Kinetic Determination of Granulocyte Alkaline Phosphatase by the GEMSAEC

Transcription

1 ANNALS OF CLINICA L A N D LABORATORY SC IE N C E, V ol. 10, N o. 3 Copyright 1980, Institute for Clinical Science, Inc. Kinetic Determination of Granulocyte Alkaline Phosphatase by the GEMSAEC LYNN CROOK, M.D., P h.d.,* NICOLAS M. SARJI, M.S.,* PAUL I. LIU, M.D., P h.d.,* R ICHARD H. G ADSD EN, PH.D.f and THOM AS R. W ILLIAM S, B.S.f Hematology* and Clinical Chemistry f Divisions, Department o f Laboratory Medicine, Medical University o f South Carolina, Charleston, SC Abstract G ranulocyte alkaline phosphatase (ALP) activity is m easured kinetically using th e G EM SA EC centrifugal analyzer and p-nitrophenylphosphate as substrate. M easurem ents o f enzym e activity are m ade at ph 9.8 utilizing 1.0 M diethanolam ine as a buffer containing 0.5 mm m agnesium chloride. G ran u lo cy tes are sep a rated from h e p a rin iz e d b lo o d u sin g dextran sedim entation, follow ed w ith ficoll-hypaque separation. T he separated cells are su sp en d ed in saline, and the red cells are lysed. M anual counting of stained sm ears of the cells isolated show ed that 95 p ercen t of these cells w ere granulocytes. After lysing the red cells, the granulocyte suspension is rinsed in 12.5 p ercen t heat inactivated serum in saline and separated by centrifugation. After 200 jl 1 of the diluent solution (12.5 p ercen t h eat inactivated serum in saline) is added to the cells, they are sonicated for seven sec at 0 in an ice bath. T h e extracts are th en assayed kinetically for ALP activity. G ranulocyte ALP activity varies from day to day in norm al individuals. T he daily variation o f ALP activity observed correlates closely w ith and is confirm ed by the Sigma H istochem ical Procedure based on Kaplow s Scoring M ethod. T he coefficient o f variation of the ALP m ethod w ithin assay perform ed on two consecutive days on a pooled sam ple is 0.75 percent and 1.38 percent, respectively, and b etw een assays 4.82 percent. Introduction K now ledge o f a lk alin e p h o sp h atase (ALP) activity o f granulocytes is a helpful diagnostic tool, esp e cially in d ifferentia tin g b e tw e e n c h ro n ic g ra n u lo c y tic leukem ia w here the ALP activity is low and leukem oid reaction w here the ALP activity o f the granulocytes is high, w ith som e exceptions in both cases.9 Principle T h e s u b s tra te, p -n itro p h e n y lp h o s - phate, in the presence of M g++ ions and at a ph of 9.8 is hydrolysed by ALP from granulocytes to p-nitrophenol and phosphate. T he p-nitrophenol lib erated is proportional to the ALP activity and is d eterm ined by m easuring the A A /t at 405 nm. ALP activity is first expressed in mu p er /80/ $00.90 Institute for Clinical Science, Inc.

2 GRANULOCYTE ALP BY A KINETIC METHOD ml, w here U is defined as the ALP activity w hich converts one (jl,mol o f substrate in one m in at standard conditions. From the ALP activity m easured, the ALP activity in m U /108 granulocytes is calculated. M ethods and M aterials Re a g e n t s Sodium chloride, 0.9 percent. T his is p rep ared by d issolving 9 g of reag en t grade sodium chlo rid e in one lite r of d istilled water. D extran, 6.0 percent. This is prepared by dissolving 6 g o f clinical grade dextran (average Mol. wt. 170,000) in 100 ml of 0.9 p ercen t NaCl. Ficoll, 9.0 percent. This is p repared by dissolving 9 g of ficoll (Mol. wt., approxim ately 400,000) in 100 m l o f d istilled water. H ypaque, 33.3 percent. T his is p repared by d ilu tin g 20 ml of 75 p ercen t hypaque-m w ith 25 ml of d istilled w ater. F icoll-h ypaque solution. This is p repared by m ixing 100 ml of 9.0 percent ficoll w ith 41.7 ml of the 33.3 p ercen t hypaque solution. H eat in a ctiva ted serum. T his is p repared by heating pooled serum from normal donors at 65 for one hour. No ALP activity is d etected after heating. H eat in a ctiva ted serum, 12.5 percent. T his is prep ared by diluting 12.5 ml of the heat inactivated serum to 100 ml w ith 0.9 percent NaCl. T his solution is referred to as the rinsing diluent. Saponin, 0.5 percent. This is prepared in the rinsing d ilu en t by dissolving 0.25 g saponin in 50 m l o f 12.5 p ercen t heat inactivated serum. This solution is referred to as th e extraction diluen t. Saponin Solution. O ne gram of saponin is dissolved in 100 ml 0.9 p ercen t NaCl to prepare 1 p ercen t saponin in saline. All solutions are refrigerated u n til used. Reaction Conditions for the GEMSAEC Kinetic Alkaline Phosphatase Determination Rotaloader IV Instructions Volume Pump Sample Flush Reagent 10 yl 50 ul 500 ]il 50 ul 200 ul 1000 u l Sample Tip: Stainless steel Well B B C Blank Switch : Water Analyzer/Control Module Instructions Reaction temperature: 30 Wavelength: 405 nm Filter position: nm Reaction mode: Auto/Rate First reading: 10 sec Reading interval: 20 sec Number of readings: Computer Instructions Header: ALK Kt 1665 Hi: 150 IR: 10 TF 1 Lo: 25 RI 20 TC 2 Sa: 1.2 NR 6 AD 4 RM: 2 SC 0 CD 0 XX: 1 St a n d a r d S o l u t io n s T h e s u b s tra te, p -n itro p h e n y lp h o s - phate, and th e buffer, d ieth an o lam in e containing 0.5 mm m agnesium chloride, ph 9.8, are from a com m ercial kit.* T hese are the sam e reagents used for alkaline phosphatase d eterm in atio n in serum. Spec ia l Appa r a t u s In table I are indicated the exact reaction conditions used on the GEM SAEC centrifugal analyzerf for determ ining the ALP activity. T h e d irectio n s and centrifu g al an a ly z er settin g s for alk alin e phosphatase determ inations are included w ith the test kit and adhere very closely to those outlined by H ausam en et a l3 and as confirm ed in our laboratory. Upon investigating the stability of the granulocytes total A LP activity in differe n t extraction m edia, h eat in activ ated norm al hum an serum is found to be the m ost suitable extractant. In figure 1 are illu stra te d d ifferen ces in ALP activity * Marked (Catalog # 15990) by Bio-Dynamics Division of Boehringer Manheim Corporation, t Electronucleonics Inc., Model #

3 2 0 0 CROOK, SARJI, LIU, GADSDEN AND WILLIAMS P rocedure IN S A L I N E IN SALINE F i g u r e 1. Effect of extraction medium on alkaline phosphatase activity after storage at -20 for 24 hours. after storage at 20 for 24 hrs using th ree extractants. W hen 0.9 p ercen t NaCl was used as the extraction diluent, ALP activity dropped to 58.9 p ercen t of the original activity on rep eated analysis o f the same extracts after a 24 h r period. Sim ilar results w ere found w h en Tris buffer, ph 8.8, was su b stitu ted for saline. W hen 12.5 perc e n t n o rm al p o o le d h u m an seru m in saline was used as the extracting diluent, ALP activity in creased to 139 p ercen t after 24 hrs. This confirms the findings of W iltshaw and M oloney.10 T he purpose of using hum an serum was to provide a more natural environm ent for the protection of th e granulocyte alkalin e phosphatase activity. S in ce som e factor in serum e n h anced th e ALP activity, the serum is heated at 65 in a dry hot air bath for one hour. R ep eated d eterm in atio n s on the same extract using the heat inactivated serum in saline show ed the ALP activity was stab ilized y ield in g percent. H ow ever, the activity d ro p p ed to 75.5 percent after th ree m onths and 53.3 percent after six m onths in 12.5 percent heat inactivated serum in saline w hen stored frozen at 20. Venous blood sam ples (10 ml) w ere collected in h ep arin ized vacutainer tubes from A m erican R ed C ross m ale blood donors b etw een the ages of 21 and 52 years o ld. After thorough m ixing o f the heparinized blood, two ml of 6.0 percent dextran solution6 were added slowly with g entle mixing. T he m ixture is allow ed to stand at room tem perature for 45 m in to allow m ost of the red cells to settle. T he plasm a supernatant, rich in leukocytes w ith som e co n tam in atin g red cells, is transferred w ith a plastic p ip et and carefully layered on top of three ml of ficollhypaque solution.2 This is centrifuged at 1200 rpm in a Damon centrifuge for 10 m in at 25 (240 g RCF). T he granulocytes and red cells settle to the bottom. T he liquid supernatant is discarded and the sedim ented cells are resuspended in five ml of saline. After thorough mixing, the granulocytes in the cell suspension are counted on a C oulter counter, M odel ZBI. M anual differentials of stained sm ears of these isolated cells show ed that 94.8 ±' 0.67 p e rc e n t (± SEM, N = 20) w ere granulocytes. To the rem ainder of the cell suspension 50 /,1 of 1.0 percent saponin in saline is added, m ixed for 15 to 30 sec and centrifuged at 2800 rpm for five m inutes at room tem perature. T he supernatant is discarded. T h e p e lle t of granulocytes is resu sp ended in five ml of the rin sin g d ilu e n t an d centrifu g e d. T h e granulocytes are rin sed tw ice w ith five ml o f rinsing diluent, centrifuged and the supernatant discarded. After the second d écan tatio n, th e sides o f th e tu b e are w iped dry and 0.2 ml of the extraction d ilu e n t is a d d e d. A fter m ix in g, th e granulocytes are sonicated for seven sec at 0 using an ice bath. T he hom ogenates are centrifuged for five m inutes at 2800 rpm. } Permission for performance of this testing was secured from each donor on forms approved by the Human Research Committee at the Medical University of South Carolina.

4 GRANULOCYTE ALP BY A KINETIC METHOD T he supernatant is rem oved and assayed for ALP activity. T he results are reported as mu ALP activity p er 108 granulocytes per m in at 30. TABLE II Alkaline Phosphatase Activity in Duplicate Extracts of Granulocytes With 1.5 Percent Heat Inactivated Serum in Saline With and Without Saponin R esults and D iscussion ALP Activity 1st Extract 2nd Extract Additional % Activity in 2nd Extract G ranulocyte ALP activity is usually high in leukem oid reaction and low in chronic granulocytic leukem ia, w ith some exceptions.9 G enerally, clinical evaluations o f th e ALP activity o f th e granulocytes are done histochem ically using Kaplow s Scoring M ethod.4'5 W hile th is is fast an d re p ro d u c ib le, it is n o t a su itab le m ethod for very low granulocyte ALP activity and the results are in terp reted subjectively. A kinetic analytical m ethod is m ore objective and leaves little chance for subjective interpretation. It offers higher sensitivity and precision as w ell as the possibility to rep eat assays. For this purpose, the GEM SAEC kinetic m ethod for d eterm in in g A LP activity in serum is adapted. O nly healthy R ed Cross male blood donors b etw een th e ages of 21 and 52 years o ld w ere in v e stig a te d sin ce fem ales in that age group exhibit higher ALP activity.7 F urther studies o f ALP extraction from the granulocytes indicated th at incorporation of 0.5 p ercen t saponin into the 12.5 percent heat inactivated serum in saline released m ost of th e ALP activity from the cells in the first extraction. This is dem onstrated by com paring the ALP activity obtained by using 12.5 p ercen t h eat inactivated serum in saline w ith and w ithout saponin. It is illustrated in table II that with saponin, m ost ALP activity is extracted in th e first extraction and only 3.2 p e r cent of the activity is left for the second extraction, w hereas w ith o u t saponin, the yield of ALP activity in the first extraction is only 22 p ercen t o f th e first saponin ex-, traction and only an additional 7.5 percent activ ity d u rin g th e second ex tractio n w ithout saponin. With 0.5 mu/ml ± SE mu/ml ± SE mu/ml ± SE percent ± i ± 0.5 saponin* Without 68.8 ± ± ± percent saponint *N = 17 Range =0-8.2 percent +N = 11 flrange =0-67 percent T h ese d ata confirm th e fin d in g s o f Rosner e t a l8 that saponin increases the yield of granulocyte ALP, b u t in contrast to his findings, no fu rth e r significant release of ALP is found after retreatm ent w ith saponin. Initial studies on a healthy m ale subject show ed diurnal variations in agreem ent w ith A nstey et al.1in order to verify this, th ree healthy m ale subjects w ere tested for four consecutive days w ith Sigm a s histochem ical procedure using Kaplow s scoring m ethod and w ith the proposed k in e tic e n z y m e assay. B oth m eth o d s show ed diu rn al variations w ith sim ilar tren d s as illu stra te d in figure 2. A com p a riso n o f th e s e d ata in d ic a te th a t a Kaplow score o f 1 is equivalent to approxim ately 6 m U /108 granulocytes p er m inute at 30. T he reproducibility of the ALP m ethod was checked by assaying 27 sam ples in duplicate b eg in n in g w ith w hole blood and perform ing the total extraction procedure. A correlation coefficient o f (P < 0.001) b etw een th e duplicates and Stud ents paired t test (t = ) indicated th a t no significant difference exists b e tw een the sam ple results. T he precision of the kinetic m ethod is dem onstrated by assaying pooled granulocyte extracts in d u p licate for two successive days. T he coefficien t of variation w ith in assay is 0.75 p er

5 2 0 2 CROOK, SARJI, LIU, GADSDEN AND WILLIAMS f i I t.* 0 b»... =Sigma Histochemical... =GEMSAEC Enzymatic > / / F i g u r e 2. Diurnal variation of normal granulocyte alkaline phosphatase activity in three normal males DAYS DAYS DAYS cent and 1.38 percent, respectively, and b etw een assay 4.82 percent. Sources of E rror The red blood cells m ust b e com pletely lysed in order to avoid high background absorbance during the assay. T he sonication tim e in an ice bath m ust be kept constant. Plastic laboratory w are should be used throughout the procedure. G ranulocyte counts m ust be done w ithin 30 m in after lysing the red blood cells to avoid granulocyte agglutination. Saponin gives an alm ost com plete extraction of ALP activity w ith a single extraction. R eference Range A lthough diurnal variations exist, the referen ce range was estab lish ed on 88 healthy R ed Cross m ale donors b etw een PERCENT OF HIGHER GRANULOCYTES ALP VALUES FIGURE 3. Cumulative frequency distribution of log granulocyte alkaline phosphatase activity o f 88 normal males plotted on normal probability paper.

6 GRANULOCYTE ALP BY A KINETIC METHOD th e ages o f 21 a n d 52 y e a rs o ld. A logarithm ic norm al distribution is dem onstrated in figure 3. T h e calcu lated refere n c e ran g e e x te n d e d from 95 to 750 m U /1 08 granulocytes p er m inute at 30. Fem ales b etw een the ages of 21 and 36 years old w ere tested and appeared to have a higher reference range; how ever, an adequate num b er of donors was not available to calculate th e reference range for fem ales. Sum m ary Total ALP activity of granulocytes is determ in ed kinetically on the GEM SA EC centrifugal analyzer. T he m ethod adapted for this purpose is sim ilar to that used for th e determ ination of ALP in serum. T he extraction p ro ced u re is sim ple, b u t requires a sonicator as a special piece of eq u ip m en t w hich may not be available in all laboratories. W ith this m ethod, diurnal v ariatio n s have b e e n show n b y us in granulocyte ALP activity and th e norm al range applicable to the m ethod has b een d eterm in ed for adult m ales. R eferences 1. A n s t e y, L., Ke p, N. H., St a f f o r d, J. L., and TANNER, R. K.: Leukocyte alkaline phosphatase activity in polycythem ia rubra vera. Brit. J. Haemat. 9:91-100, BOYUM, A.: Separation of leukocytes from blood and bone marrow. Scand. J. Clin. Lab. Invest. 21 : suppl. 97, H a u s a m e n, T. U., H e l g e r, R., R ic k, W., and GROSS, W.: Optimal conditions for the determ i nation of serum alkaline phosphatase by a new kinetic method. Clin. Chim. Acta 15: , KAPLOW, L. S.: A histochemical procedure for localizing and evaluating leukocyte alkaline phosphatase activity in smears of blood and marrow. Blood J0: , KAPLOW, L. S.: Cytochemistry of leukocyte alkaline phosphatase. Use of complex naphthol A S -phosphatase in azo dye co u p lin g techniques. Amer. J. Clin. Path. 39: , P e a c o c k, A. C., G r e c h e r, G., and H ig h s m it h, E. M.: A simplified procedure for quantitative measurem ent of alkaline phosphatase in white blood cells. Amer. J. Clin. Path. 2 9 :80-85, ROSNER, F. and L e e, S. L.: Endocrine relationships of leukocytes alkaline phosphatase. Blood 25: , R o s n e r, F., L e e, S. L., Sc h u l t z, F. S., and G o r f ie n, P. C.: The regulation of leukocyte alkaline phosphatase. Ann. N.Y. Acad. Sci. 755: , W A C H S T E IN, M.: Alkaline phosphatase activity in normal and abnormal human blood and bone marrow cells. J. Lab. Clin. Med.37 :1-1 7, W i l t s h a w, E. and M O L O N E Y, W. C.: H istochemical and biochemical studies on leukocyte alk alin e phosphatase activity. B lood i0: , 1955.