For Anti-HLA-Specific Donor Antibodies Detection By Flow Cytometry Cytotoxic Crossmatches Comparison of Methods C. Cervelli, F. Pisani, A. Aureli, R. Azzarone, M. Scimitarra, C. Battistoni, B. Di Iulio, D. Fracassi, M.A. Scarnecchia, A. Famulari, and F. Papola ABSTRACT Anti-HLA-specific donor antibodies induce rapid, irreversible destruction of the transplant (hyperacute rejection) that today happens rarely due to immunologic studiesdprospective crossmatchdof patients awaiting the kidney graft. The usual approach for pretransplant donor/recipient evaluation is based on 2 methods: (1) the cytotoxic complement crossmatch (CDC) and (2) the flow cytometric crossmatch (FCX). The CDC crossmatch is positive when complement-fixing antibodies are present, an absolute contraindication to kidney transplantation. The more sensitive FCX-positive crossmatch detects low concentrations of unable to fix performed antibodies complement. It is an “index” of possible damage due to accelerated rejection. The target of our study was to develop a cytotoxic flow cytometry crossmatch (cFCX) that detected cytotoxic antibodies move sensitively than the traditional CDC method and also was less subjective and more standardized for interpretation studying sera from 23 patients; the cFCX showed the requested efficiency characteristics even in an emergency. In addition, the new method permited one to calculate a cutoff for positivity (average value of the negative control þ 2 standard deviations), assuring an “objective” interpretation of the results that agreed with the CDC but was more sensitive and accurate allowing solution of ambiguous results for cases of “doubt”-positive CDC crossmatch. Furthermore, our aim was to correlate the effect of the strength of the anti-HLA antibodies determined by mean fluorescence intensity value of LabScreen Single Antigen beads with results of CDC, cFCX, and FCX methods. HE complement-dependent cytotoxicity (CDC) crossmatch (CDC-XM) and the flow cytometric crossmatch (FCXM) are routinely used to allocate a kidney graft. The FCXM, which is widely accepted as the most sensitive crossmatch test in current use, gates out and thus disregards dead and dying cells, reducing the need for high initial cell viability (>85% for CDC assays) and requiring less performance time. FCX assay detects the IgG versus IgM class of anti-HLA antibodies by the secondary antibody. Since IgG immunoglobulins class are believed to be responsible for early graft loss, they must be detected, rapidly. FCXM permits simultaneous detection of anti-HLA alloantibodies that bind to either T (CD3-positive cells) or B cells (CD19/ CD20-positive cells), eliminating the need for a physical separation of the cell populations. This information can facilitate the interpretation of crossmatch results due to the differential expression of HLA molecules, since T cells T display predominantly HLA class I molecules while B cells express both class I (at a higher density than T cells) and HLA class II molecules. The CDC technique, which requires viable cells, simultaneously detects IgM and IgG cytotoxic antibodies but requires more time and technical experience. Even if some authors claim that the FCXM is “too sensitive,” “inappropriately denying individuals a transplantation,” we believe that the test should be viewed From the Regional Center of Immunohematology and Tissue Typing (C.C., R.A., M.S., C.B., B.D.I., D.F., M.A.S., F.P.) and Organ Transplant Centre (F.P., A.F.), S. Salvatore Hospital, and C.N.R. Institute of Translational Pharmacology (A.A.), L’Aquila, Italy. Address reprint requests to Dott Carla Cervelli, Regional Center of Immunohematology and Tissue Typing, S. Salvatore Hospital, Lorenzo Natali street, Coppito, 67100 L’Aquila, Italy. E-mail: ccervelli@asl1abruzzo.it ª 2013 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 0041-1345/13/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.07.023 Transplantation Proceedings, 45, 2761e2764 (2013) 2761 2762 in the context of the immunologic history of the patient. A B cell- positive FCXM without evidence of anti- class II HLA antibody may can be due to the recognition of non-HLA surface molecules; it does not necessarily contraindicate kidney transplantation.1e4 For this reason we have elaborated herein a new method that sought to combine the characteristics of specificity and sensitivity of the FCXM with the possibility to detect both complement-fixing and non-fixing antibodies. MATERIALS AND METHODS Cell Suspension and Sera Peripheral blood mononuclear cells were obtained from the buffy coat of four healthy donors, who were completly typed for HLA class I and II antigens. After separation using Lympholyte medium (CEDERLANE), the phosphate-buffered saline (PBS) þ 2% fetal calf serum (FCS) washed cells were resuspended at 2  106/mL for a serologic CDC-XM and at 10  106/mL for FCXM and cytotoxic FCXM (cFCX) methods Sera, which were undiluted and 1:2, 1:4, 1:8 dilutions in the CDC technique are obtained from 23 patients, already tested by flow panel reactive antibody screening and Labscreen Single Antigen class I and/or II (One Lambda). They were used undiluted in the FCX and cFCX methods. A negative control serum (AB human serum, Euroclone) was employed in conjunction CERVELLI, PISANI, AURELI ET AL with a positive control serum prepared from a pool of sera from patients with PRA > 90% class I and/or II antibodies. CDC The CDC-XM was performed according to the “long incubation” method. Briefly, serua from patients was dispensed into wells of Terasaky microplates (1 sera mL) at 1:1, 1:2, 1:4 and 1:8 dilutions followed by 1 mL of cell suspension (w2  106 cell/mL) for a 1-hour incubation at room temperature before addition of rabbit complement (One Lambda). After a further 2 hours incubation at room temperature, the cells were stained with FluoroQuench (One Lambda) that allowed detection of both intact and lysed cells by fluorescence microscopy. cFCX To 25 mL of cell suspension (10 106 mL) were added to 50 mL of undiluted serum in a test tube, for a 30-minute incubation at room temperature. Then 200 mL of rabbit complement (One Lambda) were dispensed and 2 washed with PBS þ 2% FCS at 800 g for 5 minutes were performed after a further 30-minute incubation at room temperature. Following addition of 50 mL of IgG FITC (DAKO; at 1:40 dilution), 5 mL CD3 phycoerythrin (Becton Dickinson, BD) were added before incubation the tubes for 30 minutes at 4 C in the dark. After washing with cold PBS þ 2% FCS, 5 mL of 7 amino-actinomycin-D (BD) were added for a 10-minutes Fig 1. a) Serum with cytotoxic and non cytotoxic antibodies; Fig.1 b) serum mainly with non cytotoxic Ac.; Fig.1 c) serum with completely cytotoxic Ac; Fig.1 d) serum with cytotoxic “factors”. COMPARISON OF DSA DETECTION METHODS 2763 incubation 4 C in the dark. Finally 225 mL of PBS were added to the pellet before on the flow cytometer reading (FACSCalibur, BD). FCX Table 1. CDC, FCX, CFCX, and Flow PRA Comparison Flow pra screening Patient No. CDC FCX To 25 mL of cell suspension (10  10 cells6/mL) were added 50 mL of undiluted serum in a test tube. After a 30-minute incubation at room temperature, we performed 2 washes with PBS þ 2% FCS at 800g for 5 minutes. The final incubation included 50 mL IgG FITC (DAKO; 1:40 dilution), 5 mL CD3PerCP (BD) and 5 mL CD19PE (BD) for 30 minutes at 4 C in the dark. After 2 washes with cold PBS þ 2% FCS, 225 mL of PBS were added to the pellet for the flow cytometric analysis (FACSCalibur, BD). 1 2 3 4 5 þ þ þþ þþ þ Negative Negative Negative Negative BþT þ 6 7  Statistical Analysis 8 9 10 þþþ þþþ þ 11 12 13 þþþ þþþ þ 14 þ 15  16 17 18 19 þþþ þþþ The data acquisition and subsequent analysis were performed using CellQuest Software (Becton Dickinson). RESULTS Among 11 of 23 sera studied by the 3 crossmatch methods, the new cFCX technique demonstrated high levels of cytotoxic and noncytotoxic IgG antibodies (Ab; Table 1, Fig 1a,b,c). There were cases of noncytotoxic Abs associated with cytotoxic Abs (Fig 1a); one, of only cytotoxic Abs (Fig 1c) and a last one, mainly non complement fixing antibodies in high percentage (Fig 1b), while 5/23 sera revealed negative PRA screening without anti-HLA antibodies or very low PRA, the CDC and cFCX were positive, suggesting that the new technique showed some non-IgG, non-IgM cytotoxic factors (data not shown). Their features and clinical meanings are not clear (Fig 1d). The 23 sera were all studied for the presence/absence of anti-HLA Abs with the cytofluorimetric Flow PRA Screening method (One Lambda). Positive sera were analyzed by the Labscreen Single Antigen class I method (One Lambda) to identify individual specificities. Anti-HLA-specific donor antibodies (DSA) antibody specificities which were scored in mean fluorescence intensity (MFI), were correlated with crossmatch results obtained by CDC, FCXM, and cFCX techniques. Among our patients we observed some particular cases; the first was a patient with anti-HLA-A2 DSA with a MFI medium value of 8229 (cutoff 5000MFI), a PRA screening class I and II > 80%, a highly positive CDC-XM (1:8 dilution) and positive FCXM and cFCX. In contrast, a second patient showed only high titre non-complement fixing antibodies directed toward HLA B57 allele with an MFI value of 14500, PRA class I > 80% as well as negative CDC and cFCX but a positive FCXM. DISCUSSION The analysis of these results showed cFCX to highlight 2 antibody types: (1) complement-fixing cytotoxic, and (2) non-complement fixing, noncytotoxic antibodies, that bind to the lymphocyte surface. At the moment, the morphologic and functional difference between the 2 antibody types is unknown. A possible explanation could be that the test distinguishes different classes of IgG eg. IgG1 and IgG3 that 20 21 22 23 þþ CFCX -CD3 þ þ þ þ Negative Positive þþ Positive þþ Positive þþþ Weekly positive þ Positive þþ Positive þþ Positive þþ Weekly positive þ Positive þþ Positive þþþ Positive þþþ Negative Weekly positive Positive þþþ Positive þþ Positive þþþ Positive þþþ Positive þþ Positive þ Positive þþ Positive þþ Weekly positive þ Negative Positive (CUTOFF) Positive þþþ Positive þþþ Weekly positive þ Positive þþþ Positive þþþ Weekly positive þ Weekly positive þ Weekly positive þ Positive þþþ Positive þþþ Negative Negative Negative Negative Positive þþ Negative Class I Class II d 5% d 18% d d d d d d d 56% d 56% 96% 83% 64% 30% 97% 87% 97% 99% 60% 96% 60% 74% 93% 87% 73% d 98% 97% d 87% 93% d d d 95% 94% 96% 89% 91% 71% 21% d cFCX values  15 is considered to be positive (mean C-7.57 þ 2 DS (2  3.7) ¼ 15). CDC, cytotoxic complement crossmatch; FCX, flow cytometric crossmatch; cFCX, cytotoxic flow cytometric crossmatch; PRA, panel-reactive antibodies. þ, from 15 to 50; þþ, from 50 to 79; þþþ, from 80 to 100. are complement-fixing versus IgG2 and especially IgG4, non-complement-fixing. Five of 23 patients displayed a negative PRA screening for anti-HLA antibodies (Table 1), but revealed positive cFCX and CDC. The cFCX showed the presence of cytotoxic non-IgG and non-IgM “factors” (data not shown), whose characteristics and clinical significance are still unknown. On the basis of the guidelines already used in our laboratory for the FCXM, we established a positive cutoff based on a medium value of þ2 standard deviations above the negative control for the new assay. This cutoff permitted an “objective” interpretation of results that corresponded to those obtained with the classic method. Comparing the CDC, cFCX, and FCXM techniques, we notes (1) perfect correspondence between the classic CDC and the new cFCX, and (2) a the cFCX to be more sensitive and less subjective than the CDC method, particularly in the presence of a doubtful positive crossmatch.5 Another goal was to use Luminex Labscreen Single antigen class I to identify a cutoff of positivity of anti-HLA antibodies, thereby distinguishing “not permissive antigens” 2764 to be correlated with “supposed positivity” of the “virtual” crossmatch. For this reason we related the MFI value of the anti-HLA antibodies in DSA sera with the crossmatch results obtained by the 3 methods. Considering a mean fluorescence intensity (MFI) value of 5000 as the positive cutoff for “absolutely not permitted” antibodies, we observed that was not possible to establish an exact correspondence between the MFI and the results of the 3 crossmatches, consistent with the report of Thammanichanond et al.6 As evidenced in the 2 particular cases cited above, it was evident that the predictive value of the MFI for DSA was extremely limited and not always in accordance with the results of different tests especially the CDC. For this reason a virtual crossmatch is sometimes not advisable for clinical application. In fact, the virtual crossmatch uses data that (even if theoretically corrected) does not always reflect the attended prevision. This claim could be explained because the data are calculated using beads coated with purified antibodies that can present diversities from the native antigen expressed on the lymphocyte surface. Even as our work presents some limitations, such as the small number of the patients analyzed, and the application of the new experimental method only on T cells, we believe that the cFCX offers advantages: (1) potential applications in an emergency, (2) availability of an objective interpretation and evaluation for the simultaneous presence of cytotoxic and noncytotoxic antibodies, (3) greater sensitivity than the classical CDC, (4) little influence by cellular viability, or operator experience and (5) a new assay for the CERVELLI, PISANI, AURELI ET AL post-transplantation period to highlight cytotoxic versus noncytotoxic antibodies. In conclusion, in this study we combined 3 methods with the MFI value. The cFCX provides clinicians a useful tool to investigate the type of antibodies and their effects during the post-transplant period. REFERENCES 1. Kerman RH, Susskind B, Buyse I, Pryzblowski P, Ruth J, Warnell S, Gruber SA, Katz S, Van Buren CT, Kahan BD. 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