Intended use
NAP score is used to distinguish Chronic Granulocytic Leukaemia from other myeloproliferative disorders and Polycythaemia Rubra Vera from secondary polycythaemia.
NAP score is used to distinguish Chronic Granulocytic Leukaemia from other myeloproliferative disorders and Polycythaemia Rubra Vera from secondary polycythaemia.
Principle
At an alkaline pH, NAP hydrolyses substrate naphthol AS-BI phosphate to liberate napthol-AS. The liberated naphthol couples with a diazonium salt, Fast Blue BB to produce an insoluble complex which precipitates at the site of enzyme activity. The sites of alkaline phosphatase activity will appear as blue granules. The NAP score is the sum of the rating of 100 consecutive segmented and band neutrophils using a scale of 0 to 4+ according to the appearance and intensity of the precipitated dye.
Specimen collection and handling conditions
1. Fresh capillary/venous blood preparation, not anticoagulated is required.
2. EDTA blood is not recommended as enzyme activity is inhibited.
2. EDTA blood is not recommended as enzyme activity is inhibited.
Preparation of stock substrate solution
a) Dissolve 30mg naphthol AS phosphate in 0.5ml N,N-dimethylformamide. CAUTION: N,N-dimethylformamide is toxic by inhalation. To be prepared in fume hood.
b) Add 0.2M tris buffer, pH 9.1 to bring volume to 100ml.
b) Add 0.2M tris buffer, pH 9.1 to bring volume to 100ml.
c) Store at 4-10 deg celcius. Stable for 2 months.
Preparation of working substrate
a) Add 10mg Fast Blue BB to 10ml stock substrate solution. Mix well. CAUTION: Fast Blue BB is carcinogenic and a possible mutagen. To be prepared in fume hood.
b) Prepare fresh when needed.
Quality control
A normal control smear must be included with every batch of stain and results should fall within tolerance limits.
Procedures
1. Fix air-dried smear in buffered 60% Acetone in Citrate brought to room temperature for at least 5 mins.
2. Rinse with tap water and dry slide.
3. Using filter paper No.41, filter the freshly prepared working substrate directly onto slides. Stain for 15 mins at room temperature. Discard working solution into the toxic waste container.
4. Wash in tap water.
5. Counterstain with 0.5% neutral red for 1 min.
6. Wash and dry.
7. Count under oil immersion 100 neutrophils. The slides are examined and counted by 2 technologists and an average of 2 results is reported.
Interpretation
Sites of enzyme activity are represented by discrete bright blue granules of varying sizes. Nucleus is stained red. The enzyme is present predominantly in segmented neutrophils. Cytoplasmic granules of eosinophils do not stain while basophils cannot be differentiated from other granulocytes.
One hundred segmented or band forms of neutrophils are rated. The sum of 100 cell ratings give a score with a possible range from 0-400.Normal range: 30-100
Clinical significance
Chronic Granulocytic Leukaemia and Paroxysmal Nocturnal Haemoglobinuria are associated with abnormally low or absent staining. Conversely, leukaemoid reactions, polycythaemia vera and myelofibrosis are associated with markedly elevated NAP scores. High scores can also be obtained from neonates, pregnant women or women taking oral contraceptives.
Notes
1. Do not make the smear too thick to avoid inadequate fixation.
2. Fixed smears should be stored at -20 deg celsius if staining delayed for >5-6 hours.
3. Scoring of enzyme activity should be made in areas of slide with optimal cell morphology.
Answers to possible questions
Why must the working solution be prepared fresh before every staining?
Precipitation will occur over time once the Fast Blue BB is added to the stock substrate solution that will be indicated by a colour change from bright yellow to dull brown. Therefore it is important that the working solution be mixed and applied fast.
What are the various illnesses stated?
Please visit Wikipedia or other search engines to administer your enquiries. I'm sure you will learn more than what I'll be able to explain this way. :)
I've also taken some pictures and will seek to post them as soon as possible once I get relevant approval. Hope you've learnt something!
13 comments:
Hi
Will there be any interference from any external factors resulting in inaccurate result beside enzyme activity and how do you go about avoiding such event
Ching Wei
hi alex
why is high NAP scores usually obtained from neonates, pregnant women or women taking oral contraceptives?
-Sharon Ang
0503219H
hello alex,
the med tech has to rate each neutrophil according to size and intensity? so does it take very long for them to examine the slides?
Shu Hui
TG02
Hi alex
So, NAP score is not a very specific test. Normally what type of follow-up test will be perform if patient is suspected to have CGL/polycythaemia?
Chaur Lee
Hello Ching Wei,
Basically the rating of scores is subjective in this test, what you may rate as 3+ might be rated 4+ by someone else. I would say inexperience might lead to variation of results and that is why the reported result will always be an average of 2 results by experienced technologists. Another point to take note will be the fast and efficient mixing of the working substrate at the point of adding Fast Blue BB since precipitation will start occurring. Apply the working substrate to the smears FAST.
Hello Sharon,
Score will be moderately elevated in pregnancy, with oestrogen therapy (eg. oral contraception) and corticosteroid therapy. This may be due to the effects of oestrogen and corticosteroid on NAP causing high NAP(enzyme) activity. The enzyme activity is also high in neonates.
Hello Shu Hui,
Yes the technologist has to rate the neutrophil in terms of granular size and staining intensity. The table serves as a guide for cell rating. In the hands of experienced technologists, it usually won't take long for them screen, rate and come up with an average of 2 scores.
Hello Chaur Lee,
For CGL, the consultant might request for bcr-abl molecular studies, which is a sensitive marker for the disease since more than 95% of CGL patients have that Philadelphia chromosome translocation. For polycythaemia, consultant may go for measuring the red cell mass or JAK2 for myeloproliferative disease. JAK2 is also a molecular test.
Thanks for the queries.
-Alex
Hello!
Just a rather random question. As u said, Fast Blue BB is carcinogenic. Then, after handling it, do u guys change gloves straightaway?? Coz for my lab, they do advice me to change gloves as soon as i am done handling the carcinogenic reagents.
Thanks!
Kangting 0503331A
TG01
Hello Kangting,
Haha. I guess it's a case of personal habits here. For me, the very moment I'm done with the test, I'd change gloves and have my hands washed! I was only told to be cautious but I guess it's common sense. :P
hey.. juz wondering.. why would an elevated AP levels found in neutrolphils that have polycythaemia vera and myelofibrosis and low in Chronic Granulocytic Leukaemia and Paroxysmal Nocturnal Haemoglobinuria?
Jia Hao
Jia Hao
The various low and high NAP levels are predominantly discovered in patients having these illnesses, which are why they are linked. Body-disease phenomenon? :D
Alex
Hello Alex,
Before blood collection, does the patient need to avoid anything to prevent false positive results? Thanks
Ci Liang
TG01
hey hello alex!
erm you mentioned, "High scores of NAP can also be obtained from neonates, pregnant women or women taking oral contraceptives"...
why is that so?
Natalie (:
Hello Ci Liang,
From what I've understood, no, the patient need not avoid anything in terms of diet or drug as NAP score is all about measuring the NAP enzyme activity. But do also note that oestrogen or corticosteroid therapies or a person's normal physiology (pregnancy) do result in increased counts. AND make sure smears are prepared fresh from capillary or venipuncture as anticoagulants will inhibit enzyme activity.
Hello Natalie,
Haha. Same question from Sharon Ang! Score will be moderately elevated in pregnancy, with oestrogen therapy (eg. oral contraception) and corticosteroid therapy. This may be due to the effects of oestrogen and corticosteroid on NAP causing high NAP(enzyme) activity. The enzyme activity is also high in neonates.
-Alex
Hi
Do you have any references for this method for determining NAP activity?
Post a Comment