The use of intracellular dyes to create a multiplexed flow cytometry-based red blood cell phenotyping assay.

BACKGROUND AND OBJECTIVES: Flow cytometry can be used to phenotype red blood cell antigens, allowing for high-throughput testing while using low reagent volumes. This article utilizes intracellular dyes to pre-label red blood cells to further multiplex flow cytometry-based red blood cell antigen phenotyping. MATERIALS AND METHODS: Red blood cells were pre-labelled using the intracellular dyes V450 and Oregon Green. These dyes are detected fluorescently via flow cytometry. Four combinations of intracellular staining were used to allow four patient or donor red blood cells to be analysed in a single test well. Antigen phenotyping was then performed via flow cytometry using a previously described method. RESULTS: The intracellular dyes showed uniform staining when measured in mean fluorescence intensity and allowed the red blood cells to be clearly distinguished from one another. The presence or absence of red blood cell antigens was determined with 100% accuracy. CONCLUSION: The use of intracellular dyes allowed a fourfold increase in the throughput of our previously described flow cytometry-based red blood cell antigen phenotyping method. The described method allows up to 48 patients to be simultaneously phenotyped using a single 96-well microplate. Furthermore, additional fluorescent dyes could potentially increase the throughput exponentially.

Development of multiplexed flow cytometry-based red blood cell antibody screen and identification assays.

BACKGROUND AND OBJECTIVES: The purpose of this study was to develop a high-throughput method of performing red blood cell antibody screens and identification by utilizing flow cytometry and intracellular dyes to allow a multiplexed assay where three-cell screens can be performed in a single test well and 11-cell panels in three test wells. MATERIALS AND METHODS: Reagent red blood cells were labelled using Violet Proliferation Dye 450 (V450) and Oregon Green fluorescent dyes, which bind intracellular proteins to allow up to four cells to be interrogated in a single test well. Sixteen 3-cell screen panels and ten 11-cell identification panels were tested using sera with known antibody specificity. Antibody binding was detected using secondary anti-immunoglobulin G and anti-immunoglobulin M fluorescently labelled antibodies. RESULTS: Intracellular dyes allowed clear separation of the different screen and identification panel test cells. Three distinct populations of V450+, Oregon Green+ and negative for both stains were demonstrated in the screening panel and an additional double positive for V450 and Oregon Green was utilized to include a fourth cell in the identification panel testing to increase throughput. A total of 158 screen or identification panel RBC/serum combinations were tested against different known antibodies, and expected results were obtained with 100% concordance. CONCLUSION: This study demonstrates the successful development of a high-throughput multiplexed flow cytometry-based red cell antibody screen and identification panel assays. This method could be implemented in clinical laboratories to complement existing antibody detection methods. The multiplexing enabled via intracellular staining could be utilized to further augment other flow cytometry-based transfusion assays.

Validation of a flow-cytometry-based red blood cell antigen phenotyping method.

BACKGROUND AND OBJECTIVES: Current manual and automated phenotyping methods are based on visual detection of the antigen-antibody interaction. This approach has several limitations including the use of large volumes of patient and reagent red blood cells (RBCs) and antisera to produce a visually detectable reaction. We sought to determine whether the flow cytometry could be developed and validated to perform RBC phenotyping to enable a high-throughput method of phenotyping using comparatively miniscule reagent volumes via fluorescence-based detection of antibody binding. MATERIALS AND METHODS: RBC phenotyping by flow cytometry was performed using monoclonal direct typing antisera (human IgM): anti-C, -E, -c, -e, -K, -Jka , -Jkb and indirect typing antisera (human IgG): anti-k, -Fya , -Fyb , -S, -s that are commercially available and currently utilized in our blood transfusion services (BTS) for agglutination-based phenotyping assays. RESULTS: Seventy samples were tested using both flow-cytometry-based-phenotyping and a manual tube standard agglutination assay. For all the antigens tested, 100% concordance was achieved. The flow-cytometry-based method used minimal reagent volume (0.5-1 µl per antigen) compared with the volumes required for manual tube standard agglutination (50 µl per antigen) CONCLUSION: This study demonstrates the successful validation of flow-cytometry-based RBC phenotyping. Flow cytometry offers many benefits compared to common conventional RBC phenotyping methods including high degrees of automation, quantitative assessment with automated interpretation of results and extremely low volumes of reagents. This method could be used for high-throughput, low-cost phenotyping for both blood suppliers and hospital BTS.

ABO-adjusted calculated panel reactive antibody (cPRA): A unified metric for immunologic compatibility in kidney transplantation.

Implementation of the kidney allocation system in 2014 greatly reduced access disparity due to human leukocyte antigen (HLA) sensitization. To address persistent disparity related to candidate ABO blood groups, herein we propose a novel metric termed "ABO-adjusted cPRA," which simultaneously considers the impact of candidate HLA and ABO sensitization on the same scale. An ethnic-weighted ABO-adjusted cPRA value was computed for 190 467 candidates on the kidney waitlist by combining candidate's conventional HLA cPRA with the remaining fraction of HLA-compatible donors that are ABO-incompatible. Consideration of ABO sensitization resulted in higher ABO-adjusted cPRA relative to conventional cPRA by HLA alone, except for AB candidates since they are not ABO-sensitized. Within cPRA Point Group = 99%, 43% of the candidates moved up to ABO-adjusted cPRA Point Group = 100%, though this proportion varied substantially by candidate blood group. Nearly all O and most B candidates would have elevated ABO-adjusted cPRA values above this policy threshold for allocation priority, but relatively few A candidates displayed this shift. Overall, ABO-adjusted cPRA more accurately measures the proportion of immune-compatible donors compared with conventional HLA cPRA, especially for highly sensitized candidates. Implementation of this novel metric could enable the development of allocation policies permitting more ABO-compatible transplants without compromising equity.

Donor-Specific Human Leukocyte Antigen Antibody Formation After Distal Tibia Allograft and Subsequent Graft Resorption.

The association between donor-specific human leukocyte antigen (HLA) antibody formation and small bone allograft resorption has not been studied. We present the case of a patient treated for glenoid bone loss using a distal tibial allograft with Bankart repair who formed donor-specific HLA antibodies against the allograft and had subsequent graft resorption. X-ray and computed tomography (CT) scans were performed before and after surgery at standard checkpoints. Patient blood and serum samples were collected before and after surgery for HLA typing and HLA antibody testing. Human leukocyte antigen antibodies against the donor-specific HLA-A2 antigens were identified 6 weeks after surgery and were still detected at 5 months after surgery. At 6 months after surgery, a CT arthrogram revealed significant graft resorption. This case shows a temporal correlation between HLA antibody formation and clinical findings, potentially suggesting an association between HLA antibody formation and graft resorption. Further study is required to confirm this.

Glycine and Folate Ameliorate Models of Congenital Sideroblastic Anemia.

Sideroblastic anemias are acquired or inherited anemias that result in a decreased ability to synthesize hemoglobin in red blood cells and result in the presence of iron deposits in the mitochondria of red blood cell precursors. A common subtype of congenital sideroblastic anemia is due to autosomal recessive mutations in the SLC25A38 gene. The current treatment for SLC25A38 congenital sideroblastic anemia is chronic blood transfusion coupled with iron chelation. The function of SLC25A38 is not known. Here we report that the SLC25A38 protein, and its yeast homolog Hem25, are mitochondrial glycine transporters required for the initiation of heme synthesis. To do so, we took advantage of the fact that mitochondrial glycine has several roles beyond the synthesis of heme, including the synthesis of folate derivatives through the glycine cleavage system. The data were consistent with Hem25 not being the sole mitochondrial glycine importer, and we identify a second SLC25 family member Ymc1, as a potential secondary mitochondrial glycine importer. Based on these findings, we observed that high levels of exogenous glycine, or 5-aminolevulinic acid (5-Ala) a metabolite downstream of Hem25 in heme biosynthetic pathway, were able to restore heme levels to normal in yeast cells lacking Hem25 function. While neither glycine nor 5-Ala could ameliorate SLC25A38 congenital sideroblastic anemia in a zebrafish model, we determined that the addition of folate with glycine was able to restore hemoglobin levels. This difference is likely due to the fact that yeast can synthesize folate, whereas in zebrafish folate is an essential vitamin that must be obtained exogenously. Given the tolerability of glycine and folate in humans, this study points to a potential novel treatment for SLC25A38 congenital sideroblastic anemia.

Unrelated donor choices for allogeneic hematopoietic cell transplantation in Canada: an evaluation of factors influencing donor selection.

BACKGROUND: Utilization of unrelated donors and cord blood units (CBUs) for allogeneic hematopoietic cell transplantation continues to increase. Understanding the practices of donor selection by transplant centers is critical for unrelated donor registries and cord blood banks to optimize registry composition and inventory to meet patient need. STUDY DESIGN AND METHODS: Unrelated donor and CBU selection practices of Canadian transplant centers served by Canadian Blood Services' OneMatch Stem Cell & Marrow Network (OM) were reviewed, including HLA match level, locus of disparity, age, sex, and product choice (donor vs. CBU). RESULTS: HLA-matched donors within OM and/or international (INT) registries were preferentially investigated, underscoring the primary importance of HLA matching. In the case of HLA-mismatched donors, HLA-A disparities were most common while DRB1 mismatches were least common. Advanced age, sex, and lack of donor availability were the most frequent reasons that high-probability OM donors were overlooked in favor of INT donors. High-probability 10 of 10 HLA-matched female donors from OM were often avoided in favor of INT male donors. Use of female donors, however, increased in cases restricted to more HLA-disparate donor options. Caucasian patients were more likely to find 10 of 10 matched donors, whereas use of mismatched donors and CBUs were more prevalent among non-Caucasian patients. CONCLUSIONS: Recruitment and retention of young, male donors from diverse ethnic backgrounds may increase the usage of histocompatible OM donors for patients in need.

Optimized cell transplantation using adult rag2 mutant zebrafish.

Cell transplantation into adult zebrafish has lagged behind mouse models owing to the lack of immunocompromised strains. Here we have created rag2(E450fs) mutant zebrafish that have reduced numbers of functional T and B cells but are viable and fecund. Mutant fish engraft muscle, blood stem cells and various cancers. rag2(E450fs) mutant zebrafish are the first immunocompromised zebrafish model that permits robust, long-term engraftment of multiple tissues and cancer.

Piperine, a Pungent Alkaloid from Black Pepper, Inhibits B Lymphocyte Activation and Effector Functions.

Piperine has several well-documented anti-inflammatory properties; however, little is known regarding its effect on humoral immunity. In this study, we describe the immunosuppressive effect of piperine on B lymphocytes, which are integral to the humoral immune response. Mouse B cells were cultured in the absence or presence of non-cytotoxic concentrations (25, 50, and 100 µM) of piperine during T-dependent or T-independent stimulation. Piperine inhibited B cell proliferation by causing G0/G1 phase cell cycle arrest in association with reduced expression of cyclin D2 and D3. The inhibitory effect of piperine was not mediated through transient receptor potential vanilloid-1 ion channel (TRPV1) because piperine also inhibited the proliferation of B cells from TRPV1-deficient mice. Expression of class II major histocompatibility complex molecules and costimulatory CD40 and CD86 on B lymphocytes was reduced in the presence of piperine, as was B cell-mediated antigen presentation to syngeneic T cells. In addition, piperine inhibited B cell synthesis of interleukin (IL)-6 and IL-10 cytokines, as well as IgM, IgG2b, and IgG3 immunoglobulins. The inhibitory effect of piperine on B lymphocyte activation and effector function warrants further investigation for possible application in the treatment of pathologies related to inappropriate humoral immune responses. Copyright © 2017 John Wiley & Sons, Ltd.

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.

Piperine from black pepper inhibits activation-induced proliferation and effector function of T lymphocytes.

Piperine is a major alkaloid component of black pepper (Piper nigrum Linn), which is a widely consumed spice. Here, we investigated the effect of piperine on mouse T lymphocyte activation. Piperine inhibited polyclonal and antigen-specific T lymphocyte proliferation without affecting cell viability. Piperine also suppressed T lymphocyte entry into the S and G2 /M phases of the cell cycle, and decreased expression of G1 -associated cyclin D3, CDK4, and CDK6. In addition, piperine inhibited CD25 expression, synthesis of interferon-γ, interleukin (IL)-2, IL-4, and IL-17A, and the generation of cytotoxic effector cells. The inhibitory effect of piperine on T lymphocytes was associated with hypophosphorylation of Akt, extracellular signal-regulated kinase, and inhibitor of κBα, but not ZAP-70. The ability of piperine to inhibit several key signaling pathways involved in T lymphocyte activation and the acquisition of effector function suggests that piperine might be useful in the management of T lymphocyte-mediated autoimmune and chronic inflammatory disorders.

The reversible P2Y12 inhibitor ticagrelor inhibits metastasis and improves survival in mouse models of cancer.

Tumor cells use activated platelets to promote their proliferation and metastatic potential. Because platelet activation is largely mediated through ADP engagement of purinergic P2Y12 receptors on platelets, we investigated the potential of the reversible P2Y12 inhibitor ticagrelor, a clinical agent used in the prevention of cardiovascular and cerebrovascular events, to inhibit tumor adhesion and metastasis. In B16-F10 melanoma intravenous and intrasplenic metastasis models, mice treated with a clinical dose of ticagrelor (10 mg/kg) exhibited marked reductions in lung (84%) and liver (86%) metastases. Furthermore, ticagrelor treatment improved survival compared to saline-treated animals. A similar effect was observed in a 4T1 breast cancer model, with reductions in lung (55%) and bone marrow (87%) metastases following ticagrelor treatment. In vitro, B16-F10 cells exhibited decreased interaction with platelets from ticagrelor-treated mice compared to saline-treated mice, an effect similar to that observed with blockade of glycoprotein IIbIIIa. Similarly, B16-F10 cells co-incubated with platelets from ticagrelor-treated mice exhibited reduced adhesion to endothelial monolayers compared to those co-incubated with platelets from saline-treated animals, an effect also observed in vivo. Interestingly, pretreatment of endothelial monolayers with ticagrelor did not result in reduced tumor cell adhesion. These findings support a role for P2Y12-mediated platelet activation in promoting metastases, and provide proof-of-concept for the clinical use of ticagrelor in the prevention of tumor metastasis.

A transgenic zebrafish model expressing KIT-D816V recapitulates features of aggressive systemic mastocytosis.

Systemic mastocytosis (SM) is a rare myeloproliferative disease without curative therapy. Despite clinical variability, the majority of patients harbour a KIT-D816V mutation, but efforts to inhibit mutant KIT with tyrosine kinase inhibitors have been unsatisfactory, indicating a need for new preclinical approaches to identify alternative targets and novel therapies in this disease. Murine models to date have been limited and do not fully recapitulate the most aggressive forms of SM. We describe the generation of a transgenic zebrafish model expressing the human KIT-D816V mutation. Adult fish demonstrate a myeloproliferative disease phenotype, including features of aggressive SM in haematopoeitic tissues and high expression levels of endopeptidases, consistent with SM patients. Transgenic embryos demonstrate a cell-cycle phenotype with corresponding expression changes in genes associated with DNA maintenance and repair, such as reduced dnmt1. In addition, epcam was consistently downregulated in both transgenic adults and embryos. Decreased embryonic epcam expression was associated with reduced neuromast numbers, providing a robust in vivo phenotypic readout for chemical screening in KIT-D816V-induced disease. This study represents the first zebrafish model of a mast cell disease with an aggressive adult phenotype and embryonic markers that could be exploited to screen for novel agents in SM.

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.

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.

Process Mining Uncovers Actionable Patterns of Red Blood Cell Unit Wastage in a Health Care Network.

Packed red blood cell transfusions are integral to the care of the critically and chronically ill patient, but require careful storage and a large, coordinated network to ensure their integrity during distribution and administration. Auditing a Transfusion Medicine service can be challenging due to the complexity of this network. Process mining is an analytical technique that allows for the identification of high-efficiency pathways through a network, as well as areas of challenge for targeted innovation. Here, we detail a case study of an efficiency audit of the Transfusion Medicine service of the Nova Scotia Health Administration Central Zone using process mining, across a period encompassing years prior to, during, and after the acute COVID-19 pandemic. Service efficiency from a product wastage perspective was consistently demonstrated at benchmarks near globally published optima. Furthermore, we detail key areas of continued challenge in product wastage, and suggest potential strategies for further targeted optimization.

Calcium-dependent HLA-DQ epitope revealed by EDTA mediated inhibition of antibody reactions in the Luminex single antigen bead assay.

Complement mediated interference with the detection of antibodies targeting HLA is a known limitation of the single antigen bead (SAB) Luminex assay. Ethylenediaminetetraacetic acid (EDTA) is currently the serum treatment of choice in most histocompatibility laboratories to block complement activation by chelating calcium. The purpose of this study was to investigate a serum with an antibody reactivity to HLA-DQ6, 7, 8 and 9 molecules, in the Luminex SAB assay, that was inhibited by treatment with EDTA. Serum was from a 55-year-old highly sensitised female renal transplant candidate that contained, among others, antibodies to an epitope containing the 74EL eplet, shared by HLA-DQ6, DQ7, DQ8 and DQ9 molecules. Serum samples were treated with EDTA, dithiothreitol (DTT), or heat prior to testing by SAB assay. EDTA-treated serum was also tested after the addition of calcium chloride (CaCl2). HLA-DQ-specific antibodies were isolated by adsorption/elution method using three informative donor cells and were tested in the absence or presence of EDTA. The antibody reactivity against HLA-DQ6, DQ7, DQ8 and DQ9 in the SAB assay was significantly inhibited by treating serum and eluates with EDTA and was restored by addition of CaCl2. The study represents the first description of a calcium-dependent epitope in HLA molecules. The relevance of this finding is that the treatment of sera with EDTA could lead to false-negative reactions in the SAB assay, which may compromise virtual crossmatching.

Regulation of S100A10 by the PML-RAR-α oncoprotein.

Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia that results from the expression of the promyelocytic leukemia-retinoic acid receptor α (PML-RAR-α) oncoprotein. It is characterized by severe hemorrhagic complications due in part to excessive fibrinolysis, resulting from the excessive generation of the fibrinolytic enzyme, plasmin, at the cell surface of the PML cells. The treatment of patients with all-trans retinoic acid (ATRA) effectively ameliorates the disease by promoting the destruction of the PML-RAR-α oncoprotein. In the present study we show for the first time that the plasminogen receptor, S100A10, is present on the extracellular surface of APL cells and is rapidly down-regulated in response to all-trans retinoic acid. The loss of S100A10 is concomitant with a loss in fibrinolytic activity. Furthermore, the induced expression of the PML-RAR-α oncoprotein increased the expression of cell surface S100A10 and also caused a dramatic increase in fibrinolytic activity. Depletion of S100A10 by RNA interference effectively blocked the enhanced fibrinolytic activity observed after induction of the PML-RAR-α oncoprotein. These experiments show that S100A10 plays a crucial role in the generation of plasmin leading to fibrinolysis, thus providing a link to the clinical hemorrhagic phenotype of APL.

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.

Novel alleles in the era of next-generation sequencing-based HLA typing calls for standardization and policy.

Next-Generation Sequencing (NGS) has transformed clinical histocompatibility laboratories through its capacity to provide accurate, high-throughput, high-resolution typing of Human Leukocyte Antigen (HLA) genes, which is critical for transplant safety and success. As this technology becomes widely used for clinical genotyping, histocompatibility laboratories now have an increased capability to identify novel HLA alleles that previously would not be detected using traditional genotyping methods. Standard guidelines for the clinical verification and reporting of novelties in the era of NGS are greatly needed. Here, we describe the experience of a clinical histocompatibility laboratory's use of NGS for HLA genotyping and its management of novel alleles detected in an ethnically-diverse population of British Columbia, Canada. Over a period of 18 months, 3,450 clinical samples collected for the purpose of solid organ or hematopoietic stem cell transplantation were sequenced using NGS. Overall, 29 unique novel alleles were identified at a rate of ∼1.6 per month. The majority of novelties (52%) were detected in the alpha chains of class II (HLA-DQA1 and -DPA1). Novelties were found in all 11 HLA classical genes except for HLA-DRB3, -DRB4, and -DQB1. All novelties were single nucleotide polymorphisms, where more than half led to an amino acid change, and one resulted in a premature stop codon. Missense mutations were evaluated for changes in their amino acid properties to assess the potential effect on the novel HLA protein. All novelties identified were confirmed independently at another accredited HLA laboratory using a different NGS assay and platform to ensure validity in the reporting of novelties. The novel alleles were submitted to the Immuno Polymorphism Database-Immunogenetics/HLA (IPD-IMGT/HLA) for official allele name designation and inclusion in future database releases. A nationwide survey involving all Canadian HLA laboratories confirmed the common occurrence of novel allele detection but identified a wide variability in the assessment and reporting of novelties. In summary, a considerable proportion of novel alleles were identified in routine clinical testing. We propose a framework for the standardization of policies on the clinical management of novel alleles and inclusion in proficiency testing programs in the era of NGS-based HLA genotyping.