Naphthol AS-D Chloroacetate Esterase (AS–DCE) Stain

Assay-kits

Naphthol AS-D Chloroacetate Esterase (AS–DCE) Stain

Product code: ACMBS-44
Specifications: 5Tests, 20Tests, 100Tests (Box)
Intended use: This kit is for staining bone marrow cell and blood cell smear
Store temperature: 2℃～8℃
Expiry date: 12 Months

More Details

PrincipleMain componentsSample requirementTest procedureResult interpretationMethod limitationPrecautions

Principle

Naphthol AS-D chloroacetate can be hydrolyzed into Naphthol AS-D by Esterase to mix with diazonium salt and form insoluble red-brown precipitate in cytoplasm. Positive reaction is only found in granulocytes, hence, it is also called "Specific Esterase Stain".

Main components

Reagent composition	Main ingredient
1. Fixative (solution A)	Formaldehyde
2. Pararosaniline solution (solution B)	Parafuchsin
3. Sodium nitrite solution (solution C)	Sodium nitrite
4. Phosphate buffer (solution D)	Phosphate
5. Naphthol As-D chloroacetate colution (solution E)	Naphthol As-D chloroacetate
6. Methyl green solution (solution F)	Methyl green

Sample requirement

Fresh bone marrow cell smear and blood cell smear.

Test procedure

A. Working solution preparation

Dip stain working solution preparation (50ml volume dye vat: A dip stain working solution is 42.8ml, at the same time can place at least 8～10 smears.)
1. Preparation of diazonium salt solution: Each of 0.4 ml of solution B and solution C thoroughly mixed and let stand for 2 minutes.
2. Add 40 ml of solution D in dye vat (prepare dye vat by yourself).
3. The diazonium salt solution is poured into the dye vat, and mix.
4. Plus 2 ml of solution E into the dye vat, mix gently.

※ Note: The above is the preparation amount of the dip staining working solution for the 50ml volume dyeing vat. Each laboratory can scale up or down according to the volume of its own dyeing vat.

B. Drops stain working solution preparation (A drops stain working solution is 2.14ml)

Apparatus required: disposable plastic tube, micropipette, disposable tips, dropper
Instruction: Mix 20μl solution B and 20μl solution C thoroughly, wait a minute. Add 2ml solution D and 100μl solution E. Mix well.

※ Note: Inside package of 100 tests kit, the 20ml solution D (concentrate buffer) must be diluted to 200ml buffer solution by Distilled water, then can be used.

C. Staining procedure:

1. Dried smears then fixed with fixative (Solution A) (Return to room temperature before use and shake well) for 30 to 60 seconds, rinse with distilled water, Air dry or dry with absorbent paper.
2. Add or dip working solution at room temperature to stain for 15～20 minutes. At lower room temperature in winter, incubate in a 37 ℃ water bath. Rinse with distilled water and air dry.
3. Counterstain with solution F for 1～2 minutes. Rinse with distilled water. Dry the smear for microscopic examination.

Result interpretation

Red granules found in cytoplasm means positive.

Reference range:

1. Granule system is in positive reaction, except myeloblasts.
2. Mast cells are strongly positive reaction.
3. Other cell systems are in negative reaction.

Method limitation

Only use for morphological staining to observe.

Precautions

1. 5 Tests only use on drops stain. 20 Tests and 100 Tests can be used for drip staining and dip staining.
2. The methodology is the ICSH recommendation method.
3. Return to room temperature before use and shake well (Recommend to store solution F at room temperature) Please thoroughly mixed solution B and solution C, all containers must be clean. Working solution color is pale rose color, if it is deep red, then meaning solution B, solution C does not mix thoroughly.
4. Solution E must avoid exposure to direct sunlight.
5. Prepare working solution prior to use, within 10 minutes to use, so as not to affect the dyeing effect. Use fresh working solution to obtain good staining results.
6. Tightly cap the reagent bottle immediately after use to avoid evaporation.
7. This kit must be used by professionals and the interpretation of results.
8. Should carefully read the instruction manual before using. Can only be used before expiration date, and good personal hygiene protection.
9. After use, disposal of waste should comply with the hospital or the environmental protection department requirements.
10. Production lot number and expiration date on the package.

References

1. National Health and Family Planning Commission of the People's Republic of China Medical Administration Hospital Authority. National Clinical Laboratory Procedures [M]. 4th ed. Beijing: People's Medical Publishing House, 2014.

2. Gradwohl's Clinical Laboratory Methods And Diagnosis, 8th, ed.

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