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Aim T cells specific for tFVIIIs in HA patients can induce neutralizing FVIII antibodies called inhibitors. DCs that present tFVIII derived peptides in their HLAcII repertoire may activate T cells. We employed DC protein processing and presentation assays (DCAs) to quantify tFVIII peptides in HLAcII peptidomes and used these as measures of the immunogenic potential (IP) of tFVIIIs as a function of their associated HLAcII/tFVIII peptide density. Methods We used DCs from 28 healthy donors and 5 HA patients [2 inhibitor (Inh) positive and 3 Inh negative] in 3 DCAs. In DCA 1, DCs from healthy donors 1–12 were given an equimolar mix of 5 recombinant (r) FVIIIs. In DCA 2, DCs from healthy donors 13–24 were given a plasma derived (pd) FVIII + vWF or 1 of 2 rFVIIIs (BDT or FL) ± vWF. In DCA 3, DCs from healthy donors 25–28 and HA patients 1–5 were incubated with pdFVIII or FL, BDD, or BDT rFVIII, all + vWF (Fig. 1A). After DC lysis and DR, DQ and DP isolation, we identified tFVIII peptides by LC-MS/MS. We performed a log linear analysis of the DR bound tFVIII peptides to identify determinants of Inh risk (Fig. 1B-E) with random effects to control for potential donor- and DCA-clustering. Space limits prevent description of the DQ and DP peptides. Results The IP of tFVIIIs was greater in HA patients than in healthy donors and in Inh positive than in Inh negative patients (Fig. 1B-E). We found that BDT + vWF and FL ± vWF had lesser and greater IP, respectively, than pdFVIII + vWF; the IP of BDD + vWF was comparable to that of pdFVIII + vWF. The rFVIII mix had the greatest IP. The higher IP in HA patients versus healthy donors and in Inh positive vs Inh negative patients reflect significant correlations of 0.91 and 0.52 with the presence/absence of the FVIII Inh risk allele DRB1*15:01. Download : Download high-res image (400KB) Download : Download full-size image Conclusions DC protein processing and presentation is affected by HLAcII repertoires but not by HA patient or FVIII Inh status after accounting for correlation with DRB1*15:01. Relative to pdFVIII, rFVIIIs have different IP with BDT and FL being significantly less and greater, respectively. T.E. Howard: 4. Scientific/Medical Advisor; Company/Organization; Haplomics Biotechnology. M. Hofmann: 5. Employee; Company/Organization; CSL Behring. L.V. Dinh: 5. Employee; Company/Organization; Haplomics Biotechnology. J. Powell: 5. Employee; Company/Organization; CSL Behring. E. Maraskovsky: 5. Employee; Company/Organization; CSL Limited.

Aim Luminex Single Antigen bead assay (LSAB) derived mean fluorescence intensity (MFI) values of anti-HLA antibodies directed at the potential donor’s HLA are the primary parameter used in the virtual crossmatch to predict physical crossmatch (XM) outcome. We aimed to develop statistically validated LSAB MFI cutpoints for predicting CDC XM and FCXM results. Methods We leveraged the ASHI proficiency testing (PT) 80% consensus results of 7156 T FCXM, 6758 B FCXM, 2917 T CDC XM and 2233 B CDC XM as the reference results to investigate whether LSAB MFI of IgG anti-HLA antibodies predict validated physical XM results. LSAB MFI was determined in our laboratory using One Lambda Single Antigen HLA Class I and Class II beads. The 80% consensus results are from 8 consecutive challenges during 2013 to 2016, and 107 laboratories across USA tested sera and HLA typed cells distributed by ASHI, and reported the results to ASHI for assessment of the laboratory’s proficiency. Data analysis included: summing of MFI of IgG antibodies directed at HLA-A, B and C for investigating their association with T FCXM and T CDC XM results; summing of MFI of antibodies directed at HLA-A, B, C, DR, DQ and DP for investigating their association with B FCXM and B CDC XM results; assigning alternate ASHI challenges to the Discovery set and the Validation set; investigating the association between LSAB MFI and physical XM results by logistic regression analysis corrected for overdispersion; and identification of LSAB MFI cutpoint for maximizing the sum of sensitivity and specificity. LSAB MFI cutpoints derived from the Discovery set to predict FCXM and CDC XM results were investigated in an independent validation set. Results Table 1 demonstrates that LSAB MFI cutpoints from the Discovery set predicts XM outcomes in the Validation set (ROC AUC range from 0.974 to 0.999). Conclusions We have developed and validated LSAB MFI cutpoints for use in the virtual crossmatch to accurately predict physical T FCXM, B FCXM, T CDC XM and B CDC XM results.

Aim To test the benefits of a software tool that captures HLA antibody analyses, highlights DSA and other risk factors, and provides a comprehensive report to allow fast and accurate Virtual Crossmatches (VXMs). Methods The VXM program used embedded, tiered reviewed HLA antibody data obtained from phenotype and single antigen bead panels. HLA antibody assignments in the VXM program required 2 independent reviewers and were not based on a MFI threshold; rather, assessments included HLA reactivity patterns, confirmation on multiple assays or in multiple sera, and previous HLA exposures (pregnancies and transplants). HLA typing for each potential donor was entered and DSA specificities, if present, were highlighted with individual strength assessments. Embedded HLA typing results for each candidate and all previous allografts allowed reporting of repeated HLA mismatches. Additional alerts included recent HLA sensitization changes, dates for peak and last serum tested, and HLA allele-specific antibodies that cannot be listed as avoids. Results A comparison was performed using results from the VXM program and 251 manual VXMs performed by experienced laboratory personnel during the first quarter of 2017. The majority (82%) of these VXMs involved sensitized candidates and >50% involved 99–100% CPRA candidates. One hundred of 251 VXMs examined were limited to one assessment per patient, but for the majority of cases VXM HLA analyses were performed repeatedly for the same patient. In 7.5% of manual VXMs cases, a secondary review of HLA data improved final DSA assignments. The number of VXMs per donor ranged from 1 to 6 with an average of 1.5 patients per donor. The average time for a manual VXM per patient was 30 min, while the VXM program and embedded data required 5 min per patient. In addition, the VXM program stored all previous donor assessments to assist in the decision to accept or decline the current offer. Conclusions For centers that do not list all HLA antibodies as unacceptable for sensitized waitlist candidates, VXMs can present a complex challenge given that potential donors must be reviewed and accepted/declined within one hour of initial offer. A VXM program that uses tiered reviewed HLA data increases efficiency and reduces possible errors in assessing risk for highly sensitized candidates.

Aim Our aim is to evaluate the performance of a computerized virtual crossmatch within the Duke University Medical Center HLA laboratory. Our HLA lab has used virtual crossmatch (vXM) to select deceased donors for heart and lung transplantations for more than ten years, which has allowed us to accept donors from outside of our Organ Procurement Organization territory. The virtual crossmatches were done by manually checking the recipient’s HLA antibody profiles within our lab database against the potential donor’s HLA typing to identify if the recipient has any donor-specific antibodies (DSA) in the most recent serum tested and in historical sera. To ensure no DSA was missed, the vXM was manually done by multiple transplant professionals at different stages for each sensitized recipient when a donor offer was available. Laboratory informatics vendor HLA Data Systems, developers of the mTilda Lab Management System, has developed a stand-alone software program which performs a computerized vXM (cVXM) under the product name VxMatch. The VxMatch program directly connects to any HLA lab database to query required data for analysis. Methods We performed 56 cVXMs by using the new program for patients who had previously received manual vXMs (mVXM) over the past few months. 35 of the 56 patients have calculated panel reactive antibodies (cPRA) = >80%. Results We found a 100% concordance for identification of DSA between cVXM and mVXM. It took less than 5 min to complete the cVXM while the conventional mVXM took 30 min on average. For each identified unacceptable antigen, the cVXM program provides detailed information, including antibody specificities, mean fluorescence intensity (MFI), serum date and a graph showing the MFI changes of DSA over time. The mTilda cVXM can operate in two different modes: a technologist’s level and supervisory level. The supervisory module allows us to review each individual bead carrying potential unacceptable HLA antigen at alpha chain, beta chain, or allele level. This cVXM can also be used to identify the sera (for example the serum with peak DSA) for final XM and the ability to track the change of DSA post-transplant. Conclusion Our evaluation suggests that this new computerized vXM program is a user-friendly, reliable and powerful tool for HLA testing and management.

Aim A virtual crossmatch determines which recipients are eligible for a deceased donor kidney based on UNET unacceptable antigens. The MFI threshold for unacceptable antigens is often higher than what most labs consider to be positive (e.g. 1000 MFI). Clinicians are interested in knowing whether DSAs below the threshold are present so immunosuppression protocols can be altered or, if several are present, they should rule out a patient for transplant. What is the mechanism for reporting to clinicians the presence of potential DSAs during deceased donor kidney allocation?. Methods The Laboratory of Immunogenetics & Transplant (LIT) serves three kidney transplant centers and the local organ procurement organization. Waitlist patients are screened yearly by One Lambda Single Antigen I/II beads. Unacceptable antigen (UA) cutoffs are 3000-5000MFI, while single antigen beads >1000 MFI are assigned as positive with a few exceptions. During deceased donor evaluation, LIT technologists complete HLA-specific data entry into DonorNet and perform the matchrun. Using LIT-specific Histotrac programming, LIT runs exclusion algorithms for local protocols, choses the top candidates to crossmatch, and creates a Virtual XM report. Two centers use a protocol to avoid unacceptable antigens with additive MFIs >3000. We reviewed matchruns, virtual crossmatch exclusions, and transplants from March 2015 to Febuary 2017. Results In two years of cases, 332 kidney matchruns were evaluated: 286 local matchruns were performed while 46 import donors were evaluated. Consequently, 2288 patient-donor pairs were reviewed by virtual crossmatch. Patients were ruled out due to additive DSAs in 17 (0.7%) patient-donor pairs. Two-thirds of patients who were transplanted with a deceased donor kidney had a 0% cPRA while 5% had cPRA 98–100%. Positive virtual crossmatches prompted additional review of UA and antibody history. Conclusions The laboratory staff is responsible for completing deceased donor matchruns and generating a virtual crossmatch report, thus providing a streamlined approach to deceased donor evaluations. The virtual crossmatch report is essential for clinicians’ center-specific algorithms, induction protocols, and assessing risk for kidney candidates in the presence of potential DSA.

Background Donors specific anti-HLA antibodies (DSA) in renal transplantation have been associated with higher incidence of graft failure. Therefore, potential living donors cells are tested against recipient serum using flow cytometric crossmatch (FCXM) to determine if the presence of DSA will cause positive FCXM. Antibody determination prior to crossmatching potential donors allows for the determination of the virtual crossmatch (VXM) as either being positive or negative. Herein, we report a case where the FCXM did not correlate at all with the VXM. Methods Pre-transplant serum was tested by solid-phase immunoassays for the presence of unacceptable antibodies. Strength of the antibody is determined from mean fluorescence intensity (MFI) cut-offs established by the laboratory. Results An initial FCXM was performed with potential donor cells and recipient serum. Based on a single DSA with MFI of Conclusion Distortion of antigen integrity may have caused the B62 from not being detected by solid phase assay. Antibody testing using solid phase methods are helpful tests but one cannot completely relay on them when trying to determine antibody status of recipients waiting for transplant.

Aim We have noted that anti-B44 (44:02) detected with LABScreen® SA beads may not result in a positive crossmatch (XM). Inclusion of B44 as an avoidance antigen could unnecessarily exclude XM-compatible donors. Therefore, we sought to develop a protocol for authenticating when anti-B44 detected by SA beads should be classified for avoidance, ie, potential donor-specific antibody (DSA). We chose to use CDC PRA, C1qScreen™, and iBeads™ assays, and XM with surrogate donors (Flow and CDC) for verifying the presence or absence of anti-B44. It was felt that multiple tests may be needed, since we would be expecting negative detection results. Methods Sera from a patient with several determinations yielding anti-B44:02 (∼5000 MFI) by LABScreen® SA beads were analyzed. The sera were Flow XM negative with a potential donor, despite the positive virtual XM. Lambda™ Antigen Trays (LAT), C1qScreen™, iBeads™ (non-denatured SA beads), and XM with surrogate donors by CDC and Flow were used for confirmation of the presence or absence of the putative anti-B44 (44:02) DSA. Results Anti-B44 was not detected using LAT. C1qScreen™ and iBeads™ assays did not detect anti-B44. CDC and Flow XM were negative with the potential donor and 3 additional surrogate donors. Taken altogether, it would appear that the detected anti-B∗44:02 antibody was directed against denatured or partially denatured antigen, not in the native conformation, as normally found on cell surfaces. Conclusions Anti-B∗44:02 antibody detected on SA beads may be directed to denatured or partially denatured antigens, which have no in vivo relevance. One must carefully scrutinize apparent anti-HLA B∗44:02 antibodies (and possibly others) for the ability to bind to HLA-antigens in their native conformation and activate complement for clinical relevance. Therefore, the CDC assay remains relevant. One must be careful in the declaration of DSA, which could result in elimination of a good potential donor with the Virtual XM.