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 Occasionally other collagen disorders
¾ Drug induced, e.g. hydralazine and procainamide.
Classification of Acute Myelomonocytic Leukemias
granules, 3% or more are myeloperoxidase positive; no
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
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
M5. Monocytic leukemia: Granulocytic cells less than 10%
differentiated and poorly differentiated subtypes
depend on degree of maturation; esterase reaction
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
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.
Fixative: 10% formalin in absolute methanol. To 10 mL
37% formaldehyde, add 90 mL absolute methanol. Store at
Buffer stock: 0.2 M propanediol. Dissolve 21 g of 2-amino-2
methyl-1, 3-propanediol in distilled water and dilute to
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.
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
Counterstain for 6 to 8 minutes in filtered Mayer’s
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:
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.
¾ Following corticosteroid therapy
The NAP scores are lowered in:
¾ Paroxysmal nocturnal hemoglobinuria always
¾ Idiopathic thrombocytopenic purpura
¾ Pernicious anemia relapse sometimes
¾ Collagen disorders and refractory anemias
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
Fixative: Mix 10 mL of 37% formaldehyde with 90 mL of
Ethanol 30% (v/v) in water 100 mL
Benzidine dihydrochloride 0.3 g
Sodium hydroxide, 1.0 N 1.5 mL
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.
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
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
Peroxidase activity is indicated by blue granules in the
cytoplasm. The nucleus and background cytoplasm stain
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
Peroxidase reaction is used in differentiating acute
myeloblastic leukemia (+ve) from acute lymphoblastic
leukemia (–ve). It parallels Sudan Black B reaction; Auer
Peroxidase activity may be absent in some toxic
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.
¾ Fixative: Mix 10 mL of 37% formaldehyde with 90 mL of
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
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)
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
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
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