Special Stain

Cytochemical Reactions in Acute LeukemiaCytochemical Reactions in Acute LeukemiaCytochemical Cytochemical ReactionReaction

Cellular Cellular Element StainedElement Stained Blasts IdentifiedBlasts Identified

Myeloperoxidase Myeloperoxidase (MPO)(MPO)

Neutrophil Neutrophil primary primary granulesgranules

Myeloblasts strong positive; Myeloblasts strong positive; monoblasts faint positivemonoblasts faint positive

Sudan Black B Sudan Black B (SBB)(SBB) PhospholipidsPhospholipids Myeloblasts strong positive; Myeloblasts strong positive;

monoblasts faint positivemonoblasts faint positive

Specific esteraseSpecific esterase Cellular enzymeCellular enzyme Myeloblasts strong positiveMyeloblasts strong positive

Nonspecific Nonspecific esterase (NSE)esterase (NSE) Cellular enzymeCellular enzyme Monoblasts strong positiveMonoblasts strong positive

Periodic acid-Periodic acid-SchiffSchiff

Glycogen and Glycogen and related related substancessubstances

Variable, coarse or block-Variable, coarse or block-like positivity often seen inlike positivity often seen inlymphoblasts and lymphoblasts and pronormoblasts, pronormoblasts, myeloblasts usuallymyeloblasts usuallynegative although faint negative although faint diffuse reaction maydiffuse reaction mayoccasionally be seenoccasionally be seen

Leukocyte Alkaline phosphatase (LAP):Purpose: Distinguishing the cells of

leukemoid reactions with increase activity from these of (CML) with decreased activity.

Principle: Alkaline phosphatase Activity is present in varying degrees in the neutrophil and band form of the granulocytes /some times in B lymphocytes

Leukocyte Alkaline phosphatase (LAP)Leukocyte Alkaline phosphatase (LAP)

Positive LAP Positive LAP reactionreaction

Negative LAP Negative LAP reactionreaction

1-Leukocyte Alkaline phosphatase (LAP):Interpretation:Count 100 neutrophils and score them (0/+4), then

calculate the final score by adding the total scores.Grading:*(0) No stain *(+1) Faint stain *(+2) Moderate stain *(+3) Strong stain*(+4) Strong stain without cytoplasmic background

Normal Range: 30-185

LAP elevated in: LAP decreased in:

Leukomoid reaction. CML.

Pregnancy Paroxymal Nocturnal Hemoglobinuria.

Polycythemia vera. Sickle cell anemia.

Aplastic anemia. Hypophosphatasia.

Multiuple myeloma

Obstructive juindice.

Hodgkins` disease.

**The following diseases will not affect LAP result:1. Untreated hemolytic anemia.2. Lymphosarcoma.3. Viral hepatitis.4. Secondary polycythemia.

Peroxidase stain :Purpose: To differentiate a myelogenous or

monocytic leukemia from acute lymphocytic .leukemia

Principle: Peroxidase is present in the primary

azurophilic granules of neutrophil, eosinophil and monocyte & activity increased with maturation, no activity is found in red cells or lymphocytes.

Myeloperoxidase (MPO)Myeloperoxidase (MPO)

Bluish-black Bluish-black granulesgranules

red brown red brown precipitateprecipitate

Myeloperoxidase stain, bone marrow aspirateMyeloperoxidase stain, bone marrow aspirate

The red granular The red granular staining peroxidase staining peroxidase activity.activity.

Peroxidase stain: Red – brown peroxidase found in: neutrophil and eosinophil {promyelocyte –

Myelocyte – Metamyelocyte}Finely granular staining found in: -

MonocyteNegative stain found in: ( early Myeloblast, lymphblast, basophiles and

plasma cell)

Notes:In acute leukemia, infection &

myelodysplasia neutrophils show (-ve) stain

Increase in CML*Basophile May stain +ve in granulocytic

leukemiaPeroxidase stain show results similar to

those of sudan Black B stain

Sudan Black B: Purpose: To distinguish acute myelogenous

and monocytic leukemia from lymphocytic leukemia.

Principle: Sudan black B dye is fat soluble, then it stains fat particles (Steroles, phospholipids and neutral fats) which present in the primary and secondery granules of myelocytic and monocytic cells.

Sudan Black BSudan Black BPositive sudan Positive sudan

black B (SBB) black B (SBB) stain in a patient stain in a patient with AML ,with AML ,

Not the black Not the black staining staining cytoplasmic cytoplasmic granules in the granules in the myeloblastsmyeloblasts

Interpretation:Myelogenous cells show coarse staining

granules with faint staining pattern for myelobast and increase staining with maturation.

Auer rods are +vely stained. Monocytic cells show finely scatterd

granules.-ve lymphoctic staining except Burkitt`s

lymphoma cells, may show +ve staining vacuoles.

Acid phosphatase ( with tartrate resistance)Acid phosphatase ( with tartrate resistance)Purpose: diagnosis of hairy cell leukemia.Principle: ACP enzyme present in

myelocytic, lymphocytes, monocytic, plasma cell, and platelets in these cells ACP activity will inhibited in the presence of (L-tartarate) and give no color, while hairy cell ACP will not inhibited and give (+ve).

Acid phosphatase ( with tartrate resistance)Acid phosphatase ( with tartrate resistance)Hairy cell leukemia, TRAP stain. Acid

phosphatase reaction after incubation with tartaric acid. Granular staining is seen in the lymphocytes.

Non Specific Esterase: {with fluoride inhibition}Purpose: Differentiate myelocytic and

monocytic leukemia.Principle: WBCS contain esterases, a group

of lysosomal enzymes

Non Specific Esterase: {with fluoride inhibition}

NSEs α-naphthyl acetate positivity in M5b.Not the granular positivity in the monoblasts and immature monocytes

Interpretation(+ve) brick – red staining which found in:

Megakaryocyte and platelets, Histocyte, Macrophage, Monocyte & Lymphoblast of ALL

(-ve) for granulocytes**If fluoride added, only monocyte non

specific esterase will be inhabited.

Periodic Acid – Schiff [PAS] Reaction:

Purpose: Diagnosis of some acute lymphocytic leukemiasubtypes of AMLM 6

Principle: the stain indicates the presence of muccoproteins , glycoproteins and high molecular weight carbohydrates in blood cells.

Periodic Acid – Schiff [PAS] ReactionPeriodic Acid – Schiff [PAS] Reaction

Giant multinucleate late Giant multinucleate late normoblasts (left). Granular PAS normoblasts (left). Granular PAS positivity in proerythroblasts and positivity in proerythroblasts and homogeneous positivity in the homogeneous positivity in the later normoblastslater normoblasts

Interpretation:Normally all blood cells are (+ve) but

Erythroblasts (-ve)Diffused stain pattern (Granulocytes)

Granular stain (lymphocytes and monocytes) Plts deeply stainednRBCs (-ve) stain

In diseases: In CML, lymphosarcoma and Hodgkins` disease (+ve)

staining granules will increase. nRBCs in M6, thalassemia and other types of anemia

may give [+ve] reaction.

Positive PAS stain Positive PAS stain acute acute

megakaryocytic megakaryocytic leukemia AML, M7. leukemia AML, M7.

Positive PAS stain in Positive PAS stain in ALLALL

PAS positivity in M6. PAS positivity in M6. Not the intense staining Not the intense staining of the large abnormal of the large abnormal erythroblast.erythroblast.

Specific esterase or chloroacetatePrinciple:Principle:

Interpretation:Interpretation:Myeloid cells (+ve)Monocyte and basophile (–ve) to weak (+ve)Other cells {lymph – plasma –megakaryocyte –

nrbc } (-ve)Auer rods (+ve)

Specific esterase or chloroacetateNaphthol (AS-D) Naphthol (AS-D)

Chloroacetate Esterase Chloroacetate Esterase stain in a patient with stain in a patient with AML,M2. AML,M2. Not the Not the bright red staining bright red staining indicating that these indicating that these two blasts are of two blasts are of myeloid origin.myeloid origin.

Iron stain (Prussian Blue Reaction):

Principle:Sidrotic granules are found in the cytoplasm

of developing cells in [BM] in the form of Ferric [Fe+3].

Perls' reagent is formed of (Potassium Ferricyanide + HCL)

Sidrotic granules are found in nRBCs, some reticulocytes

Sidrotic granules are found in nRBCs, some reticulocytes