Acid Alpha-Naphthyl Acetate Esterase Stain (ANAE)

Assay-kits

Acid Alpha-Naphthyl Acetate Esterase Stain (ANAE)

Product code: ACMBS-45
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 componentsTest procedureExpected resultReference rangePrecautions

Principle

Hydrochloric acid parafuchsin reacts with Sodium Nitrite to form diazonium salt (six-azo-pararosaniline). α-Naphthyl Acetate is decomposed by esterase and produces α-naphthol, which shall combine with diazonium salt to form red brown precipitate in cytoplasm. This stain has no specificity against esterase, therefore, it is called "Non-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. α-Naphthyl acetate solution (solution E)	α-Naphthyl acetate
6. Methyl green solution (solution F)	Methyl green
7. NaF solution	NaF

Test procedure

A. Working solution preparation:

1. Dip staining working solution preparation (Amount of dip staining working solution volume is 33ml, it can put 8～10 smears into dip staining tank.)
①. Diazonium salt solution preparation: Mix well 1ml solution B and 1ml solution C, wait 2 minutes.
②. Add 30ml solution D in dye vat.
③. Poured the Diazonium salt solution into tank, mix well.
④. Then add 1ml solution E into tank, mix gently. (Add 1ml NaF solution for NaF inhibition test.)

	Solution B	Solution C	Solution D	Solution E	NaF
Dye vat A	1ml	1ml	30ml	1ml	---
Dye vat B (Inhibition test)	1ml	1ml	30ml	1ml	1ml

2. Drop staining working solution preparation (Amount of drop staining working solution volume is 1.65ml) Apparatus required: disposable tube, micropipette, disposable tips, dropper Instruction: Add 50μl solution B and solution C in a test tube, mix well and wait for 1 minute. Then, add 1.5ml solution D and 50μl solution E, mix well and wait for 2 minutes. (NaF inhibition test: Add 1 drop of solution NaF in working solution.)

	Solution B	Solution C	Solution D	Solution E	Solution NaF
Tube Α	50μl	50μl	1.5ml	50μl	---
Tube B (inhibition test)	50μl	50μl	1.5ml	50μl	1 drop

※Note: When use the 100 Tests please diluted 15ml solution D (concentrated buffer solution) into 150ml buffer solution by using Distill Water.

B. Procedure:

1. Dry cell smear, directly add working solution to run step 2 or fix smear with solution A for 30～60 seconds. Rinse with distilled water. Air dry or dry with absorbent paper.

2. Drop working solution on slide to fully cover the smear. Incubate at 37℃ for 30 minutes. Rinse with distilled water. Air dry or dry with absorbent paper.

3. Redye with solution F for 1～2 minutes. Rinse with distilled water. Air dry the smear for microscopic examination.

Expected result

Red or red-brown granules found in cytoplasm are positive.

Dot type: It is mainly seen in mature T Lymphocytes. 1～4 brown or red-brown circular, mass, and big spot granules with clear contour in cytoplasm are found in positive reaction.
Diffuse type: Red-brown and dusty granules diffusely spread are found in positive reaction. Granules may appear at a certain part of cell with obscure outline in cytoplasm.
Monocyte type: Evenly stained red-brown granules diffusely spread in cytoplasm are found in positive reaction.

Reference range

1. Monocytes system: Appeared strong positive reaction, the reaction can inhibit by NaF.
2. Granulocyte system: Every stage of Granulocytes appeared negative reaction, sometimes few Granulocytes appeared weak positive reaction, its reaction can't inhibit by NaF.
3. Megakaryocytes and Platelets appeared positive.
4. The mature T Lymphocytes appeared dot positive, B Lymphocytes and plasmacytes appeared negative reaction.
5. The Monocyte-derived Histiocytes and Macrophages appeared strong positive reaction, Gaucher cells and Sea blue histiocytes appeared positive.
6. Erythroblast appeared negative.

Precautions

1. 6 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. Tightly cap the reagent bottle immediately after use to avoid evaporation.
4. Return to room temperature before use and shake well, solution B, C should be fully mixed. All container must be clean, the normal working solution color was opaque milky yellow, if the working solution was clear and not cloudy , indicating that solution B, C is not fully mixed, which will cause dyeing failure.
5. The working solution should be prepared before use for the sake of freshness, and should be used to dye fresh cell smear in not more than 10 minutes.
6. α–Naphthyl Acetate Esterase (A–NAE) Stain kit is only used for clinical and research lab Interpretation of staining results should be made by well-trained technicians and pathologists.
7. Before use, please read instruction sheets and labels of reagents carefully. Do not use the reagents beyond the stated expiration date.
8. Wastes should be disposed with care as biohazardous materials by following local EPA guidelines.

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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