LEUCOGRAM

LEUCOGRAM. BLOOD COMPOUNDS, FUNCTIONS. BLOOD PICTURE. LEUCOCYTES. HEMATOPOIESIS.

 

Using lectures (on the web-page of the department posted the presentation text and lectures), books, additional literature and other sources, students must to prepare the following theoretical questions:

1. Main components of the blood and their structure.

2. Description of the plasma composition in term of its percentage of water, plasma proteins and inorganic salts.

3. Morphofunctional characteristic of the erythrocytes. Anisocytosis and poikilocytosis.

4. Structure and function of leukocytes normally found in the blood. Classification of the leukocytes.

5. Morphofunctional characteristic of the granulocytes (neutrophils, eosinophils and basophils).

6. Morphofunctional characteristic of the agranulocytes (lymphocytes and monocytes).

7. Description of a clot formation, including the structure and role of platelets.

8. Clinical analysis of the blood: blood picture and leukocyte’s formula.

9. Definition, origin and composition of the lymph.

10. General features of hematopoiesis.

11. Structural and functional characteristics of hematopoietic stem cell.

12. Monophyletic and polyphyletic theories of haematopoiesis.

13. Description of the sequence of events in the life of each formed element of blood, from stem cell to death, and know the organ where each event occur: Erythropoiesis. Granulopoiesis. Agranulopoiesis. Thrombopoiesis

14. Compartments and the life cycle of blood cell types.

 

Blood is fluid tissue of human body, classified as a tissue of inner environment. However, its intercellular substance is a liquid, and its cells are not in a fixed position as is the case in other tissues. The blood of adult vertebrates is a red liquid, which circulates in a closed system of tubes, the blood vessels. It is pumped from the heart into arteries, from the arteries into capillaries, and from the capillaries it flows into veins for return to the heart.

Each system of the human body plays an important part in maintaining homeostasis in the internal cellular environment, but the movement of the blood through the circulatory system is of fundamental importance.

The chief function of the blood is to maintain normal cell function by the constant exchange of nutrients and wasted with all cells.

The blood must also maintain optimum pH and temperature of the intracellular fluid if the cells’ enzyme systems are to work efficiently.

The blood transports oxygen from the lungs to the tissues, and carbon dioxide to the lungs for elimination.

It transport nutrients from the intestine to all parts of the body, and it carries certain waste products to the kidneys for excretion.

The blood distributes the heat produced in active muscles and thus aids in the regulation of body temperature.

It transports internal secretions from the glands in which they are produced to the tissues on which each exerts its effects.

The buffers in the blood help to maintain acid – base balance.

The blood also is involved in immunity to disease and in protecting the body against invading bacteria.

Blood is divisible into 2 parts, the formed elements and the plasma, in which the formed elements are suspended and in which a variety of important proteins, hormones and other substances are dissolved. Its quantity in man is estimated as about 7 per cent of the body weight.

 

Hematocrit tubes with blood. Left: Before centrifugation. Right: After centrifugation. The erythrocytes represent 43% of the blood volume in the centrifuged tube. Between the sedimented erythrocytes and the supernatant light-colored plasma is a thin layer of leukocytes called the buffy coat.

 

The cells of blood are of three major functional classes: red blood cells (erythrocytes), white blood cells (leucocytes) and platelets (thrombocytes).

Erythrocytes are primarily involved in oxygen and carbon dioxide transport, the leucocytes constitute an important part of the defense and immune systems of the body, and platelets are a vital component of the blood clotting mechanism. All these cell types are formed in the bone marrow by a process called hemopoiesis. Erythrocytes and platelets function entirely within blood vessels whereas leucocytes act mainly outside blood vessels in the tissues. Thus the leucocytes found in circulating blood are merely in transit between their various sites of activity.

Composition of plasma

Plasma contains 90 % water and 10 % solutes by volume. These solutes include plasma proteins and other organic compounds as well as inorganic salts.

Plasma contains a rich variety of soluble proteins, 7 % by volume. There are three main types: albumins, globulins and fibrinogen. Collectively, the plasma proteins exect a colloidal osmotic pressure within the circulatory system, which helps to regulate the exchange of aqueous solution between plasma and extra cellular fluid.

Albumin – this is the most abundant plasma protein (3,4-5 g/dl of blood) and is mainly responsible for maintaining the osmotic pressure of blood. Substances that are partly or completely water-insoluble (eg, lipids) are transported in the plasma in association with albumin.

The globulins are a diverse group of proteins, which include the antibodies of the immune system, and certain proteins responsible for the transport of lipids and some heavy metal ions.

Alpha, beta, and gamma globulins are globular proteins dissolved in the plasma. The gamma globulins are antibodies and are called immunoglobulins.

Fibrinogen – this protein is converted by blood-borne enzymes into fibrin during clot formation. Fibrinogen is synthesized and secreted by the liver.

Other organic compounds – Other organic molecules in plasma, 2.1 % by volume, include nutrients such as amino acids and glucose, vitamins, and a variety of regulatory peptides, steroid hormones, and lipids.

Inorganic salts in plasma, 0.9 % by volume, include blood electrolytes such as sodium, potassium, and calcium salts.

Blood is studied by spreading a drop on a slide to produce a single layer of cells (blood smear). The cells are stained, differentiated by type, and counted to reveal any disease-related changes in their relative numbers. The smears are usually stained with modifications of dye mixtures containing eosin and methylene blue, ie, Romanovsky-type mixtures.

All of the descriptions of the staining properties of blood cells refer to their appearance after staining with Romanovsky-type mixtures (e.g., Wright’s stain or Giemsa stains). Blood and their components exhibit 4 major staining properties that allow the cell types to be distinguished:

1.       Basophilia – is an affinity for methylene blue. Basophilic structures stain purple to black.

2.       Azurophilia – is an affinity for the oxidation products of methylene blue called azures. Azurophilic structures stain red-blue.

3.       Eosinophilia, or acidophilia, is an affinity for eosin. Eosinophilic structures stain yellow-pink to orange.

4.       Neutrophilia is an affinity for a complex of dyes (originally thought to be neutral) in the mixture. Neutrophilic structures stain salmon pink to lilac.

 

The formed Elements

 

Erythrocytes, or red blood cells, are the most prevalent cells in peripheral blood. The peripheral blood of an individual contains 25,000,000,000,000 (twenty-five trillion) erythrocytes, and the spleen and bone marrow contain many more.

Each cubic millimeter of blood contains approximately 5 x 10⁶ red cells. The total peripheral blood volume is approximately 5 L. Under normal conditions, these cells never leave the circulatory system. The normal concentration of erythrocytes in blood is approximately 3.9 – 5.5 million per microliter in women and 4.1 – 6 million per microliter in men. A decrease number of erythrocytes in the blood is usually associated with anemia. An increase number of erythrocytes (erythrocytosis) may be a physiologic adaptation – it is found, for example, in people who live at high altitudes, where O₂ tension is low. Erythrocytosis, which is often associated with diseases of varying degrees of severity, increases blood viscosity; when severe, it can impair circulation of blood through the capillaries. Erythrocytosis might be better characterized as an increased hematocrit, ie, an increased volume occupied by erythrocytes.

Structure. Most mammalian erythrocytes are biconcave disks without nuclei. When suspended in the isotonic medium, human erythrocytes are 7,5 μm in diameter, 2.6 μm thick at the rim, and 0.8 μm thick in the center. The biconcave shape provides erythrocytes with large surface-to-volume ratio, thus facilitating gas exchange. Erythrocytes with diameters greater than 9 μm are called macrocytes, and those with diameters less than 6 μm are called microcytes. A presence of a high percentage of erythrocytes with great variations in size is called anisocytosis.

Scanning electron micrograph of normal biconcave human erythrocytes. x3300.

 

Scanning electron micrograph of a distorted erythrocyte from a person who is homozygous for the HbS gene (sickle cell disease). x6500.

 

 

         The erythrocytes are quite flexible, a property that permits it to adapt to the irregular shapes and small diameters of capillaries. Observations in vivo show that when traversing the angels of capillary bifurcations, erythrocytes containing normal adult hemoglobin (Hb A) are easily deformed and frequently assume a cup-like shape.

Erythrocytes are surrounded by plasmalemma; because of its ready availability, this is the best-known membrane of any cell. It consists of about 40% lipid, 50% protein and 10% carbohydrates. About half the proteins span the lipid bilayer and are known as integral membrane proteins. Several peripheral proteins are associated with the inner surface of erythrocyte membrane. The peripheral proteins seem to serve as a membrane skeleton that determines the shape of the erythrocyte. They also permit the flexibility of the membrane necessary for the large changes in shape that occur when the erythrocyte passes through capillaries. Because the erythrocytes are not rigid, the viscosity of blood normally remains low.

Mature RBCs lack nuclei and cytoplasmic organelles, which they lose during differentiation. Because they lack mitochondria, the energy needed to maintain the hemoglobin in a functional state must be derived from anaerobic glycolysis. Because they lack ribosomes, the glycolytic enzymes and other important proteins cannot be renewed.

Red blood cells are structurally and functionally specialized to transport oxygen from the lungs to other tissues. Their cytoplasm contains the 33% solution of oxygen-binding protein hemoglobin – the O₂ – carrying protein that account for their acidophilia. About 1/3 of the erythrocyte mass is hemoglobin. Each hemoglobin molecule consists of 4 polypeptide subunits, each of which includes an iron-containing heme group.

Hemoglobin (Hb) exists in a variety of forms, distinguishable on the basis of the amino acid sequence of their subunits. In humans, only 3 forms are considered normal in postnatal life: HbA1 constitutes 97 %, HbA2 2 %, and HbF 1 % of the hemoglobin of healthy adults. HbF comprises around 80 % of the hemoglobin of newborns, however; this figure gradually decreases until it reaches normal adult levels at about 8 months of age. HbS is an abnormal form of HbA that is found in patients with sickle cell anemia; it differs by a single amino acid substitution in the beta chain (valine in HbS, glutamine in HbA). Unlike HbA, HbS becomes insoluble at low oxygen tensions and crystallizes into inflexible rods that deform the RBCs, giving them the characteristic sickle shape. When the rigid sickled cells pass through narrow capillaries, they cannot flexibly adapt to the passageway as normal RBCs do. They may become trapped, obstructing blood flow through the capillary, or rupture, decreasing the number of RBCs available for oxygen transport (anemia).

Combined with O₂ and CO₂, hemoglobin forms oxyhemoglobin or carbaminohemoglobin, respectively. The reversibility of these combinations is the basis for the gas – transporting capability of hemoglobin. The combination of hemoglobin with carbon monoxide (carboxyhemoglobin) is irreversible, however, and causes a reduced capacity to transport O₂.

Mature erythrocytes therefore have a limited lifespan (120 days) in the circulation before they are removed by macrophages in the spleen and bone marrow.

Erythrocytes recently released by the bone marrow into the bloodstream often contain ribosomal RNA, which, in the presence of supravital dyes (eg, brilliant cresil blue), can be precipitated and stained. Under these conditions, the younger erythrocytes, called reticulocytes, may have a few granules or a net-like structure in their cytoplasm. The process by which reticulocytes are released from the bone marrow into the circulation is not completely understood.

Reticulocytes normally constitute about 1% of the total number of circulating erythrocytes; this is the rate at which erythrocytes are replaced daily from bone marrow. Increased numbers of reticulocytes indicate a demand for increased O₂ – carriyng capacity, which may be caused by such factors as hemorrhage or a recent ascent to high altitude.

LEUCOCYTES

Leukocytes, or white blood cells, are nucleated and are larger and less numerous than erythrocytes. Leukocytes can be divided into 2 main groups, granulocytes and agranulocytes, according to their content of cytoplasmic granules.

Each of these groups can then be further divided on the basis of size, nuclear morphology, ratio of nuclear to cytoplasmic volume, and staining properties. Two classes of cytoplasmic granules occur in leukocytes, specific and azurophilic granules. Specific granules are found only in granulocytes; their staining properties (neutrophilic, eosinophilic, or basophilic) distinguish the 3 granulocytes types.

Azurophilic granules are found in both agranulocytes and granulocytes. Azurophilic granules stain purple and are lysosomes.

 

 

The 5 types of human leukocytes. Neutrophils, eosinophils, and basophils have granules that stain specifically with certain dyes and are called granulocytes. Lymphocytes and monocytes are agranulocytes; they may show azurophilic granules, which are also present in other leukocytes.

 

Unlike the RBCs, all leukocytes can leave the capillaries by passing between endothelial cells, and penetrating the connective tissue by means of the process called diapedesis. The types and levels of activity expressed by extravascular leukocytes depends upon the specific cell type.

GRANULOCYTES

Granulocytes have segmented nuclei and are described as polymorphonuclear leukocytes (PMNLs). Depending on the cell type, the mature nucleus may have from 2 to 7 lobes connected by thin strands of nucleoplasm. Granulocyte types are most easily distinguished by their size and staining properties, and by the appearance (as seen with an electron microscope) of the abundant specific granules in their cytoplasm. These granules are all membrane-limited and bud off the Golgi complex. All granulocytes have a life span of a few days, dying by apoptosis (programmed cell death) in the connoctive tissue. The resulting cellular removed by macrophages and does not elicit an inflamatory response.

Neutrophils – are the most abundant leukocytes in the blood. They usually constitute 60-72 % of the white blood cells in healthy adults. They are also found outside the bloodstream, especially in loose connective tissue. Neutrophils are the first line of cellular defense against the invasion of bacteria. Once they leave the bloodstream, they spread out, develop amoeboid motility, and become active phagocytes. Unlike lymphocytes, neutrophils are all terminally differentiated cells and so are incapable of mitosis.

Size – neutrophils in the blood are approximately 12 μm in diameter, while those in the tissues spread to a diameter of up to 20 μm.

Nucleus – neutrophil nuclei contain highly condensed chromatin both in the lobes and in the attenuated chromatin bridges between them. Most have 3 lobes; however, lobe number increases from a single horseshoe-shaped nucleus in immature neutrophils, called band neutrophils, to more than 5 lobes in aging ones. The nuclei of certain diseased neutrophils, called hypersegmented neutrophils, also have more than 5 lobes (they are typically old cells). In females, a small heterochromatic body often extends from one of the nuclear lobes. This represents the inactive X chromosome, or Barr body, and is referred to as a drumstick – like appendage because of its characteristic shape.

 

Photomicrograph of a blood smear showing 3 neutrophils and several erythrocytes. Each neutrophil has only one nucleus, with a variable number of lobes. Giemsa stain. High magnification.

 

 

Electron micrograph of a humaeutrophil stained for peroxidase. The cytoplasm contains 2 types of granules: the small, pale, peroxidase-negative specific granules and the larger, dense, peroxidase-positive azurophilic granules. The nucleus is lobulated, and the Golgi complex is small. Rough endoplasmic reticulum and mitochondria are not abundant, because this cell is in the terminal stage of its differentiation. x27,000.

 

Neutrophil cytoplasm is abundant and filled with specific membrane-bound granules. These granules are modified lysosomes and have a bactericidal function.

Azurophilic granules (primary, or type A) stain with azure dye and are diagnostic for neutrophils. These large (0.4 um) electron-dense granules comprise about 20 % of the granule population and are visible in the light microscope.

Specific granules (secondary, or type B) are smaller (0,2 μm) and may contain crystalloids. They comprise 80 % of the granule population and are not visible in the light microscope. They stain salmon pink with typical bloodstains. The less numerous azurophilic granules stain a reddish-purple. The specific granules contain alkaline phosphatase and bactericidal cationic proteins called phagocytes. Azurophilic granules contain lysosomal enzymes and peroxidase. Neutrophils also contain more glycogen than other leukocytes.

Neutrophils are short-lived cells with a half-life of 6-7 hours in blood and a life span of 1-4 days in connective tissues, where they die by apoptosis.

The primary function of neutrophils is the phagocytosis and destruction of bacteria. Neutrophils are active phagocytes of small particles and have sometimes been called microphages to distinguish them from macrophages, which are larger cells. Neutrophils are inactive and spherical while circulating but change shape upon adhering to a solid substrate, over which they migrate via pseudopodia.

Bacteria first adhere to the neutrophil surface and then are surrounded and engulfed by pseudopodia; in this way bacteria eventually occupy vacuoles (phagosomes) delimited by a membrane derived from the cell surface. Immediately thereafter, specific granules fuse with and discharge their contents into the phagosomes. Azurophilic granules then discharge their enzymes into the acid environment, killing and digesting the microorganisms.

Eosinophils – constitute only 0,5-54 % of the circulating leukocytes in healthy adults. They may leave the bloodstream by diapedesis, spread out, and move about in the connective tissues. They are capable of only limited phagocytosis, showing a preference for antigen-antibody complexes. The number of circulating eosinophils typically increases (eosinophilia) during allergic reactions and in response to parasitic (helminthic) infections, and rapidly decreases in response to treatment with exogenous corticosteroids. These cells produce substances that modulate inflammation by inactivating the leukotriens and histamine produced by other cells.

Eosinophil with its bilobed nucleus and coarse cytoplasmic granules. Giemsa stain. High magnification.

 

Photomicrograph of an eosinophil. Note its typical bilobed nucleus and coarse cytoplasmic granules. Giemsa stain. High magnification.

 

Eosinophils are usually slightly smaller than typical neutrophils, measuring about 12 μm in diameter in the blood and up to 14 μm in loose connective tissue.

 

 

 

Electron micrograph of an eosinophil. Typical eosinophilic granules are clearly seen. Each granule has a disk-shaped electron-dense crystalline core that appears surrounded by a matrix enveloped by a unit membrane. EG, eosinophil granule; N, nucleus; M, mitochondria. x20 000.

 

Eosinophil nuclei contain condensed chromatin and usually have 2-3 lobes connected by a thin chromatin bridge (bilobed nucleus). The nuclei are often partially obscured by the numerous specific granules in the cytoplasm.

Cytoplasm – The most characteristic structural feature of eosinophils is the presence of numerous large (0.5-1.5 μm in diameter), brightly eosinophilic granules (specific granules) in their cytoplasm (about 200 per cell). These granules are specialized lysosomes that lack lysozyme but contain acid phosphatase, cathepsin, and ribonuclease. In electron micrographs, each specific granule has an oblong shape and an elongated, centrally located, electron-dense crystalloid, or internum, lying parallel to its long axis. It contains a protein called the major basic protein – with a large number of arginine residues. This protein constitutes 50% of the total granule protein and accounts for the eosiniphilia of these granules. The major basic protein also seems to function in the killing of parasitic worms such as schistosomes. Between the granule membrane and the internum lies the electron-lucent matrix, or externum. Microfilaments are prominent in the eosinophil cortex.

Functions. Eosinophils kill parasitic larvae as they enter peripheral blood or the lamina propria of the gut. They help to regulate mast cell response to inflammation by releasing an enzyme that degrades the histamines released by mast cells at inflammation sites. Eosinophilic granule crystalloids have a dominant component called the major basic protein, which has a poorly understood antiparasitic function. Eosinophilic granules contain lysosomal enzymes that destroy dead parasites.

Basophils are the least numerous of the circulating leukocytes, constituting from 0 to 1% of the white blood cells of healthy adults. Like other white blood cells, basophils may leave the circulation, but they are capable of only very limited ameboid movement and phagocytosis in the tissues. Extravascular basophils are most often found at sites of inflammation and may be the major cell type at sites of cutaneous basophil hypersensitivity.

Basophils vary in diameter from 12 μm to 15 μm but are usually slightly smaller thaeutrophils. Their nuclei are less heterochromatic than other granulocytes and usually consist of 3 irregular lobes which are often obscured by the large, dark-staining cytoplasmic granules. The specific granules of basophils are their most characteristic feature. These granules have irregular shapes and vary in size; the largest are the size of the specific granules of eosinophils, the smallest nearly as small as those of neutrophils. The granules stain metachromatically and appear reddish-violet to nearly black in stained blood smears. The specific granules of basophils (like those of the mast cells of connective tissue) contain heparin and histamine, which may be released by exocytosis in response to certain types of antigenic stimuli. The granules may contain inclusions, but they appear more homogeneously electron-dense than do those of eosinophils.

 

Two leukocytes and several erythrocytes. The cell on the right is a basophil. The cell on the left is a neutrophil. In the basophil there are many cytoplasmic granules over the nucleus. Giemsa stain. High magnification.

 

A basophil with many granules covering the cell nucleus. This makes it difficult to see the nucleus clearly. Some erythrocytes were deformed during the smear preparation. Giemsa stain. High magnification.

 

Electron micrograph of a rabbit basophil. The lobulated nucleus (N) appears as 3 separated portions. Note the basophilic granule (B), mitochondria (M), and Golgi complex (G). x16,000.

 

Functions. Basophils mediate the inflammatory response and secrete eosinophil chemotactic factor. In response to certain antigens, basophils stimulate the formation of immunoglobulin E(IgE) – a class of antibodies. Subsequent exposure to the same antigen can cause a basophil and mast cell response restricted to specific organs (e.g., bronchial asthma in the lungs or a severe and systemic response such as anaphylactic shock brought on by a bee sting).

 

Agranulocytes

 

Agranulocytes have round unsegmented nuclei and are described as mononuclear leukocytes. They lack specific granules, but they contain various number of azurophilic granules (lysosomes) that bind the azure dies of the stain. This group includes the lymphocytes and monocytes.

Lymphocytes – constitute a diverse class of cells; they have similar morphologic characteristics but a variety of highly specific functions. They normally account for 20-25 % of the white blood cells in adult blood, with a considerable range of normal variation (20-45%). Lymphocytes are also found outside the blood vessels, grouped in lymphatic organs or dispersed in connective tissues. They respond to invasion of the body by foreign substances and organisms and assist in their inactivation. They also have diverse functional roles, all related to immune reactions in defending against invading microorganisms, foreign macromolecules and cancer cells. Unlike other leukocytes, lymphocytes never become phagocytic.

Photomicrograph of a large lymphocyte and several erythrocytes. The nucleus of this cell is round, and the cytoplasm is devoid of specific granules. Giemsa stain. High magnification.

 

Two small lymphocytes with their round, dark-stained nuclei. Giemsa stain. High magnification.

 

They can be classified into several groups due to distinctive surface molecules (markers), which can be distinguished only by immunocytochemical methods.

The 2 major functional classes of lymphocytes are T cells and B cells. Lymphocytes in the blood are predominantly (about 80 %) T cells.

 

 

Electron micrograph of a human blood lymphocyte. This cell has little rough endoplasmic reticulum but a moderate quantity of free polyribosomes. Note the nucleus (N), the nucleolus (Nu), and the mitochondria (M). Reduced from x22,000.

 

Lymphocytes vary from 6 to 18 μm in diameter. Most of those found in blood are small lymphocytes in the 6- to 8 μm range, making them the smallest leukocytes, comparable in size to erythrocytes. A small number of medium-sized and large lymphocytes are also ground in the circulation and probably represent lymphocytes activated by an antigen.

Lymphocyte nuclei are spheric and often flattened on one side. In small lymphocytes, the nucleus is densely heterochromatic, staining purplish-blue to black, and nearly fills the cell. In large lymphocytes, the nucleus is larger and less dense and stains reddish-purple.

Lymphocyte cytoplasm exhibits a pale basophilia and occasionally contains a few purplish azurophilic granules but lacks specific granules. In the smaller cells, the cytoplasm forms a thin rim around the nucleus; in the larger cells, it is more abundant. It contains many free ribosomes, few mitochondria, sparse endoplasmic reticulum, and a small Golgi complex. When stimulated by an antigen, lymphocytes undergo blast transformation, a process of enlargement and sequential mitotic divisions. Some of the daughter cells, called memory cells, return to an inactive state but retain the capacity to respond more quickly to the next encounter with the same antigen. Other daughter cells, called effector cells, become activated to carry out an immune response to the antigen. Effector cells may be derived from either B lymphocytes (B cells) or T lymphocytes (T cells). While circulating B and T cells are morphologically indistinguishable, they carry different cell-surface components (antigens recognized by other species) and can be identified by special procedures.

B Lymphocytes differentiate into plasma cells, which secrete specific antigen-binding molecules (antibodies or immunoglobulin’s) that circulate in the blood and lymph and serve as a major component of humoral immunity.

T Lymphocyte derivatives serve as the major cells of the cellular immune response. They produce a variety of factors, termed lymphokines (eg, interferon) that influence the activities of macrophages and of other leukocytes involved in an immune response. There are several types:

(i)               Cytotoxic (killer) cells secrete substances that kill other cells and in some cases kill by direct contact; they play the major role in graft rejection.

(ii)             Helper T cells enhance the activity of some B cells and other T cells.

(iii)           Suppressor T cells inhibit the activity of some B cells and other T cells.

The primary (central) lymphoid organs include the thymus, where lymphocyte precursors are programmed to become T cells and, in birds, the bursa of Fabricius, where lymphocyte precursors are programmed to become B cells. Humans have no bursa; our B cells appear to be programmed in the bone marrow.

         According to the electron-microscopic studies there are 4 different types:

1.                Small dark lymphocytes;

2.                Small light lymphocytes;

3.                Medium lymphocytes;

4.                Plasmocytes or lymphoplasmocytes.

         Lymphocytes vary in life span; some live only a few days, and others survive in the circulating blood for many years. Lymphocytes are the only type of leukocytes that return from tissue back to the blood, after diapedesis.

 

Monocytes are often confused with large lymphocytes, but they are larger and constitute only 3-8 % of the white blood cells in healthy adults. Monocytes are found only in the blood, but they remain in circulation for less than a week before migrating through capillary walls to enter other tissues or to become incorporated in the lining of sinuses. Once outside the bloodstream, they become phagocytic and apparently do not recirculate. Monocytes are the direct precursors to macrophages. The mononuclear phagocyte system (portions of which were formerly referred to as the reticuloendothelial system) consists of monocyte-derived phagocytic cells distributed throughout the body. Examples include the Kupffer cells of the liver and some of the macrophages of connective tissues.

 

Photomicrograph of a monocyte. This cell type has a kidney-shaped nucleus with delicately stained chromatin. The cytoplasm is slightly basophilic. Giemsa stain. High magnification.

 

Monocytes in the blood of healthy adults have a diameter of 12-15 μm, but when they attach to surfaces they flatten and spread out, often reaching 20 μm in diameter they are the largest among leukocytes).

Monocyte nuclei may be ovoid, but are usually kidney- or horseshoe shaped and eccentrically placed; unlike lymphocyte nuclei, they are rarely spherical. The chromatin is less condensed than that of lymphocyte nuclei, has a “smudgy” appearance, and stains reddish-purple. There may be 2-3 nucleoli, but these are often difficult to distinguish.

 

Electron micrograph of a human monocyte. Note the Golgi complex (G), the mitochondria (M), and the azurophilic granule (A). Rough endoplasmic reticulum is poorly developed. There are some free ribosomes (R). x22,000.

 

Cytoplasm – The faint blue-gray cytoplasm of monocytes is more abundant than that of lymphocytes and contains many small azurophilic granules, which are distributed through the cytoplasm, giving it a bluish-gray color in stained smears. In the electron microscope, one or two nucleoli are seen in the nucleus, and a small quantity of rough endoplasmic reticulum, polyribosomes. It also contains many small mitochondria, a well-developed Golgi apparatus. Many microvilli and pinocytotic vesicles are found at the cell surface.

An increase in the number of leukocytes is called leukocytosis; this occurs in most systemic and localized infectious processes, such as appendicitis or abscesses. It is a normal response to infection. On the other hand, a decrease in the number of leukocytes is called leukopenia; this may occur in certain acute and chronic diseases, such as typhoid fever or tuberculosis. Leukopenia is also a constant finding in radiation sickness, the clinical result of excessive exposure to gamma rays. For example, victims of the atomic bomb expositions were exposed to intensive radiation and as result suffered a marked depression of bone marrow function; the absolute white count in the more severe cases ranged from 1500 to zero per cu. mm. of blood. There also was anemia, due to interference with formation of red blood cells.

 

Platelets: Platelets, or thrombocytes, the smallest formed elements in the blood, are dislike cell fragments that vary in diameter from 2 to 5 μm. In humans, they lack nuclei and originate by budding from large cells in the bone marrow called megakaryocytes. They range iumber from 150,000 to 300,000 microliter of blood and have a lifespan of about 10 days. In blood smears they appear in clumps. Each platelet has a peripheral hyalomere region that stains a faint blue and a dense central granulomere that contains a few mitochondria and glycogen granules and a variety of purple granules. Dense bodies, or delta granules, are 250-300 μm in diameter and contain calcium ions, pyrophosphate, ADP, and ATP; they take up and store serotonin. Alpha granules are 300-500 μm in diameter and contain fibrinogen, platelet-derived growth factor, and other platelet-specific proteins. Lambda granules (platelet lysosomes) are 175-200 μm in diameter and contain only lysosomal enzymes. The hyalomere contains a marginal bundle of microtubules that helps to maintain the platelet’s discoid shape. The glycocalyx is unusually rich in glycosaminoglycans and is associated with adhesion, the major functional characteristic of platelets. Platelets have an important physical role in plugging wounds. They promote blood clotting and help repair gaps in the walls of blood vessels, preventing loss of blood.

Electron micrograph of human platelets. x40,740.

 

The Role of Platelets is controlling hemorrhage can be summarized as follows.

1.                 Primary aggregation – Discontinuities in the endothelium, produced by blood vessel lesions, are followed by absorption of plasma proteins on the subjacent collagen. Platelets immediately aggregate on this damaged tissue, forming a platelet plug.

2.                 Secondary aggregation – Platelets in the plug release the contents of their alpha and delta granules. ADP is a potent inducer of platelet aggregation.

3.                 Blood coagulation – Platelets release fibrinogen in addition to that normally found in the plasma. The fibrinogen is converted by the clotting factor cascade into fibrin, which forms a dense fibrous mat to which more platelets and other blood cells attach to form blood clot or thrombus.

4.                 Clot Retraction: The clot (thrombus) that initially bulges into the blood vessel lumen contracts because of the interaction of platelet actin, myosin, and ATP.

5.                 Clot Removal: Protected by the clot, the vessel wall is restored by new tissue formation. The clot is then removed, mainly by the proteolytic enzyme plasmin, formed, through the activation of the plasma proenzyme plasminogen activators. Enzymes released from platelet lambda granules also contribute to clot removal.

A marked reduction in the number of blood platelets is called thrombocytopenia, and a marked increase in the number of blood platelets is called thrombocytosis.

Products and functions of the blood cells

Cell type	Main products	Main functions
Erythrocytes	Hemoglobin	CO2 and O2 transport
Leukocytes Neutrophil (terminal cell)	Specific granules and modified lysosomes (azurophilic granules)	Phagocytosis of bacteria
Eosinophil (terminal cell)	Specific granules, pharmacologically active substances	Defense against para-sitic helmints; modulation of inflammatory processes
Basophil (terminal cell)	Specific granules containing histamine and heparin	Release of histamine and other inflammation mediators
Monocyte (not terminal cell)	Granules with lysosomal enzymes	Generation of mono-nuclear – phagocyte system cells in tissues; phagocytosis and digestion of protozoa and virus and senescent cells
B – lymphocyte	Immunoglobulins	Generation of antibody – producing terminal cells (plasmocytes)
T – lymphocyte	Substances that kill cells. Substances that control the activity of other leukocytes (interleukins)	Killing of virus – infected cells
Natural killer cell (cytoxic T cell)	Substances that pro-mote perforations in the cell membrane of target cells (thereby killing them)	Killing of some tumor and virus – infected cells
Platelet	Blood – clooting factors	Clotting of blood

 

LYMPH

 

         Lymph is a pale yellow fluid of proteiature, circulating in lymphatic capillaries and vessels. It consists of the plasma and formed elements. It is chemically quite similar to blood but contains lesser proteins. Albumine is more than globulin. Enzymes like diastase, lipase and glycolitic enzymes are present. Plasma contains neutral fats, simple sugar, NaCL, NaCO, Ca,Mg, and Fe.

         Formed elements are 98% lymphocytes and monocytes (and other leukocytes). It contains extremely small amount of erythrocytes. Lymph is formed in lymphatic capillaries of tissues and organs, where under the effect of different factors like osmotic and hydrostatic pressure, from the tissues and released various components of the lymph plasma. From the capillaries lymph flows to peripheral lymphatic vessels and from there to nodes and through ducts into the blood vessels. The chemical composition keeps on changing.

         Lymph divides into peripheral lymph i.e. the one presents up to the lymph nodes; secondary lymph i.e. the one present after lymph nodes; central lymph i.e. the one present in the thoracic duct and right lymphatic duct. The process of lymph formation is interrelated to the movement of water and other substances from blood to intercellular spaces and formation of tissue fluid.

 

CLINICAL ANALYSIS OF THE BLOOD

 

In medicine the microscopic study of the blood picture is highly important method of examination. During analysis the chemical constituency of blood, quantity of erythrocytes, leukocytes, thrombocytes, hemoglobin, erythrocyte’s resistance, speed of sedimentation are determined. In adults the formed elements of blood are located in standard proportions with another, called hemogram, or blood picture. The leukocyte’s formula (about the percentage of leukocytes in peripheral blood) has a special significance.

 

blood picture:

Count of erythrocytes in men 4,0 – 5,1 × 10¹² / l        in women 3,7 – 4,7 ×10¹² / l

Hemoglobin in men 130 – 160 g / l          in women 120 –140 g / l

Count of reticulocytes 0,2 –12%

Speed of erythrocyte’s sedimentation in men 2 –10 mm/hour in women 2 –15 mm/hour

Count of plateletes 180 –320 × 10⁹ / l

Count of leukocytes 4,0 –9,0 × 10⁹ / l

 

luekocyte’s formula:

granulocytes, %					agranulocytes, %
neutrophils			Eosinophils   0,5-5	Basophils   0-1		Lymphocytes   18-37	Monocytes   3-11
juvenile 0-1	band 1-6	segment nuclear 47-72

 

 

General features of the hematopoiesis

 

Blood and lymph are the only fluid tissues in human body. Each of them consists of cells and plasma. Plasma appears from the intercellular substance. Thus hematopoiesis and lymphopoiesis means first of all formation of their cell components, which take place in special hematopoietic organs. It is possible to say about erythropoiesis, poises of platelets and leucocytes. In each turn there are granulocytopoiesis, monocytopoiesis and lymphocytopoiesis.

Erythropoiesis is the process of erythrocytes; granulocytopoiesis – development of platelets; monocytopoiesis – development of monocytes and lymphocytopoiesis – lymphocytes development. All these poieses take place in hematopoietic organs.

 

theories of hematopoiesis.

 

The lineage of each blood cell type has been the subject of numerous theories because historically morphologists had disagreed about the origin and development of blood cells. There were two theories of hematopoiesis. The earliest one – polyphyletic theory – suggests that each mature blood cell type is derived from its own distinct stem cell. But later Russian morphologist A.A. Maximov had proved that there is the only stem cell, which can form all the mature blood cells types. This theory has gained substantial experimental support. Due to this monophyletic theory the multipotential stem cell (CFU-S – colony-forming-unit of spleen) Stem cells are defined as undifferentiated cells that can divide at a slow rate to replicate themselves and to give a rise to daughter cells forming specific, irreversibly differentiated cell types. Stem cells play a central role in hematopoiesis and, because of their importance in biomedical research.

The study of stem cells in bone marrow is possible because of experimental techniques that permit analysis of hematopoiesis in vivo and in vitro.

In vivo techniques include injecting the bone mar­row of normal donor mice into lethally irradiated mice whose hematopoietic cells have been destroyed. In these animals, the transplanted bone marrow cells develop colonies of hematopoietic cells in the spleen

In vitro investigation of hematopoiesis is made pos­sible through the use of a semisolid tissue culture medium. Made with a layer of cells derived from bone marrow stroma, this medium creates favorable microenvironmental conditions for hematopoiesis.

Data from an extensive series of experiments show that hematopoiesis occurs when suitable microenvi­ronmental conditions and stimulation by growth fac­tors influence the development of the various types of blood cells.

Pluripotential and multipotential stem cells. It is thought that all blood cells arise from a single type of stem cell in the bone marrow. Because this cell can produce all blood cell types, it is called a pluripotential stem cell. These cells proliferate and form one cell lineage that will become lymphocytes (lymphoid cells), and another lineage that will form the myeloid cells that develop in bone marrow (granulocytes, monocytes, erythrocytes, and megakaryocytes). Both these types of stem cells are called multipotential stem cells. Early in their development, lymphoid cells migrate from the bone marrow to the lymph nodes, spleen, and thymus, where they differ­entiate into lymphocytes.

 

 

Differentiation of pluripotential stem cells during hematopoiesis.

 

Progenitor and precursor cells. The proliferating multipotential stem cells form daughter cells with re­duced potentiality. These uni- or bipotential pro­genitor cells generate precursor cells (blasts) in which the morphologic characteristics differentiate for the first time (unlike these cells, stem and pro­genitor cells cannot be morphologically distinguished and resemble lymphocytes), suggesting the cell types they will become. Both pluri- and multipotential stem cells di­vide at a rate sufficient to maintain their relatively small population (in mouse bone marrow, only 0.1-0.3% of the cells are multipotential cells). Mitotic rate is accelerated in progenitor and precursor cells, producing large numbers of differentiated, mature cells (3 x 10⁹ erythrocytes and 0.85 x 10⁹ granulocytes/kg/day in human bone marrow). While progenitor cells can divide and produce both progen­itor and precursor cells, precursor cells produce only mature blood cells.

Thus hematopoiesis is the result of simultaneous, continuous proliferation and differentiation of cells derived from stem cells that undergo reductions in their potentials as differentiation progresses. This process can be observed in the in vivo and in vitro studies previously mentioned, in which colonies of cells derived from stem cells with various potentialities appeared. Colonies de­rived from a pluripotential myeloid stem cell can produce erythrocytes, granulocytes, monocytes, and megakaryocytes, all in the same colony.

In these experiments, however, some colonies ap­peared that produced only red blood cells. Other col­onies were observed that produced granulocytes and monocytes. Cells forming colonies of specific cell types are called colony-forming cells (CFC), or colony-forming units (CPU). The convention used iaming these various cell colonies is to use the initial of the cell each colony produces. Thus, MCFC denotes a monocyte-producing colony, ECFC produces eosinophils, MGCFC produces monocytes and granulocytes) and so on.

Stem cell peculiarities:

1.         It looks like a small dark lymphocyte.

2.         At first appears in the yolk sac mesoderm. (Extra embryonic origin).

3.         It is disposed in red bone marrow.

4.         Stem cell is migrating from one place to another all the time.

5.         Has a possibility to differentiate into each type of blood cell.

6.         Self-supporting cell, which can divide 100 times.

7.         It is dividing very rarely (being in G0 phase of cell cycle).

8.         Sensitive cell to regulation from outside.

There is two principally differ kinds of hematopoiesis: embryonic and postembryonic ones.

 

Differences between and embryonic postembryonic hematopoisis

	Embryonic hematopoisis	Postembryonic hematopoisis
1.	Histogenesis of blood	Blood physiologic regeneration
2.	Extracorporal (extraembryonic)	Inside human body
3.	Intravascular	Extravascular
4.	Occurs in different organs	Red bone marrow is the universal hematopoietic organ
5.	Megaloblastic erythropoiesis mesoblastic	Normoblastic

 

Due to contemporary scheme of hematopoiesis there are 6 main classes of hematopoietic cells.

I class – polipotential stem cell.

II class – hemistem cells for lymphocytopoiesis and myelopoiesis.

III class unipotential cell sensitive to exact hemopoietin (erythropoietin, leykopoietin, thrombopoietin).

IV class – blasts (young actively dividing cells).

V class – maturing cells.

VI class – an “adult” mature cells in peripheral blood.

 

HEMATOPOIETIC TISSUES

 

Hematopoietic tissues – are collections of CFUs and their progeny in various stages of maturation, suspended in reticular connective tissue stroma. It is connective tissue with special properties, which consists of reticular cells and reticular fibers. Stroma of hematopoietic organs contains capillaries of sinusoidal type and is reach with macrophages. There are myeloid (red bone marrow) and lymphoid hematopoietic tissues (in others hematopoietic organs).

Mature blood cells have a relatively short life span, and consequently the population must be continuously replaced by the progeny of stem cells produced in the hematopoietic (from Greek, haima, blood, + poie-sis, a making) organs. In the earliest stages of embryogenesis, blood cells arise from the yolk sac mesoderm. Sometime later, the liver and spleen serve as temporary hematopoietic tissues, but by the second month the clavicle has begun to ossify and begins to develop bone marrow in its core. As the prenatal ossification of the rest of the skeleton accelerates, the bone marrow becomes an increasingly important hematopoietic tissue.

After birth and on into childhood, erythrocytes, granular leukocytes, monocytes, and platelets are de­rived from stem cells localized in bone marrow. The origin and maturation of these cells are termed, re­spectively erythropoiesis (from Greek, erythros, red, -+• poiesis), granulopoiesis, monocytopoiesis, and megaka-ryocytopoiesis. The bone marrow also produces cells that migrate to the lymphoid organs producing the various types of lymphocytes.

Before attaining complete maturity and being re­leased into the circulation, the blood cells go through specific stages of differentiation and maturation. Be­cause these processes are continuous, cells with char­acteristics that are intermediate between the different stages are frequently encountered in smears of blood or bone marrow.

 

Drawing showing the passage of erythrocytes, leukocytes, and platelets across a sinusoid capillary in red bone marrow. Because erythrocytes (unlike leukocytes) do not have sufficient motility to cross the wall of the sinusoid, they are believed to enter the sinusoid by a pressure gradient that exists across its wall. Leukocytes, after the action of releasing substances, cross the wall of the sinusoid by their own activity. Megakaryocytes form thin processes that cross the wall of the sinusoid and fragment at their tips, liberating the platelets.

 

MATURATION OF ERYTHROCYTES

 

A mature cell is one that has differentiated to the stage at which it has acquired the capability of car­rying out all its specific functions. The basic process in maturation is the synthesis of hemoglobin and the formation of an enucleated, biconcave, small corpus­cle, the erythrocyte. During maturation of the erythrocytic series, several major changes occur. Cell volume decreases, and the nucleoli diminish in size until they become invisible under the light microscope. The nuclear diameter de­creases, and the chromatin becomes increasingly more dense until the nucleus presents a pyknotic ap­pearance and is finally extruded from the cell. There is a gradual decrease in the number of polyribosomes (basophilia), followed by a simulta­neous increase in the amount of hemoglobin (acidophilia) within the cytoplasm, and the mitochondria gradually disappear.

There are from 3 to 5 intervening cell divisions between the proerythroblast and the mature erythro­cyte. The development of an erythrocyte from the first recognizable cell of the series to the release of reticulocytes into the blood takes approximately 7 days. The hormone erythropoietin and substances such as iron, folic acid, and vitamin B,, are essential for the production of erythrocytes.

Stages in the development of erythrocytes and granulocytes

 

 

 

Electron micrograph of red bone marrow. Four erythroblasts in successive stages of maturation are seen (E1, E2, E3, and E4). As the cell matures, its chromatin becomes gradually condensed, the accumulation of hemoglobin increases the electron density of the cytoplasm, and the mitochondria (M) decrease iumber. x11,000.

 

Differentiation

The differentiation and maturation of erythrocytes involve the formation (in order) of proerythroblasts, basophilic erythroblasts, polychromatophilic erythroblasts, orthochromatophilic erythroblasts (normoblasts) reticulocytes, and erythrocytes.

The first recognizable cell in the erythroid series is the proerythroblast. It is a large cell with loose, lacy chromatin and clearly visible nucleoli; its cytoplasm is basophilic. The next stage is represented by the basophilic erythroblast (erythros + Greek, blastos, germ), with a strongly basophilic cytoplasm and a condensed nucleus that presents no visible nucleolus. The basophilia of these two cell types is caused by the arge number of polyribosomes involved in the syn­thesis of hemoglobin. During the next stage, polyribosomes decrease and areas of the cyto­plasm begin to be filled with hemoglobin. Staining at this stage causes several colors to appear in the cell— the polychromatophilic (from Greek, polys, many, + chroma 4- philein) erythroblast. In the next step, the nucleus continues to condense and no cytoplasmic basophilia is evident, resulting in a uniformly acido-philic cytoplasm—the orthochromatophilic (from Greek, orthos, correct, + chroma + philein) eryth­roblast. At a given moment, this cell puts forth a series of cytoplasmic protrusions and expels its nucleus, encased in a thin layer of cytoplasm. The remaining cell still has a small number of polyribo­somes that, when treated with the supravital dye bril­liant cresyl blue, aggregate to form a stained network. This cell is the reticulocyte, which soon loses its polyribosomes and becomes a mature red blood cell (erythrocyte).

Erythropoiesis principal features:

1.         Decrease in cell size (from 20 till 8 мm).

2.         Ejection (extrusion) of the nucleus.

3.         Accumulation of hemoglobin in the cytoplasm.

4.         Basophilia decrease and acidophilia increase.

 

MATURATION OF GRANULOCYTES

 

The myeloblast is the most immature recognizable cell in the myeloid series. It has a finely dispersed chromatin, and nucleoli can be seen. In the next stage, the promyelocyte (from Latin, pro, before, + Greek, myelos, marrow, + kytos) is characterized by its basophilic cytoplasm and azurophilic granules. These granules contain lysosomal enzymes and my-eloperoxidase. The promyelocyte gives rise to the 3 known granulocytes. The first sign of differentiation appears in the myelocytes where specific granules gradually increase in quantity and eventually occupy most of the cytoplasm. These neutrophilic, baso­philic, and eosinophilic myelocytes mature with fur­ther condensation of the nucleus and a considerable increase in their specific granule content. The neutrophilic granulocyte presents an inte stage whose nucleus has the form of a i (band cell; see color plate). This cell appears in the blood with strong stimulation of hematopoiesis.

The appearance of large numbers of infant neutrophils (band cells) in the blood is called shift to the left and is of clinical significance, usually indicating bacterial infection.

KINETICS OF NEUTROPHIL PRODUCTION

The total time taken for a myeloblast to emerge as a mature neutrophil in the circulation is about 11 days. Under normal circumstances, 5 mitotic divisions occur in the myeloblast, promyelocyte, and neutrophilic myelocyte stages of development.

Neutrophils pass through several functionally and anatomically defined compartments.

The medullary formation compartment can be subdivided into a mitotic compartment (~ 3 days) and a maturation compartment (~ 4 days).

A medullary storage compartment acts as a buffer system, capable of releasing large numbers of mature neutrophils upon demand. Neutrophils remain in this compartment for about 4 days.

The circulating compartment consists of neutro­phils suspended in plasma and circulating in blood vessels.

The marginating compartment is composed of neutrophils that are present in blood but do not circulate. These neutrophils are in capillaries, tempo­rarily excluded from the circulation by vasoconstriction, or—especially in the lungs—they may be at the periphery of vessels, adhering to the endothelium, and not in the main bloodstream.

The marginating and circulating compartments are of about equal size, and there is a constant inter­change of cells between them. The half-life of a neu­trophil in these 2 compartments is 6-7 hours. The medullary formation and storage compartments to­gether are about 10 times as large as the circulating and marginating compartments.

 

Section of red bone marrow with a group of erythropoietic cells (upper right) and a group of neutrophilopoietic cells (lower left). The immature granulocytes shown have mostly azurophilic granules in their cytoplasm and therefore are myelocytes. PT stain. High magnification.

 

Drawing illustrating the sequence of gene expression in the maturation of granulocytes. Azurophilic granules are blue; specific granules are pink.

Neutrophilic myelocyte from normal human bone marrow treated with peroxidase. At this stage, the cell is smaller than the promyelocyte, and the cytoplasm contains 2 types of granules: large, peroxidase-positive azurophilic granules (AG) and smaller specific granules (SG), which do not stain for peroxidase. Note that the peroxidase reaction product is present only in azurophilic granules and is not seen in the rough endoplasmic reticulum (RER) or Golgi cisternae (GC), which are located around the centriole (C). N, nucleus. x15,000.

Bone marrow with neutrophilic (arrowheads) and eosinophilic myelocytes. Giemsa stain. High magnification.

 

Neutrophils and other granulocytes enter the connective tissues by passing through intercellular junc­tions found between endothelial cells of capillaries and postcapillary venules (diapedesis). The connec­tive tissues form a fifth compartment for neutrophils, but its size is not known. Neutrophils reside here for 1-4 days and then die, whether or not they have performed their major function of phagocytosis.

Changes in the number of neutrophils in the pe­ripheral circulation must be evaluated by taking all these compartments into consideration. Thus neutrophilia, an increase in the number of neutrophils in the circulation, does not necessarily! imply an increase ieutrophil production. In-1 tense muscular activity or the administration off epinephrine causes neutrophils in the margining compartment to move into the circular compartment, causing apparent neutrophils even though neutrophil production has not increased.

Neutrophilia may also result from liberations greater numbers of neutrophils from the medul­lary storage compartment. This type of neutrophilia is transitory and is followed by a recovery period during which no neutrophils are released. The neutrophilia that occurs during the course of bacterial infections is due to an increase ieutrophil production and a shorter stay of these cells in the medullary storage compartment. In such cases, immature forms such as band cells, neutrophilic metamyelocytes, and even myelocytes may appear in the bloodstream. The neu­trophilia that occurs during infection is of longer duration than that which occurs as a result of intense muscular activity.

Granulocytopoiesis main features.

1.         Decrease in the cell size.

2.         Condensation iuclear chromatin.

3.         Changes iuclear shape (flattening – indentation – lobulation).

4.         Accumulation of cytoplasmic granules.

Functional compartments of neutrophils. 1: Medullary formation compartment. 2: Medullary storage (reserve) compartment. 3: Circulating compartment. 4: Marginating compartment. The size of each compartment is roughly proportional to the number of cells.

 

MATURATION OF LYMPHOCYTES & MONOCYTES

 

Study of the precursor cells of lymphocytes and monocytes is difficult because these cells do not con­tain specific cytoplasmic granules or the nuclear lobulation that is present in granulocytes, both of which facilitate the distinction between young and mature forms. Lymphocytes and monocytes are dis­tinguished mainly on the basis of size, chromatin structure, and the presence of nucleoli in smear prep­arations. As lymphocyte cells mature, their chromatin becomes more compact, nucleoli become less visible, and the cells decrease in size. In addition, subsets of the lymphocytic series acquire distinctive cell-surface receptors during differentiation that can be detected by immunofluorescence techniques.

Lymphocytes

 

Circulating lymphocytes originate mainly in the thymus and the peripheral lymphoid organs (spleen, lymph nodes, tonsils, etc). It is now thought, how­ever, that all lymphocyte progenitor cells originate in the bone marrow. Some of these relatively undifferentiated lymphocytes migrate to the thymus, where they acquire the attributes of T lymphocytes. Subsequently, T lymphocytes populate specific regions of peripheral lymphoid organs. Other bone marrow lymphocytes remain in the marrow, differentiate into B lymphocytes, and then migrate to peripheral lymphoid organs where they inhabit their own special impairments.

The first identifiable progenitor of lymphoid cells is a lymphoblast, a large cell capable of incorporating H-thymidine and dividing 2-3 times to form prolymphocytes. These latter cells are smaller and have relatively more condensed chromatin but none of the cell-surface antigens that mark prolymphocytes as T or B lymphocytes. In the thymus or bone marrow, these cells synthesize cell-surface receptors charac­teristic of their lineage, but they are not recognizable as distinct cell types using routine histologic proce­dures. The distinction is made by using immunocytochemical techniques.

Lymphocytopoiesis peculiarities.

1.                This type of hematopoiesis begins in red bone marrow and then continues in lymphoid tissue.

2.                Lifespan various in different types of lymphocytes.

3.                Antigenindependent development occurs in the central hematopoietic organs (red bone marrow and thymus) and antigendependent – in peripheral ones (spleen, lymph nodes and nodules).

 

Monocytes

 

The monoblast is a committed progenitor cell that is virtually identical to the myeloblast in its morphol­ogy. Further differentiation leads to the promonocyte, a large cell (up to 18 mm in diameter) with a basophilic cytoplasm and a large, slightly idented nu­cleus. The chromatin is lacy, and nucleoli are evi­dent. Promonocytes divide twice in the course of their development into monocytes. A large amount of rough endoplasmic reticulum is present, as is an ex­tensive Golgi complex in which granule condensation can be seen to be taking place. These granules are primary lysosomes, which are observed as fine azurophilic granules in blood monocytes. Mature monocytes enter the bloodstream, circulate for about 8 hours, and then enter the connective tissues, where they mature into macrophages and function for sev­eral months. From 20 to 30 hours they are “swimming” in blood the next 2-3 days they may be find in connective tissue like free macrophages.

Monocytopoiesis peculiarities.

1.         Decrease in cell diameter.

2.         Decrease in nuclear diameter.

3.         Cytoplasm basophilia decreases.

4.         Nucleus changes its shape from round to kidney-look.

 

ORIGIN OF PLATELETS

 

In adults, the platelets originate in the red bone marrow by fragmentation of the cytoplasm of mature megakaryocytes. These in turn arise by differentia­tion of the megakaryoblasts.

Megakaryoblasts

The megakaryoblast is 15-50 xm in diameter and has a large ovoid or kidney-shaped nucleus with nu­merous nucleoli. The nucleus becomes highly polyploid (it contains up to 30 times as much DNA as a normal cell) before cytoplasmic differentiation be­gins. The cytoplasm of this cell is homogeneous and intensely basophilic.

Megakaryocytes

The megakaryocyte is a giant cell (35-150 mm in diameter) with an irregularly lobulated nucleus, coarse chromatin, and no visible nucleoli. The cytoplasm contains numerous mito­chondria, a well-developed rough endoplasmic reticulum, and an extensive Golgi complex. Alpha granules and vesicles containing lysosomal enzymes (lambda granules) develop from Golgi vesicles and cisternae. With maturation of the megakaryocyte, nu­merous invaginations of the plasma membrane ramify throughout the cytoplasm, forming the demarcation membranes. This system defines areas of the megakaryocyte’s cytoplasm that will be shed as platelets after a complex and little-understood process of mem­brane fusion.

Cells of the megakaryocyte series shown in a bone marrow smear. Note the formation of platelets at the lower end of the megakaryocyte.

Section of bone marrow showing various stages of megakaryocyte development (1–4), several adipocytes (*), and blood sinusoids (arrowheads). PT stain. Medium magnification.

 

Section of bone marrow with an adult megakaryocyte and several granulocytes, mainly neutrophils in the myelocyte stage with many azurophilic granules and few, less darkly stained specific granules. A mitotic figure is indicated by an arrowhead. Giemsa stain. High magnification.

 

In certain forms of thrombocytopenic purpura, a disease in which the number of blood platelets is reduced, the platelets appear bound to the cy­toplasm of the megakaryocytes, indicating a de­fect in the liberation mechanism of these corpuscles. The life span of these corpuscles was found to be approximately 2-3 days.

 

Thrombocytopoiesis special features:

1.         Enlargement in size.

2.         Lobulation of nucleus.

3.         Development of elaborate demarcation membrane system.

Electron micrograph of a megakaryocyte showing a lobulated nucleus (N) and numerous cytoplasmic granules. The demarcation membranes are visible as tubular profiles. x4900.

HEMASTOPOIESIS REGULATION

 

Hematopoiesis depends upon the presence of suit­able microenvironmental conditions and growth factors. The microenvironmental conditions are fur­nished by cells of the stroma of hematopoietic or­gans, which produce an essential extracellular matrix, Once the necessary environmental conditions are present, the development of blood cells depends on factors that affect cell proliferation and differentia­tion. These substances are called growth factors, colony-stimulating factors (CSF), or hematopoie-tins (poietins). Growth factors, which have differing chemical compositions and complex, overlapping functions, act mainly by stimulating proliferation (mitogenic activity) of immature (mostly progenitor and precursor) cells, supporting the differentiation of im­mature cells as they mature, and enhancing the func­tions of mature cells.

These three functions may be present in the same growth factor, but they may be expressed with differ­ent intensities in different growth factors. Recently, genes for several growth factors have been isolated and cloned, permitting both the mass production of growth factors and the study of their effects in vivo and in vitro.

Growth factors have been used clinically to pro­duce an increase in marrow cellularity and blood cell counts in patients. The use of growth factors to stim­ulate the proliferation of leukocytes is opening broad new applications for clinical therapy. Potential ther­apeutic uses of growth factors include increasing the numbers of blood cells in diseases or induced condi­tions (eg, chemotherapy, irradiation) that cause low blood counts; increasing the efficiency of marrow transplants by enhancing cell proliferation; enhancing host defenses in patients with malignancies and in­fectious and immunodeficient diseases; and enhanc­ing the treatment of parasitic diseases.

Hematopoietic diseases rarely result from mal­functions of hematopoietic organ stroma. They are usually caused by suppression or enhance­ment of undifferentiated cell production, with a consequent reduction (or overproduction) of he­matopoietic cells. In some diseases, however sequential or simultaneous suppressed and en­hanced proliferation of more than one type of stem cell can occur. There are, in such cases, reduced numbers of some cell types (eg, aplastic anemia, a disorder characterized by decreased production of hematopoietic cells) coinciding with increased numbers of others (e.g., leukemia, the abnormal proliferation of white blood cells). The initial experiments transplanting normal bone marrow to lethally irradiated mice estab­lished the basis for bone marrow transplantation, now routinely used to treat some hemato-poietic-cell-growth disorders.

Abnormal bone marrow can produce diseases based on cells derived from that tissue. Leukemias are malignant clones of white blood cell precursors. They occur in lymphoid tissue (lymphocytic leukemias) and in bone marrow (myelogenous and monocytic leukemias). In these diseases, there is usually a release of large numbers of immature cells into the blood. The symptoms of leukemia’s are a consequence of this shift in cell proliferation, with a lack of some cell types and excessive production of others (which are often abnormal in function). The patient usually exhibits anemia and is prone to infection.

A clinical technique helpful in the study of leukemia’s and other bone marrow disturbances is bone marrow aspiration. A needle is intro­duced through compact bone (usually the ster­num), and a sample of marrow is withdrawn. The sample is spread on a microscope slide and stained. The use of labeled monoclonal antibod­ies specific to proteins in the membranes of pre­cursor blood cells aids in identifying cell types derived from these stem cells and contributes to a more precise diagnosis of the various possible types of leukemia.

 

STUDENT’S PRACTICAL ACTIVITIES

Task No 1. Students must know and illustrate such histological specimens.

Specimen 1. Blood smear. Giemsa stain.

In this specimen you can see such blood cells, as erythrocytes, leukocytes and thrombocytes.

This specimen demonstrates the characteristic appearance of erythrocytes in a stained smear of peripheral blood. The cells are stained pink due to their high content of hemoglobin. The ale staining of the central region of the erythrocyte is a result of its unusual biconcave disk shape. Note the total absence of cytoplasm organelles.

White blood cells are subdivided into two main classes, granulocytes (neutrophils, eosinophils, basophils) and agranulocytes (lymphocytyes, monocytes) according to the granularity of their cytoplasm and general nuclear characteristics.

Neutrophils. The most prominent feature of the neutrophil is the highly lobulated nucleus. In the mature neutrophil there are usually five lobes connected by fine strands of nuclear material, but in the less mature neutrophil the nucleus is generally not as lobulated. The salmon-pink cytoplasm of neutrophils is lightly stippled with reddish-purple granules called azurophilic granules which are merely large lysosomes often referred to as primary granules. The more numerous but much smaller specific granules are poorly stained.

Eosinophils. Eosinophils are usually slightly smaller than typical neutrophils. Characteristically, eosinophils have a belobed nucleus and the cytoplasm is packed with large, eosinophilic (dark-pink stained) specific granules of uniform size.

Basophils. Like eosinophils, basophils also have a belobed nucleus but, in general. This is obscured by numerous large, densely basophilic (deep blue) specific granules. These granules have irregular shapes and vary in size; the largest are the size of the specific granules of eosinophils, the smallest nearly as small at those of neutrophils.

Lymphocytes. Lymphocytes are the smallest cells in the white cell series, being only slightly larger than erythrocytes. Lymphocytes are characterized by a round, densely stained nucleus and a relatively small amount of pale basophilic, nongranular cytoplasm. The amount of cytoplasm varies with the state of activity of the lymphocyte, and in circulating blood there is a predominance of “small” lymphocytes; however, “medium” and “large” lymphocytes are also seen in peripheral blood. Specimen illustrates. In the medium lymphocytes, the cytoplasm is readily visible, contains purple-blue nucleus, but in the small lymphocyte the cytoplasm is almost too sparse to be seen, it forms a thin rim around the nucleus.

Monocytes. Monocytes are the largest members of the white blood cells. They are characterized by a large, eccentrically placed nucleus, which is stained less intensely than that of other leukocytes. The nucleus is usually indented, has a smudgy appearance and stained reddish-purple, a feature which becomes more pronounced as the cell matures, so as to give a horseshoe or even belobed appearance. The extensive faint blue-gray cytoplasm is filled with small lysosomes which, in light microscopy, confer a characteristic “frosted-glass” appearance.

Platelets. Are the smallest formed elements in the blood, are disk-like cell fragments. They lack nuclei and originate by budding from large cells in the bone marrow called megakariocytes. In blood smear they appear as irregular masses of basophilic (blue) cytoplasm and they tend to form clumps.

Illustrate and indicate: 1. Erythrocytes; 2.Neutrophils; 3. Eosinophils; 4. Basophils; 5. Lymphocytes; 6. Monocytes; 7. Thrombocytes.

Specimen 2. Bone marrow smear.

Stained with haematoxylin and eosin.

This bone marrow smear illustrates several stages in erythropoiesis and granulopoiesis. The proerythroblast is the first recognisable erythrocyte precursor; the cell has a large, intensely stained, granular nucleus containing one or more paler nucleoli. The sparse cytoplasm is strongly basophilic due to its high content of RNA and lack of hemoglobin. A narrow, pale zone of cytoplasm close to the nucleus represents the Golgi apparatus. Three increasingly differentiated normoblast forms can also be distinguished. An early normoblast (basophilic erythroblast) is recognized by its basophilic cytoplasm and smaller nucleus with increasingly condensed chromatin. More advanced in the maturation sequence is an intermediate normoblast (polychromatic erythroblast), the cytoplasm of which exhibits both basophilia and eosinophilia, the latter due to increasing haemoglobin content. The nucleus is also condensed and is accompanied by several small fragments called Howell-Zolly bodies, an unusual finding iormal erythropoiesis. With further haemoglobin synthesis and degeneration of cytoplasmic ribosomes, the late normoblast (orthochromatic erythroblast) stage is reached and by this time the nucleus is extremely condensed prior to being extruded from the cell.

The myeloblast is the earliest recognisable stage in granulopoiesis. Myeloblasts give rise to promyelocytes which are characterised by their content of azurophilic granules; since the azurophilic granules develop before the specific granules they were referred to as primary granules. The primary granules are merely large lysosomes. This specimen illustrates three phases of neutrophil granulocyte development. A neutrophil myelocyte is recognised by a large, eccentrically located nucleus, a prominent Golgi apparatus and cytoplasm containing many azurophilic (primary) granules. The next stage towards maturity, the metamyelocyte, is the smaller cell characterised by indentation of the nucleus and loss of prominence of the azurophilic granules. The final stage before maturity, the stab cell, has a more highly segmented nucleus approaching that of the mature neutrophil.

Platelet formation begins with the development of a large binucleated cell, the megakaryoblast. After this stage, fusion of the nuclei occurs and successive duplication of the nuclear material takes place without the formation of separate nuclei and without cell division. The resulting polyploid cell, the megakariocyte, has an enormous volume of cytoplasm.

Illustrate and indicate: 1.Basophilic erythroblast. 2.Polychromatic erythroblast. 3.Orthochromatic erythroblast. 4.Erythrocyte. 5.Promyelocyte. 6.Myelocyte. 7.Metamyelocyte. 8.Megakariocyte.

 

References:

A-Basic:

1.     Practical classes materials

http://intranet.tdmu.edu.ua/data/kafedra/internal/histolog/classes_stud/English/medical/II%20term/07%20Blood.%20Lymph.%20Hematopoiesis.htm

2.     Lecture presentations

http://intranet.tdmu.edu.ua/ukr/kafedra/index.php?kafid=hist&lengid=eng&fakultid=m&kurs=1&discid=Histology, cytology and embryology

3.  Stevens A. Human Histology / A. Stevens, J. Lowe. – [second edition]. –Mosby, 2000. – P.99-112

4.     Wheter’s Functional Histology : A Text and Colour Atlas / [Young B., Lowe J., Stevens A., Heath J.]. – Elsevier Limited, 2006. – P. 46-63

5.     Inderbir Singh Textbook of Human Histology with colour atlas / Inderbir Singh. – [fourth edition]. – Jaypee Brothers Medical Publishers (P) LTD, 2002. – P. 70-88

6.     Ross M. Histology : A Text and Atlas / M. Ross W.Pawlina. – [sixth edition]. – Lippincott Williams and Wilkins, 2011. – P. 268-310

 

B – Additional:

1.     Eroschenko V.P. Atlas of Histology with functional correlations / Eroschenko V.P. [tenth edition]. – Lippincott Williams and Wilkins, 2008. – P. 98-115

2.     Junqueira L. Basic Histology / L. Junqueira, J. Carneiro, R. Kelley. – [seventh edition]. – Norwalk, Connecticut : Appleton and Lange, 1992. – P. 251-284

3.     Charts:

http://intranet.tdmu.edu.ua/index.php?dir_name=kafedra&file_name=tl_34.php#inf3

4.     Disk:

http://intranet.tdmu.edu.ua/data/teacher/video/hist/  

5.     Volkov K. S. Ultrastructure of cells and tissues / K. S. Volkov, N. V. Pasechko. – Ternopil : Ukrmedknyha, 1997. – P. 54-68

http://intranet.tdmu.edu.ua/data/books/Volkov(atlas).pdf

http://en.wikipedia.org/wiki/Circulatory

http://www.meddean.luc.edu/LUMEN/MedEd/Histo/frames/histo_frames.html

http://www.udel.edu/biology/Wags/histopage/histopage.htm

 

Material instruction has been worked out by ass. A. O. Kotyk