α-Naphthyl Butyrate Esterase Stain (α-NBE)

Alpha-Naphthyl Butyrate Esterase Stain (Alpha-NBE)

Assay-kits

α-Naphthyl Butyrate Esterase Stain (α-NBE)

Product code: ACMBS-47
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

Under alkaline condition, α-Naphthyl Butyrate is hydrolyzed into α-naphthol by Esterase. α-naphthol shall combine with diazonium salt to form insoluble colored 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. Diazotization solution (solution B)	Parafuchsin
3. Sodium nitrite solution (solution C)	Sodium nitrite
4. Phosphate buffer (solution D)	Phosphate
5. α- Naphthyl butyrate solution (solution E)	α-Naphthyl butyrate
6. Methyl green solution (solution F)	Methyl green
7. NaF solution	NaF

Sample requirement

Fresh bone marrow cell smear and blood cell smear.

Test procedure

A. Working solution preparation:

1. Dip stain working solution preparation

(50ml volume dye vat: A dip stain working solution is 42.2 ml, at the same time can place at least 8-10 smears.)

①. Preparation of diazonium salt solution: Each of 0.1ml solution B and 0.1 ml 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 well.

④. Plus 2 ml of solution E into the dye vat, mix gently. (Add 1.3ml NaF solution for NaF inhibition test.)

Working solution preparation	Solution B	Solution C	Solution D	Solution E	NaF solution
Dye vat Α	0.1ml	0.1ml	40ml	2ml	---
Dye vat B (Inhibition test)	0.1ml	0.1ml	40ml	2ml	1.3ml

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

2. Drops stain working solution preparation (A drops stain working solution is 2ml.)

①. Apparatus required: disposable plastic tube, micropipette, disposable tips, dropper

②. Instruction: Add 5μl solution B and 5μl solution C in a test tube, mix well and wait for 1 minute. Then, add 2ml solution D and 100μl solution E , and mix well (NaF inhibition test: Add 1 drop of solution NaF in working solution.)

Working solution preparation	Solution B	Solution C	Solution D	Solution E	NaF solution
Tube Α	5μl	5μl	2ml	100μl	---
Tube B (Inhibition test)	5μl	5μl	2ml	100μl	1 drop

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

B. Staining procedure:

1. Dry cell smear and fix it in solution A (Return to room temperature before use and shake well) for 30 to 60 seconds. Rinse with distilled water. Dry with the filter paper or air dry.
2. Add or dip working solution on slide to fully cover the smear at room temperature for 60 minutes. (At lower temperature in winter, incubate at 37℃). Rinse with distilled water. Dry with the filter paper or air dry.
3. Restain with solution F for 1～2 minutes. Rinse with distilled water. Air dry the smear for microscopic examination.

Result interpretation

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

Reference range:

1. Monocyte System: are in positive reaction. The reaction can be inhibited by sodium fluoride.
2. Granulocyte System: Granulocytes at all stages are in negative reaction.
3. Mature T Lymphocytes are present with punctuated positive granule. B Lymphocytes and plasmacyte are in negative reaction.
4. Megakaryocytes and platelets are negative.
5. Histiocyte derived from monocyte and macrophages are in stronger positive reaction. Sea-blue histiocytic and Pseudo-Gaucher cells are in positive reaction.

Method limitation

Only for morphological staining observe used.

Precautions

1. 6 Tests only use on drops stain. 20 Tests and 100 Tests can be used for drip staining and dip staining.
2. 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. the normal working solution color was opaque milky white, if the working solution was clear and not cloudy , indicating that solution B, C is not fully mixed, which will cause staining failure.
3. Use fresh specimen, Otherwise, the positive rate may decrease or false negative results may occur.
4. The working solution should be prepared before use for the sake of freshness, and should be used to stainfresh cell smear in not more than 10 minutes.
5. Tightly cap the reagent bottle immediately after use to avoid evaporation.
6. This kit must be used by professionals and the interpretation of results.
7. Should carefully read the instruction manual before using. Can only be used before expiration date, and good personal hygiene protection.
8. Production lot number and expiration date on the package.
9. After use, disposal of waste should comply with the hospital or the environmental protection department requirements.

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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