Virtual crossmatching and epitope analysis in kidney transplantation: What the physician involved in kidney transplantation should know?

Solid-phase single antigen bead (SAB) assay for detection of anti-human leukocyte antigen (HLA) antibodies and high-resolution HLA typing have enabled tremendous progress in virtual crossmatch (VXM) technology in recent years. However, misinterpretation of the SAB assay may result in detrimental consequences after kidney transplantation. Meanwhile, epitope analysis could be an effective method to estimate immunizing eplets, which may provide ancillary information for better understanding of the SAB assay. To perform epitope analysis appropriately, it is necessary to understand the basic principles related to histocompatibility testing and the characteristics of the SAB assay. Therefore, knowledge of the properties and limitations of the SAB assay is critical. In this review, we aim to describe the fundamental concepts regarding immunobiological assessment, including HLA, anti-HLA antibodies, and SAB assay, and explain epitope analysis using examples.

HLA-specific memory B-cell detection in kidney transplantation: Insights and future challenges.

Humoral alloimmunity mediated by anti-human leucocyte antigen (HLA) antibodies is a major challenge in kidney transplantation and impairs the longevity of the transplanted organ. The immunological risk of an individual patient is currently mainly assessed by detection of HLA antibodies in the serum, which are produced by long-lived bone marrow-residing plasma cells. However, humoral alloimmunity is complex, and alloreactive memory B cells constitute an additional factor in the interplay of immune cells. These recirculating "silent" cells are responsible for the immunological recall response by differentiating into antibody-producing cells upon antigen re-encounter. Historically, due to the lack of appropriate and routinely applicable assays to determine the presence and HLA specificity of alloreactive memory B cells, their contribution to the humoral alloimmune response has clinically often been suspected but could not be determined. In this review, we give an overview of recent advances in techniques to detect alloreactive memory B cells and discuss their strengths and limitations. Furthermore, we summarize experiences with these techniques in alloimmunized individuals and transplant recipients, thereby emphasizing unmet needs to be addressed in future studies.

False negative single antigen bead assay: Is it always an effect of prozone?

BACKGROUND: Bead based flow cytometry and Luminex play a major role in identification of alloantibodies in renal transplant work-up. Strong sensitization events may lead to prozone phenomenon that can affect single antigen bead (SAB) assay and result in false negativity. However, this can also be due to high titer of other blocking antibodies. While methods like, heat inactivation, C1 inhibitor, Ethylene diamine tetra-acetic-acid and Dithio threitol treatment can remove interfering antibodies of complement and IgM, these methods are not optimal if false negativity is due to prozone effect, which is high titer of antibodies alone. METHODS: We hereby present a case of a highly sensitized renal transplant recipient with 64% panel reactive antibody positivity (PRA) and a subsequent negative SAB assay. This paradoxical finding hinted at SAB being a false negative result and serial dilutions were used to perform further tests. RESULTS: Serum dilutions lead to positive flow based panel reactive antibody (PRA) and flow cytometry crossmatch (FCXM), with an increasing trend in FCXM. CONCLUSIONS: In highly sensitized patients serial dilution should be considered during a transplant work-up to avoid missing any underlying antibodies. Serum dilution can be used as first option to circumvent prozone. Also, interference of other antibodies should not be labeled as prozone effect.

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.

Pretransplant human leukocyte antigen antibodies detected by single-antigen bead assay are a risk factor for long-term kidney graft loss even in the absence of donor-specific antibodies.

Clinical relevance of ELISA- and single-antigen bead assay (SAB)-detected pretransplant HLA antibodies (SAB-HLA-Ab) for kidney graft survival was evaluated retrospectively in 197 patients transplanted between 2002 and 2009 at the University Clinic Frankfurt. Having adjusted for retransplantation and delayed graft function, a significantly increased risk for death-censored graft loss was found in patients with pretransplant SAB-HLA-Ab [HR: 4.46; 95% confidence interval (CI): 1.47-13.48; P = 0.008]. The risk for increased graft loss was also significant in patients with pretransplant SAB-HLA-Ab but without SAB-detected donor-specific Ab (SAB-DSA) (HR: 4.91; 95% CI of 1.43-16.991; P = 0.012). ELISA was not sufficient to identify pretransplant immunized patients with an increased risk for graft loss. In immunized patients, graft loss was predominantly present in patients who received transplants with a mismatch on the HLA-DR locus. In conclusion, even if our study is limited due to small sample size, the results show an increased risk for long-term graft loss in patients with pretransplant SAB-HLA, even in the absence of DSA. SAB-HLA-Ab-positive patients, being negative in ELISA or CDC assay, might profit from a well-HLA-DR-matched graft and intensified immunosuppression.

Single antigen flow beads for identification of human leukocyte antigen antibody specificities in hypersensitized patients with chronic renal failure.

AIMS OF THIS STUDY: Aims of this study were to identify class I and class II antibodies in highly sensitized patients by flow cytometry single antigen bead (FC-SAB) assay and to evaluate according to donor HLA type in order to increase their kidney transplantation chance. MATERIAL AND METHODS: We analyzed 60 hypersensitive patients of 351 individuals, who applied to our laboratory for PRA test in November 2013-December 2014. Flow cytometric PRA screening and single antigen bead commercial kits were used for these analyses. RESULTS: In our study group, 19 (31.7%) of these patients were male while 41 (68.3%) patients were female. The most common acceptable antigens were A*02 (10.11%), HLA-A*23 (10.11%), HLA-B*38 (8.79%) and HLA-DRB1*03 (7.83%) in hypersensitive patients. The highest antibody reactivity on SAB was observed against HLA-A*25, HLA-B*45, HLA-DRB1*04 and HLA-DRB1*08 antigens. CONCLUSIONS: The determination of these acceptable and unacceptable antigens may increase their transplantation chance. Pre-transplant HLA antibody identifications provide prognostic information with respect to the determination of patients who are at increased risk of graft loss.

Complement-dependent cytotoxicity crossmatch in solid organ transplantation: The gold standard or golden history?

Complement-dependent cytotoxicity crossmatch (CDC-XM) has been considered for many years the standard of practice for determining compatibility in solid organ transplantation (SOT). However, as this method is laborious, time intensive and lacks sensitivity and specificity, it has been replaced in many laboratories worldwide by flow cytometry crossmatch (FCXM) and/or virtual crossmatch (vXM). With this study we intend to show the relevance of performing CDC-XM in the era of virtual crossmatching. We retrospectively analyzed 1,007 consecutive T and B cell deceased donor (DD) CDC-XMs performed in parallel using non-treated and dithiothreitol (DTT) treated sera between May 2022 and January 2023 in waitlisted patients with no donor specific antibodies (DSA) against HLA-A, B and/or DR antigens. Thirty five of 1,007 (3.5%) T cell crossmatches and 132 of 1,007 (13.1%) B cell crossmatches were positive with non-treated sera. Correlation with the vXM demonstrated no DSA in any of the positive T cell crossmatches. DSA were also absent in 126/132 positive B cell crossmatches, indicating a high rate of false positive CDC-XM. Indeed, only 4/35 T cell and 13/132 B cell CDC-XM remained positive after treatment with DTT, confirming that false positive reactivity with non-treated sera is high. Class I HLA DSA against C locus antigens were present in 17/1,007 T cell crossmatches and none were detected by CDC-XM (sensitivity = 0%). Similarly, only 6/77 B cell crossmatches with DSA targeting HLA-C, DQ and/or DP antigens were CDC-XM positive (sensitivity = 7.8%). Furthermore, only 4/6 positive B cell CDC-XM were confirmed to have complement binding potential using the C1q assay, suggesting additional false positive reactivity in 2/6 of the positive CDC-XM. Our study demonstrates that CDC-XM exhibits poor sensitivity, high false positive reactivity (especially without DTT treatment) and does not meaningfully contribute to pre-transplant compatibility testing in the context of vXM based allocation. Furthermore, the use of CDC-XM can unnecessarily delay or even prevent safe and appropriate transplant allocation.

Specificity of HLA monoclonal antibodies and their use to determine HLA expression on lymphocytes and peripheral blood stem cells.

HLA Class I and II expression are known to differ locus-to-locus, however, HLA expression on the cell-surface is frequently reported as the total amount of HLA Class I or II antigens. This is despite evidence that indicates the differential expression of HLA can influence patient outcomes post-transplantation. Although numerous commercially available HLA monoclonal antibodies (mAbs) exist to characterize HLA expression, there is currently a lack of detailed information regarding their reactivities to HLA specificities. The specificities of locus-specific HLA mAbs (nine Class I and four Class II mAbs) were evaluated by two solid-phase Luminex single antigen bead assays. The reactivity patterns of these mAbs were then confirmed by flow cytometry using lymphocytes and PBSCs (peripheral blood stem cells). Out of the 13 HLA mAbs tested, only four (one Class I and three Class II mAbs) displayed intra-locus reactivity without also reacting to inter-locus specificities. Epitope analysis revealed the presence of shared epitopes across numerous HLA loci, explaining much of the observed inter-locus reactivity. The specificity of the HLA mAbs seen in solid-phase assays was confirmed against PBSCs and lymphocytes by flow cytometry. Using this method, we observed differences in the cell surface expression of HLA-C, HLA-DR, HLA-DQ, and HLA-DP between PBSCs and lymphocytes. Our results emphasize the need to characterize the reactivity patterns of HLA mAbs using solid-phase assays before their use on cells. Through understanding the reactivity of these HLA mAbs, the cellular expression of HLA can be more accurately assessed in downstream assays.

Direct addition of EDTA to LABScreen single antigen bead suspension: A simple way to prevent complement mediated interference and streamline HLA antibody testing.

Compromised detection of HLA specific antibodies due to complement mediated interference (CMI) is a well-recognized limitation of the single antigen bead (SAB) assay. Serum treatment with EDTA prior to SAB assay testing is a common strategy used to prevent CMI, however, treatment of individual sera, especially in large clinical runs, can extend assay turnaround time and increase the risk of a sample mix-up. In this study, we describe a simplified EDTA treatment strategy that can be applied simultaneously to all sera in a testing run. This strategy effectively prevents CMI without added pipetting steps and the increased turnaround time associated with other strategies. In the novel bead treatment (BT) method, EDTA solution and SAB suspension are combined and added into the wells of a testing tray together, patient sera are then added to the SAB/EDTA mixture. This eliminates the separate EDTA pre-treatment step used in the standard procedure (ST). Parallel testing using the BT, ST, and no EDTA treatment strategies followed by antibody identification using the Rapid Optimized SAB (ROB) protocol was performed for 19 well characterized sera with known CMI at two different laboratories. Both BT and ST methods were equally effective in preventing CMI. In addition, excellent MFI correlation was observed for specificities not affected by CMI. Both negative and pooled positive control sera performed as expected using the BT method and the pooled positive control serum, specifically designed to exhibit CMI, served well as an EDTA treatment control for all sera. The modified BT protocol can be easily implemented for clinical testing and eliminates extra pipetting steps, reduces the likelihood of pipetting error, and decreases turnaround time, while effectively preventing CMI and ensuring accurate detection of HLA antibodies. This protocol was recently implemented in the Halifax HLA Laboratory and has been very positively received by the laboratory team.

The significance of pretransplant donor-specific antibodies reactive with intact or denatured human leucocyte antigen in kidney transplantation.

Antibodies recognizing denatured human leucocyte antigen (HLA) can co-react with epitopes on intact HLA or recognize cryptic epitopes which are normally unaccessible to HLA antibodies. Their specificity cannot be distinguished by single antigen beads (SAB) alone, as they carry a mixture of intact and denatured HLA. In this study, we selected pretransplant sera containing donor-specific HLA class I antibodies (DSA) according to regular SAB analysis from 156 kidney transplant recipients. These sera were analysed using a SAB preparation (iBeads) which is largely devoid of denatured HLA class I, and SAB coated with denatured HLA class I antigens. A total of 241 class I DSA were found by regular SAB analysis, of which 152 (63%) were also found by iBeads, whereas 28 (11%) were caused by reactivity with denatured DNA. Patients with DSA defined either by regular SAB or iBeads showed a significantly lower graft survival rate (P = 0·007) compared to those without HLA class I DSA, whereas reactivity to exclusively denatured HLA was not associated with decreased graft survival. In addition, DSA defined by reactivity to class I SAB or class I iBeads occurred more frequently in female patients and in patients with historic HLA sensitization, whereas reactivity to denatured HLA class I was not associated with any of these parameters. Our data suggest that pretransplant donor-specific antibodies against denatured HLA are clinically irrelevant in patients already sensitized against intact HLA.

A modeling approach for mean fluorescence intensity value harmonization and cutoff prediction for luminex single antigen bead assays of two different vendors.

Luminex single antigen bead (SAB) kits from One Lambda (OL) and Lifecodes (LC) are widely used for HLA antibody detection but have substantial differences in design and assay protocol resulting in different mean fluorescence intensity (MFI) values. Here, we present a non-linear modeling approach to accurately convert MFI values between two vendors and to establish user-independent MFI cutoffs when analyzing big datasets. HLA antibody data from a total of 47 EDTA-treated sera tested using both OL and LC SAB kits were analyzed. MFI comparisons were made for the common 84 HLA class I and 63 class II beads. In the exploration set (n = 24), a non-linear hyperbola model on raw MFI corrected by locus-specific highest self MFI subtraction yielded the highest correlation (class I r2 : 0.946, class II r2 : 0.898). Performance of the model was verified in an independent validation set (n = 12) (class I r2 : 0.952, class II r2 : 0.911). Furthermore, in an independent cohort of post-transplant serum samples (n = 11) using the vendor-specific MFI cutoffs dictated by the current model, we found 94% accuracy in bead-specific reactivity assignments by the two vendors. We recommend using the non-linear hyperbola modeling approach with self HLA correction and locus-specific analyzes to harmonize MFI values between two vendors in particular research datasets. As there are considerable variations between the two assays, using MFI conversion for individual patient samples is not recommended.

Donor-Specific Antibody Detection by Single-Antigen Bead Assay for Renal Transplantation: A 2-Year Experience from South India.

Introduction: Recipient sensitization against donor human leukocyte antigens (HLA) plays a key role in transplant rejection, and this risk is best minimized by efficient pre transplant antibody detection. Determination of antibody specificity with the highest sensitivity and degree of resolution to the allelic antigen level is achieved by using single-antigen bead (SAB) assay. Methods: This study evaluated the correlation of Luminex cross match (LXM) with SAB assay for detection of donor-specific antibodies (DSA). A total of 2075 renal transplant patients were screened for the presence of DSA by LXM, complement-dependent cytotoxicity (CDC) cross match, and 125 patients for SAB from January 2018 to December 2019. Results: There was a male preponderance among recipients (P < 0.0001), and the most affected age group was 21-40 years. HLA typing was done in 550/2075 by DNA PCR-reverse sequence-specific oligonucleotide probes (SSOP) method. HLA DSA by LXM was detected in 16.3% of recipients (338/2075). Majority 180/338 (53.2%) of the patients were class II DSA positive, (P < 0.0001). Among the class II DSA positive patients, 20/180 (11.1%) samples gave false-positive results by LXM. SAB for class I and class II HLA IgG antibodies was done in 125/338 renal transplant recipients, which included 20 recipients with false-positive class II Luminex DSA, to check whether the DSA detected were really donor specific or not. The results showed that although 20/125 patients had some antibodies detected in their serum, they were not against the donor HLA antigens, as per the HLA typing reports of the donors. When compared to SAB assay, LXM showed more discrepant results, particularly to class II DSA. Conclusion: In conclusion, LXM, if used in combination with SAB assay and HLA typing of donors if necessary for virtual cross match, will help in avoiding unnecessary exclusion of donors for renal transplant recipients and also for post transplant monitoring of recipients, especially in cadaveric donor transplants.

HLA diversity in ethnic populations can affect detection of donor-specific antibodies by single antigen beads.

Introduction: In solid-organ transplantation, human leukocyte antigen (HLA) donor-specific antibodies (DSA) are strongly associated with graft rejection, graft loss, and patient death. The predominant tests used for detecting HLA DSA before and after solid-organ transplantation are HLA single antigen bead (SAB) assays. However, SAB assays may not detect antibodies directed against HLA epitopes that are not represented in the SAB. The prevalence and potential impact of unrepresented HLA epitopes are expected to vary by ethnicity, but have not been thoroughly investigated. To address this knowledge gap, HLA allele frequencies from seven ethnic populations were compared with HLA proteins present in SAB products from two manufacturers to determine unrepresented HLA proteins. Materials: Allele frequencies were obtained from the Common, Intermediate, and Well Documented HLA catalog v3.0, and frequencies of unrepresented HLA types were calculated. Next-generation sequencing was used to determine HLA types of 60 deceased solid-organ donors, and results were used to determine if their HLA-A, -B, -C, and -DRB1 proteins were not present in SAB reagents from two vendors. Unrepresented HLA proteins were compared with the most similar protein in SAB assays from either vendor and then visualized using modeling software to assess potential HLA epitopes. Results: For the seven ethnic populations, 0.5% to 11.8% of each population had HLA proteins not included in SAB assays from one vendor. Non-European populations had greater numbers of unrepresented alleles. Among the deceased donors, 26.7% (16/60) had at least one unrepresented HLA-A, -B, -C, or -DRB1 protein. Structural modeling demonstrated that a subset of these had potential HLA epitopes that are solvent accessible amino acid mismatches and are likely to be accessible to B cell receptors. Discussion: In conclusion, SAB assays cannot completely rule out the presence of HLA DSA. HLA epitopes not represented in those assays vary by ethnicity and should not be overlooked, especially in non-European populations. Allele-level HLA typing can help determine the potential for HLA antibodies that could evade detection.

Deciphering the role of the conjugate's phycoerythrin label in complement-mediated interference occurring in HLA single antigen Luminex bead assays.

Complement-mediated interference is a well described phenomenon in single antigen bead (SAB) Luminex assay that leads to falsely low or negative results for anti-HLA antibody (Ab). In a context of high amount of Ab, the enrichment of the Ab around the bead can lead to complement cascade activation and deposition, thereafter impairing Ab detection. EDTA is now routinely used to circumvent this interference. In this report, we attempted to decipher the role of the phycoerythrin (PE) label conjugated to the secondary Ab in this interference. Indeed, PE is a huge molecule (240 kDa) that could participate to limiting access of the conjugate to its Ab target on the bead. To this purpose, 22 sera displaying complement interference without pre-treatment with EDTA were compared on SAB assay with three detection strategies: the recommended PE-conjugated secondary Ab (IgGPE), an Alexa Fluor 532-conjugated Ab (IgGAF) bearing a tiny 724 Da fluorochrome, and a biotinylated Ab followed by PE-conjugated streptavidin (IgGBiot). Complement interference occurred with the three detection methods, but its depth, defined by the percentage of MFI loss with neat serum, was the highest for IgGPE. Our study highlighted the partial role of the PE fluorochrome in complement interference in SAB assays.

Clinical significance of low pre-transplant donor specific antibodies (DSA) in living donor kidney recipients with negative complement-dependent cytotoxicity crossmatches (CDCXM), and negative flow cytometry crossmatches (FLXM) - A single-center experience.

BACKGROUND: It is controversial whether all donor-specific antibodies (DSA) detected by the solid-phase single antigen bead (SAB) assay negatively affect kidney transplantation outcomes. The study aimed to evaluate the possible clinical significance of low pre-transplant DSA in living donor kidney recipients. We analyzed a group of patients with HLA-A, B, and -DR DSA reactivities below a virtual crossmatch (VXM) value of 5000 MFI but with all VXM DSA reactivities at HLA-DQ, -DP, and -Cw, which were not typed routinely for donors prior to transplantation. We also investigated the incidence of persistent and de novo DSAs in available posttransplant SAB assays. METHODS: From the historical cohort of living donor recipients transplanted between 2014 and 2018 at our center (n = 82), 55 patients met the inclusion criteria, namely: these patients were > 18 years old with non-HLA identical sibling donors, who were not desensitized, who had available pre-transplant SAB results, and who had negative both complement-dependent cytotoxicity crossmatch (CDCXM) and flow cytometry crossmatch (FLXM) results. An additional donor HLA typing, performed for all 55 recipients, identified donor additional HLA-DQ, -DP, and -Cw DSA reactivities. These patients were then divided by SAB reactivity into three groups: 1) those with DSA-positive reactivities; 2) those with non-donor-specific anti-HLA reactivities (NDSA); and, 3) those who were anti-HLA-negative. All these recipients were followed for three years and checked for their de novo or persistent DSA. RESULTS: In the studied cohort, DSA-positive, NDSA reactive, and anti-HLA negative recipients constituted 33%, 36%, and 31% of 55 patients, respectively. Non-routinely considered pre-transplant HLA-DQ, -DP, and -Cw DSA-positive reactivities were shown in as many as 78% of DSA-positive cases (group 1) with the lowest MFI value of 319 to DP4 and the highest MFI of 5767 to DQ2. Of the pre-transplant HLA-A, B, and -DR DSA reactivities, only -DR52 DSA reactivity reached the highest MFI value of 2191. These detected DSAs did not reduce the mean estimated glomerular filtration rate (eGFR) values and did not increase the incidence of proteinuria in recipients. While the 3-year graft survival was lower in the DSA-positive group (94.4%) with one recipient who lost kidney transplant, the difference was not significantly different (p = 0.7) from the NDSA (100%) and negative (100%) groups. In terms of the incidence of de novo acute antibody-mediated rejection (AMR) at three years after transplantation, no case has been reported in the cohort. This may suggest that low DSA-positive recipients do not experience higher rejection rate. However, DSA-positive recipients had a tendency for a higher frequency of C4d deposits in peritubular capillaries (PTC) and de novo DSA. CONCLUSION: Our 3-year follow-up of patients with low pre-transplant DSA found no association with a deterioration in graft function and worse graft survival. Furthermore, we did not observe an increase in AMR in our patients with low DSA. A larger cohort and a longer follow-up period may be needed to evaluate the tendency of low DSA-positive recipients towards the higher incidence of C4d deposits in PTC and/or de novo DSA.

Eplet-Predicted Antigens: An Attempt to Introduce Eplets into Unacceptable Antigen Determination and Calculated Panel-Reactive Antibody Calculation Facilitating Kidney Allocation.

(1) Calculated panel-reactive antibody (CPRA) is a measure of sensitization based on unacceptable antigens (UAs). Determination of UAs based on single-antigen bead assays at allele or antigen levels may be inappropriate. We aimed to introduce eplets for better assessment of sensitization; (2) 900 recipients and 1427 donors were enrolled for candidate or donor pools, respectively. Eplets were from the HLA Epitope Registry. UAs were determined by anti-HLA antibodies identified using LIFECODES Single Antigen (LSA) kits. CPRA values were calculated using a simplified method of donor filtering; (3) HLA antigens containing all eplets of an HLA antigen in LSA kits (LSA antigen) were defined as eplet-predicted (EP) antigens, the reactivity of which could be predicted by that LSA antigen. High reactivity concordance was found between LSA and EP antigens. More HLA antigens were covered by EP antigens in the population than LSA antigens. CPRA values at the EP level were higher than at the allele level and lower than at the antigen level. The EP antigens facilitated UA determination for non-LSA antigens and avoided acute rejection; (4) UA determination using EP antigens can lead to more accurate assessment of sensitization, enabling a high probability of compatible organs and a low risk of adverse outcomes.

Cutting through the weeds: Evaluation of a novel adsorption with crossmatch cells and elution protocol to sharpen HLA antibody identification by the single antigen bead assay.

The single antigen bead (SAB) assay is the most used test for the identification of HLA specific antibodies pre- and post-transplant. Nevertheless, detection of spurious reactivities remains a recognized assay limitation. In addition, the presence of weak reactivity patterns can complicate unacceptable antigen assignment. This work presents the evaluation of the adsorption with crossmatch cells and elution (AXE) technique, which was designed to help differentiate weak HLA specific antibodies targeting native antigens from spurious and background SAB assay reactivity. The AXE protocol uses selected donor cells to adsorb HLA specific antibodies from sera of interest. Bound antibodies are then eluted off washed cells and identified using the SAB assay. Only antibodies targeting native HLA are adsorbed. Assay evaluation was performed using five cell donors and pooled positive control serum. AXE efficiency was determined by comparing SAB reactivity of adsorbed/eluted antibody to that of the antibodies in unadsorbed sera. A robust efficiency was seen across a wide range of original MFI for donor specific antibodies (DSA). A higher absorption/elution recovery was observed for HLA class I antigens vs. class II. Locus-specific variation was also observed, with high-expression HLA loci (HLA-A/B/DR) providing the best recovery. Importantly, negligible reactivity was detected in the last wash control, confirming that AXE eluates were not contaminated with HLA antibody carry-over. Donor cells incubated with autologous and DSA-containing allogeneic sera showed that AXE selectively adsorbed HLA antibodies in a donor antigen-specific manner. Importantly, antibodies targeting denatured epitopes or other non-HLA antigens were not detected by AXE. AXE was particularly effective at distinguishing weak HLA antibodies from background reactivity. When combined with epitope analysis, AXE enhanced precise identification of antibody-targeted eplets and even facilitated the characterization of a potential novel eplet. Comparison of AXE to flow cytometric crossmatching further revealed that AXE was a more sensitive technique in the detection of weak DSA. Spurious reactivities on the current SAB assay have a deleterious impact on the assignment of clinically relevant HLA specificities. The AXE protocol is a novel test that enables users to interrogate reactive patterns of interest and discriminate HLA specific antibodies from spurious reactivity.

Principles of Virtual Crossmatch Testing for Kidney Transplantation.

Human leukocyte antigens (HLAs) are the primary determinants of alloimmunity. A crossmatch test is a test that determines the immunologic risk of a recipient with a potential donor by ensuring that there are no transplant-relevant circulating antibodies in the recipient directed against donor antigens. Physical crossmatch (PXM) tests, such as complement-dependent cytotoxicity crossmatch (CDCXM) and flow cytometry crossmatch (FCXM), require mixing of patient serum and donor cells, are labor intensive, and are logistically challenging. Virtual crossmatch (VXM) test assesses immunologic compatibility between recipient and potential donor by analyzing the results of 2 independently done physical laboratory tests-patient anti-HLA antibody and donor HLA typing. The goal of VXM is pretransplant risk stratification-though there is no consensus on whether such risk assessment involves predicting the PXM result or the posttransplant outcome. Although the concept of VXM is not new, the advent of solid-phase assays for detecting circulating antibodies in the recipient directed against individual HLA and DNA-based methods for typing donor HLA specificities at a higher resolution makes the routine use of VXM a reality. Accordingly, VXM may be applied at different scenarios-both for sensitized and nonsensitized patients. Implementation of VXM-based approach has resulted in statistically significant reduction in cold ischemia time without an increase in hyperacute rejection episodes. Though there are considerable challenges, VXM is expected to be used more often in the future, depending on the transplant center's tolerance of immunologic risk.

Prozone phenomenon in pretransplant testing: An interesting conundrum involving solid-phase and cell-based assays.

BACKGROUND: Human leukocyte antigen (HLA) is a major determinant in deciding upon solid organ histocompatibility. Donor-specific anti-HLA antibodies (Donor-specific anti-HLA antibodies (DSAs)) are always a contraindication for solid organ transplantation, and identification of DSA becomes very crucial before transplantation to provide long-term graft survival. For identification of DSA, usually, either cell-based or HLA bead-based assay is being used in laboratories. However, both cell-based and bead-based assays have certain limitations. One such common limitation is "prozone effect," which can give false-negative results. Here, we would like to present a small pilot study to analyze the effect of the prozone phenomenon in the cell-based and HLA bead-based assays and its utility in histocompatibility testing. MATERIALS AND METHODS: In a series of four experiments, cell-based assay, flow cytometric cross-match (FCXM), and HLA bead-based flow cytometric panel reactive antibodies (PRAs) were performed. Single-antigen bead (SAB) testing was conducted as a first experiment on four known positives samples for anti-HLA antibody-antibodies. In the second experiment, these four samples were pooled together (called pooled sera in the text) and tested for FCXM and PRA. In the third experiment, known commercially available positive control sera were mixed with pooled positive sera (positive control sera + pooled sera) to prepare, what we have called "positive concoction" in the text. In the fourth experiment, the positive concoction was diluted serially (1:2, 1:4, 1:8, and 1:16) and FCXM and PRA were performed again to analyze and compare the prozone effect. RESULTS: Pooled sera did not have the expected median fluorescence intensity (MFI) values in FCXM assay, whereas the PRA was showing >90% positivity. In positive concoction, the MFI of FCXM assay was observed to be declining; however, PRA values remained almost constant. Dilutions of the pooled sera showed that MFI values of FCXM assays were increased suddenly after dilution. The highest MFI values were observed in 1:4 dilution of the sera, and then, it declined gradually, but the PRA values remained almost constant even after serial dilutions. CONCLUSION: In our experimental findings, it was clear that cell-based assay (FCXM) was more severely affected by the prozone, whereas solid-phase (flow PRA) assay remained resistant to prozone.

Single antigen testing to reduce early antibody-mediated rejection risk in female recipients of a spousal donor kidney.

Female recipients of a spousal donor kidney transplant are at greater risk of donor-specific pre-immunization, which may increase the risk of acute antibody-mediated rejection (ABMR). We assessed the incidence of early ABMR (within two weeks after transplantation), risk factors for ABMR and graft function in 352 complement-dependent cytotoxicity test-negative LURD transplant recipients, transplanted between 1997 and 2014 at the Leiden University Medical Center in The Netherlands. Risk factors for immunization were retrieved from the health records. As methods to screen for preformed donor-specific antibodies (pDSA) have developed through time, we retrospectively screened those with ABMR for pDSA using pooled-antigen bead (PAB) and single-antigen bead (SAB) assays. The cumulative incidence of rejection in the first six months after transplantation was 18% (TCMR 15%; early ABMR 3%). Early ABMR resulted in inferior graft survival and was more common in women who received a kidney from their spouse (10%) than in other women (2%) and men (<1%). The SAB assay retrospectively identified pDSA in seven of nine cases of early ABMR (78%), while the PAB detected pDSA in only three cases (33%). Seeing that early ABMR occurred in 10% of women who received a kidney from their spouse, a SAB assay should be included in the pre-transplant assessment of this group of women, regardless of the result of the PAB assay.