Luminex Single Antigen Bead Assay Research Articles

P012 Human IVIG preparations contain antibody to native and denatured HLA

Aim Human intravenous immunoglobulin (IVIG) is frequently used for treatment of antibody mediated rejection. Little is known regarding the risk of passively transferred HLA antibody (Ab) in these products. Our aim was to test commercial IVIG preparations for presence and level of HLA Ab and specificity. Methods Thirty IgG preparations derived from plasma collected in the US, Europe and Canada (CSL Behring: 6 Hizentra, 20% protein; 24 Privigen, 10% protein) were tested for HLA class I/II Ab in Luminex single antigen bead assays. Reagents were treated with Adsorb Out, then tested at various dilutions. Testing was performed with 2 vendor kits. Furthermore, preparations were tested on the C1q screening assay and after acid treatment. Results Initial testing showed HLA Ab reactivity (MFI >2000) at 1:10 but negative by 1:50. Regardless of protein concentration, IgG preparations had similar reactivity patterns. Following acid reduction, most class I beads remained positive with one vendor kit, suggesting the remaining reactivity was towards denatured and not native HLA molecules. As shown in the Table, class I Ab reactivity after acid treatment was limited to a few HLA specificities. Class I results with a second vendor kit were negative. Class II initial testing with one kit showed a similar reactivity pattern in 20/30 samples, mainly DR4+DR18, plus a few additional positive reactions; testing with the second kit gave MFI values Conclusions Ab reactivity in IgG preparations was low titer regardless of the percentage protein. All positive reactions were observed with One Lambda and not Immucor kits, and most positive class I reactions were towards denatured HLA molecules. Class II reactivity appeared to be non-specific. We conclude that these IgG preparations do not pose a risk for passively transferred HLA Ab that can potentially be harmful to the allograft. Research support and test materials provided by CSL Behring, LLC. Class I Luminex reagents provided by Immucor. M. Berger: Employee; Company/Organization; CSL Behring, LLC. M. Spycher: Employee; Company/Organization; CSL Behring AG. A. Zeevi: Grant/Research Support; Company/Organization; CSL Behring, LLC.

P028 Uncovering the invisible; pre-treatment of sera with EDTA relieves prozone effect

Aim High titer HLA antibodies in patient sera can be falsely assessed due to interfering factors, also known as the prozone phenomenon, causing the masking of such antibodies. In this study we assessed the elimination of prozone by treatment of sera with DTT, EDTA and dilution. Methods Sera from 10 patients were treated with DTT, 6% EDTA, and 1:10 dilution. Antibody screening was performed by Luminex single antigen bead (SAB) assay. Effect of each treatment on the negative and positive controls, false-positive, and false-negative beads were compared to one another as well as to the untreated serum. Results Patient sera treated with DTT, EDTA and diluted at 1:10, all showed elimination of interfering factors and resolved the prozone effect. Comparison of the three treatment approaches showed that, DTT resolved the prozone, however it appeared to cause a slight increase in the mean fluorescent intensity (MFI) of the Luminex negative control (NC) bead. EDTA treatment eliminates prozone effect and has no effect on the NC bead. The 1:10 dilution also can resolve prozone, however the sensitivity can be compromised especially for the lesser strength antibodies. Conclusion In circumstances where prozone phenomenon is suspected, such as in the pre-transplant evaluation of highly sensitized patients, or during post-transplant monitoring for detection of donor specific antibodies, DTT, EDTA, and dilution are all effective strategies in elimination of interfering factors. However in addition to a higher NC, DTT-treatment, unlike EDTA, requires an additional incubation step with the sera (45 min) which affects the overall turnaround time. Dilution of serum can affect the sensitivity of the assay and in addition, often one dilution might not be sufficient for an accurate assessment of prozone, and performing multiple dilutions can prove to be costly. Routine treatment of sera with EDTA can accurately assess antibody specificities without affecting the sensitivity, turn around time and test cost.

P004 The challenge of virtual crossmatching in the presence of weak donor specific antibodies

Aim Our laboratory uses a normalized 2500 MFI cut off in the Luminex single-antigen bead assay to define unacceptable antigens. Antibodies (Abs) between 1000 and 2500 MFI are considered weak positive, but are not used to strictly rule out potential donors. The challenge is to accurately predict flow crossmatch (FXM) results in the presence of weak donor-specific Abs (DSA). Method We identified 62 FXM performed in the presence of weak DSA. For simplicity we considered only the B-cell FXM results. We analyzed the data based on the DSA MFI values or DSA epitope profile (DSA against shared epitopes (SE), DSA against a single antigen (Ag), and multiple weak DSA (M)). Results 39/62 (64%) were positive and 22/62 (36%) were negative. All (10/10) FXM with multiple weak DSA were positive. The majority (13/17) of FXM in the presence of weak DSA against a single antigen (Ag) were negative (76%). On the other hand, most (25/34) FXM with weak DSA that shares epitopes with other antigens (SE) were positive (74%). Table 1 summarizes these results. When we analyzed the FXM results based on DSA MFI (excluding multiple DSA cases), we observed that DSA with MFI of 1,000-2,500 resulted in a positive FXM in 50% of cases (Table 2). Unexpectedly, we got more positive FXM (14) than negative (7) when the DSA was under 1,000 MFI. Further analysis revealed that 13/14 of these positive FXM were caused by Abs with reactivity to SE and in particular Abs against HLA-B and -DRB1. Conclusion MFI values of weak DSA are not a reliable predictor of FXM results. When performing a virtual XM in the presence of weak DSA, the Ab profile is a better predictor for FXM reactivity (PPV for DSA with SE is 73.5, independent of the MFI value). Abs that target broadly-shared epitopes frequently cause positive FXM. Download high-res image (105KB) Download full-size image Download high-res image (104KB) Download full-size image

The Significance of Screening for HLA Antibodies in the Long-Term Follow-up of Pediatric Liver Transplant Recipients

BackgroundPost-transplant donor-specific anti-HLA antibodies (DSA) reportedly have detrimental effects on the outcomes of organ transplantation. However, the prevalence of post-transplant DSA in the long term after pediatric liver transplantation remains unclear, and the significance of post-transplant DSA is unknown. The aim of this cross-sectional study was to determine the prevalence of and characteristics of patients with post-transplant DSA. Materials and MethodsOf the 84 pediatric liver transplant recipients who were followed up in the outpatient department of our institution, 34 patients with available HLA typing data were included after they or their parent(s) provided informed consent for DSA evaluations. Luminex single-antigen bead assays were performed, and a mean fluorescence intensity of ≥1000 was used as the cut-off for a positive reaction. ResultsNo class I DSA were detected, whereas class II DSA were detected in 11 patients (32%). There were no differences in age at transplantation, immunosuppressive drugs, or follow-up period between the DSA-positive and DSA-negative patients. The rate of positive pre-transplant complement-dependent cytotoxicity crossmatch was higher with class II DSA than without, although the difference was not statistically significant. ConclusionsThe utility of screening for class I DSA was insignificant in the long-term follow-up of pediatric liver transplant recipients. The prevalence of class II DSA was relatively high; therefore, screening for class II DSA might be justified, although a follow-up survey of the association between post-transplant class II DSA and the long-term clinical course needs to be conducted.

A reliable method for avoiding false negative results with Luminex single antigen beads; evidence of the prozone effect

Luminex single antigen bead (SAB) assays have become an essential tool in monitoring the status of antibody to the Human Leucocyte Antigen (HLA) of patients both before and after transplantation. In addition SAB data is used to aid risk stratification to assess immunological risk of humoral rejection in solid organ transplantation (CTAG/BTAG guidelines) [1]. Increasingly laboratories are reporting false negative results at high antibody titre due to a prozone effect.Here we report a case study where the prozone effect led to a false negative antibody result that could have resulted in adverse outcome. We describe a method to reliably remove the prozone effect through heat inactivation and the addition of Ethylenediaminetetraacetic acid (EDTA) to the Luminex wash buffer.

Bortezomib may stabilize pediatric renal transplant recipients with antibody-mediated rejection.

Current therapeutic strategies to effectively treat antibody-mediated rejection (AMR) are insufficient. Thus, we aimed to determine the benefit of a therapeutic protocol using bortezomib for refractory C4d + AMR in pediatric kidney transplant patients. We examined seven patients with treatment-refractory C4d + AMR. Immunosuppression included antithymocyte globulin or anti-CD25 monoclonal antibody for induction therapy with maintenance corticosteroids, calcineurin inhibitor, and anti-metabolite. Estimated glomerular filtration rate (eGFR) calculated by the Schwartz equation, biopsy findings assessed by 2013 Banff criteria, and human leukocyte antigen (HLA) donor-specific antibodies (DSA) performed using the Luminex single antigen bead assay were monitored pre- and post- bortezomib therapy. Seven patients (86% male, 86% with ≥6/8 HLA mismatch, and 14% with pre-formed DSA) age 5 to 19 (median 15) years developed refractory C4d + AMR between 1 and 145 (median 65) months post-transplantation. All patients tolerated bortezomib. One patient had allograft loss. Of the six patients with surviving grafts (86%), mean pre-bortezomib eGFR was 42 ml/min/1.73m(2) and the mean 1year post-bortezomib eGFR was 53 ml/min/1.73m(2). Five of seven (71%) had improvement of histological findings of AMR, C4d staining, and/or acute cellular rejection. Reduction in HLA DSAs was more effective for class I than class II. Bortezomib appears safe and may correlate with stabilization of eGFR in pediatric kidney transplant patients with refractory C4d + AMR.

OR34 Flow and virtual cross match exchange – A new component of the International Cell Exchange

Aim Single antigen bead assays provide investigators with a means to accurately identify the presence and relative strength of HLA antibodies through the use of MFI (Mean Fluorescence Intensity) values. However, inter-laboratory variation to the interpretation of MFI values creates the potential for discordant results. The aim of this study was to provide a medium to compare and correlate MFI data together with flow crossmatch outcomes. Methods In 2014, the International Cell Exchange program introduced the UCLA Flow and Virtual Cross Match Exchange. In three exchanges, a total of 6 cells and 12 sera were tested by 17 laboratories around the world. T and B flow cytometric crossmatch results, along with class I and class II single antigen data were reported and analyzed. Fifteen laboratories used a Luminex single antigen bead assay (LSAB) and 2 used a flow single antigen bead assay (FSAB). To further investigate the correlation between MFI values and positive flow crossmatch results, the strength of donor-specific antibodies were compared with their crossmatch outcomes. In addition, coefficients of variations (%CV) were calculated using the MFI reported for each DSA and by each laboratory. We also examined the prediction of positive T/B virtual crossmatches in relationship to the strength of DSAs identified. Results The mean class I and class II DSA MFI of the positive T and B flow crossmatches were 15355 and 14641, respectively. Among labs reporting LSAB MFI, the mean %CV was 26%. Overall, 16 (67%) T-cell and 8 (33%) B-cell flow crossmatch challenges were reported among the labs without discordance. When restricting analysis to DSA MFI greater than mean values, the T flow concordance rate improved to 79%, while B flow concordance remained poor at 31%. In contrast, high concordance rates were observed for virtual crossmatch predictions: 92% (22/24) for T- and 88% (21/24) for B-cell crossmatches. Conclusions The data suggests that the variability of cell based assays is higher than that observed in solid phase SAB test. It also suggests that variability exists in the sensitivity of flow crossmatch testing among laboratories and that this variability is correlated with DSA strength. As such, educational activities should continue to provide standards for quality control of antibody and crossmatch testing methods.

High strength HLA-DPA1 donor-specific antibodies cause positive CDC B-cell crossmatch but weak positive B-cell flow crossmatch

DPA1 antibodies were shown to be a risk of antibody-mediated rejection (AMR). We present a case showing high MFI DPA1 donor-specific antibodies (DSA) causing +ve CDC crossmatch (CxM), but -ve flow xM (FxM). At the time of deceased donor offer, we perform prospective FxM if the intended candidate displays strong DP DSA. This particular case received first kidney transplant in 2004 from a 8/10 mismatched donor. Luminex single antigen bead assay (One Lambda) revealed strong antibodies to all mismatched donor’s HLA and CREG, and thus had 100% CPRA. The patient also displayed strong antibodies to several DPs including to the alpha chains, DPA1∗01 and DPA1∗03. The patient received a deceased donor offer in May 2015. Virtual xM (VxM) identified 3 strong DSAs directed to DPA1∗01(MFI = 16282), DPA1∗03(MFI = 13167) and DPB1∗04(MFI = 19568), and predicted positive B FxM. Contrary to the prediction, the donor-specific FxM was -ve (T cell MCS = 29; B cell MCS = 110). Kidney offer was declined because of multiple high MFI DSAs. To have better insights on our failed VxM prediction, we performed pronase as well as CxMs. The pronase B FxM was weakly +ve with a MCS = 209 (+ve cutoff > 120 MCS), which is within the range relates to -ve CxM. Unexpectedly, the B CxM with and without DTT-treated sera were turned to be strongly +ve. Additional xMs with a surrogate donor cells expressing a single target DPA1∗01:03 also produced similar results, i.e., +ve pronase B FxM with MCS=178 and +ve B CxM. We confirmed DP antibodies assignment using Immucor single antigen beads, and typed previous donor and patient for DPA1 and DPB1 loci. These results ruled out DPB1∗04:01 antibody, but cannot confirm DPB1∗04:02, because DPB1∗04:02 beads in both venders’ systems were paired with DQA1∗01:03. It is more likely the patient has only DPA1∗01 and DPA1∗03 antibodies. Unambiguous assignment of DPA1 antibodies will remain challenging until the single antigen bead array includes multiple DP antigen series with different alpha and beta chain pairs. In conclusion, comprehensive antibody analyses by single antigen beads, and high-resolution typing of DPB1 and DPA1 of donor and recipient are necessary to define DPA1 antibodies. CDC xM is a critical test to determine the risk of hyper acute AMR in patients having strong DPA1 DSAs, and FxM may yield false negative results in these cases.

Evaluation of discrepancies between Luminex single antigen bead assays

Currently there are two manufacturers offering HLA antibody testing using the Luminex platform. Our laboratory incorporates both methods in our testing protocol. We have documented discrepancies and use them to help us identify samples that need follow-up verification of unacceptable antigens. More discrepancies are found with Class I than with Class II and some appear to be major differences. The purpose of this study was to examine the discrepancies with Class I single antigen testing between the two manufacturers with respect to correlation with Flow crossmatch results and MFI values. For this study discrepant samples from a single run of Class I single antigen beads were chosen. An antibody of interest was chosen for each sample and surrogate cells were found which did not have HLA detected by other Class I antibodies reported in the same sample. Flow crossmatch was performed using a BD FACsCaliber instrument on a 1024 scale. All discrepancies in this study resulted from an antibody being called by Method A that was not detected using Method B. Surrogate crossmatches were done to determine if there was a sensitivity problem with Method B or a specificity problem with Method A. Twenty-seven crossmatches were examined which included eleven different specificities. Fourteen samples were T cell positive and thirteen were T cell negative. Of the fourteen that were T cell positive, six were B cell negative, which could indicate non-HLA reactivity. Therefore, it is possible that 19 of the 27 specificities detected by Method A may not be clinically relevant. The discrepancies between methods is of concern and emphasizes the necessity of including manufacturers and MFI cutoffs when publishing Luminex single antigen bead results. Most HLA laboratories use single antigen results for determining unacceptable antigens and virtual crossmatches. More specificities were called with Method A using our current cutoff, but at least half of these did not result in a positive Flow crossmatch. Re-evaluation of the cutoff suggests that the cutoff may be different for certain loci or certain specificities. The use of both methods may have value in fine-tuning the unacceptable antigens and illuminating those specificities that should be confirmed.

OR3 Clinical relevance of preformed “acceptable” donor specific antibodies in kidney transplantation

Preformed HLA cytotoxic antibodies, specific for mismatched HLA molecules of the potential donor, represent an absolute contraindication in kidney transplantation. New techniques, like the Luminex-Single Antigen Beads assay, are very sensitive allowing detection of HLA donor specific antibodies (DSAs) at low mean fluorescence intensity (MFI) values. However, few evidence on the clinical relevance of such low “strength” DSAs have been reported. Graft outcome (follow-up 34.2 ± 19.8 months) of 99 pre-sensitized patients (%FlowPRA class I = 41 ± 33; %FlowPRA class II = 30 ± 36), transplanted between May 2007 and June 2014 on the basis of both CDC-XM and FC-XM negative results, was analyzed. Five patients were excluded from the study because of primary non-function of the graft (3 patients) or death for non-immunologic causes with non-functional graft (2 patients). Forty-seven (47%) patients did not have pre-formed HLA DSAs; the remaining 52 (53%) patients had “acceptable” DSAs (MFI ⩽ 5000) specific for HLA-A/B/C/DR/DQB molecules or anti-DPB/DQA DSAs with high MFI values (>5000). HLA class I DSAs were present in 28 patients; five of these showed DSAs against more than one donor HLA molecules. HLA class II DSAs were present in 19 patients; three of these showed anti-DPB/DQA DSAs with MFI > 5000. The remaining five patients had both HLA class I and class II DSAs; one of these had high level of anti-DP DSA. Analyzing graft outcome of the remaining 94 transplanted patients with functioning graft, we did not evidenced any significant difference between DSA positive patients and DSA negative patients (rejection: 10.4% vs. 6.5%, P = ns; graft loss: 12.5% vs. 10.9%, P = ns). In particular, among the 48 DSA positive patients, three (6.2%) had humoral rejection without graft loss; two (4.2%) had cellular rejection, one of these lost the graft. Six DSA positive patients suffered graft failure that was never due to antibody-mediated rejection related to pre-formed DSAs. The results of this study show that pre-formed DSAs with low mean fluorescence intensity values do not represent a contraindication in kidney transplantation. An accurate evaluation of the “strength” of Luminex-detected DSA allows transplanting patients with clinically “irrelevant” HLA antibodies.

Treating sera with ethylenediaminetetraacetic acid: A promising technical solution for the complement-mediated prozone effect in anti-hla antibody detection by single antigen bead assay

Aim The Luminex-single antigen bead (SAB) assay is considered the most sensitive method for the identification of anti-HLA antibodies (Ab). However, false negative results can occur due to the prozone effect (PrE) leading to underestimation of some high titer anti-HLA Ab. The aim of this study was to evaluate the ethylenediaminetetraacetic acid-EDTA’s Ca++ chelating effect on the C1 complement in eliminating the PrE in anti-HLA Ab identification by SAB. Method Ten patients with PRAs > 65% were included in the study. Neat and EDTA treated samples were tested using SAB for either HLA class I (n=14) or class II (n = 10) specificities. A mean fluorescence intensity (MFI) ⩾ 2 fold increase in EDTA treated versus neat sera was considered relevant to PrE. The PrE was confirmed with 1:10 dilution and with dithiothreitol treatment of the sera. To confirm the clinical significance of the new identified Ab, sera with defined PrE and MFI value 1000 in neat sera were further tested with AHG-CDC crossmatch against donor cells expressing the relevant HLA. Results PrE was found in 7/10 patients concerning either HLA class I (n = 4) or class II (n = 3) Ab specificities. Using an MFI = 1000 as a cut off, six ‘new’ HLA specificities were identified either HLA class I (HLA-A∗01:01, A∗34:02, A∗11:02) or class II (DQB1∗03:01, DQB1∗03:02, DQB1∗03:03). However, a significant shift of MFI value of HLA Ab class I and II was observed. More precisely, 38 HLA-class I Ab directed against HLA-A (n = 31) and HLA-B (n = 7) showed a significant mean MFI value shift from 5162 (266 lower value) to 20207. Regarding HLA class II Ab, 26 directed against HLA-DQ (n = 23) and HLA-DR (n = 3) showed a significant shift from MFI 4419 (113 lower value) to 23795. New HLA Ab specificities detected after EDTA treatment were confirmed with a strong positive AHG-CDC crossmatch. Conclusion Taking into account that selection of potential recipients pre-Tx as well as the follow up post-Tx is based to HLA Ab identification, these preliminary results highlight the importance of using EDTA treatment of the patient’s sera prior to SAB assay, in order to abolish the prozone effect in HLA antibody detection.

Deciphering allogeneic antibody response against native and denatured HLA epitopes in organ transplantation.

Anti-HLA donor-specific antibodies are deleterious for organ transplant survival. Class I HLA donor-specific antibodies are identified by using the Luminex single antigen beads (LSAB) assay, which also detects anti-denatured HLA antibodies (anti-dHLAs). Anti-dHLAs are thought to be unable to recognize native HLA (nHLA) on the cell surface and therefore to be clinically irrelevant. Acid denaturation of nHLA on LSAB allows anti-dHLAs to be discriminated from anti-nHLAs. We previously defined a threshold for the ratio between mean fluorescence intensity against acid-treated (D for denaturation) and nontreated (N) LSAB, D ≥ 1.2 N identifying the anti-dHLAs. However, some anti-dHLAs remained able to bind nHLA on lymphocytes in flow cytometry crossmatches, and some anti-nHLAs conserved significant reactivity toward acid-treated LSAB. After depleting serum anti-nHLA reactivity with HLA-typed cells, we analyzed the residual LSAB reactivity toward nontreated and acid-treated LSABs, and then evaluated the ability of antibodies to recognize nHLA alleles individually. We observed that sera can contain mixtures of anti-nHLAs and anti-dHLAs, or anti-nHLAs recognizing acid-resistant epitopes, all possibly targeting the same allele(s). Therefore, the anti-HLA antibody response can be highly complex and subtle, as is the accurate identification of pathogenic anti-HLA antibodies in human serum.

LBP33: DEFINING A CLINICALLY RELEVANT CUTOFF FOR THE IDENTIFICATION OF HLA ANTIBODIES USING THE SINGLE ANTIGEN BEAD ASSAY

The single antigen bead assay is a widely-used platform for the detection of human leukocyte antigen (HLA)-specific antibodies in both pre- and post-transplant patients. Our regional organ procurement organization recommends a cutoff value of 2000 mean fluorescence intensity (MFI) for the detection of HLA antibodies using the Luminex single antigen bead assay. This cutoff has proven effective for the prevention of HLA-mediated hyperacute rejection; however, this cutoff may be too lenient for the detection of post-transplant donor-specific antibodies. Indeed, we hypothesize that some patients have donor-specific HLA antibody-mediated organ allograft rejection even with a MFI of <2000. To test this hypothesis, we performed a retrospective analysis of all biopsy-proven cases of antibody-mediated rejection (ABMR) in the Houston Methodist Hospital System. Over the period from 02/2012 to 057/2014, we identified 75 cases with findings at least suggestive of ABMR (all kidney biopsies) in our surgical pathology database. Of these, 42 had donor-specific antibody assays run at the time of the biopsy. Six out of 42 cases (14%) showed no DSAs. To define a clinically-relevant DSA MFI cutoff, we analyzed MFI values of 75 runs of a commercially available negative control serum and MFI values of 75 post-transplant patients with negative DSAs. After normalizing the data by log-transformation, we found that the negative control runs had a mean + 3 standard deviation cutoff of 328, and the post-transplant negative DSA patients had a mean + 3 standard deviation cutoff of 969, suggesting that a cutoff of 1000 MFI would be adequate to capture approximately 99% of all negative DSA patients. We applied the 1000 MFI cutoff to the six cases with findings suggestive of ABMR but with no DSAs identified. We found that the highest MFI for donor-specific antigens in these patients was well below the 1000 MFI cutoff. Using a cutoff of 1000 would not have changed the DSA result to positive in these patients. The overall findings suggest that a cutoff of 2000 MFI has been adequate to capture all cases of HLA-mediated antibody-mediated rejection over the period analyzed at our institution.

LBP06: HLA-C ANTIBODY: HOW STRONG IS UNACCEPTABLE? DEFINING HLA-C ANTIBODY CUT-OFF AT ONE TRANSPLANT CENTER

The addition of HLA-C to UNOS cPRA calculation was the result of increased recognition of the importance of HLA-C antibody and the fact that enough data was available on the frequency of HLA-C types in donors. The unacceptable cut-off for HLA-A/B/DR/DQ antibodies, which varies drastically throughout the HLA field, was determined based on the likelihood of producing a positive crossmatch. Will the same arbitrary cut-off apply to HLA-C? HLA-C has unique antigen expression level on the cell surface and limited antigenic polymorphism, which can create artificial over representation on Luminex Single antigen beads. This is an ongoing case study from one transplant center aiming to answer these questions. Nine sera with HLA-C antibodies were identified; five of them only had HLA-C antibodies. The antibody profile and strength were characterized using Luminex single antigen bead assay (One Lambda Inc, Canoga Park, CA). Surrogate donor cells with corresponding DSA were collected and crossmatched using CDC, AHG-CDC and Flow methods. All HLA-C DSA(s) are >4000 MFI, which is the cut-off used in our center as unacceptable antigen determination and likely to produce positive CDC and/or AHG-CDC crossmatch. All CDC, AHG-CDC crossmatches were negative. Among the 4 sera with multiple loci DSA, 3 were T and B cell Flow positive and 1 was B cell positive only. Among the 5 HLA-C DSA only sera, 3 were T and B cell Flow positive and 2 were T and B cell Flow negative. Anti-Cw4 demonstrated positive flow T and B cell crossmatches with MFI’s over 4K range (5–8 K tested). Anti-Cw1, often observed as only HLA-C antibody sera together with Cw1/C15/C12, was completely negative for all T and B cell CDC, AHG-CDC and Flow crossmatches. If HLA-C unacceptable antigen cut-off is set at 4000 MFI, all of the above ‘donors’ would have been eliminated. At a center where transplants are routinely performed over positive DSA/Flow crossmatch, all of these cases would be transplantable, and furthermore, two of these cases would not require any immune modulation prior to transplant. An anti-Cw7 calculates as 49% cPRA and will cause a high percentage of donor exclusion. Is the Cw1/C15/C12 valid? Ongoing data collection is in progress for better defining HLA-C antibody and unacceptable cut-off for virtual donor exclusion.

LBP29: TRANSPLANTATION OF SENSITIZED HEART PATIENTS

In 2010, at ASHI, we presented our preliminary data of using antibody titration to determine unacceptable HLA antigens (UAG) for selection of deceased donors for heart transplant. The one-year survival rate of recipients who had “low titer” donor-specific antibody (DSA) was comparable to recipients who had no DSA or no pre-transplant sensitization. This approach has been continuously used at our transplant center. This study was conducted to evaluate the survival of recipients who received a heart transplant between 2010 and 2013 and to assess the risk of “low titer” DSA in the current cohort. All patients that received heart transplants from January 1, 2010 and December 31, 2013 were included in this study. Prior to listing, all patients were evaluated for HLA sensitization which was defined by presence of any HLA antibodies detected by Flow antibody screening and Luminex single antigen bead assay. For sensitized recipients, a titration test at 1:16 dilution was performed. Utilizing the principle of virtual crossmatch (VXM), only donors without UAGs determined at 1:16 dilution were selected for sensitized patients. All final XMs were performed by flow cytometry. A total of 183 recipients were included in this current study. Among them, 84 (45.9%) were pre-transplant sensitized. Among the 84 sensitized patients, 28 patients received organs with UAGs as determined by Abs detected in undiluted serum samples. 13 of these 28 patients had positive HLA XM by flow cytometry. The one-year survival rate of the 56 sensitized patients who received organs without UAGs and the 28 sensitized patients who received organs with UAGs were equal, that is 89.3%. The one-year survival of the 99 non-sensitized patient is 92.9% (p = 0.685, comparing to that of sensitized patients). Among the 28 pts who received organs with UAGs determined in undiluted serum samples, the 1 year survival rate of patients with a positive XM (N = 13) is 84.6%, while the 1 year survival rate for the patients (N = 15) with a negative XM is 93.3% (p = 0.58). Based on our data, we conclude that the current approach of using a 1:16 titration to determine UAGs for the transplantation of sensitized heart patients is an acceptable practice. The long term survival beyond 1 year warrants further investigation.

LBP35: STRENGTH OF DE NOVO HLA DONOR SPECIFIC ANTIBODY IS A STRONG PREDICTOR OR ITS C1Q BINDING CAPABILITY

Introduction Antibody mediated rejection (AMR) is the major complication of renal transplantation that significantly affects the renal allograft survival. Complement fixation mediated through Donor specific anti-Human Leukocyte Antigen (HLA) antibodies (DSA) is the hallmark of AMR. Different methods have been developed to detect DSA including Luminex single antigen beads (SAB) assay. However, it is still unclear how to define harmful DSA that causes poor graft outcomes. Aim We analyzed C1q assay positivity at different posttransplant time points and evaluated its relationship with different DSA characteristics including time of occurrence (early or late posttransplant), HLA class (class I, II or both) and Ab titre (mean fluorescence intensity, MFI). Method A total of 315 kidney transplant recipients between January 2007 and December 2012 were retrospectively analyzed for the presence of DSAs and their MFI using IgG SAB assay (One Lambda Inc., USA). Out of those, 39 patients with de novo DSA were further characterized using C1q assay. All 39 patients were followed for 2 years and their sera collected consequently with the protocol and/or diagnostic biopsies were analyzed by C1q assay (One Lambda Inc.). Results Most patients had at least 3 posttransplant sera during the 2-year follow up. The high MFI of HLA-IgG in all biopsy samples correlated significantly with C1q positivity. The mean MFI values of HLA-IgG for the negative and positive C1q were 2547 and 6424 ( p = 0.003), 2481 and 6130 ( p = 0.001) and 2200 and 8400 ( p = 0.009) for the sera collected at 6, 12 and 18-month posttransplant, respectively (Fig. 1). No other DSA characteristic correlated with C1q positivity. Conclusion The C1q assay is a potentially useful tool in allograft monitoring after transplantation. Our data indicates that Ab strength strongly correlates with the capacity to bind C1q. Correlation with clinical outcomes will further elaborate the relevance of C1q positivity on the renal allograft outcome.

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.

OR13: HIGH TITER ANTIBODY STRENGTH CANNOT RELIABLY BE DESCRIBED WITHOUT DILUTION

Background Luminex Single Antigen Bead (SAB) assay is commonly used to detect circulating donor specific antibody (DSA) in serum of post-transplant patients. DSA Mean Florescent Index (MFI) may be used to impute strength and titer of DSA, and to follow treatment response over time. It is not accurate to impute the strength of high titer antibody based on MFI without testing serum dilutions. Methods Serum samples were heat inactivated (HI) for 30 min at 56° to remove complement inhibition. Serum dilutions were done in phosphate buffered saline. The starting dilution was 1:50 and increased as needed. HLA antibody testing was done by the Luminex Single Antigen Bead (SAB) assay. Results Dilution of serum can have a range of effects on the MFI of the antibody (see figure ). Decreasing MFIs with dilution are the expected result of an accurate MFI measurement within the reportable range of the assay. When there is an excess of antibody relative to the amount of binding sites on the beads, this is not the case. The MFI may maintain a constant value, or it may actually increase as the serum is diluted, indicating a prozone or “hook” effect not attributable to complement. Some patient sera were diluted as much as 1:200 or 1:2,000 in order to obtain mfi values that could be used to follow trends in DSA with treatment and over time (see table). Conclusion To accurately assess the strength of a high titer antibody, serum should be diluted to concentrations that result in consistently decreasing MFIs. Extremely strong antibodies may saturate the binding sites on the beads resulting in MFI values at the upper limits of the assay without truly binding and measuring all the antibody present. Hook effects in HI sera may indicate interfering factors besides complement.

P116 : T CELL POSITIVE AND B CELL NEGATIVE FCXM WITH PRONASE TREATED DONOR CELLS OBSERVED IN AN HIV INFECTED PATIENT

Introduction Un-expected positive flow crossmatch was occasionally observed in solid organ transplanations. The un-expected crossmatch could be caused by presence of multiple factors. Viral infections could be one of those factors. In this abstract we describe a persistent un-expected T cell positive, B cell negative flow cytometry crossmatch (FCXM) observed in an HIV patient who was on waitlist for kidney transplant. Materials and methods Patient and donor HLA typing was performed by rSSOP. Percent Reactive Antibody (PRA) and antibody specificity testing were conducted by flow cytometry and Luminex Single Antigen Bead Assay (SAB), respectively. Crossmatch (XM) testing was performed by flow cytometry. Donor cells for FCXM are used with or without pronase treatment. Results Flow PRA presented single peak, clear negative histogram for both HLA class I and II, however SAB class II had a very weak positive DP17. Despite of the negative PRA, the patient’s FCXM were T cell positive and B cell negative with all deceased donors (CDO) tested in our standard method using low dose (0.5 mg/ml) of pronase. CDO3-CDO5 XM were performed only with low dose pronase treated donor cells and all results were T positive and B negative. When XM was performed with non-pronased donor cells (CDO6), both T and B cell XM became negative. The following XM were performed with both pronase treated and non-treated donor cells. All “Positive” T-XM testing with low dose pronase treated donor cells (CDO7-CDO9) were turned to “Negative” with non-pronased donor cells. B-XM results were negative but with slight shifts in both protocols. Conclusion Endogenous antigen peptides, like viral antigens are presented on CD8+ T cells and therefore activate almost only cellular immunity. Since, T cells express fewer Fc receptors; the pronase treatment might expose cryptic epitopes on T cell surface that could be reacted with virus specific antibodies present in patient’s sera probably through molecular mimicry.

P007 : HIGHLY ACCURATE PREDICTION OF ALLELIC-RESOLUTION HLA TYPING BY HAPLOTYPE ANALYSIS OF LOW-RESOLUTION HLA TYPING

Allele-specific HLA antibodies are commonly detected by Luminex single antigen bead assays in solid organ transplant recipients. However, allelic-resolution HLA typing for their donors is commonly unavailable. This causes uncertainty in reporting donor specific antibodies (DSA) and predicting virtual crossmatches (VXM). We investigate the ability of haplotype prediction analysis based on data compiled from the NMDP registry database (Maiers et al., 2007) to predict allelic-resolution HLA typing from low-resolution typing and its possible application in the clinical setting for DSA determination and VXM. One hundred and five proficiency testing samples between 2007 and 2013 with available low- and allelic-resolution typing results were selected. Low-resolution HLA typing (A, B, C, DRB1 and DQB1) was entered in the haplotype prediction analysis tool and the predicted allelic-resolution typing was compared to the actual allelic-resolution typing results. As shown in Table 1, the haplotype prediction analysis tool was able to predict allelic-resolution typing 87% over all with the highest allele prediction rate for HLA-C and -DQB1 (90%) and the lowest for HLA-DRB1 (82%). Haplotype analysis based on common allele associations is an accurate method to predict allelic-resolution typing for donors of recipients with allele-specific antibodies in the solid organ transplant setting. This method allows us to identify allele-specific DSA with greater accuracy and confidence and further aid in the management of these patients.