HAEMATOLOGYHAEMATOLOGYHAEMATOLOGYHAEMATOLOGYGabriel MbassaGabriel Mbassa

Definition and scope

Haematology is the study of blood

COURSE DESCRIPTION

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

•Pre-requisite: None•Objective: 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

•Blood• Specialized fluid tissue

circulating through vascular channels

• Carries compounds to all cells• Receives 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 7.4•Lipids 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 CELLS

• Blood 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

•Neutrophilic•Eosinophilic, &•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

LYMPHOCYTES• Lymphocytes play role in

reception of foreign molecules processing & execution of immune responses.

• Lymphocytes are heterogeneus morphologically and functionally.

• Classified as small (6-9μm) medium sized and large (9–15 μm)

• Cell size, cytoplasmic basophilia, nuclear chromatin indicate age and maturity

• Immature lymphocytes are large, basophilic and have smooth chromatin

Morphological classification

• Mature lymphocytes have decreased

• 1) Nuclear size• 2) Cytoplasmic basophilia• 3) DNA content• 4) Histone dye-binding capability• Have increased• 1) Chromatin clumping• 2) Nucleus – to-cytoplasm ratio

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

Functional classification

• Lymphocytes 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 oval• Cytoplasm is scanty to moderate• Cytoplasm varies in basophilia• Nucleus is spherical to ovoid • Nuclear membrane distinct

• Chromatin is heavily clumped.• Nucleus may be indented• Lymphocytes 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 nucleoli•Some 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 mitochondria•Small Golgi complex•Little rough endoplasmic

reticulum

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

• Various organelles are prominent• Centrioles are more conspicuous• There are few pinocytotic vesicles• Glycogen 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

Biochemistry

• many 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 techniques• Identify 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 leukemia

• Showing 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 – lymphocytes

• This 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).• Leukemia• Secretions from dry mammary

glands have increased T- lymphocytes.

• B cells are reduced in • Acute lymphocytic leukemia (Also

T cells).

B Lymphocytes• B 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-Lymphocytes

• Peripheral blood lymphocytes have 21 – 85% T cells

• T cells are identified by E-rosetting with heterologous erythrocytes

• T 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 cells• Pre-T and Pre-B cells have Tdt activity• Pre-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 cells• Null cell lack specific markers of T and B cells • Null cells respond poorly to mitogens• Null cells do not produce immunoglobulins in

vitro• Are non adherent and non phagocytic• They are precursors of T and B cells• Precursors of myeloid and erythroid cells

• Fig. 1: Human blood• A 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