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
¾ Wash for 5 to 10 minutes in tap water and counterstain
with Harris hematoxylin for 10 minutes.
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
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
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.
Add 40 mL buffer solution to 60 mL stock stain solution.
Filter using suction. This is stable for approximately
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
¾ Wash slides with 70% ethyl alcohol for 2 to 3 minutes to
¾ Wash slides in tap water for 2 minutes
¾ Allow slides to dry. Counterstain slides with Wright’s
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.
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 Hexazotized pararosaniline 3.0 mL
¾ Alpha-naphthol acetate 50 g dissolved in 2.5 mL ethylene
¾ Filter mixture through Seitz filter
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
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
¾ Thrombocytopenia occurs in most cases, often
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
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.
Peroxidase — + (including Auer
Nonspecific — + (in monoesterase
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.
¾ Leukocytes usually > 50,000/cu mm and sometimes >
¾ A complete spectrum of myeloid cells in the peripheral
blood. The levels of neutrophils and myelocytes exceed
those of blast cells and promyelocytes.
¾ Philadelphia chromosome on cytogenetic analysis of
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
¾ Serum vitamin B12 and vitamin B12 binding capacity are
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)
¾ Leukocytosis: The absolute lymphocyte count is above
5000/cu mm and in the majority of patients it is 30–
/L. Between 70 and 99% of white cells on
blood film appear as mature lymphocytes. Smudge or
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
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.
¾ Chronic lymphocytic lymphoma
¾ Benign monoclonal gammopathy
¾ Chronic cold hemagglutinin disease
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.
1. In about 98% cases, monoclonal protein occurs in
serum and/or urine. Incidence of serum paraprotein
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
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
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)
2. About half the cases have raised serum calcium levels
and elevated serum alkaline phosphatase.
4. Renal damage leads to raised blood urea and serum
IgG, IgM and IgA can be measured quantitatively by using
immunoturbidimetric or nephelometric kits available
for estimation of the immunoglobulins IgG, IgM and IgA
1. Raised red cell count, hematocrit and hemoglobin.
2. Anisocytosis and poikilocytosis in late stages.
3. Neutrophilic leukocytosis (50% cases), in some cases
4. Raised platelet count (50% cases).
6. Raised neutrophil alkaline phosphatase score.
7. Increased vitamin B12 binding capacity.
iron diminished Reticulin diminished (late stages).
9. Serum uric acid may be raised, serum iron— histamine
levels (blood and urine)—arterial oxygen saturation
2. At the onset, white cell and platelet counts are frequently
high but later leukopenia and thrombocytopenia are
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
5. Low serum folate, raised serum vitamin B12,and raised
vitamin B12 binding capacity, increased neutrophil
6. High serum urate, LDH and hydroxybutyrate dehydrogenase levels reflect the increased but largely
ineffective turnover of hemopoietic cells.
radioiron studies, by liver biopsy or splenic aspiration.
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
¾ Mild normocytic, normochromic anemia; from
¾ Moderate leukocytosis, with eosinophilia up to 10%
¾ Normal or increased platelet count
¾ Decreased serum iron and iron-binding capacity;
¾ Decreased cell-mediated immunity, antibody activity
¾ Coombs’ positive hemolysis (relatively rare)
¾ Mild hypoalbuminemia, hyperglobulinemia
¾ Low serum zinc, high serum copper.
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)
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:
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
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
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