Positive XM Research Articles

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Many Histocompatibility and Immunogenetics laboratories across the world are increasingly using the tool of Virtual crossmatching to predict positive XM based on the information about the presence of donor-specific HLA antibody (HLA-DSA) in patients. However, lab to lab variation in quantifying HLA-DSA is the major limitation to determine a cutoff concentration of DSA that can predict a positive XM. Here, we address this issue by assessing a cutoff value for HLA-DSA detected by solid-phase immunoassay (represented as mean fluorescence intensity, MFI) in a retrospective analysis that can virtually predict a positive CDC-AHG XM. We performed a retrospective study for 61 XM performed for 53 renal transplant recipients. Availability of results for HLA antibodies in patients, HLA typing of donor and patient and result of CDC-AHG XM were the inclusion criteria. The MFI values for DSA in patients were compared with the results of CDC-AHG XM. HLA class I and/or class II DSA were identified in 34 renal transplant patients. DSA strength (MFI value) significantly correlated with the CDC-AHG XM outcome (r = 0.82; p < 0.0001). An MFI value of 11500 showed 100% specificity, 93% sensitivity, 100% positive predictive value and 88% negative predictive value for predicting a positive T cell and/or B cell CDC XM. Our results showed that HLA-DSA strength correlated well the XM outcome. A precise appraisal of HLA-DSA strength (i.e. a cutoff MFI value) can significantly enhance the predictability of virtual crossmatching. The clinical significance of HLA-DSA in cases where DSA is present in strength lower than the cutoff is critical in patient management and requires assessment.

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Aim In general two types of results from anti-HLA class-1 antigen bead assays are highly predictive of cross match (XM) outcomes. Negative screen assays routinuely predict negative XMs and highly reactive donor specific single antigen beads (SAB) tests usually predict positive XMs. The laboratory, however, is faced with a number of circumstances where SAB assays do not reliably predicted XM results. In one situation, donor specific antibody (DSA) is detected but at a low level. In another circumstance, a relatively high level of DSA is observed but the XM is surprisingly negative. Methods XMs were routinely performed by CDC Amos one wash and by flow cytometry for deceased donor kidney transplantation. Anti-HLA antibody detection and quantitation are performed with screening and SABs, respectively (Gen-Probe, CT). DSA calculations are performed by addition of the net MFI of all positive beads, net MFI reactivity score (NMRS). SAB assays for DSA that detects C1q reactive classes of IgG and the standard assay (One Lambda, CA) were performed on selected patients. Results In the former situation, where low levels of DSA are detected, we show that the analysis of the DS epitopes provides a better quantitative window for predicting the outcome of the XM. Analyses including only the DSA beads had a range of 3400 NMRS where positive or negative XM results might be obtained, whereas analyses that included all beads containing the reactive DS HLA epitope had a range of “uncertainty” of 1500 NMRS. In the other circumstance we observed a relatively high level of DSA with negative XMs (n = 5 of 379 XMs involving 82 recipients). All of these potential recipients had DS antibodies that did not fix complement as determined in C1q SAB binding assays. Conclusions Though the above situations occur in less than 10% of the XMs they take up considerably more laboratory time and effort. Epitope analysis and C1q tests may reduce the effort and uncertainty of correlating solid state antibody tests with XM outcome. Norin: Gen-Probe: Speakers Bureau.

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Highly sensitized (PRA ⩾ 80%) patients have a significantly reduced probability of finding a matched renal transplant. Newer solid-phase antibody identification assays (Luminex) allow accurate detection and characterization of recipients’ comprehensive anti-HLA antibody profiles. Virtual crossmatching (VXM) utilizes the recipients’ anti-HLA antibody profile and donor’s HLA typing to predict a positive XM. Those antigens that cross-react with high mean fluorescence intensity (MFI) donor-specific antibodies (DSA) are considered unacceptable antigens (UA), a factor used for UNOS calculated PRA (cPRA) formula. As of 2008, all patients on our institution’s deceased donor kidney waitlist have been tested using solid-phase assays. Since 2009, the VXM replaced the prospective preliminary CDC crossmatches. We evaluated the impact of using VXM instead of prospective CDC crossmatches for patient selection in deceased donor renal transplants. For all patients on our institution’s UNOS deceased donor kidney waitlist, a quarterly PRA screening and a yearly single antigen bead (SAB) analysis was performed in sensitized patients. Antibodies with MFI > 3500 were identified as UA’s. A significant change in PRA (>20%) prompted a new SAB analysis. Patients with 1 strong (MFI >5000), more than 2 moderate (MFI = 2001-5000) and 3 weak (MFI = 700-2000) DSA’s were removed from the match run list. All donors were typed for the HLA-A, -B, -C, -DRB1, -DQB1, -DPB1 and DQA1 loci. The transplant rates in highly sensitized patients increased from 0% in 2006 to 14.7% in 2010 (P < 0.05) after the implementation of the VXM in 2009. In addition, the transplant rates for sensitized patients with PRA ⩾20% also increased from 14.3% in 2006 to 20.6% in 2010. For our center, the positive predictive value of the VXM was 80% and 81% for the final T-cell and B-cell FCXM’s, respectively. VCXM resulted in increased deceased donor kidney transplants, particularly in highly sensitized patients.

Effect of Implementing Anti-HLA Antibody Detection by Luminex in the Kidney Transplant Program in Chile

The development of new highly sensitive, specific technologies to detect HLA antibodies has allowed a better definition of the profile of non-permitted antigens for patients awaiting kidney transplantation. The use of calculated or virtual panel reactive antibodies (CPRA or vPRA) seeks to improve the prediction of positive crossmatches (XM), but increases the proportion of sensitized patients on the waiting list. In 2008-2009, we implemented detection of antibodies using Luminex technology and applied vPRA since 2009. The objective of this study was to evaluate the impact of these innovations in defecting patient sensitization on kidney transplant waiting lists for deceased donors and among transplanted patients. We analyzed the waiting list for 2007 through 2009 and the first semester of 2010, including the patients transplanted in these periods and the XM with deceased donors. We observed an increase in the mean peak PRA of transplanted patients from 7.2% in 2007 to 17.1% in 2010 ( P = .001), and in the proportion of patients transplanted with a peak PRA > 50% from 2.8% in 2007 to 15.7% in 2010 ( P = .0001), with no increase in the proportion of this population on the waiting lists. There was a concurrent decrease in positive XM among patients with a peak PRA > 50%. The use of vPRA and Luminex permitted a greater number of transplants of patients with peak PRA > 50% and was a good predictor of a positive XM.

Donor-Directed MHC Class I Antibody Is Preferentially Cleared from Sensitized Recipients of Combined Liver/Kidney Transplants

For patients with chronic renal and liver diseases, simultaneous liver and kidney transplantation (SLKT) is the best therapeutic option. The role of a pretransplant donor-specific antibody (DSA) in SLKT is unclear. We report the results of a retrospective review from 7/08 to 10/09 of SLKT at our institution. Monitoring of DSA was performed using single antigen bead assay. Between 7/08 and 10/09, there were six SLKT who had preformed DSA and positive XM (four class I and II DSA, one class I DSA only, one class II only). One-year patient and renal graft survival was 83%. Death-censored liver allograft survival was 100%. Acute humoral rejection (AHR) of the kidney occurred in 66% (three with both class I and II DSA and one with only class II DSA) of patients. In those with AHR, class I antibodies were rapidly cleared (p < 0.01) while class II antibodies persisted (p = 0.25). All patients who had humoral rejection of their kidney had preformed anticlass II antibodies. Liver allografts may not be fully protective of the renal allograft, especially with pre-existing MHC class II DSA. Long-term and careful follow-up will be critical to determine the impact of DSA on both allografts.

C4d staining in post-reperfusion renal biopsy is not useful for the early detection of antibody-mediated rejection when CDC crossmatching is negative

Sensitized patients (pts) may develop acute antibody-mediated rejection (AMR) due to preformed donor-specific antibodies, undetected by pre-transplant complement-dependent cytotoxicity (CDC) crossmatch (XM). We hypothesized that C4d staining in 1-h post-reperfusion biopsies (1-h Bx) could detect early complement activation in the renal allograft due to preformed donor-specific antibodies. To test this hypothesis, renal transplants (n = 229) performed between June 2005 and December 2007 were entered into a prospective study of 1-h Bx and stained for C4d by immunofluorescence. Transplants were performed against a negative T-cell CDC-XM with the exception of three cases with a positive B-cell XM. All 229 1-h Bx stained negative for C4d. Fourteen pts (6%) developed AMR. None of the 14 protocol 1-h Bx stained positive for C4d in peritubular capillaries (PTC). However, all indication biopsies-that diagnosed AMR-performed at a median of 8 days after transplantation stained for C4d in PTC. These data show that C4d staining in 1-h Bx is, in general, not useful for the early detection of AMR when CDC-XM is negative.

Heat Inactivation Can Differentiate Between IgG and IgM Antibodies in the Pretransplant Cross Match

The presence of IgG antibodies in the pretransplant cross-match (XM) test results in hyperacute rejection, but IgM antibodies are inconsequential. The XM should be able to differentiate between IgG and IgM antibodies. This study evaluated 3 methods. This study was based on 500 patients for whom XM were performed between 2004 and 2006 with all 3 techniques. Two patient sera were used: normal serum and heat inactivated serum, which was prepared by incubating patient serum at 63°C for 10 minutes to destroy IgM antibodies. The efficiencies of flow cytometry XM (FC-XM), dithiothreitol complement-dependent microlymphocytotoxicity (DTT/CDC-XM), and heat inactivation (HI-CDC-XM) to differentiate between IgG and IgM were evaluated by using both sera. Patients with positive XM, and negative HI-CDC-XM were reported as negative XM. During the study period, there were 70 patients with positive B-cell XM. Forty-nine became negative after HI-XM, and 21 remained positive. Only 34 cases became negative after DTT-CDC-XM and 36 remained positive. HI-CDC-XM was comparable to FC-XM; all patients testing negative with this technique experienced successful renal transplantations without hyperacute, accelerated, or acute rejection episodes. Our study showed that HI-CDC-XM was effective at exclude donor-specific IgM antibodies, a result which was comparable to FCXM to detect only IgG antibodies. HI is simple and rapid and does not involve any extra equipment or cost.

211: False Negative Virtual Crossmatch in Lung Transplantation

Purpose: At our institution, virtual crossmatch for thoracic organ transplantation utilizes a flow cytometry based PRA screening assay to determine the presence of anti HLA antibodies (Ab). In 15/16 cases, a negative virtual crossmatch (XM) successfully predicted a negative retrospective complement dependent cytotoxicity (CDC) XM. We determined the molecular basis of a discrepancy between the virtual cross-match and post-transplant positive CDC XM in a patient who underwent bilateral lung transplantation. Methods and Materials: Pre-transplant serum was analyzed using flow cytometry, ELISA, and single antigen bead assays. High resolution HLA typing was performed on donor tissue to assess donor HLA antigens. The presence of Ab was followed overtime utilizing flow cytometric analysis of donor cells with recipient serum. Results: Both the ELISA and flow cytometry PRA screens were negative for preformed antibody against HLA molecules. Following a positive CDC XM, single antigen Luminex based assay was performed and revealed anti-donor DR13 Ab. This Ab was eliminated using a protocol of apheresis, IVIG and anti CD20 therapy. Clinical improvement of the patient correlated with decreasing antibody titers following therapy. Conclusions: The current practice of utilizing a single method (ie flow cytometry PRA screen) to assess patients for the presence of anti HLA antibodies can result in the failure to identify clinically relevant Ab. The predictive value of the virtual crossmatch can be improved through the addition of a Luminex based assay system. A combination of plasma phoresis, IVIG and anti CD20 antibody can be used to successfully treat acute graft dysfunction associated with preformed donor specific anti HLA antibodies.

Cardiac Transplant Outcomes in Pediatric Patients with Pre-formed Anti-Human Leukocyte Antigen Antibodies and/or Positive Retrospective Crossmatch

Children undergoing heart transplantation who have preformed anti-human leucocyte antigen (HLA) panel reactive antibodies (PRA) or positive retrospective crossmatch (XM) may be at increased risk for rejection and graft failure. We assessed outcomes of transplant recipients with either positive PRA before transplant or positive retrospective XM. A review of 148 heart transplant patients between 1990 and 2006 was undertaken, identifying transplants in patients with pre-transplant PRA > 1% and/or a positive XM. Demographic information and detailed post-transplant outcomes including episodes of rejection, infection, and graft failure were recorded. There were 11 PRA positive (PRA+) transplants, 135 PRA negative (PRA-) transplants, and no PRA data on 2. There were 14 XM+ transplants, 115 XM- transplants, and no XM data on 19. Kaplan-Meier graft survival was better in XM- than XM+ patients (p < 0.015), but not different between PRA+ and PRA- Groups. Timing of first rejection and number of rejection episodes were not different between XM+ and XM- Groups or between PRA+ and PRA- Groups. Infections were not different between PRA or XM Groups. Four patients were PRA+/XM- (all PRA, 1%-10%), 7 were PRA-/XM+, and 7 were PRA+/XM+ (6 of 7 PRA >10%). Pediatric heart transplant patients whose retrospective XM is positive are at significantly increased risk for graft failure. Elevated pre-transplant PRA may not predict increased risk of graft failure, although markedly positive PRA (>10%) predicts a positive retrospective XM. Improved treatment for pediatric transplant patients with a positive retrospective XM is needed.

568: Cardiac transplant outcomes in pediatric patients with preformed anti-HLA antibodies and/or positive retrospective crossmatch

Purpose: Children undergoing heart transplantation (HTx) who have preformed anti-HLA antibodies (PRA) and/or a positive retrospective crossmatch (XM) may have increased complication rates. Pragmatic limitations preclude routine prospective donor XM. This study assessed the outcome of patients that received HTx at our institution who had either positive PRA before transplant or a positive retrospective XM.

The virtual crossmatch – A screening tool for sensitized pediatric heart transplant recipients

Heart transplantation in the setting of human leukocyte antigen (HLA) sensitization is challenging, as a time-consuming prospective crossmatch (XM) may be required, severely limiting the number of potential donors. We evaluated a 'virtual XM', defining a positive virtual XM as the presence of recipient pre-formed anti-HLA antibodies to the prospective donor HLA type, and compared the virtual XM to a standard direct XM. Bead-based flow cytometric analysis was used to identify anti-HLA antibody (Ab) present in a child listed for heart transplantation. Using recipient serum, direct-flow cytometric T- and B-cell XM were run for potential donors against whose HLA type the recipient had specific antibodies (group 1, n = 7) and for potential donors with predicted compatible HLA types by virtual XM (group 2, n = 7). Results were expressed as median channel difference (MCD) between the control and recipient serum. A positive T-cell XM was defined as MCD > 50, whereas MCD > 100 constituted a positive B-cell result. The rate of T-cell reactivity was significantly less in group 2 than in group 1 (29% vs. 100%, p = 0.02); similarly, B-cell reactivity was also less for group 2 (14% vs. 100%, p = 0.005). The virtual XM was 100% sensitive in detecting positive flow cytometric XM results for T and B cells. Although only 72% specific in predicting a negative T-cell XM, and 86% specific for negative B-cell XM, the false negatives were weakly positive and would probably have been clinically acceptable. Currently, potentially suitable donor organs are often declined for lack of a prospective XM; these organs may ultimately be allocated to more distant recipients or perhaps not used at all. While further studies are needed, virtual XM has the potential to improve availability of organs for sensitized patients and improve the overall allocation process.

Cross-reactivity between swine leukocyte antigen and human anti-HLA-specific antibodies in sensitized patients awaiting renal transplantation.

Xenotransplantation is increasingly viewed as a promising way to alleviate the problem of patients who have alloreactive lymphocytotoxic antibodies and therefore tend to accumulate on the waiting list for renal transplantation. One barrier to xenotransplantation in these patients could be the hyperacute or acute vascular rejection as a result of preexisting anti-HLA antibodies that recognize swine leukocyte antigens. The cross-reactivity of sera from 98 patients with pig lymphocytes was studied by flow cytometry. After absorption of xenoreactive natural antibodies (XNA), isotype, class, and antibody specificity causing a positive cross-match (XM) were determined. For nonsensitized patients, all of the antibody binding to pig lymphocytes was due to XNA, which were removed by pig red blood cells absorption. In contrast, in sensitized patients, after removal of XNA, pig lymphocyte XM remained positive. There was no correlation between antibody binding to pig lymphocytes and Ig isotype (IgG or IgM) or HLA class-specific antibodies. For testing evidence that class II-specific antibodies were responsible for antibody binding to pig lymphocytes, HLA class I-specific antibodies were absorbed with pooled human platelets. It was confirmed that HLA class II-specific antibodies were responsible for the positive pig XM, but the strength of the positive XM was weaker than the strength caused by HLA class I-specific antibodies. Sera with multiple specificities (plurispecific sera) displayed a greater frequency of cross-reactivity with swine leukocyte antigens (P < 0.05). Seven of 11 highly immunized patients without cross-reactivity IgG with porcine lymphocytes showed positive XM before an IgM was used. The results demonstrate the cross-reactive nature of HLA antibodies and therefore point out the need to perform a prospective XM after absorption of XNA in presensitized individuals.

INFLUENCE OF RACE ON CROSSMATCH OUTCOME AND RECIPIENT ELIGIBILITY FOR TRANSPLANTATION1

Data from the Office of the Inspector General and the United Network for Organ Sharing suggested that black end-stage renal disease patients had unequal access to organ transplantation, in that they waited twice as long as white ESRD patients for a renal transplant. We hypothesized that preTx histocompatibility factors were more influential in determining how quickly an ESRD patient was transplanted than had been realized by either the Office of the Inspector General or UNOS. To test this hypothesis we compared the crossmatch reactivity of 378 white and 227 black ESRD patients awaiting a primary renal Tx against 100 consecutive cadaveric donors (80 white, 10 black, and 10 Mexican-American). A positive XM frequency of 16% (179 positive XM/1121 total XM) was observed when white ESRD patients were crossmatched against white donors. A comparable frequency of 21% (39/186) (+) XM was observed when white ESRD patients were crossmatched against black donors. Similarly, black ESRD patients crossmatched against black donors showed a 17% (20/115) (+) XM frequency. In contrast, a significantly higher (+) XM frequency of 43% (283/655) was observed when black ESRD patients were crossmatched against white donors (P less than 0.001). When black ESRD patients with a (+) preTx blood transfusion history were crossmatched against white donors, a 55% (241/438) (+) XM frequency was observed. This (+) XM frequency was 3.2 times greater than when black ESRD patients were matched against black donors (P less than 0.001). However, when untransfused black ESRD patients were matched against white donors only a 19% (42/217) (+) XM frequency was observed. Finally, preoperatively transfused black ESRD patients displayed a higher PRA of 47 +/- 12% vs. 27 +/- 10%, P less than 0.01, and a longer median waiting time to transplant of 329 vs. 181 days, P less than 0.01, than comparable white patients. The data suggest that presentization of blacks, following preTx blood transfusions from nonblack donors, predisposes black ESRD patients to present as immunologically inappropriate recipients for predominantly white donor allografts and results in their spending a longer time on renal transplant waiting lists.

Clinical applicability of the immunomagnetic beads technique for serological crossmatching in renal transplantation

The aim of the present prospective study was to investigate the clinical applicability of the immunomagnetic (IM) beads technique for serological crossmatching (XM) in renal transplantation. The IM XM were read after various periods of incubation, and the results were compared with those obtained by the conventional Kissmeyer-Nielsen (KN) technique. A total of 132 sera from 96 potential recipients were tested against cells from 62 donors. Eight-nine KN T-cell XM-negative renal allograft transplantations were performed, and the IM XM results were related to clinical 3-month follow-up data (incidence of primary non-function, never functioning grafts, graft losses and rejection episodes). The IM technique was clearly more sensitive than KN, and sensitivity increased markedly with increasing duration of incubation. KN-, IM+ reactions were predominantly found among sera from patients with panel-reactive antibodies (PRA, 2p less than 0.01), and thus probably caused by HLA antibodies. However, positive IM XM, appearing after more than 35 min of incubation, did not influence the overall clinical outcome in the observation period. With reading after exactly 35 min of incubation, XM results obtained by IM and KN techniques correlated well. Thus, we believe, that the IM XM technique will be as safe and effective in avoidance of hyperacute rejections as the conventional assay. In the present material, the incidence of primary nonfunction was significantly (2p = 0.0023) higher among PRA+ recipients compared to PRA- patients. To conclude, we recommended the IM technique with reading after exactly 35 min of incubation for easy, fast (70 min) and reliable XM, that is always possible to perform using peripheral blood.