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Cytochemical Stains Cutochemistry Identification of chemical constituent within the cell Celluler morphology is not changed Used in differentiation

of acute leukemias 1. Enzymatic: myeloperoxidase, esterase, acid and alkaline phosphatase 2. Nonenzymatic: Sudan black B, periodic acid-Schiff, and terminal deoxynucleotidyl transferase

NAME Myeloperoxid ase

TYPE Enzyme

CONSITUENTS STAINED Marker for primary granules and Auer rods

INTERPRETAT ION Peroxidase activity produces dark brown granules in cytoplasm of granulocytes and monocytes. Neutrophil and precursors Monocyte weak positive Dark purpleblack granules in neutrophil precursors Lymphoblasts are negative Enzyme activity results in bright red granules in cytoplasm of neutrophils,

IMPORANT INFORMATIO N Myeloperoxid ase enzyme deteriorates. Stain should be done only on fresh specimens.

Sudan Black B (SBB)

Nonenzym e

Marker for phospholipids and lipids

Can be done on stored specimens Parallels myeloperoxid ase for interpretation Known as specific esterase Stable enzyme that lasts for months

Napthol AS-D Chloroacetat e Esterase

Enzyme

Marker of mature and immature neutrophils and mast cells

-Naphthyl Acetate Esterase - Naphtyl Butyrate Esterase

Enzyme

Enzyme

Marker for monocytes, megakaryocytes, and plasma cells Identifying monocytes, promonocytes, and monoblasts Present in all hematopoietic cells and found in lysosomes Marker for hairy cell leukemia

neutrophil precursor, and mast cells. Monocytes stained brown Enzyme activity results in dark, red precin cytoplasm Activity is indicated by purple to red granules Activity is indicated by purple to dark red granules in cytoplasm 100 cell count is done. Neutrophils are scored from 0 with no activity to 4 with large amount of activity. Activity results in bright fuchsia pink. Pattern of staining varies with each cell type. Is present in 90% cases of ALL

Monocytes are weakly potive. Known as nonspecific esterase (NSE) Known as nonspecific esterase (NSE) Cannot be stored Excellent marker for hairy cell leukemia Used to differentiate CML form leukemoid reaction. CML has decreased activity. Lymphoblasti c leukemias show blockly or crunchy pattern. L1 and L2, block pattern L3 negative Erythroblasts in M6 Leukemia also positive Used to differentiate AML from ALL

Acid Phosphatase Tartrate Resistant Acid Phosphatase Leukocyte Alkaline Phosphatase (LAP)

Enzyme

Enzyme

Enzyme

Neutrophil is the only leukocyte that contain this activity

Periodoc Acid-Schiff (PAS

Nonenzym e

Marker for glycoged, glycoproteins, mucoproteins, and high molecular weight carbohydrates

Terminal Deoxynucleot idyl Transferase (TdT)

Enzyme

DNA polymerase immunoperoxida se

Toluidine Blue

Nonenzym e

Binds with acid mucopolysaccha rides in blood cells

Strongly metachromati c

Useful for recognition of mast cells and basophils

Cytochemistry Flow Sheet in Acute Leukemia

BLAST CELL ON WRIGHTS STAIN

Peroxidase / Sudan Black

Acute nonlymphocytic leukemia M1 M2 M3 M4 M5 (+/-)

Acute Lymphocytic Leukemia

Esteras e

PAS

Naphthyl acetate Naphthyl Butyrate Positive MONOCYTIC M4 M5 M6

AS-D Chloroacetate Positive GRANULOCYTIC M1 M2 M3 M4 M5 (+/-)

Varaibl e L1 L2 M6

TDT L1 L2 L3

TRAP HAIRY CELL T-CELL

TDT = Terminal deoxytransaminase TRAP = Tartrate resistant acid phosphatase

Bone Marrow Analysis

Bone marrow aspiration

Erythroblasts % ANC

<50% Erythroblast

>50% Erythroblast

Blasts % ANC

Blasts % NEC

>30% blasts

<30% blasts

<30% blasts

>30% blasts

AML

MDS

MDS

M6

M1 M5

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