HEMATOLOGY 1 LECTURE

INTRODUCTION TO HEMATOLOGY LYMPHOCYTES: viral infection

● T-cell: maturation is in Thymus.
HEMATOLOGY - is the branch of biology ● B-cell: maturation is in Bone Marrow.
which is concerned with the study of blood, the → PLASMA CELL - responsible for the
blood-forming organs, and blood diseases. formation of antibodies (immunoglobulins).

BLOOD - is a specialized bodily fluid in BASOPHIL – increase of allergic reaction.

animals that delivers necessary substances EOSINOPHIL – increase of parasitic infection.
such as nutrients and oxygen to the cells and MONOCYTES – blood
transports metabolic waste products away from → MACROPHAGES – tissues
those same cells. NEUTROPHILS – bacterial infection.

TYPES OF CONNECTIVE TISSUES: PLASMA VS SERUM

● Connective tissue proper
● Cartilage PLASMA – the liquid, cell-free part of blood
● Bone that has been treated with anticoagulants.
● Blood → EDTA (purple)
→ HEPARIN (green)
→ COAGULATION (light blue)
→ SODIUM CITRATE (black)

SERUM – the liquid part of blood after

PLASMA
coagulation, therefore devoid clotting factors as
fibrinogen.
BUFFY COAT → PLAIN (red)

RED BLOOD CELL FUNCTIONS OF BLOOD:

TRANSPORTATION:
● Respiration
PLASMA – serum or liquid portion → transports oxygen (O2)
Formed Element: ● Nutrient carrier from GIT
→ BUFFY COAT ● Transportation of hormones from endocrine
→ BLOOD glands.
→ INSULIN – decrease glucose
→ GLUCAGON – increase glucose
→ MELATONIN – controls body clock
→ LUTEINIZING HORMONE – controls
estrogen and testosterone.
● Transport metabolic wastes
→ CO2 (tissue back to the lungs)

REGULATION:
● Regulates pH → 7.35-7.45
TO REMOVE CLOT: ● Adjust and maintains body temperature
→ Heparin Sulfate ● Maintains water contents of cells
→ Prostacyclin → Vasopressin and Albumin

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PROTECTION:
● WBC protects against disease by VOLUME:
phagocytosis ● about 8% of the body weight in a normal
● Reservoir for substances like water, young healthy adult, weighing about 70 kg.
electrolyte etc. → AVERAGE VOLUME: 5 liters
● Performs haemostasis → NEWBORN: 450 mL
→ HAEMOSTASIS – stoppage of bleeding. → FEMALES: 4.5 L
(platelets) → MALES: 5-6L
→ HOMEOSTASIS – maintenance of balance.
pH:
GAS TRANSPORT: → 7.35-7.45
● blood carries oxygen from lung to the tissues ● Blood is slightly alkaline
and carbon dioxide in reverse direction. ● pH in normal conditions is 7.4

TRANSPORT OF NUTRITIONAL SPECIFIC GRAVITY:

SUBSTANCES: ● total blood: 1.052 to 1.061
● Glucose, Amino Acids, Fatty Acids, Vitamins, ● blood cells: 1.092 to 1.101
Ketone Bodies, Microelements etc. ● plasma: 1.022 to 1.026
● Blood carries final products of metabolism
→ Urea, Uric Acid, Bilirubin, Creatinine etc. DIAMETER:
from tissues to kidney, where from they ● 7.2u (6.9-7.4u)
excreted with urine.
THICKNESS:
REGULATION: ● At the periphery it is thicker with 2.2 u
● Blood creates and carries local hormones and at the center it is thinner with 1 u
(hormonoids) to the target organs. ● This difference in thickness is because
of the biconcave shape.
THERMOREGULATION:
● heat change between tissues and blood. VISCOSITY:
● Blood is five times more viscous than water
OSMOTICFUNCTION: mainly due to RBC and ALBUMIN
● maintenance of the osmotic pressure in blood
vessels. ELEMENTS OF BLOOD:

PROTECTIVE FUNCTION: PLASMA: 55%

● blood has antibodies (plasma cells) and FORMED ELEMENT: 45%
leukocytes (wbc), which perform phagocytosis.
● 7% PROTEINS
DETOXIFICATION: → 58% ALBUMINS
● blood enzymes can neutralize (split) different → 38% GLUBULINS
toxic substances. → 4% FIBRINOGEN

PROPERTIES OF BLOOD: ● 91% WATER

● 2 OTHER SOLUTES:
COLOR: → IONS (Na, K, Cl, Mg, Ca)
● ARTERIAL BLOOD – bright red (oxygenated → NUTRIENTS (glucose)
blood) → WASTE PRODUCTS (CO2, Creatinine)
● VENOUS BLOOD – dark red (deoxygenated → GASES (O2, CO2)
blood, CO2 increase) → REGULATORY SUBS (Hormones)

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● PLATELETS: 25-400,000 ● NON-PROTEIN NITROGENOUS

● WBC: 5000-9000 SUBSTANCES:
→ Neutrophil: 60-70% → Ammonia
→ Lymphocytes: 20-25% → Creatine
→ Monocytes: 3-8% → Creatinine
→ Eosinophil: 2-4% → Xanthine
→ Basophil: 0.5% → Hypoxanthine
● RBC: 4.2-6.2 MILLION → Urea
→ Uric acid
PLASMA:
● ANTI-BODIES
● SOLID: 7%-8%
● WATER: 92%-93% INORGANIC SUBSTANCES:
● GASES
→ Oxygen ● SODIUM
→ Carbon Dioxide ● CALCIUM
→ Nitrogen ● POTASSIUM
● MAGNESIUM
ORGANIC SUBSTANCES: ● BICARBONATE
● CHLORIDE
● PLASMA PROTEINS: ● PHOSPHATE
→ Albumins ● IODIDE
→ Globulins ● IRON
→ Fibrinogen ● COPPER

● AMINO ACIDS: BLOOD TYPING

→ Essential amino acids BLOODTYPE ANTIGEN ANTIBODIES
→ Non-essential acids A A Anti-B
B B Anti-A
● CARBOHYDRATE AB (univ recipient A&B None
→ Glucose O (univ donor) NONE Anti-A & Anti-B

● FATS: WHITE BLOOD CELLS

→ Triglycerides
→ Cholesterol GRANULOCYTES:
→ Phospholipids
● NEUTROPHILS – bacterial infection
● INTERNAL SECRETION: ● EOSINOPHILS – Parasitic Infection
→ Hormones ● BASOPHILS – Allergic reactions

● ENZYMES:
→ Amylase
→ Carbonic anhydrase
→ Acid phosphatase
→ Alkaline phosphatase
→ Lipase
→ Esterase
→ Protease
→ Transaminase

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AGRANULOCYTES: BASIC HEMATOLOGICL TESTS:

● LYMPHOCYTES – Viral infection 1. ABO blood group system.
● MONOCYTES 2. Rh typing and weaker variants in rh
→ phagocytes system.
→ macrophages 3. Subgroups and weaker variants of A
and B: Bombay phenotype.
4. Coombs' test
5. Blood grouping and cross matching in
blood bank.
6. Investigations of transfusion reactions.
7. Care and selection of donors.
8. Screening for Australia antigen (HBS
HEMATOLOGY APPLICATIONS: AG) - as er. 6
● Medical genetics 9. Hla antigens and their significance in
● Immunology blood transfusion.
● Clinical pathology and laboratory medicine 10. Preservation of blood, principles, and its
● Blood banking and transfusion medicine application.
● Nuclear medicine 11. Screening blood for infective material in
● Oncology blood banking.
12. Blood bank administration.
BASIC HEMATOLOGICL TESTS:
● Blood collection BLEEDING DISORDERS
● Anticoagulants used in haematology, ● Mechanism of coagulation
● Normal values in haematology ● Collection and anticoagulants used in
● Basic hematological techniques coagulation studies.
→ RBC counts ● Bleeding time and clotting time.
→ Hemoglobin estimation ● Other coagulation studies PT, APTT, TGT,
→ Packed cell volume etc.,
→ Calculation of absolute indices, wbc counts- ● Platelet count or platelet function tests.
total and differential
→ Absolute eosinophil count
→ Platelet count
→ Erythrocyte sedimentation rate
→ Preparation of blood films
→ Stains used in haematology
● Morphology of red cells
● Morphology of leucocytes and platelets
● Bone marrow
→ Techniques of aspiration, preparation and
staining of films
→ Bone marrow biopsy.
● Preparation of buffy coat smears
● Reticulocyte count
● Laboratory methods used in the investigation
of deficiency anaemias
→ B12 and folate assay
→ Schilling test
→ Serum iron and iron binding capacity

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HEMATOPOIESIS ● Start of definitive hematopoiesis

→ RBC
HEMATOPOIESIS → WBC – GRAM (H, E, R), monocyte,
→ continuous, regulated process of blood lymphocytes
cell production that includes cell renewal, ● Lymphoid cells begin to appear
proliferation, differentiation, and maturation ● LIVER is the major site of hematopoiesis and
→ 1% - production retaining activity until 1-2 weeks after birth
→ 1% - cell death ● The spleen, kidney, thymus, and lymph
nodes contribute to the hematopoietic process
FUNCTION OF THE BONE MARROW ● Detectable levels of hemoglobin (Hb) F, Hb
A, and Hb A22 may be present.
● supply blood with mature cell
● mobilize the bone marrow to incorporate MEDULLARY PHASE
production (if needed)
● compensate for decreased hematopoiesis ● At 5th month of development, hematopoiesis
begins in the BONE MARROW.
STAGES OF HEMATOPOIESIS ● M:E ratio reaches adult levels of 3:1 at 21
weeks of gestation
● MESOBLASTIC PHASE ● Measurable levels of HbA1, fetal hemoglobin,
● HEPATIC PHASE and Hb A2.
● MEDULLARY PHASE ● After the first 3 weeks postpartum, the bone
marrow becomes the only normal site of blood
MESOBLASTIC PHASE cell production and remains so throughout life.

→ “YOLKSAC PHASE” AT 18 YEARS OF AGE, THE ONLY ACTIVE

● Begins during the embryonic development HEMATOPOIETIC SITES ARE:
in blood islands of the yolk sac at around 19TH
of gestation → Sternum
● Primitive erythroblasts arise from → Skull
mesodermal cells → Vertebrae
● Primitive hematopoiesis (does not contribute → Ribs
to definitive hematopoiesis) → Pelvis
● Characterized by the development of → Proximal extremities of the long bones
primitive erythroblasts that produce
hemoglobin: ADULT HEMATOPOIETIC TISSUE:
→ Portland, BONE MARROW
→ Gower-1,
→ Gower-2 RED MARROW:
- fetal hemoglobin ● Hematopoietically active
- they do not survive adult life ● Predominant type during infancy and
- doesn’t participate oxygen delivery childhood (0-4 years old red marrow)
● This phase of hematopoiesis occurs ● Composed of extravascular cords that
intravascularly. contain all developing cells (stem and
progenitor cells, adventitial cells, and
HEPATIC PHASE macrophages)
● The hematopoietic cells tend to develop in
● Begins at 4th to 5th gestational weeks specific niches within the cords:
● Characterized by clusters of developing → Normoblasts develop in small clusters
erythroblasts, granulocytes, and monocytes adjacent to the outer surfaces of the

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vascular sinuses, some normoblasts are found MARGINAL ZONE:

surrounding iron-laden macrophages → forms a reticular meshwork containing blood
→ Megakaryocytes are located close to the vessels, macrophages, and specialized B-cells.
vascular walls of the sinuses to facilitate
release of platelets RED PULP:
→ Immature myeloid (granulocyte) cells → comprised on dendritic processes that
through the metamyelocyte stage create a filter, the cords of Bill Roth stagnate
are located deep within the cords and depletes the glucose supply of RBCs that
lead to their removal.
YELLOW MARROW: → Culling cells are phagocytosed with
● Hematopoietically inactive, comprised of subsequent degradation of cells and organelles.
adipocytes (fat cells) → Pitting splenic macrophages remove
● Between ages of 5 to 7, adipocytes become inclusions or damaged surface membrane form
more abundant RBC’s.
● The process of replacing the red marrow by
the yellow marrow is RETROGRESSION LYMPH NODES:
● It is capable or reverting to active marrow in ● Formation on new lymphocytes from
cases of increased demand germinal centers
● Approximately, there is equal amount of red ● Processing of specific immunoglobulins (B-
and yellow marrow in adults cells)
● Filter particulate matter. debris. and bacteria
ADULT HEMATOPOIETIC TISSUE entering the lymph node.

LIVER: THYMUS:
● Significant role in hematopoiesis in the 2nd ● Densely populated with progenitor lymphoid
trimester and the major site during hepatic cells that migrated from the bone marrow and
stage will soon give rise to T-cells.
● Capable of Extra Medullary Hematopoiesis
(counter part of hepatic phase in adults) in STEM CELL CYCLE KINETICS &
case of bone marrow shut down CYTOKINES
Example:
→ Myelophthisic Anemia (abnormal tissue in ABBREVIATION CELL LINE
the bone marrow) CFU-GEMM Granulocyte, erythrocyte,
→ Cancer megakaryocyte, monocyte
→ Leukemic Blast CFU-S Spleen
CFU-E Erythrocyte
SPLEEN: CFU-MEG Megakaryocytes
● Removes senescent RBCs CFU-M Monocyte
● Sequesters approximately 30% of platelets CFU-GM Granulocyte, Monocyte
● 3 Types of Tissues: CFU-BASO Myeloid to Basophil
→ White Pulp CFU-EO Myeloid to Eosinophil
→ Marginal Zone CFU-G Myeloid to Neutrophil
→ Red Pulp CFU-PRE-T T-lymphocyte
CFU-PRE-B B-lymphocyte
WHITE PULP:
→ consists of scattered follicles with germinal
centers containing lymphocytes, macrophages,
and dendritic cells.

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STEM CELL THEORY

MONOPHYIETIC THEORY
→ widely accepted
→ All blood cells are derived from a single
pluripotential stem cell.

POLYPHYIETIC THEORY
→ unique stem cell Suggests that each of the
blood cell lineages is derived from
its own.

CFU-S
→ Mice spleens and bone marrows are
irradiated, afterwards, marrow cells are
intravenously injected into the mice. Colonies
of HSCs are observed after 7-8 days and were
referred to as CFU-s, which will correspond to
CFU-GEMM today

COMMON LYMPHOID PROGENITOR

● T – cell CELL CYCLE MITOSIS
● B – cell
● NK – cell INTERPHASE → correlate with G1, S,
and G2
COMMON MYELOID PROGENITOR PRE-PHASE → chromosomes
● Granulocytic, Erythrocytic, Monocytic condense
Megakaryocytic lineage (GEMM) PROMETAPHASE → centrosomes move to
opposite poles
CELL CYCLE ANAPHASE → sister chromosome
segregate
G1 (GAP 1) RNA and protein synthesis, pre- TELOPHASE → cell division
synthetic stage
→ 10 hours KINETICS
SYNTHESIS DNA synthesis
→ 9 hours ● 3 billion RBCs, 2.5 billion platelets, and
G2 (GAP 2) Pre-mitotic stage, 1.5 billion granulocytes per kilogram of body
Post-synthetic stage weight is produced by the bone marrow daily
→ 4 hours ● Stem cells exist in a ratio of 1:1000
MITOSIS → Resting stage nucleated blood cells
→ 1 hour ● Mitotic index:
→ A calculated value used to establish the
number of cells undergoing mitosis,
normally it is 1-2%.
→ Increased mitotic index indicates increased
proliferation except in
megaloblastic anemia wherein mitosis is
prolonged.

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CYTOKINE AND GROWTH FACTORS GENERAL MORPHOLOGIC CHANGES

ASSOCIATED WITH RBC MATURATION:
● CYTOKINES
→ glycoproteins that regulate the proliferation, ● Decrease in overall size
differentiation, and maturation of hematopoietic ● Decrease in size of nucleus and in N:C ratio
precursor cells. ● Nuclear chromatin pattern
→ Interleukins, lymphokines, monokines, ● Nucleoli disappear
interferon, and colony-stimulating factors. ● Decrease in basophilia
→ GRANULES: Protein from ribosome to
● POSITIVE INFLUENCE - IL- 1, 3, 6, 9 11, nucleus.
GM-CSF, and kit-ligand
RBC DEVELOPMENTAL STAGES
● NEGATIVE INFLUENCE - Transforming
growth factor B, TNF-a, Interferons. PRONORMOBLAST (RUBRIBLAST)
● Nucleus takes up much of the cell (high N:C
OVERVIEW OF HEMATOPOIESIS ratio)
● Measures 14-20 um and cytoplasm is quite
blue
● Globin production begins

BASOPHILIC NORMOBLAST
(PRORUBRICYTE)
● n:c ratio decreases to 6:1
● Nucleoli usually not visible
● measures 12-17 um and the cytoplasm
stains deep blue
● detectable level of hemoglobin synthesis
(Minute amount)

POLYCHROMATIC NORMOBLAST
ERYTHROPOIESIS (RUBRICYTE)
● N:C ratio is 4:1
GENERAL MORPHOLOGIC CHANGES ● Measures 10-15 um and the cytoplasm is
ASSOCIATED WITH MATURATION: pink, blue (Murky-gray blue)
● This is the last stage capable of Mitosis
NUCLEUS: ● 1% stage where Hb synthesis is visible
● Loss of nucleoli
● Decrease in size of nucleus ORTHOCHROMIC NORMOBLAST
● Condensation of chromatin (METARUBRICYTE)
→ RETICULOCYTES (young RBC) ● The nucleus is pyknotic
● Possible changes in shape ● Pink-orange color of the cytoplasm reflects
● Possible loss of nucleus nearly complete production of hemoglobin
● Later in this stage the nucleus is ejected
CYTOPLASM:
● Decrease in basophilia POLYCHROMATOPHILIC ERYTHROCYTE
→ basic/alkaline – attracts acid stain (RETICULOCYTE)
● Increase in the proportion of the cytoplasm ● No nucleus
→ 1/3 central pallor ● Cytoplasm is the predominant color of
● Appearance of granules hemoglobin (pink)

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● Called a reticulocyte when the remnants of HEXOSE MONOPHOSPHATE PATHWAY

the ribosomal ● Aerobic glycolysis
● RNA (reticulum) is stained with supravital → uses oxygen to produce ATP
stain - New Methylene Blue (NMB) ● NADP is reduced to NADPH
→ Nicotinamide adenine dinucleotide
ERYTHROCYTE phosphate
● No nucleus ● NADPH reduces glutathione
● Biconcave disc measuring 7-8 um in ● Reduced glutathione reduces H2O2
diameter (peroxides) – oxidize bacteria) to water
● Appears salmon pink or red with a pale ● Common enzyme that is deficient is G6PD
central palor – Glucose - 6 - Phosphate Dehydrogenase
● Has a lifespan of 120 days
METHEMOGLOBIN REDUCTASE PATHWAY
ERYTHROKINETICS ● NADPH reduces the Ferric (Fe+3) to the
Ferrous (Fe+2) in the presence of
ERYTHRON methemoglobin reductase. (reversible)
● collection of all stages of erythrocyte → Ferric – without oxygen
throughout the body. → Ferrous – with oxygen
→ Hemoglobin = iron
RBC MASS
● cells in the circulation LUEBERING-RAPAPORT SHUNT
● Generates 2,3-diphosphoglycerate
HYPOXIA ● 2,3-DPG regulates oxygen delivery to tissues
● detected by peritubular (kidney) interstitial by competing with oxygen for hemoglobin
(tissues) cells, which produces EPO ● When 2-3 DPG binds hemoglobin, oxygen is
● RBC: deliver oxygen released which enhances delivery of
oxygen to tissues
EPO (ERYTHROPOIETIN)
● a glycoprotein hormone which is the major
stimulatory cytokine for RBC RBC DESTRUCTION
→ Early release of reticulocytes
→ Inhibition of apoptosis RES – RETICULO-ENDOTHELIAL SYSTEM
→ Reduced marrow transit time
MACROPHAGE-MEDIATED HEMOLYSIS
ERYTHROCYTE METABOLISM (NORMAL EXTRAVASCULAR HEMOLYSIS)
● The spleen generates a stressful
EMBDEN-MEYERHOF PATHWAY environment for the RBCs
→ major pathway ● Glucose is low that leads to reduced
● Anaerobic glycolytic pathway glycolysis and reduced ATP production
→ production of glucose without the use of → decrease in glucose = no glycolysis, no ATP
oxygen. ● pH is low and leads to oxidation of iron
● Results in a net gain of 2 ATP molecules per ● ATP dependent cellular processes begin to
1 glucose molecule fail and lead to a loss in BC flexibility
● Generates 90% of RBC's ATP ● RBCs become trapped in the splenic sieve
● Common enzyme being deficient is pyruvate and are phagocytosed by macrophages
kinase ● Trace urobilinogen in urine, no bilirubinuria
● Urobilinogen and urobilin in stool

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MECHANICAL HEMOLYSIS OR
INTRAVASCULAR HEMOLYSIS

● Small portion of RBCs rupture in the blood

vessels due to turbulence
● Haptoglobin and Hemopexin salvage
released hemoglobin so that iron is not loss in
the urine
● Albumin temporarily holds metheme and
passes it eventually to hemopexin

EXCESSIVE EXTRAVASCULAR
HEMOLYSIS

● Traumatic physical lysis of RBCs caused

by prosthetic heart valves, malarial
parasites
● Hemoglobinemia, Hemoglobinuria, and
Hemosiderinuria eventual increase in urinary
urobilinogen
● Decreased Haptoglobin and Hemopexin

SAMPLE TEST RESULT INTRAVASCULAR EXTRAVASCULAR

SERUM Increased total bilirubin EXPECTED EXPECTED
Increase indirect bilirubin EXPECTED EXPECTED
Normal direct bilirubin EXPECTED EXPECTED
Decreased haptoglobin EXPECTED MINOR
COMPONENT
Decreased hemopexin EXPECTED MINOR
COMPONENT
URINE Increased urobilinogen EXPECTED EXPECTED
Hemoglobinuria EXPECTED NONE
WB Decrease in HGB, HCT and EXPECTED EXPECTED
RBC
Decrease in Glycated EXPECTED EXPECTED
hemoglobin

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