8/8/2019 Cytochemistry table-Enzymatic Techniques

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Enzymatic Techniques

Peroxidases A H2+ H2O2→ A+2H2O B. Esterases 

Diaminobenzidine Method Cyanide-Resistant Peroxidase Stain Naphthol AS-D Chloroacetat e EsteraseΑ-Naphthyl Acetate E

(ANAE)

eagents :xative-buffered formalin-acetone

Hexazotized new fuchsin: mix equal volumeof new fuchsin&4% Na nitri te for 1min

before use

Napththol AS-D chloroacetate sol’n:10mg Naphthol AS-D chloroacetate+ 5mLN,N-dimethylformamide

Hexazoti zed pararosaMix equal vol of the 1

sol’ns for 1min before

cubation Mixture :

repare fresh for each batch)

osphate buffer 0.07M, pH 7.4-50mL3 DAB t etrahydrochloride-37.5mg

% H2O2 emsa 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 mLfresh hexazotized new fuchsin 0.2m L

Naphthol AS-D chloroacetate sol 2mL

PO4 buffer, 0.07M pH

Α-naphthyl acetate s100mg+ 5mL ethylen

monomethyl ether.

Refrigerat e before us

ontrols :

ood film from normal donorcontrol-neutrophils

control-lymphocytes

normal blood buffy coat

strongly+ -eosinophils (brown)-  → neutrophils 

+ : normal neutrophils + : normal monocyte

histiocytes in BM or bfilm prep

terpretation :

ark brown granules in the cytoplasmgranulocytes&monocytes

onocytes: weak to moderate

anulocytes: are packed with +

anules

BCs: stain diffusely brow n (cuz of

eudoperoxidase activity in Hb)

Uses:

1.   identification of the Eosinophiliccomponent of acute myeloid&acute

myelomonocytic l eukemias2.   aid in recognition of de novo acute

eosinophilic leukemia

bright red granules in mast cells,

neutrophils&neutrophil precursors in theircytoplasm

Uses:1.   useful in demonstrating myeloid

elements on paraffin-embeddedsections such as grnul ocytic sarcoma

2.   systemic mast cell dss

Monocytes: stain red

Lymphocytes: dotlik e

ontinuation of Esterases C. Phosphatases 

Α -Naphthyl Butyrate Esterase (BE) Combination of α-naphthyl butyrate-chloroacetate Esterase Acid Phosphatase

Tartrate-ResistantPhosphatase (TR

eagents : Fixative: methanol-acetone mixt ure-30secs

Acetate buffer

Substrate sol’n

Naphthol AS-BI phosphoric acid 100m gN,N-dimethyl formamide 10mg

Stable for 2mos at 4°-10°C 

Substrate sol’n:

Acetate buffer same

100mLNaphthol AS-BI ph

acid 10mgN,N-dimethyl form

0.5mL

Stable at 4°-10°C fo

cubation Mixture :

mins at RT

07M PO4 buffer pH 6.69 38mL

sh hexazotized pararosanilin 0.4m L

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 buffer0.5mL substrate5mg f ast garnet GBC salt

Mayer’s hematoxylin-counterstain-5-20m in.

Mounting medium: Glycerin jelly

1 hr incubation

1mg fast garnet GBCsubstrate sol’n and 7L-(+ )- tart aric acid.

Adjust to pH 5.2

Filter before use&use

immediately. 

ontrols :me w/ ANAE

  normal monocytes&neutrophils i nblood film

  histiocytes or developing neutrophilsin BM

peripheral blood film normal blood film

terpretation :

zyme activity is noted as dark redts in the cytoplasm of

onocytes&histiocytes

cytoplasm of monocytes&histiocytes-dark

red ppt

neutrophils-blue granules

discrete purplish to dark red granules

T-cell acute lymphoblastic leukemia-moderate activity confined to Golgi area

Non T-cell acute leukemi a-show negative

Neoplastic hairy cell

leukemia: strongly +

Histiocytes: weak t ar

resistant acid phosphactivity

Store at 4°-10°C. Stable for 1mo. 

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