HEMATOLOGY in House Review PDF

HEMATOLOGY I
James Patrick C. Picar, RMT
OUR LADY OF FATIMA UNIVERSITY COLLEGE OF MEDICAL LABORATORY SCIENCE - QUEZON CITY CAMPUS
HEMATOLOGY
- Study of appearance, development,
physiology, kinetics and pathology of blood.
- For the purpose of assessing an individuals
general condition, diagnosis of anemia,
leukemia and other blood related disease.
OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

UNIVERSITY SCIENCE - QUEZON CITY CAMPUS
FUNCTIONS OF BLOOD
• Respiratory – hemoglobin carries Oxygen/Carbon
Dioxide Molecules
• Transport – physiologic substances (hormones),
drugs, macromolecules (CHO, CHON, Lipids)
• Excretory – glomerular filtration of waste
products, transport of CO2 to the lungs
• Buffering action – pH maintenance
*AMPHOLYTES – susbsts that can act as an
ACID or BASE (proteins)
• Defense mechanism (immunity and hemostasis) –
Igs in the plasma/serum, coagulation proteins
CHARACTERISTICS OF BLOOD
1. Volume – 7-8% of total body wt (75-85 mL/Kg)
M: 5 to 6 Liters NB: 250 to 350 mL
F: 4 to 5 Liters (Rodak)
*Solid Portion: 20g/100mL of blood
2. In Vivo – fluid form
In Vitro – coagulable (5 to 15 mins)
3. Color: In vitro - Bright red (arterial blood)
- Dark Red (venous blood)
In vivo – Dark Purplish Red (Pulmonary Artery, deoxygenated)
- Bright red (Pulmonary Vein, oxygenated)
Ergo, generally, Blood is RED
4. Osmotic Concentration – 0.85% NaCl (isotonic)
Hypertonic condition – RBC’s tend to crenate/shrink
Hypotonic condition – RBC’s swell and tends to lyse
5. pH – 7.4 (slightly alkaline)
Venous Blood – 7.35 | Arterial Blood – 7.45
Henderson-Hasslebach Formula:
pH = pKa + log (conj. base/conj. acid)
pH = 6.1 + log (20/1)
pH = 6.1 + 1.3
pH = 7.4
Conjugate Base = HCO3 (Bicarbonate)
Conjugate Acid = H2CO3 (Carbonic Acid)
**for every 1 molecule of H2CO3, there are 20 mols of HCO3
6. Specific Gravity – 1.055
7. Viscousity – 3 to 4x thicker than Water
COMPOSITION OF BLOOD
A. Formed Elements
a. Packed RBC – 44 to 45% of the blood
b. Buffy Coat - <1% of the blood
i. Platelets (uppermost layer)
ii. WBC’s
- Granulocytes (Lowermost layer)
- Agranulocyte (Middle layer)
iii. Retics (Lowermost)
B. Plasma/Serum – 55% of the blood
- 91% Water
-7.5% CHONS
- 1.5% Other solutes (electrolytes, vits.,
hormones, lipids, carbs etc.)
• PLASMA – liquid portion of the

anticoagulated/non-clotted blood.
- turbid/hazy (due to presence of
fibrinogen)
• SERUM – liquid portion of the non-
anticoagulated/clotted blood.
- straw, usually clear (absence of Fbg),
but can be physiologically turbid (chyle).
HEMATOPOEISIS
• A continuous and highly regulated process of
blood cell production.
• Includes cell renewal, proliferation,
differentiation and maturation of stem cells.

TYPES OF Human Stem Cells
1. Totipotential Stem Cells
- first type of cell produced during fertilization. Can
develop into ANY type of human cell. Dev’t of Embryo  Fetus.
2. Pluripotential Stem Cells
- present several days after fertilization
- can dev’t into any cell type BUT can NOT dev’t into
FETUS.
3. Multipotential Stem Cells
- derived from the pluripotential cells.
- limited to specific type of tissues.
Ex. BM stem cells produce all type of Blood cells, bone cartilage,
adipose
OUR cells.
LADY OF FATIMA
UNIVERSITY
COLLEGE OF MEDICAL LABORATORY
SCIENCE - QUEZON CITY CAMPUS
STEM CELL THEORIES
Monophyletic theory
- Single type of stem cell gives rise to all mature
cells. (Pluripotential stem cell)
- This theory is more widely accepted than the

Polyphyletic theory which suggests that each
blood cell linages is derived from a unique
stem cell.
Other theories of stem cell differentiation..
Stochastic Model – hematopoeisis is a random

process whereby HSCs randomly commits to
self-renewal or differentiation.
Instructive Model – the microenvironment of

BM determines whether the stem cell will self-
renew or differentiate

HEMATOPEITIC MICROENVIRONMENT
• The environment for hematopeitic cells must
allow 1) Self-renewal, 2) Proliferation and
differentiation and 3) Apoptosis
• After birth, BONE MARROW becomes the

primary site of hematopoeisis

STROMAL CELLS – general term for specialized
cells w/i the BM that provide protective and
nourishing environment to the HSCs. Includes
the ff:
- Fibroblasts - Endothelial cells
- Reticulum cells - Adipocytes
- lymphocytes - Macrophages
- All theses cells, secrete substances that make
up the extracellular matrix which are essential
for cell growth and support of the HSCs.

PHASES OF HEMATOPOEISIS
I. MESOBLASTIC/YOLK SAC PHASE
- PRIMITIVE HEMATOPOEISIS
- start at 19th day of gestation
- Mesodermal cells and Angioblasts line the
yolk sac cavity
- Mesodermal cells give rise to PRIMITIVE
ERYTHROBLASTS
- Angioblasts are cells that will later on form
the blood vessels.

PRIMITIVE ERYTHROBLASTS produce measurable
amts of Hgb INTRAVASULARLY:
• GowEr I - 2 Epsilon, 2 zeta
• GowEr II – 2 Epsilon, 2 alpha
• Portland – 2 zeta, 2 gamma
Yolk sac phase is active until 8th-12th week of

gestation.

~~*!!! REMEMBER !!!*~~
• “gTAE”. gower Two has Alpha and Epsilon
chains.
• Gower Hemoglobins have E (epsilon) chains
because they have “E” on their name - gowEr
• El Gamma Penumbra became famous because
of Pilipinas Got Talent or (PGT) |Portland Hgb
has Gamma and Zeta (PGZ).

II. HEPATIC/LIVER PHASE
- start of DEFINITIVE HEMATOPOEISIS
- begins at 4th to 5th week of gestation
- recognizable clusters of developing
erythrocytes, granulocytes and monocytes are
forming during this phase.
- extravascular hematopoeisis (since liver is
responsible). And is active 1 to 2 weeks after birth.
- decline in Primitive hematopoeisis in yolk
sac. Lymphoid cells also start to appear.
- peaks at 3rd month of gestation. Producion
of RBC is predominant
- Developing organs, such as spleen, thymus,
kidney and lymph nodes also contribute to blood
production:
• Thymus – maj. Site for T-cell production
• Spleen and Kidney – B cells
• Lymph Nodes – T and B cells.
*megakaryocyte production also begins
*alpha and gamma chain production
predominates
Hgbs produced:
1. Hgb F - 2 alpha, 2 gamma
2. Hgb A1 – 2 alpha, 2 beta
3. Hgb A2 - 2 alpha, 2 delta
III. MYELOID/MEDULLARY/BONE MARROW PHASE
- “myelo”  marrow
- starts at 5th month of gestation
- differentation of mesenchymal cells into
skelatal and hematopoeitic blood cells.
- Myeloid activity is predominant which
means granulocytes are the major cells produced
during this phase.
- by 21st week, M:E ratio reaches 3:1
- by the end of 6th month, BM becomes he
major site of hematopoeisis.
(high lvls of EPO, G-CSF, GM-CSF, Hgb F, Hgb A2 and
HgbA1.)
PEDIATRIC/ADULT HEMATOPOEISIS
• Bone Marrow is the primary site of hemostasis
from the 6th month of gestation all throughou
the life.
• Although, some pathologic conditions wherein
there is BM suppression, Extramedullary
Hematopoesis take place in compensation for
the non-functional BM.

ORGANS INVOLVED IN HEMATOPOSESIS
PRIMARY LYMPHOID TISSUE
- Bone Marrow
- Thymus
SECONDARY LYMPHOID TISSUE
- Spleen
- Lymph nodes
- GALT (Gut Assoc. Lymphoid Tissue)

I. BONE MARROW
- Considered as one of the largest organ in

the body
- Defined as the tissue located within the
cavities of the cortical bones.

*Retrogression – the process of replacing the active
marrow with adipose and eventual restriction of active
marrow to flat bones, pelvis, ribs, sternum, vertebrae,
skull and proximal portions of the long bones
Bone Marrow Cellularity
a. Normocellular – 30-70% of cells are
hematopoeitically active
b. Hypercellular/Hyperplastic - >70% is active
c. Hypocellular/Hypoplastic - <30% is active
d. Aplastic – few or no hematopoeitic cells

M:E Ratio – Myeloid:Erythroid Ratio
Normal = 3-4:1
WBCs have higher ratio because they are short lived
(1-2 days), while RBCs last for 120 days.
(Lymphocytes and Monocytes are not included in
the ratio)
a. Infection= 6:1
b. Leukemia = 25:1
c. Myeloid Hyperplasia = 15-20:1
d. Erythroid Hyperplasia = 1:25, 2:30
e. Erythroid Hypoplasia = 5:1, 6:1, 7:1
II. THYMUS
- For T cell production
- Bilobed organ at the anterior mediastenum
above the heart.
- Produces hormone thymosine that promotes
t-cell maturation.
- Size increase from birth to puberty and start
to athrophize at old age.

III. LYMPH NODES
- Outer capsule (Trabeculae) – supports MACROPHAGES
and most Lymphocytes
- Cortical region contains B-cell proliferation foci called
the germinal center
- B cells (and plasma cells), however, are located at the
medullary cords.
- Paracortex (in bet cortex and medulla) contains the T
lymphocytes and macrophages
Lymph Nodes functions:
1. Proliferation of lymphocytes
2. Processing of specific Immunoglobulins
3. Filters particulate matters/debris/bacteria that enters
the lymph nodes

IV. SPLEEN
- Largest lymphoid organ in the body
- Stores 30% of platelets
a. White Pulp – contains lymphocytes,
macrophages and dendritic cells.
b. Red Pulp – cord of Billroth. Contains
specialized macrophages for removal of the
senescent RBC’s:
CULLING- phagocytosis that leads to
eventual degradation of cell organelles.
PITTING – removal of inclusion
• SPLENOMEGALY – enlargement of spleen.
- increased spleen workload. Such as in
cases of hemoglobinopathies, thalassemias,
malarias, hodgkin’s lymphoma, myeloprliferative
disordes and chronic leukemias.
• HYPERSPLENISM – results to pancytopenia
• AUTOSPLENECTOMY – necrosis of spleen due
to entrapment of sickled RBC in cases of sickle
cell anemia.

Let’s recall..
• Normal volume of blood
• Substances that can act as an ACID or BASE
• Cause of serum turbidity
• Human stem cell capable of dev’t embryo to fetus
• Provides nourishment and protection to BM HSCs
• Site of hematopoeisis at 6th month of gestation
• Hgb chains of Gower 1
• M:E ratio for Erythroid hyperplasia
• Extramedullary site of hematopoeisis
• Other name of the red capsule
• Bones that have active marrow in adults

ERYTHROCYTES

ERYTHROPOEISIS
• Hematopoeisis focused on the production of
Erytropoeisis
• Erythropoeitin - hormone that stimulates Hgb
production
- produced mainly in the kidney
- 10-15% is produced in the liver (primary
source of EPO in the unborn)
- also help in megakaryopoeisis together
with IL 1 and 3, GM-CSF
• CFU-GEMM give rise to BFU-E
• BFU-E contains only few receptor for EPO
thus not significantly affected by this
hormone.
• BFU-E is under the influence of IL-3, GM-CSF,
TPO and kit Ligand to develop into CFU-E (the
earliest identifiable colony of RBC), which
usually take 1 week.
• CFU-E is stimulated by EPO to develop into an
Erythroblast another 1 week.

RBC Maturation Nomenclatures
• Rubricytic Nomenclature – ASPC proposed
• Normocytic Nomenclature – USA used
• Erythroblastic Nomenclature – Paul Ehrlich
proposed

Maturation Pattern
• Cellular and nuclear diameter decreases as cell
mature
• Nuclear diameter decreases more rapidly than
the cytoplasm thus N:C RATIO also DECREASES
• Chromatin coarsens and eventually clumps as the
cell mature, which will later be extruded from the
cell
• Cytoplasmic chromatia transits from blue to gray
to pink (reflects gradual cessation of ribosmal
activity and increased Hgb pigmentation)
Pronormoblast/Rubriblast/Proerythroblast
• 12 to 20 um
• N:C Ratio is 8:1
• Has 1 to 3 Nucleoli
• Fine Chromatin
• Blue cytoplasm
• Capable of mitosis – yields 2 basophilic
normoblasts within 24 hours.
• 1% BM HSCs

Basophilic Normoblast or Prorubricyte
or Basophilic Erythroblast
• 10 – 15 um
• N:C Ratio – 6:1
• 0-1 nucleoli
• Coarsening chromatin
• Deep blue cytoplasm (ribosomes and rRNA)
• Start of Hgb production (barely detectable)
• Capable of mitosis – yields 2 polychromatic
normoblasts within 24 hours.
• 1 -4 % of BM HSCs
Polychromatophilic Normoblast or
Rubricyte or Polychromatophilic
Erythroblast
• 10 – 12 um
• No nucleoli
• Clumped chromatin
• Murky gray-blue (polychromatic)
• Increased Hgb production than Basonormo
• Last stage capable of mitosis – 2 Orthochromic
normoblast within 24 hours – 30 hours
• 10 – 20% BM HSCs
Orthochromatic Normoblast/Erythroblast
or Metarubricyte
• 8 -10 um
• Condensed Pyknotic Chromatin
• Pink-Orange cytoplasm
• Last stage with nucleus (nRBC)
• Not capabale of mitosis
• Hgb production is almost complete, matures into
Retics for 48 hours
• 5 -10% HSC’s in BM
Polychromatic Erythrocyte or Reticulocyte
• 8 – 10 um
• Anucleated
• Polychromatic cytoplasm (Diffusedly
basophilic)
• But the Hgb is completely synthesized already
• Residual RNA is present - threadlike structure
in Supravital stain (NMB)

• Usually 2 days lifespan (1 day in BM, 1 day in
circulation before splenic pitting)
• Reticulocyte Count – best indicator for BM
function
Normal Range: 0.5 – 1.5 % (Adult)
2.5 – 6.5 % Newborn
• Stress or Shift Reticulocyte – retics released from the
BM in response to increased need.
• MILLER DISK – specialized eyepiece for Retic
Ct.

ERYTHROCYTE
• 6 – 8 um
• Mean Cell Volume 80 – 100 fL
• Anucleated
• SALMON PINK cytoplasm
• Biconcave disk with central pallor
• Normal Hgb conc (MCHC) 32-36%
• Ave. cell surface area 140um
• 120 lifespan
• NV:Male – 4.6-6x10^12/L (SI Unit)
Female – 4-5.4x10^12/L
RBC Membrane Deformability
• An RBC is 90fL in volume and has 140um2
surface area, with 40% excess to allow
deformability.
• Hgb concentration of >36% makes the RBC
more viscous and thus difficult to be
accommodated by the capillaries or by the
splenic pores

Characteristics of RBC membrane
1. Biconcave – for maximum surface area
2. Deformable
3. Osmotic balance
4. Provides surface for antigen receptors
5. Transport of ions and gases

Major RBC membrane components
- 52% CHON, 40% Lipids, 8% CHO
1. PROTEINS
a. Integral
b. Peripheral
2. LIPIDS
a. External surface
b. Internal surface

RBC Membrane PROTEINS
I. INTERGRAL (transmembranous) PROTEINS
- extends from the cell surface into the
cytoplasm and supports the carbohydrates:
Glycophorin A – MN blood group system
Glycophorin B – Ss blood group system
Glycophorin C – Gerbich blood group
Band 3 – ABH antigens

RBC Membrane PROTEINS
II. PERIPHERAL (cytoskeletal) PROTEINS
- makes up the skeletal framework of the
cell. Anchorage of the integral proteins
Spectrin Myosin
Ankyrin P55
Actin Bands 4.1

RBC Membrane LIPIDS
• Provides membrane pliancy or deformability
• Forms bilipid layered cell membrane
– Hydrophilic Polar “Heads” internal and external
surface of the cell
– Hydrophobic Acyl “Tails”
• Cholesterols – provides RBC membrane
endurance
• Phospholipids – provides RBC elasticity
RBC ENERGY METABOLISM
• Oxygen exchange and Oxygen-Heme binding
DOES NOT REQUIRE ENERGY
BUT the ff does:
1. Maintenance of Intracell ions
2. Maintenance of phospholipid
3. Maintenance of skeletal CON plasticity
4. Functionality of Ferrous Hgb
5. Glutathione synthesis

I. EMBDEN-MEYERHOFF PATHWAY
• MAJOR energy generating pathway (90%)
• ANAEROBIC GLYCOLYTIC PATHWAY
• Metabolism of glucose to pyruvate to
generate ATP.

II. HEXOSE MONOPHOSPHATE SHUNT
• PENTOSE PHOSPATE SHUNT
• 10% of glycolysis
• G6PD provides NADPH generation
• NADPH will reduce the oxidized Glutathione
(GSSG) to Glutathione (GSH)
• GSH is essential for reduction of oxidants that
denatures Hgb (Heinz Bodies).

III. METHEMOGLOBIN REDUCTASE
PATHWAY
• Maintains Hgb in Ferrous State (reduced)
• Oxidants such as Oxygen and H2O2, oxidizes
heme Iron to Ferric state
• NADPH reduces Fe3 (minor)
• Cytochrome b5 reductase is responsible for
65% of methemoglobin reduction.

IV. RAPOPORT-LUEBERING PATHWAY
• Generation of 2,3-BPG or 2,3-DPG
• Competes with Oxygen to Hgb bindingand
thus permits oxygen delivery to the tissue
• Increased 2,3-DPG decreased Oxygen affinity
to Hgb thus shifts the dissociation curve to the
right (Band cells, hyposegmented PMNs),

Let’s recall…
• Tissue source of EPO
• Maturation of BFU-E to CFU-E takes how long..
• Last erythropoeitic cell capable of mitosis..
• Start of Hgb synthesis occurs at what stage?
• Stain used to visualize RNA remnants in RBC
cytoplasm
• RBC membrane Protein that maintains cell shape
• RBC membrane Lipid that provides elasticity
• RBC energy metabolism pathway that generates
NADPH and reduced Glutathione
• Oxidized form of hgb is called
HEMOGLOBIN SYNTHESIS and
METABOLISM
• HEME = ring of
Hydrogen, Carbon
and Nitrogen
(Protoporphyrin
IX) + divalent
Ferrous Iron
(ferroprotoporphy
rin).

• GLOBIN = 141 to 146 chain of amino acids.
– Chromosome 16  and  chains
– Chromosome 11 other chains
 chain – 141 AA  chain – 146 AA
 chain – 146 AA  chain – 141 AA
a chain – 146 (136:ala)  chain - unknown
g chain – 146 (136:gly)
65% Hgb Synthesis - immature nRBCs

35% Hgb Synthesis - reticulocytes
Hemoglobin Protein Structure
• Primary – amino acid sequence of the polypeptide chains
• Secondary – chain arrangement in helices and non-helices
• Tertiary – arrangement of helices into pretzel-knot
configuration
• Quaternary - tetrameric molecule, describes the complete
Hgb molecule.
4 molecules of Heme
4 polypeptide chains (2, 2) [one for each]
4 molecules of Oxygen (oxyHgb) [one for each] Resting State
or
4 molecules of 2,3-DPG (deoxyHgb) [one fore each] Tensed
State

HEMOGLOBIN SYNTHESIS
• Heme synthesis start in the mitochondria,
continues in the cytoplasm and returns back
to the mitochondria to end the process.
• Globin synthesis is under genetic control.
– Chromosome 11: , ,  and  globin genes
– Chromosome 16:  and  globin genes
• Heme is then released from the mitochondria
to bond with the globin chain that is
synthesized from the ribosomes.
REMEMBER
Substrates Enzymes
“Glysa and Suzy A, “FOODS DDS”
pour here ur cup of
pronto for him”
Glycine and Succinyl CoA
Synthase (ALA)
Aminolevulinic Acid
Dehydratase (ALA)
Porphobilinogen
Deaminase (PBG)
Hydroxymethylbilane
Uroporphyrinogen Synthase (Uroporphyrinogen)
Coproporphyrinogen Decarboxylase (Uroporphyrinogen)
Protoporphyrinogen Oxidase (Coproporphyrinogen
Protoporphyrin IX Oxidase (Protoporphyrinogen)
Heme Ferrochelatase (Protoporphyrin)

HEMOGLOBIN
• Primary protein constituent of red blood cells
• Oxygen carrying capacity, delivers O2 from
lungs to tissues
– At 20mmHg, only 70% dissociates from Hb
• At full 100% saturation:
– 1g of Hb = 1.34mL of Oxygen
– 1 RBC = 600g of Hb = 800mL of Oxygen

HEMOGLOBIN METABOLISM
EXTRAVASCULAR RBC Catabolism
• Occurs after the mean life span 120 days

• Through the action of Macrophages or RES,
Liver and Spleen.

Extravascular RBC Catabolism
haemoglobin
haem globin
iron protoporphyrin
Amino acids
CO Bilirubin
transferrin Expired air (free)
Liver
conjugation
erythroblast
Bilirubin glucuronides
Urobilin(ogen) Stercobilin(ogen)
Urine
UNIVERSITY faeces
HEMOGLOBIN METABOLISM
INTRAVASCULAR RBC Catabolism
• Accounts for <10% of normal RBC destruction
• Predominant in intravascular hemolytic
anemia

Intravascular RBC Catabolism
Haemoglobin
Alpha and beta dimer

+ Haptoglobin
Liver
Unbound alpha Conversion to methemoglobin
and beta dimers
Normal heme
Kidney
metabolism Hemopexin
heme
Conversion to Hemosiderin
LRP/CD91 to tissue
Hemoglobinuria/Hemosiderinuria macrophages
UNIVERSITY SCIENCE - QUEZON CITY CAMPUS Metemalbumin
TYPES OF HEMOGLOBIN
• Embryonic Hgb – present in the unborn
• Fetal Hgb – predominant in the Fetus and
Newborns (60-95%)
• Hemoglobin A – predominant in adult (95-
97%)
– Hgb A1c – glycoslated Hgb – 3-6% normal
<6% diabetic
– Hgb A2 – Hgb A variant – 2-3%

HEMOGLOBIN VARIANTS
• Carboxyhemoglobin – Hgb + CO
– Cherry red color of RBC
– CO has 210x-240x greater affitiny to Hgb than
Oxygen.
– <3% normal, >3% to 20% -Smokers
– 15% to 20% Acute poisoning (headache, dizziness,
muscle weakness)
– 40%-50% Unconciousness to death
– Hyperbaric therapy (100% Pure Oxygen Therapy)
HEMOGLOBIN VARIANTS
• Sulfhemoglobin – Hgb + Sulfur
– Greenish derivative
– Irreversible
– Results in Hgb precipitation (Heinz Bodies)
– Cannot transport Oxygen
– Can bind Carboxyhgb to form
CARBOXYSULFHEMOGLOBIN
– <1%, <1% conc is asymptomatic (cyanosis)
– Caused by drugs (Phenacetin, Sulfonamides) and
bacteremia (Clostridium welchii)
HEMOGLOBIN VARIANTS
• Methemoglobin – Hgb + Fe3
– Ferrihemoglobin
– Brownish to bluish
– Cannot bind oxygen
– Shift to the left
– <1% normall, >30% Hypoxia and cyanosis
– Increased in chronic exposure to oxidants
(nitrites)
– Genetic dso such as HEMOGLOBIN M DISEASE also
increases MetHgb
REMEMBER! HEMOGLOBIN MIGRATION:
CELLULOSE ACETATE @ pH 8.6; C(crawl),
S(slow), F(fast), A(accelerated)
- (origin) +
A2 S F A
C D
E G
O Arab Lepore
C Harlem
HEMOGLOBIN MIGRATION: CITRATE
AGAR @ pH 6.2
- origin +
F A S C

IRON METABOLISM
• Most abundant trace element in the body
• Exists as either Ferrous or Ferric Ion
– 65% Hemoglobin
– 5% Myoglobin
– <5 Other protein compartments:
• Transferrin, Lactoferrin, Peroxidase, Cytochrome
oxidase, Riboflavin Enzymes
– 25% Storage form
• Ferritin: Major stored form, Water Soluble
• Hemosiderin: degraded ferritin, Water Insolule
PHYSIOLOGIC REQUIREMENT FOR
IRON
• Daily, 20-25mg of Ferrous iron is needed in
Erythropoeisis. (from hemoglobin catabolism)
• Adult Male, only 1mg/day is needed to be
taken in
• Adult Female, 2-2.5mg/day is needed to be
taken in (due to mensturation)
• Increased iron requirements for:
– Pregnant Individuals
– Adolescent and Children
IRON ABSORPTION
• Occurs mainly in the GIT (duodenum and
jejunum)
• Absorption depends on the ff:
– Form of iron available for absorption.
– State of iron stores
– GI Acidity
– Bone Marrow Activity

IRON TRANSPORT/STORAGE
• TRANSFERRIN – mjor transport protein of iron
• APOFERRITIN – binds Ferrous ions to reduce it

into Ferric ions for storage

GENERAL RULE ABOUT IRON REGULATION
• As serum iron increases, storage iron (ferritin)
increases and transferrin and transferrin
receptors decrease.
• As serum iron decreases, storage iron (ferritin)
decreases, and transferrin and transferrin
receptors increase
• The way to increase absorption of iron from
the GI tract is to increase the production of
transferrin (by liver) and transferrin receptors
which bind iron saturated luminal transferrin
OUR LADY OF FATIMA
UNIVERSITY
Oxygen Dissociation Curve of Hgb
• Main function of Hgb is to transport Oxygen
from the lungs to the tissue
• 1 gram of Hgb carries 1.34 mL of Oxygen mol
• BOHR EFFECT – shift in the curve due to
change in blood pH
• HALDANE EFFECT – relationship of Oxygen
affinity to Hgb to CO2 level in the tissue

Hgb-Oxygen
Dissociation Curve
• pO2 (Oxygen molecule
saturation on 50% of Hgb
molecule) – 27 mm Hgb
• 50% Saturation at less
than 27mm Hgb – Left
shift.
• 50% saturation at more
than 27mm Hgb – Right
shift
• Change in the curve DUE
to change in pH – BOHR
EFFECT
Oxygen Dissociation Curve
• Left Shift: “Wont LET go (of oxygen)”
- increased Oxygen affinity to Hgb
- increased pH
- decreased 2,3-DPG
- decreased body temperature
– Fetal Hemoglobin, Hypothermia, alkalosis,
Methemoglobinemia and
carboxyhemoglobinemia, multiple transfusion of
stored blood (depleted 2,3-DPG)
Oxygen Dissociation Curve
• Right Shift: “Wont Hold Tight”
- increased 2,3-DPG
- increased Body temperature
- decreased oxygen affinity to Hgb
- decreased pH
– High fever, acidosis, hypoxia

VENIPUCTURE
• Phlebotomy: “Phleb” – vein, “tomus” – to cut
• “Primum non nocere”
• Methods of venipuncture: Syringe, evacuated
system, butterfly
• COMPLICATIONS:
– Immediate Complications – bruising, hematoma etc
– Late Local Complications – thrombosis,
thrombophlebitis
– Delayed General Complications – parenteral
infections…

“the higher the gauge, the smaller the bore”
Routinely, 19, 20, 21
gauge needles are
used.
The most commonly

used lengths are 1
and 1.5 inches.

ADDITIVES
– Chemical agent s or solutions that maintain a particular property
of the blood. May be anticoagulant, procoagulant, anti-glycolytic
etc.
ANTICOAGULANTS
– Chemical agents or solutions that inhibit
the transformation of liquid blood into a
solid clot.
– Hematology and coagulation procedures
must be done using whole blood or
plasma
– The choice of particular anticoagulants
depends on the test procedure
– An appropriate concentration of
anticoagulant for the volume of blood
drawn is critical, significant errors may be
the outcome with an incorrect
concentration of anticoagulant.

EDTA
– The disodium, dipotassium or tripotassium salt of
ethylenediaminetetraacetic acid is the most
commonly used anticoagulant in hematology.
– Blood coagulation is inhibited by chelating or
binding calcium ions.
– Optimal concentration is 1.5 mg/dL of blood.
– This anticoagulant does not alter the morphology
of blood cells and does not form artifacts.
– Best for the preparation of blood smears
– It is unlikely to cause platelet clumping and even
aggregation of WBC.
– it does not also affect the hb level.
– Considered to be the best anticoagulant for CBC
and platelet count.

DISADVANTAGES:
– Excessive concentration of EDTA causes cell
shrinkage and result to falsely low hematocrit and
ESR.
– Increased amount of EDTA leads to degenerative
changes in the white cells such as vacoulation of
the cytoplasm, more homogenous and irregularly
shaped nuclei and poorly defined cytoplasmic
borders.
– Elevated concentration of EDTA causes swelling
and breaking up of platelets and results to
increased platelet count.

CITRATE
– Prevents coagulation of blood by binding
calcium in a soluble complex.
– Standard ratio is (9:1) (blood : citrate)
– Best anticoagulants for coagulation
studies.
– Trisodium citrate is the choice today for
many coagulation studies since it
preserves labile factors V & VIII
– Most satisfactory for platelet aggregation
studies.
– It is now used to monitor heparin therapy
– Test performed using citrate is PT, APTT,
EOFT, for correction platelet satellitism
a rare phenomenon observed in blood
smears obtained from blood
anticoagulated with EDTA.

DISADVANTAGE OF CITRATE
– If Hct exceeds .50, amount of

unbound citrate in the
citrate:plasma mixture
causes prolonged clotting
times
MAY COME AS A BLACK TOP

- 4:1 blood-anticoagulant ratio
- for Westergren Mtd of ESR
HEPARIN
– An excellent and naturally occurring
anticoagulants but it is more expensive than the
artificial one.
– Coagulation is inhibited by its interaction with
antithrombin III and subsequent inhibition of
thrombin
– Concentration is 15-30 units/ml. Of blood
– Best anticoagulant to be used when minimum
hemolysis is desired such as EOFT.
– Used also in blood transfusion and also in
exchange transfusion.

Why Doctors Order
Osmotic Fragility
EOFT- Your red Tests?
blood cells will -Doctors may order
be tested to see osmotic fragility tests
how easily they for infants with a
break. family history of
thalassemia or
hereditary
spherocytosis.

DISADVANTAGES OF
HEPARIN
• It causes aggregation of
white cells and platelets.
• A heparinized specimen
causes a blue background in
a Wright – Stained smear.

DOUBLE OXALATE MIXTURE (Balanced
Oxalate, Ammonium – potassium
Oxalate, Wintrobe fluid, Heller – Paul
fluid.)
– Suitable for hemoglobin, RBC count, WBC
count hematocrit and blood smears.
– It can also be a substitute for citrate in
case of coagulation studies.
– The proper ratio is 3:2.

PREPARATION:
• Ammonium oxalate….1.2 grams

• Potassium oxalate……0.8 grams
• Neutral formaldehyde1.0 ml
• Dist. H20………………..100 ml

ADVANTAGES OF DOUBLE
OXALATE
• Cheap
• Requires no dilution
• Easy to prepare
• Suitable for Hgb, RBC, WBC cts.
• Plasma can be used in chemistry

DISADVANTAGES OF DOUBLE OXALATE
• Decrease of packed cells

• Vacuolation of the cytoplasm
• Lobulation of lympho and mono
• Phagocytosis of oxalate crystals
• Nuclear Degeneration
• Can’t be used in blood transfusion
• Unsuitable for platelet count- clumps

• DEFIBRINATION
– Involves the removal of fibrin
from whole blood specimen.
• ADVANTAGES:
1.0 RBC and WBC morphological
Study
2.0 gives a large volume of
serum
3.0 Used for L.E. preparation

SKIN PUNCTURE
• Order of collection of specimens is as follows:
a. Tube for blood gas analysis
b. Slides, unless made from sample in the EDTA
microcollection tube
c. EDTA microcollection tube
d. Other anticoagmicrocollection tubes with
ulants (i.e., green or gray)
e. Serum microcollection tubes
LAB EVALUATION OF ERYTHROCYTES
I. RBC COUNT
M: 4.5 - 6.0 x 10^12/L (SI Unit)
F: 4.0 – 5.4 x 10^12/L (SI Unit)
Diluting Fluid: Gower’s Solution
Hayem’s Solution
NSS
Dilution Factor: 1:200

II. HEMOGLOBIN DETERMINATION
M: 13.5 – 17.5 g/dL
F: 12.0 – 16.0 g/dL
Mtds of Determination:
- Automated
- Acid-Hematin Method (Manual)
- Reagents: 0.1N HCl
- Cyanmethemoglobin Method (semi-automated)
- Drabkin’s Rgnt (Potassium Cyanide, K Ferricyanide,
Na Bicarbonate)
OUR -
LADYRead
OF FATIMA at 100% atCOLLEGE
540OFnmMEDICAL LABORATORY
III. HEMATOCRIT DETERMINATION
M: 40 – 54%
F: 35 – 49%
Mtds of Determination:
- Microhematocrit
- Macrohemaocrit
Hct = (Height of Packed Cell/Amt of Blood) x 100

Microhematocrit Method
• Capillary tube must be 7cm long, 1mm
internal bore (5cm to be filled r ¾ of tube)
• Centrifuge at 10k to 15kg for 5 mins
(>50% Hct + 5 mins)
• The EMPTY END is the one filled with SEALING
CLAY
• 1-3% of Hct is from the TRAPPED WBC,
platelets and plasma in the packed red cell
IV. RED BLOOD CELL INDICES
A. Mean Cell Volume – ave. RBC volume
MCV (fL) = Hct x 10 / RBC ct.
NV: 80 – 100fL
B. Mean Cell Hemoglobin – ave. Hgb weight/RBC
MCH (pg) = Hgb x 10 / RBC ct.
NV: 28 – 32pg
C. Mean Cell Hemoglobin Concentration – ave.
concentration of Hgb in an RBC.
MCHC = Hct x 100 /Hgb
NV: 32 – 36g/dL
V. RETICULOCYTE COUNT
• 0.5 – 1.5 % (25 – 75 x 10^3/L, SI Unit)
• Indicator of bone marrow function
• Uses supravital stain, New Methylene Blue
A. Retics Ct. = (Counted cells/1000 RBCs) x 100
B. Corrected Retic Ct. = Retic % x (Hct/45)

C. MILLER DISK
• Special eyepiece for reticulocyte counting.
• Retics % = (retics in large square x 100) /
no. RBC in small square x 9)
D. Absolute Retic ct = (Retic % x RBC ct) / 100

E. Reticulocyte Production Index
- Measures erythropoeitic activity when there is
a stress/shift reticulocyte.
Maturation Correction
Hematocrit
Factor
40 – 45 1
35 - 39 1.5
25 – 34 2
15 - 24 2.5
<15 3
RPI = corrected retic ct/maturation factor

VII. ERYTHROCYTE SEDIMENATION RATE
• Used to detect and monitor non-specific
inflammation.
Mtds. of Determination:
• Westergren Method – EDTA
– 30cm long, 2.55mm internal bore, 0-200 graduation
• Modified Westergren Mtd - CITRATE
NEVER HEPARIN – alters zeta potential
NV:
M: 0-10mm/hr
F: 0-20mm/hr
ERYTHROCYTE DISORDERS and
other RBC related abnormalities

ABNORMAL RBC MORPHOLOGY
• ANISOCYTOSIS – variation RBC size
*RDW – histogram that indicates variation in RBC
volume. (Gaussian Curve)
> Left Shift (Microcytosis)
> Right Shift (Macrocytes)
> Wider than normal curve – variation in cell size
(anisocytosis)
– Normocytes – 6-8um RBCs
– Macrocytes - >8um RBCs
– Microcytes - <6um RBCs
• POIKILOCYTOSIS – variation in RBC shape
a. Acanthocytes – RBCs with thorny, spike-like

projections on the surface
b. Blister Cells – vacuolated RBCs DEGMACYTES
c. Burr Cells (Echinocytes) – crenated RBC SEA URICHIN
d. Dacryocytes – Teardrop shaped
 Myelofibrosis w/ Myeloid Metaplasia (MMM)
a. Elliptocytes (ovalocytes) – elongated,
cigar/sausage/egg shaped RBCs
b. Keratocytes – bite cells (pacman-like), helmet, horn
cells
c. Knizocytes – pinched cells, indicative of erratic pitting.

h. Leptocytes (Codocytes) – target cells
 THALASSEMIAS
i. Megalocytes – oval macrocytes
j. Pyknocytes – contracted RBCs
k. Schizocytes – RBC fragments (fragmentocytes)
l. Schistocytes – RBC fragments
m. Sickle Cells (Drepanocytes) – boat shaped RBC
 MENISCOCYTES
n. Spherocytes – RBC w/o or DECREASED central
pallor
o. Stomatocytes – RBCs with lip-like central pallor
Abnormal RBC distribution
• Agglutination – clumping of RBCs
• Rouleaux Formation – stacking of RBCs in the

PBS

RED BLOOD CELL INCLUSIONS
a. Basophilic Stipplings – evenly distributed
basophiles that are remnants of
ribosomes/RNA. Dark granules in the RBC under
routine stains.
b. Cabot Rings – ring/loop structures inside the
RBC. Remnants of mitotic spindle.
c. Hemoglobin C crystals – rodlike opaque
structures. Abnormal hemoglobin
d. Hemoglobin H crystals – blue globules in RBC
when stained with BCB. Precipitated beta chains
of Hgb A
e. Hemoglobin SC crystals – fingerlike/quartzlike
Hgb proturuding from the RBC
f. Heinz Bodies – precipitated unstable
hemoglobin. G6PD deficiency.
g. Howel-Jolly Bodies – DNA fragments
h. Papenheimer Bodies – iron deposits

ANEMIA
• Decrease in oxygen carrying capacity of the
blood. Whether because of low RBC ct, low
hemoglobin or low hematocrit.
RELATIVE ANEMIA – anemia due to increased

plasma volume
ABSOLUTE ANEMIA – anemia due to actual
decline in RBC parameters
CLASSIFICATION OF ANEMIAS
I. According to Mechanism
A.Impaired Erythropoeisis
a. Ineffective Erythropoeisis – destruction of
progenitor cells before maturation
b. Insufficient Erythropoeisis – decreased level
of progenitor cells in the BM
B. Acute Blood Loss and Hemolysis
a. Profuse Bleeding
b. Intrinsic Defects
c. Extrinsic Defects
CLASSIFICATION OF ANEMIAS
II. According to Blood Picture (Wintrobe)
Clinically significant:
A. Microcytic , Hypochromic
B. Normocytic, Normochromic
C. Macrocytic, Normochromic

MICROCYTIC, HYPOCHROMIC ANEMIA
• Sideroblastic Anemia
• Iron Deficiency Anemia
• Thalassemia
• Anemia of Chronic Inflammation

MACROCYTIC, NORMOCHROMIC
ANEMIA
• Megaloblastic Anemia
A. Vitamin B12 Deficiency
B. Folate Deficiency
• Non-Megaloblastic Anemia
A. Aplastic Anemia
B. Chronic Liver Disease
C. Alcoholism
D. Erythroleukemia
NORMOCHROMIC, NORMOCYTIC
ANEMIA
• Hemolytic Anemias
– (Intrinsic and Extrinsic Defects)
• Aplastic Anemia
• Renal Disease
• Myelophthisic Anemia
• Parvovirus (B19) Infection
• Lead Poisoning
• Anemia of Chronic Inflammation

LABORATORY EVALUATION FOR
ANEMIA
A. CBC with RBC Indices
a. RBC ct., Hemoglobin Conc, Hematocrit
b. MCV, MCH, MCHC, RDW
B. Reticulocyte Count
C. Peripheral Blood Smear
D. BM examination
E. Other Lab Tests: (UA-Hgburia, FOBT- bleeding)..
IRON STUDIES

IRON STUDIES
• SERUM IRON CONCENTRATION
– Measure of Ferrous iron bound to serum
TRANSFERRIN.
– Reflects tissue iron supply
– NV: 10-30 umol/L
• TOTAL IRON BINDING CAPACITY
– Measure of iron bound to a transferrin molecule when
fully saturated.
– NV: 47-70umol/L

• PLASMA FERRITIN CONCENTRATION
– Plasma concentration is in equilibrium in tissue
concentrations.
– Indicator of iron stores in the body
– NV: 12-300mcg/L
• PERCENT TRANSFERRIN SATURATION
– Percentage of sites available for carrying Fe.
– NV: M- 16 to 60% F: 16 to 50%
TS = (Serum Iron Conc/TIBC) x 100

MICROCYTIC ANEMIAS

IRON DEFICIENCY ANEMIA
• Also known as SIDEROPENIC ANEMIA
• Most common anemia
• Most prevalent in early life, pregnancy,
menstruation, poor diet, malabsorption,
chronic blood loss.
• SnS: Classic Anemia features plus
KOILONICHIA (spooning of nails), PICA

STAGES OF IDA
• STAGE 1 (Iron Depletion)
– DECREASED: Ferritin
– NORMAL: Hgb, Serum Fe, TIBC
• STAGE 2 (Transport Iron Depletion)
– DECREASED: Serum Iron, Ferritin
– INCREASED: TIBC
– NORMAL: Hgb
• STAGE 3 (Functional Iron Depletion)
>>Full Blown IDA
– DECREASED: Hgb, Serum Fe, Ferritin
– OFINCREASED:
OUR LADY FATIMA TIBC
SIDEROBLASTIC ANEMIA
• Caused by blockages in the protoporphyrin
heme synthetase resulting to defective Hgb
synthesis with iron overload
RINGED SIDEROBLAST – encircling of excess iron
in the mitochondrial region of immature
erythrocytes in the BM.
SIDEROCYTES – RBCs with iron deposits. (perl’s
Prussian Blue)

• CONGENITAL
– X-linked autosomal (mostly males)
– Treated with PYRIDOXINE
• ACQUIRED
PRIMARY – irriversible and idiopathic
- concurrent
- dimorphic RBCs
- myelodysplastic syndromes: RARS, CMML etc
SECONDARY – reversible
- Drug and/or Toxin induced
LABORATORY EVALUATION FOR
• Microcytic, hypochromic
• INCREASED: Serum Fe, Ferritin, RDW
** ANISOCYTOSIS (dimorphism)
• DECREASED: TIBC

PORPHYRIA
• Group of inherited disorders characterized as
a blockage in the protoporphyrin pathway of
heme synthesis
• SnS: Photosensitivity
AB pain
CNS disorders
Porphyuria
Sideroblastosis
IRON OVERLOAD
• HEMOSIDEROSIS - accumulation of iron to
supranormal levels with deposition of iron
primarily in the macrophages of the spleen, liver,
bone marrow, and other tissues.
• Causes of hemosiderosis are
–Hemolytic anemias
–Multiple blood transfusions
–Disorders of erythropoiesis
–Inappropriate intestinal absorption of
iron
• There is usually no organ damage
IRON OVERLOAD
• HEMACHROMATOSIS - occurs when iron
accumulation has progressed to involve
widespread deposition of iron in parenchymal
tissue with resulting organ injury.
• Impaired liver function enzymes test (elevated
AST and ALT)
• Increased: Serum Fe, Ferritin
• Normal: TIBC
• REMEDY: Phlebotomy
Iron Chelating Drugs (Desferoxamine)

ANEMIA OF CHRONIC DISEASE
• Inability to use available iron for Hg
production
• Increased levels of Hepcidin reverse acute
phase reactant: increased level of this
hormone decreases GUT iron absorbtion.
• Lactoferrin and Ferritin also binds iron during
inflammation.
• Second most common anemia
ANEMIA OF CHRONIC DISEASE
INCREASED: ESR, Ferritin
DECREASED: TIBC, Serum Iron
From normocytic, normochromic; eventually,
blood picture will be microcytic, hypochromic.

MACROCYTIC ANEMIA

MEGALOBLASTIC ANEMIA
• Impaired DNA Synthesis
• Nucleus matures slower than the cytoplasm
(asynchroism)
TYPES OF MEGALOBLASTIC ANEMIA
• Vitamin B12 Deficiency
• Folate Deficiency

Cobalamin metabiolism
• In the alimentary tract
– R binders: transports Vit B12 into duodenum
• Sources: Saliva, food, peptic sectretions
– Intrinsic Factor: binds Vit B12 for absorption in the
ileum
• Source: parietal cells
– Amnionless & Cubilin: receptors for Vit B12-IF
complex
• Sources: Ileum
• In the plasma
– Transport form is METHYLCOBALAMIN
– Transport CHON: Transcobalamin II
Haptocorrins (cobalophilin)
VITAMIN B12 DEFICIENCY
• Cobalamin deficiency due to impaired
absorption in the intestine
• INTRINSIC FACTOR, secreted by parietal cells
bind Vit B12 for absorption
• Can lead to PERNICIOUS ANEMIA
• Takes 3 to 6 years to develop because of high
body stores

VITAMIN B12 EFICIENCY
• CAUSES:
Malabsorption syndrome
Deficient IF or Pareital Cells
D. latum infection
Atrophy of gastric parietal cells
H. pylori infection
Total Gastrectomy
Pericious Anemia
Vegetarianism
Increased need (pregnancy)
VIT. B 12 DEFICIENCY
• Lab Evaluation:
– Macro, Normo
– Hypersegmentation of Neutrophils
– Howel-Jolly Bodies, Basophillic Stipplings
– Increased BILIRUBIN, LD and iron levels

SCHILLING’S TEST
• Test for cobalamin absorption
• impaired absorption could indicate IF
deficiency
• Radioactive cobalamin is given orally
• 2hrs after, a non-labelled “flushing” dose is
given
• 24-hr urine is examined for Radioactive
cobalamin
FOLATE DEFICIENCY
• More common
• No CNS involvement
• Predisposes in poor diet, pregnancy,
chemotheraphy, anti-folic acid agents
(Methotrexate)

NON MEGALOBLASTIC MACROCYTIC
ANEMIA
• Alcoholism
• Liver Disease
• LAB EVALUATION:
– Increased Bilirubin and GGT
– Macro, normo

NORMOCYTIC, NORMOCHROMIC
ANEMIAS

BLOOD LOSS ANEMIAS: ACUTE
• anemia due to excessive extravasation of
blood associated with traumatic injury or after
a surgery.
• SnS: Hypovolemia, rapid puls, low BP, pallor
• Lab Eval: Normo, normo RBC
– Normal Retic, Hgb and Hct.
– Increase in Plt and WBC w/ left shift after few
hours
– Will develop to Macrocytosis later on

BLOOD LOSS ANEMIA: CHRONIC
• Gradual, long-term loss of blood; GI bleeding,
menstruation
• Lab Eval: Initially, normo, normo but will
develop into micro, hypo later on

APLASTIC ANEMIA
• Acquired/congenital hypoproliferative
disorder or reduced blood cell production.
• PANCYTOPENIA due to bone marrow failure.
• Cell chromasia and size is not affected, only
the rate of production.
• Congenital anemia has many possible cause
• Acquisition is mostly from chemical exposure
– Benzene, TNT, Insecticides…
ACQUIRED APLASTIC ANEMIA
• 80 to 85% of aplastic anemia
• Caused by exposure to toxic substances, viral
infection and drug intake.
• Can be fatal if left untreated
• Lab Eval: Normo, Normo
Pancytopenia (even retics)
Iron overload (multiple transfusion)
Toxic granulation in neutrophils
Hypocellular BM
INHERITED APLASTIC ANEMIA
• Presents disorder at early age and usually,
with physical deformity
• Fanconi Anemia
• Dyskeratosis Congenita
• Schwachmann-Diamond SYndrome

FANCONI ANEMIA
• Autosomal recessive trait

• Dwarfism, renal dse, mental retardation
• Café au lait spots (skin pigmentations)
• Strongly associated with ALL.

DYSKERATOSIS CONGENITA
• Rare congenital disorder, 600 reported cases
worldwide
• May be Autosomal dominant, x-linked and
autosomal recessive
• Abnormal skin pigmentation, oral leucoplakia
• May manifest multisystem abnormality –
Pulmonary fibrosis, liver dses, dwarfism,
microcephaly
• 40% risk of developing cancer with AML at age of
50.
– With macrocytosis and increased Hgb F
SCHWACHMANN-DIAMOND
SYNDROME
• Multisystem disorder – pancreatic
insufficiency, skeletal abnormalities and
pancytopenia

OTHER BONE MARROW FAILURE
SYNDROMES

PURE RED CELL APLASIA
• Severe decrease in Erythocyte precursor but
normal Bone marrow.
• Acquired PRCA
- PRIMARY – idiopathic or autoimmune
- SECONDRY – thymoma, hematologic
malignancy, solid tumor, infection, chronic
hemolytic anemia and drug exposure.

• CONGENITAL PRCA (Diamond-Blackfan
Anemia)
- true red cell aplasia
- normal BM, hypoplastic erythroid
precursor
- MACROCYTOSIS
- reticulocytopenia

HEMOLYTIC ANEMIA
• May be acquired or hereditary abnormality in
the basic membrane structure, erythocytic
enzyme or hemoglobin sturcture.
• All are normocytic, normochromic anemia,
with reticulocytosis

CATEGORIES OF HEMOLYTIC ANEMIA
• INTRINSIC DEFECT (Hereditary)
– Membrane Defect
– Enzyme Defect
– Hemoglobin Defect (Hemoglobinopathies)
• EXTRINSIC DEFECT
– Immune
– Non-Immune

INTRINSIC DEFECT:
HEREDITARY SPHEROCYTOSIS
• MOST COMMON MEMBRANE DEFECT
• Autosomal dominant
• Splenomegaly and spherocytes in the PBS
• Increased permeability to Sodium
• Decreased surface area to volume ratio
• Increased Osmotic fragility and Bilirubin,
MCHC
• OFT is a sensitive test

INTRINSIC DEFECT:
HEREDITARY ELLIPTOCYTOSIS
• Autosomal dominant; asymptomatic
• >25% Ovalocytes
• Caused by polarized cholesterol at the ends of
the cell instead around the pallor area
• HEREDITARY PYROPOIKILOCYTOSIS – subtype
of HE seen in black ppl.

INTRINSIC DEFECT:
HEREDITARY STOMATOCYTOSIS
• Autosomal dominant: >50% Stomatocytes
• Abnormal Sodium and Potassium
permeability.
• Causes RBC Swelling

INTRINSIC DEFECT:
HEREDITARY XEROCYTOSIS
• Permeability disorder: thermal instability of
spectrin in vitro
• Crenated cell due to increased Na intake of
the cell
• Increased MCHC

INTRINSIC DEFECT:
HEREDITARY ACANTHOCYTOSIS
• Associated with steatorrhea, neurological and
retinal abnormalities
• 50 to 100% acanthocytes
• Increased Cell Lecithin and Cholesterol ratio
• Abetalipoproteinemia
• Normal blood indices

INTRINSIC DEFECT: G6PD DEFICIENCY
• Sex-linked enzyme defect; most common
defect in Hexose Monophosphate shunt
• Increased methemoglobin and heinz bodies
due to impaired NADPH generation

INTRINSIC DEFECT:
PYRUVATE KINASE DEFICIENCY
• Autosomal recessive; most common defect in
Embden-Meyerhoff Pathway
• Lacks ATP causes impaired cell contorol in
cation pumps.
• Decreased cell deformability = decreased life
span
• Severe hemolytic anemia
• Increased reticulocytes and Echinocytes
IMMUNE EXTRINSIC DEFECT:
WARM AUTOIMMUNE H.A.
• RBCs are coated with IgG/Complement.
• In attempt to remove the coating Ab,
macrophages damages the membrane forming
SPHEROCYTES
• 60% of causes are idopathic: may be secondary to
dse (Lymphoma, CLL)
• Lab: Spherocytes, MCHC >37g/dL
• Increased OFT, Bilirubin and Retics ct.
• (+) DAT

CAIHA (Cold AIHA)
• RBCs are coated with IgM and Complement at
temp below 37C
• Antibodies are usually ati-I or anti-I
• May be idiopathic; secondary to Mycoplasma
pneumoniae infection, lymphoma and IM.
• Lab: RBC clumping, same lab result with
WAIHA

PAROXYSMAL COLD HEMOGLOBINURIA
• Least common type
• IgG biphasic Donath-Landsteiner Ab with P
specificity fixes complement to RBC in cold
temp and lyses at warmer temp
• Same lab result with WAIHA, (+) Donath
Landsteiner Test

HTR
• recepient antibodies reacting to donor cells
• May trigger DIC due to RBC lysis release of
tissue factor
• (+) DAT and Increased Hgb decreased
Haptoglobin

HDN
• Due to Rh Incompatibility (may also be due to
ABO incompatibility)

NON-IMMUNE EXTRINSIC HEMOLYTIC
ANEMIA
• Normo, normo. Anemia is cause by trauma of
RBC
• Intravascular hemolysis: schistocytes and
thrombocytopenia

MICROANGIOPATHIC HEMOLYTIC
ANEMIA
• THROBOTIC THROMBOCYTOPENIC PURPURA
– Occurs in adults
– Deficient ADAMTS13 that breaks down vWF
multimers. Unbroken multimers damage RBC and
causes CNS impairment.
• HEMOLYTIC UREMIC SYNDROME
– Occurs in children following GI infection (E. coli)
– Clots form causin renal damage
• DISSEMINATED INTRAVASCULAR COAGULOPATHY
- Excessive clotting, fibrin deposits damaging the RBCs.
MARCH HEMOGLOBINURIA
• Transient hemolytic anemia occuring after a
forceful contact of the body with hard
surfaces

Other Causes of Hemolytic Anemia
• P. falciparum, C. perfringens
• Mechanical trauma (prosthetic heart valve-
Waring Blender Syndrome)

HEMOGLOBINOPATHIES
• Qualitative or structural abnormality in globin
chain synthesis. More of amino acid
replacement/alteration
• RBC deformability and electrophoretic mobility
alteration
• HOMOZYGOUS – both globin chains are affected
• HETEROZYGOUS – only ONE globin chain is
affected
• Most common: Hgb S, Hgb C and Hgb E.

SICKLE CELL DISEASE (Hgb SS)
• Valine replacement of Glutamic Acid at the 6th
AA of both beta-globin chain
• Genes are inherited to BOTH PARENTS
(Homozygous)
• Occurs mostly in African-American, African,
Mediterranean and Middle East populations
• Apparent immunity to P. falciparum may
develop
• No Hgb A is produced. 80% Hb S and 20% Hgb F
(compensatory Hgb)
• Deoxygenation of Hgb S produces SICKLING.
Diminished Oxygen carrying capacity.
• CLINICAL FINDINGS:
- Vasoocclusion: sickled RBCs are stuck
(adhere) in the blood vessel.
- Hypersplenism  Autosplenectomy
- Tissue necrosis (hypoxia)

• LAB EVALUATION:
– Normo, normo
– Polychromasia (Reticulocytosis)
– Low M:E
– High Bili, Low Hapto (Hemolysis)
– Sickle Cells, Target Cells. nRBCs
– Pappenheimer Bodies, Howel-Jolly Bodies
– Cellulose Acetate Agar: migration with Hgb D and
G (alk)
– Citrate Agar: Separate migration Hgb S
– (+) Hgb Solubility Test

SICKLE CELL TRAIT (Hgb AS)
• Replacement of Glu with Valine in ONE BETA
chain.
• Genes are inherited from ONE PARENT only
• 60% Hgb A, 40% Hgb S, normal or increased
Hgb A2 and Hgb F
• Heterozygous (most common in US)
• Asymptomatic
• Rare anemia but normo, normo
Hemoglobin CC Disease
• Glutamic Acid at 6th AA position is replaced by
LYSINE on both beta chains.
• African American, African populaiton
• No Hgb A: 90% Hgb C, 2% Hgb A2 and 8% Hgb
F
• Asymptomatic or mild anemia

• LAB EVAL:
– Normo, normo
– Hgb C crystals (rod like.. “washington monument”
– Cellulose Acetate Agar: migrates w/ Hgb A2, E and
O
– Citrate Agar: solo migration
– (-) Hgb Solubitiy Test (RODAK)
Heterozygous Hgb C trait (Hgb AC): 60% Hgb A, 40%

Hgb C, normal Hgb A2 and F

Hgb C-Georgetown/Harlem
• Lysine replacement of glutamic acid at 6th
amino acid position in ONE beta chain
• Aspartic Acid replacement of Asparagine at
73rd amino acid in ONE beta chain (Korle Blu
Mutation)

Hemoglobin SC DIsease
• Double heterozygous hemoglobinopathy.
• One beta chain: 6th AA is Valine instead of Glu
• One beta chain: 6th AA is Lysine instead of Glu
• Less severe than sickle cell anemia
• 50% Hgb S and 50% Hgb C

• LAB EVALUATION:
– (+) Target Cells
– SC Crystals (finger like projections from RBC
membrane)
– (+) Hgb Solubility Test

Hemoglobin E
• Lysine replaces Glutamic Acid on 26th AA beta
chain.
• South East Asian, African, African-American
• HOMOGENOUS – mild anemia, target cells and
microcytes
• HETEROGENOUS – asymptomatic
• Alk Electrophoresis – migration with A2, C, O
• Acid Electropheresis – separates with Hgb C but
co-migrates with A and O
• (-) Solubility Test
Hemoglobin O
• Lysine replaces Glutamic Acid at 121st AA on Beta
chain
• Common in Middle Eastern and Indian People
• Both homozygous and heterozygous are
asymptomatic (except. Mild splemonegaly in
Homo)
• Hgb O migrates with Hgb S and G (Alk elec)
• (-) Solubility test
• When inherited with Hgb S, sever condition.

Hemoglobin D
• Glutamine replacement of Glutamic Acid at
121st amino acid position in beta chain
• Hgb D-Punjab – 3% Newestern Indians
• Hgb D-LA – 2% of African americans

Hemoglobin G-Philadelphia
• Alpha chain hgbpathy – Lysine replacement of
Asparagine at 63rd Amino acid chain
• More common than Hgb D in Africa

Hemoglobin M
• Affects alpha, beta, or gamma chain
• Histidine replacement of Tyrosine
• Increased oxidation of Iron
• Methemoglobinemia

THALASSEMIA
• Quantitative or decrease in rate of globin
chain synthesis
• MICROCYTIC, HYPOCHROMIC w/ Target Cells
• Mediterranean (beta), Asian (alpha) and
African (alpha and beta)
• Th Major – no alpha or beta chain produced
• Th Minor – abnormal amt of Hgb A, A2 or F.
But normal alpha and beta chains
BETA THALASSEMIA
1. MAJOR/HOMOZYGOUS (Cooley Anemia)
• Significant decrease in synthesis or absence of
BOTH BETA chains
• Results in INCREASED ALPHA CHAINS.
(precipitable.. Heinz Bodies)
• 90% Hgb F to compensate
• Destroyed in the BM or removed by the
spleem
• Symptomatic by 6mos of age:
Hepatosplenomegaly, stunted growth,
jaundice, prominnet facial bones, iron
overload, multiple transfusions
LAB RESULTS:
Micro, hypo Increased Serum Fe
target cells Increased Bilirubin
teardrop cells
RBC inclusions

2. MINOR THALASSEMIA
• Decreased synthesis rate of one beta chain;
other beta chain is normal
LAB EVAL:
Micro, hypo Basophilic stipplings
normal or slight elevated RBC ct.
Target cells
Slightly decreased Hgb A
Slightly increased Hgb A2

ALPHA THALASSEMIA
1. MAJOR (Hydrops Fetalis)
• ALL FOUR ALPHA GENES are DELETED.
• No normal Hgb
• 80% Bart’s Hgb (4 gammas)
• Incapale of carrying oxygen
• DEATH in utero or shortly after birth

2. Hgb H disease
3 ALPHA GENES are deleted. Decreased
Alpha chains  excessive beta chains
Hemoglobin H (4Beta) is UNSTABLE
LAB EVAL:
Heinz Bodies
Micro, Hypo
30% Hgb H, 70% Hgb A

3. Minor/Trait
2 Alpha genes are deleted
Usually asymptomatic
6% Hgb Bart in NB
Micro, hypo
Oftern assoc with elevated RBC ct. and
target cells

4. Silent Carrier
One Alpha gene is deleted.
Asymptomatic and often not diagnosed
Borderline line MCV


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HEMATOLOGY I

James Patrick C. Picar, RMT

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

• PLASMA – liquid portion of the

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

- This theory is more widely accepted than the

Stochastic Model – hematopoeisis is a random

Instructive Model – the microenvironment of

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

• After birth, BONE MARROW becomes the

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

Yolk sac phase is active until 8th-12th week of

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

- Considered as one of the largest organ in

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

65% Hgb Synthesis - immature nRBCs

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

• Occurs after the mean life span 120 days

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

Alpha and beta dimer

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

• APOFERRITIN – binds Ferrous ions to reduce it

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

The most commonly

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY

OUR LADY OF FATIMA COLLEGE OF MEDICAL LABORATORY