Detection of platelet antibodies by anti-kappa light chain facilitation of C-FDA (KC-FDA) thrombocytotoxicity

Detection of Platelet Antibodies by Anti-kappa Light Chain Facilitation of C-FDA (KC-FDA) Thrombocytotoxicity Gail E. Lizak and F. Carl Grumet

A B S T R A C T : Sensitivity of the carboxyfluorescein diacetate (C-FDA~ thrombocytotoxicity technique for the detection of antiplatelet antibodies has been enhanced by the addition of an anti-Kappa light chain antibody facilitation step. This new technique, KC-FDA, was compared with the platelet suspension immunofluorescence test (PSIFT) by titering platelet-reactive allo-anti-PlAl and antiHLA antibodies. The results show that compared to PSIFT. KC-FDA is more sensitive for detecting platelet specific antibodies (PlAl), is more or equally sensitive for detecting other antibodie~ (HLAL and is significantly faster and easier to perform. ABBREVIATIONS

C-FDA PSIFT Anti-K INTP ACD CPD FITC

carboxy-fluorescein diacetate platelet suspension immunofluorescent test anti-Kappa light chain isoimmune neonatal thrombocytopenia acid citrate dextrose citrate phosphate dextrose fluorescein isothiocyanate

EDTA PBS BSA HBSS 2-ME CAP

ethylene diamine tetraacetic acid phosphate buffered saline bovine serum albumin Hanks balanced salt solution 2-mercaptoethanol solution College of American Pathologists

INTRODUCTION As previously described, carboxyfluorescein diacetate (C-FDA) thrombocytotoxicity [1] is faster and simpler than 51 Cr thrombocytotoxicity or platelet suspension immunofluorescence (PSIFT) for detection of antiplatelet antibodies. It was also more sensitive than the 51 Cr assay for all antibodies tested, but not as sensitive as PSIFT for some types of antibodies. By the addition of a facilitation step with anti-Kappa light chain (anti-K) it has proven possible to increase the C-FDA thrombocytotoxicity sensitivity to that of the PSIFT with almost no loss of the speed and simplicity of the original C-FDA test. The present report describes this new technique which has the abbreviated designation KC-FDA.

From the Stanford University Medical Center, Stanford Universi(y Hospital, Transfusion Sereice.Stanford, CA 94305. Address requestsfor reprints to F. Carl Grumet, M.D., Associate Professor. Department of PatholoKy. 800 Welch Road, Palo Alto, CA 94304. ReceivedApril 25, 1983; acceptedJuly 27, 1983. H u m a n Immunology 8, 2 6 5 - 2 7 1 (1983) Elsevier Science Publishing Co., Inc., 1983 52 Vanderbilt Ave., N e w York, N Y 10017

265 (J 198-8859/83/$ ].00

266

G.E. Lizak and F. C. Grumet

MATERIALS AND METHODS

Sera. Pools o f sera from male AB donors were used as negative control sera and also as diluents for titration of reactive sera. Sera H O G , PHI, FRA, HES, and M C K were obtained from mothers whose platelets were typed as being PIAl negative and whose newborns suffered from isoimmune neonatal thrombocytopenia (INTP). The corresponding fathers' platelets typed as PIAl positive. AntiHLA and PIAl typing sera were from the authors' own collection of antisera, and an additional reference anti-P1Al serum, N A G , was kindly provided by Dr. R. Aster (Milwaukee Blood Center). ACD, CPD, or E D T A anticoagulated whole blood up to three days old from blood donors was used to prepare platelet suspensions which were tested immediately or after storage at - 7 6 ° C [2]. A final concentration of 104 platelets/~l was used in the C-FDA assay and 105 platelets/~l in PSIFT [3].

C-FDA. The C-FDA assay was performed as previously described [1]. Approximately 1 × 105/mm 3 fresh or frozen platelets were washed twice in AB (EDTA) plasma and resuspended on 0.4 ml of PBS containing 4 tzg/ml of carboxyfluorescein diacetate (Molecular Probes, Junction City, OR), incubated at 37°C for 15 min and then washed twice in AB plasma. O n e / A of these fresh or frozen stored C-FDA treated platelets suspended in PBS, at a concentration of 1 × 104/txl, were added to one ~l of serum preloaded into standard tissue typing trays (Robbins, Palo Alto, CA). The first incubation then took place at 37°C for 30 min followed by 10 min at room temperature. Five ~l of fresh frozen rabbit serum from 6 - 1 2 week old animals (Pel-Freez # 3 1 7 3 Rogers, AR), previously screened for nonspecific cytotoxic antibodies, were next added as a source of complement. As with any complement-dependent assay, care must be taken to select rabbit serum lots which both lack nonspecific toxicity and possess effective complement levels. Following a 90 rain room temperature incubation, test results were read under low-power fluorescent microscopy with appropriate filters for fluorescein excitation. Viability in the AB control well was established as the reference 100% standard. Wells with viability less than 50% of that standard were considered positive. KC-FDA. The KC-FDA assay was the same as the C-FDA assay, but with the anti-K facilitation step added after the (serum) incubation described above. Ten /A o f PBS were gently added to each well and trays were then spun at 500 × g for 2 rain at room temperature. The supernatant was then removed by placing an absorbent towel, cut to the tray size, on top o f the tray and applying pressure. This wash step was repeated twice. After the final wash step, one microliter of an appropriate dilution in PBS o f a goat antihuman K, free and bound (Cappel # 1 3 9 0 5 , Cochranville, PA), was added to each well and incubated one minute at room temperature. Selection of an effective facilitating antiglobulin antibody is an important step and was done emperically by screening samples from several different suppliers in checkerboard titrations using weak PIA1 and/or HLA antibodies. Five microliters o f the fresh frozen rabbit serum, diluted 1:1 in heat inactivated rabbit serum were then added and following a 90 min room temperature incubation test results were read as for the regular C-FDA assay.

PSIFT. This assay was performed as described by von dem Borne, using FITC labeled rabbit antihuman IgG F(ab)'2 (Cappel #14646, Cochranville, PA) or FITC labeled goat antihuman Ig (Tago # 2 1 9 3 , Burlingame, CA) [3].

Fluorochromasia lymphocytotoxicity. This technique was performed as described in detail earlier [ 1]. Anti-K facilitation was also characterized for lymphocytotoxicity

Platelet Antibody Detection by KC-FDA

267

and was performed as described above for thrombocytotoxicity, but with different optimal dilutions of anti-K light chain.

2-ME reduction. A 0.2 M 2-mercaptoethanol solution (2-ME) was prepared in phosphate buffered saline. Equal volumes of 2-ME and test sera were mixed together and incubated at 37°C for 30 min. As a control, equal volumes of phosphate buffered saline and sera were mixed together and incubated at 37°C for 30 min. Control sera and 2-ME treated sera were then titered against the appropriate cell following standard techniques as described above. Control sera consisted of known IgG and/or IgM anti-HLA antisera. RESULTS T o compare the newly developed KC-FDA with the other assays, several wellcharacterized anti-HLA and anti-P1Al alloantisera were titered by C-FDA and PSIFT, the latter with both anti-Ig and IgG. As seen in Table 1, KC-FDA gave higher titers than C-FDA. Table 1 also shows the comparison of the KC-FDA to PSIFT. KC-FDA titers were equal to or better than those of PSIFT except for serum Gil and Mar, both anti-HLA-Als. The reliability and reproducibility of the KC-FDA technique was shown by consistently concordant results produced in duplicate testing on different days of 20 specific antisera against a panel of platelets from 6 donors ( + / + 118, + / -1, - / + 1, - / 120: P = <0.001). For additional negative controls the sera from 62 untransfused male donors were collected sequentially and tested against 12 type O random donor platelets; all were consistently negative. T o further evaluate the KC-FDA assay, all clinical maternal samples received for investigation of I N T P over a 20 month period and originally tested by our routine C-FDA technique were retrospectively retested by KC-FDA and PSIFT whenever possible. PIAl testing of mother, father, and infant platelets had been performed with reference anti-PIA1 typing sera. In general the typing of infant platelets was not possible due to the severely decreased platelet count. O f 17 samples, 11 were from PIAl negative mothers whose infants had P1Al positive fathers, one was from a P1A1 negative mother whose infant was PIA1 negative,

TABLE 1

Comparison of titers of well-characterized antiplatelet alloantibodies in different techniques ~

Antibody (specificity) Mar (HLA-A1) Gil (HLA-A1) W e s (HLA-B7) Kin (HLA-A3) Har (HLA-A2) Moo (HLA-A2) Cas (HLA-B27) Mal (Anti-P1A~) N a g ( A n t i - P 1 M)

Target platelet antigens HLA-A1, HLA-A1, HLA-A3, HLA-A3, HLA-A2; HLA-A2; HLA-A2; HLA-A2; HLA-A3,

A3; B7 Bw63(Bw4)(Bw6) A3; B7, Bw63(Bw4)(Bw6) A l l ; B7, B18(Bw6); Cw3 A l l ; B7, B18(Bw6); Cw3 B27; (Bw4); Cw2, Cw3 B27, (Bw4); Cw2, Cw3 B27, (Bw4); Cw2, Cw3 B8, B27(Bw4)(Bw6); Cw3 A 3 ; B 7 , B7(Bw6)

P1 m = P1AI= P1A~P1 ^~= P1 ^~ + P1 ^~ + PI A~+ P1A~+ Pl^l+

C-FDA

KC-FDA

lgG' PSIFT

IgJ PSIFT

NR b NR 4 Neat 2 Neat 2 2 4

Neat Neat 8 8 32 8 4 4 32

2 2 4 4 32 Neat Neat 2 2

.l 4 8 8 32 Neat 2 2 2

"Reactions of all sera were specific. Negative control platelets lacking the appropriate antigen failed to show either significant cytotoxicity or immunofluorescence with test sera. +Nonreactive. 'Goat anti-human IgG (F(ab)'2). 2Rabbit anti-human lg (IgG, IgM, lgA).

268

G.E. Lizak and F. C. Grumet and five were from PIAl positive mothers whose infants had PIAl positive fathers. Of the 11 samples received from P1Al negative mothers with P1Al positive husbands, five had previously been identified as containing anti-PIAl antibodies by C-FDA thrombocytotoxicity and were reconfirmed by the KC-FDA assay. Since we reported earlier that PSIFT was comparable or better than C-FDA for the detection of antiplatelet antibodies (PIAl), we assumed that these 5 sera would also be positive by PSIFT [1]. (One mother, MCK, had two sample dates from two separate pregnancies in which both infants were affected by INTP.) The results of testing the 6 remaining samples are shown in Table 2, where it is seen that for platelet-specific anti-PIA1 antibodies, the KC-FDA technique is superior to either C-FDA and comparable or better than PSIFT in all cases tested. The two positive reactions of serum FRA in PSIFT with two PIA1 negative platelets were not due to anti-HLA antibodies (see below) but could not be further identified because the small serum sample available was insufficient for additional testing. Essentially no significant difference was observed between IgG and Ig (IgM, IgA, and IgG) PSIFT with the reagents available to us. To further characterize the test sera, all those listed in Table 2 were treated with 2-ME [4,5] and tested by KC-FDA. Titers of all the sera remained unaffected by 2-ME, indicating each of the anti-PIAl antibodies were of the IgG class. To eliminate the possibility of anti-HLA antibodies confounding the determination of specificity, all the sera listed in Table 2 were also screened for lymphocytotoxins against the lymphocytes of the same donors that were used for the platelet panels. Table 2 shows these results, including those of K facilitation lymphocytotoxicity. All sera lacked lymphocytotoxins except for MCK 1980. Without K facilitation only one cell, a homozygote A3 +, B7 4- reacted with this serum; with K facilitation, this serum reacted with all A3 4- lymphocytes and no others, indicating the anti-A3 specificity. A 54 donor lymphocyte panel was also negative by standard lymphocytotoxicity. The anti-A3 was not detected against the platelet target cell by any of the other methods employed. In comparing two of the anti-HLA antibodies (Moo, HLA-A2 and Wes, HLA-B7) in titration against lymphocytes the respective titers are 2 and 8 (C-FDA, KC-FDA), and 4 and 8. Titers against the same donors' platelet target cells (from Table 1), are lower, confirming that HLA antibodies are better detected on lymphocytes than platelets [1,6,7]. KC-FDA also appears to detect some CYNAP antibodies. Serum Simp contains a lymphocytotoxic anti-Bw49 and a CYNAP anti-BW50 that fails to activate and/or bind complement at the surface of Bw50 lymphocytes [8]. Simp reacted in the C-FDA technique with lymphocytes from three of three and with platelets from two of three Bw49 donors, but not with lymphocytes or platelets of two Bw50 donors. In the KC-FDA technique (with extension of the last incubation to 31/2 hr), the lymphocytes and platelets of all three Bw49 and both Bw50 donors were reactive while target cells from donors lacking those antigens remained unreactive. With respect to effort and reagent requirements, compared to KC-FDA, the PSIFT assay requires 100 times more sera, 1,000 times more platelets, and when testing 72 sera against 1 platelet, weights 90 CAP units, whereas PSIFT weights 240 CAP units [9].

DISCUSSION This report describes an anti-K facilitation step (KC-FDA) to enhance the detection of antiplatelet antibodies by the C-FDA thrombocytotoxicity technique [1]. It is well known that antibodies to certain erythrocyte and lymphocyte an-

269

v,,., +

[... Z

.< .,<

,..ca

<

va .n

I r

+

II

e~

,.... ©

U~ + + 7~

.n ~

+ + +

+ + +

o

+

©

+

~o >

+ em ~D

--~

I

r

o

g~

~n~ +

~+

+

.an +

I

a --~

I I

H

+ +

© II .< ©

I I

t~

<5 a~

~r

+

+ ..n + ~ +

+ ~q +~

¢.> ©

'

II .<

{D-

rm,

© ao

+

+ .an++

-+I I

+ I

eq

.<

0c c r-- ~2

:za:~

~<

ua

270

G.E. Lizak and F. C. Grumet tigens may be only or more easily detected by antiglobulin techniques [10-14]. An anti-Kappa antiglobulin was chosen for KC-FDA since this is the most common light chain. Most alloantibodies are polyclonal, therefore the likelihood of missing antibodies because they were only of lambda light chain would be extremely rare. Since a recent report concluded that the PSIFT was the most sensitive technique detecting antiplatelet antibodies, we compared this technique to KC-FDA [ 15 ]. Our results show that KC-FDA is more sensitive than PSIF for all the platelet specific (i.e., anti-PIA1) antibodies tested. PSIFT detected antibody in three out of five, and KC-FDA detected antibodies in the sera of all five PIAl negative mothers (6 samples) whose infants were affected by isoimmune neontal thrombocytopenia; the specificities in all cases were IgG anti-P1Al. In one case, FRA, another specificity may have been detected by PSIFT but the small quantity of maternal serum available did not allow us to characterize these "extra" reactions further. Serum drawn from the affected infant of FRA, two days after delivery, again showed the anti-PIA1 antibody but the extra PSIFT reactivity of the maternal serum was not present. In 5 out of the 6 samples platelet specific antibodies alone were detected in the serum. In one case, MCK 80, a specific anti-HLA-A3 was also detected by KC-FDA lymphocytotoxicity. Whether this is due to cytotoxic conversion (of C Y N A P antibodies) or cytotoxic enhancement (of complement fixing antibodies) through binding of the anti-Kappa reagent to the anti-HLA alloantibodies is not known. That K-facilitation can bind complement efficiently enough to permit noncomplement binding antibodies to be detected by cytotoxicity is demonstrated by the data shown for serum (Simp). Using PSIFT, yon dem Borne found antibodies in 29 out of 29 PIA1 negative cases [16], whereas we were able to detect antibody in only eight out of ten PIA1 negative cases. Using KC-FDA, however, we could detect specific antibody in all ten cases. Further, when testing multiple samples, KC-FDA is substantially faster, simpler, and more conservative of reagents than PSIFT. In conclusion, KC-FDA thrombocytotoxicity offers advantages over both PSIFT and C-FDA thrombocytotoxicity assay, retaining the versatility and advantages of the latter technique for clinical laboratory use and for such purposes as screening large numbers o f sera against panels of donor cells in genetic analysis. The availability of this new, rapid assay for detection of antibodies in isoimmune neonatal thrombocytopenia may allow a more prompt diagnosis of transfusioninduced platelet alloimmunization. Further study of this technique is thus planned for the cross-matching of patient sera with platelets of HLA matched donors to determine the utility of KC-FDA testing in such additional clinical settings.

ACKNOWLEDGMENTS

The authors wish to thank Dr. R. Aster for supplying serum samples, Dr. G. Rodey for advice with the technical aspects of the antiglobulin assay, and Edith Davis for her superb secretarial assistance. A portion of this work was supported by NIH grant number HL21662 and NOI AI 82558.

REFERENCES 1. Lizak GE, Grumet FC. A new micromethod for the in vitro detection of antiplatelet antibodies: C-FDA thrombocytotoxicity. Hum Immunol 1:87, 1980. 2. Lizak GE, Grumet FC: Storage of reagent platelets for antiplatelet antibody testing in the 51Cr platelet lysis assay. J Clin Path 32:191, 1979.

Platelet Antibody Detection by KC-FDA

271

3. von dem Borne AEGK, Verheught FWA, Baslerhof E, von Reisz LE, Brutel de la Reviere A, Englefriet CP: A simple immunofluorescence test for the detection of platelet antibodies. Brit J Hem 39:195, 1978. 4. Freeman J, Masters C, Newlands M, Mollison P: Optimal condition for these use of sulphydryl compounds in dissociating red cell antibodies. Vox Sang 30:321, 1976. 5. Rosner ER, Profsky Bi Sheth K: The influence of dialysis of 2-mercaptoethanol reduction of erythrocyte antibodies. Transfusion 14:47, 1974. 6. Lubert M, Aster RH: Expression of HLA-B12 on platelets, on lymphocytes anti in serum: a quantitative study. Tissue Antigens 9:199, 1977. 7. Aster RH, Szatkowski N, Leibert M, Duquesnoy RT: Expression of HLA-B 12, HLAB8, W4 and W6 on platelets. Transplan Proc 9:1695, 1977. 8. Lublin DM, Grumet FC: Mechanism of the CYNAP phenomenon. Evidence in the Bw49/Bw50 model for epitopes with different spatial orientation of antibody. Hum Immunol 4:137, 1982.

9. Laboratory Workload and Recording Method. College of American Pathologists, Fourth Ed., 1977. 10. Race RR, Sanger R: Blood Groups in Man. Philadelphia, PA, F.D. Davis, 1968. 11. Johnson, AH, Rossen RD, Butler WT: Detection of alloantibodies using a sensitive antiglobulin microcytotoxicity test. Identification of low levels of pre-formed antibodies in accelerated allograft rejection. Tissue Antigens 2:215, 1972. 12. Cannady WG, Reckel R, Trepode D, Shaw SM, Baldassau D, Metz LA: Sensitivity of various HLA typing techniques. Tissue Antigen 4:546, 1974. 13. Crawford MN, Pollack MS: Confirmation of Bg-HLA relationship by antiglobulin microcytotoxicity testing. Transfusion 18:731, 1978. 14. Cross DE, Whittier FC, Weaver P, Forworth U: A comparison of the antiglobulin versus extended incubation time crossmatch. Results in 223 renal transplants. Transplan Proc 9:1803, 1977. 15. Hemerhorst FM, Bossers B, De Bruin HG, Engelfriet CP, yon dem Borne AEGK: Detection of platelet antibodies: A comparison of three techniques. Vox Sang 3983, 1980. 16. yon dem Borne AEGK, van Leawen EF, yon Riesz LE, von Barrel CJ, Engelfriet CP: Neonatal alloimmune thrombocytopenia: Detection and characterization of the responsible antibodies by the platelet immunofluorescence test. Blood 57:649, 1981.