal cells
 High level of EPO
 RBC should stay in the BM for 3.5 days and 1 day
in the peripheral circulation but due to the
stated condition above, the RBC transit time in
the BM will be shorter and prematurely released
in the circulation (prolonged stay) shift/stress
reticulocytes
2. MEGALOBLASTIC
 Asynchronous maturation
 Due to abnormal development  that during
their earliest developmental stage
(pronormoblast) their development is already
abnormal  promegaloblast  2nd stage:
basophilic megaloblast (b. normoblast should
undergo normal maturation)
 MAIN PROBLEM: Impaired DNA synthesis
 Defective nuclear maturation caused by
impaired DNA synthesis (prolonged intermitotic
resting phase; block early in mitosis)
 N-C asynchrony or “maturation arrest”
o Deficient DNA synthesis (nucleus)
resulting to retarded nuclear maturation
while the RNA synthesis in the cytoplasm
is less impeded thus cytoplasm
maturation proceed normally
o While the nucleus is not yet mature, it
will not cause division but the cytoplasm
keeps on maturing and enlarging
resulting to prolonged intermitotic
resting phase leading to block in early
mitosis or the cell will not divide at all
the cell become larger appearing now as
macrocytic.
 In contrast to microcytosis
wherein the cells are small
because they undergo
extradivision.
 RULE: as the cell divides, the cell
size decreases
o NUCLEUS: control center of the cell:
control all the process including the
division of the cell.
 Not only affect RBC but all rapidly proliferating
cells  enlargement of ALL rapidly proliferating
cells like neutrophils
 MCV: >100 fL; cannot be relied alone in
determining a megaloblastic maturation
 IMPORTANT FINDINGS INDICATING A
MEGALOBLASTIC MATURATION: MCV of >100 fL
+ macro-ovalocyte and macropolycyte
 RBC is often seen as macro-ovalocytes (large and
oval) and Neutrophils as macropolycyte (large in
size with hypersegmentation) indication of
megaloblastic anemia.
 HEMATOLOGY 2

o Neutrophil: has a normal 3-4 lobes but
when hypersegmented and stil has a
normal size (9-15 um) it indicates old
cell.
 Multiple Howell-Jolly bodies and many
nucleated RBCs with karyorrhexis.
o DNA containing inclusion indicating
nuclear remnants
o Problem in DNA synthesis which
affected the nuclear maturation thus
the nucleus is prone to fragmentation
 karyorrhexis
o Due to increase karyorrhexis
 Pancytopenia, leucopenia and
thrombocytopenia

ERYTHROKINETICS
 Total erythropoiesis is increased yet there is
reticulocytopenia indicating ineffective
erythropoiesis.
 Hyperplastic marrow: more red marrow in
order to compensate for decreased amount of
A. MACROCYTOSIS WITH NORMOBLASTIC RBCs.
MARROW o A marrow which has red
 Characterized by the presence of macrocytic hematopoietic cell (hematopoietically
cells that are not megaloblastic. This may be due active) over the marrow fats
to early release of immature erythrocytes from o Hypoplastic: more of marrow fats
the bone marrow in response to acute hemolysis o Normoplastic: normally, 50:50 ratio
of red cell and fats or 25:75 in long
and/or acute blood loss.
bones
 Not a complication but a compensation
 3x erythropoiesis
 Supravital stain: reticulocyte  Decrease M:E ratio = 1:1
 Wright’s stain: Polychromatophilic cells o Normal M:E ratio is 3:1
 Reticulocytopenia: Increased RBC in the
B. MEGALOBLASTIC ANEMIA marrow but not enough number of cells in
 Characterized by enlargement of all rapidly the circulation and due to premature lysis
proliferating cells of the body including marrow  Pancytopenia
cells. The major abnormality is the diminished
capacity for DNA synthesis, resulting to “nuclear- CHEMISTRY
cytoplasmic asynchrony” or “maturation arrest”  Related to hemolysis
RNA synthesis is less impeded than is DNA o Increase serum Lactate
synthesis, hence cytoplasmic maturation and Dehydrogenase (LD)  from RBC-
derived isoenzyme
growth continue.
o Increase bilirubin (both total and
 Most common causes: Vitamin B12 deficiency
unconjugated)
and folate deficiency o Increase endogenous carbon
NOTE: monoxide (CO)  while the
hemoglobin continuously degrade,
BLOOD PICTURE:
the heme and globin will separate
 Presence of macroovalocytes and giant
and heme will further separate into
hypersegmented neutrophils.
iron and protoporphyrin IX.
 Basophilic stippling: precipitated ribosomes
Protoporphyrin IX will further
indicating a disturbed erythropoiesis and
undergo degradation releasing
erythrocyte metabolism
biliverdin and CO. In normal
 HEMATOLOGY 2

hemolysis, carbon monoxide is

exhaled but if hemolysis is increased
CO is also increased.
o Common source of CO is exogenous:
factory and automobile exhaust
 Others: different mechanism
o Increased serum muramidase 
present in granules of neutrophils
 Disturbed granulopoiesis
thus enlarged and
hypersegmented neutrophils
and easily damaged thus
releasing their enzyme
content (muramidase).
o Increased homocysteine  due to
failure convert into methionine
 Homocysteine should receive
methyl from folate to
convert into methionine, but C. CYANOCOBALAMIN (VITAMIN B12) DEFICIENCY
due to folate deficiency it  Not common to occur because the body’s daily
fails to be converted. requirement of B12 is very minimal (around 2-5
DNA = double stranded helix consisting of sugar- ug/day)
phosphate backbone and nucleotides.  When intake is stopped, this type of anemia can
be acquired after 3-6 months or a year. Because
In the formation of thymidine structure to thymine, the body’s utilization of B12 is also minimal thus
both folate and B12 are needed. Therefore deficiency B12 deficiency takes gradually.
in folate and B12 will result to impaired DNA synthesis.
 Vitamin B12 is required in the Demethylation of
The bone marrow converts
folate during DNA synthesis
deoxyuridinemonophosphate into
deoxythymidinemonophosphate using the enzyme  Folate is absorbed through the ileum with the aid
thymidylate synthethase which needs a cofactor of the intrinsic factor (IF) or Castle’s factor
tetrahydrofolate (THF) which is from dietary folate. produced by the parietal cells, and is transported
Dietary folate enters the plasma in the form of 5- by transcobalamin II (TC II) in the plasma.
methyl THF and enters again the red cell in the form of o Intrinsic factor: a protein synthesized by
5- methyl THF but before it could participate in the gastric mucosa; without it, B12 can’t be
synthesis of DNA structure, it needs to undergo absorbed
demethylation wherein there should be removal of o In ileum, cells (enterocyte) with
the methyl group. The methyl removed will be receptors for B12 are available however
transferred to homocysteine thus converting it into it is specific only to B12-IF complex. Thus
methionine by the enzyme methionine synthase
when only B12 is pass through it, it will
needing the cofactor B12.
not be recognized therefore not
TWO MOST COMMON CAUSES OF MEGALOBLASTIC absorbed.
ANEMIA: o Transcoblamin II: transports B12 to sites
1. B12 deficiency anemia where it could be metabolized or stored
2. Folate deficiency anemia like in the liver, bone marrow and other
tissues.
 RDA: 2-5 ug/day
 TWO SOURCES OF B12:
 HEMATOLOGY 2
o DIET: primarily from food of animal
origin  main source of B12
o LARGE INTESTINE: synthesized by some
of the normal flora in the intestine
(lactobacilli spp. and bacteroides spp.).
However the absorption of B12 is in the
ileum but the production comes from
the large intestine. Thus, B12
synthesized in the large intestine is not
all/readily available for consumption of
the human body.
NOTE:
ACQUISITION OF METABOLISM OF B12
 B12 comes from the diet (meat origin) once
reaches the stomach, the gastric juice is very
acidic because of the HCl produced by the
parietal cells. Thus B12 can be damaged. In
order to protect B12 from acid destruction,
salivary glands produce haptocorrin or
Transcobalamin I (TC I). While masticating the
food, B12 combines with TC I. Since TC I
protects B12, it can bypass the acidity and
digestive action of the stomach in small
intestine (duodenum;1st portion) the pH of
the chyme is neutral to alkaline thereby
destroying haptocorrin and B12 will be
released. B12 combines with Intrinsic factor
(IF) coming from the parietal cells of the
stomach and thus forming the B12-IF
complex. The complex goes down until the
jejunum and terminal part – ileum where it
will be absorbed by the intestine. Once
absorbed, IF will be detached and B12 will be
released into the circulation. B12 will then
combine with Transcobalamin II and be
brought into the liver (for storage or
metabolism), bone marrow (for utilization and
DNA synthesis) and other tissues (for
utilization being a cofactor).
IMPORTANCE OF B12
 For metabolism
 Formation of RBCs
 Maintenance of CNS (brain and spinal cord)
CONSEQUENCES OF B12 DEFICIENCY
 Ineffective DNA synthesis: Megaloblastic
anemia
 Inadequate myelin synthesis: neurologic
damage
 Hyperhomocysteinemia: cardiovascular
disease.
NOTE:
RELATED CAUSES IF VITAMIN B12 DEFICIENCY:
1. INADEQUATE INTAKE
 RDA: 2-5 ug/day from meat diet
 Common in strict vegan people
2. MALABSORPTION SYNDROME
 Any condition affecting the ileum
(inflammation): Celiac disease, ileal disease
(Crohn’s disease/regional enteritis) tropical
sprue, resection of the small bowel
 Imerslund-Grasbeck syndrome – deficiency
or absence of IF-B12 receptors
 Zollinger-Ellison syndrome – hypersecretion
syndrome  gastric hypersecretion (acid and
juices) that can degrade B12 and prevent it
from binding with IF and therefore B12 is not
absorbed
3. LACK OF AVAILABILITY OF COBALAMIN
 Blind loop syndrome
o Refers to bacterial overgrowth
o The large intestine (colon) is rich in
normal flora that synthesis vitamins
but in this case, what will proliferate
in the LI are the abnormal flora which
will utilize the available B12 at the
expense of the host. And this
abnormal flora will also destroy the
normal flora in the expense of their
capacity of producing some
important vitamins (B12)
 Infection with Diphyllobothrium latum
o D. latum is the longest tapeworm
infecting human which resides in the
ileum
o Bothriocephalus anemia: long
tapeworm that competes with the
host with the absorption of B12 and
secretes substance that inhibit
binding of IF with B12.
4. PRESENCE OF AUTOANTIBODIES
 Can be either those who destroy the intrinsic
factor or those which destroys cells(parietal)
 HEMATOLOGY 2
that produce IF  Inability of the gastric
mucosa to secrete IF (Pernicious anemia)
5. DRUGS/ SUBSTABCE-RELATED
 e.g. Neomycin and ethanol  chronic
alcoholics
6. ABNORMAL PRODUCTION OF TC-II
D. PERNICIOUS ANEMIA
 Autoimmune type of vitamin B12 deficiency
anemia
 Types of Autoantibodies:
o Anti-parietal cell antibodies: destroys
parietal cells
 Intrinsic factor: complexes with
B12 in order for it to be
absorbed
 HCl: gives acidity of the
stomach  absence can lead to
achloridria
o Anti-intrinsic factor antibodies
 IF production is normal, but IF is
inhibited by the autoantibodies
thus it cannot bind with B12 
non-absorption of B12
 2 TYPES OF ANTI-INTRINSIC
FACTOR ANTIBODY:
NOTE: DIAGNOSIS OF VITAMIN B12 DEFICIENCY
Blocking antibody: blocks the
 Normal plasma vit. B12 levels: 180-914 ng/L
binding of cobalamin to IF
Binding antibody: binds to the
cobalamin-IF complex
becoming trimolecular complex
receptors will not recognize;
and prevents the complex from
binding to receptors in the
ileum
 Clinical signs & symptoms include:
o Atrophic glossitis: tongue will atrophy
and become inflamed due easy
desquamation of surface epithelial cells
(lack of integrity because of B12 def.) 
beefy red tongue
o Atrophic gastritis and achloridria:
leading to malabsorption; when
stomach become inflamed, parietal
cells become defective and can’t
produce HCl
 Achloridria: absence of HCl;
more specific of pernicious
anemia especially when the
autoantibodies destroy the
parietal cells.
 HCl: important in digestion in
the stomach  digestion of
proteins
o Episodic abdominal pain, constipation,
and diarrhea
o Symptoms of anemia with a
combination of skin pallor and lemon-
yellow skin
o Diffuse and irregular degeneration of
the white matter of the CNS
 Memory loss, numbness and
tingling in toes and fingers
damaged nerves
 Symmetric sensation of “pins &
needles” of the distal
extremities
 In advance cases, brain may be
affected resulting to irritability,
emotional instability, or a
change in personality
(megaloblastic madness)
1. SERUM COBALAMIN ASSAY
 A microbiological assay using an organism (E.
gracilis) that requires B12 for its growth.
 Organisms used: Euglena gracilis and
Lactobacillus leichmannii
 Patient’s plasma + organism incubated for
48-72 hours at 37 degree Celsius
 If the patient’s plasma has a normal B12, it will
support the growth of the organisms
organisms will grow and cause turbidity of the
medium
o If the patient’s plasma has an
abnormal B12  growth will be
retarded  organisms will not grow
and will not cause turbidity
o TURBIDITY WILL BE COMPARED WITH
STANDARD CONTROLS
 HEMATOLOGY 2
 Highly sensitive but it is lengthy to performed
but;
o Not sensitive to patients taking
antibiotics  antibiotics are in plasma
thus when incubated with the
organisms, it can destroy or kill these
thus this test should not be done with
patients undergoing an antibiotic
therapy
 Reference value: 200-900 mg/dL
2. METHYLMALONIC ACID & HOMOCYSTEINE
ASSAY
 B12 is a cofactor of methymalony CoA mutase
in the isomerization of methylmalonyl CoA to
Succinyl CoA. Deficiency of B12 will lead to
excretion of increased amount of
methylmalonate in the urine.
 Methyl malonyl CoA  MMCoA mutase with
cofactor Vit. B12  Succinyl CoA
 Measured by:
o Methyl malonic acid assay: GC-MS
o Homocysteine assay: GC-MS; HPLC;
Fluorescence Polarization
 In both B12 and folate deficiency,
homocysteine will increase.  homocysteine
assay
3. DEOXYURIDINE SUPPRESSION TEST
 Indirect test that measures the ability of the
bone marrow cells in vitro to utilize
deoxyuridine in DNA synthesis.
 It is abnormal in both cobalamin (B12) and
folate deficiency.
4. COMPETITIVE PROTEIN BINDING
RADIOASSAY
 Method of choice for both B12 and folate
deficiency quantitation
 Principle: patient’s B12 and folate compete
with Co-B12 and -folate in binding with hog IF
and B-lactoglobulin
 Patient’s B12  hog IF  Co-B12
o Competition between the patient’s
B12 against the cobalt-labeled B12 in
binding with hog IF
o Differentiation: using the radioassay
 Patient’s folate  B-lactoglobulin  I-folate
o Competition between patient’s folate
against the iodine-labeled folate in
binding with the reagent B-
lactoglobulin
o Differentiation: using the radioassay.
5. SCHILLING’S TEST
 Specific test for Pernicious Anemia
 Provides a measure of the body’s ability to
secrete viable IF & absorb orally administered
Co-labeled B12.
 PART ONE: patient is given a physiologic dose
of B12 labeled with radioactive cobalt (0.5 -2
ug) ingested orally to see whether the patient
can absorb the B12 and after 1-2 hours;
another flushing dose is given  injection of
non-radioactive vitamin B12 (1000 ug)
intramuscularly in order to saturate the
binding sites of B12 so that the minimal
amount of B12 absorbed will not bind in the
binding site instead it will be urinated. check
B12 levels in the patient’s urine
o Patients with IF or NO pernicious
anemia: the cobalt labeled B12 given
orally in the duodenum will combine
with IF available in the patient to now
allow B12 absorption. B12 absorbed
will proceed to the plasma and those
some will be stored in the liver,
plasma protein binding and urinary
excretion.
 Patients with normal IF: excrete >7% ingested
B12
 If the first part of the test is normal: excreted
>7% B12  report it as NORMAL. But if there
is abnormal result (<7% or none excreted) that
may be due to deficient or absent IF 
proceed to PART II
o If IF is absent, there will be no
intestinal absorption of B12 thus,
ingested B12 will be excreted through
defecation.
 PART TWO: Done after 24 hours of the
conduct of first part of the test. Patient is
given an oral cobalt-labeled B12 with IF that
will allow absorption of B12 in the ileum.
Result is therefore expected to be impoved.
o Urinary excretion: improved >7% or
15%  PERNICIOUS ANEMIA
o Low cobalt in urine  Other B12
deficiency anemia
 HEMATOLOGY 2
OTHER TESTS
1. TEST FOR AUTOANTIBODIES
 For pernicious anemia
 Identify serologically what specific antibody is
produced
 More specific than schilling’s
2. GASTRIC ANALYSIS (HCl) AND SERUM
GASTRIN
 For pernicious anemia
 HCl: Achloridria due to damage in parietal
cells
 Gastrin: peptide hormone secreted by the
gastric walls which functions to stimulate
stomach to secrete juice and acids. Since there
is decrease HCl, gastrin will be stimulated to
induce more HCl production. Although serum
gastrin is plenty, there will be still no HCl since
the parietal cells are destroyed.
 DECREASE HCl and INCREASE SERUM GASTRIN
3. SERUM HOLOTRANSCOBALAMIN ASSAY
 Complementary test
 Holotranscobalamin: metabolically active
form of cobalamin (B12)
4. FECALYSIS (FOR BOTHRICEOHALUY ANEMIA)
 Operculated eggs of D. latum
E. FOLIC ACID/ PTEROYLGLUTAMIC ACID
DEFICIENCY
 Features are similar to Vitamin B12 deficiency
but no neurologic symptoms; leukopenia and
thrombocytopenia are less constant
 RDA: 50 ug folic acid or 400 ug food folate
 The clinical s/s of it is very similar with B12
deficiency except NO NEUROLOGIC SYMPTOMS
 uncommon to occur even in severe cases
 Absorption: small intestine: duodenum and
jejunum (same with iron absorption)
o Small intestine parts: 1st duodenum 
2nd jejunum  3rd ileum (B12
absroption)
 Deficiency inhibits the thymidine
 SOURCE: vegetables and poultry products (eggs)
NOTE:
RELATED CAUSES IF FOLATE DEFICIENCY
 Inadequate intake/Dietary deficiency
 Liver disease
 Alcoholism: alcoholic is hepatotoxic
 Increased demand/requirement
o Prone to babies and pregnant women
 Defective absorption
 Inadequate utilization of folate
 Presence of folate antagonists (antimalarial
drugs that inhibits utilization of folate)
DIAGNOSIS OF FOLATE DEFICIANCY
1. Microbiologic assay using Lactobacillus casei
 Serum and Red Cell folate: usually < 3 ug/L
2. Deoxyuridine Suppression Test
 Ability of BM to utilize deoxyuridine into
deoxythymidine and finally into DNA
structure
3. Quantitation of Vitamin B12 and folate
through competitive protein binding
radioassay
 Method of choice
4. Homocysteine Assay
F. TROPICAL SPRUE & GLUTEN-SENSITIVE
ENTEROPATHY
 A condition that can cause both vitamin B12 and
folic acid malabsorption as a result of intestinal
atrophy.
 Intestinal atrophy (SI and proximal intestine)
Vit. B12 and folic acid malabsorption
o Tropical Sprue: intestinal atrophy of the
small intestine
o Gluten-sensitive enteropathy: intestinal
atrophy of the proximal intestine
NOTE: Tropical Sprue & Gluten-sensitive enteropathy
THERAPY: depends on the condition
1. ORAL COBALAMIN
 B12 deficiency: B12 supplement
2. PARENTERAL CYANOCOBALAMIN
 For pernicious anemia  through IV/
injection
3. ORAL FOLATE
 HEMATOLOGY 2

 Folate deficiency: Folate supplement o HYPOPLASTIC: preferred term meaning

severe depletion of blood cells
o APLASTIC: may be confused due to the
NOTE: RECAP prefix “a” meaning absence; but in
DEVELOPMENTAL DEFECT:
anemia it means the same as hypoplastic
MICROCYTIC: abnormal cytoplasm maturation (hb
 Clinical signs include the common signs of
synthesis)
MACROCYTIC: Megaloblastic: Asynchronous anemia plus infection and bleeding seen also in
maturation due to defective DNA synthesis leukemia
 Splenomegaly & lymphadenopathy are ABSENT
in aplastic anemia but present in leukemia
NORMOCYTIC-NORMOCHROMIC ANEMIAS o In leukemia, there are many leukemic
 Synchronous maturation: not a developmental cells that causes the spleen to be
defect but a survival or proliferation problem overworked (remove cells) 
 Anemia occurs due to: splenomegaly.
o Increased destruction  Hemolytic o For ALL, the lymph nodes are also
anemia: can be INTRINSIC or EXTRINSIC infiltrated  lymphadenopathy
 Problem in the
circulation/spleen wherein a
lysis occur
 Increased in reticulocyte count
due to compensatory
mechanism of a normal bone
marrow
NORMAL BM HYPOCELLULAR HYPERCELLUL
o Decreased in proliferation or
BM AR BM
hypoproliferation 50% of red Decrease in red Increase in red
 Bone marrow defect wherein marrow and marrow but marrow but
there is decrease production of 50% of marrow increase in white decrease in
cells fats marrow(fats) white marrow
 Decreased or normal (fats)
reticulocyte count due to bone Ratio depends Seen in
marrow failure on age: hypoplastic/aplas Seen in
 Differentiated through reticulocyte count Younger child: tic anemia polycythemia
100% red vera
I. ANEMIA OF BONE MARROW FAILURE marrow
Adults: 50:50;
 Could be due defect of bone marrow cells
25:75
(damaged)
 Abnormal or lack of stimulation of the hormone
(EPO) even with normal bone marrow PATHOGENESIS
1. Direct damage to the hematopoietic
A. APLASTIC/ HYPOPLASTIC ANEMIA stem & progenitor cells decreased in
 Characterized by a severe reduction of the blood cell count
amount of hematopoietic tissues that results to o Causes: ionizing radiation, toxic
deficient production of blood cells (hypocellular chemicals (benzene)
bone marrow) o Infiltration BM: myelophthistic
anemia
 BONE MARROW: hypocellular
o competition for nutrients by
 CIRCULATION: pancytopenia
normal and abnormal cells
cancerous cells take up the
 HEMATOLOGY 2
oxygen and nutrient supply
o PANCYTOPENIA
2. Immune-mediated
destruction of marrow cells
o Antibody destruction wherein
patient produce autoantibody
that damages its own cell.
o PANCYTOPENIA
3. Low effectiveness of EPO
(cannot activate erythroid
stem cell)
o Defective/inefficient hormone
that causes insufficient
stimulation of the bone marrow
o PURE RED CELL APLASIA
4. Low production of EPO like
in renal diseases
o Normal function of EPO but low
in number thus unable to
stimulate the bone marrow
o PURE RED CELL APLASIA
A.1. IDIOPATHIC APLASTIC ANEMIA
 The cause is of unknown etiology
 The development of sign and symptoms is
hideous and gradually occurring until it becomes
a full-blown anemia
A.2. ACQUIRED APLASTIC ANEMIA
 May be the effects of exposure to certain
physical and chemical agents some drugs like
antimicrobials, anticonvulsants analgesics
 SECONDARY TO:
o Ionizing radiation  high doses
damages bone marrow
o Benzene  chemical that has toxic
effect to BM  Benzene Hypersensitive
Aplastic Anemia
o Chloramphenicol  most common
antibiotic causing AML and Aplasic
anemia  toxic to BM
A.3. CONSTITUTIONAL APLASTIC ANEMIA
 Associated with other group of congenital
anomalies or with genetic predisposition to
chronic bone marrow failure. Predisposition to
cancer and chronic bone marrow failure.
 Patient is born without aplastic anemia but has
congenital genetic abnormality that made him
predispose to suffering other
malignancies/cancer.
FANCONI’S ANEMIA 9CONGENITAL APLASTIC ANEMIA)
 Congenital bone marrow defect that leads to
decrease production blood cells
 Hereditary Disorder: Autosomal recessive trait
or pattern of inheritance
 Chromosomal defect are found in lymphocytes
and BM cells.
 With features of developmental abnormalities
that include hyperpigmentation, short stature
(inability to grow), hypogonadism, mental
retardation, strabismus (cross-eyed) and
malformation of the extremities.
 Fanconi’s syndrome: different and has no
relationship from this kind of anemia
o Defect in PCT reabsorption thus it is a
renal tubular defect wherein the
important solute in the filtrate are not
reabsorbed.
FAMILIAL APLASTIC ANEMIA
 Subset of Fanconi’s anemia.
 SCHWACHMAN – Diamond syndrome prone to
develop familial aplastic anemia
 Patients may have pancytopenia and a
hypocellular marrow without major
developmental abnormalities.
A.4. PURE RED CELL PLASMA
 Only the red cell are decreased
NOTE:
1. Transitory Arrest of Erythropoiesis/
transient aplastic crises – may occur during
the course of a hemolytic anemia often
preceded by an infection with parvovirus B19
 HEMATOLOGY 2
infects CFU-E
2. Transient Erythroblastopenia of Childhood –
associated with a history of viral infection
within the last 3 months (humoral inhibition)
3. Acquired Pure Red Cell Aplasia – associated
with thymoma
4. Congenital Red Cell Aplasia/
Congenital Hypoplastic Anemia
(Diamond-Blackfan anemia)
 Defects in BFU-E and CFU-E with
accelerated apoptosis (programmed cell
ANEMIA OF RENAL INSUFFICIENCY
death) thus decrease production of RBCs
o Progenitor cells BFU-E CFU-E
that give rise to pronormoblast
mature erythrocytes
 Most common type caused by a defect in
the erythroid-committed progenitor cell
II. MYELOPLASTIC ANEMIA
 Associated with marrow replacement by
metastatic carcinoma, multiple myeloma,
leukemia or other conditions.
ANEMIA OF ENDOCRINE DISEASE
 This is characterized by the presence of varying
number of normoblasts and immature
neutrophils. (leukoerythroblastic-anemia)
o Leukoerythroblastic reaction: increase in
immature granulocytes or myeloblast
and immature erythrocyte.
o Occasionally macrocytic but more
ANEMIA IN LIVER DISEASE
commonly normocytic-normochromic
 Marrow replacement: space occupied by cancer
cells  overcrowding and consumption of
nutrients and oxygen supply at the expense of
normal cells by the cancer cells
 In leukemia, there is decrease RBC (anemia),
decrease WBC (infection) and decrease platelets
(bleeding tendencies). Anemia that develops in
leukemia is Normocytic-Normochromic.
III. ANEMIAS OF SYSTEMIC DISORDERS
(ASSOCIATED WITH CHRONIC DISEASES)
 These include anemia of renal insufficiency,
anemia in liver diseases, and anemia of
endocrine function, among others.
 RBCs are usually normocytic-normochromic,
occasionally microcytic-hypochromic.
 In chronic disorders/inflammation: WBC are
activated causing them to release cytokines.
Increased cytokines (interleukins, TNF,
interferons)  alter iron metabolism that affects
hb synthesis (microcytic – hypochromic) but if
the effect of it is on blood cell production like will
cause decreased EPO because it inhibits kidneys
or it causes decrease in RBC production as it
directly suppresses the erythroid progenitors
(normocytic - normochromic)
 Increased cytokines and Decreased EPO
 Normocytic anemia due to renal disease wherein
the kidney will not be able to produce enough
hormones (EPO) which is the main growth factor
for erythropoiesis
 Decreased EPO (inefficient stimulation)
decreased BM response thus decreased RBCs
 Patients on dialysis is often given commercially
produced EPO and undergo frequent transfusion
of packed red blood cells
 EPO has hormonal regulation and its function in
stimulating blood production is also affected by
hormones produced by the endocrine glands like
the following:
o Thyroid, pituitary, adrenal, gonads
 More characterized by macrocytic anemia but
occasionally may be normocytic
 Liver diseases are associated with abnormal
metabolism and storage of folate
megaloblastic anemia
IV. CONGENICAL DYSERYTHROPOIETIC
ANEMIA (CDA)
 Group of anemia: inherited refractory anemia
characterized by erythroid multinuclearity,
karyorrhexis and bizarre malformations.
o Refractory means unresponsiveness to
stimulation and medication
 HEMATOLOGY 2

NOTE: o Intrinsic defects (defects of the red cell

itself)
o Abnormal membrane of RBCs:
Hereditary spherocytosis, elliptocytosis,
South-east asian ovalocytosis
o Abnormal metabolism/ metabolic
disorder: embden-meyerhof pathway or
hexose-monophosphate shunt is not
occurring normally due to enzyme
deficiency.
TYPE I: Megaloblastic changes with some binuclearity o Hemoglobin defects: RBCs are
 has nuclear bridging. synthesizing abnormal hemoglobin that
 More on macrocytic. makes the cell prone to lysis.
 Defective gene (Ch 15) o Most are inherited defects
TYPE II: HEMPAS (Hereditary Erythroblastic  Extrinsic defects (factors outside the red cell)
Multinuclearity with a Positive Acid Serum Test) o Normal RBC but the environment of it is
abnormal.
TYPE III: Giant erythroid precursor with more o Damaged blood vessels/
pronounced multinuclearity,
microcirculation
 More on macrocytic
o Plasma has antibodies or toxic agents
 Defective gene (Ch 15)
that can destroy the cells
o Most are acquired defects
V. APLASTIC ANEMIA ASSOCIATED WITH
CHEMICAL OR PHYSICAL AGENTS
 Chemical Agents: Benzene  toxic to BM
causing Toxic Aplastic Anemia and
Chloramphenicol
 Physical Agent: Ionizing Radiation  RBC (most
sensitive)  WBC  Platelets (more resistant;
last to be affective)

HEMOLYTIC ANEMIAS
 Belong to normochromic-normocytic anemia
 Normally, 1% of our RBCs are destroyed daily by
extravascular hemolysis
 Hemolysis can occur:
o Extravascular hemolysis: outside the
blood vessels  spleen (splenic
macrophages)
o Intravascular hemolysis: within
the blood vessel or circulation
o In both cases, there will be the release of
hemoglobin that may cause the loss of it.
Hemoglobin contains most of the body’s
iron (60% of total iron)
 Increased red cell destruction can be cause by:

If the iron released from the
hemoglobin is not recycled, it will
be loss. The body’s mechanism in
order to recycle the iron: Our
iron source may come from the
diet and recycled iron, thus it is
important for our body is able to
recycle iron. When RBCs are
hemolyzed, it will release
hemoglobin and it will be
degraded to release the iron.
Iron can go directly to cells for
reuse, or can be bound to
transferrin for transport or
stored in the form of ferritin and
hemosiderin.
HEMATOLOGY 2
EXTRAVASCULAR HEMOLYSIS occuring in macrophages. When the RBCs are
abnormal, the spleen function in filtering the blood and removes any abnormal or
senescent/old red cells called splenic culling.When there are alot of abnormal cells,
splenic culling/ extravascular hemolysis will be increased resulting to the release of
hemoglobin due to hemolysis of RBCs. Hemoglobin undergoes degradation by
separating into heme and globin. Globin will be recycled as amino acid and goes
back to cytoplasm and plasma as part of of the amino acid pool for further protein
synthesis. Heme undergoes further degradation releasing iron and recycled.
Protoporphyrin IX will also be released from the heme and be broken down into
endogenous carbon monoxide (exhaled) and biliverdin. Biliverdin is reduced into
bilirubin (unconjugated) and released into the plasma. So what increases in the
plasma is the unconjugated type which is water-insoluble and is immediately bound
to albumin. Unconjugated bilirubin can’t be excreted in the urine because kidney is
not able to filter it due to the following reasons: it is water-insoluble and they are
bound to albumin (HMW). This albumin-unconjugated bilirubin complex is brought
into the liver for conjugation. It is conjugated with 2 molecules of glucuronic acid
catalyzed by the enzyme UDP-glucuronyl transferase to form the conjugated
bilirubin called bilirubin diglucuronide. Conjugated bilirubin is stored in the gall
bladder and when needed it forms part of the bile. When needed for digestion it
will be secreted along with the bile into the small intestine to be acted on by some
bacterial enzyme and intestinal enzymes thus it converts into urobilinogen. ½ of the
urobilinogen will reabsorbed going back to the circulation then into the liver. The
other half will be converted into urobilin and stercobilin to be excreted in the stool.
FINDINGS: Increased plasma bilirubin, increased urine urobilinogen, increased
endogenous carbon monoxide.
INTRAVASCULAR HEMOLYSIS: The red cell is not engulf but it lyses in the circulation
releasing now hemoglobin in the circulation resulting to free hemoglobin in the
plasma. Hemoglobin is a LMW protein thus it can be easily filtered (solutes with MW
of <70,000 daltons) by the kidney and excreted into the urine resulting to
unpreservation of the hemoglobin content like iron. To prevent haemoglobin loss,
haptoglobin from the liver will bind with it forming now the hemoglobin-
haptoglobin complex (HMW) and direct into liver for degradation. But due to this,
haptoglobin is consumed. When there is continuous hemolysis, haptoglobin will be
depleted. Thus, hemoglobin becomes free again and will be degraded into heme
and globin in the plasma. Some heme will be oxidized and binds with albumin
forming methemalbumin and some will also bind with hemopexin forming heme-
hemopexin complex and these two will be brought back in to the liver for
degradation.
FINDINGS: hemoglobinuria or methemoglobinuria; Decreased haptoglobin;
increased plasma methemalbumin; increased carbon monoxide
 HEMATOLOGY 2

TYPES OF HEMOLYSIS (intravascular hemolysis)

 Increased urine urobilinogen
 EXTRAVASCULAR (MACROPHAGE-MEDIATED):
(extravascular hemolysis)
destruction of senescent red cells by splenic
macrophages. RBC Survival Studies (Radioactive Chromium
 INTRAVASCULAR DESTRUCTION (MECHANICAL (51Cr)
HEMOLYSIS): may be caused by the turbulent o RBC half-life: 25-32 days
environment in the circulation o Cr-RBCs IV blood count every 1-2 days
for 10-14 days
3 MECHANISMS ON HOW THE BODY CONSERVES IRON o Residual activity: index of the
IN HGB DURING INTRAVASCULAR HEMOLYSIS intravascular lifespan
1. Haptoglobin binds with free Hb preventing it o Detects sites of red cell destruction
from being filtered by the kidneys
2. When haptoglobin is depleted, heme is liberated
from hb and binds with hemopexin.
3. Oxidized heme binds with albumin forming
Methemalbumin

LABORATORY FINDINGS: RBCs and HgB Destruction

HEMATOLOGY:
 Reticulocyte count measured by RPI
o Reticulocytosis/
polychromatophilia
 Decreased red cell hemoglobin, Hct, and
RBC count
 Normocytic and/or occasionally
Macrocytic anemia representing the
reticulocytosis that is prematurely
released in the circulation

BONE MARROW
 Erythroid hyperplasia (decreased M:E)
 Increased Plasma hemoglobin and
bilirubin (unconjugated)
o Intravascular: highly increased
o Extravascular: slightly increased
 Decreased Serum haptoglobin
o Intravascular: severely decreased
to 0
o Extravascular: slightly decreased
 Increased plasma methemalbumin 
formed when haptoglobin can’t no longer
support hemoglobin
 Increased LHD due to hemolysis 
increased in isoenzyme LD 1 and 2

URINALYSIS
 Hemoglobinuria and methemoglobinuria
 HEMATOLOGY 2

TEST PARAMETER INTRAVASCULAR HEMOLYSIS EXTRAVASCULAR

HEMOLYSIS
H Hb, Hct, RBC count Decreased Decreased
E
M Reticulocyte count Increased Increased
A
T Bone Erythroid hyperplasia Erythroid
O marrow (decreased M:E ratio) hyperplas
L examinatio ia
O n (decrease
G d M:E
Y ratio)
RBC morphology Normocytic some macrocytic Normocytic some
macrocytic

P Free hemoglobin Increased Slightly increased

L
A Bilirubin Increased Unconjugated Increased
S Unconjugated
M
Haptoglobin Decreased/Absent Slightly decreased
A
C
H Lactate dehydrogenase Increased Slightly increased
E
M
Hemopexin Decreased Slightly decreased
I
S Endogenous CO Increased Increased
T
R
Y

U Free hemoglobin Positive Negative

R
I Urobilinogen Increased Increased
N
E Hemosiderin Positive Negative

1. HEREDITARY SPHEROCYTOSIS  It may involve a defect or deficiency in spectrin

 A defect in the RBC membrane protein
composition leading to hyperpermeability of the
cell to sodium.
 A genetic problem that leads to production of
abnormal membrane protein or cause non-/ or
deficient production of red cell protein.
 Repeated removal of parts of the unsupported
membrane results to spherocyte formation.
 Loss of unsupported lipid membrane
or a defect in the binding of spectrin to protein
4.1, ankyrin, band 3, beta-spectrin, protein 4.2.
o Proteins involves are vertical proteins
that support the red cell membrane.
o Many proteins can be involve:
combination of which or it could also
mean the interaction of these proteins
to together like the connection of
spectrin to protein bands.
 HEMATOLOGY 2
 Uncoupling of unsupported
phospholipid bilayer due
defective protein. The detached
lipids will form vesicles and
remaining part becomes smaller
cell without a central pallor
spherocytes. (HEREDITARY)
 Spherocyte does not always mean a hereditary
condition, it could also be an acquired condition
(AUTOIMMUNE ANEMIA that can lead to
spherocytosis).
 It could also be ARTEFACTUAL: in an old stored
blood, most of the red blood cells are
spherocytes.
 Hence, it is needed to differentiate the true
hereditary membrane defect (HS) from other
causes of spherocytosis like in immune
hemolytic anemia.
o IMMUNE HEMOLYTIC ANEMIA:
antibody sensitized the red blood cell. So
when this sensitized RBCs passes
through the spleen, the macrophages
will recognize it as a foreign/abnormal
cell and remove the inclusion (attached
antibody) in the cell (splenic pitting). As
the macrophage pulls off the antibodies
from the red cell, it also knits-off some
parts of the red cell. The remaining RBC
fragments will reseal and form into
spherocytes.
 Since some of these proteins take part in ion-
exchange channel, deficiency will cause
alteration or have defect on the channel thus
ion transport is affected.
o Sodium-Potassium ATPase: instead of
normal ion-exchange (normal amount
of Na (major extracellular cation) and K
(major intracellular cation) is
maintained.  Defect in transport 
will result to accumulation of sodium
inside or the cell gains more sodium
causing also the cell to gain more water
hence the cell will be bloated.
LABORATORY FINDINGS:
FINDINGS IN BOTH HEREDITARY AND ACQUIRED
 Spherocytes, microspherocytes and many
reticulocytes
o Seen in both hereditary and acquired
condition
o Reticulocytes  as compensatory
mechanism of BM
 Osmotic Fragility Test – always increased
o Since the spherocytes are non-
deformable because they are already
fully distended, they are fragile and
easily lyse.
 MCV: normal/slightly decrease
o Normocyte is larger than spherocyte,
but when it comes to volume, they are
the same. The volume of spherocyte is
closely similar to normocyte despite
the difference in size because
although the spherocyte is smaller, its
entire volume is occupied by the
hemoglobin (no central pallor) fully
distended. Unlike the normocyte
wherein it may have a normal size but
its volume is not entirely occupied
(1/3 central pallor).  SPHEROCYTE
and NORMOCYTE have the SAME
VOLUME.
o DIFFERENCE: in SIZE  spherocyte
(smaller) vs. normocyte (larger)
 MCHC is increased  no central pallor due to
diminished volume thus the entire volume will
be occupied by the hemoglobin. When
hemoglobin is measured in relation to the cell
volume, it is high. Hence, MCHC is above 36%.
o Spherocytes are cells with highest
MCHC but the volume is normal
 Autohemolysis test – positive
o When the spherocytes are incubated
for 48 hours, the more they become
sphere therefore they hemolyse on
their own.
DIFFERENTIATION:
 Direct Antiglobulin/Coomb’s Test (DAT/DCT)
– negative
o Test done in blood banking to
determine occurrence of in vivo
sensitization of RBCs
 HEMATOLOGY 2

o Determine whether the RBCs were whether the spherocytosis is hereditary,

attached with antibodies in vivo immune or other causes.
o In HEREDITARY: no antibody-  For hereditary spherocytosis and other
associated whether in vivo or in vitro anemia characterized by spherocytosis
 NEGATIVE in DAT o Spherocytes are test using OFT
o In IMMUNE HEMOLYSIS that result to because they are the most fragile cell
SPHEROCYTOSIS: there are  able to detect the presence of
antibodies-associated  POSITIVE in spherocyte in the circulation.
DAT o RESULT IN HS: 0.60 - 0.65% saline
CLINICAL SIGNS AND SYMPTOMS solution, hemolysis is already
JAUNDICE AND SPLENOMEGALY observed: spherocyte (fragile) easily
 Since it is a type of hemolytic anemia, increase lyse
in bilirubin causing the jaundice.  PRINCIPLE: The cell is placed in a hypotonic
 With the predominance of spherocytes in the saline to cause osmosis (the shift of the fluid
circulation, the spleen becomes overworked from the hypotonic saline into the red blood
in the response of removing them.  cell). If the red cell could still contain its
SPLENOMEGALY: enlargement of the spleen volume, the RBC would be able to absorb
 When the spleen is enlarged, the more it water without lysing and if not, it will easily
sequesters blood and destroys more blood. hemolyse. POSITIVE RESULT: Hemolysis
 HEREDITARY SPHEROCYTOSIS (HS): the main  RESULTS:
cause of hemolysis is the spleen. It is the o Incomplete hemolysis: 0.45%
spleen that causes the hemolytic process  Faint pink tinge supernatant
because the function of the spleen is to filter with intact red cell button
the blood thus it removes abnormal cells. o Complete hemolysis: 0.35%
Since spherocytes are abnormal cells, they  Homogenous pink/red
remove it through hemolysis. solution with the absence of
 It is the hemolytic process that will lead to the red cell button.
complications  when the bilirubin levels  Fragility means increased OFT  cells are
increased and the hemoglobin is rearbsorbed easily lyse (spherocytes)
in the kidneys wherein the heme is toxic to the  INCREASED OFT:
kidney  HS. o HS, abnormal membrane, severe G-6-
TREATMENT PDH deficiency, PK deficiency,
SPLENECTOMY is considered to Hemolytic anemia
prevent/lessen/correct the extravascular hemolysis  DECREASED OFT:
however it will not correct the spherocytosis because o Resistant to red cell lysis even when
it is hereditary and therefore the abnormal gene will placed in hypotonic solution
always be there causing the production of  With increased surface area-
spherocytes. But the spherocytes will no longer to volume ratio
hemolyse because the spleen was removed.  Hypochromic cells, target
cells/leptocytes, sickle cell
o Severe IDA (microcytic-hypochromic
LABORATORY TESTS: cell)
1. OSMOTIC FRAGILITY TEST o Thalassemia (microcytic-
 A test to identify the ability of red cells to hypochromic and target cell)
absorb water without lysing. o Sickle cell anemia: If the sickling is still
 It reflects the shape and size of erythrocyte mild and is reversible, when the sickle
(specifically the surface area-to-volume ratio) cell absorbs water it goes back to
 Not a confirmatory test for HS; it will only being a normal cell. However, if its
identify the presence of fragile cells like membrane is totally damaged, the
spherocytes. But it will not determine
 HEMATOLOGY 2
sickling becomes irreversible
therefore becomes resistant.
 AFFECTED BY:
o Surface area-to-volume ratio
 A normocyte with normal
central pallor is resistant to
lysis because the space can
bloat to extend the capacity,
so the cell can absorb more
water without hemolyzing.
But a cell without a central
pallor will immediately lyse
upon absorption of water like
spherocyte.
 If the surface area-to-volume
ratio is still high, the cell is
resistant
 If the surface area-to-volume
ratio is decrease, the cell is
fragile  easily lyse
 Cells with decreased surface
are-to-volume ratio have a
limited capacity to expand in
hypotonic solution and
therefore lyse even at a less
hypotonic concentration of
saline than the normal
biconcave cells.
o Functional state of the cell
membrane
 If the red cell membrane is
defective, it is easily
hemolyse.
 Like in HS, there is membrane
problem wherein it lack
integrity due to loss of
protein.
o Sanford Method  macroscopic
 Reagent: 0.5% saline + add
corresponding drops of
DH2O
 SPECIMEN: Heparinized or
defibrinated blood
 PROCEDURE: Prepare
different concentration of
hypotonic saline using the
following procedure:
1. Set up 12 test tubes and
label no. 14 -25.
2. Place in each tube 0.5%
NaCl solution (The number of
the tube corresponds to the
drops of 0.5% NaCl)
3. Using the same dropper,
add distilled water into each
tube until a total of 25 drops
(NaCl & water) are in each
tube.
4. Dispense to each tube one
drop of heparinized or
defibrinated blood. Note:
Deliver at the same angle
directly to saline solution.
5. Mix tube and allow to
stand at room temperature
or centrifuge for 1 minute at
2,500 rpm.
6. Examine each tube for
initial/incomplete and
complete hemolysis,
 DETERMINE THE
CONCENTRATION OF NaCl
SOLUTION WHERE INITIAL
AND COMPLETE HEMOLYSIS
OCCURRED.
 FORMULA: %NaCl = test tube
number x 0.02
o Dacie Method  more accurate
 Hypotonic saline (pH 7.4)
 Buffered at pH 7.4 using PO4
in order to prevent
interference of inconsistent
pH
 RECAP: OSMOSIS: movement
of fluid in order to attain
homeostasis/equillibrium
 Outside is hypotonic, inside is
isotonic movement of
water from outside to inside
 Hemolysis: @540 nm
 INCUBATED OFT: incubate
the sample for at least 24
hours to detect mild forms of
HS
 It is done because if the
observation is done
immediate and the HS is mild
there will be no hemolysis
seen. false negative result
 Mild forms of HS: on
prolonged incubation they
 HEMATOLOGY 2
become fully spherocyte
therefore show hemolysis
 PURPOSE: detect mild forms
of HS
2. EOSIN-5’-MALEIMIDE (EMA) BINDING TEST
 Alternative confirmatory test  accurate
3. SODIUM DODECYL SULPHATE-
POLYACRYLAMIDE GEL ELECTROPHORESIS
(SDS-PAGE)
4. OSMOTIC GRADIENT EKTACYTOMETRY
5. ACIDIFIED GLYCEROL LYSIS TEST
6. AUTOHEMOLYSIS TEST
7. HYPERTONIC CRYOHEMOLYSIS TEST
8. PCR
 single-stranded and conformational
polymorphism analysis
 alternative confirmatory test
NOTE:
Since HS is a genetic disorder, the confirmatory tests
are the genetic studies like chromosomal studies or
polymerase chain reaction (PCR).
2. HEREDITARY ELLIPTOCYTOSIS
 A defect in the skeletal protein interaction
(spectrin dimmers, ankyrin, protein 3 and 4.1)
anemia is normocytic normochromic with >25%
elliptocytes.
o Proteins involves are horizontal proteins
that support the red cell membrane.
 Clinical manifestions are mild, or none at all.
 Deficiency of glycophorin C
 ACQUIRED: <25% elliptocytes of the RBC
population
 HEREDITARY: >25% elliptocytes of the RBC
population
 Splenectomy is not considered
VARIANTS:
SOUTHEAST ASIAN OVALOCYTOSIS (SAO)
 In Southeast Asian Ovalocytosis/ (SAO), the
mutation in protein band 3 results to
increased membrane rigidity, making the red
cells resistant to Plasmodium invasion.
o Mutation: gene for band 3 increased
rigidity (not flexible) of the membrane
and resistance to invasion by malaria
o BAND 3: gives elasticity and flexibility
of the RBC membrane
 Common in southeast asian population
 Since the RBC membrane is rigid and instead
of showing a pale center area, a band is
formed at the center
 PINCER-LIKE CELLS: traverse ridge or
longitudinal slit
HERIDITARY PYROPOIKILOCYTOSIS (HPP)
 Affected red cells are heat sensitive and start
to bud & fragment at 45-46°C. Normal red
cells fragment starting at 49°C.
 Budding and fragmentation at 45-46 °C
 Normal RBC will start to fragment or bud at 49
degree Celsius  vesicle formation and
fragmentation
 But cells in HPP are heat-labile meaning that
even at lower temperature (45-46 C) they
easily bud off and fragment
 If they bud off they will form a small vesicle
which are called microspherocytes
buds/fragments from RBC
 PYROPOIKILOCYTES or MICROSPHEROCYTES:
cells that easily buds and fragment @45-46
degree Celsius
 Pyro: means heat/fire cells are resistant to
heat
SPHEROCYTIC HE
 Variant of both HS and HE
 HEMATOLOGY 2

 The patient has the abnormal gene resulting  Deficiency will lead to decreased membrane
to spherocytic and elliptocytic HEREDITARY transport mechanism hence it will lead to
 BLOOD SMEAR will show combination of dehydrated or overhydrated cell.
many spherocytes and elliptocytes  Normal cell should maintain more
concentration of sodium outside and
potassium inside but if the defect of the
3. HEREDITARY STOMATOCYTOSIS: transport protein results to:
HYDROCYTOSIS (OVERHYDRATED) & o A NET INCREASE in cellular cations (
HEREDITARY XEROCYTOSIS (DEHYDRATED)  Na and K inside)  cell becomes
 A mild hemolytic anemia characterized by too concentrated which will drive the
increased permeability and flux of both Na+& K+. water from the outside to shift inside
Intercellular water is influenced by ions,  cells gains more water appearing
primarily sodium, thus, a net increase or as STOMATOCYTE: a swollen cell/fully
decrease in cellular cations will influence the hydrated cell
shift of fluid shift into or out of the cell. o A NET DECREASE in cellular cation
almost all of the cations have left the
 ALTERED MEMBRANE TRANSPORT PROTEINS
RBC as the cations leaves the RBC,
 In phospholipid bilayer aside from cytoskeleton, even intracellular water will also shift
peripheral proteins at the inner leaflet or lining, outside the RBC  cells become
there are also proteins that traverse the bilayer dehydrated or dessicated 
which are called integral proteins, which serve as appearing as DESSICOCYTE or
ion channels or transport proteins. Integral XEROCYTE: a dehydrated cell
proteins can also be defective altering the
membrane transport mechanism which can
The blood antigens are
result into:
found on the red cell
o Overhydrated Hereditary
surface like the Rh
Stomatocytosis/Hydrocytosis antigens which add in
 Stomatocytes (hydrocytes) are the integrity of the RBC
formed due to the shift of fluid membrane. Hence their
into the cell (swollen cell) these absence (complete
cells have decreased surface-to- absence: Rh null disease)
volume ratio make the RBC
o Dehydrated Hereditary membrane incomplete
Stomatocytosis/Xerocytosis therefore it is
 Xerocytes (dessicocytes) are compromised.
formed due to the shift of fluid Net INCREASE in cellular
out of the cell (dehydrated cell). cations Decrease
surface-to-volume ratio
These cells have increased
OFT: Increase  prone
surface-to-volume ratio.
to lysis MCV: Decreased
CAUSES: Seen in Rh null
1. Mutation in the RHAG gene Net DECREASE in cellular
 RH Blood Group system: any of the antigen cations Increased
DCcEe surface-to-volume ratio
OFT: Decrease 
2. Absence of stomatin (band 7 region) resistant to lysis
3. Mutations in the PIEZO1 gene (Piezo-type MCHC: Elevated  cells
mechanosensitive ion channel component 1 hemoglobin appears
protein)
 HEMATOLOGY 2

concentrated due to 3. Rapoport-Luebering Pathway

intracellular water loss  Production of 2,3 DPG
Characterized by Mild  Regulates hemoglobin affinity to oxygen
hemolytic anemia  NOT involved in ATP production or RBC
hemoglobin protection
SUMMARY: MEMBRANE DAMAGE 4. Methemoglobin Reductase Pathway
HEREDITARY SPHEROCYTOSIS: most affected protein  Maintains hemoglobin in the reduced form
structure are the vertical ones like ankyrin and band 3

HEREDITARY ELLIPTOCYTOSIS: the proteins involved

can be the same with HS but the position of the
protein is horizontal like actin

HEREDITARY STOMATOCYTOSIS: whether in

dehydrated or overhydrated, the problem are the
transport proteins

1. GLUCOSE-6-PHOSPHATE DEHYDROGENASE
DEFICIENCY ANEMIA
 This is the most common RBC enzymopathy
associated with hemolytic anemia, inherited as
a sex-linked disorder. Red cells need G6PD to
generate NADPH and reduced glutathione
HEMOLYTIC ANEMIA DUE TO METABOLIC
(GSH). These reducing potentials are important
ABNORMALITIES (ENZYMOPATHIES)
in protecting hemoglobin from oxidation.
 G-6-PD enzyme is found in hexose
Metabolism Pathway in RBCs: monophosphate shunt which functions to
provide the reducing potential of the RBC or to
1. Embden-meyerhoff Pathway: anaerobic provide the antioxidant property of RBCs.
glycolysis  In the absence of G6PD, hemoglobin is not
 Provides 90-95% ATP protected from oxidation by peroxides. It will be
 Mature RBCs lack mitochondria thus they easily oxidized becoming
produce ATP through EMP
methemoglobin/hemoglobin  iron is in the
ferric state
2. Hexose Monophosphate Shunt or Pentose
Phosphate Pathway:  Oxidized hemoglobin easily denatures and
 Oxidative pathway precipitates as Heinz bodies.  located at the
 Provides 5-10% ATP periphery
 Production of reduced glutathione  main o Heinz bodies are found at the periphery
function attach to red cell membrane inducing
 Glutathione is the main antioxidant property cell rigidity and fragility.
of RBC  detoxifying agent o When RBC with Heinz bodies enters a
microcirculation(splenic sinusoids), a
 HEMATOLOGY 2

part of the cell which is flexible can pass

through however the part where there
is inclusion (rigid) will not and will be
detached from the RBC  the
remaining cell will be knit off producing
now a pitted red cells called bite cell,
horned cells, thorned cell or
degmacyte.
 Increased oxidation may be associated with
exposure to oxidant drugs, diabetic acidosis &
during neonatal period.
o Included in newborn screening in the
Philippines.
 G6PD Mediterranean is occasionally associated
with severe hemolytics episodes ( CLASS ENZYMATIC ACTIVITY DEGREE OF
ASSOCIATED
hemoglobin) of following consumption of fava
HEMOLYSIS
beans (favism)
I Severely deficient  Chronic hemolysis
o G6PD Mediterranean - favisms most severe
 PBS: Heinz bodies, bite cells, polychromasia II Severely deficient Acute, episodic
MAIN REACTION: Glucose-6-Phosphate (<10% residual
Dehydrogenase (cofactor NADP; when functions it activity)
becomes reduced into NADPH  reducing potential) III Moderately to mildly Acute, episodic
catalyzes the conversion of Glucose-6-Phosphate into deficient (10% to 60%
6-phosphogluconate. residual activity)
IV Mildly deficient to Absent
Importance of reduced NADP  NADPH normal (60% to 150%)
1. Protects the hemoglobin from being oxidized; the V Increased (>150%) Absent
H of NADPH will be given to hemoglobin reverse the Not all G6PD deficiency is characterized by severe
oxidation and reduces methemoglobin (oxidized hgb) hemolysis, it depends on the type (5). With hemolysis
back to reduced hemoglobin. (1-3); without hemolysis (4-5)
2. Reduce another reducing potential: Reduced
Glutathione
DIAGNOSTIC LABORATORY TESTS FOR G6PD
 Good reducing potential if it is in reduced
DEFICIENCY:
form; it can reduce back methemoglobin into
1. FLUORESCENT SPOT TEST (REDUCTION)
normal form.
 Recommended screening test): hemolysate +
 Major antioxidant property: breaks down
reagent  NADH – NAD (NADH conversion to
hydrogen peroxide (toxic) into water and
NAD results to loss of fluorescence)
oxygen; detoxify peroxides and free radicals
 Blood + Reagent: G6P + NADP  NADPH + 6-
PG
Methemoglobin production: 3.5 himeglobin daily
 RESULTS:
If accumulated can suffer from methemoglobinemia
o With fluorescence: conversion of
 abnormal hgb that can’t transport oxygen
NADP into NADPH
o Without fluorescence: absence of
NADPH; conversion of NADPH into
NADP  reaction does not occur
normally

2. ASCORBATE CYANIDE TEST

 HEMATOLOGY 2
 Measures the ability of RBCs to detoxify
hydrogen peroxide when incubated with
ascorbate. A brown color develops
(methemoglobin) in the absence of adequate
RBC G6PD.
 If the patient RBC has a normal G6PD levels,
it is able to produce NADPH which will cause
the reduction of glutathione (major
antioxidant property of red cell that will
break down hydrogen peroxide)
 DETOXIFICATION OF HYDROGEN PEROXIDE
(H2O2)
 PROCEDURE:
o RBC (hemoglobin) + ascorbate
cyanide reagent –incubated-- 
produces H2O2 (hydrogen peroxide,
a toxic radical that destroys
hemoglobin)
o RBC (Normal – G6PD) + H2O2 
Hemoglobin (normal)  pink
solution: cell is maintained in normal
state
o RBC ( Decrease – G6PD) + H2O2 
Hemiglobin/Methemoglobin 
brown solution: G6PD deficient cell
3. METHEMOGLOBIN REDUCTASE TEST
 INDIRECT TEST that determines the ability of
red cell to reduce methemoglobin back to
normal hemoglobin.
 All hemoglobin compounds
(abnormal/normal) are reversible EXCEPT
sulfhemoglobin.
 Hemiglobin is reversible.
 Detects NADPH generation by G6PD. The
transfer of H+ from NADPH to
methemoglobin results to its reduction back
to normal hemoglobin.
o If patient RBC have normal levels of
G6PD, then it is able to generate
enough NADPH (reducing potential)
o Methylene Blue can be added to
make the transfer of hydrogen to
methemoglobin faster.
o It facilitates a fast transfer of
hydrogen
4. AUTOHEMOLYSIS TEST (SCREENING TEST
FOR PK AND G-6-PD DEFICIENCIES)

Measures the spontaneous lysis of RBCs
when incubated at 37°C for 48 hours.
Depletion of Glucose and ATP during
incubation results to membrane loss,
spherocyte formation, and then ultimately,
lysis. If positive, test is repeated with addition
of Glucose and ATP to protect against
autohemolysis.
INTERPRETATIONS:
 Normal sample: 0.2-2% hemolysis; 0%-0.8% /
0.9% if with ATP or glucose
 G-6-PD deficiency: moderate high
autohemolysis, partially corrected by glucose
 PK deficiency: greatly high autohemolysis,
corrected by ATP
 Hereditary spherocytosis: Greatly high
hemolysis corrected by glucose or ATP but not
to a normal value.
2. PYRUVATE KINASE DEFICIENCY ANEMIA
 Pyruvate kinase enzyme is found in EMPathway
(anaerobic glycolysis) which is important in the
synthesis of ATP. Hence, its deficiency will lead
as well to ATP deficiency.
 PK catalyzes phosphoenolpyruvate to pyruvate.
 ATP energizes the Na-K pump which is
necessary in the maintenance of RBC
membrane
 PK deficiency results to impaired Na+-K+ pump.
Water and K+ are lost resulting to shrinkage,
distortion, speculation and premature RBC
destruction.
 RBC morphology is generally normocytic-
normochromic, with presence of
polychromasia.
NOTE:
In embden meyerhoff pathway, there are two
reaction that are ATP producing:
(1) Conversion of 1,3 – biphosphoglycerate into 3-
phosphoglycerate
(2) Conversion of phosphoenolpyruvate (PEP)
into pyruvate 
catalyzed by pyruvate kinase
There is two (2) glyceraldehyde-3-phosphate so there
will be 4 ATP molecules produced. However, if there
is a deficient enzyme like PK, ATP production will also
be deficient.
 HEMATOLOGY 2

o Without fluorescence: Normal PK:

conversion of NADH into NAD+
(oxidized form)
 If patient RBC have PK, it will
cause the conversion of the
above reaction
 Loss of fluorescence means
normal pyruvate kinase
enzyme

2. AUTOHEMOLYSIS TEST (Screening Test for

IMPORTANCE OF ATP IN RBC:
1. Active Transport: Transport protein + ATP
 The transport of ion is mostly active thus
ATP is needed like in Calcium pump and
NaK ATPase pump
 Facilitated transport: only transport protein
is needed
Thus the effect of ATP deficiency is that the transport
of ions along cell membrane becomes abnormal thus
the cell loses more potassium and losses more water
 crenated/distorted cell that is prone to
lysis/destruction
DIAGNOSTIC LABORATORY TESTS FOR PK
DEFICIENCY
1. FLUOERSCENT SPOT TEST (OXIDATION)
 Blood RBC (Hemolysate) + reagent (PEP,
NADH, LD) –incubate--  NADH  NAD+
o PEP – PK  Pyruvate –Lactate
Dehydrogenase (with cofactor NADH)
 Lactate
 Pyruvate: aerobic product
 Lactate: anaerobic product
 RESULTS:
o With fluorescence:
Abnormal/Deficient PK: Presence of
NADH (reduced form)
PK and G-6-PD Deficiency)
 For diagnosis of PK, G6PD, HS
 Incubated in sterile condition at 37 degree
Celsius for 48 hours (2 days)
 Measures the spontaneous lysis of RBCs
when incubated at 37oC for 48 hours.
Depletion of Glucose and ATP during
incubation results to membrane loss,
spherocyte formation, then ultimately, lysis.
If positive, test is repeated with addition of
Glucose and ATP to protect against
autohemolysis.
 INTERPRETATION:
o POSITIVE RESULT: Hemolysis
o 1st test: positive for hemolysis 2nd
test: add Glucose or ATP repeat
autohemolysis test
 1st test: Greatly
High/Increase Hemolysis;
2nd test: hemolysis is
corrected by ATP  PK
deficiency
 1st test: Moderate Hemolysis
partially; 2nd test: hemolysis
is partially corrected by
glucose  G6PD deficiency
 3rd test: Greatly Hemolysis;
2nd test: hemolysis is
partially corrected by ATP
and Glucose  HS
INTERPRETATIONS:
 Normal sample: 0.2-2% hemolysis; 0%-0.8% /
0.9% if with ATP or glucose
 G-6-PD deficiency: moderate high
autohemolysis, partially corrected by glucose
 PK deficiency: greatly high autohemolysis,
corrected by ATP
 HEMATOLOGY 2
 Hereditary spherocytosis: Greatly high
hemolysis corrected by glucose or ATP but
not to a normal value.
ACQUIRED INTRACORPUSCULAR HEMOLYTIC
MECHANISM
1. PAROXYSMAL NOCTURNAL HEMOGLOBINURA
(PNH)
 Acquired intrinsic defect which arises in the
bone marrow stem cells affecting RBCs, WBCs
and platelets.
o The cause of mutation is not yet
established because many factors are
involved.
o Acquired but genetic in
nature/hereditary disease
o EFFECT OF MUTATION: the mutation
has lead to deficiency or absence of
complement defense protein like the
regulatory protein C3 convertase. The
complement system is part of the
body’s defense mechanism thus it
function normally if its activation occurs
normally. The complement protein
causes lysis but RBCs are resistant to it
because it is protected by C3
convertase however in this disease, the
RBC does not elaborate these defense
proteins thus prone to hemolysis.
Another cause is the deficiency or
absence of GPI which supposedly
should function to linked complement
proteins thus deficiency will lead to
hemolysis.
 ↓ / (-): complement defense
proteins (C3 convertase)
 ↓ / (-): Glycosylphosphatidyl
Inositol (GPI)  linked protein
 Affected cells show an abnormal sensitivity to
complement in acidified plasma or low ionic
strength environment, resulting to
complement-mediated lysis.
o Affected cells (acidified plasma/Low
Ionic Strength)  COMPLEMENT-
mediated lysis
o COMPLEMENT: a thermo-labile protein
coming from complement activation
that in the presence of antibodies may
mediate hemolysis
 ETIOLOGY:
o HEMOGLOBINURIA: presence of
hemoglobin in the urine due to
hemolytic anemia (intravascular
hemolysis)  hemoglobin is directly
freed into the plasma and plasma is
filtered by the kidney hence will appear
in the urine
 Hemolysis mainly is the cause
for hemoglobinuria
o NOCTURNAL: means NIGHT.
 The intravascular hemolysis and
hemoglobinuria occurs chiefly
at NIGHT  plasma is more
acidified thus the RBCs
hemolyse.
o PAROXYSMAL: mean INTERVAL
 The lysis does not occur
everytime or constantly
anytime but it occurs at an
INTERVAL  NIGHT
 Decrease or absence
 Most cases of PNH, the patient conditions are
related to aplastic anemia indicating that the
mutation is caused by weaken bone marrow.
o In aplastic anemia, bone marrow is
defective, it has decreased function due
to infiltration of marrow fats hence it is
prone to mutation.
NOTE:
The complement system has 3 pathway: classical,
alternative, and lectin pathway in which involve the
assembly of C3 convertase that will later cleave C3 into
C3a and C3b and other fragments. C3 convertase is
injurious to RBC, that C3 may bind with the RBC and
stress it. Sooner or later, the RBC bound with
complement hemolyse. However, if the RBC have on
their surface the expose CD55 and CD59 which are
protective proteins, the RBC will be protected against
complement. CD55 and CD59 inhihits the further
activation of complement.
In PNH, CD55 (also called DAF) and CD59 are
deficient/absent, thus on continued activation of the
complement pathway, there will be formation of
membrane-attack complex which can also assemble
 HEMATOLOGY 2

on surface of RBC together with the complement  PROCEDURE: incubate RBC with the
proteins that will induce further lysis. following:
o Weak acid: complement activation
MAIN MUTATION in PNH occurs in the gene and cause the complement to attach
responsible in the production of the normal GPI  on RBC surface causing stressful
mutation of GPI environment and later hemolysis
GPI: links regulatory protein like CD55 and CD59 on o RBC + N. serum + 0.1% HCl  weak
RBC surface DEFICIENCY OF EITHER/BOTH will lead to acid  POSITIVE
prone complement-mediated lysis. o RBC + Pxt. Serum
o RBC + Inactivated serum  NO
HEMOLYSIS: complement is there
but inactivated; complement
proteins are thermo-labile thus when
heated at 56 degree Celsius will
cause its inactivation.
 Affected RBCs are abnormally sensitive to
complement-mediated lysis in acidified
serum (use of weak acid as reagent)
 Positive screening + Positive confirmatory
test  PNH
 Also test CDA II (HEMPAS)

Currently, flow cytometry procedures are available

that can test white cells for the presence or absence
of GPI-linked proteins. White cells can be examined
DIAGNOSTIC LABORATORY TEST FOR PNH
for reactivity to anti-CD48, CD55, and CD59, all of
1. SUGAR WATER SCREENING TEST which are anchored proteins. A new diagnostic
 Whole blood and normal control are procedure, FLAER (fluorescent- labeled aerolysin) has
incubated at 37 degrees Celsius in a LISS. proved effective in detecting smaller populations of
Hemolysis in the test/sample may indicate abnormal leukocytes in PNH.
complement-mediated lysis. OTHER DIAGNOSTIC TESTS FOR PNH
o LISS promotes binding of
FLOW CYTOMETRY
complement to RBC  straining;
 Able to detect which specific GPI or
while the complement is on the RBC
defense/regulatory protein is deficient or
surface, it stresses the cell until such
defective in affected cells because it made
time the RBC can no longer bear the
use of specific anti- sera
stress and hemolyse
 ANTI-SERA for: CD48, CD55, CD59, WBC and
o WB in LISS at 37°C
other blood cells
o NC in LISS at 37°C
o When blood cell react to the anti-
 RESULTS:
sera means that they possess the
o WB: with hemolysis; NC: no
following markers have normal
hemolysis  valid but should not yet
regulatory proteins
reported  proceed to another test
 GPI – linked proteins; -WBCs reactivity to
 confirmatory test like sucrose
anti-CD48, CD55 and CD59
hemoysis test and Ham’s acidified
serum test
FLAER (FLUORESCENT-LABELED AEROLYSIN)
2. SUCROSE HEMOLYSIS TEST
 It uses fluorescent dyes and has an
 Confirmatory Test
advantage of being able to detect even the
small population of affected cells
3. HAM’S ACIDIFIED SERUM TEST
 More sensitive
 Confirmatory test for PNH
 HEMATOLOGY 2
ACQUIRED IMMUNE ANEMIC OF INCREASED
DESTRUCTION (Antibody Related)
1. ISOIMMUNE HEMOLYTIC ANEMIA
 Antibodies comes from another person:
o Hemolytic Disease of the Fetus and the
Newborn (HDFN)
 Rh negative mother; Rh positive
baby
o Hemolytic Transfusion Reactions (HTR)
2. AUTOIMMUNE HEMOLYTIC ANEMIA (AIHA)
 Antibodies produced by patient’s own body
 Happens when the patient’s immune system
becomes altered to cause the production of
antibodies that will attack the patient’s own
RBC causing lysis.
 Can be drug-induced, infections, unknown
 WARM AIHA: IgG production reacts @37
degree Celsius; more common
o Associated/Pre-exist with CLL, wherein
the affected lymphocytes are the
dormant B-cells but they start
producing abnormal antibodies that will
attack patient’s own red blood cell
causing WARM AIHA
 COLD AIHA: IgM production reacts @colder
temperature (0-10 degree Celsius)
o Bind with RBCs at cold temperature,
since IgM is pentamer, more RBC can
bind thus agglutinates more.
NOTE:
WARM-ANTIBODY AIHA
- Occurs when the patient’s own immune
system produces anti-RBC antibodies (IgG
antibodies) that react most effectively at
warm temperatures
COLD-ANTIBODY AIHA
- Caused by pathologic antibodies (IgM
antibodies against the I antigen) that react
most effectively at cold temperature (0-10
degrees Celsius). Often associated with M.
pneumoniae & Epstein-barr virus/EBV
infections. Patients exposed to these
infections may develop harmful Anti-I & Anti-
I antibodies, respectively.
IgM Ab  directed against I Ag: 0-10 degree Celsius
 Harmless cold antibodies: Anti-I, Anti-H and
Anti-IH
 Harmful antibodies: produced in infections 
M. pneumonia (Anti-I); EBV (Anti-i) 
hemolytic anemia
 Reynaud’s phenomenon/acrocyanosis:
Cyanotic appearance: red- blue-black
coloration plus painful sensation on the
exposed parts of the body due to
vasooclussion caused by agglutination of the
RBC by the abnormal antibody.
The exposed body parts like ears and fingertips which
are the coldest or easily cold part of the body. The
IgM which is the Anti-I will bind to I antigen causing
now agglutination. So the blood vessels supplying the
distal tissues will be obstruction resulting to non-
oxygenation  cyanotic  may even lead to
necrosis/death of tissue.
3. PAROXYSMAL COLD HEMOGLOBINURIA (PCH)
 The hemolysis does not occur anytime but
occurs in an interval  during cold temperature
o Technically, hemolysis occurs in warm
temperature but it is called cold because
antibodies bind in this temperature.
 Caused by the binding of the Donath-Landsteiner
antibody to the patient’s red cells following
exposure to cold. Donath-Landsteiner is an Ab
specific for Pp blood group system. It is biphasic
that reacts at cold and warm temperature. At
 HEMATOLOGY 2
cold temperature, it binds to the red cell
membrane without causing lysis. Intravascular
hemolysis and hemoglobinuria occurs as the
temperature returns to normal body temp.
 It is immune because of the presence of
antibody: Donath-Landsteiner (D-L) antibody is
directed for the Pp blood group (hence, also
called  anti-P). IgG antibody type but biphasic
which reacts in both warm and cold
temperature.
o In cold temperature, it does not cause
hemolysis, it only binds to surface of the
RBC. This binding will cause the partial
activation of complement mechanism
from C1 to C4. Nothing will happen if the
patient is maintained in the cold
temperature however when exposed to
warmer/normal (37 degree Celsius)
temperature, the D-L antibody will be
detached and the complement
mechanism will completely (C3-C9) be
activated thus hemolysis will occur.
o Complement – mediated lysis by D-L
antibody
 D-L TEST
o PROBLEM: presence of antibody which is
found in the serum
o TEST: Pxt serum + Reagent cells: O and
P+( for anti-P) normal cells incubate @
4 degree Celsius transfer to 37 degree
Celsius incubation
o RESULTS:
 HEMOLYSIS: Presence of D-L
antibody
 NO HEMOLYSIS: absence of D-L
antibody
ACQUIRED EXTRACORPUSCULAR HEMOLYTIC
ANEMIAS
 Vascular OBSTRUCTION caused by fibron clots or
threads (DIC and MAHA) and by abnormal
platelet aggregates (TTP and HUS)
1. MICROANGIOPATHIC HEMOLYTIC ANEMIA
(MAHA)
 SECONDARY HEMOSTATIC
DEFECTS/COAGULATION DISORDER
 Caused by abnormal deposition of fibrin or
atheroma, or anatomic defects of the small
blood vessels resulting to reduced blood vessel
lumen and increased blood pressure. The
combination of this abnormal increase in the
force of moving cells and the smaller vessel
cause injury and fragmentation of RBCs.
 When the cell passage is small and is obstructed,
the circulation becomes limited and to
compensate for the decreased blood supply,
there will be increase in cardiac output and the
effect is increased blood pressure.
o The combination of increased pressure
pushing the red cell and the limited
circulation where the red cell should
pass through  trauma to red cells
 PBS: presence of schistocytes  hallmark of
hemolytic anemia
 Affects or occurs in the small blood vessels
wherein it further narrows due to the:
o Presence of fibrin deposits causing
obstruction
o Presence of atheroma plaques like in
diabetes mellitus or
hypercholesterolemia  thickening and
hardening of blood vessels (rough)
o Anatomic defects: blood vessel is very
small  hemolysis
2. DISSEMINATED INTRAVASCULAR
COAGULATION (DIC)
 SECONDARY HEMOSTATIC
DEFECTS/COAGULATION DISORDER
 Caused by extensive damage to vessel
endothelium or exposure to compounds that
initiate clotting resulting to fibrin deposition
along and across the vessel lumen. RBCs are
fragmented as they are pushed through the
vessel by the action of rapid circulation.
 Affects all types of blood vessels: small,
medium, large and may also affects either
male/female
 Test: Coagulation & Fibrinolysis tests
 Due to release the release of thromboplastin-
like or tissue-factor like substance, it will cause
the activation of both coagulation and
fibrinolysis, the effect of which is because of the
 HEMATOLOGY 2

continous activation of coagulation decreased  Shiga toxin attach to glomerular

coagulation factors; Increased activation of capillaries(renal damage)
fibrinolysis will lead to increased fibrin-split causing the release and
product (FSP) thus will result to bleeding activation of vWF which causes
o Decreased coagulation and Increased the platelets to adhere with
FSP them and aggregate 
OBSTRUCTION
3. THROMBOTIC THROBOCYTOPENIC PURPURA o NON-DIARRHEAL: in infant and children
(TTP) / MOSCHKOVITZ SYNDROME following vaccination
 PRIMARY HEMOSTATIC DEFECTS
 Associated with defective ADAMTS-13 which is 5. HEMOLYSIS DUE TO MECHANICAL INJURY
an enzyme that cleaves the ultralong and  Damage to the heart of aorta – RBCs fragment
ultralarge von Willebrand factor so they as they come in contact with damaged heart
become a smaller or medium sized vWF that is valves.
more effective in hemostasis. However the o Patients with artificial or prosthetic
deficiency/absence in ADAMTS-13, the vWF will heart valves
persist as large which easily binds with  Stress or March hemoglobinuria – lysis is caused
platelets. Ultralarge vWF will bind with platelets by mechanical pressure during exercise or
causing them to aggregate on them thus during marching
blood circulation, the platelet aggregates can o Also called Cadet’s,Orthostatic
detached and flow along the circulation and can hemoglobinuria/proteinuria, Cyclic
be deposited elsewhere causing now hemoglobinuria/proteinuria occurs
obstruction.  thrombosis (thrombus) red cell during the day
trauma o Hemoglobinuria: presence of
 Caused by formation of platelet aggregates (due hemoglobin in the urine due to
to activation) resulting to vascular occlusions hemolysis which is caused by stress and
and RBC fragmentation. More common in force against renal vein
young adults and is characterized by fever,  Infections & Infestations
petechiae, neurologic signs and renal disease. o PLASMODIUM (hemolytic condition;
intraerythrocytic parasite (humans),
4. HEMOLYTIC UREMIC SYNDROME (HUS) Babesia (ring forms; intraerythrocytic
 PRIMARY HEMOSTATIC DEFECTS cattles  humans)), Bartonella
 Etiology is not clear, though some cases are bacilliformis (Oroya fever), Clostridium
associated with mild febrile illnesses, certain perfringens, Infectious Mononucleos
immunizations, and gastrointestinal (EBV).
disturbances. o INFESTATIONS: spider bites 
 This is common among infants and young Parasitism outside the body
children manifested with acute hemolysis, renal  Exposure to Chemicals and Toxins
failure, neurologic signs, abdominal pain,  Thermal Injury
vomiting, and diarrhea.
 Associated with either diarrheal and non-
diarrheal condition
o DIARRHEAL: seen commonly in young
individuals caused by shiga
(Shigella/Salmonella) or verotoxin
producing organism like E.coli O15H:7
 HEMATOLOGY 2

BLOOD LOSS ANEMIA (POST-HEMORRHAGIC

ANEMIA)

NOTE:
Post-Hemorrhagic because anemia occurs after the
blood loss.
TYPES:
1. ACUTE
 The amount loss is >20% of patient’s blood
volume and the occurrence is too short like in
overt bleeding (gunshot wounds, accident,
trauma)
2. CHRONIC
 The amount of blood being is loss quite low
but on prolonged duration like GIT bleeding,
Menstruation, Repeated Pregnancy. Gradual
loss of blood.

ACUTE POSTHEMORRHAGIC ANEMIA CHRONIC POSTHEMORRHAGIC ANEMIA

It is acute when the amount of blood loss is >20% of the
patient’s blood volume.
EXAMPLE: Normal blood volume is 5 Liter  20% is 1L
If a patient loss a 1L of blood in a single incident  ACUTE
BLOOD LOSS  most patient can adjust to a blood loss of
20% however more than 20%  patient may suffer in
HYPOVOLEMIA (decrease in blood volume)
Hematocrit: PRBC/Total Volume
Loss of small amount of blood but in long duration. In
chronic blood loss, there is no disruption in the blood
volume but hemoglobin is loss therefore even iron is lost
leading to iron deficiency anemia.
Morphology: before becoming a full-blown (Stage 1and 2)
IDA: NORMOCYTIC-NORMOCHROMIC  Stage 3:
MICROCYTIC

When a patient loses blood, it will not select RBC only but
it is a loss of proportionate amount plasma and RBC. Thus
both plasma and RBC will decrease and when we get the
hematocrit immediately, it will appear normal. But there
will be sudden decrease in platelets and after an hour will
rise. There will be decrease in platelets because they will
be needing in arresting the bleeding by forming clots and
increased due to splenic mobilization which is caused by
stress.

Since there is hypovolemia, there will be vasoconstriction

which will decrease the blood flow however it should not
be remain as that for too long thus the body will adjust to
bring back blood vessel into its normal size. So in order to
 HEMATOLOGY 2

have vasodilution, it will cause a shift of fluid from

extravascular and interstitial compartment into the
circulation in order to expand it thus causing an increased
plasma volume over the red blood cell  hemodilution
(will only correct blood volume)  decreased hct and
hgb. Starting 3-5 days after the blood loss, the BM will
compensate by producing more blood cells and releasing
even immature blood cells: red blood cells
(reticulocytes/shift cells) and leukocytes (immature
neutrophil: shilling’s hemogram: shift to the left:
increased in young forms).

BLOOD MORPHOLOGY
Initial: normocytic-normochromic/ unchanged
depending on px blood pic. Later (3rd -5th day): will
become transient macrocytic  immature cells 6th day:
no macrocytosis: immature forms start to mature.

1. ACUTE POSTHERMORRHAGIC ANEMIA o Reticulocyte count and its associated

 Blood loss over a short period of time in
amounts sufficient to cause anemia. Normal
individuals are able to compensate for rapid
blood loss of up to 20% of the circulating blood
volume with few symptoms.
 Major manifestations of anemia are due to
depletion of blood volume
 RBCs are Normocytic-Normochromic followed
by transient Macrocytosis and increased
polychromasia
2. CHRONIC POSTHEMORRHAGIC ANEMIA
 Blood loss in small amounts over an extended
period of time. This results to gradual iron loss
and slower regeneration of red cells.
 At first, the anemia is Normocytic-
Normochromic and gradually becomes
Microcytic-Hypochromic
NOTE:
RETICULOCYTE COUNT
 It measure the erythropoietic activity of the
bone marrow.
corrections can be used to assess
bone marrow erythropoietic activity.
 RETICULOCYTE: 5th stage in the erythopoiesis
 first non-nucleated stage but it has
remnant of RNA; fully hemoglobinized
o Young erythrocytes that are in a
discrete, penultimate phase of
maturation where the nucleus has
been removed, however some of the
extranuclear RNA remains in the
cytoplasm.
o The term reticulocyte is derived from
the fact that the cell contains a small
network of basophilic material called
reticulum which is demonstrable only
by supravital stain.
o In the peripheral blood they are called
polychromatophilic erythrocytes.
 PRINCIPLE: Supravital staining of RNA
o The ribosomal RNA is stained
supravitally. Any non-nucleated RBC
that contains stained
particles/granulofilamentous
materials are counted as reticulocyte.
 HEMATOLOGY 2

o Supravital staining: staining cells RETICULOCYTE POLYCHROMATOPHILI

while in their living state C CELLS
o STAINS: Most commonly used: New STAIN: Supravital stain Without granules 
Methylene Blue and Brilliant Cresyl The granules are arranged RNA is not precipitated
Blue; Others: Janus Green, Nile Blue in “reticulum pattern” Non- but the cell will be seen
sulfate nucleated; with 2 or more as polychromatophilic
 METHOD OF STAINING: Wet method and Dry blue-stained (polychromasia)
Method (schilling’s method) particles/granulofilamento showing a lot of colors
 SPECIMEN NEEDED: EDTA-anticoagulated us  precipitated RNA (grayish, bluish,
blood or capillary blood greenish) due to
 PROCEDURE: mixture of the cell.
a. Mix equal amounts of filtered stain COMPOSITION OF
and EDTA-anticoagulated blood or WRIGHT’S STAIN:
fresh capillary blood in a small test Methylene blue (basic
tube dye) and eosin(acidic
b. Incubate mixture at room dye)  reticulocyte
temperature/using incubator for 10 – have hemoglobin
15 minutes (eosin) and RNA(MB)
o Purpose of incubation: to colors are mixed
promote the staining of RBC.
Since the RBCs are alive, they
resist staining due to intact
membrane.
c. After incubation, mix thoroughly and
prepare a wedge smear.
d. Examine smear using 100x objective
(OIO) using the routine light
microscope method. Select an area NOTE:
where erythrocytes are close but not METHODS OF COUNTING
overlapping and reticulocytes appear 1. LIGHT MICROSCOPE
to be well stained.  Reticulocytes are enumerated among 1000
RBCs in areas where RBCs are close but not
APPEARANCE OF RBCs and RETICULOCYTES: overlapping and reticulocytes appear to be
 MATURE RBCs: light to medium green without well stained.
granules
 RETICULOCYTES: light green with granules/ 2. MILLER DISK METHOD
granulofilamentous that stain deep blue.  Still made use of light microscope but with
additional component attached to one of the
e. Count the number of reticulocytes in oculars (miller disk).
1000 red cells. Reticulocytes should  The calibrated Miller disc appears in the field
also be counted as erythrocytes. with 2 squares: a large square and a small
f. Calculate as follows: square inside the large square which is 1/9 of
𝑵𝒐. 𝒐𝒇 𝒓𝒆𝒕𝒊𝒄𝒖𝒍𝒐𝒄𝒚𝒕𝒆𝒔 × 𝟏𝟎𝟎 the size of the larger square.
% 𝑹𝒆𝒕𝒊𝒄𝒖𝒍𝒐𝒄𝒚𝒕𝒆𝒔 =
𝟏𝟎𝟎𝟎 𝑹𝑩𝑪  Reticulocytes are counted in the large square
while the RBCs are counted in the small
square in successive fields on the film until a
total of 500 RBCs have been counted.
 Counterstain is not advisable
 Reticulocytes have lower specific gravity 
after incubation, the reticulocyte appear to
 HEMATOLOGY 2
settle in the upper portion  always mix to
avoid false decrease count
 Patient with hyperglycemia: Increase glucose
 inhibit staining  false decrease
 TWO SQUARES:
o LARGER SQUARE: for reticulocyte
counting
o SMALLER SQUARE: for RBC counting
(mature and immature) Size: 1/9 of
the large square
 REFERENCE VALUES:
o Adults: 0.5 – 1.5%
o Newborn: 2.0-6.0%
𝑹𝒆𝒕𝒊𝒄 %
𝑻𝒐𝒕𝒂𝒍 𝒏𝒐. 𝒐𝒇 𝒓𝒆𝒕𝒊𝒄𝒔 𝒊𝒏 𝒍𝒂𝒓𝒈𝒆 𝒔𝒒𝒖𝒂𝒓𝒆 × 𝟏𝟎𝟎
= artifacts will glisten.
𝑻𝒐𝒕𝒂𝒍 𝒏𝒐. 𝒐𝒇 𝑹𝑩𝑪𝒔 𝒊𝒏 𝒔𝒎𝒂𝒍𝒍 𝒔𝒒𝒖𝒂𝒓𝒆 × 𝟗
NOTE: INCLUSIONS & ARTIFACTS CONFUSED WITH
RETICS
 HOWELL-JOLLY BODIES
- Seen in Romanowky and methylene blue stain
but reticulocyte (RNA) will not be seen in
romanowsky stain.
 PAPPENHEIMER BODIES
- Pappenheimer bodies are present in
romanowsky, supravital stain and Prussian
blue stain (Siderotic granules) but reticulocyte
are negative in Prussian blue.
 HEMOGLOBIN H INCLUSIONS
- Hemoglobin H have no pattern; just
distributed in the red cell making the cell as
pitted golf ball whereas reticulocyte have
reticulum pattern
 HEINZ BODIES
- Most confusing because Heinz bodies are not
seen in romanowsky like the reticulocyte’s
RNA and are both present in supravital stain.
To differentiate, observe closely. Heinz bodies
are often few and found at the periphery
while reticulocyte RNA follows a reticulum
pattern
 ARTIFACTS
- Dust particles  move the the fine
adjustment and if the focus is lost. The
- OUT OF FOCUS: refractile  artifacts
disappearance  retics
 HEMATOLOGY 2

NOTE:
ASSOCIATED CORRECTION:
1. ABSOLUTE RETICULOCYE COUNT HEMOGLOBINOPATHIES
 Actual number of reticulocytes in 1 Liter of  Hemoglobinopathies are results of structural
whole blood defects in the globin component of hemoglobin.
 Formula: The structural defect result from the alteration
𝑅𝑒𝑡𝑖𝑐 % 𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 (× 1012 /𝐿) × 1000 of the DNA genetic code for the chains leading to
𝐴𝑅𝐶 =
100 the production of abnormal hemoglobins known
as hemoglobin variants.
 Reference value: 25,000 -75,000/uL  NOMENCLATURE:
o Denotes the affected polypeptide chain
2. CORRECTED RETIC COUNT / RETICULOCYTE
o The sequential amino acid number(s)
INDEX
affected (primary structure)
 This corrects the reticulocyte count to a
normal hematocrit to allow correction for the o The helix number involved (secondary
degree of patient anemia. The percentage of structure A-H).
the reticulocytes may appear increased o The nature of the abnormality (amino
because of early release in the circulation or acid substitution, deletion, addition or
because of a decrease in the number of globin chain fusion).
mature RBCs in the circulation.
 Also called Hematocrit Index or Hematocrit
correction
 Formula:  Disorder in structure
𝐻𝑐𝑡 (𝐿/𝐿)
𝐶𝑅𝐶 = 𝑅𝑒𝑡𝑖𝑐 % ×  Results from the alteration of the DNA genetic
0.45 𝐿/𝐿 code for the chains conditions characterized by
 Reference value: 1% (depends on the degree
qualitative structural abnormalities of the globin
of anemia)
polypeptide chains that result from alteration of
3. RETICULOCYTE PRODUCTION INDEX OR the DNA genetic code for those chains. Structural
SHIFT CORRECTION (RPI) abnormalities may involve amino acid
 Index calculated to correct for the presence of STRUCTURAL DEFECTS ON A OR B CHAIN RESULTING
shift reticulocytes that otherwise falsely TO HB VARIANT:
increased the visual reticulocyte count. 1. SUBSTITUTION
 General indicator of the rate of erythrocyte  Ex.: Hb Hb S: β 6(A3) Glu val
production INCREASE above normal in
anemias
2. DELETION
 Formula:
𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑅𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 𝐶𝑜𝑢𝑛𝑡  some amino acid is deleted
𝑅𝑃𝐼 =  reason of thalassemia
𝑀𝑎𝑡𝑢𝑟𝑎𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒 (𝑑𝑎𝑦𝑠)
 Reference value: 1 if hct is 0.45  Ex.: Hb Gun Hill: (β91 to β 95)0
Patient’s Hct (%) Correction factor/
Maturation time 3. FUSION
(days)  Hb Lepore-Baltimore: (G(1-50) B(86-146)) caused
40-45 1.0 by cross over during mitosis resulting to fusion
35-39 1.5
25-34 2.0 4. ELONGATION
15-24 2.5 5. Hb CONSTANT SPRING (a+31c)
<15 3.0
Clinical Significance of Reticulocytes: Increased in
hemolytic and hemorrhagic anemias

NOTE:
REASON FOR DELETION & ELONGATION: mutation in
genetic code
REASON OF HEMOGLOBINOPATHIES: mutation of
gene
NOMENCLATURE:
 COMMON NAME
 Morphology: sickling Hb (Hb S)
 Place where they were discovered: (Hb
Boston)
 SCIENTIFIC NAME
 Gives structural defect of Hb
 Ex: Sickling Hb Hb S: β 6(A3) Glu val
 B: affected polypeptide chain
 6: sequential amino acid number(s) affected
(primary structure).
 A3: Helix number involved (secondary
structure: A-H); not necessary as long as
primary structure is indicated Glu:nature of
the abnormality (substitution, deletion,
addition or globin chain fusion)
 PRIMARY STRUCTURE: sequence or
arrangement of amino acid
o Hemoglobinopathies commonly
affected the B chain.
NOTE:
 HEMOGLOBIN ELECTROPHORESIS:
Laboratory test for variant identification and
separation.
o For variant identification
o Follows general principle
electrophoresis.
 PRINCIPLE: utilizes an electric chamber that
has both cathode & anode electrons.
o Hb like proteins are amphoteric.
o Proteins are somewhat amphoteric
wherein its negative charge is
neutralized by the + charge thus it can
show a net Negative & Positive
charges thus it can behave depending
on the pH of the medium.
HEMATOLOGY 2
CELLULOSE ACETATE ELECTROPHORESIS (PH 8.4-8.6)
 For the detection&preliminary identification
of both normal abnormal Hbs particularly, Hbs
A, F, S & C.
 PROCEDURE: a small quantity of red cell
hemolysate is placed on the cellulose acetate
membrane between the center & the
cathode. An electrical field is created in the
chamber through the use of a power supply&
the ones with greatest charges migrates
towards the anode.
 Hemoglobin molecules have a net NEGATIVE
charge at alkaline pH, & therefore migrates
towards the anode
 BASIS OF MIGRATION: net Negative charge
because all of them are negative but not all
have the same number of negative charges
Note: Hb that has the greatest no. of Net
charges migrates fastest towards the anode
farthest from the point of application but the
Hb with few Negative charges migrates slower
 Owing to the variations in amino acid content
of different Hbs the net charge of each varies&
this determines their rate of mobility in the
electrical field.
 MAJOR Hb: A (adult), F (fetal), S (most
common abnormal Hb that causes sickling of
RBC) &C (second most common)
 BANDS: indicates the migration
 FAST Hb: any other Hb that migrates faster
than A such as H & I
of
A – migrates fastest, followed by F
C – slowest followed by S
Arrangement: CSFA
 HEMATOLOGY 2

CITRATE AGAR ELECTROPHORESIS (PH 6-6.2)

 PURPOSE: For the separation of Hbs that
migrate together on cellulose acetate
 Also useful in detecting small amounts of
either HbA or F in the presence of large
amount of others& in revealing small amounts
of adult Hbs A & S present at birth in cord
blood
 PRINCIPLES:
o Samples are place at the center
between cathode & anode & apply
external electricity& migration will
occur
o Hbsare identified by their migration
toward the anode & cathode&
comparing the migration to that of
known control samples
 BASIS: Hbsare separated based on the
interactions among Hb variants, agar & citrate
buffer in addition to the altered electrical
charge of the various Hbs at acid Ph.

S & C migrates alone

F & A migrates towards the cathode
S & C migrates towards the anode
Arrangement: FASC

NOTE:
SUMMARY OF CHARACTERISTICS OF HBF:
 Resistant to both acid elution & alkaline
denaturation.
 In electrophoresis, it is slower than HbA

FUNCTION:
 Has high affinity to O2 thus when it goes to the
tissues, it will not readily release the O2.
 Decreased affinity to 2-3
Diphosphoglycerate: molecule that regulates
affinity of Hb to O2.
 HEMATOLOGY 2

CRITERIA CELLULOSE ACETATE ELECTROPHORESIS CITRATE AGAR ELECTROPHORESIS

pH pH 8.0 – 8.6 ( Turgeon) pH 6.0-6.2 (Bishop)
pH 8.4 - 8.6 (Bishop)
Principle A fresh hemolysate made from a packed RBC Citrate agar electrophoresis is performed at an
( Basis/bases of sample is applied to a cellulose acetate plate acid pH after abnormal haemoglobin is detected
hemoglobin using a buffer of alkaline Ph and electrophoresis on cellulose – acetate electrophoresis.
separation) is performed. After electrophoresis, the Hemoglobin F, with the fastest cathodial
membrane is stained and cleared. The patient’s mobility, is also the most soluble, probably
haemoglobin migration is compared with that because it is most resistant to denaturation at pH
of a control test interpretation. A rough 6. Adult hemoglobins with solubility similar to
estimate of proportions of different that of haemoglobin A, such as D, E, G, O, I and so
hemoglobins may be made using a forth, move with haemoglobin A. The relatively
densitometer. The order of electrophoretic insoluble haemoglobin S moves behind
mobility, from slowest to fastest, is haemoglobin A, and the even more insoluble
hemoglobins C, S, F and A. There are several haemoglobin C moves behind haemoglobin S.
mnemonic devices for remembering the
migration pattern, such as accelerated, fast,
slow, and Crawl. Hemoglobin that migrates
beyond haemoglobin A is termed fast
haemoglobin.
Hemoglobin Bart’s and haemoglobin H both
migrate here. Any abnormal haemoglobin or
any normal haemoglobin A2 and F, should be
confirmed. If there is abnormal haemoglobin,
confirm with citrate agar electrophoresis and
solubility test if haemoglobin S is suspected. If
an increased amount of A2 or F occurs, then
quantify the amount.
Hemoglobin D and G comigrate with
haemoglobin S in this method.
Importance/ Hemoglobin electrophoresis by cellulose An important factor in determining the mobility
purpose in acetate is useful in identifying and quantifying of hemoglobin
haemoglobin haemoglobin variants and abnormal quantities is solubility and provides ready separation of Hbs
separation of haemoglobin fractions. It is rapid and that migrate together on cellulose acetate: S
reproducible and separates the major Hb from D and G, and C from E and O.
variants S, D, G, C, and E from Hb A.
Quantification of the major bands is easily
accomplished.

SEVERAL ABNORMALITIES OF HEMOGLOBIN  SIDEROBLASTIC ANEMIA: problem in the

utilization of iron due to deficiency in
1. HEME IS ABNORMAL
protoporphyrin 9
 Iron is deficient thus affecting Hb synthesis
leading to IDA
 Deficient Protoporphyrin IX because of enzyme
2. GLOBIN IS ABNORMAL
defectalong the synthesis leading to excess
 Ex: Hb A1 - 2 A w/ 4 genes & 141 amino acids &
iron&iron will not be utilized in the absence of
2 B chains w/ 2 genes & 146 amino acids
protoporphyrin 9. Unused iron will precipitate
 2 FACTORS:
into siderotic granules leading to sideroblastic
o Alpha Thalassemia
anemia
 HEMATOLOGY 2
o B Thalassemia
VARIANTS CAUSED BY B-CHAIN SUBSTITUTION
- Most common cause of hemoglobinopathies
HEMOGLOBIN S (SICKLING HEMOGLOBIN)
 Results from the substitution of glutamic acid by
valine on the 6th position of the B-chain
 Inheritance may be homozygous (SS) known as
sickle cell anemia, or heterozygous (AS) known
as sickle cell trait.
 Hb S is fully soluble. Sickling occurs when oxygen
decreases at the tissue level because of the
formation of tactoid /fluid crystals.
 SICKLE CELL ANEMIA
ORGANS AFFECTED BY SICKLE CELL ANEMIA
- Anemia is usually severe;
(mainly related to vaso-occlusion)
characterized by normocytosis
(normocytic-normochromic)
- Blood Smear shows: Polychromasia;
Sickle cells, target cells, ovalocytes,
schitocytes; Howell-Jolly and
Pappenheimer bodies
- Growth and sexual maturation lag
behind that of normal adolescents
- Electrophoresis reveals HbS 80-90%
and Hb F 10-20%
NOTE:
 Sickle cell crises: Any situation that
produces excessive deoxygenation of
RBCs may cause a crisis. Every organ in the
body is affected and include pain of many
types.
 Vaso-occlusive crises
o Occur when rigid sickle cells
increase blood viscosity leading to
development of microthrombi &
microinfarctions in the joints,
extremities and in major organs.
o “Hand-foot syndrome” or
dactylitis refers to painful
swelling of the back of the
affected hands and feet due to
obstruction caused by many
sickle cells.
 Bone, Joint, and other crises: Pain occurs
in the joints where sickle cells accumulate.
Sickle cells may occlude the marrow
sinusoids, and the lungs, which are the
site of frequent infarcts in adults.
 Infectious crises: Due to abnormal splenic
function and depression of immune
function, patient becomes susceptible to
infections. In young patients,
Streptococcus penumoniae appears to be
the major infectious agent.
 Splenic sequestration crises: The spleen
enlarges and sequesters more blood and
hypovolemia. This may lead to shock and
death
 Aplastic crises: Infection and fever cause
temporary reduction in RBC count, Hb,
Hct and Reticulocytes
 Liver (hepatomegaly and liver
malfunction)
 Heart (Cardiomegaly & heart failure due
to iron deposition)
 Spleen (Splenomegaly, Abnormal splenic
function & Splenic infarction)
 Skin (ulcers/scars); Kidneys (infarcts &
hematuria); and the lungs
 SICKLE CELL TRAIT
- Hb A is present in higher percentage
and compensates for HbS. Patients
usually have no symptoms unless in
cases of extreme tissue hypoxia
- Electrophoresis: Hb= S 30-45%
 Hb S-THALASSEMIA
- Doubly heterozygous condition in
which the mutant genes for both
Hb S and Thalassemia are inherited
by a single individual. Hb S-a is
clinically insignificant while Hb S-B
has clinical features similar to,
although not as severe as sickle cell
anemia.
NOTE:
Other sickling Hbs: C Harlem; S Travis; C
Zinguinchor
 HEMATOLOGY 2

Hemoglobin C (B6 glulys): Second most
common hemoglobin variant
 Hb C DISEASE (CC)
- Characterized by a mild to moderate
normocytic-normochromic anemia
with numerous target cells. Under
low oxygen tension or when
dehydrated, Hb CC tend to
crystallize into hexagonal or rod-
shaped crystals with blunt ends. The
crystals does not alter the red cell’s
shape but maked the cells rigid.
 Hemoglobin O-Arab (B 121 Glu -> Lys) Rare,
found in Israel, Bulgaria, Egypt, Romania,
Jamaica, Kenya
 Hb G- Philadephia (a 68 Asn->Lys)
 Hb Gun Hill (B 91-95) Leu His Cys Asp Lys -> 0
 Hb Constant Spring (CS) a+31c (142 Gln) Due
to elongation of the a-chain (31 amino acids)
 Hb Lepore-Baltimore( (1-50) B (86-146)) Due
to fusion of B
 Hb C-Harlem/ Hb C: Georgetown 2 amino
acids substitution (B 6 Glu ->Val; Asp ->Asn)
 Hb Chesapeak: has increased affinity with
oxygen
 Hb Kansas: has decreased affinity with oxygen

 Hb TRAIT (AC)
- A heterogygous hemoglobinopathy.
Clinical symptoms are similar with
Hb SS, though milder and with fewer
complications. Often, the red cells
may appear with folded cytoplasm.
Hb SC crystals often appear
fingerlike or with projections.
Electrophoresis reveals equal
amounts of Hb S and C
LABORATORY TESTS FOR UNSTABLE HEMOGLOBIN
NOTE:
1. HEART DENATURATION/HEAT INSTABILITY
 IbM (M-Saskatoon; M-Boston; M-Iwate; M-  Blood sample is incubated at 50 degrees
HydePark; M-Mulwaukee): These are caused celcius for 3 hrs.
by structural defects (amino acid substitution)
in either the a or b chain. The structural defect 2. ISOPROPANOL PRECIPITATION
in the globin chain results to non-protection of
 Blood sample is incubated with 17%
iron from oxidation (methemoglobinemia).
isopropanol at 37°C
Patients appear cyanotic (with lavender-blue
 PRINCIPLE: Normal hemoglobins have intact
skin) due to tissue hypoxia. Babies may also
bonds and remain in the solution when
show clubbed fingers at birth. Blood appears
heated or incubated with Isopropanol.
brown with numerous Heinz bodies.
Unstable hemoglobins have weaker bonds
 Hb Barts (4 y-chains) and H (4-B chains):
and easily denature or precipitate under these
Found in patients with a-Thalassemia
conditions.
 Hb Barts disease: leads to stillbirth and
hypoxia because Hb Bart’s affinity to O2 is 3. HEINZ BODY STAINING
high
 requires supravital staining (e.g. crystal violet)
 Hb H diseases: leads to anemia

OTHER SIGNIFICANT ABNORMAL HEMOGLOBINS:

 Hemoglobin D Hb D-Punjab/Hb D: Los
Angeles (B121 glu->Gln)
 Hemoglobin E (B 26 Glu ->lys)
 HEMATOLOGY 2
NOTE:
 The HbS GENE, when present in homozygous
form, is an undesirable mutation, so a
selective advantage in the heterozygous form
must account for its high prevalence and
persistence. Malaria is possibly the selecting
agent because a concordance exists between
the prevalence of malaria and Hb S. Sickling
might protect a person from malaria by either
(1) accelerating sickling so that parasitized
cells are removed or (2) making it more
difficult for the parasite to metabolize or to
enter the sickled cell.
 SICKLE CELL TRAIT (AS) confers partial
protection against lethal Plasmodium
falciparum malaria. Multiple mechanisms for
this have been proposed, with a recent focus
on aberrant cytoadherence of parasite-
infected red blood cells (RBCs). Here we
investigate the mechanistic basis of AS
protection through detailed temporal
mapping. We find that parasites in AS RBCs
maintained at low oxygen concentrations stall
at a specific stage in the middle of intracellular
growth before DNA replication. We
demonstrate that polymerization of sickle
hemoglobin (HbS) is responsible for this
growth arrest of intraerythrocytic P.
falciparum parasites, with normal hemoglobin
digestion and growth restored in the presence
of carbon monoxide, a gaseous antisickling
agent. Modeling of growth inhibition and
sequestration revealed that HbS
polymerization-induced growth inhibition
following cytoadherence is the critical driver
of the reduced parasite densities observed in
malaria infections of individuals with AS. We
conclude that the protective effect of AS
derives largely from effective sequestration of
infected RBCs into the hypoxic
microcirculation.
ADDITIONAL INFORMATIONS:
Hb S (Sicking Hb)
 The most well-known hb variant
characterized by substitution of glutamic acid
by tRNA to valine on the 6th amino acid
position of the β- chain.
 Glutamic: CAG Hb S: β 6(A3) Glu  val
 Valine: GUG
 The effect of alteration from glutamic acid to
valine is a negative charge wherein glutamic is
highly negatively chargecompared to valine
 thus leading to loss of negative charges& Hb
becomes less negative
 In electrophoresis in cellulose acetate, S
migrates slowly than A & F due to substitution
of amino acid 6 Inheritance: Homozygous (SS)
– sickle cell anemia
 Heterozygous (AS): sickle cell trait
 EFFECTS OF HB S:
o Blacks who are suffering from this are
protected from being infected from
developing severe falciparum malaria
(Resistance). Hb C, E, B-Thalassemia
&G-6PD deficiency also confer
protection in terms of resistance but
not immunity meaning these patients
may still be infected but may not
proceed to chronic stage.
o Falciparum are intracellular species&
when present in RBC& when RBC is
sickle the parasite inside also dies
does no complication arise
o Falciparum metabolizes Hb, when Hb
crystalizes it forms into a crystal&
falciparum may no longer metabolize
the Hb
o RBC w/ falciparum will be
phagocytosis before it causes severity
HOMOZYGOUS (SS) SICKLE CELL ANEMIA
 Severe anemia because the patient does not
synthesize the normal Hb A
 On electrophoresis:
o Hb S: 80 -90%
o Hb F: 10 -20 %
o Hb A2: Normal
 Blood Smear: Polychromasia (Reticulocytes
are increased) ; Sickle cells, target cells,
ovalocytes, schistocytes ; Howell-Jolly &
Pappenheimer bodies
 HEMATOLOGY 2
 Increased RDW
 Retarded growth&sexual maturation&patient
suffers sickle cell crises Main cause of Sickle
Cell Crises: sickling
 Normally when Hb S is fully oxygenated, Hb S
is fully soluble
 But when Hb S is w/ reduced O2, Hb S
becomes insoluble wherein it polymerizes
(change in molecular arrangement) &forms
into tactoid crystals/fluid crystals (elongated
& thin, pointed at both ends) leading to
sickling &rigid cell membrane thus it is no
longer adjustable leading to vassoocclusion
thus it cannot easily pass through the small
circulations
 Sickling is reversible but repeated sickling
leads to permanent sickling due to permanent
damage to RBC membrane
 Causes vasso-obstruction
 VASOOCCLUSIVE crises occurs commonly on
small circulations like in the fingers &toes thus
hand-foot syndrome / dactylitis (inflamed or
swollen, cyanotic due to absence of
circulation) occurs
 VASSOOCCLUSION also occurs in bone&joint
causing pain
 SPLENIC CIRCULATION is also minute, since
spleen also serves as a filter of blood thus its
circulation is too small, that only deformable
red cell may pass through. Due to increase
activity of spleen&vassoocclusion, spleen also
enlarges (splenomegaly) causing
sequestration of more blood leading to
hypovolemic shock. When spleen is
enlarged&bone marrow is affected thus both
are abnormal in function leading to decrease
blood production to the marrow (aplastic
crises) thus the immune system is defective &
the patient becomes prone to infection
(infectious crises) as a result of all of this organ
damage occur (marrow lungs)
 The no. 1 cause of death is infection& in
children the most common is S. pneumoniae
SICKLE CELL TRAIT (AS)
 Predominant Hb is A, only 30-45% is Hb S
 Patient usually have no symptoms&sickling is
uncommon to occurunless in cases of extreme
tissue hypoxia because S is minimal in
 concentration
Hb S – THALASSEMIA
 Hb S-A
 Hb S-B – more severe
OTHER SICKLING HBS:
 Abnormality: Hb S: β 6(A3) Glu val
+ unique B-
substitution
 Hb C-Harlem, C-Ziguinchor, S-Travis
NOTE:
HEMOGLOBIN C
 A beta structural defect, where glutamic acid
is substituted w/ lysine
 Composition: B 6 (A3) Glu Lys
HETEROZYGOUS: Hb C Trait (AC)
 RBCs – slightly hypochromic
 Less abnormalities
HEMOGLOBIN SC DISEASE
 Signs & symptoms are milder than SS but
more severe than sickle cell
 Electrophoresis: C = S = 50 – 50%
 Cytoplasm appears folded like a pocketbook
cells (RBC w/ cytoplasm folded)
 Hb SC easily crystalize forming a fingerlike
projection w/ more than 1 protrusions where
one is long&protrude away. It also in the form
of hand in gloves crystals or pointing – finger
or Washington monument crystal
OTHER ABNORMAL HEMOGLOBINS
1. Hb METHEMOGLOBIN
 M-Saskatoon, Boston, Iwate, HydePark,
Milwaukee = all have different amino acid
substitution but the effect is the same &not
protected from oxidation
 Methemoglobinemia w/ congenital cyanosis
due to overwhelming oxidation even at baby
stage& the blood appears chocolate brown
 Easily precipitate&forms into Heinz bodies
2. Hbs WITH ABNORMAL AFFINITY TO OXYGEN
a. Hb w/ Increase Affinity
 Ex.: Hb Chesapeake (a 93 Arg Leu) –
affects affinity of Hb to O2 &O2
will shift to left
 HEMATOLOGY 2
b. Hb with Decrease Affinity
 Ex.: Hb Kansas (B 120 Asp Thr
)–
shift to the right
NOTE: Abnormal Hb are connected by weak bonds
thus they easily precipitate & flocculate.
TEST FOR UNSTABLE HB
1. ISOPROPANOL PRECIPITATION TEST
 When heated, 17% Isopropanol at 37 C
normal Hb do not precipitate but few will
do&unstable Hb will easily precipitate.
 Purpose of Isopropanol: weaken the bonds
2. HEAT DENATURATION/INSTABILITY TEST
 At 50°C for 3 hours, normal Hb will not
flocculate but unstable Hb will
3. HEINZ BODY STAINING
 If ppt & flocculation happens in vivo forming
Heinz bodies which are not easily seen in
normal wrights blood smear
 Supravital staining is employed (brilliant
cresyl blue & crystal violet).
TESTS FOR SICKLING HEMOGLOBINS
SICKLE CELLS are distorted cells appearing as thin,
dense and elongated cells with both ends pointed.
SICKLING is caused by the PRECIPITATION of an
ATYPICAL HEMOGLOBIN (commonly hemoglobin S)
which DISTORTS the RED BLOOD CELLS into a SICKLE
or CRESCENT SHAPE. HEMOGLOBIN S is fully soluble
when oxygenated but becomes insoluble when the
oxygen level is decreased. It first POLYMERIZES then
forms into CRYSTALS which cause the red cell to
become RIGID.
SCREENING TESTS:
 PRINCIPLE: HbS forms an insoluble tactoid
crystals when oxygen supply to the red cell is
decreased. When Hb S is deoxygenized
outside the cell into the plasma it will cause
the solution to be TURBID. POSITIVE RESULT:
1. Sickle RBC 2. Turbidity
 Degree of sickling or turbidity depends on the
concentration of Hb S in the RBC
1. SEALED WHOLE BLOOD METHOD (SCRIVER
& WAUGH)
 PRINCIPLE: RBC take on a SICKLE-LIKE SHAPE
when oxygen supply to the red cell is
decreased. HbSforms INSOLUBLE TACTOID
CRYSTALS when exposed to LOW OXYGEN
TENSION.
 PROCESS: Perform IN VIVO. Using a RUBBER
BAND, the rubber band is TIED at the BASE of
the FINGER or at the BALL of the finger
intended to be prick& maintain it for at least
5-10 minutes to cause obstruction in order to
obstruct the circulation thus decreasing the
oxygen& Hb S will form into crystals. Prick
the deoxygenated finger & drop into the
center of the slide &cover w/ cover slip. Seal
the sides w/ petroleum jelly or paraffin to
prevent entry of atmospheric O2 which
contains abundant O2 which may cause the
sickled cell to revert to normal shape.
Incubate for 1 hour at room temperature
&examine microscopically if still none,
incubate for 2 hours until 3rd hour. Perform
interval microscopic examination.
 POSITIVE RESULT: 1. Sickle RBC (Hb S) but
does determine if heterozygous AS or
homozygous SS REPORTING: positive or
negative
 DEGREE of sickling depends on the
CONCENTRATION of the Hb S in the RBC
 Readings are made at an HOURLY INTERVAL
for 2-3 HOURS
 POSITIVE RESULT is DIAGNOSTIC but DOES
NOT DISTINGUISH Hb S trait & Hb S anemia
2. SODIUM METABISULFATE METHODS
(DALAND & CASTLE)
 REAGENT: 2% Sodium metabisulfide or
sodium bisulfate which are reducing agent
which bind &remove O2 from the blood.
 PRINCIPLE: a drop of blood is mixed w/ a
drop of 2% sodium metabisulfite (a reducing
agent) on a slide, & the mixture is sealed
under a coverslip. The hemoglobin inside the
RBCs becomes deoxygenated causing
polymerization& the resultant sickle cell
formation
 PROCESS: add a drops of blood& the reagent
into the center of the slide.Cover the slide w/
 HEMATOLOGY 2
coverslip&seal the sides. Observe
microscopically.
 POSITIVE RESULT: sickled RBC deoxygenated
by reducing agent.
3. DITHIONITE SOLUBILITY TUBE TEST
 REAGENT: sodium hydrosulfite (dithionite)
removes O2
 SPECIMEN: whole blood w/ EDTA heparin or
sodium citrate in order to prevent clotting
 PRINCIPLE: Red cells are lysed by saponin
allowing Hb to escape. Sodium dithionine
binds&removes oxygen from the test
environment. Hb S polymerizes in the
deoxygenated state&forms a precipitate in a
high-molarity phosphate buffer solution. The
tactoid refract or deflect light& make the
solution turbid.
 PROCESS: anticoagulated whole blood +
sodium dithionite. Saponin lyses the RBC
releasing now Hb. Hb binds w/ sodium
dithionite which removes O2 from solution
causing Hb to crystalizes but polymerization
happens outside the RBC thus it will not
cause sickling but turbidity of solution& is
determined by refraction&deflection of the
solution in the presence of light. Observe
specimen for turbidity by holding the tube
2.5 cm in front of a newsprint or a card
reader w/ thin black line
 RULE: if the solution is observed in the
solution then the solution is not turbid thus
negative result but if lines are no longer
visible then it is positive for tactoid
crystals&sickling.
 POSITIVE RESULT: turbidity
 Turbidity indicates presence of sickling Hb
regardless of any genotype
 SCREENING METHOD OF CHOICE
4. HEMOCARD Hb A & S
 PRINCIPLE: Hb A& S contains monoclonal Ab
(IgG), which specifically bind to the amino
acids at or near the 6th position of the B-
chain of the Hb S&A
 Hb S monoclonal Ab will react w/ the B-chain
of Hb S but not w/ the Hb A
 Hb A monoclonal Ab will react w/ the B-chain
of the Hb A but not w/ Hb S
 Abs are adsorbed to suspended metal sol
particles giving the reagent raspberry-like
color
NOTE:
 After electrophoresis, densitometry is
performed to determine the density of the
bands created by the migrating proteins
converted into concentration.
 In cellulose Acetate, at an alkaline pH, Hbs are
negatively charge thus they migrate towards
the anode.Hb S together w/ D & G upon
migration appears as a band of protein thus
densitometry is performed to determine the
concentration of Hb S.
 On Citrate Agar, at an acidic pH, Hb S migrates
towards the anode thus citrate agar is more
confirmatory
EXPERIMENT MODULE:
Some hemoglobins that aggregate and have reduced
solubility are capable of polymerizing and crystallizing
within the red cell causing a distortion of cell shape
(sickle shape). Hb S (Sickling Hb), when fully
oxygenated is fully soluble. Polymerization and
formation into tactoid crystals occur only when
oxygen is decreased at tissue level.
 SPECIMEN NEEDED: Capillary blood
A. SCRIVER AND WAUGH METHOD
a. Place a rubber band around the base of
the middle finger and allow stayingin
place for 5 minutes.
b. Make a finger puncture on the ball of the
finger and place a drop of capillary blood
on a slide.
c. Immediately cover with a coverslip and
seal edges with petroleum jelly.
d. Incubate the preparation at room
temperature. Observe for red cell sickling
at hourly intervals for 2, 3 hours, or after
24 hours if desired.
e. Microscopic examination (400x): If more
than 10% of the cells are sickled, the result
is positive.
B. SICKLE CELL SLIDE TEST
 In the absence of the HbS solubility test, the
sickle cell slide test is useful in detecting
sickle cells in patients who have either sickle
cell disease or sickle cell trait.
 HEMATOLOGY 2
a. Weigh 0.1g of sodium metabisulphite and
transfer to a test tube capable of holding
15 ml of water.
b. Add 5 ml of distilled or deionized water,
stopper, and mix until the chemical is
fully dissolved. The chemical can only be
used within the day it was suspended
(within 8 hrs)
c. Deliver one drop of patient’s capillary
blood or well mix venous blood on a slide
and add an equal volume of freshly made
reagent, mix and cover with a cover glass.
Exclude any air bubbles.
d. Place the slide in a plastic box or Petri
dish with damp piece of blotting paper or
tissue at the bottom to prevent drying of
the preparation (moist chamber). Leave
at room temperature.
e. After 10-20 minutes, examine the
preparation microscopically for sickle
cells. Focus the cells first with the 10x
objective and examine for sickling using
the 40xobjective. Examine several fields.
Sickling usually takes place in one part of
the preparation than the other.
f. Sickle cells usually appear crescent shape
with pointed ends or holly leaf shape.
Report as “sickle cell test positive” when
crescent shape cells are seen, or ‘sickle
cell test negative” when cells appear
rounded or oval shape.
C. TUBE SOLUBILITY TEST
 The principle of solubility method was based
on turbidity created when Hb S is incubated
with sodium dithionate.
a. Add 20 uL of blood with 2 ml of the
reagent prepared in procedure B (2% w/v
sodium metabisulphite or sodium
dithionite)
b. Mix and stand at room temperature for 5
minutes.
c. Examine the tubes using a white board
with black lines as the background in
ambient light settings.
d. Interpret results as positive if the black
lines are not visible
OSMOTIC FRAGILITY TEST (OFT)
 A test to identify the ability of red cells to
absorb water without lysing.
 It reflects the shape and size of erythrocyte
(specifically the surface area-to-volume ratio)
 Not a confirmatory test for HS; it will only
identify the presence of fragile cells like
spherocytes. But it will not determine
whether the spherocytosis is hereditary,
immune or other causes.
 For hereditary spherocytosis and other
anemia characterized by spherocytosis
o Spherocytes are test using OFT
because they are the most fragile cell
 able to detect the presence of
spherocyte in the circulation
o RESULT IN HS: 0.60 - 0.65% saline
solution, hemolysis is already
observed: spherocyte (fragile) easily
lyse
 PRINCIPLE: The cell is placed in a hypotonic
saline to cause osmosis (the shift of the fluid
from the hypotonic saline into the red blood
cell). If the red cell could still contain its
volume, the RBC would be able to absorb
water without lysing and if not, it will easily
hemolyse. POSITIVE RESULT: Hemolysis
 RESULTS:
o Incomplete hemolysis: 0.45%
 Faint pink tinge supernatant with
intact red cell button
o Complete hemolysis: 0.35%
 Homogenous pink/red solution
with the absence of red cell
button
 Fragility means increased OFT cells are easily
lyse (spherocytes)
 INCREASED OFT:
o HS, abnormal membrane, severe G-6-
PDH deficiency, PK deficiency,
Hemolytic anemia
 DECREASED OFT:
o Resistant to red cell lysis even when
placed in hypotonic solution
 With increased surface area-to
volume ratio
 Hypochromic cells, target
cells/leptocytes, sickle cell
o Severe IDA (microcytic-hypochromic
cell)
 HEMATOLOGY 2
o Thalassemia (microcytic-hypochromic
and target cell)
o Sickle cell anemia: If the sickling is still
mild and is reversible, when the sickle
cell absorbs water it goes back to
being a normal cell. However, if its
membrane is totally damaged, the
sickling becomes irreversible
therefore becomes resistant.
 Affected by:
o Surface area-to-volume ratio
 A normocyte with normal
central pallor is resistant to
lysis because the space can
bloat to extend the capacity,
so the cell can absorb more
water without hemolyzing.
But a cell without a central
pallor will immediately lyse
upon absorption of water like
spherocyte.
 If the surface area-to-volume
ratio is still high, the cell is
resistant
 If the surface area-to-volume
ratio is decrease, the cell is
fragile  easily lyse
 Cells with decreased surface
are-to-volume ratio have a
limited capacity to expand in
hypotonic solution and
therefore lyse even at a less
hypotonic concentration of
saline than the normal
biconcave cells.
o Functional state of the cell
membrane
 If the red cell membrane is
defective, it is easily
hemolyse.
 Like in HS, there is membrane
problem wherein it lack
integrity due to loss of
protein.
 SANFORD METHOD  MACROSCOPIC
o REAGENT: 0.5% saline + add
corresponding drops of DH2O
o SPECIMEN: Heparinized or
defibrinated blood
o Determine the concentration of nacl
solution where initial and complete
hemolysis occurred.
o FORMULA: %NaCl = test tube
number x 0.02
o PROCEDURE: Prepare different
concentration of hypotonic saline
using the following procedure:
a. Set up 12 test tubes and label no.
14 -25.
b. Place in each tube 0.5% NaCl
solution (The number of the tube
corresponds to the drops of 0.5%
NaCl)
c. Using the same dropper, add
distilled water into each tube
until a total of 25 drops (NaCl &
water) are in each tube.
d. Dispense to each tube one drop
of heparinized or defibrinated
blood.
NOTE: Deliver at the same angle
directly to saline solution.
e. Mix tube and allow to stand at
room temperature or centrifuge
for 1 minute at 2,500 rpm.
f. Examine each tube for
initial/incomplete and complete
hemolysis,
 DACIE METHOD  MORE ACCURATE
o Hypotonic saline (pH 7.4)
 Buffered at pH 7.4 using PO4
in order to prevent
interference of inconsistent
Ph
 RECAP: OSMOSIS: movement
of fluid in order to attain
homeostasis/equilibrium
 Outside is hypotonic, inside is
isotonic  movement of
water from outside to inside
o HEMOLYSIS: @540 nm
o INCUBATED OFT: incubate the sample
for at least 24 hours to detect mild
forms of HS
 It is done because if the
observation is done
immediate and the HS is mild
there will be no hemolysis
seen  false negative result
 HEMATOLOGY 2

 Mild forms of HS: on

prolonged incubation they
become fully spherocyte
therefore show hemolysis
 PURPOSE: detect mild forms
of HS.