Hematopoiesis
Multipotential stem cells - can
- Process of blood cell production,
differentiation & development
(how they mature to a non-functional cell
to functional mature cell)
- Describes the process of the
maturation of blood cells as they
progress from being precursor stem
cells (immature, cannot phagocyte)
in the bone marrow to fully
functioning mature cells found in
the blood
(Circulating cells dapat ang functional,
capable of transporting o2 and co2
immunity)
- A regulated (balance, to maintain
homeostasis) process of blood cell
production that includes cell
renewal, proliferation,
differentiation, and maturation
(When haematopoiesis becomes
unregulated it will develop Hema-
pathological mechanisms)
Hematopoietic System:
1. Blood
2. Bone Marrow (site of production)
3. Liver
4. Spleen
5. Lymph nodes
6. Thymus
7. Endothelial cells
Theories on blood cell formation:
1. Monophyletic or Unitarian theory
- suggests that all blood cells are
derived from a single progenitor cell
2. Polyphyletic or Dualistic theory -
suggests that each of the blood cell
lineages is derived from its own
unique stem cell
Types of human stem cells:
1. Totipotential stem cells - can form all
human cell types but are only present
within the first few hours after fertilization
2. Pluripotential stem cells - can give
rise to all cell types, present after
fertilization
- ex. embryonic stem cell
3.
develop into more than 1 cell type, derived
from pluripotent but are limited to form
specific types of tissues (can give rise to
blood cells only, limited on the system
concerned)
- ex. hematopoietic stem cell (HSC)
Development of blood-forming tissues
Phases of hematopoiesis:
1. Mesoblastic phase (yolk sac phase)
- Comes from the mesoderm,
concerned in erythropoiesis
- Begins: 16 days AOG; ends: 2nd
month AOG (age of gestation) or 2-8
wks of life
(before the second month come ends,
before the mesoblastic declines, the stem
cells travels to the liver (livers develops))
- Origin of HSCs: Yolk sac & Aorta-
Gonad Mesonephros (AGM) region
- Embryonic hemoglobins detected:
Portland, Gower 1, Gower 2
2. Hepatic phase
- Liver now takes over
- Begins: 4-6 weeks AOG; ends: 7th
month AOG
- HSCs also appear in the placenta,
umbilical cord blood, spleen &
persist until birth. (Involved in blood
cell formation as well)
- Lymphoid progenitors derived from
the liver begin to seed in the newly
developed thymus.
- Megakaryocyte production also
begins.
- Hgbs detected: Hgb Fetal, (adult
hemoglobins) Hgb A1, Hgb A2
(pag matanda na di nakakakeep up yung
bone marrow, extra medullary
hemtopoiesis begins)
3. Medullary phase (definitive/adult
phase)
- Begins: 5th month AOG; ends:
lifetime
- Sites of production: skull, sternum,
pelvis, ribs, vertebrae & proximal
ends of femur
Hematopoietic stem cells (HSC):
• Are rare perhaps 1 in 20 million
nucleated cells in the BM
 • Essential for the maintenance of
hematopoiesis thus by definition are
multipotent
• Capable of self-renewal (different
from mitosis)
• Immunophenotype CD34+ , CD38-
(Cluster of differentiation)
Other markers:
Erythroid lineage: CD71
Myeloid lineage: CD33
B lymphoid: CD10
T lymphoid: CD7, CD5
(Surface proteins serves as markers to
identify the cell, it is difficult to
differentiate the stem cells when it comes
to their appearance, flow cytometry
machine to identify cluster differentiation
because they express different proteins)
• Capable of asymmetric & symmetric
division
Symmetric: two same progenitor cells or
it can produce two stem cells
Asymmetric: can produce stem cell or
progenitor cells
** progenitor – cell line
• Cell differentiation is via committed
progenitors
• BM HSCs spend most of the time in
“quiescence” (resting phase)
If the stem cells are active, and it becomes
abnormal, they are susceptible to
mutation (nucleophiles attack or mutagen)
• Pluripotent stem cells to multipotent
stem cells to progenitor cells to
committed progenitor cells to lineage
specific progenitor cells.
(Self-renewal gagawa ng kapareho niya)
❑ Multipotent cells - most immature,
capable of self-renewal &
differentiation
❑ Progenitor cell - like a stem cell but
differentiates into a more specific
type of cell
❑ Intermediate cells - committed
progenitor cells destined to develop
into distinct cell lines
❑ Mature cells - most developed with
specific functions
Hematopoietic growth factors:
- glycoprotein hormones that regulate
the proliferation & differentiation of
hematopoietic progenitor cells &
function of mature blood cells
1. Interleukins - cell signaling molecules
& are part of the cytokine family
(stimulates a colony stimulating factor)
- IL1 stimulates production of GM-
CSF, G-CSF, M-CSF & IL6 from a
variety of cells within the BM
including stromal cells
- IL3 (target HSC), IL4 (target
activated B-cells), IL6 (target
activated B-cells, plasma cells)
act early multipotential cells
- IL5 (eosinophilic CSF)
- IL7 (lymphocytic CSF)
Stromal cells: bone marrow supporting
cells
2. Colony Stimulating Factors (CSF) -
glycoproteins that stimulate production of
white blood cells
- Granulocyte CSF (G-CSF) stimulates
the differentiation of granulocyte
precursors (myeloblast) & also the
activity of mature granulocytes
- Granulocyte-Macrophage CSF (GM-
CSF) stimulates the differentiation &
maturation of granulocytes &
macrophages & also the function of
these cells once mature.
3. Erythropoietin (EPO) - released mostly
from the kidneys & travels in the blood
through the BM where it acts on erythroid
precursors to stimulate RBC production.
4. Thrombopoietin (TPO) - produced
principally by the liver, stimulates
megakaryopoiesis
5. Tumor Necrosis Factor has similar to
IL1
 Lineage Specific Hematopoiesis:
Erythropoiesis
- Erythropoiesis is triggered by tissue
hypoxia

Erythropoietin
• glycoprotein hormone produced by
the kidneys
• about 46,000 D in molecular weight
• EPO gene is located on chromosome
7
• normal serum levels is at 20
mU/mL but can increase of up to
20,000 mU/mL in response to
anemia
• targets & stimulates the CFU-E &
BFU-E promoting the proliferation &
differentiation
• has anti-apoptotic activity

NORMOBLAS RUBRIB ERYTHROBL

TIC LASTIC ASTIC
NK – natural killer cell

Pronormoblas Rubribla Proerythrobla

t st st

Lineage Specific Hematopoiesis:

1. Erythropoiesis Basophilic Prorubric Basophilic
2. Leukopoiesis - Granulopoiesis, normoblast yte erythroblast
Monopoiesis & Lymphopoiesis
3. Megakaryopoieis

General morphological changes in blood

Polychromatic Rubricyt Polychromatic
cell maturation:
normoblast e erythroblast
- Decrease in cell size big to small
- Decrease in size of the nucleus
- Condensation of nuclear chromatin
- Cytoplasm changes from blue gray
to pink Orthochromic Metarubr Orthrochromi
normoblast icyte c erythroblast

Polychromatic Reticuloc Polychromatic

erythrocyte/R yte erythrocyte/R
eticulocyte eticulocyte

Erythrocyte Erythroc Erythrocyte

yte
 ❑ 12-17 µm, only slightly smaller than
pronormoblast
❑ the chromatin begins to condense
revealing clumps along the
periphery of the nuclear membrane
❑ 4:1
❑ dark blue cytoplasm
❑ gives rise to 2 daughter cells
❑ detectable Hgb synthesis occurs
❑ BM transit time is slightly more
- The blueness or basophilia is due to than 24 hours
the acidic components of the
cytoplasm such as the cellular
amount of RNA & cytoplasmic
organelles that attract the stain
methylene blue. As theses organelles
disappear the blueness fades.
- The pinkness or eosinophilia is due
to the basic components that attract
the acid stain eosin. The basic
component primarily in the RBC is
the Hgb

1. Rubriblast (Pronormoblast)
❑ 12-19 µm
❑ large round nucleus containing1-2
nucleoli with fine chromatin pattern
❑ 4:1
❑ dark blue cytoplasm (basophilic)
❑ gives rise to 2 daughter cells
❑ begins to accumulate the
components necessary for Hgb
production, ex. iron
❑ BM transit time: slightly more than
24 hours

3. Rubricyte (Polychromatic
normoblast)
❑ 11-15 µm
❑ nucleus is round with reduced size
❑ chromatin is quite condensed or
increasingly clumped
❑ 4:1
❑ gray-blue cytoplasm bec of the
mixing of acidophilic & basophilic
❑ the first stage in which redness
associated with stained Hgb can be
seen & Hgb synthesis is increasing
❑ gives rise to 2 daughter cells
❑ BM transit time is approx. 24 hours

2. Prorubricyte (Basophilic normoblast)


4. Metarubricyte (Orthrochromic
normoblast)
❑ 8-12 µm
❑ nucleus is completely pyknotic
(tightly condensed chromatin
pattern) which will be later extruded
from the cell
❑ 0.5:1
❑ pink-orange cytoplasm indicates
large amounts of Hgb
❑ Hgb production continues on the
ribosomes with mRNA produced
earlier
❑ BM transit time is approx. 48 hours
5. Reticulocyte (Polychromatic
erythrocyte)
❑ 8-8.5 µm
❑ bluish to salmon pink cytoplasm
❑ completes Hgb production from
residual mRNA using the remaining
ribosomes
❑ cytoplasmic protein machinery is
dismantled
❑ reduced receptors for adhesive
molecules that hold developing RBC
in the BM
❑ BM transit time: 2-3 days spent as
reticulocyte in the BM & 1 day in
the peripheral blood
6. Erythrocyte
❑ 6-8 µm (average 7.2 µm)
❑ salmon pink with a central pallor
that occupies 1/3 of the cell
❑ length of time is 120 days
Hemoglobin and Hematocrit
Determination
Hemoglobin Determination
• screening procedure that is helpful
in the diagnosis of many diseases
associated with anemia
• it is used to monitor the effects of
drug & radiation therapy
• it is used as an indicator of the
patient’s prognosis in certain
disease states
• Hgb is measured as oxyhemoglobin
• 1 g Hgb can carry 1.34 mL oxygen &
3.47 mg iron
oxyhemoglobin, deoxyhemoglobin
800mL oxygen sa buong RBC
I. Colorimetric Method
A. Direct visual colorimetry
1. Tallquist method
2. Dare’s method
3. Acid Hematin
a. Sahli-Hellige
b. Sahli-Adam’s
c. Sahli-Haden
d. Hayden Hausser
e. Newcomer’s 4. Alkaline Hematin
f. Osgood-Haskin • uses an alkaline reagent, NaOH, to
4. Alkaline Hematin produce a stable solution of hematin
B. Photoelectric method (machines) • disadvantage: Hgb F is alkali-
1. Oxyhemoglobin method resistant thus may produce a
2. Cyanmethemoglobin method significant source of error
(MHbCN) or Hemiglobin cyanide method
(HiCN) – reference method B. Photoelectric colorimetry
1. Oxyhemoglobin Method
II. Specific gravity a. Photometric Sodium Carbonate method
A. Copper sulfate method • uses 0.007 N NaOH & 0.1% Na2CO3
III. Gasometric method to measure oxyhemoglobin
IV. Chemical method b. Photoelectric Oxyhemoglobin
- Measures the amount of iron • pulse oxygen saturation & pulse
rate can be measured through the
finger
1. Tallquist Method using photoelectric oxyhemoglobin
- filter paper monitor
• procedure- blood is collected on a
type of chromatography/absorbent
paper then the color is compared to the
chart
• source of error is 30%-50%

2. Dare’s Method 2. Cyanmethemoglobin method

• consists of a glass plate & an
eyepiece
• source of error is 30%-50%
3. Acid Hematin
• principle: Hgb in the blood is
converted to acid hematin upon the
addition of 0.1 N HCl which is
brown in color & diluted drop by
drop with distilled water until it
matches the color standard in the
comparator blood
(MHbCN) or Hemiglobin cyanide method
(HiCN)
• best method for Hgb determination
because it measures all forms of
Hgb except sulfhemoglobin.
• uses Drabkin’s reagent to measure
Hgb, it is composed of NaHCO3,
KCN, K3Fe(CN)6
• the ferricyanide oxidizes the iron in
the Hgb from ferrous to ferric state
to form methemoglobin (Hi) or
hemiglobin or ferrihemoglobin then
potassium cyanide further oxidizes
it to produce cyanmethemoglobin or
hemiglobin cyanine (HiCN), a very
stable compound with absorbance
oat 540 nm.

Types of Hemoglobinometer:
a. Sahli-Hellige
b. Sahli-Adam’s
c. Sahli-Haden
d. Hayden Hausser
e. Newcomer’s
f. Osgood-Haskin Precautions:
 • Drabkin’s is sensitive to light & a • uses double oxalate anticoagulant
toxic chemical • requires 1 mL blood
• Highly elevated WBC & platelets • tube is centrifuged for 30 mins at
may cause turbidity & falsely elevate 3,500 rpm
results • L1 (height of PRBC) / L2 (height of
• Lipemia WB) x 100
• Hgb S & Hgb C are resistant to • Time consuming
hemolysis causing turbidity • Glass tube
• Lupus erythematosus prep, check
Alternative to drabkins: Sodium Lauryl buffy coat smear
Sulfate (SLS), this is less toxic

Hemoglobin reference values

Conventional SI (g/L)
(g/dL)
Male 14-18 140-180
Female 12-16 120-160
Newborn 16.5-21.5 165-215
Multiply by 10

Newborn: mabilis pa cell turnover

Female: because of monthly period Simula baba pataas, to prevent bubbles

II. Specific gravity B. Adam’s Method

A. Copper Sulfate method • microhematocrit method
• a drop of blood is added to the • uses 75 mm long capillary tube with
CuSO4, with known specific gravity 1.2 mm bore
1.053, if the drop falls within 15 • specimen can be capillary blood or
seconds it is estimated to have 12.5 whole blood
g/dL Hgb • capillary tube is centrifuged for 5
• used in screening blood donors & mins at 10,000-12,000 rpm
not for patients • 0.05 mL
• prone to inaccuracy
** blue – plain, red – anticoagulant
III. Gasometric method (heparin)
• indirect Hgb method based on the EDTA use blue
principle that 1 g Hgb can carry Excess anticoagulant causes cell
1.34 mL oxygen shrinkage
• Van Slyke method & Haldane &
Smith method
Conventional SI (g/L)
IV. Chemical method (g/dL)
• indirect method based on the Male 40-54 0.40-0.54
principle that 1 g of Hgb can Female 35-49 0.35-0.49
approximately carry 3.47 mg iron Newborn 48-68 0.48-0.68
• Wong & Kennedy method

Hematocrit Determination (Hct) Falsely increased? Falsely decreased?

• ratio of volume occupied by the RBC
to the volume of whole blood in the
sample
• denotes the % of rbc in a known
volume of blood
• fluctuations in Hgb will also be
reflected in Hct
• used to detect anemia, polycythemia
• 2 general methods: Wintrobe &
Adam’s
** 55% plasma, 45% formed elements
Under centrifugation - Falsely increased
Improper sealing - Falsely decreased
Dehydration - Falsely increased
Tissue juice contamination - Falsely
decreased
Hemoconcentration - Falsely increased
Hemolysis - Falsely decreased
Excess anticoagulant - Falsely decreased
Blood loss - Falsely decreased
“Trapped plasma” - Falsely increased

A. Wintrobe Method
• Macrohematocrit method (large mL
of blood)
 Nuclear expulsion or extrusion

RBC Membrane Structure and RBC

Metabolism - Biconcave
- 6-8 micra, 7.2 micra is the average
Rubriblastic terminology - 1/3 central pallor
- Limitation: don’t have an idea on - Similar with nucleus of lymphocyte
what cytoplasm looks like (size)
Erythroblastic terminology - 120 days lifespan
- Has sense of description - No cytoplasmic organelles
- Travels 300 miles
*** Always look at the nucleus and - Flexible
cytoplasm when looking at the cells - May antioxidant and rich in
enzymes

Mature erythrocyte: Anucleated

Feature Advantage Disadvantage
Uncomplicated cell Simple passage of oxygen Fragile, so relatively
membrane susceptible to damage
Anucleated, lack of Flexibility to penetrate the Unable to synthesize
enzymes capillaries essential protective &
maintain membrane
integrity
Membrane lacks HLA Relatively easy to transplant ---
molecules
All features Highly specialized Short lifespan
an anion channel. Protein 4.2
Anucleated enhances this interaction.
- Para mas maraming hemoglobin at ** Cytoskeletal proteins: Skeleton (nasa
magpalit ng shape ilalim ng cell membrane)
- Can’t generate ATP efficiently and - Spectrin (look like helix), actin,
protect itself ankyrin, Protein band 4.1, Protein
** blood groups lang may HLA band 4.2
- Pag wala mabilis ma hemolyzed ang
RBC, labas pasok lang ang
electrolytes
- Hold the cell membrane in place
** Spectrin is 2 dimers: alpha and beta
spectrin

• RBC membrane composition: 50%

CHON, 20% phospholipids, 20% Band 3 Anion transporter
cholesterol & 10% CHO Glycophorin A Transports negatively
• Integral proteins (nag traverse sa charged sialic acid
phospholipid bilayers): Band 3, (associated MNS blood
Glycophorin A, Glycophorin C, Rh group system)
Associated glycoprotein (regulates for water
• Cytoskeletal proteins: Spectrin balance)
dimers form a loosely wound helix. Glycophorin B
The tail end is linked to Glycophorin Glycophorin C
C by protein band 4.1, which Rh Antigens Gas transporter, ion
stabilizes the association of spectrin transport
with actin. At the head end, β- Blood group
spectrin attaches to ankyrin which antigens
connects to Band 3, a
transmembrane protein that acts as Rh antigens: most complicated

Genetic disease which has no cytoskeletal
proteins: hereditary erythrocytosis
RBC metabolic processes requiring
energy:
• maintenance of intracellular cations
• maintenance of membrane
phospholipid
• maintenance of skeletal protein
plasticity
• maintenance of functional ferrous
Hgb
• protection of cell proteins from
oxidative phosphorylation
• initiation and maintenance of
glycolysis
• synthesis of glutathione
Biochemical pathways of RBC:
1. Embden-Meyerhof Pathway
2. Hexose Monophosphate or Pentose
Phosphate Pathway (sidearm)
3. Rapoport-Luebering Shunt
4. Methemoglobin reductase pathway
1. Embden-Meyerhof pathway
- main anaerobic glycolytic pathway
- uses glucose & generates ATP
- maintains cell energy
- in this pathway glucose is
catabolized to pyruvate, consuming
2 molecules of ATP per molecule of
glucose & maximally generating 4
molecules of ATP per molecule of
glucose, for a net gain of 2
molecules of ATP
- dito kumukha enery ang RBC
2. Hexose Monophosphate Pathway or
Pentose Phosphate Pathway
- Aerobic/oxidative glycolysis
- detoxifies accumulated peroxide, an
agent that oxidizes heme iron,
proteins & lipids
- this pathway diverts G6P to pentose
phosphate by the action of G6PD, in
the process the oxidized NADP is
converted to the reduced form
NADPH.
- NADPH is then able to reduce the
oxidized form of glutathione (GSSG)
to its reduced form GSH using
glutathione reductase
- Heinz body forms from G6PD
deficiency
- protects itself from oxidation and
stress
- sidearm
- Glutathione: has antioxidation to
fight oxidative stress, free radicals
- Enzyme of importance: glucose-6-
phosphate dehydrogenase (kapag
wala ito, walang generation ng gluta,
mas mabilis mamatay ang RBC)
- Heinz body: G6PD deficiency
3. Methemoglobin Reductase Pathway
- prevents oxidation of heme iron
- within the Embden
- makes sure that iron is reduced to
its ferrous form (enzyme)
Ferric: di nakakapagdala ng O2, kaya
kailangan maging ferrous
4. Rapoport-Luebering Pathway
- generates 2,3 DPG (very important
protein) which regulates oxygen
affinity to hemoglobin
- regulatory protein
** 2,3 diphosphoglycerate abnormal hemoglobin known as
hemoglobinopathies

Hemoglobin Ontogeny, Structure &

Function

Hemoglobin - Qualitative hemoglobinopathy:

• conjugated protein Hgb synthesis is normal or near
• tetrameric protein with 4 subunits normal rate but the Hgb molecule
• 4 heme groups and 4 polypeptide (structural defect) & its function
chains (qualitative defect) (kapag yung
• concentration within rbc is 34 g/dL amino acids nagkakapalit palit)
• molecular weight: 64,000 Daltons - Quantitative defect: reduced or no
• Heme consists of protoporphyrin IX production of globin chains, but
(9 ring) with a central ferrous atom: does not affect amino acid sequence
ferroprotoporphyrin IX (9 ring) – pag of globin chains (nawalan ng chains)
may iron na
• Globin chains: 2 identical pairs of
unlike polypeptide chains with a Common seen Qualitative
total of 574 amino acids hemoglobinopathy
• globin chains are coiled into helices Molecular Hgb Common β-Chain
Abnormal Name Substituti
ity on
Amino Hgb S (most 6th
Acid common) glutamic
Substituti acid to
on - Sickling valine
cells
- Lifelong
abnormalit
y
- Genetic
- Middle
eastern,
Africa
Hgb C (2nd most 6th
common) glutamic
acid to
lysine
Hgb D- Los 121st
Angeles glutamic
acid to
glycine
Hgb D- Punjab
- Thalassemia Hgb E (3rd most 26th
2 identical pairs of unlike polypeptide common) glutamic
chains - Asian acid to
- 1 alpha pair (2 alpha) lysine
- 1 beta pair (2 beta)
Hgb O- Arab 121st
- 4 chains
glutamic
Polypeptide chains:
acid to
- 141 amino acids: alpha, zeta
lysine
- 146 amino acids: beta, gamma,
Amino Hgb Gun Hill 91st to
delta & epsilon
Acid 97th → 0
- arranged into specific sequence &
Deletion
any derangements can lead to
Amino Hgb Constant α+31
Acid Spring
Elongatio
n Vegetable: Ferric (Fe 3+)
Global Hgb Lepore- δ(1-50)
Chain Baltimore β(86-146)
Fusion - Combinatio
n of 2
globin
chains
Heme Synthesis: Iron Absorption
• Fe absorption occurs in the
duodenum
• Fe2+ also called heme iron easily
goes inside the cell while Fe3+ is
reduced by the enzyme cytochrome
B reductase or ferrireductase & is
taken up by divalent metal
transporter 1 (DMT1)
• Once inside the duodenal cell: iron
may be used for synthesis of various
enzymes; maybe stored in as ferritin
(storage form of iron); or exported
into the circulation
• For iron to enter the circulation, it
must be oxidized by hephaestin &
exit through ferroportin (fpn 1).
Another oxidizing protein is
ceruloplasmin.
• The released Fe3+ can now bind to
its carrier protein called transferrin
(Fe transport protein) which delivers
it to cells requiring Fe, especially
erythropoietic cells
• The process of Fe absorption is
regulated by hepcidin
Iron homeostasis
- Diet (can maintain and recycle iron);
meat, vegetables
- Iron is absorbed in small intestine
Meat: Ferrous (Fe 2+) and it easily
absorbed
- In the apical portion of the small
intestine, there are enzymes called
duodenal cytochrome reductase, it
will reduce the ferric into ferrous, it
can now bind to Divalent metal
transporter 1 (DMT-1), once inside
the cell the iron can now be used by
the cell itself in making enzyme.
- Free iron is toxic, lagi dapat naka
bind kay protein
- Exit basolateral membrane, it needs
to be oxidized, gagawin siyang ferric,
a protein known as Hephaestin an
oxidase, it will exit through
ferroportin portal
- Ceruloplasmin: copper tansport
protein, also acts as ferrooxidize,
that will oxidize iron
- Si iron dapat mag bind kay
Transferrin: transport protein for
iron
- Tightly regulate by the Liver organ
for absorption
- Liver produces protein hormone
known as Hepcidin, which
regulates iron absorption, it blocks
ferroportin, hindi na makakaexit si
iron and it will remain in duodenal
cell which will eventually shed off
- Ferritin: storage form of iron
Hepcidin
- regulatory protein hormone
produced by the liver
- can block ferritin
- inflammation
- it decreases Fe absorption by
negatively modulating ferroportin
- it also regulates cellular Fe
mobilization/transport stores
- involved in inflammation
- hepcidin deficiency causes Fe
overload & mutation in the HFE
gene causes hemochromatosis
 STAGE HEMOGLOBIN
Embryonic Gower 1 (2 Epsilon,
2 Zeta)
Gower 2 (Alpha,
Epsilon)
Portland (Zeta,
Gamma)
Hgb F: Alpha,
Gamma
Fe homeostasis Adult hgb:
A1: Alpha, Beta
A2: Alpha, Delta
Birth Hgb F (60-90%)
Hgb A1 (10-40%)
Adult Hgb A1 (>95%)
Hgb A2 (<3.5%)
Hgb F (1-2%)

- Kahit maraming kinakain na iron 1-

2 milligrams per day lang naabsorb
- Anemia: the absorbtive process
changes kasi kulang na iron supply
- Hemochromatosis: too much iron
na di nareregulate

Heme synthesis: Porphyrin IX or Defects in Porphyrin Synthesis:

Porphyrin ring Synthesis • Can be inherited or acquired
• Bone marrow & the liver are the • Inherited enzymatic defects are
main producers of porphyrin known as porphyrias (genetic), this
• synthesis involves a series of leads to accumulation & excretion of
enzymatic steps which occurs in the porphyrins or their precursors
mitochondrion & cytoplasm of the • Acquired enzymatic defects are due
cell to lead poisoning, alcohol & certain
• The first step in the mitochondrion drugs.
involves the condensation of glycine • Lead blocks the enzymes delta-
with succinyl coenzyme A (CoA) aminolevulinic acid synthase, ALA-
catalysed by the enzyme ALA- dehydratase & heme synthase
synthase, this leads to the (acquired)
formation of delta-aminolevulinic
acid. PORPHYRIAS ENZYME
• A series of enzymatic reactions DEFICIENCIES
follow in the cytoplasm leading to Sideroblastic delta-Aminolevulinic
porphobilinogen →
anemia (X- Acid synthase
uroporphyrinogen → linked)
coproporphyrinogen → the final
ALAD-deficient delta-Aminolevulinic
steps go back to the mitochondrion)
porphyria Acid dehydratase
protoporphyrinogen IX →
Acute Porphobilinogen
protoprotphyrin IX ring which in
intermittent deaminase
incorporated with Fe2+ atom by the
porphyria
enzyme ferrochelatase
Congenital Uroporphyrinogen
erythropoietic III cosynthase
porphyria
Porphyria Uroporphyrinogen
cutanea tarda decarboxylase
Hereditary Coproporphyrinogen
coproporphyria oxidase
Variegate Protoporphyrinogen
porphyria oxidase
Erythropoietic Ferrochelatase
porphyria
• can be acute or chronic
• can be neurologic or cutaneous
manifestation
• port-wine red color urine
• Fe accumulates in the cell & become
sideroblasts
Globin Chains:
• Polypeptide chains composed of:
- 141 aa: alpha, zeta
- 146 aa: beta, gamma, delta &
epsilon
• Chromosome 16 has the alpha
genes (2) & zeta gene
• Chromosome 11 has the epsilon
gene, gamma genes (2), delta gene &
beta gene
Alpha: simula embryo
Hgb F: tuloy tuloy, pagdating ng ilang
buwan bumababa, 1-2% sa adult
- Gamma papalitan ni beta
- Thalassemia: result of deletion
2,3 Diphosphoglycerate
- Regulates the oxygen affinity to
hemoglobin, to release O2 to tissues
Oxygen dissociation curve
- Habang pataas nang paatas yung
oxygen sa katawan, nasasaturate
yun hemoglobin
- Shift to the right: red dots, mabiilis
na pagrelease ng oxygen, kahit di pa
saturate yung hgb, narerelease na
yung O2sa tissues; decrease pH;
acidosis, mataas na temperature,
mataas 2,3 DPG, mataas na PCO2
- Shift to the left: mas nagkakaroon
ng saturation, increase pH;
alkalosis, mababa temperature,
mababa 2,3 DPG, mababa PCO2
Relaxed
Tight: deoxygenated
*** Hemoglobin Dissociation Curve
Shift to the right- low pH, high pCO2, high
temperature, high 2.3 DPG
Shift to the left- high pH, low pCO2, low
temperature, low 2.3 DPG
RBC Catabolism:
As an erythrocyte ages, the following
processes occur:
1. The membrane becomes less
flexible.
2. The concentration of cellular
hemoglobin increases.
3. Enzyme activity, particularly
glycolysis, diminishes.
2 sites of hemolysis:
1. Extravascular hemolysis: involves
the macrophages of the reticule
endothelial system
2. Intravascular hemolysis: involves
the direct lysis of RBCs in the blood
vessels, happens at a low
concentration
Extravascular
- Normal
- Old RBC (senescent, aging,
imperfect) is going be taken down by
macrophage, marerelease si heme
and globin (balik sa circulation)
 - Heme: Protoporphyrin will be
converted to a pigment bilirubin,
pupunta sa liver for conjugation,
where in unconjugated bilirubin
becomes conjugated bilirubin,
pwede na lumabas sa stool and
urine. Pag di nalabs si bilirubin,
magkakaroon ng jaundice
VARIATIONS IN SIZE, SHAPE,
HEMOGLOBIN CONTENT & INCLUSIONS
OF THE RED BLOOD CELLS
6-8 micra (normal size)
7.2 um (average)
Terms:
Anisocytosis- variation in size
Microcytic: mas maliit pa sa nucleus ng
lymphocyte
Macrocytic

Hemoglobin Variants
1. Methemoglobin: oxidized Hgb (ferric
state) thus cannot bind O2
- brownish to bluish color
- causes left shift in the dissociation
curve
- if levels >30% px become cyanotic &
hypoxic
- seen in nitrites & low levels of
methemoglobin reductase
- high levels are present in Hgb M
disease
- peak absorbance 620-640 nm at pH
7.1
- treatment includes removal of
substance or administration of
ascorbic acid or methylene blue
microscopic guide:
the size of a normal rbc is
approx. the same size as the
nucleus of a small lymphocyte
Poikilocytosis- variation in shape
Anisochromia:
Normochromic
Hypochromic
Polychromatophilic
Anisocyte:
Microcytes- small RBC <6 um, MCV <80
fL
Seen in: IDA (iron deficiency anemia),
Thalassemia, Sideroblastic Anemia,
Anemia of Chronic Disease or Anemia of
Inflammation, Hemoglobinopathies

2. Sulfhemoglobin: irreversible oxidation

caused by sulfonamides, phenacetin,
acetanilide or phenazopyridine
- greenish pigment
- results in ineffective oxygen
transport thus px become cyanotic
- toxic level is at 0.5 g/dL
- cannot be measured by
cyanmethemoglobin method
- absorbance at 620 nm Macrocytes- large RBC >8 um, MCV >100
fL, cells appear filled with Hgb
3. Carboxyhemoglobin: CO + Hgb Seen in: Megabloblastic anemias (B12
- binding capacity of CO for Hgb is deficiency anemia, and folate deficiency
200x stronger than that of oxygen anemia: problem in DNA), macrocytic
for Hgb anemias, liver disease, myelodysplastic
- its release is 10,000x slower syndromes
- reference interval: 0.2-0.8%
(endogenously)
- exogenous from
gases/exhaust/smoking
- smokers have 4-20% of total Hgb
can be HgbCO
- levels of 10-15% can cause dizziness
& headaches
- >50% may cause coma &
convulsions
- cherry-red color
- absorbance at 541 nm
- hyperbaric oxygen treatment is
recommended
 • Alcoholic liver disease
Poikilocyte:
SPHEROCYTE
- Small, round dense with fairly
uniform size with no central pallor
- Cells have decreased membrane
surface: volume ratio
- less deformable, fragile cell & short
life span
- indication of hemolytic anemia
SEEN IN:
• Hereditary spherocytosis (Spectrin
mutation) (not common in Asian)
• Immune hemolytic anemia
• Extensive burns
• Prolonged storage of blood ** Rh system: most complex
• Blood transfusion RhAg proteins: help regulate ions (labas
pasok), kung walang ganito ang isang tao,
magkakaroon siya ng Rh null disease

SICKLE CELL / DREPANOCYTE /

MENISCOCYTE
- thin, dense elongated RBC pointed
at each end
SEEN IN: Sickle cell anemia

ELLIPTOCYTE / OVALOCYTE
- cigar-, egg-, sausage- rod-, pencil-
shaped
SEEN IN:
• Hereditary elliptocytosis-
(Protein band 4.1 mutation)
• Iron Deficiency Anemia
• Thalassemia major
• Megaloblastic Anemia
BURR CELL / ECHINOCYTE /
• Myelophthisic Anemia
CRENATED CELL
- with blunt or pointed, short
projections that are usually evenly
spaced
- can be seen in anticoagulated blood
several hours old, in stored blood
due
to decreased ATP
SEEN IN:
• Uremia
• Pyruvate Kinase Deficiency
• Gastric CA, peptic ulcer
• Renal insufficiency

STOMATOCYTE (mouth cell, kissing

cell)
- RBC with slit-like central pallor
resembling a mouth
- high intracellular (concentration)
Na, low K (mabilis magburst/swell)
SEEN IN:
• Hereditary stomatocytosis
• Rh null disease
TEARDROP CELL / DACROCYTE
- with single pointed extension
resembling a teardrop or pear
SEEN IN:
• Myelophthisic anemia: infiltration
• Primary Myelofibrosis
• Thalassemia
• Megaloblastic anemia
Blister cells
- contains 1 or more vacuoles
resembling blisters
- SEEN IN:
• severe burns
• traumatic interaction of blood
vessels and circulating blood such
as fibrin deposits
• pulmonary emboli in sickle cell
anemia
• Microangiopathic hemolytic anemia
SCHISTOCYTE / SCHIZOCYTE
- fragmented RBC due to rupture in
the peripheral circulation
SEEN IN:
• Microangiopathic Hemolytic Anemia
• Burns
• Traumatic cardiac hemolysis
• Renal graft rejection
Keratocyte / Helmet cell
- a schistocyte with 1 or more
hornlike projections
Seen in:
• Disseminated Intravascular
Coagulation
SPUR CELL / ACANTHOCYTE
- small, dense RBC with few
irregularly spaced projections with
varying length
- change in the ratio of plasma lipids
SEEN IN:
 • Severe Liver Disease
• Abetalipoproteinemia
• Mc Leod Syndrome
• Following heparin administration
• Neonatal hepatitis
• Post-splenectomy

HOWELL-JOLLY BODIES
- dense, round blue-purple granule
that is usu. 1 per cell; occasionally
multiple (almost towards the
TARGET CELL / CODOCYTE (bulls eye
peripheral of the cell)
cell)
- made of DNA nuclear fragment (may
- with Hgb concentrate at the center
natira pa)
& around the periphery resembling
- Fuelgen reaction (+)
a target
SEEN IN:
- increased surface area to volume
• Hypersplenism
ratio
• Megaloblastic anemia
SEEN IN:
• Hemolytic anemia
• Hemoglobinopathies- C, SC, SS (E;
common in Asian)
• Thalassemia
• Liver diseases

HEINZ BODY
RBC inclusions:
- round, refractile inclusions not seen
BASOPHILIC STIPPLING
in Wright’s stain
- blue-purple granules distributed
- supravital stain
throughout the cytoplasm
- On supravital: round, granule
- made of precipitated RNA
attached to inner membrane
(aggregates of ribosomes)
- made of denatured Hgb
SEEN IN:
- bite cell
• Lead poisoning
SEEN IN:
• Thalassemia
• G6PD def. A.
• Hemoglobinopathies
• Unstable Hgbs- Zurich, Köln
• Abnormal heme synthesis (def. in
• Oxidizers
pyrimidine 5’ nucleotidase)
**
Supravital stains: New methylene blue,
and brilliant cresyl blue
Uses supravital stain: Heinz body,
Hemoglobin H, reticulocyte

PAPPENHEIMER BODIES
- clusters of small, light blue
granules often seen near the
periphery of the cell
- made of iron
- Perl’s Prussian Blue (+) (iron stain)
SEEN IN:
• Sideroblastic anemia: hereditary,
kulang sa enzymes para magawa si
porphyrins
• Hemoglobinopathies
• Hypersplenism
• Megaloblastic anemia
**
Hemoglibin SS: Disease
Hemoglobin AS: Trait
Hemoglobin S disease: homozygous
Hemoglobin S trait: heterozygous
HEMOGLOBIN SC crystals:
- finger-like or quartz-like crystal of
dense hemoglobin protruding from
the RBC membrane
- “Washington monument” shape
SEEN IN: Hgb SC disease

CABOT RING
- blue rings or figure-eights
- remnant of mitotic spindle
SEEN IN:
• Megaloblastic anemia
• Myelodysplastic syndrome

POIKILOCYTE SECONDARY TO
ABNORMAL HGB CONTENT:
Hemoglobin C crystals
- hexagonal crystals of dense Hgb
formed within the rbc membrane
- SEEN in Hgb C disease not in Hgb
AC (hemoglobin A and C) disease;
post-splenectomy
MIDTERMS

Granulopoiesis
- formation of granulocytes
- neutrophil, eosinophil, basophil
- monocytes: very fine granules,
agranular, but not granulocytes
- GCSF: targets the CFU-G

Myeloblast
Maturation series:
Size: varies from 15-20 µm (18-20)
Nucleus:
• delicate round to oval with
prominent nucleoli (2-5)
• nuclear chromatin is finely reticular
Cytoplasm:
• contains RER
• loose chromatin
• 15 hours
• a developing golgi apparatus
• no visible granules yet;
• (+) Auer rods: fused lysosomes, red
to pink, needle-like, pathologic
** (leukemia)
leukocyte termintology: 1 system of
nomenclature
Rbc terminilogy: normoblastic,
rubliblastic, erythroblastic

Pool:

N:C: 4:1
Location: BM 0-2%

Stem pool: hematopoietic stem cell

Proliferation/mitotic pool: precursor
even progenitors whose main role is to
proliferate and undergo mitotic division
(myeloblast, promyelocyte, myelocyte:
because of their capability to undergo cell
division)
Maturation/storage pool: walang
capability of mitosis, nasa bone marrow
and eventually release to circulation.
These are reserved (metamyelocyte, band,
neutrophil)
Promyelocyte
Peripheral blood Size- 14-20 µm
- Di lahat narerelease for circulation Nucleus:
- Marginating pool: neutrophils in the • round to oval
interstitial tissue sides. Pwedeng • nucleoli begin to fade
bumalik kapag kailangan • 24 hours
Cytoplasm:
• basophilic
 • primary granules (azurophilic
granules) become dominant:
important for bactericidal actions
Primary/Azurophilic Granules:
• Myeloperoxidase: leukemia staining,
to identify kung ang leukemia ay
myeloid or lymphoid, very dark stain
• Chloroacetase esterase
• Lysosomal enzymes (acid hydrolase,
acid phosphatase, B-glucoronidase
• Elastase
• Arylsulfatase
• Cationic antibacterial proteins

N:C- 3:1
Location: BM 2-5% Metamyelocyte
Size: 10-15µm
Nucleus:
• Indented nucleus, kidney-bean
shape
• no more nucleoli
• chromatin is coarse clumped
Cytoplasm:
• pale blue to pink
• few primary granules
• predominantly secondary granules
N:C: 1:1 to 1.5:1
Location: BM 13-22%

Myelocyte
Size: 12-18 µm
Nucleus:
• round to oval
• may have 1 flattened side near the
well-developed Golgi apparatus
• nucleoli are no longer visible
• chromatin is coarse & more
condensed
• 4 days
Cytoplasm
• slightly basophilic
• primary granules are few to
moderate
• secondary granules (specific
granules) are increasing: allows to Band (Stab cell)
identify what kind of granulocyte it Size: 10-15µm
is Nucleus:
• lilac staining • C or S shaped, horse shoe shaped
• neutral neutrophil • coarse clumped chromatin
• last mitotic stage Cytoplasm:
Secondary/Specific Granules: • pale blue to pink
predominant • few primary granules
• Lysozyme: for digestion • abundant secondary granules
• Lactoferrin • marami na sa bone marrow
• Collagenase • mayroon na sa circulation (pero di
• Plasminogen activator madalas)
• Aminopeptidase • tumataas kapag may serious
Tertiary Granules: infections
• Gelatinase • capable of phagocytosis
N:C- 1:1
N:C- 2:1 Location- BM 17-33%
Location: BM 5-19% Peripheral blood 0-5%
 Mitosis: 7 days, 7 days maturation pool
bago i-release sa circulation
- Takes about 14 days, wherein the
last 6-7 days are spent in the
maturation and storage pool

Eosinophilic series

Eosinophil myelocyte
- Earliest recognizable precursor

Polymorphonuclear Neutrophil
Size: 10-15µm
Nucleus:
• 2-5 lobes connected by thin
filamentsof coarse clumped
chromatin
• 7-10 hours
Cytoplasm: Eosinophil metamyelocyte
• pale blue to pink
• few primary granules
• abundant secondary granules
N:C- 1:1
Location: BM 3-11%
Peripheral blood 50-70%

Eosinophil band

Neutrophil Function:
Phagocytosis
Bactericidal Activity

Eosinophil
Eosinophil-specific Granules: Monoblast
Major basic protein (MBP)- toxic parasites - strongly positive for CD 33 & weakly
& cells, neutralizes heparin & induces positive for CD 34 markers; CD 4
release of histamine from basophils, positive
sumisira sa cell ng helminths ng parasites - low numbers in the BM
• Acid hydrolase - only function is mitosis
• Peroxidase - very similar to myeloblast
• Phospholipase - immature cell
• Cathepsin Size- 12-18µm
• Eosinophil cationic protein Nucleus- round to ovaleccentric
• Eosinophil-derived neurotoxin - 1-2 nucleoli
- fine chromatin
Eosinophil function: Cytoplasm- deeply basophilic
Antihelminthic activity N:C- 4:1
Phagocytosis Location- BM
Allergic response

Charcot-leyden crystals
- Sputum Promonocyte
- Bronchial lavage - similar in size to blast but may have
some granulation
- can be motile & participate in
phagocytosis but they lack the
activity seen in mature cells
Size- 12-20µm
Nucleus- irregularly-shaped (elongated,
folded, indented)
- nucleoli may/may not be visible
(0-2)
- fine chromatin
Cytoplasm- blue-gray with fine azurophilic
granules
Basophil granules: - vacuoles variable
Histamine N:C- 2:1 to 3:1
Heparin Location: BM <1%
Peroxidase
Eosinophilic chemotactic factor A
Functions:
Immediate hypersensitivity reactions (in
tissue, MAST cells)

Monocyte
Monopoiesis - great morphologic variability
 - tends to marginate along vessel position for 1 hour (standing,
walls undisturbed)
Size- 12-20µm
Nucleus- variable, maybe round, horse- Diagnostic importance:
shoe or kidney-shaped, • non-specific measurement used to
indented or curved with lacy detect & monitor an inflammatory
chromatin with small clumps or response, in which there is an
folds increase in plasma concentration of
Cytoplasm- blue-gray, may have acute phase reactants
pseudopods, maybe quite • commonly used as a general
irregular screening test
- many fine granules giving • useful in monitoring the course of
‘ground glass’ appearance an existing inflammatory disease or
- absent to numerous for differentiating similar diseases
vacuoles • used as an index of the presence of
- pseudopods active infection
N:C- 2:1 to 1:1 • c- reactive protein (capsular
Location: BM- 2% polysaccharide)
Peripheral blood- 3-11%

✓ ESR is directly proportional to RBC

mass
✓ ESR is inversely proportional to
plasma viscosity

Monocyte Granules: (antigen presenting 3 Factors Affecting ESR:

cell) 1. Erythrocytes
Acid hydrolase 2. Plasma composition: most important
Arylsulfatase factor
Non-specific esterase 3. Mechanical or Technical factors
Peroxidase
Acid phosphatase
Functions: 1. Erythrocytes
Phagocytosis ❑ Normal rbc tend to be more or less
Antigen processing separated, they are negatively
Cell-mediated immunity charged (zeta potential) &
therefore repel each other, this is
known as the zeta potential.
❑ Settle at a slower rate, because they
want to be separated
• reduction in the zeta potential that
is due to the increase of
surrounding plasma protein
concentration, leads to increased
rouleaux formation & larger rbc
mass which leads to faster rate of
fall

Macrophage: nagccompose ng reticulo

endothelial system

(2 folder) 57:45

Erythrocyte sedimentation rate

• rate of settling of the rbc in an
anticoagulated blood
• it is the measure of the distance &
speed of fall of rbc in the plasma
• the distance in mm is the rate of the - Nawala ang zeta potential because
rbc falling of positive charge
• it is reported in mm/hr
• the procedure: allowing a specific ❑ refers to the size & mass of rbc, the
amount of blood to sit in a vertical larger the size the faster its rate of
fall
 • ex. macrocytes fall faster than 3. Presence of clots or bubbles- INVALID
microcytes results, repeat set-up remember: ESR
• aggregated or agglutinated rbc leads should be performed within 2 hrs of blood
to increased cell mass & more rapid collection if blood has been refrigerated
sedimentation rate due to delays, the test may be set-up
• anisocytosis & poikilocytosis reduce within 6 hrs
the ability of the rbc to form
aggregates & thereby tend to falsely 2 general ESR methods:
lower ESR
• in severe anemia, due to the Factors Wintrobe Westergren
decreased concentration of rbc, the Method Method
ESR is markedly elevated
Length of 115 mm or 300 mm or 30
2. Plasma composition tube 11.5 cm cm
❑ single most important factor in
Bore 3 mm 2.5 mm
determining the ESR
diameter (internal)
❑ rouleaux & aggregate formation of
of tube 5.5 mm
the rbc are controlled primarily by
(external)
the concentration or levels of acute
phase reactants Calibrati 0-100 mm 0-200 mm
❑ contributes to change in positive on/grad
charges uation of
✓ ESR is increased in increased levels tube
of acute phase reactants because of
increased rouleaux & aggregate Amount 1.0 mL 2.4 mL
formation of blood
❑ Ex. of acute phase reactants: used
fibrinogen, alpha-1 globulin, alpha-2 Anticoag Double 3.8% sodium
globulin, beta globulin ulant of oxalate citrate
❑ High levels of albumin lowers the choice
ESR, low levels of albumin increases
ESR Referenc 0-9 mm/hr 0-15 mm/hr
e values (male) (male)
3. Mechanical or Technical factors 0-20 mm/hr 0-20 mm/hr
❑ tube must be exactly perpendicular (female) (female)
❑ a tilt of 3o can cause 30% error, the *>50 y/o 0-20
rack holding the tube should be mm/hr (male)
stable & free from any movement >50 y/o 0-
❑ increase temperature will increase 30 mm/hr
ESR (female)
❑ the length & bore of the tube will Advantag Requires More sensitive
also affect the sedimentation rate es & smaller method
disadvan amount of especially for
tages blood serial study of
chronic
Other tests inflammation
can be
performed Disposable
using this tubes
tube such as
3 Stages of Sedimentation: Hct, LE Requires
1. Lag- first 10 mins, initial period of preparation larger amount
aggregation or rouleaux formation of blood
2. Decantation- 40 mins, period of fast Considered
settling & at a constant rate less sensitive
3. Packing- last 10 mins, period of final due to
settling, the sedimentation is slow shorter tube
length
Sources of Error:
1. Excess anticoagulant EDTA falsely
increased ESR
2. reading >60 mins falsely increased
ESR
<60 mins falsely decreased
ESR
 2. Sedimat 15 (Polymedco)- uses infrared
measurement, can measure 8 specimens
with results after 15 mins

Bone Marrow Examination

BONE MARROW
- The Wintrobe tube has 2 • 3.4% to 5.9% of body weight
calibrations 10 cm to 0 & 0 to 10 • 1600 g to 3700 g
cm. Which calibration is for ESR, for • volume of 30-50 mL/kg
Hct? (answer: 0-10) 2 types of marrows:
a. red marrow- hematopoietically
active
b. yellow marrow- inactive marrow
composed of adipocytes
• Lower skull, vertebrae, shoulder,
ribs, sternum, pelvis & proximal
ends of long bones
• Cellularity:
➢ Childhood- 80%
➢ 30-70 y/o- 50%
➢ >70 y/o- reduced
- Westergren plastic and glass

Other manual ESR methods:

1. Bray’s method- uses 1.3% sodium
citrate, uses Bray’s tube which is
flat-bottomed & calibrated on sides
like Wintrobe, can also be used for
Hct & LE prep, reading is done every
5 mins for 30 mins & the total fall
after 1 hr
2. Cutler method- uses 3% sodium citrate,
uses Cutler tube which has 5 mL capacity
& graduation of 0-50 mm
3. Linzenmeier method- uses 3% sodium
citrate, uses Linzenmeier tube which is 65
mm in length, 5 mm diameter & has a • Contains hematopoietic cells,
capacity of 1 mL (with a mark at 18 mm) stromal cells, & blood vessels
4. Micro methods- • Stromal (supporting) cells include:
a. Smith- used for infants & children - endothelial cells- form the lining
- uses 5% sodium citrate - adipocytes- regulates volume & may
b. Landau- uses 5% sodium citrate secrete cytokines
- macrophages- phagocytosis, provides
iron, tightly regulated by hepcidin
- lymphocytes
Automated ESR Method: - osteoblasts & osteoclasts
1. Ves-Matic Sytem- measures the
change in opacity of a column of - reticular adventitial cells
blood as sedimentation progresses (fibroblasts)
with the use of optoelectronic - form an incomplete layer of cells on
sensor, 1 mL blood is collected the abluminal surface of the vascular
using special tubes by the sinuses
manufacturer which contain sodium - extend long, reticular fibers into the
citrate, results are obtained in 20 perivascular space that form a
minutes & is comparable to supporting lattice the developing
Westergren method hematopoietic cells
a. Mini-Ves- 4 samples at one time
b. Ves-Matic- 20 samples at one time
c. Ves-Matic 60- 60 samples at one
time

The hematopoietic cells develop in specific
niches within the cords.
- Erythroblasts develop in small
clusters, and the more mature forms
are located adjacent to the outer
surfaces of the vascular sinuses; in
addition, erythroblasts are found
surrounding iron-laden
macrophages (nurse cell)
- Megakaryocytes are located
adjacent to the walls of the vascular
sinuses, which facilitates the release
of platelets into the lumen of the
sinus.
- Immature myeloid (granulocytic)
cells through the metamyelocyte
stage are located deep within the
cords.
- As these maturing granulocytes
proceed along their differentiation
pathway, they move closer to the
vascular sinuses.
Indications for bone marrow studies:
1. Diagnosis of neoplasia
2. Staging of neoplasia
3. Diagnosis of bone marrow failures
4. Diagnosis of metabolic disorders
5. Diagnosis of infections
6. Monitoring of treatment
Contraindications:
1. Coagulopathies
2. Vitamin K deficiency: need to make the
four coagulation factors (vitamin k
dependent factors)
** thrombocytopenia
Sites:
1. Posterior superior iliac crest
(POSIC): most common site
2. Anterior superior ilia crest
3. Sternum
4. Anterior medial surface of the tibia-
children < 2y/o
5. Spinous process of the vertebra
Materials:
• Prior to bone marrow aspiration or
biopsy, venous blood is collected for
CBC.
• Disposable bone marrow specimen
collection tray
Types of needle:
1. For aspiration- University of Illinois:
bone marrow
2. For aspiration & biopsy- Jamshidi
needle, Westermann-Jensen, Vim
Silvermann: kukuha ng buto
guage: 14
Bone marrow aspiration:
• used to examine the types &
proportions of hematologic cells & to
look for morphologic variance
• 1-1.5 mL is aspirated &
immediately transferred to slides for
smearing
• another sample maybe collected &
transferred to suitable stopper tubes
for cytogenetic analysis, molecular
diagnosis or immunophenotyping
using flow cytometry
• anticoagulated specimens may also
be used for bone marrow films
 • *** heparin (6mL bone marrow) - observes for maturation gaps,
misdistribution of maturation stages &
Bone marrow biopsy/Trephine biopsy: abnormal morphology
• it is a solid core of bone & marrow - performs differential count of 300-
tissue 1000 nucleated cells
• demonstrates bone marrow - computes M:E ratio (1.5:1 to 3.3:1)
architecture: the spatial relationship - normal marrow cells:
of hematologic cells to fat, - osteoblasts
connective tissue & bony stroma - osteoclasts
• particularly important for evaluating - adipocytes
diseases that characteristically - endothelial cells
produce focal lesions such as - reticular cells
Hodgkin’s & non-Hodgkin’s
CELL DISTRI CELL DISTRI
lymphoma, multiple myeloma,
BUTION BUTION
metastatic tumors, amyloid &
granulomas Myeloblast 0-3% Rubriblast 0-1%
• obtained first before aspiration to
avoid any disruption of marrow Promyeloc 1-5% Prorubricyt 1-4%
architecture ytes e
• the specimen is placed on gauze to Myelocyte 6-17% Rubricyte 10-20%
absorb excess blood & immediately s
imprinted by touching the slide to
the specimen Metamyel 3-20% Metarubric 6-10%
ocytes yte
Managing the bone marrow specimen:
Neutrophil 9-32% Lymphocyt 5-18%
• Direct marrow films are made
ic band es
immediately then air dried
• Imprints (touch preparation) Segmente 7-30% Plasma 0-1%
• Crush smears d cells
• Concentrate smears- maybe used neutrophil
for sparse nucleated cells or very s
few; anticoagulated blood is
transferred in a Wintrobe tube & Eosinophil 0-3% Monocytes 0-1%
centrifuged at 2500 g for 10 ic &
minutes eosinophil
- layers: fat (1-3%), plasma (varies), ic
buffy coat/ME layer (5-8%), PRBC precursor
• Histologic sections fixed using s
Zenker’s glacial acetic acid, 10% Basophils 0-1% Histiocytes 0-1%
formalin for 6-18 hours or B5 fixative & mast
for 1-2 hours cells
• Staining- Wright’s or Wright’s-
Giemsa for marrow films Megakary 2- M:E ratio 1.5:1-
- Perl’s Prussian blue for iron ocytes 10/LPO 3.3:1
stores
- H&E for marrow biopsy

Examining the bone marrow aspirate,

imprint or smear:
A. Low power magnification
- classifies the observed area as to
normo-, hypo-, hypercellular
- searches for abnormal/tumor cells
(tumor cells’ nuclei are hyperchromic &
are often found near the edges of the
smear)
- the erythrocytic maturation stages
stain more intensely & their margins are
more sharply defined
- evaluates, examines & estimates
megakaryocytes(2-10/LPO)

B. High power magnification

- observes myelocytic & erythrocytic
maturation stages
Automated Cell Counting
Instrumentation in Hematology
• Electronic cell counting provides
more accurate & precise results
than manual methods.
• Most hematology analyzers are
multiparameter cell counters which
provide 8-10 or more parameters,
including RBC, WBC, Platelet
counts, H & H, indices, red cell
distribution width (RDW) & mean
platelet volume (MPV).
• RDW (red cell distribution width)
provides an estimate of rbc
anisocytosis (change in size).
Reference value: 11.5-14.5%
• MPV (mean plate volume) is
determined from the platelet
histogram curve.
Reference value: 6.8-10.2 fL
Main principles:
1. Electrical impedance
2. Radiofrequency or RF resistance
3. Optical scatter/flow cytometer
1. Electrical impedance or low
voltage direct current (DC)
Coulter principle
• based on the detection &
measurement of changes in
electrical resistance produced by
cells as they traverse a small
aperture suspended in an ionic
solution.
• As each cell passes through the
opening, the electrical resistance
between the 2 electrodes increases
because cells are poor conductors of
electricity.
• As resistance increases, voltage
increases.
• A voltage pulse of short duration is
produced for each cell that passes
through the aperture.
• The magnitude of voltage is
proportional to the cell volume or
size, & the number of voltage pulses
is proportional to the frequency of
particles passing through the
aperture.
• Pulses are collected & sorted
(channelized) according to their
amplitude by pulse height analyzers.
• The data are plotted on a frequency
distribution graph or size
distribution histogram, with relative
number on the y-axis & size on the
x-axis.
• The histogram depicts the volume of
distribution of cells counted.
** Cells are poor conductor of electricity
• RBC & WBC are counted in
duplicate or triplicate. Each of the
duplicated counts must agree within
a standardized range of deviation
from each other.
• The MCV is often determined
directly from voltage-pulse heights
from the RBC count or histogram
curves.
• The Hemoglobin of the sample is
obtained spectrophotometrically
from the WBC dilution.
• Platelets are counted in duplicate or
triplicate in the rbc aperture bath.
Particles ranging from 2-20 fL in the
rbc bath are sorted as platelets &
plotted as a platelet histogram.
• RBC indices are computed
parameters commonly obtained from
automated cell counters.
▪ The Hct is computed
from the RBC count &
MCV & calculated from
this formula: Hct %=
(RBC x MCV) ÷ 10
▪ The MCH is computed
from the MCV & the
MCHC & calculated
from this formula (note
that 1 µg = 1 pg): MCH=
(MCV x MCHC) ÷ 100
▪ The MCHC is computed
from the Hgb & Hct &
calculated from this
formula: MCHC %=
(Hgb ÷ Hct) x 100
** cell will resist electricity so it will emit
voltage pulse
• Common errors:
▪ Missing parameters- any rbc
or wbc count grossly out of
range must be repeated. The
rule of three must agree & any
one of the indices being
“singly” out of range must be
considered suspicious.
 ▪ Carryover- results from the
incomplete removal of all
WBCs in the counting
chamber between counts.
(Previous sample nadagdag sa
new sample)
▪ WBC counts exceeding
instrument linearity limits
result in increased cell
turbidity & may falsely
increase Hgb, MCH & MCHC.
▪ High patient glucose
concentrations >400 to >600
mg/dL result in intracellular
hyperosmolality in RBCs &
may cause high MCV & Hct
with a low MCHC.
▪ Extremely microcytic cells
may be overestimated by the
instrument.
▪ Cold agglutinins in high titer
give a falsely high MCV, low
RBC counts & very high
MCHCs. Warming the blood at
37oC may eliminate this
problem. A good clue is to look
for RBC clumping in the thin
area on the smear.
▪ Lipemia may produce
turbidity in the WBC aperture
bath & falsely increase Hgb,
MCH & MCHC.
2. Radiofrequency or RF resistance to a
high voltage electromagnetic current
flowing between both electrodes
simultaneously.
• The total volume of the cell is
proportional to the change in DC,
the cell interior density (e.g. nuclear
volume) is proportional to pulse size
or change in RF signal.
• Conductivity is measured by this
high-frequency electromagnetic
probe, is attenuated by nucleus to
cytoplasm ratio, nuclear density &
cytoplasmic granulation.
• Two different cell properties, such as
impedance & conductivity, can be
plotted to create a two-dimensional
distribution cytogram or scatterplot.
• Such plots display the cell
populations as clusters, with the
number of dots in each cluster
representing the concentration of
that cell type.
3. Optical scatter/flow cytometer- a
hydrodynamically focused sample stream
is directed through a quartz flow cell past
a focused light source.
• The light source is generally a
tungsten-halogen lamp or a
helium-neon laser.
• As the cells pass through the
sensing zone & interrupt the beam,
light is scattered in all directions.
• Light scatter results from the
interaction between the process of
absorption, diffraction (bending
around corners or surface of a cell),
refraction (bending because of
change in speed) & reflection
(backward scatter of rays caused by
an obstruction).
• The detection of scattered rays &
their conversion into electrical
signals is accomplished by
photodetectors (photodiodes &
photomultiplier tubes) at specific
angles.
• Lenses fitted with blocker bars to
prevent non-scattered light from
entering the detector are used to
collect scattered light.
• A series of filters & mirrors separate
the varying wavelengths & present
them to the photodetectors.
• Photodiodes convert light photons
to electronic signals proportional in
magnitude to the amount of light
collected.
• Photomultiplier tubes are used to
collect the weaker signals produced
at 90-degree angle & multiply the
photoelectrons into stronger, useful
signals.
• Analogue-to-digital converters
change the electronic pulses to
digital signals for computer analysis.
• Forward-angle light scatter (0o)
measures cell volume or size. This is
diffracted light.
• Orthogonal light scatter (90o) or
side scatter provides information on
cell granularity & lobularity.
• Forward low-angle scatter (2-3o)
can relate to size or volume
• Forward high-angle scatter (5-15o)
also correlate with cell volume &
refractive index or with internal
complexity or internal cellular
components.

Blood cell histograms
• Provided by many high-volume
instruments to give representation
of size distributions of the
different cell populations.
• The volume (fL) is plotted against
the relative frequency of platelets,
RBCs & WBCs.
1. WBC histogram
- provides a count & plot of cells in the
WBC aperture bath larger than 45 fL.
Normal WBC histograms have three
distribution peaks:
• 45-90 fL represents small,
mononuclear population of cells (ex.
lymphocytes)
• 90-160 fL represents a minor
population of larger mononuclear
cells (ex. monocytes). An increase
in this range can also signify
abnormal cell types such as
immature precursor cell types found
in patients with leukemia.
• 160-450 fL represents normal
mature types of granulocytes.
Abnormal WBC histogram patterns
- The lower threshold is 45 fL but the
histograms will extend lower to
detect abnormalities. A region code
(R) flags signal irregularities in the
WBC distribution & will appear next
to the differential parameters that
are in error.
• R1- warns of increased
interference in the area left of the
lymphocyte peak (approx. 35 fL),
which is typically caused by sickled
RBCs, nucleated RBCs or clumped
& giant platelets being counted in
the WBC aperture bath.
• R2- warns of excessive overlap of
cell populations at the
lymphocyte/mononuclear cell
boundary (approx. 90 fL) caused by
the presence of abnormal cell types,
such as atypical lymph, blast or
plasma cells.
• R3- caused by excessive overlap of
cell populations are the
mononuclear/granulocyte
boundary (approx. 160 fL) which is
due to the increased presence of
immature granulocytes like
metamyelocytes & band.
• R4- caused by the extension of the
cell distribution past the upper end
of the WBC threshold (approx. 450
fL). Most commonly seen in when
granulocytes are very high.
2. RBC histograms represent the cells
counted in the RBC dilution, in the size
range of 36-360 fL, which are sorted &
plotted as frequency against cellular
volume.
• Normal RBC histogram- a single
peak should be found normally
between 70 fL & 110 fL & the peak
should coincide with the MCV.
• Abnormal histograms result when
the MCV of the curve falls outside of
the normal range of 80-100 fL or
when the RDW is >14.5
▪ increased MCV shifts the curve to
the right & decreased MCV shifts
the curve to the left
▪ increased RDW is reflected by an
increase in the “width” of the area
beneath the curve
▪ in some disorders, there may be two
populations of RBCs (microcytes &
macrocytes)
3. Platelet histograms represent cells in
the RBC counting baths that are in the
size range of 2-20 fL.
• Cells in this range are counted, &
their frequency is plotted against
cellular volume.
• Atypical platelet histograms can
result in some disorders when large
platelets are present.
 • too large drop creates a long/thick
film
• too small drop creates a short/thin
film
• too slow spread accentuates poor
leukocyte distribution by pushing
larger cells such as monocytes &
granulocytes to the very end & sides
of the film

Buffy coat smear: leucopenia

** freshly collected
EDTA blood:
• smears can be made within 2-3
hours after collection
• >5 hours will produce artifacts-
echinocytic, spherocytic, necrobiotic
WBCs & vacuolated PMNs.
• Platelete satelitism

COVER GLASS TECHNIQUE

- thought to contain more even
distribution but difficult to master
& labeling problems
Macroscopic examination
✓ Quality of the smear
✓ Any abnormalities present
✓ Bluer film than normal suggests
increased plasma proteins or
rouleaux
✓ Grainy appearance may indicate
RBC agglutination
✓ Holes in the film could mean high
lipid contents
✓ Markedly increased WBC & platelet
counts can be detected from the
blue specks out at the feather edge

Wedge smear

SPUN SMEAR
- uses the more easily handled &
labeled glass slide & has the
advantage of even distribution
of the white cells & red blood cells free of
distortion
- this is an automated method
Stains:
1. Basic dyes- Methylene Blue- has the
affinity for acidic components like
the nucleus
2. Acidic dyes- Eosin/azure- has the
affinity for basic components like
the cytoplasm
3. Mixture of acidic & basic- stains
neutrophilic materials
Romanowsky’s Group of Stains:
1. Wright Stain
- composed of methyl alcohol,
methylene blue & eosin
- the most satisfactory in general
staining
- pH 6.4-6.8
- uses 0.05M sodium phosphate as
buffer
- staining reaction is pH dependent
2. Leishman stain
- same component but the
polychroming is longer
3. Giemsa stain
- with azur dyes, glycerine, eosin &
methylene blue
- good for inclusion bodies (12-18
hours) as well as blood parasites
Panoptic Stain:
1. Jenner Giemsa
2. May-Grunwald Giemsa
- these are combination of
Romanowsky’s & another group of stain
- composed of eosinated methylene
blue
Methanol- Fixative
Methylene blue- oxidized product
Azure B trimethylthiomin
Eosin- derived from xanthine
POSSIBLE CAUSES IF…?
- RBCs appear gray
- WBCs are too dark
- Eo granules are gray
- RBCs are too pale or red
- WBCs are barely visible
Causes of first scenario: (basic)
1. Stain or buffer too alkaline (most
common)
2. Inadequate rinsing
3. Prolonged staining
4. Heparinized blood sample
5. Thick films
Causes of Second Scenario: (acid)
1. Stain or buffer too acidic (Most
Common)
2. Underbuffering (too short staining)
3. Over rinsing
4. Mounting of coverslip before it dries
5. Very thin smear
Microscopic examination
Low power objective (10x)
✓ Assess the overall film quality,
color & cell distribution
✓ Feathered edge & lateral edge
can be checked for WBC
distribution
✓ Check for fibrin strands
✓ Check for rouleaux formation
or agglutination
✓ Scan for large, abnormal cells
such as blasts, reactive
lymphocytes or parasites
High power objective (40x)
✓ Select the correct area of the
film in which to begin the
differential count & evaluate
cellular morphology
✓ Obtain the WBC estimate by
scanning 10 HPO fields:
❑ Average number of WBC
are multiplied by 2000
❑ Can be useful for
internal QC between the
machine reading &
estimate
Oil immersion objective (100x)
✓ Perform WBC differential
count & platelet estimate or
platelet count
✓ Observe blood cell morphology
 ✓ RBC inclusions & WBC
inclusions can be seen if
present
✓ Reactive or abnormal cells can
be observed more in detail
Optimal area of examination
✓ Between the thick area or “heel” and
the very thin feathered edge
✓ RBCs appear uniform & singly
distributed, few overlapping, normal
appearance
Performing a differential count and
examining cell morphology
✓ Slide is scanned, counting &
identifying 100 WBCs
✓ Reported in relative counts or
absolute counts
✓ “Serpentine” or “battlement” pattern
is used for identification
✓ For very low WBC counts,
centrifugation may be used or count
✓ Reporting of any morphological
changes are done semi-
quantitatively
❑ Slight, moderate or
marked
❑ 1+, 2+ 3+
1. RBC
✓ Size- normocytic?,
microcytic?, macrocytic?,
anisocytosis?
✓ Shape- poikilocytosis?
✓ Color- Hgb content?,
normochromic?,
hypochromic?
✓ Count
✓ RDW- anisocytosis?
✓ Inclusions
✓ Arrangement
2. WBC
✓ Total count- leukocytosis?
Leukopenia?
✓ Differential count- philia? –
cytosis? –cytopenia?
✓ Abnormal cell morphology-
blasts? Immature stages?
Inclusions?
3. Platelets
✓ Count- thrombocytosis?
Thrombocytopenia?
✓ Platelet Estimate:
= number of platelets in 10
OIO x 20,000 approximates platelets/mm3
there are approximately 200
RBCs per OIO
✓ Abnormality
Systematic approach in peripheral blood
smear examination
1. Red blood cells
2. White blood cells
3. Platelets
4. Compare with the automated results
Summarizing Red Blood Cell Parameters
Step 1. Examine the Hgb or Hct for
anemia.
Step 2. Examine the RBC indices.
Step 3. Examine the morphology for
pertinent abnormalities.
A specimen from an adult male patient
yields the following CBC results
WBCs—3.20 x 109/L
RBCs—2.10 x 1012/L
HGB—8.5 g/dL
HCT—26.3%
MCV—125 fL
MCH—40.5 pg
MCHC—32.3 g/dL
RDW—20.6%
PLT—115 x 109/L
Differential in percentages: 100
Neutrophils—43
Bands—2
Lymphocytes—45
Monocytes—10
Morphology: hypersegmentation of
neutrophils, anisocytosis, macrocytes, oval
macrocytes, occasional teardrop, Howell-
Jolly bodies, and basophilic stippling
Smear shows:
Macrocytic, hypochromic red cells,
poikilocytosis seen, oval macrocytes &
occasional tear drop cells
presence of Howell-Jolly bodies &
basophilic stippling,
slight/moderate/marked
anisocytosis observed & RBC count is
decreased.
WBC count is moderately decreased,
presence of hypersegmented neutrophils.
Platelet count is slightly decreased.
Non-Malignant Leukocyte Disorders
I. Quantitative Disorders
A. leucocytosis & leukopenia (-
cytopenia)
1. Neutrophilia/--penia
2. Eosinophilia
3. Basophilia
4. Monocytosis/-penia
II. Qualitative Disorders
A. Morphologic alterations- in the
nucleus or cytoplasm
1. Toxic granulation
2. Döhle bodies
3. Hypersegmentation
4. Pelger-Huët anomaly
5. May-Hegglin anomaly
6. Chédiak-Higashi syndrome
7. Alder-Reilly inclusions
B. Functional abnormality
1. Chronic granulomatous
disease - neutrophilia with shift to the left
2. Myeloperoxidase deficiency
3. Leukocyte adhesion Neutrophilia Neutropenia
disorder
4. Lazy leukocyte syndrome
5. Job’s syndrome bacterial infections Congenital:
6. Congenital C3 deficiency (pyogenic, Kostmann’s
localized/generalize Syndrome
I. Quantitative abnormalities d)
• leukocytosis→ increased in the inflammation or Acquired: drugs
concentration or percentage tissue necrosis (myelosuppressive,
of circulating wbcs in the peripheral toxicity or immune
blood mediated change)
➢ increased movement from
maturation pool to circulation
➢ increased movement of
marginating cells to metabolic disorders chemicals, radiation
circulation
➢ increased activity of BM acute overwhelming
proliferative pool hemorrhage/hemol infections/inflamma
➢ Relative count- percentage of ysis tion
type of WBC
➢ Absolute count- gives the
drugs Cyclic neutropenia
actual number of the WBC per
(corticosteroids,
liter of blood
lithium)
- relative count x total
WBC count treatment with Benign- starvation,
myeloid growth anorexia
Neutrophils Reference range: factors
Relative count- 50%-70%
Absolute count- 2-7.5 x 109/L physiologic immune disorders
response to stress, (autoimmune,
1. Neutrophilia burns or surgery hypersensitivity)
• absolute neutrophil count >7.5-8.0
x 109/L
• shift to the left- increased numbers
of young forms in the circulation
such as bands & metamyelocytes
2. Neutropenia
• absolute neutrophil count < 2.3 x
109/L
• agranulocytosis <0.5 x 109/L
Eosinophil Monocyte
Relative count- 1%-3% Relative count- 3%-11%
Absolute count- 0-0.6 x 109/L Absolute count- 0.4-1.3 x
Eosinophilia 109/L
Monocytosis

allergic reactions- asthma, hay

fever
parasitic infections chronic bacterial infections
(TB, SBE, brucellosis, typhoid)
graft versus host disease
(GVHD) Inflammatory responses
drug sensitivity GVHD
protozoan infections
hypereosinophilic syndrome Recovery from neutropenia
Pulmonary syndromes Connective tissue disease
(Loeffler’s syndrome,
Eosinophilic pneumonia)
certain skin diseases
treatment with GM-CSF

Basophil
Relative count- 0-1%
Absolute count- 0-0.2 x 109/L
Basophilia

Hypersensitivity or
anaphylactic reactions

ulcerative colitis

chronic inflammatory
conditions
II. Qualitative disorder
1. Toxic granulation
- altered primary granules; appears
as large, dark blue to purple
staining granules
- commonly found in severe infections
or other toxic conditions
- clinically significant because they
reflect poorer prognosis
- peroxidase positive
2. Döhle bodies
- small, round to oval, light blue
accumulations of ribosomal RNA
- 1-5 µm in diameter & gray to light
blue in Wright stain
- associated with wide range of
physical or chemical insults such as
burns, infections, surgery,
pregnancy & the use of GM-CSF
- transient in that they are seen most
often during the first 1-3 days after
an insult after which they tend to
disappear; reflects sudden storage
pool release
3. Toxic valuolization
- caused by phagocytosis.
- autophagocytosis of granules is
caused by drugs such as antibiotics
(sulfonamides, chloroquine), alcohol
or radiation
- clinically significant when
associated with toxic granulation,
degranulation or Döhle bodies
- caused by making a smear from
blood that has been held for a long
time
4. Hypersegmentation/hypersegmented
neutrophil
- commonly seen in long term chronic
infections
- may reflect borderline vit B12 or
folate deficiency, in some it may
reflect a degenerative process such
as old segmenters
5. Pelger-Huët anomaly
- autosomal dominant neutrophil
hyposegmentation
- most nuclei are band-shaped, have
2 segments, peanut-shaped,
dumbbell-shaped or pair of
eyeglasses
- chromatin is clumped & overly
mature in contrast to the immature
shape of the nucleus; & the
population of the neutrophils is
uniform
6. May-Hegglin anomaly
- rare autosomal dominant condition
in which the neutrophils contain
basophilic inclusions of RNA
(resembling Döhle bodies) in the
cytoplasm
- larger, more spindle-shaped than
oval,
- permanent & have been found in all
leukocytes
- associated with thrombocytopenia with
giant platelets
7. Alder-Reilly inclusions
- autosomal recessive trait in
which decreased
mucopolysaccharide (lipids)
degradation results in the
cytoplasm of most cells.
 - appear as large coarse azurophilic /
metachromatic (deep blue to purple)
granules resembling toxic granules
- may be seen in lymphocytes &
monocytes
8. Chédiak-Steinbrinck-Higashi
Syndrome
- rare autosomal recessive trait with
abnormally large peroxidase-positive
lysosome granules.
- affected individuals may show
partial albinism (relative pigmentary
dilution),
hepatosplenomegaly
- are more susceptible to bleeding
tendencies, infections due to reduced
chemotaxis & bactericidal activity &
neutropenia
B. Functional
1. Chronic Granulomatous Disease
(CGD)
- is a disorder caused by the inability
of phagocytes to produce superoxide
& reactive oxygen species
- results in the predisposition to
bacterial & fungal infections & the
formation of granulomas that can
obstruct hollow organs such as the
stomach, intestines & urinary tract
- The majority of cases are X-linked
recessive (60-65%) while (35-40%)
are autosomal recessive while some
are due to point mutations.
- PMNs demonstrate a weak or
negative staining with nitroblue
tetrazolium reduction test (NBT),
<10% of the blood granulocytes.
2. Myeloperoxidase deficiency/Alius-
Grignaschi anomaly
- is probably the most common
neutrophil abnormality inherited in
an autosomal recessive manner but
usually not a severe functional
abnormality
- MPO mediates oxidative destruction
of bacteria by H2O2
- The discovery of MPO is credited to
automated hematology analyzers
that use MPO to identify cells
- The MPO gene is located on CHR 17
& up to 10 mutations have been
described.
3. Leukocyte Adhesion Disorders (LAD)
- results in the inability of PMNs &
monocytes to adhere to endothelial
cells & to transmigrate from the
blood to the tissues.
- The basic defect is a mutation in the
genes responsible for the formation
of adhesion molecules, including
integrins & selectins
4. Lazy leukocyte syndrome
- rare condition in which both
random & directed movement of
cells are defective & there are
recurrent mucous membrane
infections.
- Granulocytes do not respond to
chemotactic factors.
5. Job’s Syndrome
(hyperimmunoglobulin E)
- rare neutrophil motility disorder
(directional motility is impaired)
- cells respond very slowly to
chemotactic agents
6. Congenital C3 deficiency
- rare autosomal recessive trait in
which asymptomatic carriers have
half the
normal C3 activity
Lipid storage diseases affecting
monocyte-macrophage
1. Gaucher disease
- caused by deficiency of β-
glucocerebrosidase (removes glucose
from sphingolipid-glucosylceramide)
leading to
accumulation of cerebroside in
macrophage
- described as large, 1-3 eccentric
nuclei with wrinkled cytoplasm
2. Niemann-Pick disease
 - another inherited anomaly in lipid
metabolism
- deficiency in sphingomyelinase (an
that separates phosphoryl choline
from sphingomyelin) leading to
accumulation of sphingomyelin in
macrophage
- Pick cell has foamy cytoplasm
Calculating the dilutions for the pipette:
Bulb Units = Dilution Factor
Blood Unit
RBC: 100 = 200; The dilution is 1:200
5
WBC: 10 = 20; The dilution is 1:20
5

Diluting Fluids
Characteristics of an Ideal Diluting Fluid:
1. Isotonic- for RBC; Hypotonic- for
Hemocytometry WBC
- numerical evaluation of formed 2. Inexpensive/economical
elements or the estimation of the 3. Easy to prepare & secure
number of blood cells in a known 4. With preservative action
volume of blood 5. Does not initiate the growth of
molds
Consists of the ff. materials: 6. With buffer action
1. Thoma pipettes- RBC & WBC 7. Non-allergenic, non-corrosive
2. Diluting fluids 8. With high specific gravity
3. Counting chamber 9. Stable

Thoma Pipettes RBC Diluting Fluids:

- parts: stem & bulb 1. Dacie’s (Formol Citrate)- keeps for a
long time; formalin acts as the
Characteristic RBC WBC preservative; preserves the morphology;
best diluting fluid
Size of the bulb Large Small
Color of the bead Red White 40% solution of 10 mL
formaldehyde
Volume of the 100 10
bulb 3% w/v trisodium citrate 990 mL
Upper 101 11
Calibration/Mark 2. Hayem’s- usually develops yeast
colonies & produces clumping of cells but
can stand for long periods of time & non-
corrosive

Mercuric chloride 0.5 g

Sodium sulfate, 5g
crystalline 2.65 g

anhydrous
Sodium chloride 1g
Distilled water 200 mL
How to use the Thoma pippetes:
1. Aspirate blood to 0.5 (most common)
or 1.0 mark 3. Gower’s- prevents rouleaux formation &
2. Dilute with diluting fluid to 101 for precipitation of protein
RBC or 11 for WBC Glacial acetic acid 0.85 g
3. Mix using figure 8 motion.
4. Discard the volume in the stem Sodium sulfate 6.25 g
(approx. 4 drops) since this contains
diluting fluid. Distilled water 100 mL
5. Use blood from the bulb in charging
the hemocytometer.
Sodium chloride 0.85 g Counting Chamber or Hemocytometer
• consist of a thick rectangular
Sodium sulfate 40.5 g colorless glass slide
• in the center are ruled areas
Glycerin 33.3 g separated by moats from the rest of
the slide & two raised transverse
Distilled water 100 mL bars
• the ruled portion may be in the
center of the central area (single
4. Toisson’s Fluid- has high specific chamber) or there may be an upper
gravity; stains WBC but supports the & lower ruled portion (double
growth of fungi chamber)
• Types: 1. Fuchs-Rosenthal
Sodium chloride 1g
2. Speirs-Levy
Sodium sulfate 8g 3. Improved Neubauer

Glycerin 30 g

Distilled water 180 mL

Methyl Violet 0.025 g

5. Normal Saline Solution (NSS)- stable;

acts as a preservative; used in emergency
cases; prevents rouleaux formation
Sodium chloride 0.85 g 1. Fuchs Rosenthal
• the ruled area measures 4x4 mm &
Distilled water 100 mL the depth is 0.2 mm
• the central ruling is divided into 16
6. Bethel’s smaller squares of 1 mm2
• larger than the Neubauer
Sodium sulfate, crystalline 5g • used for low cell counts, such as
Sodium chloride 1g eosinophil count, CSF fluid,
leukopenic counts
Glycerine 20 g
Sodium merthiolate, 2 mL
1:1000
Distilled water 200 mL

7. 3.8% Sodium Citrate

Sodium citrate 3.80 g
Distilled water 100 mL 2. Speirs-Levy
• consists of 4 platforms per counting
area
WBC Diluting Fluid • each ruled area consists of 10
1. 1%-3% Acetic Acid with Gentian squares each measuring 1x1 with a
Violet total area of 10 mm2, arranged in 2
Glacial acetic acid 2g horizontal rows of 5 squares which
are further subdivided into 16
Gentian violet 1 mL
squares
Distilled water 100 mL • the depth of the chamber is 0.2 mm
2. 1% HCl
Hydrochloric acid 1 mL
Distilled water 100 mL
3. Tuerk’s
Glacial acetic acid 2 mL

Methyl violet 1 mL
3. Improved Neubauer
Distilled water 100 mL • has 2 identically ruled platfoms with
a raised ridge on both sides of the 2
 platforms on which a cover glass is be ignored
placed
• the space between the top of the
platform & the cover glass is 0.1 mm

Each platform has:

• 9 large squares, each square is
1 mm wide x 1 mm long; the entire
ruled area is 9 mm2
• the volume of the entire ruled area
is 0.9 uL
• the volume of 1 large square is 0.1
uL
• the 4 corner large squares are
subdivided into 16 intermediate
squares or tertiary squares; these
squares are for WBC counts

• the middle large square is divided

into 25 tertiary or intermediate
squares, each of these tertiary or
intermediate squares is further
subdivided into 16 smaller squares
• the middle large square has a
volume of 0.1 uL
• the volume of each of the 25 squares
is 0.004 uL for a total of 0.02 uL for
5 squares

Counting of cells:
✓ Cells that touch the top & left lines
should be
counted
✓ Cells that touch the bottom & right
lines should