Acid Phosphatase-387

Reagent Preparation

Reagents are provided ready for use. Prepare Fixative solution by combining 25 mL. Citrate solution, to 65 mL acetone, and 8 mL. formaldehyde. Place in a glass bottle and seal tightly. Prewarm sufficient deionized water at 37 ºC.

Procedure

  1. Fix slides in room temperature Fixative solution for 30 seconds. Rinse thoroughly in deionized water.
  2. To each of 2 test tubes add 0.5 mL Fast Garnet GBC Base solution and 0.5 mL Sodium Nitrite solution.
  3. Mix by gentle inversion for 30 seconds. Let stand for 2 minutes.
  4. Label two 100 mL beakers A and B and add the following while mixing.
  A B
Water (prewarmed to 37 ºC) 45 mL 45 mL
Diazotized Fast Garnet GBC sol. 1.0 mL 1.0 mL
Naphthol AS-BI Phos. sol. 0.5 mL 0.5 mL
Acetate solution 2.0 mL 2.0 mL
Tartrate solution 1.0 mL
  1. Transfer solutions to labeled Coplin jars and warm solutions in a water bath to 37 ºC before adding slides.
  2. Add slides and incubate for 1 hour at 37 ºC in the dark.
  3. Rinse thoroughly in deionized water.
  4. Counterstain in Gill’s 3 Hematoxylin solution (GHS-3) for 2 minutes.
  5. Rinse several minutes in tap water and allow to air dry.

Results

Stained blood films are evaluated based on the presence or absence of tartrate-resistant enzyme. In the absence of tartaric acid, most leukocytes demonstrate granular sites of staining. In the presence of tartaric acid, an occasional granule may be observed.

Acid Phosphatase-387

Reagents

     
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