Mitigating the Prozone-Like Effect in HLA Antibody Testing: A Case Report of Therapeutic Plasma Exchange for Antibody-Mediated Rejection Post-Heart Transplantation.

Therapeutic plasma exchange (TPE) is a cornerstone treatment for antibody-mediated rejection (AMR) post-organ transplantation, aiming to eliminate pathogenic donor-specific HLA antibodies (DSA). However, limitations in HLA antibody interpretation due to the prozone-like effect (PLE) can lead to inaccurate assessment of treatment efficacy. We present a case of a heart transplant recipient with suspected AMR, where an unexpected increase in DSA levels post-TPE prompted investigation into PLE. Solid-phase Luminex assays were employed to detect HLA antibodies. Serum was run neat as well as after treatment with ethylenediaminetetraacetic acid (EDTA). Nephelometry was used to detect complement levels. Laboratory analysis of pre-TPE serum revealed higher DSA levels with EDTA treatment, characteristic of PLE. Complement measurements supported complement-mediated interference in the pre-TPE sample. This case underscores the importance of being aware that PLE can occur in HLA testing and can impact the interpretation of TPE efficacy for AMR.

Loss of heterozygosity leading to incorrect HLA typing for platelet-transfusion refractory patient.

BACKGROUND: Management of platelet-transfusion refractory (PR) patients due to anti-HLA antibodies includes the provision of HLA-matched (HLAm) platelets (PLT) or PLTs that are negative for HLA antigens corresponding to the recipient antibodies. Obtaining HLAm PLTs is predicated on accurate HLA antigen typing of the recipient and donor. Here, we present the clinical implications of a case involving loss of heterozygosity (LOH) in a patient presented for PR workup. STUDY DESIGN AND METHODS: HLA typing was performed by three methods: (1) Real-time PCR; (2) Sequence-specific oligonucleotide (SSO) typing test; and (3) Next-Generation Sequencing (NGS). Cytogenomic SNP microarray was utilized to assess LOH. RESULTS: A 30-year-old female with newly diagnosed acute myelogenous leukemia was found to be PR secondary to HLA sensitization. A peripheral blood (PB) sample, containing 93% myeloid blast cells, was sent for HLA typing for the provision of HLAm PLTs. HLA typing revealed homozygosity at the HLA-A locus but was heterozygous at the -B and -C loci. After chemotherapy, HLA typing on a new PB sample, devoid of blast cells, identified HLA-A locus heterozygosity, which was subsequently confirmed by real-time PCR and NGS. Cytogenomic SNP microarray analysis demonstrated LOH of the HLA-A locus on chromosome 6p in the pretreatment sample but not in the posttreatment sample. CONCLUSION: In hematologic patients with high tumor burden, HLA homozygosity should be viewed with suspicion for potential LOH. Therefore, HLA testing should be repeated, preferably with a non-hematological source (e.g., buccal swab) or following successful reduction of the tumor burden.

The impact of belatacept on third-party HLA alloantibodies in highly sensitized kidney transplant recipients.

Recent evidence suggests that belatacept reduces the durability of preexisting antibodies to class I and class II human leukocyte antigens (HLAs). In this case series of 163 highly sensitized kidney transplant candidates whose calculated panel-reactive antibody (cPRA) activity was ≥98% to 100%, the impact of belatacept on preexisting HLA antibodies was assessed. Of the 163 candidates, 72 underwent transplantation between December 4, 2014 and April 15, 2017; 60 of these transplanted patients remained on belatacept consecutively for at least 6 months. We observed a decrease in the breadth and/or strength of HLA class I antibodies as assessed by FlowPRA in belatacept-treated patients compared to controls who did not receive belatacept. Specifically, significant HLA antibody reduction was evident for class I (P < .0009). Posttransplant belatacept-treated patients also had a clinically significant reduction in their cPRA compared to controls (P < .01). Collectively, these findings suggest belatacept can reduce HLA class I antibodies in a significant proportion of highly sensitized recipients and could be an option to improve pretransplant compatibility with organ donors.

Evidence of Chronic Allograft Injury in Liver Biopsies From Long-term Pediatric Recipients of Liver Transplants.

BACKGROUND & AIMS: A substantial proportion of pediatric liver transplant recipients develop subclinical chronic allograft injury. We studied whether there are distinct patterns of injury based on histopathologic features and identified associated immunologic profiles. METHODS: We conducted a cross-sectional study of 157 stable, long-term pediatric recipients of transplanted livers (70 boys; > 6 years old at time of transplantation; mean, 8.9 ± 3.46 years after liver transplantation) who underwent liver biopsy analysis from August 13, 2012, through May 1, 2014. Participants had received livers from a living or deceased donor and had consistently normal results from liver tests. Liver biopsy specimens were scored by a central pathologist; an unsupervised hierarchical cluster analysis of histologic features was used to sort biopsy samples into 3 clusters. We conducted transcriptional and cytometric analyses of liver tissue samples and performed a systems biology analysis that incorporated clinical, serologic, histologic, and transcriptional data. RESULTS: The mean level of alanine aminotransferase in participants was 27.6 ± 14.57 U/L, and the mean level of γ-glutamyl transferase was 17.4 ± 7.93 U/L. Cluster 1 was characterized by interface activity (n = 34), cluster 2 was characterized by periportal or perivenular fibrosis without interface activity (n = 45), and cluster 3 had neither feature (n = 78). We identified a module of genes whose expression correlated with levels of alanine aminotransferase, class II donor-specific antibody, portal inflammation, interface activity, perivenular inflammation, portal and perivenular fibrosis, and cluster assignment. The module was enriched in genes that regulate T-cell-mediated rejection (TCMR) of liver and other transplanted organs. Functional pathway analysis showed overrepresentation of TCMR gene sets for cluster 1 but not clusters 2 or 3. CONCLUSION: In an analysis of biopsies from an apparently homogeneous group of stable, long-term pediatric liver transplant recipients with consistently normal liver test results, we found evidence of chronic graft injury (inflammation and/or fibrosis). Biopsy samples with interface activity had a gene expression pattern associated with TCMR.

A diagnostic 'C' saw: the ups and downs of C1q testing.

PURPOSE OF REVIEW: The present review will focus on recently published data of solid organ allograft recipients reporting that patients with de-novo donor-specific HLA antibodies (DSA) that fix complement in vitro have a significantly higher risk for antibody-mediated rejection (AMR) and/or graft loss compared to patients whose de-novo DSA do not fix complement or patients who present with preexisting complement fixing DSA. RECENT FINDINGS: HLA DSAs that fix complement in vitro appear to be a key indicator for rejection and failure of kidney, heart, and lung allografts from studies performed around the world. The majority of these studies are population based and retrospective in nature. Although these studies seemingly indicate that in-vitro complement activating DSAs represent a higher clinical risk than noncomplement fixing DSAs, the majority have not accounted for false-negative reactions attributable to the so-called prozone/interference phenomenon. In the limited number of published studies addressing that concern, high mean fluorescence intensity (MFI) value noncomplement fixing DSAs correlate as well as complement fixing DSAs with AMR and graft loss. Combined with the cost of additional testing, these observations bring into question whether there is sufficient clinical applicability to warrant routine testing for complement fixing antibodies. SUMMARY: Complement fixing DSAs are clearly associated with AMR and/or loss of transplanted allografts. However, under appropriate testing conditions, complement fixing capability typically correlates with MFI values of the DSAs. As such, the routine implementation of in-vitro assays to determine whether DSAs fix complement is of questionable value especially when considering additional issues such as cost of testing, logistics, and whether the test results factor into individualized patient care.

Comparison of de novo IgM and IgG anti-HLA DSAs between belatacept- and calcineurin-treated patients: An analysis of the BENEFIT and BENEFIT-EXT trial cohorts.

Preventing conversion of donor-specific anti-HLA antibodies (DSAs) from an IgM-to-IgG could a way to prevent chronic rejection. We evaluated whether belatacept-treated patients (belatacept less-intensive [LI] or more-intensive [MI] regimens) have a lower rate of conversion than do cyclosporine A (CsA)-treated patients. We included 330 HLA-mismatched patients from 2 phase 3 trials with either (a) complete donor/recipient HLA-A, -B, -DR, and -DQ loci typing or (b) incomplete HLA typing with IgG DSAs detected pretransplant or posttransplant. IgM and IgG DSAs were tested with single antigen beads at 0, 6, 12, 24, and 36 months posttransplant. The overall (preexisting or de novo) rates of IgM- and IgG-positive DSAs were 29% and 34%, respectively. The pretransplant IgM and IgG DSA-positive frequencies were similar between treatment groups. The IgG-positive dnDSA rate was significantly higher in the CsA-treated group (34%) compared with the belatacept-LI (8%) and belatacept-MI (11%) (P < .001) groups. In IgM-positive dnDSA patients, the IgG-positive dnDSA rate of conversion was 2.8 times higher in the CsA group than in the combined belatacept groups (P = .006). However, the observed association between belatacept treatment and more limited conversion of IgM-to-IgG dnDSAs was based on a limited number of patients and requires further validation.

Stability of anti-A blood group titers among blood group B renal transplant candidates.

BACKGROUND: As deceased donor kidney allocation is based in part on blood type compatibility, group B candidates are disadvantaged due to their disproportionate representation on the wait list compared to the group B donor pool. To mitigate this discrepancy, group B candidates can receive group A2 or A2 B donor kidneys if their anti-A titers are below a predetermined cutoff. Currently, eligibility is reverified quarterly to UNet based on individual center protocols, which can vary due to a lack of set guidelines for monitoring ABO titers in these patients. Our goal was to assess the stability of anti-A titers in blood group B renal transplant candidates over time to provide data that could aid in the development of standardized ABO titer protocols. STUDY DESIGN AND METHODS: Titers performed between January 2011 and December 2015 were assessed for 191 group B patients with two or more documented titers. RESULTS: Fifty patients (26%) were ineligible, as the first titer exceeded the cutoff of 8. Of the remaining 141 patients, 19 (13%) became ineligible as the second titer exceeded 8. Thirty-nine patients (28%) had no change in titer between samples, while 71 (50%) had a titer change that never exceeded 8. Only 12 patients (8.5% of total) experienced a titer change that affected eligibility after the second test. CONCLUSION: Although patients experience some variability in anti-A titers over time, in most cases, stability did not affect candidate eligibility. Our results indicate that regular testing beyond the second titer may be unnecessary and represent test overutilization.

De novo DQ donor-specific antibodies are associated with worse outcomes compared to non-DQ de novo donor-specific antibodies following heart transplantation.

BACKGROUND: Antibody-mediated rejection (AMR) resulting from de novo donor-specific antibodies (dnDSA) leads to adverse outcomes following heart transplantation (HTx). It remains unclear what role dnDSA to specific HLA antigens play in adverse outcomes. This study compares outcomes in patients developing dnDSA to DQ antigens with those developing non-DQ dnDSA and those free from dnDSA. METHODS: The present study was a single-center, retrospective analysis of 122 consecutive HTx recipients. The primary outcome was a composite of death or graft dysfunction. RESULTS: After 3.3 years of follow-up, 31 (28%) patients developed dnDSA. Mean time to dnDSA was 539 days. Of 31 patients, 19 developed DQ antibodies and 12 developed non-DQ antibodies. Compared to non-DQ dnDSA, DQ antibodies presented with higher MFI values (P=.001) were more likely persistent (P=.001) and appeared later post-HTx (654 vs 359 days, P=.035). In a multivariable analysis, DQ dnDSA was associated with increased risk of the primary endpoint (HR 6.15, 95% CI 2.57-14.75, P=.001), whereas no increased risk was seen with non-DQ dnDSA (P=.749). CONCLUSIONS: dnDSA to DQ antigens following HTx are associated with increased risk of death and graft dysfunction.

Becoming a chef in the human leukocyte antigen kitchen: interpretation and modification of human leukocyte antigen-antibody assays.

PURPOSE OF REVIEW: Fluorescence-based human leukocyte antigen (HLA) antibody detection methods, including flow cytometric crossmatch and single antigen bead assays revolutionized HLA antibody identification and assessment of immunological risk in transplant candidates and patients. Nevertheless, these assays are not flawless and their interpretation can be complex. This review highlights the limitations of the single antigen bead and flow cytometric crossmatch assays and discusses protocol modifications and interpretive approaches to address these issues. RECENT FINDINGS: Several limitations of HLA antibody detection methods have been identified in recent years. Protocol variability, denatured epitopes, and interfering factors can all significantly impact the identification of clinically relevant HLA antibodies. A number of solutions to address these challenges have been developed. These include pretreatment of sera, method standardization, and protocol modifications. In addition, HLA epitope-based analysis approaches to improve interpretation of antibody test results have been introduced. SUMMARY: In the 50 years, since Patel and Terasaki first developed the crossmatch assay there have been remarkable advances in HLA antibody testing methodology. However, with these advances, new problems emerged and solutions had to be developed. As the technology continues to evolve, our methods and ability to interpret results must keep pace to provide transplant patients with the best possible care.

Class I and II HLA antibodies in pediatric patients with thalassemia major.

BACKGROUND: HLA alloimmunization is a potential complication of red blood cell (RBC) transfusion with detrimental consequences for future organ or hematopoietic stem cell transplantation. STUDY DESIGN AND METHODS: The study aimed to determine the prevalence and specificity of HLA antibodies among pediatric patients with thalassemia major (TM) and antibody changes over time while on leukoreduced chronic transfusion therapy. HLA antibodies were measured at two or more time points in children and young adults ages 3 to 21 years with TM. HLA Class I and II antibodies were measured by FlowPRA screening. Positive screening assays were confirmed with LabScreen single-antigen bead assays for antibody specificity. RESULTS: HLA antibodies were detected in 10 of 19 (53%) subjects: seven of 19 (37%) with HLA Class I and II antibodies, two of 19 (11%) with only HLA Class I antibodies, and one of 19 (5%) with only HLA Class II antibodies. Subjects with HLA antibodies were older (14.6 years vs. 7.1 years, p = 0.05), predominantly male (80%), and more likely to have a remote history of nonleukoreduced transfusions (p = 0.057). Median time between testing was 3.7 years. De novo HLA antibodies were detected in two of 11 patients who initially had negative panel-reactive antibody screens, while one subject lost detection of Class II antibody. Two of seven patients with HLA antibodies had antibodies to self-HLA. CONCLUSION: HLA antibodies have a high prevalence in TM patients and may be associated with nonleukoreduced transfusions and older age. For such patients, antibody identification will be useful if subsequent organ or stem cell transplantation is needed.

Deceased donor multidrug resistance protein 1 and caveolin 1 gene variants may influence allograft survival in kidney transplantation.

Variants in donor multidrug resistance protein 1 (ABCB1) and caveolin 1 (CAV1) genes are associated with renal allograft failure after transplantation in Europeans. Here we assessed transplantation outcomes of kidneys from 368 African American (AA) and 314 European American (EA) deceased donors based on 38 single-nucleotide polymorphisms (SNPs) spanning ABCB1 and 16 SNPs spanning CAV1, including previously associated index and haplotype-tagging SNPs. Tests for association with time to allograft failure were performed for the 1233 resultant kidney transplantations, adjusting for recipient age, sex, ethnicity, cold ischemia time, panel reactive antibody, human leukocyte antigen match, expanded-criteria donation, and APOL1-nephropathy variants in AA donors. Interaction analyses between APOL1 with ABCB1 and CAV1 were performed. In a meta-analysis of all transplantations, ABCB1 index SNP rs1045642 was associated with time to allograft failure and other ABCB1 SNPs were nominally associated, but not CAV1 SNPs. ABCB1 SNP rs1045642 showed consistent effects with the 558 transplantations from EA donors, but not with the 675 transplantations from AA donors. ABCB1 SNP rs956825 and CAV1 SNP rs6466583 interacted with APOL1 in transplants from AA donors. Thus, the T allele at ABCB1 rs1045642 is associated with shorter renal allograft survival for kidneys from American donors. Interactions between ABCB1 and CAV1 with APOL1 may influence allograft failure for transplanted kidneys from AA donors.

Red blood cell transfusions are associated with HLA class I but not H-Y alloantibodies in children with sickle cell disease.

Blood transfusions can induce alloantibodies to antigens on red blood cells (RBCs), white blood cells and platelets, with these alloantibodies affecting transfusion and transplantation. While transfusion-related alloimmunization against RBC antigens and human leucocyte antigens (HLA) have been studied, transfusion-related alloimmunization to minor histocompatibility antigens (mHA), such as H-Y antigens, has not been clinically characterized. We conducted a cross-sectional study of 114 children with sickle cell disease (SCD) and tested for antibodies to 5 H-Y antigens and to HLA class I and class II. Few patients had H-Y antibodies, with no significant differences in the prevalence of any H-Y antibody observed among transfused females (7%), transfused males (6%) and never transfused females (4%). In contrast, HLA class I, but not HLA class II, antibodies were more prevalent among transfused than never transfused patients (class I: 33% vs. 13%, P = 0·046; class II: 7% vs. 8%, P = 0·67). Among transfused patients, RBC alloantibody history but not amount of transfusion exposure was associated with a high (>25%) HLA class I panel reactive antibody (Odds ratio 6·8, 95% confidence interval 2·1-22·3). These results are consistent with immunological responder and non-responder phenotypes, wherein a subset of patients with SCD may be at higher risk for transfusion-related alloimmunization.

Translational applications of flow cytometry in clinical practice.

Flow cytometry has evolved over the past 30 y from a niche laboratory technique to a routine tool used by clinical pathologists and immunologists for diagnosis and monitoring of patients with cancer and immune deficiencies. Identification of novel patterns of expressed Ags has led to the recognition of cancers with unique pathophysiologies and treatment strategies. FACS had permitted the isolation of tumor-free populations of hematopoietic stem cells for cancer patients undergoing stem cell transplantation. Adaptation of flow cytometry to the analysis of multiplex arrays of fluorescent beads that selectively capture proteins and specific DNA sequences has produced highly sensitive and rapid methods for high through-put analysis of cytokines, Abs, and HLA genotypes. Automated data analysis has contributed to the development of a "cytomics" field that integrates cellular physiology, genomics, and proteomics. In this article, we review the impact of the flow cytometer in these areas of medical practice.

Hiding in plain sight: Misinterpretation of immunogenic DPB epitopes within G/P groups. Short running title: The impact of HLA G and P codes on DPB.

The clinical impact of HLA DP antibodies is poorly understood, resulting in variable clinical strategies for transplant candidates and recipients with donor-directed HLA-DP antibodies. Complicating matters further, the DPB naming convention is not based on allelic homology and requires sequence alignments to identify potential immunogenic epitopes. Historically, G and P codes, which consolidated alleles that were identical over Exon 2, were used to simplify the reporting of HLA Class II typing as differences outside of Exon 2 have not been considered immunogenic (i.e., able to induce an antibody response). Herein, we present four cases demonstrating that polymorphisms at codons 96R/K and 170I/T, in Exon 3 of DPB, are targets for alloantibody recognition. These regions "hide in plain sight" due to the current use of G/P code-level typing, potentially leading to incorrect compatibility assessments (i.e., virtual crossmatches) and misinterpreted antibody responses. The unintentional crossing of an HLA-DPB donor-specific antibody (DSA) in a solid organ or hematopoietic stem cell transplant may lead to unforeseen deleterious clinical outcomes. Our data underscore the complexities of DPB histocompatibility assessments and highlight the need for adaptable systems that align with evolving research and clinical outcomes.

Identification of Antibodies to DQß:DRα Interisotypic Heterodimers in Human Sera.

BACKGROUND: HLA class II antigens, DR, DQ, and DP, comprised an α and ß chains, which typically combine, within the same isotype, to form the major histocompatibility complex:peptide complex. Interisotypic pairing is not commonly observed. Although reports of DQß:DRα heterodimers exist, the pairing was reported to be unstable and, therefore, not studied to any extent. METHODS: DQß:DRα single antigens were produced through transfectant cell lines and used to identify and characterize positive reactive human sera by a multiplex bead-based assay. RESULTS: Stable DQß:DRα transfectants were constructed. Cell surface staining with class II-specific monoclonal antibodies revealed that some DQB1 alleles appear to be more efficient in expressing DQß:DRα heterodimers. Interestingly, alleles within the same serological group varied in their efficiency of forming dimers on the cell surface. For example, DQß0601:DRα had the highest transfection and cell membrane expression efficiency among 16 common DQB1 alleles tested. In contrast, DQß0603:DRα-positive transfectants demonstrated minimal surface expression. Assembly of DQß0601:DRα was not affected by the presence of a DQα chain. DQß0601:DRα and DQß0603:DRα single-antigen beads were used to screen human sera. Positive sera were identified that reacted to the unique epitopes of DQß0601:DRα protein on the cell surface of the transfectants. CONCLUSIONS: Our studies have demonstrated that unique DQß:DRα heterodimers can be formed and are stably expressed on the cell surface. Such antigenic combinations, presented on single-antigen beads, demonstrated that patient sera can react with such heterodimers. Investigations on the potential clinical roles of antibodies against such interisotypic heterodimers are now possible.

HLA class I peptide polymorphisms contribute to class II DQß0603:DQα0103 antibody specificity.

Antibodies reactive to human leukocyte antigens (HLA) represent a barrier for patients awaiting transplantation. Based on reactivity patterns in single-antigen bead (SAB) assays, various epitope matching algorithms have been proposed to improve transplant outcomes. However, some antibody reactivities cannot be explained by amino acid motifs, leading to uncertainty about their clinical relevance. Antibodies against the HLA class II molecule, DQß0603:DQα0103, present in some candidates, represent one such example. Here, we show that peptides derived from amino acids 119-148 of the HLA class I heavy chain are bound to DQß0603:DQα0103 proteins and contribute to antibody reactivity through an HLA-DM-dependent process. Moreover, antibody reactivity is impacted by the specific amino acid sequence presented. In summary, we demonstrate that polymorphic HLA class I peptides, bound to HLA class II proteins, can directly or indirectly be part of the antibody binding epitope. Our findings have potential important implications for the field of transplant immunology and for our understanding of adaptive immunity.

Technical aspects of HLA antibody testing.

PURPOSE OF REVIEW: Since the landmark studies of Patel and Terasaki, pretransplant identification of donor-directed HLA alloantibodies (DSAs) has been a critical prelude to renal allograft transplantation. Pretransplant, DSAs may be an acceptable risk or an unconditional contraindication to transplantation depending on the particular donor : recipient combination. Posttransplant, DSAs are associated with episodes of acute rejection, chronic rejection, and graft loss. Thus, monitoring for such antibodies is an important aspect of patient care. RECENT FINDINGS: The development of solid-phase antibody detection assays significantly enhanced our ability to identify HLA antibodies, taking virtual crossmatching from concept to reality. At the root of these detection assays are two questions that have been asked for almost 50 years: are donor-directed HLA antibodies present and, if so, are they clinically relevant? While the technology related to solid-phase antibody detection has seemingly allowed the first question to be answered with exquisite sensitivity and specificity, can the same be said for question 2? SUMMARY: Solid-phase antibody detection assays have clear benefits over historical approaches to antibody identification, but are not flawless. In fact, the limitations of these assays are frequently ignored. Herein, the strengths and weaknesses of solid-phase antibody detection are highlighted.

COVID-19 infection and vaccination rarely impact HLA antibody profile in waitlisted renal transplant candidates- a multicenter cohort.

Although rare, infection and vaccination can result in antibodies to human leukocyte antigens (HLA). We analyzed the effect of SARS-CoV-2 infection or vaccination on HLA antibodies in waitlisted renal transplant candidates. Specificities were collected and adjudicated if the calculated panel reactive antibodies (cPRA) changed after exposure. Of 409 patients, 285 (69.7 %) had an initial cPRA of 0 %, and 56 (13.7 %) had an initial cPRA > 80 %. The cPRA changed in 26 patients (6.4 %), 16 (3.9 %) increased, and 10 (2.4 %) decreased. Based on cPRA adjudication, cPRA differences generally resulted from a small number of specificities with subtle fluctuations around the borderline of the participating centers' cutoff for unacceptable antigen listing. All five COVID recovered patients with an increased cPRA were female (p = 0.02). In summary, exposure to this virus or vaccine does not increase HLA antibody specificities and their MFI in approximately 99 % of cases and 97 % of sensitized patients. These results have implications for virtual crossmatching at the time of organ offer after SARS-CoV-2 infection or vaccination, and these events of unclear clinical significance should not influence vaccination programs.