Neutrophil alkaline phosphatase (NAP) stain stands as a valuable diagnostic tool, offering insights into the functionality of neutrophils, a critical component of the immune system. This enzymatic stain specifically targets alkaline phosphatase, an enzyme abundantly present in the cytoplasm of mature neutrophils. The intensity of the stain reflects the level of NAP activity, providing a measure of neutrophil maturity and overall health.
NAP staining serves as a diagnostic aid in various hematological conditions, particularly those involving neutrophil dysfunction. It plays a crucial role in differentiating between reactive neutrophilia, a normal response to infection or inflammation, or chronic myeloid leukemia which has a higher number of immature neutrophils or blast cells.
Principle of NAP stain
Neutrophil alkaline phosphatase (NAP) stain relies on the principle of diazotization and coupling reactions to visualize the activity of alkaline phosphatase (ALP), an enzyme abundant in mature neutrophils. The intensity of the stain reflects the level of ALP activity, providing a measure of neutrophil maturity and overall health.
The staining procedure begins with the application of a diazonium salt solution to blood smears. Diazonium salts are highly reactive compounds that readily form azo dyes when coupled with aromatic compounds. In this case, the diazonium salt reacts with naphthol AS-BI phosphoric acid, a substrate for ALP.
ALP, when present in the cytoplasm of NAP-positive neutrophils, catalyzes the hydrolysis of naphthol AS-BI phosphoric acid, releasing naphthol. This naphthol then reacts with the diazonium salt, forming an insoluble blue-violet or reddish brown precipitate (depending on the dye) used within the neutrophil cytoplasm.
The intensity of the stain depends on the amount of ALP present in the neutrophil. Mature neutrophils, with their high ALP content, exhibit strong staining, while immature neutrophils, with lower ALP activity, show weaker staining. This differential staining pattern allows for the assessment of neutrophil maturity and the identification of potential abnormalities in neutrophil function.
Method differs slightly according to the manufacturer’s protocol.
- Fresh unstained peripheral blood smear slide, a positive control slide from a hamster and a negative control slide from mice
- Freshly prepared fixative: 1:9 ration of 40% formaldehyde: absolute methanol
- Naphthol AS phosphate
- 0.2 mol/L Tris buffer pH 9.0
- Fast Blue BB salt (coupling azo dye)
- Neutral red, 0.02% aqueous solution (counterstain)
- Filter paper
- Prepare stock substrate solution: Dissolve 30 mg naphthol AS phosphate in 0.5 mL N,N-dimethylformamide and add 100 mL 0.2 mol/L Tris buffer.
- Prepare working buffered substrate solution: Warm 40 mL of stock substrate solution to room temperature and add 24 mg of Fast Blue BB salt. Mix well until the salt is fully dissolved. Filter the mixture using a filter paper and funnel before use.
- Fix all the blood smear slides in the freshly prepared fixative.
- Rinse the slide with slow running tap water for 2 – 3 minutes.
- Incubate the slides in working buffered substrate solution for 15 minutes.
- Rinse the slide with slow running tap water for 1 – 2 minutes.
- Air dry the slides.
- Incubate the slides in counterstain for 3 minutes.
- Rinse the slides briefly and allow to air dry.
- Mount the slide with Depex and cover the zone of morphology with a cover slip.
- This slide is now ready for viewing.
NAP scores are used to grade the intensity of the NAP stain on a scale of 0 to 4+. A NAP score of 0 indicates no staining, while a NAP score of 4+ indicates the strongest staining. NAP scores are interpreted in the context of the patient’s clinical presentation and other laboratory findings. The NAP score has a possible scoring of 0 – 400.
In general, a NAP score of 3+ or 4+ is considered normal, indicating that the neutrophils are mature and functioning normally. A NAP score of 2+ or less may suggest impaired neutrophil maturation or function. This could be due to a variety of factors, including chronic myeloid leukemia (CML).
However, it is important to note that NAP scores should not be interpreted in isolation. The presence of immature neutrophils, as indicated by a low NAP score, must be considered in conjunction with other laboratory findings, such as a complete blood count (CBC) and a bone marrow biopsy, to make an accurate diagnosis.
Presence of alkaline phosphatase is based on the precipitates of blue or red granules in the neutrophils. A positive control (hamster) and a negative control (mice) is used as comparison for the measurements of the NAP score. Assessment of NAP scoring is as follows:
- Count 100 neutrophils (including band forms)
- Rate the NAP score for each neutrophil as below and add the score for 100 neutrophils.
|0||Absence of stained granules||Negative|
|1||Occasional stained granules in the cytoplasm||Positive|
|2||Moderate number of stained granules||Positive|
|3||Numerous stained granules in the cytoplasm||Strong positive|
|4||Numerous stained granules crowding and overlying the nucleus in the cytoplasm||Heavy positivity|
3. Normal range: 40 – 100.
4. A high score may indicate a leukemoid reaction for e.g. severe infections such as sepsis and pneumonia and tissue necrosis such as from burns or pancreatitis.