FlowPRA Beads Research Articles

P047 A cold case: When single antigen beads assay do not respond

Case history: We evaluated a live donor (D1) and recipient (P1) pair for potential kidney donation however their flow crossmatches (FXM) continuously result in B-cell positive. P1 was tested negative by single antigen beads and no donor specific antibody (DSA) was evident. Patient has been transfused once in 2014 and treated with adalimumab for ankylosing spondylitis in the past. Methods: Autologous FXM of D1 was conducted. A series of FXM with surrogate donors was conducted. Supplemental single antigen beads, FlowPRA beads and Luminex-based mixed screening beads were also conducted but results are questionable. Results: After the first allo FXM, a repeat was done to rule out B cell positive FXM whether it was reproducible. Since patient was screened negative by single antigen beads assay and supplemental beads, absence of DSA could not explain why B cell FXM can be positive. When patient serum subject to two other surrogate donors (D2 “O” and D3 “A”) interestingly B cell FXM has become negative. To rule out blood type issue and non–HLA factors, autologous FXM of D1 and allo-FXM with zero PRA serum (P2 “A”), and with positive PRA serum (absence of DSA, P3 “A”) were performed and all were found negative, but remained positive with diluted serum of P1. Patient P1 was tested again in 3 months however the same result remains. The final FXM was then focused on testing with surrogate donors (D4 and D5) who carry the same class II antigens as D1. The result became positive with D5. Although no clear evidence can confirm the antibody to DR11 to be allele specific, the collective results infer that suspected DSA DR11 is not distinguishable by FlowPRA screening beads and mixed beads and not detectable by single antigen and supplemental beads. Conclusion: This is the first case in our experience to discover such a situation with “cold bead” or specificity-unproven DSA. Transplantation is not recommended. Correlation between different assays and diligent testing to clarify unreasonable positive FXM is very important especially in cases of autoimmune disease.

P045 Kidney transplant with historic but no current DSA – A case study

Presence of donor-specific HLA antibodies (DSA) at the time of transplant is associated with poor outcome. DSA may be present in historic serum samples but not at the time of a kidney offer. Going forward with the transplant holds the risk of a memory B-cell response upon re-exposure to a specific HLA antigen or epitope. A patient with X-linked Alport syndrome was investigated for a kidney transplant. Anti-HLA class II antibodies were observed following pregnancy, which were specific to her husband’s mismatched DR and DQ antigens. A year later, she received a blood transfusion and had an episode of aseptic peritonitis while on dialysis. Class I HLA antibodies appeared but were observed over a period of 7 months. Thereafter, anti-HLA antibody screening using FlowPRA beads and identification using Luminex single antigen beads showed disappearance of class I antibodies but persistence of a single anti-HLA-DR antibody developed after prior pregnancy. Three years later, a kidney from a DCD donor was offered and transplanted despite presence of historic DSA against 2 HLA-B antigens. A CDC crossmatch proved negative using a current serum whereas a flow cytometric crossmatch using 3 DSA-containing historic sera gave positive but inconsistent results (T + B+, T-B+, T-B-). One week after transplant, historic DSA reappeared (3000 MFIs). Despite absence of biopsy-proven rejection, short-term treatment comprising plasmapheresis, rituximab and low-dose IVIg was initiated. DSA quickly disappeared from circulation with only an HLA-DR-specific reactivity from the previous pregnancy. Without any further desensitization treatment, the patient has not demonstrated re-appearance of DSA nor experienced antibody-mediated rejection (AMR) as demonstrated upon for-cause and protocol biopsies, up to 8 months posttransplant at the time of abstract submission. This case demonstrates that, although DSA may reappear following re-exposure to a sensitizing HLA antigen/epitope, acceptable transplant outcome can be achieved without AMR. Time between DSA development and transplant could be a factor in identifying acceptable HLA mismatches.

P015 Removal of selected non-specific reactivity in single antigen bead assays via FBS pre-treatment

Aim Non-specific reactivity is a known complication of single antigen bead (SAB), solid phase assays. Several approaches have been tried to reduce these reactions but are mostly related to Class I (eg: iBeads, acid treatment). No such treatments exist for Class II. Presently, the current lot of Class II SABs (LS2A01, Lot#: 011) has shown strong non-specific reactivity with some DRB1∗04 and DRB1∗16 bearing beads in patients who are otherwise non-sensitized; MFI values up to 4000. Previously, we reported that pre-treatment of patient serum with fetal bovine serum (FBS) effectively reduced non-specific binding to HLA Class I and Class II FlowPRA beads. In this study we sought to determine if FBS treatment would be effective in reducing/eliminating this non-specific class II reactivity. Methods Sera from 20 non-sensitized patients, each exhibiting the DR4/DR16 pattern, were pre-treated with FBS. Briefly, 5ul of FBS was added to 95ul of patient serum; incubated for 30 min at 37 °C; centrifuged and then used in the SAB assay. As controls, we treated sera from patients with well documented HLA antibody including DR4/DR16 (N = 10) and patients without HLA antibody (N = 10). Results Our results showed that in 100% (20/20) of the effected patients the DR4/DR16 reactivity was eliminated or significantly reduced. (Fig.). Importantly, patients with bona fide HLA antibody had no change in MFI values and additional reactivity was not observed in patients without HLA antibody following FBS treatment. Download : Download high-res image (339KB) Download : Download full-size image Conclusions In this study, we have demonstrated that the non-specific DR4/DR16 reactivity pattern was eliminated by pre-treatment with FBS. We speculate that this pattern is a result of reactivity against bovine peptides (possibly derived from culture media) present in the grove of the DR4/DR16 alleles. Pre-treating sera with FBS provides an exogenous source of soluble target proteins that interact with the antibodies thereby preventing their subsequent interaction with the beads.

How do we make the best choice in assays used to monitor HLA antibodies in renal recipients awaiting transplant? A comparison of flowpra beads with luminex single-antigen beads

Introduction Solid-phase HLA antibody assays are sensitive and specific to identify antigen and allele-level specificities. Protocol changes to test frequency and the addition of FlowPRA bead (FPB) screening to monitor HLA antibodies in renal patients required post-implementation monitoring. This study correlated Luminex single-antigen bead (SAB) and FPB test results to monitor the effectiveness of these changes. Methods SAB for antibody ID and FPB for screening are performed quarterly on actively-waitlisted renal recipients. This study evaluated agreement based on current SAB positive cutoff MFI values: 3000 for HLA-A and -B, 5000 for HLA-C and 1000 for all Class II specificities. Class II results with cutoff ⩾3000 were included to gauge sensitivity. The current FPB positive cutoff is ⩾5% over the negative control. Results 615 serum samples from 261 patients tested within a 15-month window after FPB implementation were evaluated for agreement by patient number and number of samples tested. Patient Numbers : SAB and FPB Class I results did not agree in 84 patients (32.2%); 63.1% of patients had false positive (FP) and 36.9% had false negative (FN) results. With a MFI ⩾1000 cutoff, Class II results did not agree in 119 patients (45.6%); 0.8% of patients had FP and 99.2% had FN results. With a MFI ⩾3000, Class II results did not agree in 49 patients (18.8%); 18.4% of patients had FP and 81.6% had FN results. Sample Numbers : SAB and FPB Class I results did not agree in 162 serum samples (26.3%); 60.5% of samples had FP and 39.5% had FN results. With a MFI ⩾1000 cutoff, Class II results did not agree in 312 samples (50.7%); 11.9% of samples had FP and 88.1% had FN results. With a MFI ⩾ 3000 cutoff, Class II results did not agree in 128 samples (20.8%); 33.6% of samples had FP and 66.4% had FN results. Conclusion HLA antibody monitoring is important to anticipate donor-specific reactivity in renal transplant recipients. If ID and screening assays are used to monitor these levels, an acceptable degree of correlation between methods is important. The FPB assay is not as sensitive or specific as SAB, especially in detecting weak, clinically-significant specificities. This study identified weakness in our protocol, leading us to evaluate other HLA antibody assay options.

P032 : DSA IN THE ABSENCE OF KNOWN SENSITIZING EVENTS: WHAT TO DO?

Donor-specific HLA antibodies (DSA) carry a risk of antibody-mediated rejection (AMR) in kidney transplants. However, the relevance of DSA seen in unsensitized patients prior to transplant is unclear. We report a case of DSA in an unsensitized male investigated for potential live kidney transplantation. HLA typing of the patient and potential donor was performed by low/intermediate and high resolution SSP. HLA antibodies were screened on serial serum samples using Flow PRA beads, Luminex single antigen beads (SAB), Luminex phenotype beads, Flow phenotype beads and Flow single antigen beads. Crossmatching was performed by CDC and flow cytometry. A 41-year old male without prior sensitization history was investigated for a potential unrelated live kidney transplant (HLA mismatches: A1, B44, Cw4, DR7, DR13, DQ2, DQ6). Initial HLA antibody screening using Flow PRA beads appeared negative but Luminex SAB testing revealed antibodies to A25, A32, DR16 and mismatched DQ6 (5/6 beads including donor DQB1 ∗ 06:04/DQA1 ∗ 01:02-associated bead, MFIs ∼7000). Serum testing using Luminex phenotype beads, Flow phenotype beads and Flow single antigen beads all suggested reactivity against the mismatched DQ6 antigen. Interestingly, DTT-treated serum showed similar Luminex SAB reactivity patterns as untreated serum but fetal bovine serum-absorbed serum showed reactivity only to mismatched DQ6 beads and uncovered very weak class II reactivity on Flow PRA beads. The T- and B-cell CDC crossmatches were negative. Both T- and B-cell flow crossmatches were negative although the B-cell flow crossmatch showed a slight median channel shift of 70, albeit lower than our validated cut-off of 150. No prozone effect was observed on serum dilution. The clinical relevance of this HLA-DQ6 DSA is unclear in the context of negative crossmatches and the patient has not been transplanted yet. Still, despite no known sensitizing event, pretransplant DSA could lead to AMR, as recently suggested, and could represent an immunological risk.

P039 : CORRELATION OF FLOW HLA ANTIBODY SCREENING AND LUMINEX SINGLE ANTIGEN BEAD ASSAY

Introduction Solid phase HLA antibody detection is more sensitive and specific compared to traditional CDC tests. Different solid phase methods use different platforms. FlowPRA uses 30 beads coated with HLA antigens of 30 individuals representing the most common HLA antigens. Luminex single antigen bead assay (LSA) uses up to 100 beads, each of which is coated with antigen molecules of a single specificity, and has much better coverage of the HLA antigens. Aims This study was to determine a correlation between FlowPRA and LSA in regarding if a negative FlowPRA would correlated to a negative LSA assay. Methods We use FlowPRA for screening and LSA for identification. Every new heart and lung transplant candidate is evaluated for presence of HLA antibodies by performing both FlowPRA and LSA up front. The routine cutoff of LSA is MFI = 1000. For this study we retrospectively reviewed records of FlowPRA and LSA performed in 100 new heart transplant candidates evaluated between April – December 2013. Results Of these 100 heart transplant candidates, 37 had either class I and/or class II LSA detectable antibodies. Among these 37 patients, 32 had LSA class I antibodies, 14 had LSA class II antibodies, and 9 had both class I and II antibodies. Among these 32 patients with positive LSA class I antibodies, only 20 demonstrated a clear positive FlowPRA. Class I FlowPRA of the other 12 patients with positive LSA appeared more likely to be negative. Among the 14 class II LSA positive patients, 7 patients’ FlowPRA appeared more likely to be negative. The majority of these HLA antibodies detected by LSA, but that did not cause a clear positive FlowPRA, had an MFI under 2000. A few of these antibody bindings were confirmed to be not reactive to antigens on cell surface in crossmatch. Conclusion The antigen density on LSA beads may be higher than on the FlowPRA beads which may explain that some antibodies may be detected by LSA but not FlowPRA. It could also not be excluded that some of the antibodies detected only by LSA might be non-specific which need further clarification. The clinical relevant of the weak antibodies detected only by LSA warrantees further studies. It is meaningful to test the HLA antibody using a combination of different detection platforms, especially for patients who will be transplanted using donors selected based on a virtual crossmatch.

1006-LB

Aim Minimizing lot-to-lot variability should be a primary goal for all manufacturers of diagnostic products. Nonetheless, it is the responsibility of each laboratory to verify that a new lot or reagent performs as expected. Here we discuss concerns related to quality control of the FlowPRA test by One Lambda, Inc. Methods Initial testing of the new lot (#18) of FlowPRA Class II screening beads was performed using samples with and without HLA antibodies as determined with the previous lot (#17) of FlowPRA. All samples were run according to manufacturers’ recommended specifications with the exception of using 40ul of serum instead of 20 ul. Samples were analyzed on a FACS Canto II instrument. Results Testing of sera that contained known HLA antibodies were comparable between lots. However, unexpected positive reactions consistently were observed with lot #18 when HLA antibody negative sera were tested. As a follow up, samples from 10 such patients were tested by single antigen beads (SABs, LabScreen, One Lambda, Inc.). In addition, historic samples, shown to be devoid of HLA Class II antibody (FlowPRA Lot #17), were retested against the new lot of FlowPRA beads. Of concern was the observation that all 10 historic 0% PRA samples were now positive with the new bead lot. Importantly, testing by SABs confirmed the absence of Class II antibodies. Conclusions Our results clearly demonstrate a problem with lot #18 Class II FlowPRA beads. Lot #18 differs from the previous lot by a single bead. Although this bead carries a new phenotype, it added only one new allele (DPB*02:02) to the collection of alleles compared to FlowPRA lot #17. The unexpected reactivity is most likely due to non-HLA antibodies since the possibility of multiple patients expressing a solitary DPB*02:02 antibody is not likely. Such false positive reactivity should have been identified by the manufacturer. Failure to do so ultimately translates into wasted time, effort and expense. More importantly, patient care may be compromised by reporting of false positive results.

32-OR

Aim To present two patients with unreal anti-HLA-Cw antibodies. Methods Anti-HLA antibodies were tested with LABScreen single antigen beads (SAB), PRA beads, iBeads or Flow PRA beads (One Lambda) using standard protocols from the vender. Results Case #1: A 47-year-old non-sensitized male was found to have antibodies (MFI = 1300-6300) to all HLA-Cw except self HLA-Cw7 with Luminex SAB. There were no antibodies to HLA-A, -B and class II. The strength of antibodies did not raise substantially after increasing the serum to beads ratio 5 times higher than standard protocol. Class I II Luminex PRA were all clearly negative. Mocked FCXMs with surrogate cells were all negative. The anti-Cw antibodies are likely not real so negative PRA was reported. The patient was transplanted with a kidney from a 4/6 HLA-ABDR mismatched deceased donor. T-AHG XM was negative with present of “unreal” DSA to Cw12 (MFI = 4293) and Cw15 (MFI = 2305). Since then the patient has had great graft function in 3-year follow up post-transplantation. Case#2: A 22-year-old non-sensitized native male with ESRD due to malignant hypertension was found to have strong antibodies (MFI = 10,000-17,000) to HLA-Cw12, Cw15, and self HLA-C∗01:02 with Luminex SAB. Treatment with DTT and absorption with empty bead didn’t reduce the binding. None of the strong SAB-defined antibodies to HLA-Cw can be deduced from Luminex PRA panel. Flow Class I PRA was negative. Surrogate FCXM with Cw1 homozygous cells was clearly negative. Those anti-Cw antibodies also bond to denatured antigens and can be absorbed out with beads carrying regular or denatured single antigen of Cw7 or Cw17. There were still some moderate binding (MFI = 4000-5000) for HLA-Cw1, Cw12, Cw15 with HLA class I iBeads, which carry less denatured antigens than the regular SAB. Conclusions Anti-HLA-Cw antibodies defined by SAB could possibly be “unreal” antibodies, which may bind denatured antigens on the beads and may not be a contraindication to kidney transplantation.

180-P

Aim Single antigen bead (SAB) assay allows in-depth characterization of HLA antibody specificities. In addition to detection of antibodies against natural HLA antigens, the SAB may also detect antibodies against denatured HLA antigens. Recently, we observed antibody reactivity to all DR antigens (pan-DR) in serum samples of two transplant candidates using the Luminex SAB assay. This study was to characterize the pan-DR antibodies detected by Luminex SAB on two serum samples. Methods Serum samples received from two transplant candidates were analyzed in the study. Initial antibody screening and specificity tests were performed using Flow PRA and Luminex SAB. Additional tests using Flow SAB, Flow phenotype bead assay and Flow T and B cell crossmatch were performed to confirm the presence of HLA antibodies. Results Flow PRA was clearly negative for both samples, while Luminex SAB surprisingly revealed antibody reactivity to all DR antigens. No antibodies to DQ and DP were detected. However, no HLA antibodies were detected by Flow phenotype bead assay. In the Flow SAB assay, patient #1 showed reactivity with majority of the DR antigen beads, while patient #2 showed no any binding reactivity. The crossmatch with a donor cell carrying DR4, DR15, DR51, and DR53 was completely negative for both serum samples. No specific antibody removals were observed on serum samples adsorbed using the donor cells in a Luminex SAB assay. Conclusions Flow PRA and Flow phenotype beads are coated with natural HLA antigens extracted from cell lines, while SABs are coated with recombinant HLA antigens. The antibody binding detected by SAB, but not by FlowPRA and Flow phenotype, may indicate the presence of antibodies against denatured antigens. Our results suggest that the pan-DR antibodies detected in these patients were possibly antibodies against denatured HLA antigens, more likely to a shared epitope uncovered on denatured DR antigens. Antibodies to denatured antigens would not result in crossmatch reactivity.

6-P

Aim VADs are increasingly used as a bridge to transplantation and are implicated as a risk factor for allosensitization. We investigated in a matched case-control study the association between VAD and PRA changes (peak & significant increase ⩾20%). Methods We analyzed records of VAD recipients at our institute from 00-09 (n = 384) and included patients with pre-VAD PRA testing and another PRA test within 6 months post-VAD (n = 100). PRA testing was performed by screening flowPRA beads (One Lambda, CA). A control group was constructed from heart transplant candidates evaluated in the same time frame with no VAD (n = 830) for whom we had ⩾3 PRA tests within 8 months pre-transplant (n = 445). Two controls were randomly matched for each VAD case based on yr of initial PRA test. Results Follow-up was longer in the controls (median:103 vs 33; p Table 1 ). Multivariable models adjusted for gender, race, pre-VAD PRA, duration of VAD and blood transfusion yet the associations remained unchanges. Of VAD patients, that had a significant increase in C1 PRA and single antigen testing performed 81% (35/43) showed development of new HLA antibody specificities post-VAD. Conclusions Our results suggest that VAD is associated with CI allosensitization independent of other risk factors. These findings have important clinical implications and warrant prospective studies to better understand VAD associated allosensitization.

Public Epitope Specificity of HLA Class I Antibodies Induced by a Failed Kidney Transplant: Alloantibody Characterization by Flow Cytometric Techniques

Patients whose kidney grafts fail develop alloantibodies that react with many HLA molecules. We analyzed the epitope specificity of HLA class I alloantibodies in the sera of 55 patients who had been sensitized by kidney grafts, and investigated the immunogenicity of various polymorphic epitopes. HLA class I alloantibodies were detected and characterized by flow cytometry (FlowPRA beads). Potential "immunizing epitopes" were identified by comparing the amino acid sequences of HLA class I antigens/alleles of the donor, recipient and the antibody-reactivity pattern. In the 55 anti-HLA class I-positive patients, 82 different antibody reactivity patterns were identified; all but 5 (94%) were determined by a "public epitope" of donor HLA-A and/or -B molecules. Forty-five of 50 patients who showed HLA-A Res-MMs with their donors produced HLA-A antibodies, but only 31 of 51 subjects with HLA-B Res-MMs produced HLA-B antibodies (P=0.001; O.R.=5.81). The antibody patterns were specific for a "single" epitope of the mismatched donor molecules in 91% of patients. Forty-three of the 120 (36%) mismatched HLA-A and/or -B epitopes were positively correlated with antibody production. The polymorphic determinants of higher immunogenic capacity were b80N (Bw6-associated) and ab82-83LR (Bw4-associated) public epitopes. The humoral immune response against a kidney graft mainly produces HLA class I antibodies specific for "public epitopes" of mismatched donor molecules. A "single" donor-epitope may determine the production of a spread antibody pattern. In renal transplantation, epitope matching is better than HLA antigen matching for avoiding or minimizing development of HLA antibodies.

The repercussions of implementing flow cytometry as a single HLA antibody screening technique in prospective renal transplant recipients

In an effort to replace the complement-dependent cytotoxicity test (CDC) with a more sensitive single technique we looked at flow cytometry as a possible replacement. The Flow PRA Bead technique (One Lambda) performed well in our laboratory. Although as expected this technique was more sensitive and specific than CDC, there remained 11 samples from eight patients which were flow negative, CDC positive. The results of various antibody identification tests on these samples prompted us to alter the positive selection criteria which we had been using on our routine screening with the Flow PRA Beads and persuaded us that the initial CDC result was correctly positive in nine of the 11 samples.

Pitfalls and ambiguities in antibody screening in patients with cardiac assist devices: The role of flow PRA beads

Pitfalls and ambiguities in antibody screening in patients with cardiac assist devices: The role of flow PRA beads

C4d]FlowPRA Screening—A Specific Assay for Selective Detection of Complement-Activating Anti-HLA Alloantibodies

On waiting lists, transplant candidates are routinely screened for potentially harmful complement-fixing alloantibodies using complement-dependent cytotoxicity (CDC) panel-reactive antibody (PRA) testing. We have recently developed a novel cell-independent assay for assessment of complement-activating panel reactivity ([C4d]FlowPRA), which is based on selective flow-cytometric detection of alloantibody-triggered C4 complement split product deposition to human leukocyte antigen (HLA)-coated FlowPRA beads. Serum specimens selected from 120 transplant candidates were evaluated by [C4d]FlowPRA (HLA class I vs II) in comparison with FlowPRA IgG alloantibody screening (HLA class I vs II), a method detecting both complement- and noncomplement-activating alloantibodies, and with CDC-PRA on separated T (T-CDC) or B cells (B-CDC, evaluation on platelet-absorbed sera). For each assay, ≥10% PRA reactivity was considered positive. Comparing complement-dependent PRA assays with standard FlowPRA, the specificity calculated for [C4d]FlowPRA (HLA class I: 92%; class II: 100%) was found to be superior to that of CDC testing (T-CDC-PRA: 79%; B-CDC-PRA: 86%). Because noncomplement-activating alloreactivities were not detected for both techniques, low sensitivities were calculated ([C4d]FlowPRA HLA class I: 61%; class II: 31%; T-CDC-PRA: 70%; B-CDC-PRA: 55%). Compared with CDC-PRA, [C4d]FlowPRA gave a high specificity (HLA class I compared with T-CDC: 89%, HLA class II compared with B-CDC: 95%) but, at least in part because of false-positive CDC results, a modest sensitivity (66% and 38%, respectively). For both HLA classes, we found a highly significant association between absolute [C4d]FlowPRA and CDC-PRA levels ( p < 0.0001). Our results suggest that detection of C4 split product deposition to FlowPRA beads may represent an attractive HLA-specific and time-effective alternative to CDC-PRA screening.

Class I and Class II antibodies in multiparous primary renal transplant candidates detected using Flow PRA beads

Class I and Class II antibodies in multiparous primary renal transplant candidates detected using Flow PRA beads

The detection and definition of IgM alloantibodies in the presence of IgM autoantibodies using flowPRA beads

We have developed a flow cytometry–based screening method using FlowPRA (One Lambda) human leukocyte antigen (HLA) class I panel beads and FlowPRA (One Lambda) HLA class I specificity beads for the detection and definition of immunoglobulin (Ig)M HLA–specific antibodies in the presence of IgM autoantibodies. Forty-six autoantibody-positive patients who were on the waiting list for a renal transplant (56 sera) were tested in parallel with FlowPRA (One Lambda) HLA class I beads and FlowPRA (One Lambda) control beads. Sera that were positive for IgM HLA class I antibodies were subsequently tested with FlowPRA HLA class I specificity beads to determine the HLA specificities. Thirteen of the 46 patients were positive for IgM HLA class I–specific antibodies. Eleven of the 13 had previous failed transplants and 2 were awaiting a primary transplant. For 9 of the 13 positive patients, IgM HLA class I specificities were defined. We have demonstrated the presence of IgM HLA–specific antibodies in patients with IgM autoantibodies. This study demonstrates the value of FlowPRA HLA class I panel and specificity beads for the detection and definition of IgM HLA class I–specific antibodies.

Flow cytometry based detection of HLA alloantibody mediated classical complement activation

Complement-dependent cytotoxicity (CDC) panel reactive antibody (PRA) testing is used to assess recipient presensitization and post-transplant alloantibody formation in transplant recipients. However, CDC test results can be affected by false-positive reactions brought about by autoantibodies or antilymphocyte reagents. As an alternative to the CDC-PRA assay, detection of HLA alloantibodies using HLA antigen-coated microbeads (FlowPRA test) was recently established. FlowPRA testing, however, does not distinguish between (presumably more harmful) complement-fixing and noncomplement-fixing alloantibodies. In this study, we established a novel assay allowing flow cytometric detection of HLA alloantibody dependent classical complement activation using the FlowPRA test. For the detection of complement activation, FlowPRA beads were incubated with sera from highly sensitized dialysis patients (CDC-PRA reactivity >60%) and then stained for C4 (C4d, C4c) and C3 (C3d, C3c) fragments, as well as C1q deposition using indirect immunofluorescence. We demonstrate alloantibody induced induction of C4 fragment, and in parallel C1q deposition to HLA class I or class II beads. As shown by immunoblotting, C4 staining was not due to the presence of preformed C4 fragment–IgG/M complexes. Indeed, C4 fragment deposition in our in vitro system was demonstrated to result from de novo complement activation. First, inactivation of C4 by treatment of sera with methylamine, which inhibits cleavage of the internal thioester, completely abolished C4 fragment deposition. Second, C4 fragment deposition was not observed in the evaluation of C4-free immunoadsorption eluates obtained from highly sensitized dialysis patients. After supplementation with complement, however, eluates induced C4 deposition. Deposition of C4 split products and C1q was temperature-dependent with maximum binding after incubation at 4 °C for 60 min. In contrast, maximum C3 fragment deposition was found at 37 °C. At this temperature, C3 deposition occurred in an alloantibody and C4-independent fashion, presumably as a result of alternative complement activation. In summary, we describe a novel cell-independent and easy-to-perform PRA test that permits flow cytometry based detection of alloantibody induced classical complement activation. Future studies will have to evaluate its possible relevance as an alternative to CDC-PRA testing in clinical transplantation.

Conundrums with FlowPRA™ beads

In 1969, a study by Patel and Terasaki persuaded the renal transplant community that a pre-transplant cross-match should always be performed between donor and recipient to detect HLA antibodies and prevent hyperacute allograft rejection. Although the role of the cross-match among nonsensitized patients is controversial, its importance among sensitized recipients is undeniable. Over the past 30 years, more sensitive techniques, such as the flow cytometric cross-match (FCXM), were developed to identify low levels of antibodies undetectable by other approaches. The clinical relevance of a positive FCXM, however, has been hotly disputed, with some investigators maintaining that the FCXM is 'too sensitive' and rules out acceptable donor-recipient combinations. An alternative explanation is that the FCXM is non-specific, and, at least in certain situations, identifies non-HLA antibodies that are clinically irrelevant. Recently, a solid phase immunoassay utilizing purified HLA Class I or Class II molecules bound to microparticles (FlowPRA) was developed. Ideally, use of the FlowPRA for the identification of HLA antibodies in recipient sera would help ascertain whether a positive FCXM with donor cells was truly the result of an HLA-specific antibody. As shown here, this may not always be true. In this study, two unexpected serum patterns were observed. Pattern 1: FlowPRA beads were positive (with an associated HLA Class I specificity) and the FCXM with cells expressing the HLA antigen(s) to which the antibody was directed, was negative. Sequence analysis of the HLA antigens reactive with this unexpected antibody suggests that the epitope recognized resides on the floor of the groove, a site generally not expected to generate antibody activity. Pattern 2: FlowPRA beads were negative yet the FCXM was T and B cell positive. Further analysis of the FlowPRA negative/FCXM positive sera using a flow cytometric cell-based panel reactive antibody (PRA) approach revealed those sera to have specific anti-HLA Class I activity. We suspect that both types of antibodies described above have clinical relevance. Thus, a negative or positive FCXM (when the FlowPRA against donor antigens is positive or negative, respectively) is not always a straightforward interpretation.

Sensitization of renal transplant candidates by cryopreserved cadaveric venous or arterial allografts.

Cryopreserved cadaveric venous or arterial allografts are used in ESRD patients as an alternative to synthetic grafts for hemodialysis access. We evaluated the effect of these allografts on the PRA against HLA class I and II antigens in 11 ESRD patients awaiting a kidney transplant. Flow Cytometry using purified antigen coated beads (Flow PRA Beads) was used to determine PRA against HLA class I and II antigens. Patients with no antibody prior to allograft implantation were all positive for HLA class I and II antibodies after implantation. Patients with anti-HLA class I and II antibodies prior to allografting, developed higher antibody titers. Of the 11 patients that received cryopreserved cadaveric allografts no deaths were reported. Two grafts were removed due to thrombosis consistent with rejection. Recipients of cadaveric venous or arterial allografts elicit an HLA antibody response attesting to the antigenicity of cryopreserved cadaveric allografts.

A comparison of enzyme-linked immunoabsorbent assays and flow cytometry techniques for the detection of HLA specific antibodies

LATM, Quikscreen (QS), and B-Screen (QSB) are ELISA-based tests for the detection of HLA specific antibodies. FlowPRA beads are microparticles coated with HLA antigens for the detection of HLA specific antibodies by flow cytometry. The aim of this study was to evaluate the sensitivity and specificity of the LATM, QS, QSB, and FlowPRA screening tests. One hundred sixty-three sera from renal transplant patients were tested using LATM, FlowPRA, QS, and QSB. Discrepant results were further investigated using complement dependent cytotoxicity, QuikID, and PRA-STAT. When QS was compared with LATMI and FlowPRAI for the detection of HLA class I specific antibodies the overall concordance was 82.8% with no particular specificity missed by any one test. Comparing QSB with LATMII and FlowPRAII, for the detection of HLA class II specific antibodies, there was 90.7% concordance. Although the overall concordance was better for class II specific antibodies, QSB failed to detect antibodies to HLA-DQ in a number of samples from different patients. Of the methods tested, flow cytometry using FlowPRA beads appeared to be the most sensitive and specific, missing the least number of specificities. However, the ELISA methods offer the advantage of being more suitable for testing large numbers of samples in a more time- and cost-effective manner.