CYTOCHEMISTRYI. Enzymatic TechniquesA. PeroxidasesA H2O +H2O2→A+2H2O

A.1. Diaminobenzidine MethodReagents: Fixative-buffered formalin-acetoneIncubation mixture: (prepare fresh for each batch

Phosphate buffer 0.07M,pH 7.4-50mL3,3 DAB tetrahydrochloride-37.5mg3% H2O2

Giemsa stain, 10.0 mL

Procedure:Controls: blood film from normal donor

Positive control (neutrophils)Negative controls (lymphocytes)

Interpretation: Dark brown granules in the cytoplasm of granulocytes and monocytes

Monocyte: weak to moderateGranulocytes: are packed with positive granulesRBCs-stain diffusely brown because of pseudoperoxidase activity in Hb

A.2. Cyanide-Resistant Peroxidase Stain:Uses: as proposed by Gabbas and Li

1. identification of the eosinophilic component of acute myeloid and acute myelomonocytic leukemias

2. aid in recognition of de novo acute eosinphilic leukemia

Incubation Medium: In addition to DAB method4.9 mg NaCN (sodium cyanide) is added and titrate to pH 7.4 with 1N HCl

Procedure: same with DABIncubation mixture-5 minutesControls: Normal blood buffy coat

Eosinophils should be strongly positive (brown)Neutrophils-negative

B. Esterases: are enzymes that hydrolyze aliphatic and aromatic esters at acid or neutral pH

B.1. Naphthol AS-D Chloroacetate EsteraseUses:

1. Useful in demonstrating myeloid elements on paraffin-embedded sections such as granulocytic sarcoma

2. systemic mast cell disease

Hexazotized new fuchsin: mix equal volume of new fuchsin and 4% Na nitrite for 1 min before use

Naphthol AS-D chloroacetate solution: 10mg Naphthol AS-D chloroacetate dissolve in 5mL of N,N-dimethyl formamide.Store at 4°-10°C. Stable for 1 month.

Phosphate buffer 0.07M, pH 7.73Mayer’s hematoxylin-10 min. Counterstain.Procedure:

3. Incubate films at room temp for 10 minutes using:0.07M phosphate buffer-38.0mLFresh hexazotized new fuchsin-0.2mLNaphthol AS-D chloroacetate sol-2.0mL

Controls: Normal neutrophils-positive controlInterpretation:

B.2. Alpha-Naphthyl Acetate Esterase (ANAE)T-helper lymphocytes-focal dotlike staining with long incubationT-lymphoblasts of acute leukemia-focal staining is weak or negative

Hexazotized pararosanilin: Mix equal volume of the first 2 solution for 1 minute before use.

Phosphate buffer, 0.07M pH 6.64Alpha-naphthyl acetate solu: 100mg dissolve in 5.0mL ethylene glycol monomethyl ether. Refrigerate before use.

Controls:Positive controls: Normal monocytes or histiocytes in bone

marrow or blood film prep.Monocytes-stain red-brownLymphocytes-dotlike

B.3. Alpha-Naphthyl Butyrate Esterase (BE)Use to differentiate acute myelomonocytic, acute monocytic and

chronic myeolomonocytic leukemias from other acute nonlymphocytic leukemias and dysmyelopoietic syndromes.

Incubation mixuter: 45mins. at room temp.

0.07M phosphate buffer pH 6.69-38.0mLFresh hexazotized pararosanilin-0.4mLAlpha-naphthyl butyrate solution-2.0mL

Control: same with ANAEInterpretation: Enzyme activity is noted as dark red precipitates in the cytoplasm of monocytes and histiocytes

B.4. Combination of Alpha-naphthyl butyrate-chloroacetate Esterase

Procedure:BE incubation mixture at room temp. for 30 min.CE incubation mixture at room temp. for 5 min.

Controls: normal monocytes and neutrophils in blood filmhistiocytes or developing neutrophils is bone marrow

Interpretation:Cytoplasm of monocytes&histiocytes-dark red pptNeutrophils-blue granules

C. PhosphatasesC.1. Acid phosphatase

Reagents:Fixative: methanol-acetone mixture-30 secondsAcetate bufferSubstrate Solution

Naphthol AS-BI phosphoric acid-100.0 mgN,N-dimethyl formamide-10mgStable for 2 months at 4°-10°C

45 min.-incubation mixture: 50mL acetate buffer0.5mL substrate5 mg fast garnet GBC salt

Mayer’s hematoxylin-counterstain-5-20 min.Mounting medium Glycerin jellyControl: peripheral blood filmInterpretation: discrete purplish to dark red granulesT-cell acute lymphoblastic leukemia-moderate activity confined to Golgi area

Non T-cell acute leukemia-show negative

C2. Tartrate-Resistant Acid Phosphatase (TRAP)Substrate solution:

Acetate buffer same in #1-100mLNaphthol AS-BI phosphoric acid-10mg

N,N-dimethyl formamide-0.5mLStable at 4°-10°C for 2 monthsIncubation mixture-1 hour incubation1.0mg of fast garnet GBC dissolve in 10.0 mL of substrate

solution and 75.0 mg of L-(+)-tartaric acid. Adjust to pH 5.2.Filter before use and use immediatelyControl: normal blood filmInterpretation:

Neoplastic cells of hairy cell leukemia are strongly positiveHistiocytes-may have weak tartrate-resistant acid

phosphatase activity

C3. Leukocyte Alkaline PhosphataseReagents:

Fixative: formalin 40.0mLMethanol 360mLStore in freezer.

Tris buffer 0.2M pH9.1Trizma base 48.44mgDistilled H2O 200mLAdjust pH to 9.1 with 1N HCl

Substrate solution:Dissolve 0.6g of naphthol-AS-BI phosphate in 10mLN.N-dimethylformamide. Add 0.2M Tris buffer to 2L. Store in

refrigerator.

Working solution:50mg of fast blue BBN dissolve in 50mL of substrate, prepared fresh

and filter before use.

Procedure:Cold fixatives (4°-10°C)-30 secondsWorking solution-20 minutesNuclear fast red-counterstain-20 minutes

Control:Slides taken from pregnant women in their last trimester

Interpretation: Count 50 segmented neutrophilsScore staining reaction from 0-4+

0=no granules1+=very few granules2+=moderate # of granules scattered throughout the cell3+=numerous granules starting to coalesce

4+=cytoplasm packed with granules=