The best-known method is testing a control sample along side the routine specimen in each batch of test. Control material is either obtained commercially or prepared individually, but its stability and homogeneity should be

 

for 20 to 30 minutes

¾ Wash for 5 to 10 minutes in tap water and counterstain

with Harris hematoxylin for 10 minutes.

Interpretation

In blood cells a positive PAS reaction usually indicates

presence of glycogen. This is shown by digestion with

diastase and consequent loss of staining.

Neutrophils react at all stages of development, the most

strongly in the mature stage (similarly for eosinophils).

The glycogen is not in the granules but in the background

cytoplasm. Myeloblasts contain a few small PAS-positive

granules. Monocytes have a faint staining reaction in the

form of fine granules. Lymphocytes may contain a few small

or large granules. Normoblasts are normally PAS negative.

In erythroleukemia and in thalassemia some of the

erythroid precursors are PAS positive. In acute lymphoblastic leukemias, the lymphoblasts often contain large

coarse clumps of PAS positive material (block positivity).

Sudan Black B Stain (Sheehan and Storey)

Principle: Sudan black B stains phospholipids and other

lipids. It appears to stain both azurophilic and specific

granules in neutrophils, whereas the peroxidase is

found only in azurophilic granules. In early forms, late

myeloblasts and early promyelocytes, the Sudan black B

reaction is therefore, parallel to the peroxidase in its utility

in separating acute lymphoblastic from acute myeloblastic

leukemia.

Reagents

Stock solution of stain: Dissolve 0.3 g of Sudan black B

powder in 100 mL ethyl alcohol.

Buffer solution: Dissolve 16 g crystalline phenol in 30

mL ethyl alcohol. Add this to a solution of 0.3 g hydrated

disodium hydrogen phosphate (Na2HPO4. 12H2O)

dissolved in 100 mL distilled water.

Working Stain Solution

Add 40 mL buffer solution to 60 mL stock stain solution.

Filter using suction. This is stable for approximately

2 months.

Procedure

¾ Fix air dried films in formalin vapor for 10 minutes.

Slides need not be freshly made.

¾ Wash slides in running tap water for 10 minutes

¾ Place slides in working stain solution (in Coplin jar) for

60 minutes

¾ Wash slides with 70% ethyl alcohol for 2 to 3 minutes to

remove excess dye

¾ Wash slides in tap water for 2 minutes

¾ Allow slides to dry. Counterstain slides with Wright’s

stain or hematoxylin.

Interpretation

Cytoplasmic granules stain faintly in neutrophil precursors

and strongly in mature neutrophils with a brown black color.

Eosinophilic granules are brown but often show a central

pallor. Monocytes have scattered fine brown-black granules.

Lymphocytes and lymphoblasts are negative, but at least

some myeloblasts contain Sudan black positive granules.

The peroxidase and Sudan black B reactions show

roughly similar patterns in the various cell types. These

techniques are most useful in distinguishing myeloblasts

from lymphoblasts when large numbers of primitive blast

forms are present in acute leukemias.

Nonspecific Esterase (Yam et al)

Alpha-naphthol acetate esterase.

Reagents

Fixative: Buffered formalin and acetone.

Formaldehyde, 37%; 25 mL, Na2 HPO4, 20 mg, KH2PO4

100 mg; distilled water, 30 mL; acetone 45 mL.

Buffer: Sorensen’s phosphate buffer (M/15. pH = 7.6)

Incubation mixture: Add in the following manner:

¾ Buffer 44.5 mL.

¾ Hexazotized pararosaniline 3.0 mL

¾ Alpha-naphthol acetate 50 g dissolved in 2.5 mL ethylene

glycol monomethyl ether

¾ Filter mixture through Seitz filter

¾ Harris hematoxylin.

Procedure

¾ Place air dried blood or marrow films in fixative for

30 seconds at 4°C. Wash in running tap water

¾ Place slides in incubation mixture for 60 minutes. Wash

in running tap water

¾ Counterstain with Harris hematoxylin for 10 minutes.

268 Concise Book of Medical Laboratory Technology: Methods and Interpretations Interpretation

Alpha-naphthol acetate esterase activity is found in

monocytes but not in neutrophils or neutrophil precursors,

other granulocytes or lymphocytes. It may be found, however,

in activated or atypical lymphocytes, in imprints of active

lymphoid tissue, and probably in the poorly differentiated

lymphocytes of some lymphomas.

Acute Leukemias: Laboratory Diagnosis

Routine Hematologic Investigations

¾ A normocytic, normochromic anemia

¾ The white cell count may be decreased, normal or

increased up to 2 lakh/cu mm

¾ Thrombocytopenia occurs in most cases, often

extremely low in AML

Blood Film Examination

Shows variable numbers of blast cells. In AML the blasts

may show Auer rods and other abnormal cells may

be present, e.g. promyelocytes, myelocytes, agranular

neutrophils, pseudopelger cells, myelomonocytic cells.

In erythroleukemia, many normoblasts may be seen but

these may be seen in smaller numbers in the other forms.

The differentiating features of various blast cells have been

discussed elsewhere. In leukemias one may see typical and

atypical blasts.

Bone marrow is hypercellular with a marked proliferation of leukemic blast cells, which typically amount to

over 75% of the marrow cell total. In ALL marrow may be

difficult to aspirate due to increased reticulin fiber.

Differentiation of ALL from AML

In most cases, the clinical features and morphology on

routine stains separate ALL from AML. In ALL blasts,

show no differentiation whereas in AML some evidence of

differentiation, to granulocytes is often seen in the blasts of

their progeny. Special cytochemical staining techniques just

described are needed when cells are undifferentiated.

Cytochemistry ALL AML

Peroxidase — + (including Auer

rods)

Sundan black B — +

Nonspecific — + (in monoesterase

cytic types)

PAS + (coarse) + (fine)

Acid phosphatase —

(T cell ALL)

Other Investigations

Tests for disseminated intravascular coagulation (DIC)

are positive in promyelocytic leukemia. Lumbar puncture

shows raised spinal fluid pressure, it contains leukemic

cells in patients with meningeal leukemia.

Chronic Myeloid Leukemia

Laboratory Investigations

Diagnostic Features

¾ Leukocytes usually > 50,000/cu mm and sometimes >

5 lakh/cu mm

¾ A complete spectrum of myeloid cells in the peripheral

blood. The levels of neutrophils and myelocytes exceed

those of blast cells and promyelocytes.

Additional Features

¾ Philadelphia chromosome on cytogenetic analysis of

blood or bone marrow

¾ Bone marrow is hypercellular with granulopoietic

predominance (especially myelocytes)

¾ Neutrophil alkaline phosphatase score invariably low

¾ Increased circulating basophils

¾ Normocytic, normochromic anemia

¾ Platelet count is usually increased but may be normal

or decreased

¾ Serum vitamin B12 and vitamin B12 binding capacity are

increased

¾ CML is said to be undergoing blast transformation when

percentage of myelocytes and promyelocytes exceeds

20% in peripheral smear or 30% in bone marrow.

Chronic Lymphocytic Leukemia (CLL)

Laboratory Findings

¾ Leukocytosis: The absolute lymphocyte count is above

5000/cu mm and in the majority of patients it is 30–

3000 × 109

/L. Between 70 and 99% of white cells on

blood film appear as mature lymphocytes. Smudge or

smear cells are also present

¾ Normocytic, normochromic anemia in later stages

¾ Thrombocytopenia occurs in many patients.

Bone marrow aspiration: Shows lymphocytic replacement

of normal marrow elements. Lymphocytes comprise

25–95% of all the cells.

Reduced concentration of serum immunoglobins: They are

found in most cases, particularly with advanced disease.

Leukemia can be differentiated from leukemoid reaction: By

taking into consideration, the clinical picture, neutrophil

alkaline phosphatase score (high or normal in leukemoid

reaction but low in leukemia). In addition, cytochemical

stains may be used for differentiation.

Clinical Hematology 269

Paraproteinemias

Causes

¾ Multiple myeloma

¾ Macroglobulinemia

¾ Malignant lymphoma

¾ Chronic lymphocytic lymphoma

¾ Benign monoclonal gammopathy

¾ Chronic cold hemagglutinin disease

¾ Rarely with carcinomas.

Multiple Myeloma

Multiple myeloma (can be solitary myeloma called

plasmacytoma in extramedullary sites) is a neoplastic

proliferation of plasma cells. Characterized by lytic bone

lesions, plasma cell (myeloma cell) accumulation in the

bone marrow and the presence of monoclonal protein in

serum and in about half the cases in urine also.

Laboratory Diagnosis

1. In about 98% cases, monoclonal protein occurs in

serum and/or urine. Incidence of serum paraprotein

IgG 66%

 Ig A 33%

 Ig M or

 Ig D 1%.

 The normal immunoglobulins are depressed. The

Bence-Jones proteins found in urine are free light

chains, either kappa or lambda of the same type as

serum paraprotein.

2. The bone marrow shows more than 10% plasma, cells

and often with abnormal ‘myeloma cells’.

3. 60% of patients have osteolytic areas 20% show

generalized bone rarefaction or osteoporosis, which

may cause pathological fractures. 20% show no such

lesions. Most often two of the three diagnostic features

stated above are present.

Other Laboratory Findings

1. Normochromic, normocytic anemia is usual.

Marked rouleaux formation is seen. Neutropenia and

thrombocytopenia seen in advanced cases. There

may be myeloma cell spillover in the peripheral blood

or there may be (very rare) plasma cell leukemia.

Leukoerythroblastic changes (immature cells of both

myeloid and erythroid series in the peripheral blood)

are occasionally found.

2. About half the cases have raised serum calcium levels

and elevated serum alkaline phosphatase.

3. ESR is markedly raised.

4. Renal damage leads to raised blood urea and serum

creatinine levels.

5. Serum albumin is low.

The monoclonal peak (M peak, spike) is found by immunological and electrophoretic techniques. Immunoglobulins

IgG, IgM and IgA can be measured quantitatively by using

immunoturbidimetric or nephelometric kits available

for estimation of the immunoglobulins IgG, IgM and IgA

separately.

Polycythemia Vera

Laboratory Diagnosis

1. Raised red cell count, hematocrit and hemoglobin.

2. Anisocytosis and poikilocytosis in late stages.

3. Neutrophilic leukocytosis (50% cases), in some cases

basophilia.

4. Raised platelet count (50% cases).

5. Reticulocyte count raised.

6. Raised neutrophil alkaline phosphatase score.

7. Increased vitamin B12 binding capacity.

8. Bone marrow is hypercellular (generalized hypercellularity), with prominent megakaryocytes. Storage

iron diminished Reticulin diminished (late stages).

9. Serum uric acid may be raised, serum iron— histamine

levels (blood and urine)—arterial oxygen saturation

normal (92%).

Myelosclerosis

Laboratory Diagnosis

1. Anemia is usual.

2. At the onset, white cell and platelet counts are frequently

high but later leukopenia and thrombocytopenia are

common.

3. A leukoerythroblastic blood picture is seen. The red

cells characteristically show ‘tear drop’ poikilocytes.

4. Bone marrow is usually unobtainable by aspiration.

A trephine biopsy may show a hypercellular marrow

with an increase in reticulin pattern. Increased megakaryocytes are frequently seen. In some cases, there is

increased bone formation.

5. Low serum folate, raised serum vitamin B12,and raised

vitamin B12 binding capacity, increased neutrophil

alkaline phosphatase.

6. High serum urate, LDH and hydroxybutyrate dehydrogenase levels reflect the increased but largely

ineffective turnover of hemopoietic cells.

270 Concise Book of Medical Laboratory Technology: Methods and Interpretations 7. Extramedullary hemopoiesis may be documented by

radioiron studies, by liver biopsy or splenic aspiration.

Hodgkin’s Disease

This is a neoplastic disorder of lymphoreticular tissue and

four morphologic types are known, viz.

1. Lymphocyte predominance (5–13%).

2. Nodular sclerosis (40–50%).

3. Mixed cellularity (35–40%).

4. Lymphocyte depletion (5–10%).

Laboratory Findings in Hodgkin’s Disease

Early in Course

¾ Mild normocytic, normochromic anemia; from

depressed erythropoiesis

¾ Moderate leukocytosis, with eosinophilia up to 10%

¾ Normal or increased platelet count

¾ Increased ESR

¾ Decreased serum iron and iron-binding capacity;

normal or ↓ marrow iron

¾ Decreased cell-mediated immunity, antibody activity

normal.

Later in Disease

¾ Lymphopenia

¾ More severe anemia

¾ Coombs’ positive hemolysis (relatively rare)

¾ Thrombocytopenia

¾ Mild hypoalbuminemia, hyperglobulinemia

¾ Hypercalcemia

¾ Hyperuricemia

¾ Low serum zinc, high serum copper.

QUALITY CONTROL IN HEMATOLOGY

Quality control in medical laboratories encompasses a

set of procedures, which ensure that reliable and timely

test results are received by the users of laboratory service.

Reliability implies both precision and accuracy.

There are four components of quality assurance program:

¾ Internal quality control (IQC)

¾ External quality assurance (EQA)

¾ Standardization

¾ Proficiency surveillance.

Internal Quality Control

Since now most of the laboratories are dependent on

automated machine, it has become extremely important to

maintain good internal quality control, which is done by:

Testing Control Sample

The best-known method is testing a control sample along

side the routine specimen in each batch of test. Control

material is either obtained commercially or prepared

individually, but its stability and homogeneity should be

ensured.

Control Chart (Levy-Jennings or L-J Chart)

In this process when a batch of samples is dispensed (after

being run along a control sample), the mean and standard

deviation of each diameter is obtained and linear graphs

are ruled, showing the +2 standard deviation (SD) limits.

Statistically, not more than 1 in 20 samples should fall

outside these limits if the system is in control.

Cusum Analysis

¾ Chronic myeloid leukemia ¾ Paroxysmal nocturnal hemoglobinuria always ¾ Idiopathic thrombocytopenic purpura ¾ Infectious mononucleosis ¾ Pernicious anemia relapse sometimes ¾ Collagen disorders and refractory anemias

 

Interpretation: Positive LE cells in blood are found in:

¾ Systemic lupus erythematosus (70–80%)

¾ Rheumatoid arthritis (10%)

¾ Occasionally other collagen disorders

¾ Malaria

¾ Drug induced, e.g. hydralazine and procainamide.

Classification of Acute Myelomonocytic Leukemias

FAB Classification

M1. Myeloblastic leukemia without maturation: Nongranular blasts with occasional Auer rods or azurophilic

granules, 3% or more are myeloperoxidase positive; no

maturational changes.

M2. `Myeloblastic leukemia with maturation: Maturation

to promyelocytic stage: 50% of marrow cells are blasts

or promyelocytes, later stages variably present, often

with bilobed nuclei, Pelger-Huet anomaly or decreased

granulation.

Clinical Hematology 265

M3. Hypergranular promyelocytic leukemia: Predominant

cell is heavily granulated promyelocytes; bundles of

Auer rods common in cytoplasm or free on smear.

M4. Myelomonocytic leukemia: Promonocytes and

monocytes comprise 20% of nucleated cells in marrow,

blood or both; myeloblasts plus promyelocytes are

20% of marrow cells; monocytic cells have strong

nonspecific esterase reaction inhibited by fluoride;

esterase activity in myelocytic cells persists after

fluoride exposure.

M5. Monocytic leukemia: Granulocytic cells less than 10%

differentiated and poorly differentiated subtypes

depend on degree of maturation; esterase reaction

inhibited by sodium fluoride.

M6. Erythroleukemia: Marrow has 50% erythropoietic

forms, often with bizarre morphology or megaloblastic

changes (show PAS+ve granules), myeloblasts and

promyelocytes 30% or more; abnormal megakaryocytes

present.

Laboratory Diagnosis of Leukemias

Cytochemical Methods for Staining Leukocytes

Neutrophil Alkaline Phosphatase (Kaplow’s Method)

Principle: The enzyme, located in the neutrophil specific

granules, is exposed to the substrate (a naphthol

phosphate) in the presence of a diazonium salt (fast

blue or fast violet) at an alkaline pH 9.5. The substrate is

hydrolyzed by the enzyme, releasing a phosphate and an

aryl naphthol amide. The latter is immediately coupled to

the diazonium salt, forming an insoluble azo dye.

Reagents

Fixative: 10% formalin in absolute methanol. To 10 mL

37% formaldehyde, add 90 mL absolute methanol. Store at

10 to 20°C.

Buffer stock: 0.2 M propanediol. Dissolve 21 g of 2-amino-2

methyl-1, 3-propanediol in distilled water and dilute to

1000 mL. Store at 4°C.

Working: 0.05 M propanediol pH 9.4 to 9.6. Add 70 mL 0.1

N HCI to 250 mL of stock buffer and dilute to 1000 mL with

distilled water. Store at 4°C.

Substrate mixture: Dissolve 5 mg of naphthol ASBI

phosphate or naphthol AS-MX phosphate or naphthol AS

phosphate in 0.2 to 0.3 mL dimethyl formamide in a dry

flask and add 60 mL of 0.05 M propanediol buffer and 40

mg of fast blue salt RR, BB, or BBN (or fast red violet LB).

Shake well, filter into a Coplin jar and use immediately.

Counterstain: Mayer’s hematoxylin. Add 1 g hematoxylin

to 500 mL distilled water. Heat just to boiling and add

another 500 mL distilled water. Add 0.2 g sodium iodate

and 50 g of aluminum potassium sulfate. Shake well, filter

and store in brown bottle at room temperature.

Procedure

Use freshly made blood films. If venous blood is used,

heparin should be the anticoagulant, as the enzyme

activity diminishes rapidly in EDTA.

Fix air dried blood films in 10% formal methanol for

exactly 30 seconds at 0 to –10°C.

Wash in gently running tap water for 30 to 60 seconds.

Air dry slides, then place them in substrate mixture for

exactly 10 minutes. Wash in gently running tap water again

for 30 to 60 seconds.

Counterstain for 6 to 8 minutes in filtered Mayer’s

hematoxylin.

Wash in running tap water for 2 minutes. Air dry.

Positive controls are run with each batch of slides. Women

in last trimester of pregnancy are good controls, because

their scores are high normal or somewhat increased.

Scoring procedure: Examine 100 mature neutrophils in

the thin part of the film, where red cells barely touch each

other and score each as follows:

Unstained cells 0

Cells stained faintly diffusely, or a few discrete granules 1

Cells with moderate number of granules 2

Cells with granules filling the cell 3

Cells staining deeply, almost obscuring the nucleus 4

Adding the scores for 100 cells can give a possible range

of 0 to 400. The normal range with this method is 20 to 100.

The NAP scores are raised in:

¾ Bacterial infections

¾ Myocardial infarction

¾ Trauma

¾ Diabetic acidosis

¾ Polycythemia vera

¾ Myelosclerosis

¾ Following corticosteroid therapy

¾ During pregnancy

¾ Use of oral contraceptives.

The NAP scores are lowered in:

¾ Chronic myeloid leukemia

¾ Paroxysmal nocturnal hemoglobinuria always

¾ Idiopathic thrombocytopenic purpura

¾ Infectious mononucleosis

¾ Pernicious anemia relapse sometimes

¾ Collagen disorders and refractory anemias

¾ Hypophosphatemia.

266 Concise Book of Medical Laboratory Technology: Methods and Interpretations Peroxidase (Myeloperoxidase, Kaplow’s Method)

Principle: In the presence of hydrogen peroxide, peroxidase

in leukocyte granules oxidizes benzidine from a colorless

form to blue or brown derivative, which is localized at the

site of the enzyme.

Reagents

Fixative: Mix 10 mL of 37% formaldehyde with 90 mL of

absolute ethanol:

Incubation mixture

Ethanol 30% (v/v) in water 100 mL

Benzidine dihydrochloride 0.3 g

Zn SO4. 7H2O, 0.132M

(3.8% w/v) 1.0 mL

Sodium acetate

(Na2C2H3O2. 3H2O) 1.0 g

3% hydrogen peroxide 0.7 mL

Sodium hydroxide, 1.0 N 1.5 mL

Safranin O 0.2 g

Reagents are mixed in the stated order. A precipitate forms

after adding zinc sulfate but dissolves after other reagents

are added. The pH is not critical between 5.8 and 6.5. The

mixture is filtered and may be kept in a closed container

and reused for a period of several months.

Procedure

Freshly made films or imprints are used. Peroxidase is

unstable in the light, but unfixed films are satisfactory for

as long as 3 weeks if kept in the dark. Heparin/oxalate/

EDTA can be used as an anticoagulant.

Place slides in fixative for 60 seconds at room

temperature. Wash in gently running tap water.

Place slides in incubation mixture for 30 seconds at

room temperature. Wash in gently running tap water for

30 to 60 seconds.

Allow slides to dry, and examine under the microscope.

The slides may be counterstained with Wright’s stain

or with 1% aqueous cresyl violet if greater nuclear detail is

wanted.

Interpretation

Peroxidase activity is indicated by blue granules in the

cytoplasm. The nucleus and background cytoplasm stain

red.

In neutrophilic series peroxidase becomes positive

in late myeloblasts and on till mature neutrophil. In

eosinophils specific granules contain peroxidase. Basophils,

lymphocytes and erythroid cells do not stain. Monocytes

stain less intensely than do neutrophils, and the granules

are smaller.

Peroxidase reaction is used in differentiating acute

myeloblastic leukemia (+ve) from acute lymphoblastic

leukemia (–ve). It parallels Sudan Black B reaction; Auer

rods are positive with both.

Peroxidase activity may be absent in some toxic

neutrophils in infection.

Periodic Acid-Schiff (PAS) Reaction

Principle: Periodic acid (HIO4) is an oxidizing agent that

converts hydroxy groups on adjacent carbon atoms to

aldehydes. The resulting dialdehydes are combined

with Schiff’s reagent to give a red colored product. A

positive reaction is, therefore, seen with polysaccharides,

mucopolysaccharides, and glycoproteins.

Reagents

¾ Fixative: Mix 10 mL of 37% formaldehyde with 90 mL of

absolute ethanol

¾ Periodic acid, 5 g, is dissolved in 500 mL of distilled

water. Stored in dark bottle and is good for 3 months

¾ Schiff’s reagent: Dissolve 5 g of basic fuchsin in 500 mL of

hot distilled water and filter after it has cooled. Saturate

with sulfur dioxide gas by bubbling for 1 hour. Extract the

FAB classification of lymphoblastic leukemias

Observation L1 L2 L3

Consistency of appearance Homogeneous Heterogeneous Homogeneous

Cell size Uniformly small Large, but variable Uniformly large

Nuclear shape Regular, little clefting Irregular, clefted, indented Regular, rounded

Nucleoli None or inconspicuous One or more large One or more, prominent

Amount of cytoplasm Scant Variable, often abundant Abundant

Other findings T d T* usually T d T usually May have B cell

Myeloperoxidase negative markings

(T d T—Terminal deoxynucleotidyl transferase)

Clinical Hematology 267

solution with 2 g of activated charcoal for a few seconds

in a hood and immediately filter through Whatman No.

1 filter paper into a dark bottle. The solution is kept for

2 to 3 months.

¾ Harris hematoxylin.

Procedure

¾ Place air-dried blood and marrow films or imprints in

fixative for 10 minutes. Wash briefly with tap water

¾ Control slides are exposed to digestion with saliva

(diastase) for 30 minutes. Place slides in periodic acid

for 10 minutes. Wash briefly with tap water and blot dry

¾ Immerse slides in Schiff’s reagent for 30 minutes

¾ Rinse slides in several changes of sulfur dioxide water

6. Transfer the buffy coat to Wintrobe’s hematocrit tube and centrifuge again for 5–10 minutes. 7. Transfer the buffy coat and an equal volume of plasma to a small tube, mix well, and prepare smears. Dry rapidly and stain with Giemsa’s or Leishman’s

 

¾ Lipid storage disease

¾ Hemolytic anemia

¾ Hypochromic anemia

¾ Recovery from agranulocytosis.

Basophilia

¾ Chronic myeloid leukemia

¾ Myelosclerosis

¾ Polycythemia vera

¾ Hypersensitivity states

¾ Myxedema

¾ Iron deficiency anemia (some cases)

¾ Hemolytic and toxic anemias of long standing

¾ Preleukemia (some cases).

Morphologic forms of Lymphocytes

Virocyte

(Also called stress lymphocytes, Downey type cells, or

atypical lymphocytes)

1. These are small atypical cells that appear in viral

diseases such as mononucleosis, viral hepatitis, viral

pneumonia, and viral upper respiratory tract infections.

2. These may also be found in numerous nonviral

conditions:

a. Fungoid and protozoid nonviral conditions

b. Autoimmune states

c. Allergic reactions

d. After transfusions and tissue graft.

3. When seen in stress response, these are called stress

lymphocytes.

4. May be found in apparently healthy children.

5. Up to 10% of all lymphocytes, can be considered

normal.

Transformed Lymphocytes

1. Examples

a. Lymphocyte cells that may be seen in macroglobulinemia.

b. Turk cells and Reider cells that are seen in acute

lymphatic leukemia.

c. Vacuolated lymphocytes that are seen in lipidosis.

2. Culturing of lymphocytes in laboratory:

a. Stimulates small lymphocytes to transform into

large atypical cells which produce immunoglobulin.

b. Transformation response is impaired in culturing

of lymphocytes from patients with:

• Hodgkin’s disease

• Lymphatic leukemia

• Lymphocytosis

• Agammaglobulinemia.

c. Transformation response increased in sarcoidosis.

3. Other uses of transformation test are to determine

histocompatibility of recipient and donor for tissue

grafts:

a. Lymphocytes from donor not related to recipient

stimulate the production of up to 3% of transformed lymphocytes in recipient.

b. Lymphocytes from sibling react less strongly.

c. No reaction occurs on cultures from fraternal

twins.

262 Concise Book of Medical Laboratory Technology: Methods and Interpretations Arneth Count

Neutrophils can be divided into five main groups according

to the number of lobes in their nuclei.

Group 1 One lobe, even if it shows indentation and

thinning out at one or more places.

Group 2 Two lobes connected by one thin filament.

Group 3 Three lobes connected by two thin filaments.

Group 4 Four lobes connected by three thin filaments.

Group 5 Five or more lobes connected by four or more

thin filaments.

Using the above-mentioned classification, count 100

neutrophils and the number in each group is to be

expressed as percentage. Usual normal values:

Group 1 2 3 4 5

Number 5 35 42 16 2

Interpretation: In acute infections, there is rapid turnover

of neutrophils and in the process younger neutrophils with

lesser number of lobes are released into circulation, thus

increasing the number of cells in groups 1 and 2 (shift to

the left).

In macrocytic megaloblastic anemias, the neutrophil

production rate is slow, hence cells with hypersegmented

nuclei are released into circulation (shift to the right).

However, Arneth count is now no longer in use.

Arneth Index

The percentage of cells in groups 1, 2 and ½ of 3 is about

60 (normal range 51–65). Only 2–5% fall in group 1.

Neutropenia and Agranulocytosis

Discussed elsewhere.

Lymphopenia

¾ Severe pancytopenia

¾ Congestive heart failure

¾ Adrenocorticosteroid therapy (transient)

Eosinopenia

Drug/Hormone Therapy

¾ Adrenocortical steroids

¾ Adrenaline

¾ Ephedrine

¾ Insulin.

Response to Stress

¾ Acute infections

¾ Traumatic shock

¾ Surgical operations

¾ Severe exercise

¾ Burns

¾ Acute emotional stress

¾ Exposure to cold.

Endocrine Diseases

¾ Cushing’s disease

¾ Acromegaly

¾ Pheochromocytoma.

Miscellaneous

¾ Aplastic anemia

¾ Discoid lupuserythematosus.

Basophilopenia

¾ neutrophil leukocytosis or leukemoid reaction associated

with:

• Infection

• Neoplasia

• Tissue necrosis

• Acute anemia

• Allergic conditions

• Hyperthyroidism

• Myocardial infarction

• Cushing’s syndrome

• Following prolonged corticosteroid therapy.

Leukemoid Reactions

Excessive leukocytic response to a stimulus and/or

immature cell spilling over in peripheral blood.

Neutrophilic

¾ Hemolytic crises

¾ Hemorrhage

¾ Hodgkin’s disease

¾ Infections

• Tuberculosis

• Other bacterial infections

• Congenital syphilis

¾ Burns

¾ Eclampsia

¾ Mustard gas poisoning

¾ Vascular thrombosis and infarction

¾ Marrow replacement and myeloid metaplasia.

Lymphocytic

¾ Infectious lymphocytosis

¾ Infectious monocytosis

Clinical Hematology 263

¾ Pertussis

¾ Varicella

¾ Tuberculosis.

Eosinophilic

¾ Visceral larva migrans.

Bone Marrow Plasmacytosis

Acute Infections

¾ Rubella

¾ Rubeola

¾ Varicella

¾ Infective hepatitis

¾ Scarlet fever.

Chronic Infections

¾ Tuberculosis

¾ Syphilis

¾ Fungal.

Allergic States

¾ Serum sickness

¾ Drug reactions.

Collagen—Vascular Disorders

¾ Acute rheumatic fever

¾ Rheumatoid arthritis

¾ Systemic lupus erythematosus.

Neoplasms

¾ Disseminated carcinoma

¾ Hodgkin’s disease

¾ Multiple myeloma.

Others

¾ Cirrhosis of liver.

WHITE BLOOD CELLS

Neutropenia and Agranulocytosis

Neutropenia is the reduction in number of circulating

neutrophils below 2500 cells/cu mm.

Blood Picture of Drug-induced Neutropenia

¾ Neutropenia with no anemia or thrombocytopenia

¾ In some cases, there may be lymphopenia and

monocytopenia also

¾ Neutrophils may show toxic and degenerative changes

¾ ESR is usually raised.

Bone Marrow

¾ Absence of granulocytic precursors with normal

erythropoiesis and a normal number of megakaryocytes

(sometimes depleted)

¾ Toxic granulation in developing granulocytes

¾ Granulocytic hyperplasia implies recovery.

Causes of Neutropenia

Drugs

1. Drugs that cause aplastic anemia.

2. Drugs that induce selective neutropenia:

 Antipyretic analgesics—Amidopyrine.

 Antithyroid drugs—Thiouracil, methimazole, carbimazole.

 Antihistamines—Promethazine, chlorpheniramine,

mepyramine, etc.

 Tranquillizers and antidepressants —Chlorpromazine,

meprobamate, imipramine, amitri-ptiline, etc.

 Antibacterials—Tetracycline, streptomycin, ristocetin,

salazopyrin, sodium methicillin, etc.

 Anticoagulants—Phenindione, dicoumarol.

 Antituberculars—Isoniazid, PAS, thiacetazone.

 Antimalarials—Primaquine, amodiaquin.

 Miscellaneous—Procainamide, penicillamine, metronidazole, etc.

Other Causes of Neutropenia

1. Chronic idiopathic neutropenia (agranulocytosis)

neutrophil count = 500–2000 cells/cu mm— absolute

or relative lymphocytosis.

2. Infections

• Acute viral

– Rubeola

– Hepatitis.

• Bacterial

– Typhoid

– Brucellosis

– Rickettsial

– Protozoan—malaria

– All grave infections

• Bacteremia

• Miliary tuberculosis.

3. Marrow aplasia: All causes of aplastic anemia.

4. Due to known cause or myelophthisis

• Leukemia

• Neoplasia.

5. Nutritional deficit

• Folic acid or Vitamin B12 deficiency causes

megaloblastic or macrocytic anemia also.

264 Concise Book of Medical Laboratory Technology: Methods and Interpretations 6. Hypersplenism: Congestive or infiltrative.

7. Miscellaneous

• SLE

• Anaphylaxis

• Antileukocyte antibodies

• Immunodeficiencies

• Pancreatic exocrine deficiency

• Cyclic neutropenia (familial/sporadic).

Symptomatic neutropenia occurs usually in aplastic

anemia, drug-induced neutropenia, hypersplenism and

idiopathic neutropenia, and acute leukemia.

Laboratory Diagnosis Of Infectious Mononucleosis

Blood Picture

¾ There is both absolute and relative lymphocytosis, large

numbers of them atypical

¾ Hemoglobin value and platelet counts are normal

¾ Initial leukopenia due to reduction in number of

neutrophils, the neutrophil alkaline phosphatase count

is often low

¾ Lymphocytosis is maximum at about the tenth day

¾ Lymphocytosis (atypical) as described by Downey and

Mckinley

• Type I—Monocytoid lymphocytes

• Type II—Plasmacytoid lymphocytes

• Type III—Blastoid lymphocytes

¾ ESR is raised in 50% cases

¾ Wasserman reaction may be positive in 3–10% cases

¾ ELISA test is available

¾ Latex and particle agglutination tests are also available.

Paul Bunnel Test for Heterophile Antibody

This is based upon the presence of antisheep red cell

hemagglutinins in unusually high titers in the sera of these

patients. It is positive in about 80–90% of cases. It remains

positive for a variable period of time. In addition to the said

antibodies, at least 2 other types of agglutinin for sheep red

cells occur in human serum. They are:

1. An antibody present in low titers in normal persons

and in malignant lymphomas. This antibody is

absorbed by guinea pig kidney but not by ox cells.

2. An antibody occurring following the injection of horse

serum and in serum sickness. This is absorbed both

by guinea pig kidney and ox cells. This antibody of

infectious mononucleosis is not absorbed by guinea

pig kidney but is absorbed by ox cells.

Lupus Erythematosus (LE) Cell/Phenomenon

Method

1. Draw 5–10 mL of venous blood. Place in a 50 mL flask

containing 20–30 glass beads 3–5 mm in diameter or

clear metal paper clips. Swirl or shake gently for 10–15

minutes to defibrinate the blood.

2. Let stand 15 minutes (preferably at 37°C).

3. Transfer blood and a few beads to a test tube or

container and mix on a rotator or by inverting for 30

minutes.

4. Let stand at room temperature (preferably at 37°C) for

1 hour.

5. Centrifuge at 2000–3000 rpm for 5–10 minutes.

6. Transfer the buffy coat to Wintrobe’s hematocrit tube

and centrifuge again for 5–10 minutes.

7. Transfer the buffy coat and an equal volume of

plasma to a small tube, mix well, and prepare smears.

Dry rapidly and stain with Giemsa’s or Leishman’s

stain.

8. Examine smears for clumps of platelets and neutrophilic

leukocytes where the typical LE cell is most likely to

be found. Look for neutrophils containing ingested

homogeneous blue to magenta colored bodies (LE

cell) or a group of neutrophils encircling (garlanding)

such a body (LE phenomenon).

It should be differentiated from ‘tart cell’ in which though

neutrophil may show an inclusion that is not homogeneous

and is of the same color and appearance of their nuclei.