American Journal of Hematology 38:314-320 (1991)

Simple Method for Differentiating Between HLA and Platelet-Specific Antibodies by Flow Cytometry J. Freedman and A. Hornstein Department of Immunohernatology, St. Michael's Hospital, University of Toronto, Toronto, Canada

Since platelets express both platelet-specific and class IHLA antigens, serum antiplatelet reactivity assessed by most platelet antibody techniques could be due to antibodies with either or both specificities. Flow cytometric analysis of sera for detection of antiplatelet antibody commonly employs a purified platelet preparation as target cells. A method is described for investigating sera for platelet antibodies by flow cytometry using a mixture of platelets and lymphocytes. The mixture of lymphocytes and platelets as target cells has the advantage of confirming the presence of the HLA antibodies in reactive sera. The concomitant use of platelets and lymphocytes treated with citric acid, pH3, or with chloroquine (to remove or alter surface HLA antigens without affecting platelet specific antigens) may further assist in identifying antiplatelet antibodies in alloimmunized patients. These techniques may also be useful in platelet crossmatching procedures. Key words: alloantibodies, platelets, lymphocytes

INTRODUCTION

An ideal test for antibody detection should be reproducible, specific, rapid, require a small amount of sample, easy to perform at low cost and biohazard, and easy to interpret. Given the availability of a flow cytometer, flow cytometric analysis for detection of antiplatelet antibodies meets many of these requirements. An increasing number of laboratories are beginning to apply the advantages of flow cytometry to platelet serology e.g., for serum antibody detection and platelet crossmatch. The analysis of sera containing multispecific antibodies, particularly the distinction between anti-HLA and platelet-specific antibodies, remains a challenge. A method is described here which permits recognition and distinction of anti-HLA and platelet-specific antibodies by the simultaneous use of both platelets and lymphocytes as target cells in flow cytometric analysis. METHODS Preparation of Platelet-Lymphocyte Mixture

Whole blood from HLA-typed, group 0, normal volunteers was collected into K,EDTA and diluted 1: 1 with 0.9% NaC1, pH 5.5; 9 ml of the anticoagulated blood was layered over 4 ml of Ficoll-Hypaque Solution (Pharmacia LKB, Uppsala, Sweden) in 15 ml conical polystyrene tubes. The tubes were then centrifuged at 300g for 30 min 0 1991 Wiley-Liss, Inc.

at 22°C. After centrifugation, the layers of mononuclear cells were harvested, resuspended in 0.9% NaCl containing 1% EDTA, and centrifuged for 1 min at 500g. The cell pellet was resuspended, fixed in 0.5% paraformaldehyde solution (PFA) [ 11 for 5 min, and then centrifuged at 500g for 10 min and resuspended in 2 ml 1% bovine serum albumin/phosphate buffered saline solution (BSA/ PBS), pH 6.8, containing 0.02% sodium azide, and stored at 4°C. This method generally yielded approximately 10 x 10'lml lymphocytes and 200 x 106/ml platelets. The cell pellet from a sample aliquot prepared in the same way was treated with either chloroquine or acid buffer (see below) prior to PFA fixation. Removal of Class I HLA Antigenicity From the Membrane of Platelets and Lymphocytes

Treatment of cells with citric acid buffer pH 3.0. The method described by Sugawara et al. was utilized for acid modification of HLA antigens of platelets [2] and of lymphocytes [3]. Briefly, a pH3.O solution was made with citric acid-Na2HP04 buffer, (equal volumes of 0.263 M citric acid and 0.123 M Na,HPO, buffer), Received for publication November 20, 1990; accepted July 11, 1991. Address reprint requests to Dr. John Freedman, Blood Bank, St. Michael's Hospital, 30 Bond Street, Toronto, Ontario, Canada M5B 1W8.

Technique: Antiplatelet Antibodies by Flow Cytometry

containing 1% BSA. Pelleted cells were incubated with 0.5 ml acid solution at 4°C for 5 min; the reaction was then stopped by adding the same citric acid buffer with pH adjusted to 6.5 and the mixture centrifuged at 500g for 10 min. The cells were washed and fixed in PFA as described above. The cells were stored at a concentration of approx. 200 X lo6 platelets/ml and 10 X lo6 lymphocytes/ml at 4°C in 1% BSA/PBS containing 0.02% sodium azide; the treated cells were used within 2 weeks of preparation. Chloroquine treatment of the cells was performed by the method of Nordhagen and Flaathen [4]. Briefly, 2 ml of a 0.4 M solution of chloroquine diphosphate (Sigma Chemical Company, St. Louis, MO) was added to a pellet of platelet-lymphocyte mixture containing approximately 1 X lo9 cells; after incubation at 4°C for 2 h with constant gentle agitation, the cells were washed twice and resuspended at 200 X lo6 platelets and 10 X lo6 lymphocytes per ml. The cells were fixed in PFA and stored at 4°C as described above. Control cells were prepared at the same time by incubating the cell pellet with PBS pH7, in place of either chloroquine or citric acid. Staining Procedure

Equal volumes (50 pl) of cells (untreated or treated as described above) and serum were incubated in 12 X 75 mm polystyrene tubes containing 0.1 ml BSAiPBS at 22°C for 30 min. The cells were then washed twice in PBS, resuspended in 100 p1 PBS and 100 pl of fluorescein isothiocyanate (F1TC)-conjugated F(ab’), goat antihuman IgG (heavy and light chain specific; Cappel Laboratories, Organon Teknika Corp., Westchester, PA), appropriately diluted in 1% BSAiPBS, added. Following incubation at 22°C in the dark for 30 min, the cells were washed twice in PBS and promptly analyzed in a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). The stained cells can be kept at 4°C in the dark for up to 5 days without adverse effect on analysis. On some occasions the cells were stained with phycoerythrin (PE)-conjugated goat antihuman IgM (TAGO, Burlingame, CA), either alone, or in doublelabeled studies with FITC-conjugated goat antihuman IgG. Anti-CD3 (Leu 4) and anti-CD45 (HLe-1) were purchased from Becton Dickinson, and anti-CD41 (platelet membrane glycoprotein (GP) IIb/IIIa) was purchased from AMAC Inc., Westbrook, ME. All monoclonal antibodies were used undiluted at a concentration of 20 pl per test; patients’ sera were used undiluted. Flow Cytornetry Analysis

The labeled platelet/lymphocyte mixture was analyzed using a 15 mW argon FACScan flow cytometer operating at 488 nm at 15 mW power, using Consort 30 and LYSYS software (Becton-Dickinson). Data acquisition

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of 20,000 events was gated on forward and right (side) angle light scatter with gains adjusted so as to include platelets and lymphocytes. Since the average platelet/ lymphocyte mixture prepared by the method described above contained approximately 20-fold more platelets than lymphocytes, some platelets were excluded during acquisition by increasing the forward-scatter threshold, allowing acquisition of sufficient number of lymphocytes for analysis (minimum 2000 events); debris was eliminated by the increased forward scatter threshold. Live gating during acquisition was used to exclude the larger monocytes, granulocytes, and aggregates of cells. The fluorescence channels (FLI and FL2 for FITC and PE conjugates respectively) were set at logarithmic gain and the detector adjusted to have the peak channel (mode) of the negative control platelets fall in the first log decade. Two-color compensation for FITC and PE was performed using Calibrite beads (Becton-Dickinson). The distinction of platelet and lymphocyte populations was confirmed by use of fluorochrome-labeled monoclonal antiplatelet and antilymphocyte antibodies (FITC-conjugated anti-GPIIbiIIIa and PE-conjugated anti-CD3, respectively); under these conditions, over 98% of gated cells stained with these markers. Once the appropriate analysis gate was set for given donor cells, the same gate was used for all patient sera incubated with those cells. In all experiments, at least three negative control sera (from normal, healthy, never-transfused, Group AB , males) were included; positive control was a pool of 10 sera from tranfused patients with strong polyspecific anti-HLA antibodies. Positive reactions were defined as a mean channel shift with test serum over negative control serum of at least 2 SD of the negative controls. Determinations with fifty normal sera indicated a significant shift (>2SD) to be represented by a ratio of > 1.4 for mean fluorescence channel of testicontrol. Since histograms sometimes were asymmetrical, positive reactivity additionally required a ratio of > 1.4 for the modes of the histograms of test/control. RESULTS

Following incubation of test serum (or negative control serum) with the platelet/lymphocyte mixture, acquired events were first analyzed ungated. Figure 1 shows a typical light scatter profile of the platelet/lymphocyte mixture. The platelets were clearly separated from the lymphocytes on the basis of size (forward light scatter; FSC), and analysis gates were placed around the platelet (A) and lymphocyte (B) populations. The cells had been stained with a FITC-conjugated anti-IgG and the dot plot in Figure 2A shows again that the platelet and the lymphocyte populations (small or large cells, or left and right populations on the FSC axis, respectively) incubated with a negative control serum are separable on the

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FSC Fig. 1. Dot plot of forward light scatter (FSC) versus side,

or right angle, light scatter (SSC) profile of platelet (A) and lymphocyte (B) mixture.

sions this necessitated separate analysis markers (represented in the figures by the horizontal line) for the two cell populations. The distinction between the nonspecific antibody binding or fluorescence (FLI) of the negative control (Fig. 2A) and the increased fluorescence intensity in FLI of bound IgG antibody when the cells were incubated with an anti-HLA-containing serum (Fig. 2B), was easily made. In Figure 2A only 2% of the events were in the analysis window above the horizontal line; in contrast, 97% of the events were above the line in Figure 2B. Fluorescence mean or mode channel numbers can be calculated by single parameter histogram analysis. Figure 2C shows overlaid histograms of FL1 for the platelets (unshaded histograms) and lymphocytes (shaded) shown in Figures 2A and 2B; in each case, the histogram on the left (a or c for platelets and lymphocytes respectively) represents FITC-anti-IgG binding to the cells incubated with negative control serum, and the histogram on the right (b or d for platelets or lymphocytes respectively) represents bound fluorescent anti-IgG following incubation of the cells with anti-HLA-containing serum. The greater height of the platelet histograms in the vertical axis reflects the greater number of platelets than lymphocytes in the mixture. Simultaneous detection of serum TgM antibodies is accomplished by two-color analysis with double-labeling using FITC-anti-IgG and PE-antiIgM and analyzing FL1 vs. FL2. Dual parameter analysis (FSC vs. fluorescence) using known antileukocyte (anti-HLe- 1; CD45), antiplatelet (anti-GP IIb/IIIa; CD41) or anti-HLA (anti-HLA Class 1) antibodies, is shown in Figure 3. The lymphocytes, but not the platelets, showed increased fluorescence with anti-CD45 (Fig. 3A). In contrast, platelets, but not lymphocytes, showed increased fluorescence with antiGP IIb/IIIa (Fig. 3B). The small number of large cells in Figure 3B staining weakly positive with anti-GP l1biIIIa may represent either aggregated platelets or contaminating monocytes; double-labeled studies with FITC-labeled anti-GP IIb/IIIa and PE-labeled anti-CD14 (specific for monocytesimacrophages) suggested that they were monocytes, although the possibility of platelets adherent to monocytes cannot be excluded. When patient’s serum containing anti-Pl*’ was tested, there was no reactivity with leukocytes (Figs. 6 b and e); the same was true for patient sera containing anti-Bak”, -Bakb, or -Brb. When the preparation was incubated with anti-HLA (Fig. 3C), as expected, both lymphocytes and platelets stained positively; similar results were obtained with sera from alloimmunized patients containing known anti-HLA antibodies. In order to distinguish between platelet-specific and anti-HLA antibodies concomitantly present in a serum, monoclonal antibodies were also tested using the platelet/ lymphocyte mixture pretreated with either citric acid or chloroquine. Figure 4 shows the relative fluorescence

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Fig. 2. Forward scatter (FSC) vs. fluorescence (FL1) of FITC-anti-lgG stained platelet/lymphocyte mixture incubated with either negative control serum (A) or serum containing anti-HLA antibodies (B).The overlay histogram of FL1 (C) shows platelets (a and b) and lymphocytes (c and d) incubated with either negative control serum (a and c) or with anti-HLA (b and d), stained with FITC-anti-lgG.

basis of size, but show similar background fluorescence intensity on the FL1 axis. Generally, as seen in Figure 2A, the lymphocytes gave a slightly higher background fluorescence than did platelets when incubated with normal human sera and second antibody; on rare occa-

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Fig. 4. Overlay histogram of fluorescence of untreated (A) and citric acid-treated (B) platelets stained with FITC-labeled negative isotype control (), anti-HLA A2 (..-. . ), anti-HLA Class I (. ), and anti-GP llblllla ( ).

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intensities of FITC-conjugated anti-GP IIb/IIIa, antiHLA Class I and anti-HLA-A2 with untreated (Fig. 4A) and with citric acid-treated (Fig. 4B) platelets expressing the corresponding antigens. The reactivity of anti-GP IIb/IIIa was not affected by citric acid treatment of the platelets. In contrast, however, the reactivity of antiHLA Class I and anti-HLA A2 was markedly decreased with citric acid-treated platelets. Reactivity of anti-HLA antibodies against untreated lymphocytes was also completely abrogated on testing against citric acid-treated lymphocytes. The reactions of sera from alloimmunized patients containing only anti-HLA antibodies were similarly nonreactive w.ith citric acid-treated platelets and lymphocytes, whereas sera containing both anti-HLA and platelet-specific antibodies showed disappearance of reactivity against treated lymphocytes, but not of the

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reactivity against treated platelets. Thus, sera which showed increased fluorescence against both untreated platelets and untreated lymphocytes could be distinguished into (a) only anti-HLA antibodies (nonreactive with citric acid-treated platelets and lymphocytes), or (b) combinations of anti-HLA plus platelet-specific antibodies (nonreactive with citric acid-treated lymphocytes, but reactive with citric acid-treated platelets). Non-HLA lymphocyte-specific antibodies could be defined which were reactive with untreated and citric acid-treated lymphocytes but not with either untreated or treated platelets. Figure 5 shows a comparison of anti-HLA Class I monoclonal antibody reactivity against cells treated with chloroquine versus the same cells treated with citric acid. It is evident that citric acid-treatment of the cells resulted in a better inhibition of anti-HLA reactivity than did

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shows examples of patterns observed with patient sera. In those cases where antibody was detected on both platelets and lymphocytes, approximately 65% showed the same degree of reactivity (fluorescence intensity) with both cell types, 15% showed stronger fluorescence intensity with platelets and in 20% the reactions were stronger with lymphocytes. DISCUSSION

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chloroquine treatment of the cells. This was true for all of four anti-HLA antibodies tested and for all donor cells tested. Furthermore, citric acid treatment of the cells requires less incubation time ( 5 min vs. 2 h), and citric acid stripping resulted in less nonspecific binding of antibody than was seen with chloroquine-treated cells. With chloroquine-treated cells a double peak was often seen, presumably reflecting lack of complete removal of strong HLA antigens. Prolongation of incubation time with chloroquine (up to 3 hours), or incubation at 22" or 37°C rather than 4°C resulted in better inhibition of HLA reactivity but caused increase in nonspecific fluorescence. The same anti-HLA and platelet-specific antibodies were used in weekly retesting with the same donor cells. With the preparation and storage conditions described in Methods, the untreated PFA-fixed platelet/lymphocyte mixture remained satisfactory for use for at least 3 months; beyond that time there was progressive increase in nonspecific antibody binding, particularly to lymphocytes, and the cells were in no instances usable by 6 months. The citric acid-treated cells were not used for more than 3 weeks, as cells stored for longer gave high background fluorescence resulting in false positive readings. Antibodies were detected in the sera of 36/80 (45%) patients who had received repeated random donor platelet transfusions. Of those sera containing antibodies, in 32 patients (89%) the antibody was reactive against both platelets and lymphocytes, in 3 patients (8%) antibody was detectable only against lymphocytes and one serum (3%) showed antibody only against platelets. Figure 6

A simple flow cytometric technique for detection of antiplatelet antibodies and distinction between anti-HLA and platelet-specific antibodies is described. Two important advantages of the flow cytometer were applied i.e., the ability to simultaneously acquire data on two different cell populations (platelets and lymphocytes), and the ability of performing two parameter analysis. The purpose of testing the sera with lymphocytes as well as with platelets was to confirm the presence of anti-HLA when the sera were reactive with platelets. Simultaneous use of platelets and lymphocytes was also advantageous in reducing time, work, reagents and amount of serum required compared to performing the tests separately; this may be of particular advantage when a panel of donors is used to determine antibody specificity. Testing of sera with lymphocytes in addition to platelets may increase the sensitivity of detection of anti-HLA antibodies. The density of Class I HLA antigens. and antigenic expression, can vary considerably from person to person as well as between lymphocytes and platelets of the same individual [ 5 ] . Several patient sera tested in the current study reacted only with lymphocytes, and the reactions become negative when tested with acid-treated HLA-stripped lymphocytes of the same donor, indicating that they were due to anti-HLA antibodies; nonetheless, the sera were nonreactive against untreated platelets from the same donor i.e., the presence of anti-HLA antibodies would not have been detected if platelets only had been used in the assay. The clinical significance of the lymphocyte-only anti-HLA antibodies is at present unknown. The majority of patient sera tested showed reactivity with both untreated platelets and lymphocytes. While the strength of reactivity or fluorescence intensity (assessed by mean fluorescence channel number compared to that of the negative control serum) was usually similar with both platelets and lymphocytes, in a few cases reactivity was distinctly stronger against one of the cell types, likely reflecting differing antigenic density on the different cell types. Using only platelets or only lymphocytes as the target cell, some sera might have been considered nonreactive or of equivocal reactivity. Nonetheless, antibodies giving weak reactions in vitro can potentially be quite significant in vivo and the current observations may explain some cases of apparent immune refractoriness to platelet transfusion where no

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Fig. 6. Patterns of reactivity (contour plots, smoothed x l ) of patients’ sera with untreated platelets and lymphocytes (A, B, and C) and with citric acid-treated platelets and lymphocytes (0,E, and F); A and D show a serum containing only anti-HLA (anti-HLA A3), 6 and E show a serum containing only anti-PI”’, and C and F show a serum containing both anti-HLA A1 and anti-Pl”’. Analysis markers were set based on negative control sera.

antibody has been detected in tests where only lymphocytes, or only platelets, have been employed as target cells. Use of anti-IgG and anti-IgM in double-labeled studies allows for investigation of antibody isotype switch, or failure of switch, from IgM to IgG, in antiplatelet antibody development; such studies may be useful in elucidating the natural history of antiplatelet antibodies in refractoriness to platelet transfusion. HLA immunization appears to be much more frequent than immunization to platelet-specific antigens. Plateletspecific antibodies may, however, be present together with anti-HLA antibodies. Absorption/elution techniques used to distinguish between HLA and platelet-specific antibodies are cumbersome. Attempts to block HLA antigens with potent monoclonal anti-HLA or with antilymphocyte globulins are unsatisfactory approaches to defining anti-HLA antibodies in a mixture of anti-HLA and platelet-specific antibodies [6,7]. Alternative ap-

proaches have included chloroquine [4,8] or acid treatment of platelets [2,9] and lymphocytes [3], to remove HLA antigens from cell membranes. Lucas [ 101 recently reported that, in a survey of 29 laboratories using different antibody detection assays, the use of chloroquine-treated platelets was of only limited value in accurately identifying anti-HLA and platelet-specific antibodies. Decary [ l I], on the other hand, in another multicenter study, found that chloroquine treatment was a reliable and reproducible method, helpful in discriminating anti-HLA and platelet-specific antibodies; she commented, however, that there were still problems with the chloroquine technique, especially when testing multispecific and high-titer anti-HLA antibodies. A potential problem of using chloroquine-treated cells in a fluorescence assay in particular is the high rate of cell death with chloroquine treatment [9], with consequent diffuse fluorescence of the dead cells. The viability of acid-treated

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platelets has been reported to be 83%, in contrast to 52% for chloroquine-treated platelets [9]. In the current studies, acid treatment of platelets and lymphocytes was superior to chloroquine treatment in removing surface HLA antigens (Figs. 4 and 5 ) . Thus, flow cytometric analysis is a useful technique in detection of antiplatelet antibodies. It is rapid and allows for many samples to be tested inexpensively. Simultaneous analysis of platelets and lymphocytes (particularly when both untreated cells and acid-treated cells are tested) allows distinction between anti-HLA and plateletspecific antibodies. This approach may have application not only in antibody detection and identification, but also in platelet crossmatching. REFERENCES I . Dunstan RA: Use of fluorescence flow cytometry to study the binding of various ligands to platelets. J Histochem Cytochem 33:l 176, 1985. 2. Kurata Y, Oshida M, Take H, Furubayashi T, Nakao H, Tomiyaina Y, Kanayama Y, Nagao N, Okubo Y, Yonezawa T, Tarui S: New approach to eliminate HLA class I antigens from platelet surface without cell damage: Acid treatment at pH 3.0. Vox Sang 57:199, 1989. 3. Sugawara S, Abo T, Kumagai K: A simple method to eliminate the

antigenicity of surface class I MHC molecules from the membrane of viable cells by acid treatment at pH 3 . J Immunol Methods 100:83, 1987. 4. Nordhagen R , Flaathen ST: Chloroquine removal of HLA antigens from platelets for the platelet immunofluorescence test. Vox Sang 48:156, 1985. 5 . Janson M, McFarland J , Aster RH: Quantitative determination of platelet surface alloantigens usjng a monoclonal probe. Human Immunol 15:251, 1986. 6. Scomick JC, LeFor WM: Antibodies to crossreactive HLA antigens: Evaluation by cytotoxicity, flow cytometry, and inhibition of monoclonal antibody binding. Transfusion 43:235, 1987. 7. Santoso S, Mueller-Eckhardt G, Santoso S, Kiefel V , MuellerEckhardt C: HLA antigens on platelet membranes. Vox Sang 51:327, 1986. 8. Masel DS, Blumbeg N, Heal JM: A chloroquine elution technique for platelet serology. Transfusion 28: 132, 1988. 9. Kurata Y , Oshida M, Take H, Furubayashi T, Mizutani H, Tomiyama Y, Yonezawa T, Tarui S: Acid treatment of platelet as a simple procedure for distinguishing platelet-specific antibodies from antiHLA antibodies: Comparison with chloroquine treatment. Vox Sang 59:106, 1990. 10. Lucas GF: A survey of platelet serology in UK laboratories (1987): An assessment of the efficacy of using chloroquine-treated platelets to distinguish between platelet-specific and anti-HLA antibodies. Clin Lab Haematol 12:185, 1990. 11. Decary F: Report on the Second Canadian Workshop on platelet serology. Curr Stud Hematol Blood Transfus 54:1, 1988.

Simple method for differentiating between HLA and platelet-specific antibodies by flow cytometry.

Since platelets express both platelet-specific and class I HLA antigens, serum antiplatelet reactivity assessed by most platelet antibody techniques c...
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