NCCLS Document H22-P

Proposed Standard Vol. 4 No. 14

Histochemical Method
for Leukocyte Alkaline
Phosphatase

* This document is no longer being reviewed

as part of the NCCLS consensus process.
However, because of its usefulness to a
limited segment of the clinical laboratory
community, NCCLS is continuing to make the
document available for its informational
content.

Procedure for a semi-quantitative assay for determining leukocyte alkaline phosphatase; criteria for
scoring the assay and interpreting the results.

ABC
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Histochemical Method for
Leukocyte Alkaline Phosphatase

Message to the User

This proposed standard describes the semi-quantitative, histochemical method for determining leukocyte
alkaline phosphatase (LAP). The method includes procedures for preparing fixative, buffer, and
counterstains; for staining blood smears; scoring criteria for determining LAP activity; and guidance for
interpreting the LAP activity score.

The subcommittee hopes that this proposed standard method will improve the reliability and interpretation
of this clinically useful, widely used test.

This document is the result of much effort by the members of the Subcommittee on Cellular Enzymology.
Now, we look to our membership--the clinical laboratory community--to participate in the consensus
process. A questionnaire is appended to this document so that you can give us your views on the value
of this project and its execution as represented by this standard. A proposed standard is the first stage in
the NCCLS consensus process, and it is, therefore, especially important that we have your responses to
the first three questions relating to the document's scope, utility, and scientific validity.

Your comments will help ensure that the document reflects your needs as a member of the clinical
laboratory community and will help the subcommittee in related efforts in the future.

Kouichi R. Tanaka, M.D., Chairholder

Subcommittee on Cellular Enzymology

October 1984

i
 NCCLS Document H22-P
Proposed Standard ISSN 0273-3099

Histochemical Method for

Leukocyte Alkaline
Phosphatase

VOLUME 4 NUMBER 14

Kouichi R. Tanaka, M.D.

Ernest Beutler, M.D.
Leonard S. Kaplow, M.D.
Robert E. Plapinger, Ph.D.
Akira Yoshida, Ph.D.

ABC
 H22-P

© 1984. The National Committee for Clinical Laboratory Standards. All rights reserved.
This publication, or parts thereof, may not be reproduced in any form without
permission of NCCLS.

Approved by Board of Directors as:

Proposed Standard
December 1983
Published:
October 1984

NCCLS VOL. 4 NO. 14 iv

 H22-P

TABLE OF CONTENTS

PAGE

MESSAGE TO THE USER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i

COMMITTEE MEMBERSHIP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi

FOREWORD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

1.0 PURPOSE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2.0 PRINCIPLE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

3.0 APPARATUS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

4.0 REAGENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

5.0 SOLUTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

6.0 STAINING PROCEDURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

7.0 SCORING PROCEDURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

8.0 INTERPRETATION OF RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

9.0 COMMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

RELATED NCCLS PUBLICATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

NCCLS VOL. 4 NO. 14 v

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AREA COMMITTEE ON HEMATOLOGY

Robert V. Pierre, M.D. Mayo Clinic Center

Chairholder Rochester, Minnesota

SUBCOMMITTEE ON CELLULAR ENZYMOLOGY

Kouichi R. Tanaka, M.D. Harbor-UCLA Medical Center

Chairholder Torrance, California

Ernest Beutler, M.D. Scripps Clinic & Research Foundation

La Jolla, California

Leonard S. Kaplow, M.D. Veterans Administration Hospital

West Haven, Connecticut

Robert E. Plapinger, Ph.D. FDA Center for Devices and Radiological

Health
Rockville, Maryland

Akira Yoshida, Ph.D. City of Hope National Medical Center

Durate, California
St. Louis, Missouri

Onno W. van Assendelft, M.D., Ph.D. Centers for Disease Control

Atlanta, Georgia

Mary G. Hoffman NCCLS

Staff Liaison Villanova, Pennsylvania

NCCLS VOL. 4 NO. 14 vi

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FOREWORD

Neutrophilic leukocytes contain an alkaline phosphomonesterase with variable activity

in normal subjects and in patients with hematological and nonhematological disorders.
Leukocyte alkaline phosphatase is a well established term and thus will be used,
although we recognize that in the peripheral blood the enzyme is virtually restricted to
neutrophil polymorphonuclear cells.

There is no relationship between leukocyte alkaline phosphatase and serum alkaline

phosphatase. The function of leukocyte alkaline phosphatase is still unknown, but
normal leukocyte alkaline phosphatase depends on an intact pituitary adrenal axis.

Clinical interest in leukocyte alkaline phosphatase was stimulated initially by the

observation that the leukocytes of patients with chronic granulocytic leukemia usually
had low levels of enzymatic activity in contrast to high values seen in leukocytosis of
other disorders. Quantitative methods of assay are available but are not practical.
Semiquantitative assay by a variety of azo dye histochemical methods has made
determination of leukocyte alkaline phosphatase a widely used clinical test. This
document describes a standard histochemical method which should serve as a
benchmark.

KEY WORDS

Leukocyte alkaline phosphatase (LAP); phosphatase activity; LAP, histochemical

method; granulocytic leukemia; leukocytosis; LAP, scoring criteria.

NCCLS VOL. 4 NO. 14 vii

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HISTOCHEMICAL METHOD FOR LEUKOCYTE ALKALINE PHOSPHATASE

1.0 PURPOSE

Estimating leukocyte alkaline phosphatase activity is most useful clinically in

differentiating chronic granulocytic leukemia from leukocytosis as seen in severe
infections, polycythemia rubra vera, and myelofibrosis with myeloid metaplasia.

2.0 PRINCIPLE

The substrate is hydrolysed by enzymatic activity at pH 9.5 releasing phosphate

and an aryl naptholamide. The aryl naphtholamide immediately couples with the
diazonium salt present in the incubation mixture forming an insoluble azo dye at the
theoretical sites of enzyme activity.

3.0 APPARATUS

Routinely used, clean glassware

4.0 REAGENTS

(1) naphthol AS-BI phosphate, sodium salt

(2) fast violet B salt (purified), 6-benzamido-4-methoxy-m-touidine, diazonium

chloride

(3) 2-amino-2-methyl-1,3-propanediol

(4) 37% formaldehyde

(5) absolute methanol

(6) absolute acetone

(7) 0.1 N hydrochloric acid

(8) hematoxylin powder

(9) sodium iodate

(10) aluminum potassium sulfate, A1K(SO4)2 * 12 H20

(11) 0.03 M sodium citrate, Na3C6H5O7 * 2 H20 - (4.41 g/500 mL

H2O)

NCCLS VOL. 4 NO. 14 1

 H22-P

(12) 0.03 M citric acid H2O - 3.15 g/500 mL H2O

5.0 SOLUTIONS

5.1 Fixative

Sixty percent acetone buffered to pH 4.2 to 4.5 with 0.03 M citrate

buffer. Add 168 mL of 0.03 M citric acid to 32 mL of 0.03 M sodium
citrate and then add slowly, while stirring, 300 mL of absolute
acetone. Store at room temperature.

5.2 Buffer

5.2.1 Stock--0.2 M propanediol. Dissolve 21 g of 2-amino-2 methyl-

1, 3-propanediol in distilled water and dilute to 1000 mL with distilled
water. Store in the refrigerator.

5.2.2 Working--0.05 M propanediol, pH 9.4 to 9.6. To 250 mL of

stock propanediol, add 70 mL of 0.1 N HC1 and dilute to 1000 mL
with distilled water. Store in refrigerator, but warm to room
temperature before use.

5.3 Counterstain

5.3.1 Add 1 g of hematoxylin to 500 mL distilled water.

5.3.2 Heat to the boiling point and dilute to 900 mL with distilled
water.

5.3.3 Add 0.2 g of sodium iodate and 50 g of aluminum potassium

sulfate, and shake the mixture well.

5.3.4 Dilute to 1000 mL with distilled water and store in a brown

bottle at room temperature. Counterstain remains stable for 3 to 4
months.

5.3.5 Filter just prior to use.

5.3.6 Instead of the above preparation, a working solution of

reagents (4), (8), (9), and (10) is available as Mayer's hematoxylin.

6.0 STAINING PROCEDURE

6.1 Use fresh, thin smears of peripheral blood. If venous blood is drawn,
anticoagulate with heparin, (Do not use EDTA.) If staining will be

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 H22-P

delayed longer than 12 h after blood collection, fix smears and store
in a freezer during the interim.

6.2 Fix blood smears 10 seconds at room temperature in 60% acetone

citrate, pH 4.2.

6.3 Wash in slowly running tap water for 30 to 60 seconds.

6.4 Carefully air dry or blot dry.

6.5 Place dried slides for exactly 15 min at room temperature in freshly
prepared substrate mixture in a Coplin jar. Prepare the substrate
mixture by placing about 5 mg of naphthol AS-BI-PO4 in a dry 125 mL
Erlenmeyer flask to which 60 ML of 0.05 M working propanediol
buffer (pH 9.4 to 9.6) and approximately 40 mg of diazonium salt are
added. Shake well, and filter into a Coplin jar; use immediately after
preparation.

7.0 SCORING PROCEDURE

7.1 Scan the smear (-450x) and select an are where the
erythrocytes are barely touching one another. The sites of
phosphates activity are represented by granulation in the cytoplasm
varying from pale pink to red. The counterstained nuclei appear blue
gray (see 9.4).

7.2 Using the oil immersion lens, rate 100 consecutive

segmented neutrophilic leukocytes from zero to four plus on the basis
of the intensity of the precipitated dye. The criteria for scoring are
summarized in the table. The sum of the 100 ratings is considered
the score for a particular smear. Thus, scores may range from a
minimum of zero to a maximum of 400. It is important to score only
polymorphonuclear leukocytes. Do not include lymphocytes,
monocytes, basophilis, and eosinophils in the score.

NCCLS VOL. 4 NO. 14 3

 H22-P

TABLE

SCORING CRITERIA

Precipitated Azo Dye in Cytoplasm

Cell Amounta Size of Granule Intensity of Staining

Rating (%)
0 None ----------------- None
1+ < 50 Small Faint
2+ 50-80 Small to medium Moderate
3+ 80-100 Medium to large Strong
4+ 100 Medium and large Brilliant

Percentage of volume of cytoplasm occupied by azo dye precipitate.

a

8.0 INTERPRETATION OF RESULTS

8.1 In humans, alkaline phosphatase activity is found only in some

mature and band form neutrophils, and in a very rare lymphocyte
(weak reaction). All other cells of the peripheral blood are negative.

8.2 The normal human has little neutrophil alkaline phosphatase, and the
normal average score is 60. The range of normal scores is wide,
varying from 15 to 100. Each laboratory should establish its normal
range.

8.3 Increased activity is seen, for example, in infectious leukocytoses,

polycythemia vera, pregnancy or use of oral contraceptives, and most
cases of multiple myeloma and active Hodgkin's disease.

8.4 Abnormally low or absent staining is seen in chronic granulocytic

leukemia, paroxysmal nocturnal hemoglobinuria, and hereditary
hypophosphatasia.

8.5 The leukocyte alkaline phosphatase activity score is not diagnostic of

any disease.

NCCLS VOL. 4 NO. 14 4

 H22-P

9.0 COMMENTS

9.1 Store the diazonium and naphthol salts in the freezer, preferably in a
dessicator. The diazonium salts are potentially carcinogenic and
should be handled with care. Avoid contact with skin or inhalation.

9.2 If the diazonium salt does not dissolve in the aqueous buffer solution,
it has probably undergone decomposition; check its "coupling power"
at alkaline pH with naphthol AS-BI or B naphthol.

9.3 In scoring, select areas on the slide where the erythrocytes are
relatively thinly spread out and barely touch or overlap one another.
Eosinophils are unstained. They may be difficult to identify and
should not be included in the 100 cells selected for scoring.

9.4 Other naphthol salts are also available and may give equally good
results. Particularly recommended are naphthols AS-CL, AS-TR, AS-
AN, or AS-E phosphates. All these diazonium salt fast violet B, and
blue dyes when coupled with fast blue RR, BB, or BBN. Fast violet B
salt is not the ideal compound to use as the diazonium coupler, but
has been found to be the best alternative to fast red violet salt LB,
which was originally recommended but is no longer available
commercially.

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REFERENCES

1. Ackerman, G.A. Substituted naphthol AS phosphate derivatives for the

localization of leukocyte alkaline phosphatase activity. Lab. Invest. 11: 563-
567, 1962.

2. Leonard, B.J., Israëls, M.C.G., and Wilkinson, J.F. Alkaline phosphatase in the
white cells in leukaemia and leukaemoid reactions. Lancet 1: 289-292, 1958.

3. Kaplow, L.S. Cytochemistry of leukocyte alkaline phosphatase. Amer. J. Clin.

Path. 39: 439-449, 1963.

4. Kaplow, L.S. Leukocyte alkaline phosphatase cytochemistry: applications and

methods. Ann N.Y. Acad. Sci. 155: 911-918, 1968.

5. Kaplow, L.S. Leukocyte alkaline phosphatase in disease. CRC Crit. Rev. Clin.
Lab. Sci., 1971 pp. 243-278.

6. Okun, D.B. and Tanaka, K.R. Leukocyte alkaline phosphatase. Amer. J. Hemat.
4: 293-299, 1978.

NCCLS VOL. 4 NO. 14 6

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An (*) following the order code indicates that the document is

no longer being reviewed as part of the NCCLS consensus
process. However, because of its usefulness to limited
segments of the clinical laboratory community, NCCLS is
continuing to make this document available for its informational
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