LABScreen Single Antigen Beads Research Articles

P023 Getting on target: Development of the novel, prozone-resistant, dual antibody rapid test (DART) for the labscreen single antigen bead (SAB) assay

A major limitation of the SAB assay is the prozone effect, whereby the detection of high titer C’ fixing HLA antibodies is compromised due to C’ split product (C3b/C4b) deposition and interference with the reporter anti-IgG-PE antibody. Strategies to minimize prozone include serum treatment with heat, DTT, EDTA or titration. While effective, these treatments may compromise HLA antibody binding and detection. Here we describe the Dual Antibody Rapid Test (DART), a modification of the rapid optimized SAB (ROB) protocol, in which we use an IgG-PE/C3d-PE antibody cocktail to simultaneously detect bead-bound IgG and C3d, allowing for detection of HLA antibodies independent of the prozone effect. 20 prozone positive sera (10 class I & 10 class II), identified by titration, were tested by ROB protocol, ±EDTA, using 3 reporter antibody cocktails: 1. IgG-PE 2. C3d-PE 3. IgG-PE/C3d-PE (DART). MFI values were then compared. Results: IgG -ve (n = 735) vs IgG + ve (n = 1185) reactions were identified using a 1000 MFI IgG EDTA cutoff (Fig. 1). IgG + ve reactions were classified based on ΔMFI (IgG EDTA-IgG) as follows: 1. no prozone (ΔMFI < 3 K; n = 737) 2. slight prozone (ΔMFI 3–5 K; n = 49) 3. moderate prozone (ΔMFI 5–10 K; n = 93) and 4. marked prozone (ΔMFI > 10 K; n = 306). AntiC3d-PE staining showed no C3d deposition on IgG -ve beads (C3d MFI 52 ± 91) and a small amount of C3d fixed in the no prozone group (C3d MFI 475 ± 895). In contrast, increasing amounts of C3d were detected in the slight, moderate and marked prozone groups (C3d MFI 3935 ± 2082, 5805 ± 2530 and 12,466 ± 2912, respectively). Importantly, by combining IgG-PE and C3d-PE (DART), all IgG +ve specificities were detected regardless of their ability to create a prozone effect. DART MFI correlated best with IgG EDTA MFI for prozone +ve and with IgG MFI for prozone -ve specificities. DART protocol accurately detects HLA antibodies independent of the prozone effect. Implementation of DART is an easy way to overcome the prozone effect without compromising HLA antibody detection.Download : Download high-res image (333KB)Download : Download full-size image

P190 Bortezomib desensitization efficacy in pediatric cardiac transplantation: A single center study

Aim Bortezomib (BTZ) is a short-lived proteasome inhibitor, having the ability to lyse plasma cells responsible for the production of antibodies (Ab). It has been reported to have inconsistent success when used as a desensitization treatment for the reduction of HLA Ab in the context of pre and post solid organ transplantation. Furthermore, a variety of undesirable toxic side effects associated with this drug have been reported. In this work we assessed the efficacy of desensitization treatment using BTZ in a small pediatric cardiac transplant cohort. Methods Pre, during and post BTZ desensitization treatment sera were screened for HLA Ab using One Lambda LabScreen Single Antigen Beads (SAB) according to manufacturer’s instructions. SAB was performed with neat sera and dilutions in PBS. Analysis was performed using Fusion v.3.0 software. Results 10 cardiac transplant patients, 2 pre and 8 post, were monitored for BTZ desensitization efficacy by assessing HLA Ab surrounding the time of treatment. For the two pre-transplanted patients no significant change in HLA Ab profile was observed before and after treatment. Among the 8 post- transplanted patients analyzed, 4 demonstrated an increase in several HLA Ab for both Class I and II, whether DSA or not, while no significant change in HLA Ab profile was noted in 3 of these patients. One post-transplanted patient had a single DSA positive DQ7 demonstrating 11,000 MFI pre-treatment which decreased to become negative after BTZ treatment. Overall, BTZ treatment did not appear successful in lowering the level of HLA Ab in 9 out of the 10 patients. Conclusions BTZ treatment does not appear to have significant impact on long or short term reduction of HLA Class I or II Ab in this pediatric transplant patient cohort. Further studies need to be performed to determine the ability of BTZ to reduce HLA Ab in transplant patients. Moreover, these studies will further demonstrate or challenge the proposed mechanisms of action described for this drug and perhaps will help to discover currently unidentified mechanisms by which it exerts its actions.

P214 Significance and limitations of sera treated with molecular weight centrifugal column

Aim Our lab routinely treats high PRA patient serum with molecular weight centrifugal columns (SC) to remove high molecular weight interference from testing, especially in cases where prozone effect is suspected. Treated serum is used in Luminex single antigen bead (SAB) testing and/or flow cytometric crossmatching and results are correlated with test results using untreated sera. In this study our goal is to define the limitations and significance of sera treated by molecular weight centrifugal column. Methods 124 patients had complement dependent cytotoxicity (CDC) and SAB/SC testing performed between January 2015 and February 2017. Protocol treats 250 μl of serum using the PALL 300 K Nanosep Centrifugal Device. Samples were centrifuged at 14,000 g for 15 min and collected from the reservoir for testing. Specificities determined using One Lambda LABScreen single antigen bead (SAB) Luminex assay and the GEN TRAK 60 well cell tray (CDC: 3 W plus AHG). Analysis included correlation of SAB/SC and CDC including evaluation of regraft status and donor specific antibody (DSA). Results 100/124 patients were CDC positive. All CDC specificities were also identified by SAB/SC testing. 46 were regrafts, and 38 (83%) had DSA identified by both CDC and SAB/SC. 9 of these regraft patients had the prozone effect eliminated in SAB/SC testing with CDC specificities matching. 24/124 patients were CDC negative. Eight of these were regrafts, four had DSA identified by SAB/SC, three were also negative by SAB (SC); all had some reactivity on SAB, but it was weak or against putative cryptic antigens. Conclusions Our initial idea for use for the 300 K Nanosep centrifugal column (SC) was to treat sera to remove interference of high molecular weight substances including the IgM pentamers and eliminate any potential prozone effect. The concern is that IgG specific HLA antibody might also be lost in the process. Results confirm that all allo HLA antibody and DSA identified by CDC were preserved and positively identified by SAB/SC. 9 patients had prozone effect alleviated with SC treatment. One regraft patient had flow crossmatch results using SC sera that confirmed DSA remained positive. SAB/SC results MFI do not correlate to standard SAB results MFI. Interpretation should include pattern and history.

It’s about time: The development and validation of a rapid optimized single antigen bead (ROB) assay protocol for LABScreen

The LABScreen single antigen bead assay (SAB) is a method widely used for the identification and monitoring of human leukocyte antigen (HLA) antibodies in patients pre-and post-transplant. While accurate testing of patient samples is key for optimal patient care, time can also be important, especially during deceased donor workups or post-transplant assessments. Here we describe the development and validation of the Rapid Optimized SAB (ROB) protocol, a modified version of the One Lambda LABScreen SAB (OLSAB) procedure, which reduces assay time from 85 to 25min (>70% reduction) without impacting assay quality or sensitivity. Optimization steps included shortened centrifugation cycles and reduced serum and secondary antibody incubation times in combination with increased secondary antibody concentration. Linear regression analysis of baseline median fluorescence intensity (MFI) values showed excellent correlation between the ROB and OLSAB protocols (r2>0.98) for both class I and class II antibodies in 58 sera tested in two HLA laboratories. Importantly, the ROB protocol demonstrated a trend towards improved inter-laboratory MFI concordance when compared to the OLSAB procedure (r2=0.9816 vs 0.9451), especially for HLA antibody specificities in the 500–2000 MFI range (r2=0.7824 vs 0.6313). Implementation of the ROB protocol will expedite HLA antibody testing and may improve reproducibility of the SAB assay.

P131 Limitations of virtual crossmatch for deceased kidney transplantation in highly sensitized patients

Aim The revised kidney allocation system (KAS) implemented to increase opportunity for highly sensitized (>98% cPRA) patients awaiting deceased donor kidney transplantation has significantly increased organ offers for these patients. Concomitantly, we observed an increase in unexpectedly positive cellular crossmatches not predicted by virtual crossmatch (VCXM). We hypothesized that unexpected crossmatches were concentrated among highly sensitized patients and resulted from technical factors of alloantibody testing limiting VCXM efficacy. Methods Histocompatibility testing results were analyzed pre-KAS (12/2013 – 11/2014) and KAS (12/2014 – 12/2015) for living (pre-KAS n = 76, KAS n = 103) and deceased donor (pre-KAS n = 277, KAS n = 375) kidney transplantation. Cellular crossmatch was performed by flow cytometry (FCXM). Anti-HLA antibody testing was performed using LABScreen Single Antigen Bead (SAB) assay, with mfi >3,000 used as VCXM cutoff for identifying alloantibodies expected to cause positive FCXM. Results KAS implementation increased FCXM performed for highly sensitized patients for deceased donor kidney transplantation (6.0% vs. 1.1%) with increased unexpected positive FCXM (13.9% vs. 10.1%). Similar trends were not observed for living donors. Highly sensitized patients accounted for 29.4% of VCXM failures. Patients with cPRA 80–97% or>98% showed significant deviation from VCXM performance characteristics. Root cause analysis of unexpected FCXM results ( n = 79) demonstrated that only 31.7% were due to limitations in UNET (unlisted avoid antigens, allelic antibodies, or antibodies against HLA-DP). Technical limitations including positive FCXM from multiple weak DSA (21.5%), false negative SAB due to prozone (7.6%), and non-HLA antibodies (11.4%) were responsible for a majority of definable VCXM failures. Patients with cPRA >80% accounted for 70.0% of technical VCXM failures (excluding non-HLA) despite being only 12.4% of tests performed. Conculsions New KAS policy significantly increases deceased donor kidney offers to highly sensitized patients. While VCXM is highly useful for organ allocation, highly sensitized patients present significant challenge in accurate prediction of compatibility warranting prospective cellular crossmatching to ensure optimal outcomes.

P024 Automated solid-phase HLA antibody assay using the BioTek ELx50 microplate washer

Aim Our laboratory currently uses the manual method to perform the wash steps of the Luminex-based single antigen bead (SAB) assay for HLA antibody analysis. Automation of the wash steps using an automated microplate washer with a vacuum filtration module could be beneficial by reducing tech-to-tech variability and hands-on time. The goal of this study was to investigate the use of this instrument toward the automation of the SAB assay. Methods 5 well-characterized samples with antibody profiles containing both HLA Class I and Class II specificities were analyzed in duplicate within run and on 3 separate runs by means of the LABScreen SAB assay (One Lambda, Inc.). Tests were performed with the BioTek ELx50 microplate washer (BioTek Instruments, Inc.) with a MultiScreen 96-well filter plate with a 1.2 μm low protein-binding Durapore membrane (EMD Millipore Corp.), which performed all wash buffer dispensations, aspirations and incubations. The acquisition was performed on the same Luminex 100 instrument. The results obtained with the microplate washer were compared to results obtained using the manual method. Calculations of % coefficient of variation (CV%) were adapted from the NCCLS global consensus guidelines. Results The BioTek ELx50 microplate washer showed consistent results in comparison with the manual method. The MFI CV% values for each sample within run and between run was calculated. No well failures were observed and antibody strength specificities assignments were comparable in all samples. In addition, antibody specificities (MFI > 2000) showed similar MFI and CV% values between the manual and BioTek ELx50-washed samples. Conclusions We have demonstrated the ability of the BioTek ELx50 microplate washer to automate the LABScreen SAB assay. Automation using this microplate washer speeds processing, improves washing efficiency, increases sensitivity and improves accuracy by eliminating tech to tech variability.

P032 Determining the minimum EDTA concentration necessary to eliminate prozone effect in the single antigen bead (SAB) assay

Aim A major limitation of the single antigen bead (SAB) assay is its susceptibility to interference by complement activation, which inhibits binding of anti-IgG-PE to HLA specific antibodies. This interference, known as prozone effect, can reduce MFI values leading to false negative reactions. Schnaidt et. al. (2011) described a method to eliminate prozone by treating sera with EDTA to chelate calcium, preventing complement activation. However, anecdotal evidence suggests that the method is not always effective. This may be due to differences in EDTA concentration used by different labs given the original description is ambiguous and lacks sufficient detail to follow the protocol. The goal of this study was to determine the minimum EDTA concentration required to eliminate prozone. Methods Six well characterized, prozone +ve sera (3 class I and 3 class II HLA) were selected. Prozone +ve specificities were defined as >5000 MFI increase in SAB assay reactivity upon titration. Sera were treated with several concentrations of dipotassium EDTA (1.2–6 mM) or PBS control and were tested in parallel using LABScreen SAB assay. MFI values of prozone +ve specificities were then compared. Results A total of 52 prozone +ve specificities (28 class I and 24 class II HLA) were identified in titration studies. Prozone was eliminated for all 52 specificities when sera were treated with either 6 mM or 3 mM EDTA (Fig. 1). In contrast, treatment with either 2 mM or 1.2 mM EDTA was ineffective (Fig. 1). Prozone −ve specificities were not affected by EDTA treatment at any concentrations used. Conclusion EDTA treatment of sera is effective at eliminating prozone if appropriate EDTA concentrations are used. Interestingly, our experiments demonstrate that the threshold at which EDTA is effective is very distinct (3 mM is effective while 2 mM is ineffective). Since EDTA forms a 1:1 chelate complex with calcium, the concentration threshold of EDTA required to eliminate prozone is consistent with physiologic serum calcium concentration (2.1–2.6 mM). Download : Download high-res image (369KB) Download : Download full-size image

P072 De novo anti-HLA-class II donor specific antibodies are associated with patient mortality after lung transplant failure

Aim The development of donor-specific HLA antibodies (DSA) after lung transplantation has been implicated in acute and chronic rejection. The diagnosis of lung antibody mediated rejection (AMR) is based upon histology, DSA testing and clinical assessment of graft function. Post-transplant DSA monitoring has become more and more common as a guide toward diagnosis and sometimes institution of AMR treatment. In this study, we aimed to assess DSA with biopsy surveillance and associate with patient mortality after lung transplant failure (MLTF). Methods We retrospectively selected 127 patients, transplanted between Jan, 2004 and July, 2015 whose DSA were tested for clinical indications post-transplantation. Protocol biopsies were performed for these patients at 1, 3, 6, 9, and 12-months after transplant for clinical suspicions. DSA were monitored using LabScreen single antigen bead assays. Biopsy HE however, no association was found for Class I DSA. No significant relationship between DSA and histological changes of AMR in lung transplant biopsies was found in this cohort. Conclusion HLA Class II DSA is associated with graft dysfunction and mortality in pulmonary transplant patients although we did not find the correlation with biopsy findings. Our study suggests that post-transplant protocol DSA surveillance may add value to assess allograft function and to treat graft rejection at the early stage.

P044 C1q status and titer of de novo donor specific antibodies are not predictors of allograft survival

De novo donor-specific antibodies ( dn DSA) that develop after renal transplantation are independent predictors of allograft loss. However, it is unknown if dn DSA C1q status or titer at the time of first detection can independently predict allograft outcomes. In 70 renal transplant recipients who developed dn DSA we performed LABScreen single antigen bead assays using: neat, neat with ethylenediaminetetraacetic acid (EDTA), three dilutions after EDTA treatment (1:16, 1:64, 1:1024) and the C1q assay (neat sera). Histologic and clinical outcomes were correlated with the C1q assay or dn DSA titer. C1q positivity correlated with dn DSA titer (p dn DSA (p dn DSA development, but did not predict T-cell mediated rejection (TCMR) or antibody mediated rejection (ABMR). De novo DSA titer correlated with glomuerulitis, interstitial inflammation, tubulitis, peritubular capillaritis, and C4d scores, and predicted TCMR (p dnDSA development or other known predictors of post-dnDSA graft loss (i.e. clinical phenotype or non-adherence) neither C1q status nor dn DSA titer were independent predictors of a composite outcome of a 50% decline in eGFR or allograft loss (Fig. 1). Therefore, at present the additional cost and time associated with these approaches is not justified in routine practise. However, in cases where a renal biopsy is not possible dn DSA titer correlates with TCMR, and AMBR, and may assist in risk stratification. Download high-res image (239KB) Download full-size image

Defining the non-sensitized patient: a comparison of HLA antibody screening and identification methods

Aim Luminex Single Antigen beads (SAB) are widely used to identify HLA specificities and assign avoid antigens for kidney transplantation. However in our centers, “non-sensitized” waitlist patients are monitored using less sensitive screening kits. To confirm the lack of HLA sensitization, we evaluated sera by SAB. In this study, we compared several antibody screening and identification kits to uncover test discrepancies and investigate possible false reactions. Methods During initial evaluation and listing, patients were defined as “non-sensitized” when the Class I/II FlowPRA screening beads (One Lambda) were negative. Presumed non-sensitized waitlist patients are screened monthly by the Lifecodes Lifescreen kit (Immunocor) and screened yearly by FlowPRA screening beads. To ensure that no HLA antibodies were missed by this protocol, 116 patient samples were tested by One Lambda Class I/II Labscreen Single Antigen beads (1000 MFI cutoff). When discrepancies between the three assays were identified, sera were tested by the Immunocor ID assay and/or One Lambda Labscreen PRA beads. Additional analyses by surrogate crossmatches are ongoing. Results Of the 116 samples tested from non-sensitized patients, 22% were positive by the Labscreen Class I SAB and 33% were positive by the Labscreen Class II SAB but negative by the screening kits. The positive specificities spanned all HLA loci, but there were some beads that were positive more than expected. Most notably, the DRB1∗04:04 bead was positive in 10% of cases (12/116) with a range of 1080–2524 MFI, and in three cases the patient’s HLA type was DR4. Other specificities observed three or more times included: A80, B45, B76, Cw15, DR18, DP1, DP11, bead DQA1∗01:04/DQB1∗06:04. In four cases, patients presented with new UNet unacceptable antigens (⩾3000 MFI), and in three cases this led to a positive cPRA (4–17%). Conclusions Unexpected positive specificities were detected by the Labscreen SAB assay in otherwise non-sensitized kidney waitlist patients. Our data are consistent with other studies that compared multiple test assays to interrogate false reactions. Unexpected SAB reactions should be investigated with additional screening assays and/or surrogate crossmatches. In some cases, common false positive beads can be ignored during test interpretation.

LBP34: ANTI-HUMAN GLOBULIN (AHG) ENHANCED C1q ASSAY IMPROVES DETECTION OF DONOR SPECIFIC HLA ANTIBODIES (DSA) IN HEART TRANSPLANT RECIPIENTS WITH ANTIBODY MEDIATED REJECTION

Aim The C1qScreen assay detects complement binding HLA antibodies. Recent studies in cardiac transplant recipients suggest that the presence of C1q positive donor specific antibodies (DSA) is associated with increased risk of antibody mediated rejection (AMR) early post-transplant. However, in some patients with documented AMR, C1q binding DSA are not detected suggesting poor sensitivity of the C1qScreen. In this study we compared the ability to detect DSA by the standard C1qScreen vs the anti-human globulin (AHG) enhanced C1q assay in a cohort of 37 heart transplant recipients with AMR. Methods 37 heart transplant recipients with biopsy proven AMR were studied. Sera were collected at the time of AMR and screened for the presence of DSA using the standard C1qScreen, the AHG enhanced C1q and the LABScreen single antigen bead (SAB)-IgG assay. The number of DSA detected and the average DSA MFI were compared. Results The C1qScreen failed to identify DSA in 14/37 (38%) of patients with biopsy-proven, SAB-IgG positive and/or C4d positive, AMR (Fig. 1). In contrast, the AHG enhanced C1q assay identified DSA in the majority of patients (32/37; 86.5%), approaching the rate of DSA detected by the SAB-IgG assay (34/37; 92%). The average number of DSA/patient identified was also improved with the AHG enhanced C1q assay (2.47 vs 1.56 DSA/patient; AHG enhanced C1q vs C1qScreen). Finally, the mean MFI of DSA was significantly increased (22,191.3 +/− 7454 AHG enhanced C1q vs 11,993.6 +/− 7608 C1qScreen; p 〈 0.001; Fig. 2) confirming that the sensitivity of the AHG enhanced C1q assay is improved. Conclusions The C1qScreen failed to detect DSA in a significant number of heart transplant recipients with biopsy proven AMR and thus, cannot be used to accurately detect pathogenic DSA. The AHG enhanced C1q assay was more sensitive, identifying DSA in the majority of AMR patients and improving detection of low level antibodies. Based on our findings, the AHG enhanced C1q assays may be useful in post-transplant monitoring to detect pathogenic DSA.

LBP11: THE TEST OF TIME. A MULTICENTER EVALUATION OF THE RAPID OPTIMIZED SINGLE ANTIGEN BEAD (ROB) PROTOCOL FOR LABSCREEN

The LABScreen single antigen bead (SAB) assay is routinely used for HLA antibody detection and allows for testing of multiple patient samples. Despite this, standard SAB assay is time intensive, taking 1.5–2 hr to perform, which is not optimal for urgent testing. Utilizing a multicenter study we compared the rapid optimized SAB (ROB) protocol (Liwski et al. ASHI 2014), with a 30 min turn-around time (TAT), to standard SAB LABScreen method. Ten HLA labs (BCM, DCI, Edmonton, Halifax, Georgetown, Winnipeg, METC, PSBC, SWI, UPMC) participated in the study. Five ASHI 2014 AC-1 survey sera (AC 460-464) were tested in each lab with standard SAB LABScreen (SSL) vs the ROB protocol. Class I LS1A04 (lot 8) and class II LS2A01 (lot 10) HLA kits were used. Mean fluorescence intensity (MFI) values were collected. Protocols were compared with respect to the mean MFI, specificities reaching consensus (⩾90% labs; 2000 MFI cutoff) and coefficient of variation (%CV). HLA antibody reactivity patterns were similar between the protocols with an excellent MFI correlation (r2>0.99) and signal to noise differential. Average MFI was slightly reduced with the ROB protocol (by 5.0%). The number of specificities reaching consensus (⩾90% labs; 2000 MFI cutoff) was similar between protocols (97 SSL vs 94 ROB). When the ROB protocol cutoff was lowered to 1500 MFI, the number of consensus specificities improved to 99, including all 97 SSL specificities (Fig. 1). Importantly, the average %CV of raw bead MFI was significantly improved with the ROB protocol (ROB: 21.2+/−3.4 vs SSL: 34.4+/−5.6; p< 0.001; Fig. 2). A slight increase in background was noted with the ROB protocol (AC 462 serum) in some labs. However, this had no impact on antibody assignment and improved with one additional wash step. ROB protocol reduces SAB LABScreen assay time by 70% and improves precision without compromising sensitivity or specificity. Implementation of the ROB protocol will expedite antibody testing in urgent cases and improve TAT for routine testing.

Changes in successive measures of de novo donor-specific anti-human leukocyte antigen antibodies intensity and the development of allograft dysfunction.

Many patients develop de novo donor-specific anti-human leukocyte antigen antibodies (dnDSA) after transplantation. Despite development of dnDSA, not all patients will immediately fail. This study analyzes dnDSA intensity and longitudinal trends as prospective clinical parameters to assess subsequent allograft function. Twenty-four patients with dnDSA onset in the first 2 years after transplantation received antibody monitoring by LABScreen single antigen beads. Estimated glomerular filtration rate (eGFR) was recorded at time of dnDSA onset and up to 24 months thereafter. The dnDSA mean fluorescence intensity (MFI) of the stable function patient group (n=8; eGFR decline ≤ 25%) was compared with the impaired function patient group (n=16; eGFR decline>25%) using first year peak MFI (pMFI), eight month MFI change (ΔMFI), and eighteen month MFI trend (MFI slope). Both groups showed similar dnDSA characteristics (time to onset after transplantation, class I/II distribution, and initial MFI). Between groups, MFI trends were analyzed. Impaired patients showed a higher pMFI during the first year (median pMFI, 13,055 vs. 2,397; P=0.007). Longitudinal analysis revealed that ΔMFI was strongly associated with dysfunction. Both a ΔMFI increase greater than 20% as well as a stronger increase (ΔMFI>50%) were followed by graft dysfunction in almost all patients and could significantly differentiate between stable and impaired function patients (P=0.001 and P=0.04, respectively). Our study suggests that tracking dnDSA intensity, particularly in the early period after onset, is important to estimate the impact of dnDSA on the allograft and could, therefore, determine help on how best to monitor patients with dnDSA.

P030 : THE EFFECTS OF HYPOTONIC DIALYSIS AND SPIN COLUMNS ON SERUM ANTI-HLA IgG LEVELS

Aim Our laboratory recently noticed an influx in positive allo- and auto-crossmatches presumably due to non-HLA interfering factors. We investigated the use of hypotonic dialysis (HD) and size exclusion spin columns (SC) to remove these factors while mitigating the effects on IgG antibodies. Methods 50 kDa dialysis membranes and 300 kDa size exclusion spin columns were used to remove IgM and other interfering factors from serum. Sera from six patients with moderate-titer antibody were treated three ways: DTT (5 mM @ 37 °C for 30 min), HD using Spectra/Por® Float-a-lyzer G2 according to the product insert, and NanoSep 300 SC (Pall Life Sciences) according to product insert. The IgG fractions of these separation methods were tested for IgG levels using OneLambda LabScreen single antigen (SA) bead kits. Class I (CI) and Class II (CII) antibody levels were analyzed separately using a two-tailed t -test, due to the inherent variability seen in the CI SA assay. Only allele specificities above our laboratory’s positive cutoff (MFI > 1000) were investigated. Results Luminex SA CI results showed variability between patients tested. No significant change in MFI was seen for CI DTT vs dialyzed samples ( p = 0.128) or for DTT vs SC-treated samples ( p = 0.209). CII samples showed a significant change for both methods: DTT vs dialyzed ( p = 0.016) and DTT vs SC ( p = 1.38x10-6). Spin columns decreased the MFI for all 181 CII specificities investigated for the six patients tested. Fig. 1 illustrates how the MFI for one patient’s CII sample was affected by all studied methods. Conclusions We have determined that SC treatment alters patient serum too significantly for our laboratory to use. HD appears to be potentially useful, though it does often raise the MFI of some existing specificities and may increase the amount of unacceptable antigens detected. More testing is required to determine the clinical relevance of MFI increases. Flow XMs will also need to be performed with dialyzed serum to determine if sera treated in such a way may be used in crossmatches.

OR08: IT’S ABOUT TIME. THE DEVELOPMENT OF THE RAPID OPTIMIZED SINGLE ANTIGEN BEAD (ROB) LABSCREEN® PROTOCOL TO EXPEDITE HLA ANTIBODY TESTING

Background The LABScreen single antigen bead (SAB) luminex assay is routinely used for HLA antibody detection. Compared to flow or ELISA based assays, SAB assay allows for relatively rapid testing and analysis of multiple patient samples. Despite this, SAB assay is time intensive and not optimal for use in urgent cases. The goal of this study was to develop a rapid SAB protocol without compromising assay sensitivity. Methods Initially, positive control (PC) sera were used to test the effects of varying centrifugation time (1 vs 5 min), serum incubation (15 vs 30 min), IgG -PE incubation (5 vs 30 min), serum volume and IgG-PE concentration on SAB assay results. Based on this data, the Rapid Optimized SAB (ROB) protocol (70% assay time savings) was developed and compared to the standard SAB (SS) assay by parallel testing of 26 sera (17 pos/9 neg) in Halifax Lab. Protocols were compared with respect to MFI (Pearson correlation coefficient) and HLA antibody assignment (2000 MFI cutoff). Pilot evaluation with 3 ABH proficiency testing (PT) sera was performed in Santa Casa Lab. Results Reducing centrifugation from 5 to 1 min had no impact on SS assay results. When incubation times with serum, IgG-PE, or both were reduced, mean PC MFI were decreased compared to the SS protocol (by 25%, 26% and 47% respectively). In contrast, mean PC MFI were significantly enhanced (> 50%) when higher IgG-PE concentration was used. Next, the ROB protocol was formulated and compared to the SS protocol using 26 sera. HLA antibody reactivity patterns were similar with both protocols for all sera with excellent MFI correlation ( r 2 = 0.99). The ROB protocol had higher mean MFI (by 10.3%) compared to the SS protocol resulting in slight increase in positive rxns (1007/3167 vs 985/3167; 31.8% vs 31.1%; MFI > 2000). ROB+/SS- rxns ( n = 33; 0.97/ panel) had SS MFI range of 1122–1980, while ROB-/SS + rxns ( n = 10; 0.29/panel) had ROB MFI range of 1565–1703. No false positive rxns were seen in 9 antibody negative sera. ROB vs SS MFI correlation with 3 ABH PT sera in Santa Casa Lab was excellent ( r 2 = 0.99) with a single ROB+/SS- rxn (2612 vs 1978 MFI). Conclusion ROB protocol correlates well with the standard LABScreen protocol and reduces assay time by 70%. ROB protocol implementation will expedite HLA antibody testing in urgent cases and improve turn-around time for routine testing.

Incidence and Impact of Anti-HLA-DP-Antibodies in Renal Transplantation.

Background: The role of HLA-DP-antibodies in renal transplantation is still poorly defined, whereas the clinical importance of DQ-antigens is widely accepted. This study aims to describe the incidence of donor- and non-donor specific antibodies against HLA-DP antigens in patients with renal transplantation. Methods: In a cohort of 112 kidney transplant patients, 97 recipients and their donors were typed for DP antigens by high-resolution sequence-specific oligonucleotides. Sera taken pre-transplant and at 1,3,6,9,12 months and annually post-transplant were retrospectively tested for anti-HLA-DP-antibodies using LABScreen single antigen bead. Results: In 43 (44.3%) patients, anti-HLA-DP-antibodies were found. 67.4% (n=29) were present in the pre-transplant sample and 32.6% (14) were exclusively found post-transplant. The median time from transplantation to antibody detection in the serum was 16.3 months. 71.4% (26) of the antibodies had a mean fluorescence intensity (MFI) between 1000 and 2000. 28.6% (17) had a MFI greater than 2000. There was no difference in MFI-levels between preformed and de novo anti-DP-antibodies. 50% (7) of the de novo anti-DP-antibodies accompanied de novo donor-specific antibodies (DSA) against HLA Class I (1), Class II (3) or both (3). 14% (2) were donor-specific antibodies. The 36 months graft survival was significantly reduced with de novo anti-DP-antibodies (p=0.012) (Figure 1): 50% (3) of the patients losing the graft within 36 months post-transplant had non-donor-specific anti-DP-antibodies. In two patients these antibodies accompanied donor-specific anti-DR- and DQ-antibodies.Figure: No Caption available.Conclusions: Antibodies against HLA-DP antigens are common in a cohort of renal transplantation and might have a harmful effect on the graft.

Identifying Surrogate Endpoints for DSA Treatment Trials Based on Data from a 10-Year Longitudinal Study of Primary Renal Transplant Recipients.

Transplant recipients that develop DSA post-transplant are at the highest risk for allograft loss, leading to increasing interest in designing studies to treat DSA. This analysis aims to identify surrogate endpoints following DSA onset that could aid clinical trial design and clinical management. Methods: All HLA-mismatched patients receiving a primary renal transplant between 9/1999 and 2/2008 were anayzed. All patients who were positive for DSA pre-transplant, died, or were lost to follow-up were excluded. Serum was collected pre-transplant and sequentially post-transplant at 1, 3, 6, 9, 12 months and annually, thereafter. The sera were retrospectively tested for the presence of HLA IgG, IgM, and IgG3 antibodies via LABScreen single antigen beads. Results: 46 patients were found to have de novo DSA. The actual 1-year, 3-year, and 5-year death-censored allograft survival following DSA appearance in the cohort was 93%, 69%, and 67%, respectively. Surrogate predictors of allograft loss at 3 years post-DSA were selected (Table). By themselves, acute rejection in 1-year post-DSA and 25% decline in estimated glomerular filtration rate (eGFR) at 1-year post-DSA were the best surrogate markers. When combined, the surrogate endpoint of acute rejection (with or after DSA) OR 25% decline in eGFR was highly predictive of allograft loss at 3 years post-DSA (HR 8.2 95%CI 1.8-36.9). Of note, this combined endpoint was not significantly predicted by noncompliance, IgG3 DSA at DSA onset, IgM DSA, or DSA MFI at DSA onset.Table: No Caption available.Conclusion: The combined endpoint of developing acute rejection (Mixed or AMR) within 1-year post-DSA or 25% eGFR decline at 1-year post-DSA may serve as a surrogate endpoint to evaluate treatment at 1-year post-DSA appearance to predict 3-year post-DSA allograft loss.

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Aim Both bead and cell-based assays for HLA antibody detection can be impacted by non-specific background staining. In some (but not all) instances, adsorption of serum with control beads prior to testing reduces background. Herein we demonstrate that the addition of small amounts of FBS to patient sera can reduce or eliminate non-specific background activity. Methods Briefly, 5ul of heat-inactivated FBS was added to 50ul of patient or control serum. Samples were incubated from 0-30 minutes and then centrifuged before testing in bead or cell-based assays. Results Informative patient’s sera were treated with FBS and tested by FlowPRA, LABScreen Single Antigen Beads and flow crossmatch. Following treatment, 3 general patterns were observed:1) No effect on sera exhibiting well-defined HLA antibody specificities; 2) Improved resolution of true HLA antibodies in bead and cell based assays among sera with questionable (but real) antibodies; 3) Elimination of spurious activity among sera devoid of HLA antibody activity. FBS Treatment of beads alone had no effect.[figure1] Conclusions Many variables can contribute to high background staining in HLA antibody testing for which there are few remedies. These high backgrounds can confound/preclude the detection of true HLA antibody. Here we describe a simple procedure that can significantly reduce non-specific binding. FBS treatment allows easy resolution of true positive results from spurious background staining without any apparent loss in sensitivity and specificity. While its mechanism is unknown, we speculate that FBS treatment provides target antigens that block the binding of naturally occurring heterophile antibodies to beads and cells.

For Anti-HLA-Specific Donor Antibodies Detection By Flow Cytometry Cytotoxic Crossmatches Comparison of Methods

Anti-HLA-specific donor antibodies induce rapid, irreversible destruction of the transplant (hyperacute rejection) that today happens rarely due to immunologic studies—prospective crossmatch—of patients awaiting the kidney graft. The usual approach for pretransplant donor/recipient evaluation is based on 2 methods: (1) the cytotoxic complement crossmatch (CDC) and (2) the flow cytometric crossmatch (FCX). The CDC crossmatch is positive when complement-fixing antibodies are present, an absolute contraindication to kidney transplantation. The more sensitive FCX-positive crossmatch detects low concentrations of unable to fix performed antibodies complement. It is an “index” of possible damage due to accelerated rejection. The target of our study was to develop a cytotoxic flow cytometry crossmatch (cFCX) that detected cytotoxic antibodies move sensitively than the traditional CDC method and also was less subjective and more standardized for interpretation studying sera from 23 patients; the cFCX showed the requested efficiency characteristics even in an emergency. In addition, the new method permited one to calculate a cutoff for positivity (average value of the negative control + 2 standard deviations), assuring an “objective” interpretation of the results that agreed with the CDC but was more sensitive and accurate allowing solution of ambiguous results for cases of “doubt”-positive CDC crossmatch. Furthermore, our aim was to correlate the effect of the strength of the anti-HLA antibodies determined by mean fluorescence intensity value of LabScreen Single Antigen beads with results of CDC, cFCX, and FCX methods.

Impact of alloantibody strength in crossmatch negative DSA positive kidney transplantation

ObjectivesThe clinical relevance of pre-transplant “low-level” donor specific anti-HLA antibodies (DSAs) in crossmatch negative kidney transplant recipients remains unclear. To determine what level of DSA associates with antibody mediated rejection (AMR) could be the way to measure the clinical relevance of pre-transplant “low-level” donor specific anti-HLA antibodies (DSAs) in crossmatch negative kidney transplant recipients. Design and methodsA retrospective analysis of 221 patients from October 2008 to December 2009 was included in this study. Sera were obtained pre-transplant and two weeks post-transplant and tested for DSA using LABScreen single antigen beads. ResultsAmong the 221 patients, 11 experienced AMR within 200days after transplant (5%). Pre-transplant DSA was associated with AMR at multiple mean fluorescence intensity (MFI) cutoffs (500, 1000, 2000, 3000, 5000; p=0.003, 0.001, 0.007, 0.003, and 0.003, respectively). No correlation was seen between acute T-cell mediated rejection (CMR) and pre-transplant DSA at any of the same MFI cutoffs. There was an increased risk of AMR with higher levels of pre-transplant DSA. Finally, an increase in DSA MFI from pre- to two weeks post-transplant was indicative of a higher probability of AMR. ConclusionOverall, this data supports using the single antigen bead to detect “low-level” DSA both pre- and post- as having a positive and persistent DSA may be predictive of higher AMR rates and poorer graft survival.