Learning objectives 

  • This module deals with
    • Hemoglobin and its properties
    • Types of hemoglobin
    • Regulation and concentration of hemoglobin
  • Hemoglobin is the iron-containing oxygen-transport metalloprotein present in the red blood cells of all vertebrates that carries oxygen from the lungs to the tissues and carbon dioxide from the tissues back to the
  • The oxygen carrying capacity of hemoglobin is 60% more than that of
  • It also functions as a buffer in the regulation of acid base
  • A hemoglobin molecule is a complex substance consisting of conjugate protein composed of a pigment heme and a protein,
  • The protein portion of each of these chains is called “globin”. The a and b globin chains are very similar in structure. In this case, α and ß refer to the two types of
  • The globin is a conjugated protein and heme contains iron in ferrous
  • Hemoglobin contains four polypeptide chains namely two alpha and two beta Each of the four chains unites with a heme group resulting in a hemoglobin molecule.
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  • Hemoglobin (Hb) is synthesized in a complex series of steps.
  • The heme part is synthesized in a series of steps in the mitochondria and the cytosol of immature red blood cells, while the globin protein parts are synthesized by ribosomes in the cytosol.
  • Production of Hb continues in the cell throughout its early development from the proerythroblast to the reticulocyte in the bone marrow. At this point, the nucleus is lost in mammalian red blood cells, but not in birds and many other Even after the loss of the nucleus in mammals, residual ribosomal RNA allows further synthesis of Hb until the reticulocyte loses its RNA soon after entering the vasculature.

Heme synthesis 

  • Heme is synthesized in a complex series of steps involving enzymes in the mitochondrion and in the cytosol of the cell. The first step in heme synthesis takes place in the mitochondrion, with the condensation of succinyl CoA and glycine to form delta-amino levulinate. This molecule is transported to the cytosol where a series of reactions produce a ring structure called protoporphyrin
  • Many enzymes concerned with heme synthesis are intra-mitochondrial, limited to erythroid precursors including
  • The ALA synthetase is the rate-limiting enzyme of the Hb synthesis, present within the mitochondria.

Globin synthesis 

  • After heme is synthesized within the mitochondria, 4 heme molecules combine with 4 globin polypeptides to form one molecule of
  • The globin molecule of haemoglobin differs among the species, whereas there is no difference in the heme portion.
  • The amount of Hb in the blood is influenced by age, sex, muscular activity, season, excitement Erythropoietin stimulates RNA and DNA synthesis, the cell division, heme synthesis and hemoglobin production.
  • At an oxygen pressure (PO2) of 100 mm of Hg in the lung, the Hb forms loose andreversible combination with oxygen, the oxyhemoglobin, but at low oxygen pressure of 40 mm of Hg at tissue level, it readily releases oxygen to the tissues for complex metabolic process.
Species Hb (g/dl)
Dog 12-18 (15)
Cat 8-15 (12)
Cow 8-15 (11)
Sheep 8-16 (12)
Goat 8-14 (11)
Horse 11-19 (15)
Pig 10-16 (13)
  • Based on physiological functions, the hemoglobins are typed as adult hemoglobin and fetal hemoglobins. Electrophoretically, the Hbs are classified as HbA, HbB, HbC and HbF. Human beings show three types of Hb, HbA (98%), HbA2 (2%) in the adult and HbF in fetus and new born. HbA has 2 µ -chains and 2 ß -chains; HbA2 is represented by 2 µ and 2 delta – chains. HbF has 2 µ -chains and 2 γ -chains.
  • In adult sheep HbA (2 µ, 2 ß) is electrophoretically fast and has higher O2 affinity than HbB with 4γ chains. Sheep having HbA or HbB under anemia or hypoxic condition develop another type of Hb, the HbC, which partially or completely replaces the Such a change is also observed in goat. HbC is the naturally occurring Hb in sheep during growth period. HbF has higher affinity for O2. In many animal species fetal hemoglobin (HbF) is replaced by the adult types within 4 to 8 weeks after birth. In adult cat the HbA and HbB are found in the same erythrocyte.
  • Some of the Hb variants, HbS, HbC, HbE are associated with specific hematologic disorders. HbS is responsible for sickle-cell anaemia in Negro race. HbC and HbE cause failure of synthesis of alpha or beta chains thus results in alpha or beta thalassemia.



  • Hemoglobin has an important physiological relationship with Oxygen forms loose and reversible combination with hemoglobin called oxyhemoglobin when the erythrocytes passes through the pulmonary capillaries. Since there are four ferrous atoms in the hemoglobin molecule, four molecules of oxygen are transported by a molecule of hemoglobin.
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  • As blood is transported through the systemic capillaries hemoglobin loses its oxygen to the tissues and becomes hemoglobin Hemoglobin shows progressive increase in the affinity for O2 after the first two molecules of O2 are taken up by the heme.
  • The oxygen carrying capacity of the hemoglobin is dependent on the pigment it contains

which in turn depends on the iron content for oxygen combining capacity.

  • The amount of iron present in the blood is minute about 0.334% of the hemoglobin molecule or 04 to 0.05% of the blood itself. Each gram of Hb combines with a maximum of 1.34 ml of oxygen.
  • In the lung (PO2 100 mm Hg) the oxygen binds with Hb which shows 97% saturation. Onehundred ml of blood containing approximately 15 grams of Hb, can carry approximately

19.4 ml of oxygen.

  • In the tissue capillaries, (PO2 40 mm Hg) Hb is 72% saturated and contains 14 ml ofoxygen per 100 ml of blood, which indicates oxygen release from the Hb into the tissues. Thus under normal resting conditions about 5 ml of oxygen is transported by each 100 ml of blood during each cycle to the tissues. During heavy exercise this is increased to about 15 times normal. The oxygen hemoglobin dissociation curve is “S” shaped or sigmoid shaped.


Myoglobin (Muscle hemoglobin)


  • It is a true hemoglobin and functions to store oxygen in the muscle. It contains only one heme and a polypeptide chain. It contains only one iron atom and can therefore store only one molecule of O2.
  • Its molecular weight is approximately 17,000, which is four times less than Hence it

can pass through glomerulus.

  • The oxygen dissociation curve with myoglobin is hyperbolic (very steep). The appearance of myoglobin in the urine is referred to as myoglobinuria or azoturia, which is a very characteristic symptom of monday morning sickness in


Carboxyhemoglobin (HbCO)


  • Hb has 200 times more affinity for carbon monoxide than


Hb+ CO ® HbCO


  • Carbon monoxide firmly attaches with the Fe++ molecules of heme, thus interferes with the transport of O2 as oxy 0.1% of CO in inspired air will convert 20% of Hb into HbCOwithin 30 to 60 minutes.
  • Oxygen under higher partial pressure is the only means to reverse the




Methemoglobin (ferrihemoglobin)


  • It is formed by the oxidation of ferrous iron to ferric iron. During the circulation of blood about 1% of methemoglobin is formed by the oxidation of ferrous iron to ferric iron. Ferrihemoglobin cannot combine with oxygen, hence is useless as a respiratory pigment in the blood. It produces dark colored blood. Glutathione (GSH) in the erythrocytes prevents the excessive oxidation of ferrous iron into ferric
  • Chemicals like nitrates, sulphanamides, aminophenol and acetanilide cause increased concentration of methemoglobin in the blood. Horse blood at normal conditions shows significant amounts of methemoglobin. The normal blood of dog and cat has about 1% of methemoglobin.


  • When white light is passed through a solution of hemoglobin or one of its derivatives, certain wavelengths are The resulting spectrum is termed as absorption spectra; the region of absorption is known as absorption bands.
  • They can be seen by examining the solution with a
  • When white light is examined spectroscopically, a series of colours known as spectrum (VIBGYOR) is
  • When sun light is examined, certain black vertical lines called as Fraunhofer’s lines are found at definite places in the spectrum; these lines are designated as A, B, C, D, E, etc. In lamp light, these lines are not
  • When hemoglobin solution or its derivatives are examined in certain concentrations spectroscopically, absorption bands of definite size, appearance, and position are
  • Hence, spectroscopic examination helps to identify these pigments in solution. E.g. Dilute oxy-Hb solution shows two absorption bands between line D and E; adding a reducing agent (produces reduced-Hb) gives one band at line
  • Carboxy-Hb shows two bands but adding a reducing agent does not produce a single
  • Met-Hb shows a band between line C and



Learning objectives


  • This module deals with
    • production of leukocytes
    • types of leukocytes
    • fate and concentration of leukocytes


  • The process of formation of leukocytes is known as Leukopoiesis. Leukocytes are produced from the pleuripotent stem










  • Leukocytes are large sized, nucleated cells, lesser in number and do not have hemoglobin content. These cells show polymorphic forms, which differ morphologically and functionally.
  • Majority of the WBCs is larger than RBCs; while RBCs are present in millions per ml of blood, the WBCs are present in thousands per ml of
  • The ratio of WBC to RBC varies from 1:100 in chicks to 1:1300 in goats, 1:600 in dog and cat, 1:800 in cattle 1:1000 in horse 1:1200 in sheep and 1:700 in


Types of leukocytes


  • Based on the staining nature of the cytoplasmic granules when subjected to Leishman’s stain, leukocytes are classified as granulocyteswhich are further classified into three groups the neutrophils, eosinophils and
  • Some of the leukocytes have cytoplasmic granules but they are non-visible even after staining with Leishman’s stain, hence they are known as agranulocytes. The lymphocytes and monocytes form two groups of this




Site of production of WBCs


  • The granulocytes and the megakaryocytes are produced from the myelocytes of the bone marrow hence often referred as myeloid series of blood
  • The lymphocytes and the plasma cells are formed from the lymphoid tissues such as lymph nodes, spleen and lymphoid tissues of the bone marrow, gut
  • The monocytes are produced by the mononuclear phagocytic system (MPS) cells of spleen and bone marrow (reticuloendothelial system cells).


  • Neutrophils are produced in the bone marrow from the extravascular neutrophilic myelocytes. They are numerously found in the blood of most
  • The nucleus of mature cells is divided into lobes, usually five lobes and are stained blue or purple by Leishman’s stain. They have abundant and fine granular cytoplasm which take up the neutral
  • These granules store lysosomes, which contfain hydrolytic enzymes, proteolytic enzymes and lipases to digest the invading
  • The oxidative enzymes of the lysosome produce hydrogen peroxide, which attack the bacterial cell wall to cause bactericidal
  • Neutrophils are highly motile, responds to chemotaxis and actively phagocytic, thus serve as a first line of defence against invading organisms, (bacteria, viruses and cellular remnants).
  • At the onset of infection neutrophils produce pyrogens which act on thermo-regulator centre of the brain results in fever. This rise in body temperature slows the reproduction process of bacteria and viruses
  • Immature forms of neutrophils are characterized by unsegmented or less number of nuclear lobes  and  are  referred  to  as  juvenile  or  band    Pseudo  neutrophils   and heterophils are comparable to neutrophils, which are present in rabbit and poultry respectively.
  • Heterophils contain large rod or spindle shaped granules, which are acid in reaction and stain red or pink with eosin.






  • It indicates more number of neutrophils in the circulation.
  • Physiological neutrophilia occur in conditions like exercise, emotion, pregnancy, lactation and
  • Abnormal or pathological neutrophilia may be due to acute inflammation following injury, surgery, burns, arthritis and acute infection by pyrogenic bacteria.
  • Shift to left is a term used to describe an increase in the number of immature neutrophils (Band cells) in the circulation which is characteristic of bacterial infections. The shift to left is clinically helpful in diagnosis of traumatic reticulopericarditis (TRP).






  • It indicates reduction in neutrophils in the circulation, which is very common in viral infections and chronic infections like TB, brucellosis and protozoal and fungal
  • Injection of antiinflammatory drugs (cortisol) and antibacterial drugs (chloramphenicol and sulphanamides) may result in




  • They are large cells which contain large cytoplasmic granules that stain red or purple eosin stain. They have bilobed nucleus, connected by a thin
  • The cytoplasmic granules contain enzymes, which are rich in oxidases‚ and peroxidases. Eosinophils are produced from the bone marrow and are highly motile, but less phagocytic.
  • Eosinophils function to detoxify the proteins of the parasites, phagocytise antigen- antibody complexes, the inflammatory products of the mast cells and
  • These cells have antiheparin‚ and anti- histaminic substances, thus act as an anti- inflammatory and anti- allergic agent. Eosinophils release profibrinolysin‚ which is then activated to fibrinolysin‚ and causes the dissolution of old blood




  • Eosinophilia: It is increased number of eosinophils in the circulation. This condition is common in G.I. parasitic infections, allergic disorders like bronchial asthma, allergic rhinitis, in skin diseases like eczema and dermatitis, drug reactions following penicillin and sulphanamides
  • Eosinopenia: It is decreased number of eosinophils in circulation. This condition occurs following stress , administration of ACTH or cortisol




  • These cells are found in the blood in lower concentration. They have irregular shaped nucleus and the granules are stained blue by the basic dye of Leishman’s stain. They originate in the bone marrow and have slight or no
  • Extravascularly, basophils enlarge and become tissue mast cells. Both basophils and mast cells release heparin (anticoagulant)‚ and vasodilator substances histamine, serotonin and bradykinins. Thesevasodilators causes increased blood flow and reddish colour (hyperemia) at the site of infection followed by increased permeability of the plasma and proteins from the blood vessels, a process calledinflammation. Basophils and mast cells have receptors for immunoglobin






  • Basophilia: It is increase in basophils, and it is seen in allergic conditions and hematological


  • These cells are characterised by well defined centrally placed nucleus, surrounded by cytoplasm. According to the size and the ratio of nucleus to cytoplasm, these cells are further divided into 3
    • Small lymphocytes: These are small sized cells with a large notched
    • Medium lymphocytes: These are medium sized, which have notched nucleus surrounded by thin rim of
    • Large lymphocytes: These have comparatively small sized nucleus with broad rim of
  • Lymphocytes are actively motile cells with amoeboid movement and are mainly concerned with the development of immunity against specific disease organisms. They have no phagocytic




  • Lymphocytosis: This condition indicates increased number of lymphocytes in the circulation, which is common in viral infection (eg) infectious
  • Lymphopenia: This condition indicates decreased number of lymphocytes in circulation and is seen in cases of TB, acute stress and glucocorticoid injection.


  • These are the largest of WBCs, characterised by kidney or bean shaped
  • Monocytes are actively motile, pinocytic and also highly phagocytic, which may migrate into the tissue spaces and become macrophages. These cells are much more powerful phagocytes than neutrophils by their ability to engulf much large sized particles, necrotic tissues and more number of
  • Monocytes leave the blood and are attracted to the tissues by chemotaxis and lymphokines and they become macrophages, example reticular cells of lymph nodes, spleen, bone marrow and Kupffer cells of the liver






  • This condition indicates increased number of monocytes in the circulation and is seen in hematological malignancies, endocarditis, typhoid, TB, brucellosis and prolonged blood parasitic infections. Eg:









Total leukocytes (x103/ cu.mm)  

Differential Leukocyte Count (%)









Band Mature




0-3(0.8) 60-77


12-30 (20) 3-10 (5) 2-10 (4) Rare




0-3 (0.5) 35-75


20-55 (32) 1-4 (3) 2-12 (5) Rare


4-12 (8) 0-2




45-75 (58) 2-7 (4) 2-20 (9) 0-2 (0.5)


4-12 (8) Rare 10-50


40-75 (62) 0-6 (2.5) 0-10 (5) 0-3 (0.5)


4-13 (9) Rare 30-48


50-70 (56) 0-4 (2.5) 1-8 (5) 0-1 (0.5)


5.5-12.5 (9) 0-2




25-70 (44) 1-7 (4) 0-11 (4) 0-3 (0.5)


11-22 (16) 0-4 (1) 28-47


39-82 (53) 2-10 (5) 1-11 (3) 0-2 (0.5)
Chicken 20-30 25-30 55-60 10 3-8 1-4




  1. It is very difficult to assess the life span of leukocytes because the WBCs move between blood and
  2. The life span of granulocytes is normally 9 days but once they are released into circulation from bone marrow their life span is 4 to 8
  3. During infection, the life span in “B” lymphocytes live for 3-4 days only but “T” lymphocytes may live for 1-3 years in tissues and return to circulation many times.
  4. The monocytes also have short transit time in the blood (24 hours) before migrating to the tissues where they become tissue macrophages and can live for months or even


  • Leukocytosis:
    • It is an increase in the number of leukocytes which is an indication of presence of some



  • Leukocytosis may be physiological (related to time of day, meal, exercise, epinephrine, stress) or pathological. Bacterial infection shows leukocytosis with neutrophilia, whereas viral infections result in
  • Leukemia:
    • Pathologically, cancer of leukocyte producing tissues results in abnormally high white cell count which is known as


  • Plasma cells are white blood cells which produce large volumes of
  • They are transported by the blood plasma and the lymphatic system and originate from the bone marrow.
  • They are otherwise known as plasma B cells , plasmocytes , and effector B cells .
  • They are present in lymph nodes ,spleen and diffuse lymphoid tissue of alimentary and respiratory


  • Plasma cells are large lymphocytes round in shape containing a granular cytoplasm which stains with basic
  • The nucleus is eccentric in position and typically represents clumps of chromatin in a radiating manner, resembling a “cart -wheel” or clock face in
  • They have a considerable nucleus-to-cytoplasm
  • The cytoplasm contains a conspicuous Golgi apparatus and abundant endoplasmic reticulum .
  • Immunoglobulins are localised in the endoplasmic reticulum where it sometimes forms aggregates called as Russel


  • When specific B-lymphocytes are stimulated by specific antigen, they enlarge and become lymphoblast, some of which further differentiate to immature plasmablasts which finally forms, the plasma


  • These cells are rarely seen in the circulation, which are formed by the lymphoid tissues (spleen).
  • They play a very important function in body defence
  • They are involved in the production of humoral
  • Plasma cells are concerned with the synthesis, storage and release of immunoglobulins at a very rapid
  • A plasma cell can only synthesize an antibody of a single specimen, either IgM or IgG or IgA except in primary immune response when a plasma cell producing IgM initially, may later switch over to the synthesis of IgG
  • Mature plasma cells are end cells and survive only a few weeks and die after a few cell division.


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