Neutrophilic Leukocyte Alkaline Phosphatase (NAP) Stain

Neutrophilic Leukocyte Alkaline Phosphatase (NAP) Stain
Neutrophilic Leukocyte Alkaline Phosphatase (NAP) Stain

Assay-kits

Neutrophilic Leukocyte Alkaline Phosphatase (NAP) Stain

  • Product code: ACMBS-50
  • 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

Neutrophilic-Leukocyte Alkaline Phosphatase stain is based on diazotization-coupling principle. At pH 9.2~9.8, Alkaline Phosphatase hydrolyzes Naphthol AS-BI Phosphoric Acid into α-naphthol. The stable diazonium salt and naphthol shall form insoluble colored precipitate in cytoplasm.

Main components

Reagent compositionMain ingredient
1. Fixative (solution A)Formaldehyde
2. FBB-alkaline solution (solution B)FBB salt
3. Sodium nitrite solution (solution C)Sodium nitrite
4. Naphthol AS-BI alkaline solution (solution D)Naphtoquine ΑS-B
5. Neutral red solution (solution E)Neutral red

Sample requirement

  • Fresh bone marrow cell smear and blood cell smear (Do not use anticoagulant blood!).

Test procedure

A. Working solution preparation

Dip stain working solution preparation (50ml volume dye vat: A dip stain working solution is 43ml, at the same time can place at least 8-10 smears.)

  • 1. Preparation of diazonium salt solution: Each of 1 ml of B and C solution thoroughly mixed and let stand for 2 minutes.
  • 2. Add 40 ml of distilled water in dye vat (prepare dye vat by yourself).
  • 3. The diazonium salt solution is poured into the dye vat, and mix. 4. Plus 1 ml of D solution 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 2.15ml.)

  • Apparatus required: disposable tube, micropipette, disposable tips, dropper
  • Instruction: Mix 50μl solution B with 50μl C, wait for 2 minutes. Add distilled water 2ml, and then add 50μl solution D. Mix thoroughly.

C. Staining procedure

  • 1. Dried smears then fixed with fixative (Solution A) (Return to room temperature before use and shake well) for 30 ~60 seconds, rinse with distilled water, wait until dry.
  • 2. Add or dip in the working solution for 15 minutes (If the room temperature is low in winter, incubate in 37℃ water bath), rinse with distilled water 10 to 30 seconds, and then dried.
  • 3. Counterstain with solution E (Be sure to shake well before use!) for 1~2 minutes. Rinse with distilled water. (The flush time should not be too long, otherwise the nuclear hypo chromatic.) Dry the smear for microscopic examination.
  • 4. Calculate points: Result of the reaction, reported with point value. Count 100 neutrophilic granulocytes and segmented neutrophils under oil lens. Record the individual points in the classification. The sum of all positive cell point is the total point value.

Result interpretation

NΑP positive granules are blue.

  • Interpretation standard:
  • (-)0 point: No positive granules are found in cytoplasm.
  • (+)1 point: Few or sporadic positive granules are found light blue in cytoplasm.
  • (++)2 points: Medium number of granules are in cytoplasm or blue color appears diffusely.
  • (+++)3 points: Many granules are in cytoplasm or dark blue color appears diffusely.
  • (++++)4 points: Full of large granules in cytoplasm or dark blue color appears diffusely.
  • Reference value:
  • Positive reaction is found in mature neutrophils (rod and segmented neutrophils). The NAP total point values of a healthy adult is between 35~100. It is suggested that each lab should establish its own reference value, since conditions among labs vary a lot.
ClassificationNeutrophil numbersTotal point values
(-)0 point120
(+)1 point1818
(++)2 points4080
(+++)3 points2266
(++++)4 points832
Total100 cells196 points

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. Return to room temperature before use and shake well (Recommend to store solution E at room temperature). Please thoroughly mixed B and C solution, all containers must be clean, normal working solution color is light yellow or golden yellow, if the working solution was light brown or dark brown, indicating that solution B and solution C is not fully mixed or reagent failure, resulting in dyeing failure.
  • 3. Should be used for fresh smear NΑP staining, placed too long will be the enzyme activity will be reduced, the use of anticoagulant smear positive results are unstable.
  • 4. The working solution should be used within 10 minutes.
  • 5. The need for infection with fever or normal peripheral blood smear as a positive control.
  • 6. The working solution after dyeing, the water rinse to be full, the film on the residual working fluid rinse, otherwise it will affect the solution E on the nucleus of the complex effect, solution E complex, with distilled water, wash the film can be washed Too long may lead to poor re-staining of the nucleus.
  • 7. Due to the working fluid on the smear has a certain effect of filming, may lead to smear adhesion is not strong, it is recommended to pay attention to the flow of water when the intensity, so as not to smear off.
  • 8. This kit should be used by professionals and the results of the interpretation.
  • 9. Before using the instructions should be read in detail, used within the validity period, and do personal hygiene protection.
  • 10. After the hospital or environmental protection departments should be used to dispose of waste.
  • 11. Production date, production lot number and expiration date see outside packaging.

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.

3. Reference standard: Histochemical Method for Leukocyte Alkaline Phosphatase H22-P, Vol 4, No. 14, National Committee for Clinical Laboratory Standards.

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