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A. Peroxidases Diaminobenzidine Method A H2+H2O2→ A+2H2O Cyanide-Resistant Peroxidase Stain B. Esterases Naphthol AS-D Chloroacetate Esterase Hexazotized new fuchsin: mix equal volume of new fuchsin&4% Na nitrite for 1min before use Napththol AS-D chloroacetate sol’n: 10mg Naphthol AS-D chloroacetate+5mL N,N-dimethylformamide Α-Naphthyl Acetate Esterase (ANAE) Hexazotized pararosanilin: Mix equal vol of the 1st 2 sol’ns for 1min before use Store at 4°-10°C. Stable for 1mo.
Reagents : Fixative-buffered formalin-acetone
Incubation Mixture: (prepare fresh for each batch) Phosphate buffer 0.07M, pH 7.4-50mL 3,3 DAB tetrahydrochloride-37.5mg 3% H2O2 Giemsa stain, 10.0 mL
same w/ DAB method in addition: 4.9mg NaCN (sodium cyanide) is added and titrate to pH 7.4 with 1N HCl
PO4 buffer 0.07M, pH 7.73 Mayer’s hematoxylin-10min. Counterstain. Incubate films at RT for 10mins using: 0.07M PO4 buffer 38 mL fresh hexazotized new fuchsin 0.2mL Naphthol AS-D chloroacetate sol 2mL
PO4 buffer, 0.07M pH 6.64 Α-naphthyl acetate sol’n: 100mg+5mL ethylene glycol monomethyl ether. Refrigerate before use.
Controls : blood film from normal donor + control-neutrophils - control-lymphocytes Interpretation: Dark brown granules in the cytoplasm of granulocytes&monocytes
Monocytes: weak to moderate Granulocytes: are packed with + granules RBCs: stain diffusely brown (cuz of pseudoperoxidase activity in Hb)
normal blood buffy coat strongly+-eosinophils (brown) - → neutrophils
+: normal neutrophils
+: normal monocytes or histiocytes in BM or blood film prep
bright red granules in mast cells, neutrophils&neutrophil precursors in their cytoplasm Uses: 1. identification of the Eosinophilic component of acute myeloid´ myelomonocytic leukemias 2. aid in recognition of de novo acute eosinophilic leukemia
Monocytes: stain red-brown Lymphocytes: dotlike
Uses: 1. useful in demonstrating myeloid elements on paraffin-embedded sections such as grnulocytic sarcoma 2. systemic mast cell dss
Continuation of Esterases
Α-Naphthyl Butyrate Esterase (BE) Combination of α-naphthyl butyratechloroacetate Esterase
C. Phosphatases Acid Phosphatase Fixative: methanol-acetone mixture-30secs Acetate buffer Substrate sol’n Naphthol AS-BI phosphoric acid 100mg N,N-dimethyl formamide 10mg Tartrate-Resistant Acid Phosphatase (TRAP) Substrate sol’n: Acetate buffer same in AP 100mL Naphthol AS-BI phosphoric acid 10mg N,N-dimethyl formamide 0.5mL
Stable for 2mos at 4°-10°C Stable at 4°-10°C for 2mos
Incubation Mixture: 45mins at RT
0.07M PO4 buffer pH 6.69 38mL fresh hexazotized pararosanilin 0.4mL α-naphthyl butyrate sol’n 2mL
BE incubation mixture at RT for 3omins CE inc mixture at RT for 5mins
45 min.-inc mixture: 50mL acetate buffer 0.5mL substrate 5mg fast garnet GBC salt Mayer’s hematoxylin-counterstain-5-20min. Mounting medium: Glycerin jelly
1 hr incubation 1mg fast garnet GBC+1mL substrate sol’n and 75mg of L-(+)- tartaric acid. Adjust to pH 5.2 Filter before use&use immediately.
Controls : same w/ ANAE
normal monocytes&neutrophils in blood film histiocytes or developing neutrophils in BM
peripheral blood film
normal blood film
Interpretation: Enzyme activity is noted as dark red ppts in the cytoplasm of monocytes&histiocytes
cytoplasm of monocytes&histiocytes-dark red ppt neutrophils-blue granules
discrete purplish to dark red granules T-cell acute lymphoblastic leukemiamoderate activity confined to Golgi area Non T-cell acute leukemia-show negative
Neoplastic hairy cell leukemia: strongly + Histiocytes: weak tartrateresistant acid phosphatase activity