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 > 30x10 15 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
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 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
Mature lymphocytes have decreased
1) Nuclear size
2) Cytoplasmic basophilia
3) DNA content
4) Histone dye-binding capability
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.
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
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
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
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).
Secretions from dry mammary glands have increased T- lymphocytes.
B cells are reduced in
Acute lymphocytic leukemia (Also T cells).
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.
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