WHITE BLOOD CELL COUNT

INVESTIGATIONS OF LEUKOCYTES LABORATORY DIAGNOSIS OF LEUKEMIAS

 

WHITE BLOOD CELL COUNT

 

The WBC count determines the number of leukocytes per cubic millimeter of whole blood. The counting is performed very rapidly by electronic devices. The WBC may be performed as part of a CBC, alone, or with differential WBC count. An elevated WBC count is termed leukocytosis; a decreased count, leukopenia. In addition to the normal physiological variations in WBC count, many pathological problems may result in an abnormal WBC count .

 

 

Causes of Altered White Blood Cell Differential by Cell Type

 

Cell Type

Increased Levels

Decreased Levels

Neutrophils

Stress (allergies, exercise, childbirth, surgery)

Extremes of temperature

Acute hemorrhage or hemolysis

Infectious diseases

Inflammatory disorders (rheumatic fever,

gout, rheumatoid arthritis, drug reactions, vasculitis, myositis)

Tissue necrosis (burns, crushing injuries, abscesses

Malignancies

Metabolic disorders (uremia, eclampsia, diabetic ketoacidosis, thyroid crisis, Cushings syndrome)

Drugs (epinephrine, histamine, lithium, heavy metals, heparin, digitalis, ACTH)

Toxins and venoms (turpentine, benzene)

Leukemia (myelocytic)

Bone marrow depression (viruses, toxic chemicals, overwhelming infection,

Feltys syndrome, Gauchers disease, myelofibrosis, hypersplenism, pernicious

anemia, radiation)

Anorexia nervosa, starvation, malnutrition

Folic acid deficiency

Vitamin B12 deficiency

Acromegaly

Addisons disease

Thyrotoxicosis

Anaphylaxis

Disseminated lupus erythematosus

Drugs (alcohol, phenylbutazone

[Butazolidin], phenacetin, penicillin,

chloramphenicol, streptomycin, phenytoin

[Dilantin], mephenytoin

[Mesantoin], phenacemide [Phenurone],

tripelennamine [PBZ], aminophylline,

quinine, chlorpromazine, barbiturates, dinitrophenols, sulfonamides, antineoplastics

Bands

Infections

Antineoplastic drugs

Any condition that causes neutrophilia

 

None, as bands should be absent or present only in small numbers

Basophils

Leukemia

Hodgkins disease

Polycythemia vera

Ulcerative colitis

Nephrosis

Chronic hypersensitivity states

None, as normal value is 01%

Eosinophils

Sickle cell disease

Asthma

Chorea

Hypersensitivity reactions

Parasitic infestations

Autoimmune diseases

Addisons disease

Malignancies

Sarcoidosis

Chronic inflammatory diseases and

dermatoses

Leprosy

Hodgkins disease

Polycythemias

Ulcerative colitis

Autoallergies

Pernicious anemia

Splenectomy

Disseminated lupus erythematosus

Acromegaly

Elevated steroid levels

Stress

Infectious mononucleosis

Hypersplenism

Cushings syndrome

Congestive heart failure

Hyperplastic anemia

Hormones (ACTH, thyroxine, epinephrine)

Monocytes

Infections (bacterial, viral, mycotic, rickettsial, amebic)

Cirrhosis

Collagen diseases

Ulcerative colitis

Regional enteritis

Gauchers disease

Hodgkins disease

Lymphomas

Carcinomas

Monocytic leukemia

Radiation

Polycythemia vera

Sarcoidosis

Weils disease

Systemic lupus erythematosus

Hemolytic anemias

Thrombocytopenic purpura

Not characteristic of specific disorders

Lymphocytes

Infections (bacterial, viral)

Lymphosarcoma

Ulcerative colitis

Bantis disease

Feltys syndrome

Myeloma

Lymphomas

Addisons disease

Thyrotoxicosis

Malnutrition

Rickets

Waldenströms macroglobulinemia

Lymphocytic leukemia

Immune deficiency diseases

Hodgkins disease

Rheumatic fever

Aplastic anemia

Bone marrow failure

Gauchers disease

Hemolytic disease of the newborn

Hypersplenism

Thrombocytopenic purpura

Transfusion reaction

Massive transfusions

Pernicious anemia

Septicemia

Pneumonia

Burns

Radiation

Toxic chemicals (benzene, bismuth, DDT)

Antineoplastic agents

Adrenal corticosteroids (high doses)

 

 

 

 

WHITE BLOOD CELL COUNT

 

The WBC count determines the number of leukocytes per cubic millimeter of whole blood. The counting is performed very rapidly by electronic devices. The WBC may be performed as part of a CBC, alone, or with differential WBC count. An elevated WBC count is termed leukocytosis; a decreased count, leukopenia. In addition to the normal physiological variations in WBC count, many pathological problems may result in an abnormal WBC count .

 

Causes of Altered White Blood Cell Differential by Cell Type

 

Cell Type

Increased Levels

Decreased Levels

Neutrophils

Stress (allergies, exercise, childbirth, surgery)

Extremes of temperature

Acute hemorrhage or hemolysis

Infectious diseases

Inflammatory disorders (rheumatic fever,

gout, rheumatoid arthritis, drug reactions, vasculitis, myositis)

Tissue necrosis (burns, crushing injuries, abscesses

Malignancies

Metabolic disorders (uremia,eclampsia, diabetic ketoacidosis, thyroid crisis, Cushings syndrome)

Drugs (epinephrine, histamine, lithium, heavy metals, heparin, digitalis, ACTH)

Toxins and venoms (turpentine, benzene)

Leukemia (myelocytic)

Bone marrow depression (viruses, toxic chemicals, overwhelming infection,

Feltys syndrome, Gauchers disease,myelofibrosis, hypersplenism, pernicious

anemia, radiation)

Anorexia nervosa, starvation, malnutrition

Folic acid deficiency

Vitamin B12 deficiency

Acromegaly

Addisons disease

Thyrotoxicosis

Anaphylaxis

Disseminated lupus erythematosus

Drugs (alcohol, phenylbutazone

[Butazolidin], phenacetin, penicillin,

chloramphenicol, streptomycin, phenytoin

[Dilantin], mephenytoin

[Mesantoin], phenacemide[Phenurone],

tripelennamine [PBZ], aminophylline,

quinine, chlorpromazine, barbiturates,dinitrophenols, sulfonamides,antineoplastics

Bands

Infections

Antineoplastic drugs

Any condition that causes neutrophilia

 

None, as bands should be absent or present only in small numbers

Basophils

Leukemia

Hodgkins disease

Polycythemia vera

Ulcerative colitis

Nephrosis

Chronic hypersensitivity states

WHITE BLOOD CELL COUNT

INVESTIGATIONS OF LEUKOCYTES LABORATORY DIAGNOSIS OF LEUKEMIAS

 

WHITE BLOOD CELL COUNT

 

The WBC count determines the number of leukocytes per cubic millimeter of whole blood. The counting is performed very rapidly by electronic devices. The WBC may be performed as part of a CBC, alone, or with differential WBC count. An elevated WBC count is termed leukocytosis; a decreased count, leukopenia. In addition to the normal physiological variations in WBC count, many pathological problems may result in an abnormal WBC count .

 

 

Causes of Altered White Blood Cell Differential by Cell Type

 

Cell Type

Increased Levels

Decreased Levels

Neutrophils

Stress (allergies, exercise, childbirth, surgery)

Extremes of temperature

Acute hemorrhage or hemolysis

Infectious diseases

Inflammatory disorders (rheumatic fever,

gout, rheumatoid arthritis, drug reactions, vasculitis, myositis)

Tissue necrosis (burns, crushing injuries, abscesses

Malignancies

Metabolic disorders (uremia, eclampsia, diabetic ketoacidosis, thyroid crisis, Cushings syndrome)

Drugs (epinephrine, histamine, lithium, heavy metals, heparin, digitalis, ACTH)

Toxins and venoms (turpentine, benzene)

Leukemia (myelocytic)

Bone marrow depression (viruses, toxic chemicals, overwhelming infection,

Feltys syndrome, Gauchers disease, myelofibrosis, hypersplenism, pernicious

anemia, radiation)

Anorexia nervosa, starvation, malnutrition

Folic acid deficiency

Vitamin B12 deficiency

Acromegaly

Addisons disease

Thyrotoxicosis

Anaphylaxis

Disseminated lupus erythematosus

Drugs (alcohol, phenylbutazone

[Butazolidin], phenacetin, penicillin,

chloramphenicol, streptomycin, phenytoin

[Dilantin], mephenytoin

[Mesantoin], phenacemide [Phenurone],

tripelennamine [PBZ], aminophylline,

quinine, chlorpromazine, barbiturates, dinitrophenols, sulfonamides, antineoplastics

Bands

Infections

Antineoplastic drugs

Any condition that causes neutrophilia

 

None, as bands should be absent or present only in small numbers

Basophils

Leukemia

Hodgkins disease

Polycythemia vera

Ulcerative colitis

Nephrosis

Chronic hypersensitivity states

None, as normal value is 01%

Eosinophils

Sickle cell disease

Asthma

Chorea

Hypersensitivity reactions

Parasitic infestations

Autoimmune diseases

Addisons disease

Malignancies

Sarcoidosis

Chronic inflammatory diseases and

dermatoses

Leprosy

Hodgkins disease

Polycythemias

Ulcerative colitis

Autoallergies

Pernicious anemia

Splenectomy

Disseminated lupus erythematosus

Acromegaly

Elevated steroid levels

Stress

Infectious mononucleosis

Hypersplenism

Cushings syndrome

Congestive heart failure

Hyperplastic anemia

Hormones (ACTH, thyroxine, epinephrine)

Monocytes

Infections (bacterial, viral, mycotic, rickettsial, amebic)

Cirrhosis

Collagen diseases

Ulcerative colitis

Regional enteritis

Gauchers disease

Hodgkins disease

Lymphomas

Carcinomas

Monocytic leukemia

Radiation

Polycythemia vera

Sarcoidosis

Weils disease

Systemic lupus erythematosus

Hemolytic anemias

Thrombocytopenic purpura

Not characteristic of specific disorders

Lymphocytes

Infections (bacterial, viral)

Lymphosarcoma

Ulcerative colitis

Bantis disease

Feltys syndrome

Myeloma

Lymphomas

Addisons disease

Thyrotoxicosis

Malnutrition

Rickets

Waldenströms macroglobulinemia

Lymphocytic leukemia

Immune deficiency diseases

Hodgkins disease

Rheumatic fever

Aplastic anemia

Bone marrow failure

Gauchers disease

Hemolytic disease of the newborn

Hypersplenism

Thrombocytopenic purpura

Transfusion reaction

Massive transfusions

Pernicious anemia

Septicemia

Pneumonia

Burns

Radiation

Toxic chemicals (benzene, bismuth, DDT)

Antineoplastic agents

Adrenal corticosteroids (high doses)

 

 

 

 

WHITE BLOOD CELL COUNT

 

The WBC count determines the number of leukocytes per cubic millimeter of whole blood. The counting is performed very rapidly by electronic devices. The WBC may be performed as part of a CBC, alone, or with differential WBC count. An elevated WBC count is termed leukocytosis; a decreased count, leukopenia. In addition to the normal physiological variations in WBC count, many pathological problems may result in an abnormal WBC count .

 

Causes of Altered White Blood Cell Differential by Cell Type

 

Cell Type

Increased Levels

Decreased Levels

Neutrophils

Stress (allergies, exercise, childbirth, surgery)

Extremes of temperature

Acute hemorrhage or hemolysis

Infectious diseases

Inflammatory disorders (rheumatic fever,

gout, rheumatoid arthritis, drug reactions, vasculitis, myositis)

Tissue necrosis (burns, crushing injuries, abscesses

Malignancies

Metabolic disorders (uremia,eclampsia, diabetic ketoacidosis, thyroid crisis, Cushings syndrome)

Drugs (epinephrine, histamine, lithium, heavy metals, heparin, digitalis, ACTH)

Toxins and venoms (turpentine, benzene)

Leukemia (myelocytic)

Bone marrow depression (viruses, toxic chemicals, overwhelming infection,

Feltys syndrome, Gauchers disease,myelofibrosis, hypersplenism, pernicious

anemia, radiation)

Anorexia nervosa, starvation, malnutrition

Folic acid deficiency

Vitamin B12 deficiency

Acromegaly

Addisons disease

Thyrotoxicosis

Anaphylaxis

Disseminated lupus erythematosus

Drugs (alcohol, phenylbutazone

[Butazolidin], phenacetin, penicillin,

chloramphenicol, streptomycin, phenytoin

[Dilantin], mephenytoin

[Mesantoin], phenacemide[Phenurone],

tripelennamine [PBZ], aminophylline,

quinine, chlorpromazine, barbiturates,dinitrophenols, sulfonamides,antineoplastics

Bands

Infections

Antineoplastic drugs

Any condition that causes neutrophilia

 

None, as bands should be absent or present only in small numbers

Basophils

Leukemia

Hodgkins disease

Polycythemia vera

Ulcerative colitis

Nephrosis

Chronic hypersensitivity states

None, as normal value is 01%

Eosinophils

Sickle cell disease

Asthma

Chorea

Hypersensitivity reactions

Parasitic infestations

Autoimmune diseases

Addisons disease

Malignancies

Sarcoidosis

Chronic inflammatory diseases and

dermatoses

Leprosy

Hodgkins disease

Polycythemias

Ulcerative colitis

Autoallergies

Pernicious anemia

Splenectomy

Disseminated lupus erythematosus

Acromegaly

Elevated steroid levels

Stress

Infectious mononucleosis

Hypersplenism

Cushings syndrome

Congestive heart failure

Hyperplastic anemia

Hormones (ACTH, thyroxine, epinephrine)

Monocytes

Infections (bacterial, viral, mycotic,rickettsial, amebic)

Cirrhosis

Collagen diseases

Ulcerative colitis

Regional enteritis

Gauchers disease

Hodgkins disease

Lymphomas

Carcinomas

Monocytic leukemia

Radiation

Polycythemia vera

Sarcoidosis

Weils disease

Systemic lupus erythematosus

Hemolytic anemias

Thrombocytopenic purpura

Not characteristic of specific disorders

Lymphocytes

Infections (bacterial, viral)

Lymphosarcoma

Ulcerative colitis

Bantis disease

Feltys syndrome

Myeloma

Lymphomas

Addisons disease

Thyrotoxicosis

Malnutrition

Rickets

Waldenströms macroglobulinemia

Lymphocytic leukemia

Immune deficiency diseases

Hodgkins disease

Rheumatic fever

Aplastic anemia

Bone marrow failure

Gauchers disease

Hemolytic disease of the newborn

Hypersplenism

Thrombocytopenic purpura

Transfusion reaction

Massive transfusions

Pernicious anemia

Septicemia

Pneumonia

Burns

Radiation

Toxic chemicals (benzene, bismuth, DDT)

Antineoplastic agents

Adrenal corticosteroids (high doses)

 

 

 

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.

 

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.

 

 

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

 

The mechanism of phagocytosis

Phagocytosis  is active devourment of the solid substances by cells. Cells, which are capable of phagocytosis, are called phagocytes. There are poly phagocytes (neutrophils) and mononuclear phagotyces (monocytes).

Phagocytes must be selective of the material that is phagocytized; otherwise, normal cells and structures of the body might be ingested. Whether phagocytosis will occur, depends especially on three selective procedures. Firstly, most natural structures in the tissues have smooth surfaces, which resist phagocytosis. But if the surface is rough, the likelihood of phagocytosis is increased. Secondly, most natural substances of the body have protective protein coats that repel the phagocytes. Conversely, most dead tissues and foreign particles have no protective coats, which make them subject to phagocytosis. Thirdly, the immune system of the body develops antibodies against infectious agents such as bacteria. The antibodies then adhere to the bacterial membranes and thereby make the bacteria especially susceptible to phagocytosis. To do this, the antibody molecule also combines with the C3 product of the complement cascade, which is an additional part of the immune system discussed in the next chapter. The C3 molecules, in turn, attach to receptors on the phagocytic membrane, thus initiating phagocytosis. This selection and phagocytosis process is calledopsonization.

 

 

Stages of phagocytosis:

 

I          Conjugation stage. Phagocyte moves to direction of not self agent (chemotaxis).

 

II          Adhesion stage. Phagocyte interacts with the agent. There are two mechanisms:

 

1) without receptor: electrostatic and hydrophobic interaction (phagocyte is negatively charged, positive particles);

2) with receptor. On the surface of macrophages there are receptors for opsonin-substances that can interact with bacteria.

 

III          Devourment stage. Its steps:

            invagination of phagocyte membrane on the contact place;

            the formation of phagosome, which contains the agent;

            the formation of phagolysosome: consolidation of phagosome with lysosomes (secondary granules).

 

IV          Digestive stage. Its steps:

            The disposal of bacteria intercellular cytolysis with the help of germicide systems of phagocytes (myeloperoxidase system, which produces hypochloride ion ClO-, free radicals and peroxides O30, HO20, OH0, lisocim, lactoferin, non-enzymatic cationic proteins, lactic acid).

            Digestion hydrolysis of killed bacteria with the help of hydrolytic enzymes.

  Eosinophils constitute only 0,5-54 % of the circulating leukocytes in healhty 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 (helmintic) 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.

 

 

 

Basophils vary in diameter from 12 μm to 15 μm but are usually slightly smaller than neutrophils. 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.

 

 

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.

 

 

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.

 

 

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 indistingushable, 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 immunoglobulins) 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 ammune 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.

 

 

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.

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 lectrone 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.

 

Life of white blood cells

 Lifespan of white blood cells are not constant. It depends upon the demand in the body and their function. Lifespan of these ceils may be as short as half a day or it may be as long as 3-6 months. However, the normai lifespan of white biood cells is as follows:

Neutrophils         2-5 days

Eosinophils         7-12 days

Basophils           12-15 days

Monocytes         2-5 days

Lymphocytes       1/2-1 day

The amount in peripheral blood 4-9 109/liter.

The decrease of leucocytes amount is called leucopenia, the increase  leucocytosis.

There are 2 types of leucocytes:

V          Physiological  is normal, physiological reaction of the organism in some irritations. There are following types, dependently on their causes:

1)     emotional leucocytosis (occurs in result of emotional stresses);

2)          myogenic (occurs in result of intensive physical exercises);

3)          static (occurs in result of change of the position of the human body from horizontal to vertical);

4)          alimental (occurs during or after eating);

5)          painful (occurs during strong painful feelings);

6)          leucocytosis of pregnant;

7)          leucocytosis of newborn.

 

II       Pathological (reactive) it is connected with the pathological process in the organism. Its reasons:

1)  infectious diseases;

2)  inflammatory processes;

3)  allergic reactions;

4) intoxications of endo- and exogenous origin.

 

The difference between physiological

and reactive leucocytosis

 

Physiological leucocytosis:

1) it is redistributing (leucocytes from the parietal pool are moving into circulation);

2) it has transient character (it is normalizing fast after the cause disappears);

3) leukogram does not change (the correlation between different forms persists);

4) degenerative forms of leucocytes do not appear.

 

Reactive leucocytosis is connected with the increase of proliferation and maturating of leucocytes in red bone marrow or increase of moving of reserve leucocytes from RBM to the blood. During pathological leucocytosis the correlation between different forms of leucocytes is disturbed.

Percentage ratio between different forms of leucocytes is called leukogram (formula of Arnet-Shilling).

  Differential white blood cell count (Differential leukocyte count)

 

Physiological values of leukocyte count: 5-10 x 109/L blood

Neutrophil granulocytes

Physiological values: 2-7.5 x 109/l (60-70%)

Increased number - neutrophilia: bacterial infections, trauma, scorch, bleeding, inflamations, infarction, polymialgy, myeloproliferative disorders, reaction to certain medications (e.g. chorticosteroides). Significantly increased in leukemia, disseminated malignant diseases and complicated childhood infections.

Decreased number - neuthropenia: viral infections, brucellosis, thyphoid, Kala-azar, TBC, sepsis, lupus erithematodes, rheumatoid arthritis, avitaminosis B12  i bone marrow dissorders. Medications like carbamazepine or sulphonamides can decrease a number of neuthrophils. 

Band neutrophils (stab neutrophils) cells are younger forms of cells presented with kidney-shape, curved nucleus and not segmented, lobar nucleus. Usually they are represent 3-5% of leukocytes. Increased value indicates a higher demand and expenditure of neutrophils, and is called left shift (referring to ratio of immature to mature forms of neutrophils).

Lymphocytes

Physiological values: 1.3-3.5 x 109/l (20-40%).

Increased number - lymphocytosis: viral infections (EBV-Epstein Barr virus, CMV-cytomegalovirus, rubeola), toxoplasmosis, pertusis, brucellosis, chronic lymphatic leukemia.

Decreased number - lymphopenia: corticosteroid treatment, lupus erithematodes, uremia, legionella disease, AIDS, bone  marrow infiltration (tumor), after chemotherapy and radiotherapy.

Subclases: CD4: 537-1571/mm3 (decreased in HIV infection); CD8: 235-753/mm3; CD4/CD8 ratio: 1.2-3.8.

 

Eosinophil granulocytes

Physiological values: 0.04-0.44 x 109/l (1-4%).

Increased number - eosinophilia: asthma i allergic disease, parasitic infestations, skin diseases (especially pemphigus), urticaria, egzema, malignant diseases (including eosinophilic leukemia), irradiation, Loeffler syndrome, recovery after infections. Hypereosinophilic syndromecan be observed in terminal organ damage (restrictive cardiomyopathy, neuropathy, hepatosplenomegaly), withincreased eosinophile number for more than 6 weeks (>1.5 x 109/l).

Eosinophilia-myalgi syndrome muscle pain (myalgia), joint pain (arthralgia), increased body temperature, rash, arms swelling and intense eosinophilia.

Monocytes

Physiological values: 0.2-0.8 x 109/l (2-6%).

Increased number - monocytosis: acute and chronic infection (TBC, brucellosis, protozoal infections), malignant diseases (acute myeloid leukemia, Hodgkin lymphoma), myelodisplasia.

Basophil granulocytes

Physiological values: 0.01 x 109/l (0.5-1%).

Increased number - basophilia: viral infections, urticaria, myxedema, after splenectomy, chronic myeloid leukemia, malignant disease, systemic mastocytosis (urticaria pigmentosa), hemolysis, policitemia rubra vera.

 

Healthy human has instant white blood cell count and any changes in it is the signal to different sicknesses. The disturbance in correlation between immature and mature forms of neutrophils is called shift of leukogram. There is shift to the left and shift to the right.

Shift to the left is characterized byincrease in the content of immature neutrophils. Myelocytes appear in blood, the amount of metamyelocytes increase. This happens during leucocytosis.

Shift to the right is characterized with domination of mature neutrophils with big amount of segment (5-6) on the background of disappearance of immature forms. This testifies the development of inflammatory process.

 

 

Production of leucocytes

 

Granulopoiesis

The maturation process of granulocytes takes place with cytoplasmic changes characterized by the synthesis of a number of proteins that are packed in two organelles: the azurophilic and specific granules. These proteins are produced in the rough endoplasmic reticulum and the Golgi complex in two successive stages. The first stage results in the production of the azurophilic granules. In the second stage, a change in synthetic activity takes place with the production of several proteins that are packed in the specific granules. These granules contain different proteins in each of the three types of granulocytes and are utilized for the various activities of each type of granulocyte.

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 is characterized by its basophilic cytoplasm and azurophilic granules. These granules contain lysosomal enzymes and myeloperoxidase.

The promyelocyte gives rise to the three known types of granulocyte. The first sign of differentiation appears in the myelocytes, in which 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.

 

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, five mitotic divisions occur in the myeloblast, promyelocyte, and neutrophilic myelocyte stages of development.

Neutrophils pass through several functionally and anatomically defined compartments:

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

2-      A medullary storage compartment. Neutrophils remain in this compartment for about 4 days.

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

4-      The marginating compartment is composed of neutrophils that are present in blood but do not circulate. These neutrophils are in capillaries and are temporarily excluded from the circulation by vasoconstriction, orespecially in the lungsthey 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 two compartments is 67 h. The medullary formation and storage compartments together are about 10 times as large as the circulating and marginating compartments.

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 14 days and then die by apoptosis, regardless of whether they have performed their major function of phagocytosis.

 

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 nuclear lobulation, both of which facilitate the distinction between young and mature forms of granulocytes. Lymphocytes and monocytes are distinguished mainly on the basis of size, chromatin structure, and the presence of nucleoli in smear preparations.

 

Lymphocytes

Circulating lymphocytes originate mainly in the thymus and the peripheral lymphoid organs (eg, spleen, lymph nodes, tonsils). However, all lymphocyte progenitor cells originate in the bone marrow. Some of these lymphocytes migrate to the thymus, where they acquire the full attributes of T lymphocytes. Subsequently, T lymphocytes populate specific regions of peripheral lymphoid organs. Other bone marrow lymphocytes differentiate into B lymphocytes in the bone marrow and then migrate to peripheral lymphoid organs, where they inhabit and multiply in their own special compartments.

The first identifiable progenitor of lymphoid cells is the lymphoblast, dividing two or three times to form prolymphocytes.

 

Monocyte

The monoblast is a committed progenitor cell that is almost identical to the myeloblast in its morphological characteristics. Further differentiation leads to the promonocyte, a large cell (up to 18 m in diameter) with a basophilic cytoplasm and a large, slightly indented 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 h, and then enter the connective tissues, where they mature into macrophages and function for several months.

  

Origin of Platelets

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

Megakaryoblasts

The megakaryoblast is 1550 m in diameter and has a large ovoid or kidney-shaped nucleus with numerous nucleoli. The nucleus becomes highly polyploid (ie, it contains up to 30 times as much DNA as a normal cell) before platelets begin to form. The cytoplasm of this cell is homogeneous and intensely basophilic

 

Megakaryocytes

The megakaryocyte is a giant cell (35150 m 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. Platelets have conspicuous granules, originating from the Golgi complex, that contain biologically active substances, such as platelet-derived growth factor, fibroblast growth factor, von Willebrand's factor (which promotes adhesion of platelets to endothelial cells), and platelet factor IV(which stimulates blood coagulation).

 

 

CLINICAL LABORATORY DIAGNOSTICS OF HEMOBLASTOSIS

 

Leukemia appears to be a clonal disease resulting from the abnormal uncontrolled proliferation of a single stem cell from which a new clone of cells develops. For some unknown reason, these abnormal cells have a selective growth advantage over normal cells. Present data indicate that these cells are functionally different and biochemically abnormal.

Etiology is Unknown: Possible Causes:

A. Viruses are the proven etiological agent in some animal leukemias, and may well be the causative agent in some human leukemias as well.

B. Marrow damage due to irradiation increases the frequency of some leukemias, but not others.

C. A variety of chemicals and drugs have been implicated as possible etiological agents of leukemia, especially benzene.

D. Possible genetic factors have been implicated, especially in Chronic Lymphocytic Leukemia.

Leukemia Classification

Leukemias are classified into 2 major groups. Chronic in which the onset is insidious, the disease is usually less aggressive, and the cells involved are usually more mature cells. Acute in which the onset is usually rapid, the disease is very aggressive, and the cells involved are usually poorly differentiated with many blasts.

Both acute and chronic leukemias are further classified according to the prominent cell line involved in the expansion:

1. If the prominent cell line is of the myeloid series it is a myelocytic leukemia (sometimes also called granulocytic).

2. If the prominent cell line is of the lymphoid series it is a lymphocytic leukemia

Therefore, there are four basic types of leukemia.

I. Acute myelocytic leukemia AML - (includes myeloblastic, promyelocytic, monocytic, myelomonocytic, erythrocytic, and megakaryocytic)

II. Acute lymphocytic leukemia ALL - (includes T cell, B cell, and Null cell)

III. Chronic myelocytic leukemia CML - (includes myelocytic and myelomonocytic)

IV. Chronic lymphocytic leukemia CLL - (includes plasmocytic multiple myeloma, Hairy cell, prolymphocytic, large granular cell lymphocytic, Sezarys syndrome, and circulating lymphoma)

 

 

Acute leukemias can occur in all age groups

n  ALL is more common in children

n  AML is more common in adults

Chronic leukemias are usually a disease of adults

n  CLL is extremely rare in children and unusual before the age of 40

n  CML has a peak age of 30-50

Age Distribution: optimum ages for development:

1. ALL: 3‑4 years old

2. AML: 15‑20 years old

3. CLL: 50 years old

4. CML: 20‑50 years old

5. Monocytic: middle age (rarely before 30)

Comparison of acute and chronic leukemias:

Acute Chronic

Age all ages usually adults

Clinical onset sudden insidious

Course (untreated) 6 mo. or less 2-6 years

Leukemic cells immature >30% blasts more mature cells

Anemia prominent mild

Thrombocytopenia prominent mild

WBC count variable increased

Lymphadenopathy mild present; often prominent

Splenomegaly mild present; often prominent

 

Acute Leukemia

Is a result of:

Malignant transformation of a stem cell leading to unregulated proliferation and

arrest in maturation at the primitive blast stage. Remember that a blast is the most immature cell that can be recognized as committed to a particular cell line.

 

 

Clinical features

Leukemic proliferation, accumulation, and invasion of normal tissues, including the liver, spleen, lymph nodes, central nervous system, and skin, cause lesions ranging from rashes to tumors. Failure of the bone marrow and normal hematopoiesis may result in pancytopenia with death from hemorrhaging and infections.

 

Pathophysiology of the clinical manifestations of acute leukemias

1. Marrow failure due to infiltration

-anemia -fatigue, pallor,

-thrombocytopenia -bleeding, spontaneous bruising

-neutropenia-infections, sepsis

2. Infiltration of other organs

-liver, spleen, lymph nodes (particularly in ALL)

-lymphadenopathy

-hepatosplenomegaly

-mediastinal masses(T-ALL)

-gums gum hypertrophy (monocytic subtype of AML)

-bone pain, esp. in children with ALL

-any organ or tissue

-skin-leukemia cutis

-soft tissue -chloromas

-testis

-CNS

-solid organs

3. Leukostasis (only seen with WBC >>50x109 /L)

-CNS -strokes

-lungs -pulmonary infiltrates, hypoxemia

4. Constitutional symptoms

-fevers, sweats are common

-weight loss uncommon

other

-exposure of substances that can initiate coagulation can cause DIC

 

Lab evaluation

The lab diagnosis is based on two things:

1. Finding a significant increase in the number of immature cells in the bone marrow including blasts, promyelocytes, promonocytes (> 30 % blasts is diagnostic)

2. Identification of the cell lineage of the leukemic cells

Peripheral blood:

Anemia (normochromic, normocytic)

Decreased platlets

Variable WBC count:

The degree of peripheral blood involvement determines classification:

n  Leukemic increased WBCs due to blasts

n  Subleukemic blasts without increased WBCs

n  Aleukemic decreased WBCs with no blasts

Classification of the immature cells involved may be done by:

Morphology an experienced morphologist can look at the size of the blast, the amount of cytoplasm, the nuclear chromatin pattern, the presence of nucleoli and the presence of auer rods (are a pink staining, splinter shaped inclusion due to a rod shaped alignment of primary granules found only in myeloproliferative processes) to identify the blast type:

n  AML the myeloblast is a large blast with a moderate amount of cytoplasm, fine lacey chromatin, and prominent nucleoli. 10-40% of myeloblasts contain auer rods.

n  ALL in contrast to the myeloblast, the lymphoblast is a small blast with scant cytoplasm, dense chromatin, indistinct nucleoli, and no auer rods.

 

 

 

myeloblastsjpglymphoblast

Myeloblast with auer rods Lymphoblasts

 

Auer rods in AML

 

Cytochemistry

Cytochemistry help to classify the lineage of a leukemic cell (myeloid versus lymphoid).

Myeloperoxidase is found in the primary granules of granulocytic cells starting at the late blast stage. Monocytes may be weakly positive.

myeloperoxidase

 

Sudan black stains phospholipids, neutral fats and sterols found in primary and secondary granules of granulocytic cells and to a lesser extent in monocytic lysosomes.

sudan black

 

Rare positives occur in lymphoid cells. Nonspecific esterase is used to identify monocytic cells which are diffusely positive. T lymphocytes may have focal staining.

esterase

 

Acid phosphatase may be found in myeloblasts and lymphoblasts. T lymphocytes have a high level of acid phosphatase and this can be used to help make a diagnosis of acute T-lymphocytic leukemia.

adid ptase

 

Leukocyte alkaline phosphatase is located in the tertiary granules of segmented neutrophils, bands and metamyelocytes. The LAP score is determined by counting 100 mature neutrophils and bands. Each cell is graded from 0 to 5. The total LAP score is calculated by adding up the scores for each cell.

 

alk ptase

 

Chronic Lymphocytic Leukemia

 

Neoplastic proliferation of small mature-appearing lymphocytes

>99 % are B-cell derived

Older patients (> 40)

Disease may be discovered incidentally

Fatigue, weakness, weight loss, anorexia, and/or recurrent infections may occur

Variable splenomegaly and non tender lymphadenopathy

Leukemic counterpart of small lymphocytic

Lymphoma

Is a result of:

Malignant transformation of a stem cell leading to unregulated proliferation and

arrest in maturation at the primitive blast stage. Remember that a blast is the most immature cell that can be recognized as committed to a particular cell line.

 

tissue with lesion of lymph nodes and organs, characterized by growth of the giant cells called Reed-Sternberg- Beresovsky cells, large and small atypical cells (Hodgkin,s cells) and inflammatory infiltration.

Hodgkin disease: a giant mononuclear cell with a large nucleolus (arrow) and wide cytoplasmic layer (Hodgkin cell), surrounded by small and medium-sized lymphocytes.

Hodgkin disease: giant binuclear cell (ReedSternberg giant cell).

Hodgkin's lymphoma, also known as Hodgkin lymphoma and previously known as Hodgkin's disease, is a type of lymphoma, which is a cancer originating from white blood cells called lymphocytes.

Patients with Hodgkin's lymphoma may present with the following symptoms:

     Lymph nodes: the most common symptom of Hodgkin's is the painless enlargement of one or more lymph nodes, or lymphadenopathy. The nodes may also feel rubbery and swollen when examined. The nodes of the neck and shoulders (cervical and supraclavicular) are most frequently involved (8090 % of the time, on average). The lymph nodes of the chest are often affected, and these may be noticed on a chest radiograph.

     Itchy skin

     Night sweats

     Unexplained weight loss

     Splenomegaly: enlargement of the spleen occurs in about 30 % of people with Hodgkin's lymphoma. The enlargement, however, is seldom massive and the size of the spleen may fluctuate during the course of treatment.

     Hepatomegaly: enlargement of the liver, due to liver involvement, is present in about 5 % of cases.

     Hepatosplenomegaly: the enlargement of both the liver and spleen caused by the same disease.

     Red-coloured patches on the skin, easy bleeding and petechiae

 due to low platelet count (as a result of bone marrow infiltration, increased trapping in the spleen etc. i.e. decreased production, increased removal)

     Systemic symptoms: about one-third of patients with Hodgkin's disease may also present with systemic symptoms, including low-grade fever; night sweats; unexplained weight loss of at least 10 % of the patient's total body mass in six months or less, itchy skin (pruritus) due to increased levels of eosinophils in the bloodstream; or fatigue (lassitude).

     Cyclical fever: patients may also present with a cyclical high-grade fever known as the Pel-Ebstein fever or more simply "P-E fever". However, there is debate as to whether or not the P-E fever truly exist

Definitive diagnosis is by lymph node biopsy (usually excisional biopsy with microscopic examination). Blood tests are also performed to assess function of major organs and to assess safety for chemotherapy.

 

 

Bone Marrow Biopsy

Occasionally, a disease of the blood cell system cannot be diagnosed and classified on the basis of the blood count alone and a bone marrow biopsy is indicated. In such cases it is more important to perform this biopsy competently and produce good smears for evaluation than to be able to interpret the bone marrow cytology yourself.

Although the bone marrow cytology findings from the aspirate are sufficient or even preferable for most hematological questions, it is regarded as good practice to obtain a sample for bone marrow histology at the same time, since with improved instruments the procedure has become less stressful, and complementary cytological and histological data are then available from the start. After deep local anesthesia of the dorsal spine and a small skin incision, a histology cylinder at least 1.5cm long is obtained using a sharp hollowneedle (Yamshidi). A Klima and Rossegger cytology needle is then placed through the same subcutaneous channel but at a slightly different site from the earlier insertion point on the spine and gently pushed through the compacta. The mandrel is pulled out and a 5- to 10-ml syringe body with 0.5ml citrate or EDTA (heparin is used only for cytogenetics) is attached to the needle. The patient should be warned that there will be a painful drawing sensation during aspiration, which cannot be avoided. The barrel is then slowly pulled, and if the procedure is successful, blood from the bone marrow fills

the syringe. The syringe body is separated from the needle and the mandrel

reintroduced. The bone marrow aspirate is transferred from the syringe to a Petri dish. When the dish is gently shaken, small, pinhead-sized bone marrow spicules will be seen lying on the bottom. A smear, similar to a blood smear, can be prepared on a slide directly from the remaining contents of the syringe. If the first aspirate has obtained material, the needle is removed and a light compression bandage is applied. If the aspirate for cytology contains no bone marrow fragments (punctiosicca, dry tap), an attempt may be made to obtain a cytology smear from the (as yet unfixed) histology cylinder by rolling it carefully on the slide, but this seldom produces optimal results.

The preparation of the precious bone marrow material demands special care. One or two bone marrow spicules are pushed to the outer edge of the Petri dish, using the mandrel from the sternal needle, a needle, or a wooden rod with a beveled tip, and transferred to a fat-free microscopy slide, on which they are gently pushed to and fro by the needle along the length of the slide in a meandering line. This helps the analyzing technician to make a differentiatial count. It should be noted that too

much blood in the bone marrow sample will impede the semiquantitative analysis. In addition to this type of smear, squash preparations should also be prepared from the bone marrow material for selective staining. To do this, a few small pieces of bone marrow are placed on a slide and covered by a second slide. The two slides are lightly pressed and slid against each other, then separated. The smears are allowed to air-dry and some are incubated with panoptic Pappenheim staining solution (see previous text). Smears being sent to a diagnostic laboratory (wrapped individually and shipped as fragile goods) are better left unstained. Fresh smears of peripheral blood should accompany the shipment of each set of samples.

 

 

 

Cell composition in the bone marrow: normal values (%)

 

 

 

 

 

Squash preparation and meandering smear for the cytological analysis of bone marrow spicules

 

 

Bone marrow cytology. a Bone marrow cytology of normal cell density in a young adult (smear from a bone marrow spicule shown at the lower right; magnification ×100). b More adipocytes with large vacuoles are present in this bone marrow preparation with normal hematopoietic cell densities; usually found in older patients.

 

 

Normal bone marrow findings. Normal bone marrow: megakaryocyte (1), erythroblasts (2), and myelocyte (3)

 

Bone Marrow: Medullary Stroma Cells

1.    Fibroblastic reticular cells form a firm but elastic matrix in which the blood-forming cells reside, and are therefore rarely found in the bone marrow aspirate or cytological smear. When present, they are most likely to appear as dense cell groups with long fiber-forming cytoplasmic processes and small nuclei. Iron staining shows them up as a group of reticular cells which, like macrophages, have the potential to store iron. If they become the prominent cell population in the bone marrow, an aplastic or toxic medullary disorder must be considered.

2.    Reticular histiocytes (not yet active in phagocytosis) are identical to phagocytic macrophages and are the main storage cells for tissue bound iron. Because of their small nuclei and easy-flowing cytoplasm, they are noticeable after panoptic staining only when they contain obvious entities such as lipids or pigments.

3.    Osteoblasts are large cells with wide, eccentric nuclei. They differ from plasma cells in that the cytoplasm has no perinuclear lighter space (cell center) and stains a cloudy grayish-blue. As they are normally rare in bone marrow, increased presence of osteoblasts in the marrow may indicate metastasizing tumor cells (from another location).

4.    Osteoclasts are multinucleated syncytia with wide layers of grayish-blue stained cytoplasm, which often displays delicate azurophilic granulation. They are normally extremely rare in aspirates, and when they are found it is usually under the same conditions as osteoblasts. They are distinguished from megakaryocytes by their round and regular nuclei and by their lack of thrombocyte buds.

Bone marrow stroma. a Spindle-shaped fibroblasts form the structural framework of the bone marrow (shown here: aplastic hematopoiesis after therapy for multiple myeloma). b A macrophage has phagocytosed residual nuclear material (here after chemotherapy for acute leukemia). c Bone marrow osteoblasts are rarely found in the cytological assessment. The features that distinguish osteoblasts from plasma cells are their more loosely structured nuclei and the cloudy, busy basophilic cytoplasm. d Osteoclasts are multinucleated giant cells with wide, spreading cytoplasm.

 

 

Lymph Node Biopsy

 

These procedures, less invasive than bone marrow biopsy, are a simple and often diagnostically sufficient method for lymph node enlargement or other intumescences. The unanesthetized, disinfected skin is sterilized and pulled taut over the node. A no. 1 needle on a syringe with good suction is pushed through the skin into the lymph node tissue. Tissue is aspirated from several locations, changing the angle of the needle slightly after each collection, and suction maintained while the needle is withdrawn into the subcutis. Aspiration ceases and the syringe is removed without suction. The biopsy harvest, which is in the needle, is extruded onto a microscopy slide and smeared out without force or pressure using a cover glass (spreader slide). Staining is done as described previously for

blood smears.

Normal lymphadenogram