special stain
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Special Stain. Cytochemical Reactions in Acute Leukemia. Leukocyte Alkaline phosphatase (LAP):. Purpose: Distinguishing the cells of leukemoid reactions with increase activity from these of (CML) with decreased activity. - PowerPoint PPT PresentationTRANSCRIPT
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