DIFFERENTIAL LEUCOCYTE COUNT

PRINCIPLE

A blood film is stained with Leishman’s stain and scanned under high power, from one end to the other. As each WBC is encountered, it is identified until 100 leukocytes have been examined (Wintrobe’s 13ed, pg. 135). The percentage distribution of each type of WBC is then calculated.

Gross qualitative irregularity in distribution is the rule: polymorphonuclear neutrophils and monocytes predominate at the margins and the tail; lymphocytes predominate in the middle of the film. This separation probably depends on differences in stickiness, size and specific gravity of the different types of cells. Other systems of counting, such as the ‘battlement’ count, are more elaborate but may minimize error owing to variation of distribution of cells between the centre and the edge of the film.

ERYTHROCYTES

biconcave disc with diameter of 7-8 micron Normal appearance : Central 1/3rd pallor and peripheral 2/3rd dark pink Life span : approx. 120 days

THROMBOCYTES:

develops from megakaryocyte. 2- 4 microns in diameter Stains blue with leishman’s stain Non-nucleated Spherical to oval Usually gets aggregated (if anticoagulant is not used) Life span of 8-12 days

LEUCOCYTES: Granulocytes : neutrophil, eosinophil, basophil Agranulocytes : small and large lymphocytes, monocytes Life span: granulocytes : 4-8 hrs in circulation, 4-5 days in tissues Monocyte : 10-20 hours (guyton)/ upto 72 hours (ganong) in circulation, months in tissues Lymphocyte : circulate back and forth between blood and lymph, remain active for weeks to months

NEUTROPHIL : About 10-14 microns Numerous, fine, purplish to pink granules which are not distinct Segmented nucleus (polymorphonuclear cells) with condensed chromatin 50-70% Clear or Pinkish cytoplasm In women, 2–3% of the neutrophils show an appendage at a terminal nuclear segment. This ‘drumstick’ is about 1.5 mm in diameter and is connected to the nucleus by a short stalk. It represents the inactive X chromosome and corresponds to the Barr body of buccal cells. Toxic granulation is the term used to describe an increase in staining density and possibly number of granules that occurs regularly with bacterial infection and often with other causes of inflammation. In blood films spread without delay, the presence of vacuoles in the neutrophils is usually indicative of severe sepsis, when toxic granulation is

usually also present. Vacuoles will develop as an artefact with prolonged standing of the blood before films are made.

Dohle bodies are small, round or oval, pale blue-grey structures usually found at the periphery of the neutrophil. They consist of ribosomes and endoplasmic reticulum. They are seen in bacterial infections but also following tissue damage, in inflammation, following administration of G-CSF and during pregnancy.

Granules in neutrophil : 1. Primary (Azurophilic) Granules Formed during the promyelocyte stage Last to be released

(exocytosis) Contain: o Myeloperoxidase o β-glycerophosphatase o β -Glucuronidase Apolactoferrin o β -Galactosidase Lysozyme o β -Glucosaminidase o Cathepsins o Defensins o Elastase o Proteinase-3 o Others

2. Secondary (Specific) Granules Formed during myelocyte and metamyelocyte stages Third to be

released Contain: o β2-Microglobulin o Collagenase

o Lactoferrin o Histaminase o Heparinase o Others

3. Tertiary (gelatinase) Granules Formed during metamyelocyte and band stages Second to be released

Contain: • Gelatinase • Lysozyme • Acetyltransferase • others

4. Secretory Granules (Secretory Vesicles) Formed during band and segmented stages First to be released (fuse to plasma membrane) Contain (attached to membrane): • Alkaline phosphatase • Vesicle-associated membrane-2 • others

Neutrophilia: Physiological:

1. Physical stimuli : Cold, heat, exercise, pain, labor, pregnancy 2. Emotional stimuli Inflammation: Panic, rage, severe stress, depression

Pathological :

1. Acute infections especially pyogenic 2. Tissue necrosis (myocardial infarction, burns, trauma, infarction) 3. Drugs, hormones, and toxins : epinephrine, glucocorticoids, smoking tobacco, vaccines, venoms 4. Inflammation : gout, hepatitis, myositis, nephritis, pancreatitis,rheumatoid arthritis 5. Hematologic disorders: chronic hemolysis or hemorrhage, asplenia, myeloproliferative disorders

Neutropenia :

1. Neonates : relative percentages can be as low as 18% to 20% of leukocytes in the first few months of life and do not begin to climb to adult values until after 4 to 7 years of age. (rodak 4ed 135)

2. The most common cause of agranulocytosis is drug toxicity. Certain drugs, such as alkylating agents and antimetabolites used in cancer treatment, produce agranulocytosis in a predictable, dose-related fashion. (robbins 9ed, 582)

3. Some chronic infections causing splenomegaly, such as tuberculosis, brucellosis, typhoid fever, malaria, and kala azar, probably cause neutropenia because of splenic sequestration and marrow suppression.(william’s 8ed, ch 65)

4. Disease states associated with ineffective hematopoiesis : aplastic anemia, megaloblastic anemia 5. Increased peripheral utilization, which can occur in overwhelming bacterial, fungal, or rickettsial

infections (robbins 9ed, 582)

EOSINOPHIL : Eosinophils are a little larger than neutrophils, 12–17 microns in diameter. They usually have two nuclear lobes or segments (may be trilobed), The cytoplasm is packed with distinctive, spherical, red/orange (eosinophilic), coarse, refractile,

uniform sized granules which usually do not cover nucleus. The underlying cytoplasm, which is usually obscured by the granules, is pale blue.

The chromatin pattern is the same as that in the neutrophil, but the nucleus tends to be more lightly stained

1-4% Granules : 1. Primary Granules Formed during promyelocyte stage Contain: Charcot-Leyden crystal protein

2. Secondary Granules (crystalloid) Formed throughout remaining maturation Contains:

Major basic protein

Eosinophil cationic protein

Eosinophil-derived neurotoxin

Eosinophil peroxidase

Others

3. Small Lysosomal Granules

Acid phosphatase

Arylsulfatase B

Catalase

Elastase

4. Lipid Bodies

Archidonic acid

Leukotriene synthase

Lipoxygenase

Cyclo oxygenase

5. Storage/secretory Vesicles : plasma proteins (albumin) Eosinophilia : 1. Parasitic infestations: hookworm, tapeworm, roundworm, filariasis, hydatid disease

(Infect vs. infest. There are two main differences between infect and infest. First, infection involves germs or viruses, while infestation involves a menacingly large number of pests or parasites (e.g., mosquitos or rats). Second, an infection applies to a body, while an infestation applies to a place.)

2. Allergic diseases : Allergic rhinitis, Atopic dermatitis, Urticaria/angioedema, Asthma 3. Eosinophilic esophagitis 4. Bronchiectasis 5. Eosinophilic leukaemia Eosinopenia: 1. Increased corticoids : ACTH injection, glucocorticoid therapy, Cushing’s syndrome

2. Acute pyogenic infections

3. Aplastic anemia

BASOPHIL : 1. Basophils are the rarest (<1%) 2. The distinctive, large (very coarse), variably sized, dark blue or purple metachromatic granules of the

cytoplasm often obscure the nucleus 3. Slightly smaller than neutrophils 4. The chromatin pattern, if visible, is clumped

5. The granular constituents are water soluble, some granules may stain only faintly or not at all or may be lost from the cell during preparation. If any granules have been dissolved during the staining process, they often leave a reddish purple rim surrounding what appears to be a vacuole.

Granules : 1. Histamine 2. Serotonin 3. Heparin 4. Platelet activating factor 5. Eosinophil chemotactic factor of anaphylaxis (mast cells) Basophilia : 1. Certain viral infections: chickenpox, influenza, small pox 2. Basophils are present in increased numbers in myeloproliferative neoplasms and are especially

prominent in chronic myelogenous leukaemia; in the latter condition, when basophils are more than 10% of the differential leucocyte count, this is a sign of impending accelerated phase or blast crisis

3. Allergy or inflammation: Ulcerative colitis, Erythroderma, Juvenile rheumatoid arthritis Basopenia : 1. Elevated levels of glucocorticoids 2. Aplastic anemia 3. Primary or acquired immunodeficiency disorders 4. Thyrotoxicosis 5. During ovulation

MONOCYTE : 1. Monocytes are the largest normal cells in the blood, usually measuring from 15 to 22 µm in dia. 2. The nucleus is of various shapes—round, kidney-shape, oval, or horse shoe shaped —and frequently

appears to be folded 3. The chromatin is arranged in fine strands with sharply defined margins. The chromatin pattern is

looser than in the other leukocytes and has sometimes been described as lacelike or stringy. 4. Their cytoplasm is blue-gray with fine azure granules often referred to as azure dust or a ground-glass

appearance, and is frequently vacuolated, especially in films made from blood anticoagulated with EDTA.

5. N:C ratio less than or equal to 1. 6. 2-8% Monocytosis : 1. Protozoal infections : malaria, kala azar 2. Hematologic Disorders : myeloid leukemias, polycythemia vera, post spleenectomy 3. Connective tissue diseases : Rheumatoid arthritis, Systemic lupus erythematosus, Sarcoidosis 4. Infections : Mycobacterial infections, Subacute bacterial endocarditis, Syphilis 5. Infectious mononucleosis (viral infection) Monocytopenia : 1. High glucocorticoids 2. Aplastic anemia 3. Hairy cell leukemia, lymphocytic leukemia

Monocyte Destinations : Differentiation into macrophages: • Liver (Kupffer cells) • Lungs (alveolar macrophages) • Brain (microglia) • Skin (Langerhans cells) • Spleen (splenic macrophages) • Intestines (intestinal macrophages) • Peritoneum (peritoneal macrophages) • Bone (osteoclasts) • Synovial macrophages (type A cell) • Kidneys (renal macrophages) • Reproductive organ macrophages • Lymph nodes (dendritic cells)

LYMPHOCYTES 1. 20-40% 2. small lymphocytes: 7 to 10 µm and large lymphocytes : 10 to 15 µm 3. The small lymphocyte, the predominant type (80%) in normal blood, is round and contains a relatively

large, round, densely stained nucleus (ink-spot appearance). The cytoplasm is scanty and stains pale to dark blue.

4. In the large lymphocytes, the N:C ratio is >1. Condensed chromatin. The nucleus is usually round but may be oval or indented. The cytoplasm may contain a few azurophilic granules.

Lymphocytosis : 1. Physiological : infants 2. Chronic infection : tuberculosis, syphilis, brucellosis, leprosy 3. Viral infections : Epstein-Barr, Cytomegalovirus, Human immunodeficiency virus, Herpes, Rubella,

Toxoplasma gondii, Adenovirus, Dengue fever virus, Varicella zoster virus 4. Lymphocytic leukemia Lymphocytopenia : 1. Aplastic anemia 2. Corticosteroid and immunosuppressive therapy 3. Widespread irradiation 4. Most acute infections