Natural killer lymphocytes (NK cells) are the first line of defense (innate immunity) against viral infections and leukemia since they do not require activation to deliver a lethal hit to infected/aberrant cells. In contrast, T lymphocytes require stimulation by a foreign/neo - antigen, which may take days before they are active against the pathogen (adaptive immunity). A number of receptors on activated NK cells that kill the prototypical leukemia target cell line, K562, have been identified. To date, the receptor(s) by which freshly isolated unstimulated NK cells (naïve, nNK) kill K562 has not been fully elucidated. We provide peptide sequence and immune-blot data from ligand pull down experiments that moesin, a protein that typically links the inner leaf of the plasma membrane to the cytoskeleton, additionally, in NK cells, localizes to the cell surface where it may bind to its ligand, TOMM40 (aka Haymaker, HYMKR), on leukemia cells thereby initiating their destruction. Flow cytometry experiments with a mouse monoclonal antibody (Mab) to a moesin peptide (554 to 565) were performed. Moesin was detected on the surface of CD3-, CD16+nNK cells but was not detected on the surface of freshly isolated unstimulated CD3+, CD16- T cells or CD19+, CD16- B cells from healthy subjects. Moesin, is therefore another marker that distinguishes unstimulated CD3-, CD16+ NK cells from other non-activated lymphocytes. The anti -moesin peptide Mab was highly effective (>95% inhibition) in blocking target cell cytolysis by CD16+ lymphocytes demonstrating that moesin-HYMKR interaction appears to be necessary for most of the observed cell death of K562 caused by unstimulated NK cells.

The reliability of single antigen bead (SAB) assays and their use in predicting a negative cell based cross match (CBXM) is essential in the era of expanded organ sharing. A wide range of accuracy (80-95%) in predicting negative CBXM has been reported. We hypothesized that in SAB assays an antibody against an HLA eplet that was common among a number of different HLA alleles would be distributed among all of the shared eplet positive SABs. This would reduce binding to the donor specific SAB resulting in an under-estimate of antibody strength. We tested this proposal in adsorption studies using, instead of lymphocytes, a novel reagent, single-SAB (sSAB). Properties of SAB assays were examined that provided a basis for conducting adsorption - elution experiments with the sSABs. We found that incubation of sera with sA*02:01 or sB*42:01 not only depleted reactivity to these alleles but also depleted reactivity to beads that shared the reactive eplet. Anti-eplet strength from SAB data (sum of the MFI of eplet positive SABs (MFI-s) was compared with CBXM out comes in two case studies and with 99 proficiency testing sera. In these validation studies, an MFI-s above 11,000 was associated with a positive FCXM. This approach was placed into clinical practice for listing unacceptable antigens that shared a common eplet. CDCXMs (n = 3261) and FCXMs (n = 1012) were performed on patients listed in UNOS for deceased donor kidneys. All CDCXMs were negative and all FCXMs except one were negative. We conclude that summation of eplet strength provides a highly reliable method of predicting prospective negative CBXMs resulting in substantial savings of time and effort. Based on shared eplet summation data, CMS/NYSDOH has accepted our bead based XM (BBXM) method (aka, virtual XM) performed prior to transplant as fulfilling the regulation that XM results be available before kidney transplantation. (c) 2022 The Author(s). Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics. This is an open access article under the CC BY-NC-ND license (http://creativecommons. org/licenses/by-nc-nd/4.0/).

Cell surface HLA class I consists of trimers, i.e., alpha - heavy chain, beta - 2 - microglobulin, and a peptide, termed closed conformers (CC) on non-activated lymphocytes. HLA class I and class II may also exist, respectively, as alpha-chain only or alpha and beta - chain only on activated cells termed open conformers (OC). We extend previous studies using an OC-specific monoclonal antibody that demonstrate LABScreen HLA class I and II single antigen beads (SABs) contain a mixture of open and closed conformers. LIFECODES SABs have bound CC only. More HLA class I and class II LABScreen SABs were reactive than LIFECODES SABs due to the presence of OC on LABScreen SABs. We hypothesized that antibody against OC on HLA B antigens would not be detected in cell based cross matches (XMs) with typical lymphocyte targets since anti-HLA OC antibodies would not react with native HLA CC on the cell surface. To test this hypothesis, we performed flow cytometry XM (FCXM) assays with sera of sufficient strength that most laboratories would likely predict positive FCXMs. Sera that reacted strongly with LABScreen SABs (>13,000 MFI) but weakly or not at all with LIFECODES SABs (<1000 MFI) gave negative T and B cell FCXMs. In contrast, sera that reacted with LIFECODES SABs (>13,000 MFI) but weakly with LABScreen SABs (<2100 MFI) exhibited positive FCXMs. Detection of antibodies directed against OC in SAB assays, may lead to inappropriate listing of unacceptable antigens, a decision not to XM or pre-or post - transplant desensitization procedures.

BACKGROUND:We investigated the utility of an automated chemiluminescent SARS-CoV-2 IgG antibody assay platform in quantifying the amount of binding antibodies present in donated convalescent plasma.METHODS:A total of 179 convalescent plasma units were analyzed for the presence of SARS-CoV-2 IgG antibodies using the Beckman-Coulter chemiluminescent immunoassay (CLIA) platform. The equipment-derived numerical values (S/Co ratio) were recorded. Aliquots from the same units were subjected to enzyme-linked immunosorbent assay (ELISA) that detects IgG antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 S1 protein. The relationship between ELISA titers and CLIA S/Co values was analyzed using linear regression and receiver operating characteristics (ROC) curve.RESULTS:Twenty-one samples (11.7%) had S/Co values of less than 1.0 and were deemed negative for antibodies and convalescent plasma had S/Co values between >1.0 and 5.0 (70/179, 39.1%). Fifteen units (8.4%) had negative ELISA titer. The majority of the units (95/179. 53.1%) had titers ≥1:1024. The sensitivities of ELISA to CLIA were comparable (90.5% vs 88.3%, respectively; p=0.18). There was positive linear correlation between CLIA S/Co values and ELISA IgG titer (Rho = 0.75; Spearman's rank = 0.82, p-value = <0.0001). The agreement between the two methods was fair, with a κ index of 0.2741. Using the ROC analysis, we identified a CLIA S/Co cutoff value of 8.2, which gives a sensitivity of 90% and a specificity of 82% in predicting a titer dilution of ≥1:1024.CONCLUSION:The utility of automated antibody detection systems can be extended from simply a screening method to a semi-quantitative and quantitative functional antibody analysis. CLIA S/Co values can be used to reliably estimate the ELISA antibody titer. Incorporation of chemiluminescent-based methods can provide rapid, cost-effective means of identifying anti-SARS-CoV-2 antibody titers in donated plasma for use in the treatment of COVID-19 infection.

Early in the SARS-CoV-2 pandemic, convalescent plasma (CP) therapy was proposed as a treatment for severely ill patients. We conducted a CP treatment protocol under the Mayo Clinic Extended Access Program at University Hospital Brooklyn (UHB). Potential donors were screened with a lateral flow assay (LFA) for IgM and IgG antibodies against the SARS-CoV-2 S1 receptor-binding domain (RBD). Volunteers that were LFA positive were tested with an ELISA to measure IgG titers against the RBD. Subjects with titers of at least 1:1024 were selected to donate. Most donors with positive LFA had acceptable titers and were eligible to donate. Out of 171 volunteers, only 65 tested positive in the LFA (38.0%), and 55 (32.2%) had titers of at least 1:1024. Before our donation program started, 31 CP units were procured from the New York Blood Center (NYBC). Among the 31 CP units that were obtained from the NYBC, 25 units (80.6%) were positive in the LFA but only 12 units (38.7%) had titers of at least 1:1024. CP was administered to 28 hospitalized COVID-19 patients. Patients who received low titer CP, high titer CP and patients who did not receive CP were followed for 45 days after presentation. Severe adverse events were not associated with CP transfusion. Death was a less frequent outcome for patients that received high titer CP (>1:1024) 38.6% mortality, than patients that received low titer CP (≤1:1024) 77.8% mortality.

A disproportionate incidence of death has occurred in African Americans (Blacks) in the United States due to COVID-19. The reason for this disparity is likely to be multi-factorial and may involve genetic predisposition. The association of human leukocyte antigens (HLA) with severe COVID-19 was examined in a hospitalized population (89% Black, n = 36) and compared to HLA typed non-hospitalized individuals (20% Black, n = 40) who had recovered from mild disease. For additional comparison, HLA typing data was available from kidney transplant recipients and deceased donors. Hospitalized patients were followed for 45 days after admission to our medical center with death as the primary end-point. One HLA allele, B53, appeared to be more prevalent in the hospitalized COVID-19 patients (percent of positive subjects, 30.5) compared to national data in US Black populations (percent of positive subjects, 24.5). The percent B53 positive in non-hospitalized COVID-19 patients was 2.6, significantly less than the percent positive in the hospitalized COVID-19 patients (p = 0.001, Fisher's exact test) and less than the 8 percent positive listed in national data bases for US Caucasian populations. Significantly greater deaths (73 percent) were observed in HLA B53 positive hospitalized COVID-19 patients compared to hospitalized COVID-19 patients who were B53 negative (40 percent). Multi-variate analysis indicated that HLA B53 positive Black hospitalized COVID-19 patients were at a 7.4 fold greater risk of death than Black COVID-19 patients who were B53 negative. Consideration for accelerated vaccination and treatment should be given to HLA B53 positive Black COVID19 patients.

To the Editor: We read with interest the paper by Visentin et al1 entitled “Reassessment of the clinical impact of preformed donor-specific anti-HLA-cw antibodies in kidney transplantation.” Using single antigen flow beads (SAFBs) from One Lambda, Inc, this paper highlights and adds to their prior work2 demonstrating the pathogenicity of anti-HLA antibodies directed against native, trimeric (α-chain, β2-microglobulin, peptide) HLA class-I molecules (nHLA), whereas those antibodies directed against “denatured” (α-chain only, dHLA) class-I molecules appear benign. In their current paper, antibodies directed solely against dHLA-cw did not predict positive T cell flow crossmatches and were not associated with adverse outcomes posttransplant. Sera were considered reactive with only dHLA if positive with standard SAFBs (contain both nHLA and dHLA), but were negative with iBeads (devoid of dHLA). Unfortunately, as the authors point out, iBeads are no longer commercially available. There is, however, a commercially available beadset shown to be devoid of monomeric, denatured variants (Lifecodes (LC), Immucor Transplant Diagnostics, Inc.). We have compared the reactivity of the One Lambda, Inc product (LABscreen LS) with LC beadsets utilizing the IgG2a monoclonal antibody, TFL-006.3 This antibody has been shown to react with a specific epitope on the α-chain of all class-I HLA molecules normally hidden by β2-microglobulin in trimeric HLA-I molecules4; it can be blocked by the peptide 117AYDGKDY123 shared by all class-I HLA molecules. Specifically, TFL-006 reacted with only 10% of HLA A iBeads (MFI range 66-1400) and 0% of both HLA B and HLA C iBeads.4 Similarly, TFL-006 reacted with no LC beads for HLA -A, -B, and -C,5 a result indicating that this brand of beadsets is devoid of monomeric HLA class-I α-chains similar to iBeads, although some peptide-free dimers (α-chain with β2-microglobulin) may be present. This lack of reactivity of LC beadsets with TFL-006 has been verified in subsequent studies by our group.3 In comparing the reaction of the sera of kidney transplant waiting list patients to both LS and LC beadsets, we show greater reactivity (numbers of positive antigens as well as MFIs) with the former.3 These data suggest that reactions with dHLA on LS beadsets may inflate the MFI and/or result in false positivity, thereby potentially denying compatible organs and/or resulting in inappropriate desensitization procedures. Most recently, we confirmed these findings in a different group of patients’ sera. Furthermore, three patients’ sera that were reactive to a specific allele on LS beads (MFIs of 14 307-17 865) but were completely negative with LCs beads of the same allele produced negative B and T cell flow cross-matches, a result mirroring the data of Visentin et al (Ravindranath MH, Filippone EJ, Callender CJ, Arosa FA, Das B, Ou Y, and Norin AJ, unpublished data). We commend Visentin et al for highlighting the lack of pathogenicity of anti-dHLA antibodies and for pointing out the presence of these variants on the most commonly used brand of beadsets, LS. Although iBeads are no longer available, there is a commercially available brand of beadsets also devoid of dHLA molecules. The authors of this manuscript have conflicts of interest to disclose as described by the American Journal of Transplantation. MHR received grant funding from Immucor paid to his institution and contributing to his salary; AJN has been on the speakers’ bureau of Immucor; EJF and BD have no conflicts.

Solid phase anti-HLA antibody tests have become an important tool in predicting the compatibility of donor-recipient pairs for kidney transplantation. Additionally, single antigen bead (SAB) assays provide valuable information for the management of post-transplant immunotherapy. However, there can be limitations to SAB assays, for example, the presence of donor specific IgM antibodies and/or binding of high levels of compliment may block binding of donor specific IgG antibodies (DSA) to the SAB. This could possibly result in an under estimation of DSA. The following case is an example of this phenomenon. A 48 years old female patient (HLA Type: A1 A29 B53 B72 DR8 DR13) was transplanted with a deceased donor kidney (HLA Type: A2 A33 B53 B58 DR8 DR11 DQB7 DQA04 DQA05 DPB01:01 DPB04:02) in 2017. The patient was presensitized (pregnancy) to a donor antigen A2 (MFI of 4 A2 beads = 3904) with an associated epitope 144TKH (MFI = 5101, includes A68, A69). On day 3 post-transplant, there was no detectable antibodies to donor antigens due to the sponge effect of the transplanted kidney. On day 6 antibodies to A2 (MFI = 5000) and the associated epitope 144TKH (MFI = 8946) reappeared. Additionally, a low level of de novo antibodies to B*58:01 (MFI = 716) and DPB1*04:02 (MFI = 864) was detected. On day 12, antibodies were detected to only A*02:01 (MFI = 615), while a significant increase in B58 antibody was observed (MFI = 9025). At this time, a biopsy indicated antibody mediated graft rejection; creatinine was 10 mg/dl. Accordingly, the patient's antibody was further evaluated by an AMOS one wash CDC cross match with frozen cells and sera from day 6 and 12. On day 6, CDC-XM was negative, but on day 12 the CDCXM was positive. The level of DSA to class I of MFI 9640 would typically not give a positive CDC cross match. To determine if there was interference of IgM and/or compliment, the 12-day serum was treated at 63C for 13 min to inactivate these molecules. The heat-treated serum demonstrated an MFI to A*02:01 of MFI = 20,567 compared to an MFI of 615 before heat inactivation. Unfortunately, treatment for antibody-mediated rejection was not initiated soon enough to save the kidney. Although it is rare to observe IgM and/or compliment interference with SAB assays (4 out of 3020 cases, 0.1%), post-transplant assessment of DSA should be performed with heat inactivated sera or other methods to eliminate the under estimation of the strength of IgG antibodies. A.J. Norin:2. Consultant; Company/Organization; ICON PLC, Stony Brook Pathologist. 3. Speaker's Bureau; Company/Organization; immucor, one lambda.

Post transplant epitope analysis of anti-donor specific antibody (DSA) using single antigen bead (SAB) assays when combined with flow cytometry cross matches (FCXM) may provide enhanced ability to direct the management of a patient’s immunosuppressive therapy. This proposal is illustrated by the following case report. A 57 year old female (HLA, A2, A31, B51, B53, DR13, DR15) received a deceased donor kidney transplant (TX) (Donor HLA, A1, B13, B37, C6, DR7, DR15, DQ2, DQ6). The patient was sensitized (cPRA 97%) from a prior kidney TX. Pre-TX DSA was detected to B*37:01, MFI = 645 with a possible epitope 156DA, the sum of which is the MFI- score (MFI-s) = 4515). T and B cell FCXMs were negative. The patient did not receive plasmapheresis (PPE) prior to TX and DSA was not detected one day after transplantation. On day 8, DSA was detected to the A*01:01 SAB (MFI = 591) with and MFI-score of 3992 to the possible epitope 163R that includes A1. The patient’s DSA increased to 2451, A*01:01, MFI-s of 14,936, on day 12 but the creatinine (Cr) decreased from 4.1 to 2.5 mg/dL over the next 14 days so treatment was withheld. On day 30 an increase in Cr from 2.3 mg/dL to 3.7 mg/dL was noted. SAB testing indicated DSA to B*37:01, as well as the A1 bead. A C3d test and a FCXM test was performed (with frozen donor cells).The DSA did not bind C3d and the FCXM was negative. Typically the FCXM would be positive with this level of DSA (14,936). In view of the negative FCXM, further epitope analysis was performed as the patient’s post transplant serum likely contained antibodies with several overlapping specificities. In this case the donor antigen A1 did not express the epitope that was broadly expressed on other beads, 163R, but rather an epitope that was restricted to A1 alleles only 163RG with an MFI-s = 2787. This explained why the FCXM was negative. PPE, IVIG and Rituxan therapy was initiated but only 5 PPE were given since the DSA was of relatively low strength. The patient’s DSA was undetecTable 10 days later and her Cr stabilized to 2.1 mg/dL. This case illustrates how post-transplant assessment of anti-HLA antibody with SAB/ epitope analysis, combined with FCXM, can provide important information on the specificity and strength of the DSA for use in the management of recipients that develop antibodies after kidney transplantation. A.J. Norin:2. Consultant; Company/Organization; ICON PLC, Stony Brook Pathologists. 3. Speaker’s Bureau; Company/Organization; Immuncor, One Lambda.

Optical bead microarrays using Luminex have largely replaced anti-HLA antibody screening tests using cell based complement dependent cytotoxicity assays. One unintended consequence of this technologic development was the loss of the ability to distinguish between antibodies that fix complement from those that do not. This situation may have been addressed by the introduction of several assays that use the bead microarrays in conjunction with a reagent that detects whether components of the complement cascade are bound to the anti-HLA antibody – HLA complex on a reactive bead. One controversy centering on this technology has been the suggestion that the binding of complement in bead assays is dependent to a greater extend on the strength of the anti-HLA antibody rather than its complement binding activity. To address this issue we examined complement binding in single antigen bead (SAB) assays in sera that had multiple epitope specificities of different strengths. Patient sera and sera from proficiency surveys were used. SAB assays of these sera were compared to C1q/C3d – SAB assays. The bead with the highest net MFI for each identified epitope in the microarray was recorded and compared to the same bead in C1q/C3d – SAB assays. We detected both strong and weak anti-HLA antibodies in SAB assay that were detected in C1q/C3d – SAB assays. In some sera complement bound to one set of epitope reactive SABs but not to another epitope set of SABs. Importantly, this was not dependent on the strength of the antibody. For example, in one patient’s sera, an epitope defined antigen bead, B∗42:01 (epitope 65GK), had an MFI of 12,989 (normalized score) in the SAB assay but only 99 MFI in the C1q – SAB assay. In the same serum another epitope defined bead (80 N), A∗23:01 with an MFI of 10,445 in the SAB assay exhibited an MFI of 9,162 in the C1q – SAB assay. We also observed the latter finding for HLA class 2 SABs and with the C3d assay. Complement binding to SABs is not directly related to strength of the antibody as determined in pairwise comparative studies using sera that contained antibodies with different epitope specificities. C1q/C3d - SAB assays are therefore useful in evaluating the complement binding properties of pretransplant and post transplant anti-HLA antibodies.

We previously published a study (1998–2008) of 624 patients showing the association of pre-transplant DSA with poor kidney allograft survival. The aim of the current study was to assess development of post-transplant DSA in patients with well-functioning grafts >8 years after transplantation in comparison to patients with failed grafts. HLA typing (A, B, C, DR, DQ) of recipients and donors was performed by standard serologic and/or DNA (SSP or SSO) methods. Anti-HLA antibody specificity was determined by single antigen beads (Lifecodes) on a Luminex platform. Though donors were not typed for DPB1 or DPA1 the de novo development of antibodies to DP alleles was presumed to be donor specific. Of 624 patients that were originally enrolled 131 were available for evaluation. 67 recipients had functioning kidney allografts 8 to 18 years after transplantation with >10 years follow up in 50% of these patients. The remaining 64 patients had graft failure (return to dialysis) with a median survival of 7.5 years. Of these patients, 44 (68%) had evidence of post-transplant DSA to HLA class 1 and/or class 2 as detected 0.1 to 5 years prior to graft failure. Most DSA positive recipients had antibodies to both Class 1 and 2 (32, 50%), whereas only 3 patients had DSA to Class 1 and only 9 patients had DSA to Class 2. Of patients with functioning grafts only 32% had evidence of DSA. In a new cohort transplanted in 2012–2015, that received identical induction and maintenance immunosuppression as the earlier cohort, similar DSA results were observed. Approximately, 33% of recipients with functioning kidney allografts had evidence of DSA whereas 65% of patients with failed grafts had DSA. In many cases, in both cohorts, the detection of a relatively low level of DSA occurred without concomitant clinical signs of rejection (e.g., a rise in creatinine). As demonstrated in many studies of shorter duration the development of DSA was associated in this 18 year study with kidney allograft failure long after antibody has been detected and in the absence of standard clinical markers of rejection. Patients that exhibit persistent levels of DSA without clinical signs of rejection may benefit from enhanced immunosuppressive therapy to prevent damage to the kidney tubules and glomeruli that eventually lead to graft failure.

A 54 year old African American female with 4 previous pregnancies received a kidney transplant from a living non related donor in November, 2001. Anti-HLA antibodies were not detected by Flow Cytometry cross matches (FCXM) that were negative at the time of the 2001 transplant. The patient's transplanted kidney failed in Aug 2014 due to chronic rejection and the patient underwent a nephrectomy. At that time donor specific antibody (DSA) against the HLA of the first donor was not detected. The patient was subsequently XM against her daughter in November, 2014. Anti-HLA antibody using screening beads/Luminex technology was not detected and the FCXM was negative. The patient received Hepatitis A and B vaccinations in November, 2014 followed by two subsequent vaccinations in January and March of 2015 (with seroconversion). In February 2015, anti-HLA antibody was detected on routine screen and DSA was detected by single antigen bead (SAB) assays against a Class 1 HLA in common with the first and second donor (the daughter). No other identifiable sensitizing events, e.g., infection or blood transfusion were noted. The patient received 9 plasmapheresis treatments, QOD and IVIG. SAB tests indicated a reduction in DSA during the course of therapy. DSA was not detected after the 8th plasmapheresis treatment and the FCXM was negative. The patient received a kidney transplant 2 days later. Her creatinine came down from 9.9 mg/dl at transplant to an average of 1.35 mg/dl at 3–4 weeks after surgery. Conclusions and recommendations: These results suggest that caution should be exercised when considering vaccination of a previously transplanted patient even when the patient has not developed anti-HLA antibodies to potential recall antigens. Frequent anti-HLA antibody detection assays should be performed following vaccination to avoid unexpected positive XMs. Sensitization by viral vaccines may be overcome by standard desensitization protocols that are in current use.