UNIVERSITY OF SANTO TOMAS

FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 
HEMATOLOGY

THE BLOOD

 GENERAL CHARACTERISTICS
 is in fluid form in vivo
 coagulates in vitro within 5-10 minutes
 7-8% of total body component or 75-85 mL blood per kg body mass
 20 grams solid per kilogram mass
 Adult Males : 5-6 L
 Adult Females : 4 – 5 L
 Newborns : 250 – 350 mL
 pH : 7.35 – 7.45
 Venous Blood has pH of 7.35
 Arterial Blood has pH of 7.45
 Specific Gravity : 1.045 – 1.066
 FUNCTIONS
 Respiratory – most important function
 Nutritional
 Excretory
 Buffering Action
 Maintenance of Body Temperature
 Transportation of Hormones & other endocrine secretions that regulate cell function
 Body Defense Mechanism
 BLOOD COMPOSITION
 55% Plasma
 91% Water
 7% Blood Proteins
- 58% Albumin
- 38% Globulin
- 4% Fibrinogen
 2% Nutrients, Hormones, Electrolytes
 45% Cellular Components / Formed Elements
 Buffy Coat – WBCs and Platelets
 Red Blood Cells
 Noncellular Components <1%
 Chylomicrons
 Blood Dust
- small refractive particles in the circulating blood, probably lipid material
associated with fragmented stroma from red blood cells
 Hemoconia
- small particles of lipids formed by fragmentation of the stroma of erythrocytes
- Tyndall Effect : hemoconia appearing as bright points in DF Microscopy
 PLASMA
 Liquid portion of blood in natural free-flowing state - that is UNCLOTTED BLOOD
 Normally appears hazy and pale yellow
 Contains all coagulation proteins
 SERUM
 Liquid Portion of coagulated blood
 Normally appears transparent and pale yellow
 Lacks Fibrinogen
 BLOOD HOMEOSTASIS

ONTOGENY OF HEMATOPOIESIS

 STEM CELLS
 Origin of Blood Cells  Hematopoietic Stem Cells
 Totipotential Stem Cells
 Present in the first few hours after an ovum is fertilized
 Most versatile type of stem cell
 Can develop into any human cell type
 Pluripotential Stem Cells
 Present several days after fertilization
 Can develop into any human cell type except that they cannot develop into a fetus
 Multipotential Stem Cells
 Derived from Pluripotential Stem Cells
 Limited to specific types of cells to form tissues
 Example : Bone marrow stem cells
 I – EMBRYONIC PHASE / MESOBLASTIC PHASE
 Excluding the lymphocytes, embryonic blood cells originate from the mesenchymal tissue that
arises from the mesoderm.

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
 
 Cells from the mesoderm migrate to the yolk sac
 Some of these cells become primitive erythroblasts in the central cavity of yolk sac
 Others, termed angioblasts, surround the cavity of yolk sac and form blood vessels
 Major Anatomic Site progresses from Yolk Sac to Hepatic Phase to the Bone Marrow.
 Yolk Sac Phase
 Hematopoiesis begins as erythrocyte precursors appear at 2 weeks of gestation
 Primitive Hematopoiesis : inside yolk sac structures called as Blood Islands
- Begins on day 19 and continues until week 8 of gestation
- Generates Erythrocytes, Macrophages, and Platelets
- Primitive Eryhtroblasts : Megaloblastic; formed intravascularly; nucleated
- Contain Embryonic Hemoglobins (Gower and Portland)
 MESOBLASTIC PERIOD
 Definitive Erythropoiesis : begins 1-2 days later than Primitive Hematopoiesis
- Starts with the formation of self-renewing HSCs in Aorta-Gonad-Mesonephros
region (a mesodermally derived intraembryonic region)
- Definitve Erythroblasts : non-nucleated; formed extravascularly in the liver
 II – FETAL HEPATIC PHASE
 Begins at 5 – 7 weeks of gestation; major site at 2nd month of gestation
 Predominates from about 2nd to 5th month of gestation
 Characterized by recognizable clusters of erythroblasts, granulocytes, and monocytes that
colonize the fetal liver, spleen, thymus, placenta, and ultimately the bone marrow
 Reaches peak at third month of fetal month
 Predominant type of Hemoglobin is Hemoglobin F
 Production of Megakaryocytes begins
 Thymus becomes major site of T-Cell production
 Kidneys and Spleen produce B-Cells
 III – MEDULLARY PHASE / MYELOID PHASE
 Starts prior to fifth month of fetal development
 5th fetal month – bone marrow assumes ultimate role as primary site of hematopoiesis
 HSCs and mesenchymal cells migrate into the core of the bone
 Gradually reach 3:1 myeloid:erythroid ratio
 HEMATOPOIESIS TREND
 Overall Cell Size as cells mature decreases except for megakaryocitic cell line
 Nuclear:Cytoplasm Ratio or the amount of space occupied by nucleus in relation to the space
occupied by the cytoplasm decreases as cells mature
 Chromatin pattern becomes more condensed as cells mature
 Monocytes have a lacy pattern which becomes finer as they mature
 Nucleoli will not be visible in mature cells. This is realted to the ribosomal RNA rate of synthesis
 Loss of basophilia
 Granulation increases with maturity
 Vacuolation increases with maturity

HEMATOPOIETIC ORGANS AND TISSUES

 BONE MARROW
 Found within cavities of all bones
 3.5 – 6.0% of total body mass
 Two forms : Yellow Marrow and Red Marrow
 Retrogression : yellow marrow repaces active marrow
 Results in restriction of active marrow to flat bones (the sternum, vertebrae, pelvis, ribs, &
skull) and epiphysis of long bones
 Yellow Marrow is able to revert to active marrow when there is increased demand for blood
production.
 Red Marrow
 Composed of HSCs and Macrophages arranged in extravascular cords
- Cords are located in spaces between the vascular sinuses and are supported by
trabeculae of spongy bone.
 Erythroblasts
- Mature Forms located adjacent to outer surface if vascular sinuses
- Develop in small clusters; Found surrounding iron-laden macrophages
(Erythroblastic Islands)
- Erythroid Cells account for 5-38% of nucleated cells
 Megakaryocytes
- Located adjacent to the walls of vascular sinuses, which facilitates release of
platelets into the lumen of sinus
- Develop into platelets in approximately 5 days
- Protrude through the vascular wall as small cytoplasmic processes to deliver
platelets into the sinusoidal blood
 Immature Granulocytic Cells
- Located deep within the cords
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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
 
- Move closer to the vascular sinuses as they mature and differentiate
- 23 – 85% of nucleated cells
- Maturing cells spend an average of 3 to 6 days in proliferating pool.
- If needed, released from storage pool; average life span of 6-10 hrs
 Hematopoietic Microenvironment
 Aka niche
 Contains Stromal Cells which secrete a semifluid extracellular matrix that serves to
anchor developing hematopoietic cells in the bone cavity.
- Adipocytes
o play a role in regulating the volume of the marrow in which active
hematopoiesis occurs
o secrete cytokines or growth factors
- Endothelial Cells
o Production of soluble growth and differentiation factors
o regulate the flow of particles entering and leaving hematopoietic spaces
in the vascular sinuses
o Expresison of homing receptors
- Fibroblasts
o Production of integral membrane proteins
o Reticular Adventitial Cells
o cytokine production
- Macrophages
o Phagocytosis
o secrete various cytokines that regulate hematopoiesis
- Osteoblasts
o Bone-forming cells
o Production of extracellular matrix components
- Osteoclasts
o Bone Resorption
- Lymphocytes
o secrete various cytokines that regulate hematopoiesis
o account for 1-5% of nucleated cells in the normal bone marrow
 Extracellular Matrix
- fibronectin,collagen, laminin, thrombospondin, tenascin, and proteoglycans (such
as hyaluronate, heparan sulfate, chondroitin sulfate, and dermatan).
 THYMUS
 originates from endodermal and mesenchymal tissue
 Derived from third and fourth pharyngeal pouches
 located in the upper part of the anterior mediastinum at about the level of the great vessels of the
heart
 populated initially by primitive lymphoid cells from the yolk sac and the liver.
 This increased population of lymphoid cells physically pushes the epithelial cells of the
thymus apart; however, their long processes remain attached to one another by
desmosomes.
 In adults, T cell progenitors migrate to the thymus from the bone marrow for further maturation.
 It consists of two lobes, each measuring 0.5 to 2 cm in diameter, and is further divided into
lobules
 the cortex (a peripheral zone)
- characterized by a blood supply system that consists only of capillaries.
- Its function seems to be that of a “waiting zone” densely populated with
progenitor T cells.
- When progenitor T cells migrate from the bone marrow and first enter the
thymus, they have no identifiable CD4 and CD8 surface markers (double
negative), and they locate to the corticomedullary junction
- Under the influence of chemokines, cytokines, and receptors, these cells move to
the cortex and express both CD4 and CD8 (double positive).
- Subsequently they give rise to mature T cells that express either CD4 or CD8
surface antigen as they move toward the medulla
 the medulla (a central zone)
- contains only 15% mature T-cells
- seems to be a holding zone for mature T cells until they are needed by the
peripheral lymphoid tissues
- a subset of epithelial cells (medullary thymic epithelial cells) present self-antigen
to developing T-cells and causing tbeir deletion in they are reactive to the self-
antigen
 Gradually atrophies loses up to 95% of its mass during the first 50 years of life
 SPLEEN
 Highly vascularized organ (receives 350mL blood/min) with the major functions of removing aging
and damaged blood cells (culling) and particles (pitting)
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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
 
 Culling – removal of senescent/abnormal rbcs in the blood by the spleen
 Pitting – does not destroy the erythrocyte
- Plucking of particles from intact erythrocytes
- Results in formation of spherocytes
 Stores platelets. About 1/3 are sequestered in the spleen
 Contains largest collection of lymphocytes and macrophages
 The only lymphoid organ with a majority of B lymphocytes than T-lymphocytes
 Organized into three zones
 White Pulp
- Initiates immune reactions involving cellular and humoral immunity
- Lymphoid cells form a cylindrical cuff around splenic arterioles which are mainly
T-cells (Periarteriolar Lymphatic Sheath)
o At branchpoints are lymphoid nodules containing B-cells
- Activated B-Cells are found in the germinal center
 Red Pulp
- Reservoir of platelets sequestering 1/3 of circulating platelet mass
- Composed primarily of vascular sinuses separated by cords of reticular cell
meshwork (cords of Billroth) containing loosely connected specialized
macrophages.
- Sinusoids are lined by discontinous epithelium allowing passage of cells between
cords and sinuses
- Sinuses are lined by macrophages which are lossely connected creating a filter
through which the blood can seep
o Trap red cell inclusions and senscent rbcs
o In normal adult, up to 2L of blood per minute will filter through the spleen
 Marginal Zone
- surrounds the white pulp and forms a reticular meshwork containing blood
vessels, macrophages, memory B cells, and CD4+ T cells.

HEMATOPOEITIC GROWTH FACTORS

 Erythropoietin
o Glycoprotein hormone produced by the peritubular cells of the kidneys and Kupffer cells of the
liver
o Gene is encoded in Chromosome 7
o Target : CFU-E, late BFU-E, CFU-Meg
o Production is stimulated by hypoxia
o Functions
 Accelerates rate of mRNA and protein synthesis
 Decreases time of maturation of metarubricytes
 Stimulates release of reticulocytes from the bone marrow
 Increases rate of enucleation
 Interleukin 3
o Produced by Activated T-cells
o Targets Eosinophils, monocytes, and a wide-variety of progenitor cells
 Granulocyte-Colony Stimulating Factor
o Produced by Monocytes, Fibroblasts, and Endothelial Cells
o Targets CFU-G and Granulocytes
 Macrophage-Colony Stimulating Factor
o Produced by G-CSF producing cells
o Targets CFU-M and Monocytes
 Granulocyte-Macrophage-CSF
o Produced by T-cells, Eosinophils, Monocytes, Fibroblasts, and Endothelial Cells
o Targets various CFU cells and Granulocytes

ERYTHROPOIESIS

 Occurs in the cords of BM specifically in erythroblastic islands

 Nutritional and Regulator Factors
o EPO
o Iron
o Vitamins (Vitamin B12)
o Hormones

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
 

 PRONORMOBLAST / RUBRIBLAST
 The nucleus takes up much of the cell (N:C ratio of 8:1). The nucleus is round to oval, containing
one or two nucleoli.
 The purple red chromatin is open and contains few, if any, fine clumps.
 The cytoplasm is dark blue because of the concentration of ribosomes.
 The Golgi complex may be visible next to the nucleus as a pale, unstained area.
 may show small tufts of irregular cytoplasm along the periphery of the membrane
 undergoes mitosis and givesrise to two daughter pronormoblasts.
 More than one division is possible before maturation into basophilic normoblasts.
 begins to accumulate the components necessary for hemoglobin production.
 The proteins and enzymes necessary for iron uptake and protoporphyrin synthesis are produced
 Globin production begins
 BASOPHILIC NORMOBLAST / PRORUBRICYTE
 The chromatin stains deep purple-red
 the cytoplasm may be a deeper, richer blue than in the pronormoblast—hence the name
basophilic for this stage
 Detectable hemoglobin synthesis occurs
 many cytoplasmic organelles, including ribosomes and a substantial amount of messenger
ribonucleic acid (RNA; chiefly for hemoglobin production), completely mask the minute amount of
hemoglobin pigmentation.
 POLYCHROMATIC NORMOBLAST / RUBRICYTE
 No nucleoli
 This is the first stage in which the pink color associated with stained hemoglobin can be seen
 color produced is a mixture of pink and blue, resulting in a murky gray-blue
 the last stage in which the cell is capable of undergoing mitosis
 ORTHOCHROMIC NORMOBLAST / METARUBRICYTE
 The nucleus is completely condensed / pyknotic
 increase in the salmon-pink color of the cytoplasm reflects nearly complete hemoglobin
production
 Late in this stage, the nucleus is ejected from the cell
 nucleus moves to the cell membrane and into a pseudopod-like projection
 loss of vimentin, a protein responsible for holding organelles in proper location in the
cytoplasm, is probably important in the movement of the nucleus to the cell periphery.
 enveloped extruded nucleus, called pyrenocyte is then engulfed by bone marrow
macrophages
 macrophages recognize phosphatidlyserine on the pyrenocyte surface as an “eat me”
flag
 POLYCHROMATIC ERYTHROCYTE / RETICULOCYTE
 No nucleus
 the cell is the same color as a mature RBC, salmon pink. It remains larger than a mature cell,
however.
 The shape of the cell is not the mature biconcave disc but is irregular in electron micrographs
 The polychromatic erythrocyte resides in the bone marrow for 1 day or longer and then moves
into the peripheral blood for about 1 day before reaching maturity.
 During the first several days after exiting the marrow, the polychromatic erythrocyte is retained in
the spleen for pitting of inclusions and membrane polishing by splenic macrophages, which
results in the biconcave discoid mature RBC
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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
 
 The cytoplasmic protein production machinery is simultaneously being dismantled.
 Endoribonuclease, in particular, digests the ribosomes.
 ERYTHROCYTE
 7 to 8 µm in diameter, with a thickness of about 1.5 to 2.5 µm.
 salmon pink-staining cell with a central pale area that corresponds to the concavity
 central pallor is about one third the diameter of the cell.
 Mature RBCs remain active in the circulation for approximately 120 days
 Senescent RBCs (those that lived 120 days) are removed from circulation and culled by the
spleen
 The cell’s main function of oxygen delivery throughout the body requires a membrane that is
flexible and deformable—that is, able to flex but return to its original shape

HEMOGLOBIN

 Globular Protein; Conjugated protein

 Consists of two different pairs of polypeptide chains and four heme

groups

 tetramer of four globin polypeptide chains with a heme molecule

attached to each chain

 95% of RBC’s dry weight

 Primary Structure : Amino Acid Sequence

 Variations in AA Sequence: result to Qualitative Hemoglobin

Disorders / Hemoglobinopathies

 Hb S = HbA1 β chain ( 6th aa Glutamic Acid  Valine)

 Causes rbc to become stiff and crescent-shaped

 Hb C = HbA1 β chain (6th aa Glutamic Acid  Lysine)

 Most common; may cause mild hemolytic anemia if homozygotic

 Hb M = HbA1 α chain (58th aa Histidine  Tyrosine)

 Favors formation of methemoglobin

 Hb E = HbA1 β chain (26th aa Glutamic Acid  Lysine)

 Most prevalent worldwide; third most common

 Mostly in SEA

 Hb D (Punjab) = HbA1 β chain (121st aa Glutamine  Glutamic Acid)

 Secondary Structure : 8-helices separated by 7 non-helical segments

 Tertiary Structure : Folding; Pretzel-Like Configuration

 Quaternary Structure : two α-β dimers of globin

 α1 will bind to β2 and α2 will bind to β1

 HEME

 Protoporphyrin IX – ring of carbon, hydrogen, and nitrogen atoms with a central atom Fe2+

 GLOBIN

 Each Globin chain comprises of about 141 to 146 aa

 Alpha Series

 Zeta  alpha | 141 aa | Short arm Chrom16

 Beta Series

 Epsilon  Gamma  Beta | 146 aa | Short Arm Chrom 11

 Functions

 Oxygen Transport

 1.34 mL O2 is bound by each gram of hemoglobin

 Oxygen Tension in tissue is directly related to Oxygen-Hemoglobin Affinity

 P50 – Partial Pressure of Oxygen needed to saturate hemoglobin by 50%.

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
 
 P50 is inveresly related to Oxygen Affinity of Hemoglobin

 Oxygen-Dissociation Curve
 Hb-Oxygen Curve is Sigmoidal.
 Mb-Oxygen Curve is hyperbolic
 Affected by H+, temperature, and 2-BPG
o Increased factors = shift right  increased affinity to Oxygen
o Bohr Effect – effect of hydrogen ion concentration and carbon dioxide in
oxygen’s affinity for hemoglobin

 CO2 and Nitrogen Transport

 DYSHEMOGLOBINS

 CARBOXYHEMOGLOBIN

 Carbon Monoxide

 200x greater affinity than oxygen

 Reversible by increasing oxygen concentration

 Cherry-red color ; measured at 541 nm

 Toxic at 10 to 15%

 Smokers will have 4-20% Carboxyhemoglobin

 50% = coma and convulsions

 METHEMOGLOBIN

 Formed when Ferrous iron is oxidized to Ferric state

 Reversed by administrationg strong reducing substances such as ascorbic acid

 Can be hereditary (Hb M)

 Can be congenital (Methemoglobin Reductase deficiency)

 Brownish to bluish; measured at 620-640 nm at pH 7.1

 1% of total hemoglobin in normal adults

 > 30% will yield to hypoxia / cyanosis

 SULFHEMOGLOBIN

 sulfur content in the blood builds up (ingestion of sulfur-containing drugs or exposure to

sulfur chemicals)

 irreversible

 greenish color ; mauve when mixed with blood

 Measured at 620 nm

 ANALYTICAL METHODOLOGIES

 Points to Remember

 Per gram of Hb = 1.34 mL Oxygen = 3.47 mg Ferrous iron

 34 g/dL within RBC

 Classical Methods

 Copper Sulfate / Specific Gravity Method

 1.053 specific gravity of blood

 Iron Content / Kennedy Wong’s Method

/
Hb =
. /

 Oxygen Combining / Van Slyke / Gasometric Method

/ /
Hb =
. /

 Colorimetric Methods
 Direct Matching

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
 
 Blot & Match with color
 Tallquist & Dare’s Method
 Acid Hematin
 0.1 N HCl
 HbF resists acid elution
 Principle : Acid Elution / Kleihauer-Betke
 Alkaline Hematin
 0.1 N NaOH
 Principle : Precipitation
 Oxyhemoglobin
 Photometric
 0.007 N NH4OH
 Read at 540 nm
 Cyanmethemoglobin
 Photometric
 Most reliable except that it cannot measure sulfhemoglobin
 Uses Drabkin’s Reagent
o Sodium Bicarbonate = lysing agent
o Potassium Ferricyanide = oxidizes ferrous to ferric
o Potassium Cyanide = stabilizes ferric iron = Cyanmethemoglobin
 Read at 540 nm
 Automated Analyzers utilize Sodium Lauryl Sulfate = SLS-MetHb
 METABOLISM AND BIOSYNTHESIS
 BIOSYNTHESIS
 FACTORS
 Adequate supply and delivery of iron
 Adequate protoporphyrins
 Adequate globin chains
 Ontogeny
 Globin synthesis is first detected in
the primitive erythroid precursors of
the yolk sac at about 3 weeks
gestation
 Only during the first three months of embryonic life do the ζ and ε globin chains
appear (Gower-1, Portland, Gower-2 Hemoglobins)
 During the second and third trimesters of fetal life and at birth, the predominant
hemoglobin is HbF (with 2 α and 2 γ globin chains)
 By 6 months of age through adulthood, the predominant hemoglobin is Hb A.
Small amounts of HbA2 and Hb F are seen as well.
 Globin Synthesis
1. Short-Arm Chromosome 16 controls B-like cluster globin genes responsible for
the biosynthesis of B-globin inside the polyribosomes.
2. Short-Arm Chromosome 11 controls the a-like cluster globin genes responsible
for the biosynthesis of a-globin inside the polyribosomes.
3. Globin Chains are relased from the ribosome at the cytosol where it will meet the
heme.
 Heme Synthesis | First and Last stages occur in the mitochondria
1. (MITO) Glycine reacts with Succinyl CoA  Aminolevulinic Acid
 Catalyzed by Aminolevulinic acid Synthase.
 The action of ALA Synthase is influenced by Erythropoietin and Vitamin
B6.
 ALA synthase is the rate limiting step
2. Aminolevulinic Acid goes out of the Mitochondria  Porphobilinogen
 via Aminolevulinic acid dehydrogenase.
3. Four molecules of Porphobilinogen condense  Hydroxymethylbilane
 Via Porphobilinogen deaminase.
4. Hydroxymethylbilane undergoe cyclization-isomerization  Uroporphyrinogen III
 Via Uroporphyrinogen III Synthase
5. Uroporphyrinogen III decarboxylation-dehydrogenation  Coproporphyrinogen III
 Uroporphyrinogen decarboxylase
6. Coproporphyrinogen III back in Mitochondria  Protoporphyrinogen IX
 Coproporphyrinogen oxidase.
7. Protoporphyrinogen IX  Protoporphyrin IX
 Protoporphyrinogen oxidase
8. Iron is added to Protoporphyrin to form Heme.
9. Heme goes out of the cytoplasm and meets with globin chains which are
released from the ribosomes.
10. Assembly of Hemoglobin as tetramer.
 Individual alpha and beta chains quickly and spontaneously form alpha-
beta dimers.
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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
 
 Two heme molecules bind to each alpha-beta dimer.
 Two dimers quickly form a tetramer and assume final three dimensional
shape.
11. Insertion of 2,3-bisphosphoglycerate.
 Regulation
 Heme, when produced, will inhibit transcription of the ALA synthase gene leading
to a decrease in further production of heme. (Negative Feedback Mechanism)
 Heme will also inhibit other enzymes used in its biosynthesis such as ALA
dehydrase and PBG deaminase.
 Ferrochelatase Enzyme is inhibited by heme feedback mechanism or substrate
inhibition by protoporphyrin IX.
 When Heme level is low, ALA Synthase synthesis will be induced and the
translation of globin mRNA will be blocked until such time that heme level returns
to normal and is ready for hemoglobin assembly.
 When there is low levels of hemoglobin or if the hemoglobin molecule is
incapable of transporting oxygen, hypoxia results. This will then be detected by
the peritubular cells of the kidney which then will increase Erythropoietin
production.
 EPO will accelerate synthesis of erythrocyte components including hemoglobin
by increasing erythrocyte production.
 Reference Intervals for Hb
o Men: 14 to 18 g/dL
o Women: 12 to 15 g/dL
o Newborns: 16.5 to 21.5 g/dL
 Catabolism
 Extravascular
 Hemoglobin is disassembled once an erythrocyte is phagocytized and digested
by macrophages of the reticuloendothelial system.
 Hemoglobin is disassembled to heme (further disassembled to iron and
protoporphyrin) and globin.
 Iron is transported in the plasma by transferrin
 Globin is catabolized in the liver into its constituent amino acids
 Protporphyrin ring is broken by heme oxidase enzyme.
 Alpha carbon leaves as Carbon Dioxide
 Ring becomes bileverdin then bilirubin.
 Bilirubin is carried by the plasma albumin to the liver for conjugation with
glucoronide.
 Bilirubin glucoronide is excreted into the gut and released in the feces as
stercobilinogen.
 Some bilirubin glucoronide is moved to the kidney and converted to urobilinogen
which is excreted in the urine. Some urobilinogen are reabsorbed.
 Intravascular
 When there is intravascular destruction of erythrocytes, hemoglobin is directly
released into the bloodstream and dissociates into alpha and beta dimers.
 The dimers are bound quickly by haptoglobin to prevent urinary excretion. The
complex formed is removed from circulation via hepatocytes.
 Unbound dimers (due to decreased levels of plasma haptoglobin after which they
are removed from circulation together with the complexed haptoglobin) are
rapidly filtered by glomeruli in the kidneys and are reabsorbed by the renal
tubular cells then converted to hemosiderin.
 Hemoglobin that did not dissociate will be oxidized to methemoglobin whose
heme group will be taken up by the transport protein hemopexin. Heme groups in
excess will combine with albumin to form methemalbumin until more hemopexin
is available.
 Regulation
 Oxygen-binding affinity of hemoglobin is regulated by the organic phosphate 2,3-
bisphosphoglycerate via the Luebering-Rapaport Shunt.
 Will be inserted in the central cavity of hemoglobin molecule and bind to the beta
globin chains.
 Once inserted, the hemoglobin is converted to its T (tense) Form characterized
by the beta-chains being farther from each other.
 Oxyhemoglobins are stimulated to deliver oxygen.
 In the same manner, when oxygen binds, 2,3-BPG is released. Hemoglobin is
converted to its R (relaxed) Form. The conversion is referred to as respiaratory
movement.

Page 9 of 23 
 
UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
IRON
Iron Chemistry
 Elemental Symbol Fe derived from its Latin name
Ferrum
 Atomic Number 26 with Molecular Weight of 56
g/mol
 Ion Forms are Fe2+ and Fe3+
 Body contains approximately 3g to 4g Iron
Iron Kinetics
 Iron in its reduced state is transported inside
enterocytes of duodenum/jejunum via Divalent
Metal Transporter 1 (DMT-1).
2+
 Dietary Iron may be Heme Iron (Fe ) or Non-
Heme Iron (Fe3+). Heme Iron is mostly acquired
from meat while non-heme iron is acquired
from legumes and leafy vegetables.
 Non-Heme Iron is reduced to Heme Iron via the
duodenal cytochrome b reductase. The uptake
of iron from non-animal sources is enhanced by
ascorbic acid and inhibited by tannic acid or
tannates.
 Heme may also enter enterocytes as hemin
and is acted upon by heme oxygenase to
liberate Fe2+.
 Ferroportin will transport out Fe2+ to the
bloodstream.
o The activity of Ferroportin is regulated
by Hepcidin ( a short protein with 25 aa
produced predominantly by
hepatocytes) which also regulates iron
absorption in the gut.
o Hepcidin stands for Hepatic
Bacteriocidal Protein and is known as
the master iron regulatory hormone. Its
synthesis is controlled by Chromosome
19.
 In the cell membrane of gut cells, it will be
converted to Fe3+ by Hephaestin (as a cofactor
Laboratory Tests
Routine Tests
1. Serum Iron Concentration
 Spectrophotometric Method
 Low Levels Indicate Iron Deficency, Inflammations, and Pre-Menstrual State
 High Levels Indicate Iron Overload, Pregnancy, and recent iron ingestion
2. Total Iron Binding Capacity / Transferrin Test
 May be performed as chemical test (indirect) or via immonologic method (direct)
 Low Levels indicate High Body Iron Stores, Malnutrition, and Chronic Diseases.
 High Levels indicate Low Body Iron Stores and High Estrogen States
3. Transferrin Saturation
 Percent of Transferrin filled with Iron
 Best serum marker of increased body iron
Compartment % Total Body Iron
Hemoglobin Iron 70%
Storage Iron
Ferritin 25%
Hemosiderin
Myoglobin Iron 5%
Other Sources <1%
Serum 0.1%
of Ferroportin) to allow it to be recognized by
and be able to bind to Transferrin.
o In the plasma, Fe2+ is oxidized to Fe3+
by Ceruloplasmin.
 Iron in its oxidized state may also be stored as
Ferritin in the liver, bone marrow, or spleen.
Ferric ion will bind to the protein apoferritin to
form Ferritin which possess an Iron-Phosphate-
Hydroxide Core.
 Transferrin, a protein primarily synthesized by
the liver, possess two diantennary
carbohydrate chains capable of binding two
Ferric ions. It contains 95% of serum iron and is
usually about 30% of transferrin is saturated
with Fe3+.
o Transferrin synthesis is diminished
when there is iron overload.
 Transferrin receptors found in the cell
membrane mediates the uptake of transferrin
via endocytosis (receptor-mediated because of
the need of transferrin receptors). Endosomes
will release the ferric iron if the pH is 5.5.
 Soluble Transferrin Receptors have been
discovered and is potent for the diagnosis of
iron deficiency but is not currently routinely
available. These are cell surface receptors
which are truncated and is found in the
circulation.
o STfRs are high when there is iron
deficiency.
Page 10 of 23 
UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
 
 Used as a screen for iron overload

 Combines Serum Iron and TIBC

 % Transferrin Saturation = (serum iron / TIBC) x 100

4. Serum Unsaturated Iron-Binding Capacity

 Iron Binding Sites not occupied; spectrophotometrically determined

 Equal to difference of TIBC and Serum iron

5. Ferritin Measurement

 Immonologic Method of Determination

 Reflects Iron Stores

 Low Serum Levels indicate Iron Deficiency with high specificity

 High Serum Levels indicate Iron overload or may be caused by Tissue Release (due to hepatitis,
leukemia, or lymphoma) or Acute Phase response to tissue damage, infection, or cancer.

Other Tests

1. RBC Protoporphyrin

 Uses Hematofluorometer

 Measures excess protoporphyrin

 Zinc is allowed to react with free protoporphyrin

 Zinc that reacted with the protoporphyrin is measured in the fluorometer

2. Hemosiderin

 Stained with Prussian Blue

 Normally, mature RBCs must not contain iron

 Prussian Blue stains as well Siderocytes (reticulocytes in the Bone Marrow with iron) and
Sideroblasts (nucleated RBCs/precursors in the Bone Marrow containing iron)

3. Serum Transferrin Receptors

 Not routinely available

 Immonologic Method

ERYTHROCYTE METABOLISM

 Embden-Meyerhoff Pathway
o Glycolysis
o Net ATP : 2 ATP
 Hexose-Monophosphate Shunt
o Production of Reduced Glutathione
 Luebering-Rapoport
o Allows 2,3-BPG to accumulate (which enhances delivery of oxygen to tissues)
 Methemoglobin-Reductase
o Prevents oxidation of ferrous iron in heme
o Prevents precipitation (formation of Heinz Bodies)

Page 11 of 23 
 
UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
 
ERYTHROCYTE MORPHOLOGY

 Average volume = 90 fL
 Average surface area = 140 micrometer square
 Biconcave
 40% excess surface area for membrane deformability to allow passage through narrow capillaries
 Plasma membrane is 5 micrometers thick and is 100x more elastic than latex and a tensile strength
greater than that of steel
 Membrane is 8% CHO, 52% CHON, and 40% Lipid
o Acanthocytosis = altered membrane lipid
o Transmembrane Proteins
o Cytoskeletal Proteins / Peripheral Proteins – do not penetrate bilayer
 Spectrin – membrane elasticity and mechanical stability
 Hereditary Elliptocytosis / Ovalocytosis
 Hereditary Spherocytosis – spectrin, ankyrin, band 3, protein 4.2
 Cation pumps maintain high levels of intracellular potassium ions

ERYTHROCYTE ABNORMALITIES AND DEFORMITIES

Page 12 of 23 
 
UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
 
ERYTHROCYTE INCLUSIONS

RBC INDICES

Page 13 of 23 
 
UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
 
ANEMIA

 decrease in the oxygen-carrying capacity of the blood.

ANEMIA DUE TO BLOOD LOSS

 Anemia as a result of acute blood loss or chronic blood

loss
 Etiology
 Acute
 Traumatic Injury
 Accident
 Post-surgery
 Chronic
 Heavy Menstruation
 GI tract disorders

DISORDERS OF IRON AND HEME

 Result in Microcytic Hypochromic RBCs

IRON-DEFICIENCY ANEMIA

 Pica is associated with IDA

 Etiology
 Decreased intake
 Increased usage
 Rapid growth
 Menstruation
 Pregnancy and Lactation
 Excessive loss
 Chronic Blood Loss
 Malabsorption
 Celiac Disease

Normal Decreased Decreased

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
 
ANEMIA OF CHRONIC DISEASES / ANEMIA OF INFLAMMATION

 Acquired Anemia
 2nd most prevalent after IDA
 Result of increases acute phase reactants which slows iron
release
o Hepcidin decreases iron release from macrophages
o Lactoferritin competes with transferrin for plasma
irons; rbcs lack lactoferritin receptors
o Ferritin binds iron; rbcs lack ferritin receptors
o MCV MCH NORMAL
o Hgb 9-11 d/dL
o Bmwill show increased iron stores in
macrophages

SIDEROBLASTIC ANEMIA

 Decreased activity of D-ALA

 Ringed sideroblasts are formed by mitochondria
containing nonferritin iron that circles metarubricytes
 Hereditary – severe anemia is seen
o X-linked / autosomal
 Acquired
o Refractory anemia
o Drugs
o Toxins
 Iron Studies
o Serum Fe : increased
o Ferritin : increased
o TIBC : Decreased
o % Sat : Decreased / Normal

HEMOCHROMATOSIS

Iron overload
Increased iron stores (ferritin and hemosiderin)
Acquired
o Multiple Transfusion
o Chronic Liver Disease
o Alcohol Abuse
o Dietary / Supplementary Iron Overload
 Hereditary / Inherited
o Type I  Hemojuvelin  protein;
 HFE gene related modulates hepcidin
 Classical  Hallmark : no / low hepcidin
 Bronze diabetes activation
 Autosomal recessive o Type 3
o Type 2  Mutation of transferrin receptor
 Juvenile hemochromatosis 2 gene
 2a  2 Hemojuvelin gene  Appears in midlife
mutations on chrom1q21 o Type 4
 2b  homozygosity mutation  Mutation of Ferroportin-1 gene
of Hepcidin Antimicrobial  Autosomal dominant
peptide gene on chrom19q3
MACROCYTIC ANEMIAS

MEGALOBLASTIC ANEMIAS

 Impaired DNA synthesis; N:C asynchrony; hypercellular BM

 Macrocytic; normochromic
 Vit. B12 / Cobalamin deficiency (including infections : H.pylori, D.latum, and Blind Loop Syndrome)
o Low when folic acid is low
o Result in neurologic symptoms
o MMA and Homocysteineis increased
o Low intrinsic factor – pernicious anemia
 IF binds cobalamin until it reaches ileum
 Folate deficiency
o Homocysteine is increased
 Schilling Test
o Determine cause of cobalamin deficiency
 Part I : detect malabsorption
 Measure urine

Page 15 of 23 
 
UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
 
 High excretion = unable to absorb
 Part II : lack of intrinsic factor
 Pernicious anemia : increased radiolabelled vitamin b12
 Pernicious anemia is characterized by achlorydia

BONE MARROW FAILURE SYNDROMES

 Hypoproliferative disorders
 Inadequate HSCs

APLASTIC ANEMIA

 Pancytopenia, Hypocellular BM
 Acquired Aplastic Anemia
o Idiopathic (70% of cases)
o Secondary (10-15%)
 Viruses such as EBV and HIV
 Idiosyncratic
 Iatrogenic – Benzene, Cytotoxic Drugs, Radiation
 Paroxysmal Cold Hemoglobinuria
 AI Disorders
 Pregnancy
 Inherited
o Fanconi’s Anemia
 Chromosomes are susceptible to breakage
 Accelerated telomere shortening
 FANCA mutations – autosomal recessive
 FANCB – X-linked
o Dyskeratosis Congenita
 Chromosomes have short telomeres
 Abnormalities in skin pigmentation
o Shwachman-Diamond Syndrome
 Autosomal recessive
 Neutropenia and/or anemia and thrombocytopenia
 Decreased pancreatic enzymes

PURE RED CELL APLASIA

 Affects Erythroid cells only

 Acquired
o Primary is idiopathic or autoimmune
o Secondary is associated with tumors infections drugs and chemicals
 Congenital
o Diamond-Blackfan
 Autosomal dominant
 Slowly progressive Refractory Anemia w/o leukopenia/thrombocytopenia
 Macrocytosis
 Notable characteristic : Elevated Adenosine Deaminase
 Elevated HbF
 Present of fetal membrane “I” antigen
o Congenital Dyserythropoietic Anemia
 Disorders Leading to ineffective erythropoiesis
 Autosomal recessive
 Indirect hyperbilirubinemia
 Type I – apparent at birth; not fatal
 Type II – most common
 + hemolysis in acidified serum test like in PNH
 React strongly with anti-I/i
o Anemia of Chronic Kidney Diseases
 Unable to produce EPO

HEMOLYTIC ANEMIAS

INTRINSIC DEFECTS HEMOLYTIC ANEMIAS

 Hereditary Spherocytosis : Increased MCHC and RDW

o Negative DAT
o Increased Osmotic Fragility
o Autosomal Dominant
 Hereditary Elliptocytosis
o Mutations for genes encoding spectrin or band 4.1
o Increased thermal sensitivity
 Acanthocytosis
Page 16 of 23 
 
UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
 
o Spur Cell Anemia
o Membrane lipid defect
o Splenomegaly and jaundice
 Neuroacanthocytosis
o Abetalipoproteinemia
o McLeod’s Syndrome
 Paroxysmal Nocturnal hemoglobinuria
o Lack Glycosylphosphatiylinositol-anchored proteins, CD55 and CD59
o Susceptibility to complement lysis
o Hemoglobinuria, intravascular hemolysis
o Reticulocytosis
 G6PD-Deficiency
o Heinz Bodies
o X-linked disorder
o Asymptomatic unless exposed to oxidants
 Pyruvate Kinase Deficiency
o Autosomal Recessive
o ATP depletion
o Increased 2,3-BPG

EXTRINSIC DEFECTS HEMOLYTIC ANEMIA

 Non-Immune Mediated
o Microangiopathic Hemolytic Anemia
 Schistocytes
 Decreased haptoglobin
 Increased unconjugated bilirubin
 Due to obstructed blood vessels from microclots or endothelial damage
o Disseminated Intravascular Coagulopathies
 Activation of coagulation cascade
o Thrombotic Thrombocytopenic Purpura
 Long vWF multimers triggering platelet activation
 <10 Hgb; < 20 x109 / L PLT ; Schistocytes
 Normal PT and aPTT
o Hemolytic Uremic Syndrome
 Microangiopathic HA with thrombocytopenia and renal involvement
 Enterohemorrhagic E. coli; Shigella dysnteriae
 Decreased RBC, Hgb, Plt
 BUN and Crea is elevated
 Protein, casts, blood + in urine
o Mechanical Injury
o Malaria and Babesia
o Clostridium perfringens
 Immune-Mediated
o AIHA
 WAIHA
 IgG
 Extravascular Hemolysis
 + DAT
 Cold Agglutinin Disease
 IgM
 Maybe secondary to Mycoplasma pnemoniae and viral infections
 If blood has cooled before analysis = abnormal CBC
o Hgb normal
o Agglutinates on PBS if high-titer Cold-agglutinin disease is present
 Heating dissociates agglutinins = + for the disease
 Paroxysmal Cold Hemoglobinuria
 Anti-P autoantibody (Donath-Landsteiner Antibody)
o Biphasic antibody
o Full-blown hemolysis at 37C
 Drug-Induced
 Alloimmune HA
 Transfusion Rxn

HEMOGLOBINOPATHIES

 Caused by structural changes in the hemoglobin molecule

Page 17 of 23 
 
UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
 

SICKLE CELL DISEASE

 Hgb S Homozygous (Hb SS Disease)

 Drepanocytes
 Dithionite Solubility = + (soluble)
 Normocytic, normochromic anemia
 No Hb A is produced; approximately 80% Hb S & 20% Hb F
 Hgb S migrates with hemoglobins D and G on alkaline hemoglobin electrophoresis; can differentiate
using acid electrophoresis.
 Immunity to P. falciparum
 Tissue necrosis

SICKLE CELL TRAIT

 Hb AS
 Apparent immunity to P. falciparum

HEMOGLOBIN CC

 lysine replaces glutamic acid at position 6 on both beta chains

 characterized by intracellular rodlike C crystals
 Hgb C migrates with hemoglobins A2, E, and O on alkaline hemoglobin electrophoresis; can differentiate
hemoglobins using acid electrophoresis.
 The heterozygous Hgb C trait patient is asymptomatic, with no anemia;
o the one normal beta chain is able to produce approximately 60% Hgb A and 40% Hgb C, with
normal amounts of Hgb A2 and Hgb F.

HEMOGLOBIN SC

 a double heterozygous condition where an abnormal sickle gene from one parent and an abnormal C
gene from the other parent inherited.
 No Hgb A is produced; approximately 50% Hgb S and 50% Hgb C are produced.
o Compensatory Hgb F may be elevated up to 7%.
 symptoms less severe than sickle cell anemia but more severe than Hgb C disease.

HEMOGLOBIN E

 when lysine replaces glutamic acid at position 26 on the beta chain.

 Homozygous condition results in mild anemia with microcytes and target cells; heterozygotes are
asymptomatic.
 migrates with hemoglobin A2, C, and O an alkaline hemoglobin electrophoresis

HEMOGLOBIN D

 Punjab
 when glycine replaces glutamic acid at position 121 on the beta chain.
 migrates with Hgb S and Hgb G on alkaline hemoglobin electrophoresis.

THALASSEMIAS

 Quantitative Hemoglobin Disorders

 Microcytic, Hypochromic rbc

α-Thalassemia

 Silent Carrier | -α/αα

o Normal
o One alpha gene deletion
 α-Thalassemia Minor / Trait
o Homozygous | (-α/-α)
Page 18 of 23 
 
UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
 
o Heterozygous | (--/αα)
o May be confused with iron deficiency anemia
 Hgb H disease | (--/-α)
o Second most severe form of alpha thalassemia
o Results in accumulation of excess unpaired gamma or beta chains.
 Born with 10-40% Bart's hemoglobin (γ4).
 Gradually replaced with Hemoglobin H (β4).
o Adults have about 5-40% HbH.
o RBCs are microcytic, hypochromic with marked poikilocytosis. Numerous target cells.
o HbH vulnerable to oxidation. Gradually precipitate in vivo to form Heinz-like bodies of denatured
hemoglobin. Cells been described as having "golf ball" appearance, especially when stained with
Brilliant Cresyl Blue
 α-Thalassemia Major (--/--)
o Bart’s Hgb / Hydrops Fetalis
o Hb Bart's has high oxygen affinity so cannot carry oxygen to tissues. Fetus dies in utero or
shortly after birth. At birth, you will see severe hypochromic, microcytic anemia with numerous
nRBCs.

State Genotype Genes Features

Normal / 4 normal

Hetero + / –  3 Essentially normal

-thal-2

Hetero ° / – – 2 Micro / Hypo

-thal-1
Mild Anemia
Homo + – / –  2
Bart’s 2-8% (at birth)
-thal-1
Hb H <2%

+ + ° – / – – 1 Moderate Micro/Hypo anemia: Barts

Hb-H Disease <10%, Hb H <40%

Homo ° ––/–– 0 Hb A 0%, Bart’s 70-80%

Hydrops
Portland 10-20%

β-Thalassemia

 Thalassemia Major / Cooley’s Anemia

 Homozygous
 Absence of beta chain
 Splenomegaly early onset
 Thickening of skull
 Thin cortex of long and flat bones (Hair-on-end appearance on X-Ray)
 Profound anemia stimulates an increase in EPO prodt’n by kidneys & results in massive (but
ineffective) erythroid hyperplasia
 Marked bone changes & deformities occur due to massive BM expansion
 Thalassemia Intermedia
 Patients able to maintain minimum Hb (7 g/dL or greater) without transfusion dependence.
 Expression of disorder falls between thalassemia minor and thalassemia major.
 There is an increase in both HbA2 production and HbF production.
 Peripheral blood smear picture is similar to thalassemia minor
 Have varying symptoms of anemia, jaundice, splenomegaly and hepatomegaly.
 Have significant increase in bilirubin levels.
 Anemia usually becomes worse with infections, pregnancy, or folic acid deficiency.
 May become transfusion dependent.
 Tend to develop iron overloads as result of increased gastrointestinal absorption.
 Usually survive into adulthood.
 Thalassemia Minor
 Heterozygous
 Decreased rate of synthesis of one of the beta chains; other beta chain normal
 Caused by heterozygous (from one parent) mutations that affect β globin synthesis.
 β Chains production and thus Hb-A production is more reduced than the silent carrier Hb-A.
 Usually presents as mild, asymptomatic hemolytic anemia unless patient in under stress such as
pregnancy, infection, or folic acid deficiency.
 Have one normal β gene and one mutated β gene.
 Anemia usually hypochromic and microcytic with slight aniso and poik, including target cells and
elliptocytes; also may see basophilic stippling.
 Rarely see hepatomegaly or splenomegaly.
 Have high HbA2 levels (3.6-8.0%) and normal to slightly elevated HbF levels.
Page 19 of 23 
 
UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
 
 Normally require no treatment.
 You have to make sure are not diagnosed as IDA.
 Mentzer index
 Differentiate IDA and thalassemia
 MCV/RBC count
 < 13 = Thalassemia
 > 13 = IDA
 Silent Carrier
 Are various heterozygous (from one parent) β gene mutations that produce only small decrease
in production of β globin chains.
 Patients have nearly normal alpha/beta chain ratio and no hematologic abnormalities.
 Have normal levels of HbA2.

LEUKOPOIESIS

 GRANULOCYTES
 Begins with Common Myeloid Progenitor Cells (CFU-GEMMs)
 CFU-GEMMs differentiate into Granulocyte-Macrophage Progenitors (GMPs) for Neutrophils and
Monocytes while Eosinophil-Basophil Progenitors for Eosinophils and Basophils
 GMPs or EBPs differentiate into Myeloblasts
 Neutrophil Maturation
 Myeloblasts make up 0-3% of nucleated cells
in BM
 Type I = 8:1 N:C ; no visible granules
 Type II = Primary Granules visible
 Type III – darker granule, more purple
cytoplasm
 Promyelocytes
 1-5% nucleated cells
o Paranuclear halo / hof /Evenly
basophilic
o Full of priamry granules
 Myelocytes
 Final stage capable of cell division
 Production of primary granules ceases
 Manufacture of secondary granules
begins
 Dawn of Neutrophilia
 Patches of grainy pale pink cytoplasm
 Metamyelocytes
 3-20% of nucleated cells
o Synthesis of tertiary granules
 Bands
 9-32% of nucleated cells in BM
 0 – 5% of nucleated cells in peripheral
blood cells
 Begin synthesis of secretory granules
 Segmented Neutrophil
 7-30% of nucleated cells in the marrow
 Continue forming secondary granules
 Half life 7 days
 Once in the peripheral blood,
neutrophils are divided randomly into a
circulating neutrophil pool (CNP) and a
marginated neutrophil pool (MNP).
 Eosinophil Maturation
 Myeloblasts dedicated to eosinophilic lineage
has not been established
 IL-5 is critical for eosinophil growth and
differentiation
 Eosinophilic Promyelocyte
 identified cytochemically due to the presence of Charcot-Leyden crystal protein in
their primary granules.
 Eosinophilic Myelocyte – earliest recognizable eosinophil
 characterized by the presence of large (resolvable at the light microscope level),
pale, reddish-orange secondary granules, along with azure granules in blue
cytoplasm.
 The nucleus is similar to that described for neutrophil myelocytes.

Page 20 of 23 
 
UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
 
Transmission electron
micrographs of eosinophils reveal
that many secondary eosinophil
granules contain an electron-
dense crystalline core
 Eosinophilic Metamyelocytes and bands
resemble neutrophil
 Mature Eosinophils
 cytoplasm contains characteristic
refractile, orange-red secondary
granules
 half life of 18 hrs
 The tissue destinations of
eosinophils under normal
circumstances appear to be
underlying columnar epithelial
surfaces in the respiratory,
gastrointestinal, and genitourinary
tracts.
 Survival time of eosinophils in
human tissues ranges from 2 to 5
days
 regulate mast cell function
through the release of major
basic protein (MBP) that causes
mast cell degranulation
 Basophils
 0% and 2% of circulating leukocytes and
less than 1% of nucleated cells in the
bone marrowImmature basophils
 have round to somewhat
lobulated nuclei with only slightly
condensed chromatin.
 Nucleoli may or may not be
apparent.
 The cytoplasm is blue and
contains large blue-black
secondary granules
 Primary granules may not be
seen
 Granules are water soluble
 Mature Basophils
 The cytoplasm is colorless and
contains large numbers of the
characteristic large blue-black
granules.
 basophils can be induced to
produce a mediator of allergic
inflammation known as granzyme
B
 Mast cells can induce basophils
to produce and release retinoic
acid, a regulator of immune and
resident cells in allergic diseases
 MONONUCLEAR CELLS
 MONOCYTES
 2% and 11% of circulating leukocytes,
with an absolute number of up to 1.3 X
109/L.
 Monoblast  Promonocyte  Monocyte
 Macrophages
 Under normal circumstances, promonocytes undergo two mitotic divisions in 60 hours to
produce a total of four monocytes.
 LYMPHOCYTES
 See Immunology

Page 21 of 23 
 
UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
 
HEMATOLOGICAL PROCEDURES

CELL COUNT

THE COUNTING CHAMBER

 Neubauer Counting Chamber

o see improved neubauer
o central secondary square divided into 16 tertiary squares
 Improved Neubauer Counting Chamber
o On the middle third of the chamber there are 3 parallel platforms extending across the
slide and separated bt moats.
o Central Platform is 0.1 mm lower than the lateral platforms. It is subdivided bt a
transverse grrove into halves each wider thwan the 2 platforms
 Each halves consist of a primary square measuring 9mm sq. and is subdivided
into 9 secondary squares each meaasuring 1 x 1 mm
 The four corner secondary squares are used for WBC counting
 The central secondary square is divided into 25 tertiary squares each measuring
0.2 mm.
 Each of the tertiary squares are divided into 16 smaller squares (Total of 800
smaller squares inside the central secondary square)
 RBCs are counted on four corner tertiary squares and central tertiary square of
the central secondary square (Five Smaller Squares = 80 smaller squares)
o Thick-Coverslip must be used to achieve the 0.1 mm space between the slip and ruled
platform
 Fuchs-Rosenthal Counting Chamber
o Larger than Neubauer
o for low cell counts such as Eosinophil Count, Spinal Fluid Count, and leukopenic blood
count
o Ruled Area measures 4x4 mm
o Depth is 0.2 mm
o Central Ruling us dicided into 16 smaller squares
 Speirs-Levy Counting Chamber
o 4 sections; two on each side
o Each ruled area consists of 10 squares each measuring 1x1 mm with total area of 10
mm2
o Squares arranged in two horizontal rows of five squares which are further subdivided into
16 squares
o Depth is 0.2 mm

One section of Speirs‐Levy 

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
 
 GENERAL FORMULA FOR CELL COUNTS

 RBC COUNTING
 THOMA RBC-DILUTING PIPETTE (0.5, 1, 101 mark)
o Bulb (100 units of volume)
o Red Bead
o Smaller Bore
 Make 1/200 Dilution
o 0.5 mark – RBC
o 101 mark - diluent
 Count in 5 smaller squares of the central tertiary square (Area = 1/5 mm2)
 Short Formula : Cells Counted x 10 000
 Normal Values
o Males: 4.5 to 6.0 M cells / cu. mm or 4.5 – 6.0 x 1012 cells / L
o Females: 4.0 to 5.5 M cells / cu. mm
 Diluting Fluids (Isotonic)
o Dacie’s Fluid / Formol Citrate – best rbc diluting fluid
 40% formaldehyde
o Hayem’s Fluid
 Allows growth of yeasts and produces clumping (cirrhosis px)
 Non corrosive but contains mercuric chloride
o Gower’s Fluid
 Prevents rouleaux; ppt protein in cases of hyperglobulinemia and
hemoglobinemia
o Toisson’s
 High sp. gravity
 Supports growth of fungi
 Stains WBCs
o Bethell’s
o NSS
 Used in cases of ER
 Particularly in excessive rouleaux, and autoagglutinated cells
 Stable and serves as preservative
 0.85 g NaCl on 100 mL distilled water
o 3.8% sodium citrate
 Discard 5-6 drops then charge on chamber
 Stand counting chamber for 5-10 mins
 Count on HPO
 Cell difference between squares is 20 or less
 WBC COUNTING
 THOMA-WBC DILUTING PIPETTE (up to 11 mark)
o Bulb = 10 units
o White Bead
o Larger Bore
 Make 1/20 dilution
 Shortcut : cells counted x 50
 Normal Value = 5000-10 000 cells / cu. mm or 5 – 10 x 10 9 cells / L
 Discard first 2-3 drops
 Charge on chamber. Stand 5-10 mins
 Count using LPO
 Cell difference between squares must not exceed 12
 Diluting Fluids (Hypotonic to lyse RBCs)
o 1 – 3% Acetic acid
o 1% HCl
o Turk’s Diluting Fluid
 In cases of Leukocytosis, use an RBC pipette
 In cases of severe Leukopenia, smear the buffy coat

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