HAEMATOLOGYHAEMATOLOGYHAEMATOLOGYHAEMATOLOGYGabriel MbassaGabriel Mbassa

Haematology is the study of blood

COURSE DESCRIPTION

BLS 110 Hematology: 1 credit (20 Lect., 20 Pract.)

Pre-requisite: NoneObjective: To impart on students the basic knowledge on haematology

Course contents:Theory; Introduction to haematology.

Haematopoiesis.Blood cells. Haemoglobin and

haemoglobinopathies, anaemias,Blood grouping systems, blood coagulation,

blood transfusion practices and blood transfusion reactions.

Practical: Blood group typing, Cell counting, Determination of Heamatological parameters

BloodSpecialized fluid tissue circulating through vascular channels

Carries compounds to all cellsReceives waste products of metabolism for transport to organs of excretion

Maintains haemostasis & defense of body.

Blood cells form in bone marrow

Blood makes 7-8% of body weight

Extracellular substances (plasma) make 45-65% of blood

Blood cells (called formed elements) form 35-55%.

Haematology basis for diagnosis

Numerical values of all the parameters in the blood are kept within narrow physiological limits in healthy animals

Blood parameter levels may are altered in disease, increase above or decrease below physiological values

Deviations from physiological values of healthy animals allow diagnosis of presence of disease

Each disease changes blood values in its own way, diagnosis is done by blood analysis

Common diseases detected by blood analysis; blood parasites (intracellular & extracellular), helminthes, diseases affecting liver, kidney, other organs, using functional tests

Functions of blood Supply nutrients to cells &

tissues Supply of oxygen to & removal

of CO2 from tissues & cells Remove breakdown products,

taking them to excretory organs

Transport surplus metabolites to storage organs

Regulate water & electrolyte metabolism

Regulate body temperature Contain body's defenses

against foreign substances & pathogenic organisms

Blood is pumped from central pumping organ Heart, in birds & mammals divided into two halves (double heart);

Arterial part (left), carrying oxygenated blood

Venous (right) part with blood rich in carbon dioxide

Arterial & venous halves of heart each consist of atrium & ventricle, supplying separated vascular systems;

1. Systemic (large) circulation

2. Pulmonary or small circulation

Blood Composition Blood consists of;

Plasma, viscous fluid that coagulates

Blood cells; Platelets (non cellular), erythrocytes, leukocytes

Plasma; aqueous solution of Proteins (albumin, fibrinogen,

globulins) & blood sugar Inorganic substances, Na, K,

Ca, Mg ions, which maintain chemico-physical properties of blood. Bicarbonate & phosphate ions buffer extreme high or low pH (Co2, lactic acid).

Final pH of blood is 7.4Lipids occur in fine suspension

Plasma carries nutrients, hormones, enzymes, antigens, antibodies, & antitoxins for neutralization of foreign protein & bacterial toxins

Blood clots as it leaves vessels, a vital protective mechanism.

Coagulation depends on ability of fluid fibrinogen to interact with thrombin & transform into a delicate elastic network of fibrin

Coagulation is initiated by breakdown of thrombocytes, involves 30 factors.

Blood in container clots unless anticoagulant is added

Coagulation; 1-2 min in birds, 10-15 pig, 10-20 in equine

After clot cells are trapped in fibrin, leave clear fluid called serum, which contains antibodies

If blood is mixed with an anticoagulant & left undisturbed blood cells will settle down

The duration of the process is called sedimentation rate (SR)

Erythrocyte SR (ESR) is diagnostic in equine, with large red blood cells (RBC)

That means mean corpuscular volume (MCV) of > 30x1015 l (30 femtolitres or 30 fl)

ESR not diagnostic in small ruminants (MCV < 30)

The clear fluid remaining is plasma

2. BLOOD CELLSBlood cells are also called formed

elements of blood.Blood is fluid connective tissue having

cells (35-55% of blood) and extracellular fluid intravascular (blood plasma) (45-65% of blood)

Total amount of blood in man is 5 L (7‑8% bwt)

Blood cells are;1. Red blood cells or erythrocytes

2. White blood cells or leukocytes

3. Platelets or thrombocytes.

Erythrocytes & platelets functions in blood stream

Leukocytes function temporarily in blood & leave by walls of capillaries & venules to settle in connective tissues & lymphoid organs.

Some leukocytes return to the blood stream, but most end their lives in tissues

Platelets are not true cells in higher vertebrates. They lack nucleus as erythrocytes

In fish, amphibians, reptiles & birds platelets are nucleated cells, called thrombocytes

Leukocytes are eukaryotic cells containing nucleus.

Five types of leukocytes occur, classified on basis of presence or absence of intracytoplasmic granules as granular & non-granular (or agranular) leukocytes

Granular leukocytes further subdivided according to stainability of their cytoplasmic granules into

NeutrophilicEosinophilic, &basophilic granulocytes

Non-granular leukocytes comprise monocytes and lymphocytes

Leukocytes divided are also divided on basis of shape of nucleus as

1. mononuclear (leukocyte with non-lobed nucleus)

2. polymorphonuclear (leukocyte with multi-lobed nucleus) leukocytes

LYMPHOCYTESLymphocytes play role in reception of foreign molecules processing & execution of immune responses.

Lymphocytes are heterogeneus morphologically and functionally.

Morphological classificationClassified as small (6-9μm) medium sized

and large (9–15 μm)Cell size, cytoplasmic basophilia, nuclear

chromatin indicate age and maturityImmature lymphocytes are large,

basophilic and have smooth chromatin

Mature lymphocytes have decreased1) Nuclear size2) Cytoplasmic basophilia3) DNA content4) Histone dye-binding capabilityHave increased1) Chromatin clumping2) Nucleus – to-cytoplasm ratio

When small lymphocytes are activated these properties reverse and the cells undergo multiplication (lymphoblastogenesis) in vivo and in vitro.

Functional classificationLymphocytes are grouped on basis of

immune responses.Lymphocytes involved in cell mediated

immunity and immunoregulatory functions are called thymus derived, thymus – dependent, thymus processed or T-lymphocytes (T-cells).

Lymphocytes concerned with formation of humoral antibodies are thymus independent, bursa – derived (in birds), bone marrow derived (mammals) or B-lymphocytes (B cells).

Peripheral blood lymphocytes are 80 – 95% T and B types

Lymphocytes that make 5-20% of peripheral blood lymphocytes are non-T, non- B or null cells. These have different markers and functions.

Many subpopulations of lymphocytes are found in peripheral blood because of production, release and recirculation of lymphocytes at different stages of maturation & immunocompetence.

Different lymphoid organs have different types of lymphocytes

stained on blood smears by any Romanowsky stains

Lymphocytes are round to ovalCytoplasm is scanty to moderateCytoplasm varies in basophiliaNucleus is spherical to ovoid Nuclear membrane distinct

Chromatin is heavily clumped.Nucleus may be indentedLymphocytes show slow amoeboid

movements to being motile Nucleus presents patchy, dense clumps of

heterochromatin (small lymphocytes).Moderate dispersed chromatin (medium

and large sized lymphocytes).

Fairly dispersed chromatin (lymphoblasts)

Distinct nuclear membrane, and nuclear pores

Not frequent nucleoliSome nuclear pockets occur in leukemia

Thoracic lymph duct show nuclear body proximal to nucleolus

Nucleolus has electron dense area surrounded by fibrillar material

Small lymphocytes have very scanty cytoplasm

Numerous free cytoplasmic ribosomes

Few mitochondriaSmall Golgi complexLittle rough endoplasmic reticulum

Medium sized lymphocytes have some extra cytoplasm (moderate to abundant).

Various organelles are prominentCentrioles are more conspicuousThere are few pinocytotic vesiclesGlycogen granules are seen in some cells

There are also microfilaments and microtubules (uropod lymphocytes)

Lysosomes are rare.Some granules of variable size appear (5-

15% of T-lymphocytes) called azurophilic granules

Azurophilic granules are peroxidese negative. Some cells present large spherical refractile structure, called Gall body.

Some cells show aggregates of reddish – purple granules (Kurbff body).

Ribosome and polyribosome population vary with stage of cellular maturity and activity

Free ribosomes are not engage in protein synthesis.

Polyribosomes are engaged in protein synthesis antibody formation in B-cells and plasma cells.

Lymphokine production in T cells

On scanning electron microscopy

Lymphocytes are globular Surfaces are smooth or have some villi

Surfaces may have fingerlike projections

Biochemistrymany hydrolytic and oxidative enzyme in

lymphocytes.Pre-B and Pre-T cells have terminal

deoxyribonucleotidase (Tdt)B-lymphocytes, plasmablasts and plasma

cells stain for 5’ nucleotidase.

T cells and activated B cells contain lysosomal enzymes; acid phosphatase, β-glucoronidase, and acid α-naphythyl acetate esterase. These enzymes core not present in mature B cells

Lymphocytes lack cytoplasmic alkaline phosphatase, peroxidase, sudan black reactivity and lysozyme.

Lymphocytes derive energy by glucose metabolism (glycolytic pathway)

The pentose phosphate pathway is not common, but stimulated under aerobic conditions.

Lymphocytes synthesize protein, glycogen, and fatty acids.

Characterization of B and T lymphocytes Detection of surface antigens by

monoclonal antibodies & flow cytometry.

Demonstration of surface or cytoplasmic immunoglobulins by fluorescence and autoradiography

Detection of immunologic and non immunologic receptors by using heterologous erythrocytes (E-rosettes)

Detection of receptors for Fc of Ig or complement components (C3b and C3d receptors) by rosettle formation with antibody coated eryrothcytes (EA- rosetes) or antibody-complement – coated erythrocytes (EAC-rosettles)

Immunofluorescence & autoradiography

Bacterial adherence to lymphocytes (in conjuction with fluoresent antibody technique).

Helix prometia antigen chracterizes bovine and equine T-lymphocytes

Cytochemical reactions

Usefulness of techniquesIdentify functional subpopulations of

peripheral blood and lymphoid tissues in health and disease.

Classification of immunodeficiency disorders

Detection and classification of lymphoid malignancies

Characterization of leukemic lymphocytes.Immunologic classification of leukemiaShowing phenotypic characteristics of

lymphoblasts and immature lymphocytes

Quantitative Evaluation of lymphocyte Populations

T-lymphocytes predominate in the thymus gland and peripheral blood, thoracic duct lymph.

B-lymphocytes predominate in bone marrow and spleen.

T-lymphocytes account for 70% of all lymphocytes in peripheral blood

B-lymphocytes constitute 20% of peripheral blood lymphocytes.

The remaining 10% are Null – lymphocytesThis distribution varies with species and

under various physiological conditions and affected by techniques of typing T or B-lymphocytes.

The flow cytometer (fluorescent activated cell sorter) is by far the most accurate in computing lymphocyte populations and sub-populations over the rosetting methods.

Flourescent antibody techniques using antithymocyte antibody produce higher results for T-lymphocytes that rosetting methods.

Peanut agglutinin (PNA) receptor is a marker for cells in the thymic cortex immature thymocytes – mice and man.

It also marks bovine, ovine, caprine and equine T-lymphocytes

Surface Ig is a reliable marker for B cells, but is labile and requires care Ig marker is to be differentiated from T-cells (few), macrophages, suppressor macrophages, suppressor cells (CD8+) and helper T-cells (CD8),

CD8+ and CD4+ cells binds sIg via Fc receptors.

Rosetting B cells produces higher values for B cells than B cells identified by surface markers.

T cell, and B cell populations in peripheral blood and lymphoid tissues vary with age and health.

B cells are few in fetuses, steadily increase postnatally to adult values.

B cells, T cells and null cells decline with age.

Increased B cells and decreased T cells indicate disease

B cells increase in Bacterial diseases (eg mastitis).LeukemiaSecretions from dry mammary glands have

increased T- lymphocytes.B cells are reduced in Acute lymphocytic leukemia (Also T cells).

B LymphocytesB lymphocytes constitute 20% of

circulating peripheral blood lymphocytes.

B cells are identified by presence of surface immunoglobulins and receptors for complement C3b C3d.

Complement receptors are absent in plasma cells.

Most human B lymphocytes have surface IgM (monomeric) and IgD.

Most canine lymphocytes have surface IgG (70%).

The rest have IgM.

Human B cells exhibit B-cell specific Ia like antigens on surface membrane. Ia antigens are glycoproteins linked genetically to major histocopatibility complex with limited tissue distribution.

Ia-antigens occur also in macrophages, early erythroid and myeloid precursors.

Human B cells rosette with mouse erythrocytes.

B-cells stain for 5’nucleotidase (only in plasma membrane).

B cells do not stain with acid hydrolytic enzymes.

Ia – like antigens are detected on horse, cow, sheep, pig B-lymphocytes by monoclonal antibodies.

T-LymphocytesPeripheral blood lymphocytes have 21 –

85% T cells T cells are identified by E-rosetting with

heterologous erythrocytesT cells are also identified by

antithymocyte antibodies.Mature T cells form rosettes with sheep

erythrocytes (most species),

T cells from canines form rosettes with guinea pig and human type O erythrocytes

Feline T-cells rosette with guinea pig, rat, and mouse red cells.

Porcine T cells rosette with rabbit erythrocytes

Equine T cells rosette with pig erythrocytes

T-cells are also identified using specific markers (surface markers).

T-lymphocytes have receptors for Fc portions of some immunoglobulins.

Sub-sets of T lymphocytes with surface receptors for IgG, IgM, and IgA are called TG, Tm and TA cells.

Tm cells reach up to 85% of all lymphocytes, typically small to medium sized, with smooth surface, poor cytoplasmic organelles, no cytoplasmic granules.

These constitute helper T cells (CD4+)TG cells make 5-15% of T cells cells TG cells are large in size, contain

azurophilic granules, show villous surface, abundant cytoplasm and well developed organelles.

TG cells constitute suppressor T cells (commonly called CD8).

T cells are also identified by mitogenic responses to non specific stimulatory agents such as plant lectins, phytohemagluttinin (PHA) and concanavalin (Con A).

T cells are identified by cytochemical markers acid hydrolytic enzymes, such as acid phosphatase, β-glucuronidase, acid α-naphthy acetate estarase – CD8 cells.

Pre-T, Pre-B and Null cellsPre-T and Pre-B cells have Tdt activityPre-B cells show cytoplasmic IgM, without

surface Ig and complement receptors Mature B cells show cytoplasmic and surface Ig

including complement receptors.Null cells resemble morphologically and

cytochemically TG cellsNull cell lack specific markers of T and B cells Null cells respond poorly to mitogensNull cells do not produce immunoglobulins in

vitroAre non adherent and non phagocyticThey are precursors of T and B cellsPrecursors of myeloid and erythroid cells

Fig. 1: Human bloodA two―lobed neutrophil (a) and a

small lymphocyte (b). There is slight variation in size of the normal erythrocytes. Wrights stain x 400

Fig. 3: Human blood: Nucleus of the neutrophil (a) has not constricted into distinct lobes. Neutrophils with more or less band‑form of nucleus are called band neutrophils and are more immature than the lobed or segmented neutrophils. Band neutrophils comprise 3 to 5 % of leukocytes in normal blood. Blood platelets (b) and a small lymphocyte (c). Wrights stain x 400

Human blood: Blood smear showing erythrocytes, platelets and large lymphocycte. Wrights –Giemsa x 400