α–Naphthyl Acetate Esterase Stain (α-NAE) (Fast Blue Method)

Alpha–Naphthyl Acetate Esterase Stain (Alpha-NAE) (Fast Blue Method)

Assay-kits

α–Naphthyl Acetate Esterase Stain (α-NAE) (Fast Blue Method)

Product code: ACMBS-46
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

α-Naphthyl Acetate is decomposed by esterase and produces α-naphthol, which shall combine with diazonium salt to form dark brown precipitate in cytoplasm. α -NAE is present in monocytes, granulocytes and lymphocytes. It is a kind of neutral non-specific esterase. Therefore, it is also called a nonspecific esterase stain. The positive of mononuclear cells can be inhibited by sodium fluoride, so it is usually used as sodium fluoride inhibition test as the same time of α-NAE staining.

Main components

Reagent composition	Main ingredient
1. Fixative (Solution A)	Formaldehyde
2. α-Naphthyl acetate solution (Solution B)	α-Naphthyl acetate
3. Buffer solution (Solution C)	Phosphate
4. Fast blue powder (Reagent D)	Fast blue
5. Hematoxylin solution (Solution E)	Hematoxylin
6. NaF solution	NaF

Sample requirement

Fresh bone marrow cell smear and blood cell smear.

Test procedure

A. Working solution preparation:

6 Tests and 20 Tests kit working solution preparation (Amount of drop stain working solution volume is 2.05ml for 1 smear.)

1. Apparatus required: micropipette, disposable tips, dropper.
2. Operation: Add 50μl solution B and 2ml solution C into 1 bottle (0.002g/bottle) reagent D, Tighten the cap and shake well for 2 minutes, so that the reagent D is fully dissolved, stand for about 1 minute, ready to stain (NaF inhibition test: add NaF solution of 1 drops).

Working solution preparation	Solution B	Solution C	Solution D	Mix well	NaF solution	Remark
Tube Α	50μl	2ml	1 bottle (0.002g/bottle)	Shake for 2minutes, the Reagent D is fully dissolved, and then standing for 1 minute	---	Staining 1 smear
Tube B (Inhibition test)	50μl	2ml	1 bottle (0.002g/bottle)		1 drop	Staining 1 smear

100 Tests kit working solution preparation (Amount of dip stain working solution volume is 10.25ml, can do dip stain 5 smears.)

①. Inside package of 100 Tests kit, the 25ml solution C (concentrate buffer) must be diluted to 250ml buffer solution by distilled water, then can be used.

②. Apparatus required: Clean staining jar with cover, micropipette, disposable tips, dropper.

③. Operation: Add 250μl solution B and 10ml solution C (After diluted) in staining tank, then mix well. Aspirate 1～2 ml mixed solution and add into 1 bottle (0.01g/bottle) reagent D. Tighten the cap and shake well wait for 2 minutes, so that the reagent D is fully dissolved. Then all the reagent D added in the staining tank, mix well andthen stand for about 1 minute, ready to stain (NaF inhibition test: add 250μl NaF solution).

Working solution preparation	Solution B	Solution C (Diluted)	Reagent D	NaF solution	Remark
Staining tank Α	250μl	10ml	1 bottle (0.01g/bottle)	---	On the left is provided in preparation dip stain working solution for 20ml staining tank. Each laboratory according to their staining jar size, by this ratio to geometric expansion or reduction dip working solution preparation quantity.
Staining tank B (Inhibition test)	250μl	10ml	1 bottle (0.01g/bottle)	250μl

B. Staining procedure:

1. Dry the smear. Drop solution A(Return to room temperature before use and shake well) on slide to fully cover the smear fixed for 1～3 minutes, and then rinse with distilled water. Air dry or dry with filter paper.
2. Add or dip working solution on slide to fully cover the smear. Incubate at 37℃ for 60 minutes. Rinse with distilled water. Air dry or dry with filter paper.
3. Counterstain with solution E for 3 minutes. Rinse with distilled water. Air Dry the smear for microscopic examination.

Result interpretation

In the cytoplasm has dark gray or brownish black diffuse or granular precipitation as positive, the nuclei were indigo.

Reference range:

In normal cells, monocytes showed diffuse flocculent positive. After adding sodium fluoride, the positive esterase was inhibited. Granulocyte system, megakaryocytes and lymphocytes showed fine granular positive, was not inhibited by sodium fluoride.

Method limitation

Only for morphological staining observe used.

Precautions

1. 6 Tests and 20 Tests only use on drops stain. 100 Tests is suitable for more than 5 slides of dip or drops staining.
2. Return to room temperature before use and shake well. All containers must be clean.
3. Prepare working solution prior to use. For more than 5 minutes not use, otherwise will gradually lose staining power and influence staining effect. Use fresh smear to obtain good staining results.
4. The color of the normal working fluid is yellow. Precipitation or browning of the color indicates that the working fluid is not fresh. It must be re-formed after investigation.
5. Kit after use, should promptly put back 2℃～8℃ low temperature storage, so as not to affect the next use effect.
6. This kit must be used by professionals and the interpretation of results.
7. Should carefully read the instruction manual before using, and good personal hygiene protection.
8. After use, disposal of waste should comply with the hospital or the environmental protection department requirements.
9. Production lot number and expiration date on the package.

References

1. The PRC Ministry of Health Medical Administrative Department. National Clinical Laboratory Procedures (M) version 4. Beijing: People's Medical Publishing House, 2014.

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

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