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Background: Antenatal titration testing is traditionally performed using a manual tube test. Tube testing has limitations; it is a manual, time-consuming method with wide interobserver variability. Gel-based testing is an attractive alternative because it is more precise and can be automated. This study's objective was to summarize the published literature that assessed the relationship between titrations performed by tube and gel for anti-D alloimmunized pregnancies., Study Design and Methods: A comprehensive literature search was performed. Articles were selected if research was original and compared at least five pairs of anti-D titration tests performed by gel and tube. Differences in the number of dilutions between gel and tube methods were compared overall by study and cell type using linear models., Results: A total of 512 articles were identified; eight were included, and titer data from 384 tube and gel pairs were abstracted. The median anti-D titer in tube was 8 (range 0-2048) and by gel was 64 (range 0-4096). Anti-D gel titration results were 2.1 (95% CI; 1-3.3) additional dilutions greater than in tube. Most studies utilized double-dose reagent cells for testing. At a tube titer of 16, the sensitivity and specificity of gel titrations is maximal (91% and 94% respectively) at a gel titer of 64., Conclusion: Overall, titrations performed by gel were two dilutions higher than the corresponding tube titer. For titrations, double-dose reagent cells should be considered to standardize practice. A rigorous prospective study is needed to compare tube titrations with gel titrations using a standardized process., (© 2021 AABB.)

Background: The monocyte monolayer assay (MMA) is an in-vitro assay that can predict the outcome of blood transfusion of antigen positive units when serologically compatible blood is not available., Materials and Methods: Fifty-four patients testing positive by the antibody screening test using gel agglutination were further examined by the alloantibody identification panel to determine alloantibody specificity. After determining and categorizing the antibodies, patients' samples were examined using the MMA to determine the clinical significance of the detected alloantibodies. We also tested 2 seeding methods (24-well cell culture plates versus 8-well chamber-slides) and 3 visualization/staining techniques (unstained phase contrast, Leishman and Giemsa staining)., Results: 35 out of the 54 cases (64.8%) had a monocyte index of >5% which is predictive of occurrence of hemolytic reaction after transfusion; 23 cases with antibodies known to be clinically significant [anti-C, anti-E, anti-c, anti-K, anti-Fy(a), anti Fy(b), anti-JK(b)], 2 with Anti-M specificity, 7 cases with autoantibodies and 3 cases with multiple antibodies. On the other hand, 19 out of the 54 (35.2%) cases included in the study showed a monocyte index of <5% which is predictive of absence of hemolytic reaction after transfusion. The 8-well chamber-slides were better than the 24-well culture plates, as the latter showed a lot of un-phagocytosed RBCs in the background. Also, Leishman staining was better than Giemsa staining with better and clearer differentiation between the RBCs, monocytes and phagocytic vacuoles., Conclusion: MMA can be used as a surrogate cross-match test for the selection of blood units in cases where antigen-negative blood units are not available., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: There are only a few reports evaluating the applicability of endothelial-damage markers analysis by immunohistochemistry (IHC) in kidney allograft samples. This study analyzed the expression of Caveolin-1 (Cav), von Willebrand factor (Vwf), and T-cadherin (Cad) in kidney biopsies and their association with antibody-mediated injury., Methods: In this retrospective study, 114 cases with antibody-mediated changes (Banff, 2020) and 72 with interstitial fibrosis/tubular atrophy were selected. IHC for Cav, Vwf and Cad was performed and evaluated according to their qualitative expression in peritubular capillaries. The cases were grouped according to the presence of microvascular inflammation (MVI), donor-specific antibodies (DSA), C4d positivity and antibody-mediated rejection (AMR). A level of significance < 0.05 was adopted., Results: Vwf expression was associated with MVI (p < 0.001), DSA (p = 0.016), C4d (p < 0.001) and AMR (p < 0.001), and was higher in DSA+/C4d+ cases despite MVI (p < 0.001). The expression of Cad correlated with MVI (p = 0.015), C4d (p = 0.005) and AMR (p = < 0.001). Cad was more expressed in chronic AMR compared with acute/active cases (p = 0.001). Cav expression was associated with MVI (p = 0.029) and AMR (p = 0.016) and was also higher in chronic AMR (p = 0.049). A combined score of Vwf and Cad was higher in AMR when compared with C4d without rejection and IF/TA cases (p < 0.001)., Conclusion: Vwf, Cad and Cav expression shows association with antibody-mediated injury and may be helpful to support AMR diagnosis.

Background: The cellular infiltrate in protocol liver biopsies (PB) following pediatric liver transplantation remains mostly uncharacterized, yet there is increasing concern about the role of inflammation and fibrosis in long-term liver allografts. We aimed to define cell types in PB and to analyze their relationship with donor-specific antibodies (DSA) and histological phenotype., Methods: PB were performed at least 1 year after transplantation. We identified 4 phenotypes: normal, fibrosis, inflammation, inflammation with fibrosis. Cell types were counted after immunostaining for CD3, CD4, CD8, CD68, CD20, MUM1, and FoxP3., Results: Forty-four patients underwent 1 PB between 2000 and 2015. Eleven percent (5/44) of PB displayed normal histology, 13.6% (6/44) fibrosis, 34.1% (15/44) inflammation, and 40.9% (18/44) inflammation and fibrosis. The main cell types in the portal tracts and lobules were CD3+ and CD68+ cells. Frequency of de novo DSA was 63% (27/44). The presence of CD8+ cells in the lobules was associated with fibrosis. Inflammation and fibrosis in PB were associated with the presence of circulating de novo DSA, number of de novo DSA, and C1q binding activity when compared to other phenotypes., Conclusions: T cells (CD3+) and macrophages (CD68+) were the most prevalent cell-types in PB. In the presence of inflammation, portal tracts were enriched in CD3+, CD20+ but displayed fewer CD68+. This coincided with the presence and number of de novo DSA. How these cellular and humoral actors interact is unclear, but peripheral DSA may be a marker of immune cellular activity in the seemingly quiescent allograft.

Objectives: The determination of unacceptable antigens in patients on kidney transplant waiting list is a critical laboratory investigation in sensitized patients. The Luminex single antigen bead (SAB) assay has high sensitivity and accuracy. However, several countries have not yet implemented SAB testing for waitlisted patients because of limited financial resources. In Thailand, specificities of HLA antibodies are identified by using a phenotypic bead assay. The aim of this study was to evaluate the performance of the phenotypic bead assay for determining HLA antibody specificities when compared with the SAB assay., Methods: A total of 254 sera from patients awaiting kidney transplantation were included. Of 254 sera, 206 and 171 were positive for HLA class I and II antibodies, respectively. Antibody specificities of sera that were tested with both phenotypic and SAB assay were analyzed. The performances of the phenotypic bead assay were compared with those of the SAB assay as the gold standard by using estimation of pooling sensitivity, specificity, and accuracy., Results: The sensitivity, specificity, and accuracy of the phenotypic bead assay for determining HLA class I antibodies was 53.9%, 93.0%, and 78.1%, respectively. The sensitivity, specificity, and accuracy of the phenotypic bead assay for determining HLA class II antibodies were 57.3%, 94.9%, and 81.4% respectively., Conclusion: In waitlisted kidney transplant patients, the phenotypic bead assay had high specificity and moderate accuracy when compared with the SAB assay. However, the low sensitivity of the test suggests that the use of the phenotypic assay for determining HLA specificities should be applied with caution in sensitized patients., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Red blood cell (RBC) alloantibody titration is a quasi-quantitative method to assess antibody concentration and is considered a useful means of estimating maternal alloimmunization during pregnancy. Traditionally, titration is performed using conventional tube test (CTT). The gel microcolumn agglutination-based method (GMA) has been proven reliable for many immunohematology tests. Our study compared CTT with GMA of two different, commercially available GMA systems for RBC alloantibody titration., Methods: Serum samples with significant RBC-alloantibodies were evaluated in our study. Each sample was titrated concurrently with CTT, with ID-DiaMed-GmbH, Cressier, Switzerland (GMA1), and with DG Gel Coombs Diagnostic Grifols, Passeig Fluvial, Spain (GMA2)., Results: One hundred thirty-seven titration tests including 50 anti-D, 25 anti-Kell, 10 anti-E, 9 anti-Jka, 8 anti-c, 5 anti-Cw, 5 anti-Fya, 7 anti-M, 6 anti-Kpa, 3 anti-Lua, 1 anti-e, 3 anti-G, and 2 anti-Cha were performed and evaluated. Samples tested by CTT versus GMA1 and GMA2 generated mostly equal or higher titers by GMAs. The results of both comparisons were in good agreement (W = 0.91, p < 0.0001, and W = 0.92, p < 0.0001, respectively). For all antibody specificities, the mean absolute difference in titers ranged from 1 - 3 for both GMA1 and GMA2 versus CTT. Samples tested by GMA1 vs. GMA2 were in almost perfect agreement (W = 0.95, p < 0.0001)., Conclusions: Although both GMAs were found slightly more sensitive than CTT for alloantibody titration, the differences were not significant and the agreement between all methods was very good, possibly indicating GMA as a suitable alternative to CTT in RBC antibody titration.

Background: Anti-red blood cell (RBC) alloantibodies consisting of only the immunoglobulin G (IgG) 4 subtype are typically considered clinically insignificant. A US Food and Drug Administration-approved monoclonal anti-human globulin (16H8) is nonreactive with IgG4, which has been considered a benefit to avoid testing interference from IgG4. However, 16H8 also does not recognize two natural IgG3 variants (IgG3-03 and IgG3-13). Thus, 16H8 may miss clinically significant alloantibodies in some settings., Study Design and Methods: Novel mouse anti-human IgG hybridomas were generated and screened for reactivity with 32 human variants of anti-KEL1 across different IgG subtypes, as well as mutants to allow epitope mapping. Anti-IgG reactivity was determined using KEL1+ RBCs bound by each IgG variant as targets. Binding of anti-IgG was determined by flow cytometry., Results: 16H8 recognized an epitope involving amino acid 419, which is glutamate in IgG4, IgG3-03, and IgG3-13, explaining the lack of 16H8 reactivity with these subtypes/isoallotypes. A new monoclonal antibody (PUMA8) was isolated that, like 16H8, was nonreactive with IgG4 but without blind spots for known variants of IgG1, IgG2, or IgG3. PUMA8 recognized an epitope containing arginine at position 355, which is glutamine in IgG4. However, a recently described new IgG4 variant with an arginine at position 355 results in PUMA8 reactivity., Conclusion: PUMA8 represents an alternative to 16H8 that avoids IgG4 but without blind spots for IgG3 variants. However, PUMA8 reacts with one recently described IgG4 variant. In addition to relevance to immunohematology, these studies highlight the importance of patient variation with regards to assay performance in an era of personalized medicine., (© 2020 AABB.)

Background: Subclinical T cell-mediated rejection (subTCMR) is commonly found after liver transplantation and has a good short-term prognosis, even when it is left untreated. Donor-specific antibodies (DSA) are putatively associated with a worse prognosis for recipient and graft after liver transplantation., Methods: To assess the immune regulation in subTCMR grafts, gene expression of 93 transcripts for graft injury, tolerance, and immune regulation was analyzed in 77 biopsies with "no histologic rejection" (NHR; n = 25), "clinical TCMR" (cTMCR; n = 16), and subTCMR (n = 36). In addition, all available subTCMR biopsies (n = 71) were tested for DSA with bead assays., Results: SubTCMR showed heterogeneous and intermediate expression profiles of transcripts that were upregulated in cTCMR. Graft gene expression suggested a lower activation of effector lymphocytes and a higher activation of regulatory T cells in grafts with subTCMR compared to cTCMR. DSA positivity in subTCMR was associated with histological evidence of more severe graft inflammation and fibrosis. This more severe DSA+ associated graft injury in subTCMR was converged with an upregulation of cTCMR-associated transcripts. In nonsupervised analysis, DSA positive subTCMR mostly clustered together with cTCMR, while DSA negative subTCMR clustered together with NHR., Conclusions: T cell-mediated rejection seems to form a continuum of alloimmune activation. Although subTCMR exhibited less expression of TCMR-associated transcript, DSA positivity in subTCMR was associated with an upregulation of rejection-associated transcripts. The identification of DSA positive subclinical rejection might help to define patients with more inflammation in the graft and development of fibrosis.

Solid-phase assays for human leukocyte antigens (HLA) antibody detection have clearly revolutionized the field of HLA diagnostics and transplantation. The key advantages are a high sensitivity and specificity for the detection of HLA antibodies compared with cell-based assays, as well as the potential for standardization. Solid-phase assays enabled the broad introduction of tools such as "virtual crossmatching" and "calculated panel reactive antibodies," which are essential components in many organ allocation systems, kidney-paired donation programs, and center-specific immunological risk stratification procedures. The most advanced solid-phase assays are the so-called single antigen beads (SAB). They are available now for more than 15 years, and the transplant community embraced their significant advantages. However, SAB analysis and interpretation is complex and many pitfalls have to be considered. In this review, we will discuss problems, limitations, and challenges using SAB. Furthermore, we express our wishes for the improvements of SAB as well as their future use for immunological assessment and research purposes., (© 2019 Steunstichting ESOT.)

The negative impact of donor specific HLA alloantibodies in solid organ transplantation is well known and understood within the histocompatibility and immunogenetics community. However the influence of donor-specific antibodies in the outcome of haematopoietic stem cell transplantation is less well regarded. As donor choices have evolved from HLA matched siblings and extremely well matched unrelated donors to mismatched cord blood and haplo-identical-related donors, we are now identifying more patients with antibodies reactive against their donor mismatches. The clinical significance of the antibodies that can be detected has not yet been fully elucidated., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Monitoring of donor-specific HLA antibodies (DSA) has become part of the clinical routine in kidney transplantation. This paper gives a brief overview on data from the Collaborative Transplant Study (CTS) and the Heidelberg Transplant Center on the clinical relevance of post-transplant DSA monitoring in patients undergoing renal transplantation. The obtained findings underline the importance of DSA monitoring in the post-operative course in immunologically high-risk patients and patients with deterioration of graft function. Especially in patients with a pre-activated immune system, a gap in the immunosuppressive therapy appear to lead to persistence, reappearance or de novo occurrence of strong, complement-activating DSA, resulting in severe antibody-mediated rejection (AMR) and, without timely intervention, in AMR-related graft loss., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Patients on organ transplant waiting lists are evaluated for preexisting alloimmunity to minimize episodes of acute and chronic rejection by regularly monitoring for changes in alloimmune status. There are few studies on how alloimmunity changes over time in patients on kidney allograft waiting lists, and an apparent lack of research-based evidence supporting currently used monitoring intervals., Methods: To investigate the dynamics of alloimmune responses directed at HLA antigens, we retrospectively evaluated data on anti-HLA antibodies measured by the single-antigen bead assay from 627 waitlisted patients who subsequently received a kidney transplant at University Hospital Zurich, Switzerland, between 2008 and 2017. Our analysis focused on a filtered dataset comprising 467 patients who had at least two assay measurements., Results: Within the filtered dataset, we analyzed potential changes in mean fluorescence intensity values (reflecting bound anti-HLA antibodies) between consecutive measurements for individual patients in relation to the time interval between measurements. Using multiple approaches, we found no correlation between these two factors. However, when we stratified the dataset on the basis of documented previous immunizing events (transplant, pregnancy, or transfusion), we found significant differences in the magnitude of change in alloimmune status, especially among patients with a previous transplant versus patients without such a history. Further efforts to cluster patients according to statistical properties related to alloimmune status kinetics were unsuccessful, indicating considerable complexity in individual variability., Conclusions: Alloimmune kinetics in patients on a kidney transplant waiting list do not appear to be related to the interval between measurements, but are instead associated with alloimmunization history. This suggests that an individualized strategy for alloimmune status monitoring may be preferable to currently used intervals., (Copyright © 2019 by the American Society of Nephrology.)

Background: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal alloantibodies against fetal human platelet antigens (HPAs), mostly caused by anti-HPA-1a. Population-based screening for FNAIT is still a topic of debate. Logistically and financially, the major challenge for implementation is the typing of pregnant women to recognize the 2% HPA-1a-negative women. Therefore, there is need for a high-throughput and low-cost HPA-1a-typing assay., Study Design and Methods: A sandwich ELISA was developed, using a monoclonal anti-GPIIIa as coating antibody and horseradish-peroxidase-conjugated recombinant anti-HPA-1a, as detecting antibody. The ELISA results were compared to an allelic discrimination PCR-assay. In phase I, samples from unselected consecutive pregnant women were tested with both assays. Phase II was part of a prospective screening study in pregnancy and genotyping was restricted to samples with an arbitrary set, OD < 0.500., Results: The ELISA was optimized to require no additional handling (swirling or spinning) of stored tubes. During phase I, 506 samples were tested. In phase II, another 62,171 consecutive samples were phenotyped, with supportive genotyping in 1,902. In total 1,585 HPA-1a negative and 823 HPA-1a positive women were genotyped. The assay reached 100% sensitivity with a cut-off OD from 0.160, corresponding with a 99.9% specificity and a false-HPA-1a negative rate of 0.03., Conclusion: A high-throughput, low-cost, and reliable HPA-1a phenotyping assay was developed which can be used in population-based screening to select samples for testing of presence of anti-HPA-1a. Because plasma from tubes of 3- to 6-days-old samples can be used, this assay is applicable to settings with suboptimal conditions., (© 2019 AABB.)

Although the electronic cross-matching of blood has been widely applied in some developed countries and regions, concern over the risk of undetected red blood cell (RBC) antibodies has limited its application in mainland China.This study was performed to explore the missed detection rate of RBC antibodies in a Chinese population from 2011 to 2016. If the results of 2 consecutive tests of ABO/RhD blood group identification were consistent and antibody screening results were negative, electronic cross-matching of the blood was performed. In addition, traditional serological cross-matching of blood (polybrene method) and a parallel experiment for electronic cross-matching of blood were performed to analyze the missed detection of unexpected RBC antibodies in blood donors and recipients.Using the polybrene method, 40,228 blood samples were tested by parallel traditional serological cross-matching of blood; among these samples, blood compatibility was found in 40,222 cases, primary incompatibility (incompatibility of the donor's erythrocytes with the recipient's serum) was found in 6 cases, and no secondary incompatibility was found. Identification of antibody specificity was performed using panel cells, and all unexpected RBC antibodies were confirmed as anti-Mur alloantibodies in the MNS system.Further improvements in the erythrocyte antigenic spectrum, especially the Mur antigen in Asian populations, are expected to ensure the safety of implementing electronic cross-matching in China.

Background: Erythrocyte isoimmunization or alloimmunization is a late complication of transfusion, in which antibodies against erythrocyte antigens other than the ABO system are developed. Its prevalence is variable, groups of patients with low prevalence (2%) and others of high risk with more than 50% have been described. These antibodies can have serious clinical repercussions in transfused patients., Objective: To know the prevalence of erythrocyte isoimmunization, the risk factors for its development and the types of antibodies developed in the user population of two hospitals in Mexico., Methods: Retrospective study. The database of the Blood Bank and the Transfusion Service of two hospitals was analyzed for the search of transfused patients who developed isoantibodies from 2012 to 2016, analyzing their background to determine the risk factors, prevalence and type of antibodies., Results: An isoimmunization prevalence of 0.97% was found in 5 years; the main antibodies found were: anti-E, anti-K, anti-C, anti-Dia, anti-c, anti-D and anti-Fya. The associated risk factors for the development of isoimmunization were: transfusion history, pregnancy and female sex, as a finding it was found that group O is a protective factor., Conclusions: Erythrocyte alloimmunization in the population studied corresponded to a low prevalence. The main antibodies found were against Rh, Kell and Diego system antigens, with a different distribution than published in other international series. No previous report was found about the finding on group O as a protective factor for isoimmunization.

Background: Graft immunocomplex capture fluorescence analysis is an attractive method to detect intragraft donor-specific anti-HLA antibodies. In ABO-incompatible transplantation, anti-A and B antibodies are also considered to be important donor specific antibodies (ABO-DSA). Therefore, it is useful to monitor intragraft ABO-DSAs to assess antibody-mediated rejection., Methods: To capture A and B antigens, anti-Band III, von Willebrand factor (VW), and plasmalemma vesicle-associated protein (PLVAP) beads were produced. The allograft specimen was homogenized in a lysis buffer. Subsequently, A and B antigens were captured by anti-Band III, VW, or PLVAP beads. The immune complexes were then detected by phycoerythrin-conjugated anti-human IgG antibodies and analyzed using a Luminex system., Results: Although Band III and VW beads yielded false positives and false negatives, PLVAP beads captured A and B antigens with high sensitivity (91.7%) and specificity (100%) when an index > 1.5 was considered positive. The proximity in A and B antigens and PLVAP expression was confirmed using immunohistochemical evaluation. Furthermore, sodium dodecyl sulfate polyacrylamide gel electrophoresis supported that PLVAP is an A and B antigen carrier protein., Case Report: Biopsies were conducted following an ABO-incompatible renal transplant (type A to O) and evaluated for ABO-DSA. Graft immunocomplex capture fluorescence analysis was demonstrated as follows: 3.19 (1 h, serum creatinine [s-Cr] 3.95 mg/dL, titer IgG 1:512, glomerulitis [g] 0, peritubular capillaritis [ptc] 0, complement 4d [C4d] 1); 1.8 (4 d, s-Cr 2.29 mg/dL, titer 1:256, g 0, ptc 0, C4d 3); 1.2 (22 d, s-Cr 1.58 mg/dL, titer 1:128, g 0, ptc 2, C4d 3). This result indicated that the remnant ABO-DSA were adsorbed and subsequently removed from the allograft successfully., Conclusions: This novel application could be used to detect intragraft ABO-DSAs, which could lead to a correct diagnosis and shed light on the ABO-DSA kinetics following ABO-incompatible transplantation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Immunocomplex capture fluorescence analysis has recently been applied as a method for detection of intragraft donor-specific anti-major histocompatibility complex (MHC) antibodies (DSA) in humans. Although intragraft DSA in humans is an intense topic of investigation, there is no report to assess intragraft DSA in murine organ transplantation., Methods: A model of presensitized mouse cardiac transplantation by donor splenocytes was used. To capture mouse MHC, anti-MHC class I/II antibodies were immobilized on Luminex beads. The MHC/DSA complexes were captured by the Luminex beads followed by detection of phycoerythrin-conjugated antimouse IgG antibodies where DSA had already reacted with the allograft in vivo., Results: Luminex beads were capable of detecting class I DSA in the cardiac allograft, though results for class II DSA were negative. Immunohistochemical investigation revealed that cardiac allografts had abundant MHC class I expression but only minor expression of MHC class II. Furthermore, MHC/class II DSA complexes were successfully detected in splenocytes and serum from a presensitized recipient., Conclusions: These data suggested that graft immunocomplex capture fluorescence analysis can be also applied in murine cardiac transplantation. This novel application in mice would accelerate our comprehension of DSA through mechanistic studies., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: The ABO transplantation barrier can be breached if antibody is reduced to low levels. Current serologic methods involve testing natural RBCs against dilutions of plasma to determine antibody levels, but these methods are poorly standardized and inherently error prone with consequent large inter- and intra laboratory variation. We evaluated the feasibility of using antigen-standardized kodecytes and undiluted plasma as an alternative method for antibody measurement in patients preparing for ABO-incompatible kidney transplantation., Study Design and Methods: A panel of five kodecytes, bearing defined levels of synthetic blood group A type 2 antigen was developed (kodecyte assay) to show reaction patterns against undiluted plasma that were indicative of anti-A and anti-A,B levels. This panel was evaluated against the contemporary method of testing dilutions of plasma against A 1 cells to determine titer (A 1 cell assay) in both column agglutination and tube techniques. Evaluation samples included reference standards, 102 group O plus 23 group B donors, and 40 pre- and post-plasmapheresis samples from five prospective ABO-incompatible kidney transplant patients., Results: Comparisons between the kodecyte and A 1 cell assays found greater than 90% correlation for all samples. Tube and column agglutination technology platform differences were observed with A 1 cells and kodecytes. Discordant samples were generally found to have high ratios of IgG:IgM or vice versa., Conclusions: The kodecyte assay is a simple method that requires no sample dilution, and an optimized two-cell kodecyte panel is potentially capable of informing ABO-incompatible kidney transplantation decisions based on antibody levels., (© 2019 AABB.)

Cytotoxic flow cytometric crossmatch (cFCXM), identified by detecting complement-mediated cytotoxic cell death in addition to the capability of showing the alloantibodies binding onto lymphocytes at the same time, can reduce the necessary time and workload in evaluating alloantibodies. More data from clinical samples are needed for cFCXM to be accepted by tissue typing laboratories. In this study, we compared cFCXM with complement-dependent lymphocytotoxicity and standard flow cytometric crossmatch in 41 renal pretransplant patients. A comparison of the obtained data was performed using Spearman's correlation test. We found that cFCXM showed no statistically significant differences with complement-dependent lymphocytotoxicity and flow cytometric crossmatch. We believe that cFCXM can be used in clinical laboratories in the near future following intra-laboratory validation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Aim: Antibody assessment during pretransplantation term is important to detect donor specific antibodies. These donor-specific antibodies are determined by various crossmatch methods (flow cytometric [FCXM], complement-dependent cytotoxic [CDCXM], and Luminex [LMXM]). Recently, single antigen bead (SAB) assays have been used for the assessment of hypersensitized patients. The aim of this study was to compare sensitivity and specificity of the 3 crossmatch methods in reference to the SAB method., Method: In this study, 69 hypersensitized patients with high class I and/or class II panel reactive antibodies were tested using the flow cytometric SAB method. Serum samples were cross-matched by 3 crossmatch methods with the cells of a volunteer healthy individual, and the results were evaluated according to HLA and cross-reactive epitope groups (CREGs)., Results: Sensitivity was found to be better with T FCXM (0.91) and class I LMXM (0.87). Specificity of peripheral blood lymphocyte CDCXM method (1.0) was found to be better than the other 2 methods (0.33 and 0.57, respectively). Sensitivity of class II LMXM (0.88) was found to be better than the others (0.42 for B CDCXM and 0.82 for B FCXM, respectively). The specificity of the B CDCXM, B FCXM, and class II LMXM was similar (0.44, 0.44, and 0.33, respectively). CREGs results were similar to HLA results., Conclusion: Although CDCXM has high specificity for the detection of anti-HLA antibodies, it has low sensitivity. To increase sensitivity, FCXM or LMXM methods may be used with the CDCXM test. These results will be beneficial for laboratories and clinicians during graft survival and patient health assessment., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purpose: The single antigen bead (SAB) test contributes to conventional cellular and solid phase crossmatch tests in renal transplantation. However, the determination of anti-HLA antibodies of the patients may not reflect the pathologic features of these antibodies. Highly sensitized patients produce antibodies against a number of HLAs; therefore, their transplantation chance decreases. In this study, we aimed to evaluate SAB and C1q test results of highly sensitized patients., Method: In this study, 33 end-stage renal failure patients with >80% panel reactive antibody were included. Of the patients, 58% (n = 19) were female, and 42% (n = 14) were male. The mean age was 46.2 ± 12.4. All of the serum samples were inactivated by heat before use. SAB and C1q tests were performed according to the manufacturer's instructions., Results: We obtained statistically significant results between the positive bead counts and raw mean fluorescence intensity (MFI) values of 2 tests (P < .01 for class I and II). There was a statistically significant difference between the 2 tests in terms HLA-A, -C, -DR, and -DP MFI values, whereas HLA-B and -DQ MFI values were similar for the 2 tests., Conclusion: The difference of raw MFI values between the 2 tests may be due to the fact that the C1q test detects only IgG1 and IgG3 antibodies, whereas the SAB test can detect all IgG subtypes. We considered that anti-HLA-B and -DQ antibodies have high complement-fixing features; these antibodies should be investigated selectively due to the similarity of anti-HLA-B and -DQ antibody MFI values in the 2 tests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Single-antigen bead (SAB) platform permits the identification of antibodies not detectable by complement-dependent lymphocytotoxicity test, but their clinical significance is not completely understood. The aim of this study was to evaluate whether the presence of pretransplant SAB-detected antibodies is associated with the development of allograft failure. This is a single-center cohort study with 10-year follow-up in which 573 kidney recipients with negative pretransplant complement-dependent lymphocytotoxicity crossmatch who received transplants at the Kidney Transplant Center of Policlinico, Milan, from deceased donors between 1996 and 2005 were evaluated. Pretransplant plasma samples were retrospectively analyzed by SAB assay. Survival analyses were performed to assess the risk of allograft failures by SAB-detected antibodies. Pretransplant antibodies were found in 160 (28.0%) recipients, of whom 42 subsequently developed an allograft failure for a survival rate of 70.9% (95% confidence interval [CI), 63.5-78.4). Among those without antibodies, 58 (14.0%) returned to dialysis with a survival rate of 84.7% (95% CI, 81.0-88.4). In Cox regression analyses, patients with SAB-positivity had 2-fold higher risk of allograft failure than those who were SAB-negative (hazard ratio, 2.07; 95% CI, 1.39-2.79). Results did not change after adjustment for putative confounders. In conclusion, in this single-center cohort, 10-year allograft survival rate was significantly influenced by the presence of SAB-detected antibodies., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Although induction of durable mixed chimerism is required for murine skin allograft tolerance (TOL), renal allograft TOL has been achieved after induction of only transient mixed chimerism in nonhuman primates (NHPs) and humans. To better define the level/duration of chimerism required for stable renal allograft TOL, we retrospectively analyzed these parameters and compared them with transplant outcomes in NHP combined kidney and bone marrow transplant recipients., Methods: Peripheral blood levels and duration of myeloid or lymphoid chimerism were retrospectively analyzed in 34 NHP combined kidney and bone marrow transplantation recipients which were divided into 3 groups: TOL, n = 10; chronic antibody-mediated rejection (CAMR), n = 12; and T cell-mediated rejection (TCMR), n = 12., Results: All 4 of the recipients that failed to develop any chimerism lost their allografts due to TCMR after discontinuation of immunosuppression (56 ± 3 d). Among 30 recipients who successfully developed multilineage chimerism, 10 achieved long-term immunosuppression-free survival without rejection (1258 ± 388 d), 12 eventually developed CAMR (932 ± 155 d), and 8 developed TCMR (82 ± 10 d). The maximum level but not duration of lymphoid chimerism was significantly higher in TOL recipients compared with both CAMR (P = 0.0159) and TCMR (P = 0.0074). On the other hand, the maximum myeloid chimerism was significantly higher in TOL than in TCMR (P = 0.0469), but not in CAMR. Receiver operating characteristic analyses revealed that lymphoid chimerism levels of 3.1% or greater could reliably predict long-term immunosuppression-free renal allograft survival (P < 0.0001)., Conclusions: This retrospective study confirmed that induction of chimerism is essential for long-term immunosuppression-free survival, which best correlates with lymphoid chimerism levels higher than 3.1%.

Results of 773 actual flow crossmatches (aFXMs) and virtual flow crossmatches (vFXMs) performed for living and deceased donor kidney transplantation in our center were analyzed retrospectively and evaluated for their concordance. Prediction of vFXMs was based on antibody identification using single antigen bead assay and locally established mean fluorescence intensity cutoff point compared with donor HLA antigens. The vast majority of aFXMs were in concordance with vFXMs with an overall concordance of 97%. Twenty-three predicted to be negative showed positive aFXMs; 12 of them had 0% calculated panel-reactive antibody, and 11 were found in patients with multiple non-donor-specific HLA antibodies. Three predicted positive vFXMs yielded negative aFXMs; 2 of them had allele-specific antibodies. CONCLUSIONS: vFXMs based on precise characterization of antibody specificities detected by single antigen bead assay using our cutoff point accurately predicted FXMs in the majority of patients and can be used safely to allocate kidney offers without performing physical crossmatches in selected patients., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Flow cytometric crossmatch (FCXM) is widely used in many centers as part of pretransplant risk assessment to detect donor-specific anti-HLA antibodies. The limited number of crossmatches that can be performed during on-call work-up for deceased donors within reasonable time remains the main obstacle to accommodating the majority of highly sensitized listed patients to be tested by the standard tube FCXM method. This limitation often directs the organs to nonsensitized patients and deprives highly sensitized patients who could be compatible if their sera were included in the crossmatch test. The goal of this study is to optimize a 96-well tray FCXM protocol that allows more sera to be crossmatched without prolonging the overall procedural time while maintaining quality and sensitivity of the assay. The new method was validated against use of the standard tube method and included total of 63 crossmatches performed simultaneously by both methods using 20 donors' cells with patients' sera, pooled positive controls tested on different dilutions, and commercial negative control. In the new protocol we modify various assay parameters including tube platform, incubation time, amount of reagent antibody cocktail, and cell volumes. An overall concordance of 98% was achieved with the protocols with slight improvement in sensitivity (2 negative B-cell reactions converted to positive in presence of weak donor-specific antibodies and mild T-cell reactivity could be picked up at 1:80 diluted positive control by the tray method only). The median channel fluorescence values of the 2 methods were essentially equivalent for both T and B crossmatches (r 2 of 0.98 and 0.97, respectively). In conclusion, 96-well tray assay has the potential to increase the probability of highly sensitized patients receiving transplants by allowing increased number of crossmatches to be performed with significant reduction in turnaround time and assay cost. Furthermore, the enhanced sensitivity of the assay will provide more accurate information about sensitization status and strength of donor-specific antibodies to treating physicians, allowing them to choose the best therapeutic option and to provide better patient care., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The impact of solid-phase immunoassay for HLA antibody detection on the field of transplantation has been extremely significant by providing the most sensitive and precise method for characterization of HLA antibodies. However, despite all the benefits, technical limitations and inherent artifacts represent significant challenges, particularly with Luminex-based single-antigen bead (SAB) assay. Discordant results between antibody detection (screening assay) and identification (SAB) is not uncommon. Positive SAB assay in the context of negative screening testing is well documented and attributed to altered tertiary structure of HLA molecules exposing new epitopes or detection of naturally occurring antibodies. However, there are few reports that addressed the opposite scenario when negative SAB appeared in the context of positive screening assay. In such discrepant results, unmissed HLA antibody has to be excluded with certainty by other tests; however, with the availability of variable assays it may be difficult to choose the best combinations that clarify discrepancy without adding more confusion. Here we describe the results of correlation between 2 antibody screening solid-phase immunoassays (LABScreen Mixed using Luminex and FlowPRA Screen) on conventional flow cytometry and compare their outcomes with SAB and crossmatch results., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The diagnosis and the treatment of rare phenotypes remain a problematic situation in many countries especially in Tunisia. Individuals with rare phenotype may develop clinically significant red cell antibodies directed against the high incidence Antigens they lack. A 35 years old patient was referred to our laboratory to explain a high incidence (twelve) of recurrent miscarriage during the first and second terms of pregnancy. This patient was grouped as O Rhesus: 1, -2, -3, 4, 5 K:-1. In her plasma we identified a pan-reactive anti-PP1PK antibody (anti-Tja) recognized to be responsible of spontaneous recurrent abortions. The red cell phenotype was P1 and Tja negative. More investigations concluded to the absence of auto and other allo-antibodies association. Therapeutic plasmapheresis from early stages was suggested for the future pregnancy to remove anti-public antibodies in order to maintain normal placenta functions. The Anti-Tja antibody, naturally occurring in patients with rare p phenotype, has the ability to induce recurrent spontaneous miscarriages and to cause immediate hemolytic transfusion reactions. Despite the absence of compatible donors in her family, this patient is not in an impasse situation because two donors with the same phenotype were identified when investigating a first case in 2013.

Background: About one-fourth of patients with hemophilia A (HA) develop alloantibodies against factor (F) VIII, as the main treatment challenge. Here, we assessed the relationship between interleukin-10 (IL-10), tumor necrosis factor alpha (TNF-α), FII and FV polymorphisms and risk of inhibitor formation in patients with severe HA., Methods: We divided 39 patients with severe HA in two groups of case (n: 19) and control (n: 20). Genotyping was performed by multiplex amplification tetra arms refractory mutation systempolymerase chain reaction (ARMS-PCR) and PCR-restriction fragment-length polymorphism (PCR-RFLP)., Results: TNFα rs1800629 G>A polymorphism decreased the risk of inhibitor development in codominant and dominant inheritance pattern. Moreover, TNFα rs1800629 A allele, decrease the risk of inhibitor formation, while IL10 rs1800896 A>G, FV rs6025 G>A, and FII rs1799963 G>A polymorphisms were not associated with risk of inhibitor development., Conclusion: It seems that TNFα rs1800629 G>A polymorphism decreased the risk of inhibitor formation in Iranian patients with HA., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The detection and the quantification of specific antibodies represent essential tools for the diagnosis and for the biological monitoring of immune humoral response in many clinical situations in particular in autoimmune diseases or in the context of immunotherapy using monoclonal antibodies. This article focuses on the development of a specific antibody measuring method (Patent n°PCT/IB2014/064437). The principle of this method is based on the combined use of a monoclonal antibody as standard and the protein G as immunoglobulins detecting agent. We performed a complete analytical validation of this method for the quantification of antibodies in three different applications: autoantibodies, alloantibodies and therapeutic monoclonal antibody. The results showed good performances compatible with the use of these assays as diagnostic tools. This method allows avoiding the use of products from human origin as reagent that causes ethical and infectious concerns but also storage and long term stock management problems. Moreover, this approach is particularly useful when no commercial reagent is available, especially in the case of rare diseases., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Monocyte monolayer assay (MMA) is a compatibility testing method for evaluating the clinical significance of red blood cell (RBC) alloantibodies. Time-consuming monocyte isolation procedures and requirement for fresh monocytes have limited application of the MMA. The aim of this study was to develop and assess the utility and efficacy of cryopreserved buffy coat (BC)-derived monocytes for MMA application., Study Design and Methods: Peripheral blood mononuclear cells (PBMNCs) were isolated from BC or peripheral blood (PB) and pooled and BC PBMNCs were cryopreserved. Monocytes from pooled PBMNCs were incubated with anti-D-sensitized, anti-Scianna2 (Sc2)-sensitized, anti-AnWj-sensitized, or anti-Jr a -sensitized RBCs or lipopolysaccharide (LPS). MMA phagocytic index (PI) and membrane integrity were determined microscopically, and cytokine release was measured by Luminex technology., Results: PBMNC isolation rates from fresh BC and PB were not comparable (67.4 ± 6.3 and 75.8 ± 7.7% respectively, p = 0.024). There was no significant difference in PBMNC membrane integrity (fresh PB, 100%; fresh BC, 100%; cryopreserved BC, 95.2 ± 1.2%), postwash recovery (fresh PB, 85.9 ± 3.1; fresh BC, 86.9 ± 6.7; cryopreserved BC, 84.8 ± 5.1), or monocyte PI (fresh PB, 82 ± 10; fresh BC, 77 ± 11; cryopreserved BC = 80 ± 6). Monocytes from pooled cryopreserved BC PBMNCs reacted with RBCs sensitized with anti-D and RBC alloantibodies, including anti-Sc2, anti-Jr a , and anti-AnWj., Conclusions: Monocytes from pooled cryopreserved BC PBMNCs can be used reliably to evaluate phagocytic responses of sensitized RBCs and to assess clinical significance of RBC alloantibodies., (© 2018 AABB.)

Insulin-dependent diabetes may occur in patients with cancers who are treated with checkpoint inhibitors (CPIs). We reviewed cases occurring over a 6-year period at two academic institutions and identified 27 patients in whom this developed, or an incidence of 0.9%. The patients had a variety of solid-organ cancers, but all had received either anti-PD-1 or anti-PD-L1 antibodies. Diabetes presented with ketoacidosis in 59%, and 42% had evidence of pancreatitis in the peridiagnosis period. Forty percent had at least one positive autoantibody and 21% had two or more. There was a predominance of HLA-DR4, which was present in 76% of patients. Other immune adverse events were seen in 70%, and endocrine adverse events in 44%. We conclude that autoimmune, insulin-dependent diabetes occurs in close to 1% of patients treated with anti-PD-1 or -PD-L1 CPIs. This syndrome has similarities and differences compared with classic type 1 diabetes. The dominance of HLA-DR4 suggests an opportunity to identify those at highest risk of these complications and to discover insights into the mechanisms of this adverse event., (© 2018 by the American Diabetes Association.)

Background: Blood transfusion through the intraosseous route is gaining popularity in emergency medicine. Pretransfusion peripheral blood (PB) samples are usually not available in these patients, leading to discrepancies in blood group typing and a possible delay in transferring to group-specific blood products. The aim of this study was to assess the feasibility of ABO and D typing and red blood cell alloantibody screening in marrow (BM) samples., Study Design and Methods: Direct and reverse ABO typing, D typing, and a two-cell alloantibody screen were performed in EDTA-anticoagulated BM samples with standard manual column agglutination techniques. EDTA-anticoagulated PB samples were used as controls., Results: The mean age of the study subjects (n = 71) was 47 years (range, 1-82 years). All ABO groups and both D+ and D- types were represented. In all subjects, concordant results were observed for all analyses in BM and PB samples. In 15 (21%) of the samples, a discrepancy of one reaction strength step (1+) was observed in at least one of the analyses (Cohen's weighted κ = 0.993); this did not affect interpretation of the results., Conclusion: Blood group typing and alloantibody screening are feasible in BM samples, providing proof-of-concept that intraosseous samples for blood group serologic analyses can be collected from emergency patients before intraosseous blood transfusion. This will enable a timely transfer to group-specific blood products and enable conservation of the valuable universal-donor blood products., (© 2018 AABB.)

Haploidentical donors are now increasingly considered for transplantation in the absence of HLA-matched donors or when an urgent transplant is needed. Donor-specific anti-HLA antibodies (DSA) have been recently recognized as an important barrier against successful engraftment of donor cells, which can affect transplant survival. DSA appear more prevalent in this type of transplant due to higher likelihood of alloimmunization of multiparous females against offspring's HLA antigens, and the degree of mismatch. Here we summarize the evidence for the role of DSA in the development of primary graft failure in haploidentical transplantation and provide consensus recommendations from the European Society for Blood and Marrow Transplant Group on testing, monitoring, and treatment of patients with DSA receiving haploidentical hematopoietic progenitor cell transplantation.

Background: The Kidd-null phenotype, Jk(a-b-), occurs in individuals who do not express the JK glycoprotein. Jk(a-b-) individuals can make an antibody against the Jk3 antigen, a high-incidence antigen present in more than 99.9% of most populations. This presents many challenges to the blood bank including identification of the antibody, masking of other antibodies, and how to provide transfusion support given the rarity of Jk3-negative blood products. Kidd antibodies may cause acute and delayed hemolytic reactions as well as hemolytic disease of the fetus and newborn (HDFN). In this article, we present a series of four practical cases of pregnant women with the anti-Jk3 alloantibody that demonstrate a range of clinical presentations of Kidd-related HDFN., Study Design and Methods: We retrospectively reviewed the clinical and blood bank records for four patients and their newborns encountered at institutions in Tennessee, Missouri, Hawaii, and Guam with an anti-Jk3 identified during pregnancy., Results: Two cases showed no significant evidence for HDFN, while two cases were of mild-to-moderate severity requiring early delivery due to elevated middle cerebral artery (MCA) flow velocities but requiring only phototherapy for hyperbilirubinemia. No intrauterine or neonatal transfusions were necessary. Anti-Jk3 alloantibody titers ranged from 2 to 128., Conclusion: Clinical manifestations of anti-Jk3 HDFN are generally mild to moderate. Anti-Jk3 titers were not found to correlate directly with HDFN severity. We suggest a titer of 16 to 32 as a cutoff for implementing enhanced monitoring of fetal MCA flow velocities, as such titers may be indicative of elevated HDFN risk., (© 2018 AABB.)

Introduction Studies show that rates of blood transfusion associated with general surgical laparoscopy are low. Currently, there are no national guidelines in the UK regarding blood group and antibody screening (G&S) for patients undergoing emergency laparoscopy. The aim of this study was to assess whether using G&S before emergency laparoscopic general surgery routinely is worthwhile by identifying rates of perioperative transfusion. Methods Data were collected retrospectively on all emergency laparoscopic procedures at a single district general hospital between January 2014 and 31 December 2016. Emergency laparoscopic general surgical cases were included and gynaecological cases excluded. Records were reviewed to ascertain whether G&S was performed, whether antibodies were detected and whether patients were transfused. Results A total of 562 emergency laparoscopic cases were performed. The median age was 28 years (range: 6-95 years). Laparoscopic appendicectomy (n=446), diagnostic laparoscopy (n=47) and laparoscopic cholecystectomy (n=25) were the most common procedures. Of the total patient cohort, 514 (91.5%) and 349 (70.1%) had a first and second G&S respectively while 30 (5.3%) had no G&S. Four patients (0.71%) had antibodies detected. One patient (0.18%) received a transfusion. This patient had undergone laparoscopic repair of a perforated duodenal ulcer and there was no major intraoperative haemorrhage but he was transfused perioperatively for chronic anaemia. Conclusions These results demonstrate a low rate of blood transfusion in emergency laparoscopic general surgery. The majority of these patients had a low risk of major intraoperative haemorrhage and we therefore argue that G&S was not warranted. We propose a more targeted approach to the requirement for preoperative G&S and the use of O negative blood in the event of acute haemorrhage from major vessel injury.

Background: Lifelong transfusions are life savers for thalassaemia patients but are associated with many complications. Alloimmunization is a major problem for blood banks. Antigens of foreign red blood cells induce the formation of antibodies in patients suffering from thalassaemia. The purpose of this study was to examine the frequency of red cell alloantibodies and to express the type of these antibodies in thalassaemia patients., Methods: Patients that have received multiple transfusions were included in this study. Those with the positive Coombs test (DAT) results were excluded from the study and remaining patients were screened for antibodies. A panel of known blood group antigens was used for the patients who had a positive antibody screening test because they had alloantibodies in their serum. First, three cell panel was applied. If the screen was positive then eleven cell panels was used to identify the specific antibody. Both the cell panels were applied at room-temperature, liss (low ionic strength saline) and coombs phase., Results: Three hundred & two patients were selected out of which 65.6% (n=198) were males and 34.4% (n=104) females. Patient's age ranged from 1.5 years to 26 years ±5.40 years. All of the patients were given regular red cell transfusion at 2-4 weeks interval. They were given non leukodepleted transfusions. It is not the practice in any thalassaemia Centre in Pakistan to give phenotypically matched blood for Kell, Kidd, Duffy or any other minor group antigens to patients on regular blood transfusion. Alloimmunization was positive in 12 (4.0%) of the 302 patients studied. Male were 66.67% (n=8) and female were 33.33% (n=4). Samples of these positive patients were further tested to determine specificity of alloantibodies. Anti Cw was most common, detected in 4 out of 12 (1.3%) patients. Anti K, k, S and Lua were detected in 2 out of 12 (0.7%) each., Conclusions: Thalassemia major patients on regular blood transfusions can develop red cell alloantibodies. Detailed pretransfusion screening would add towards better management of these patients.

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against human leukocyte antigens (HLA). Histocompatibility guidelines are based on laboratory tests of anti-HLA immunity, which presents either as pre-existing or de novo generated HLA antibodies that constitute a major transplantation barrier. Current tests are built on a single-antigen beads (SAB) platform using a fixed set of ~100 preselected recombinant HLA antigens to probe transplant sera. However, in humans there exist a far greater variety of HLA types, with no two individuals other than identical twins who can share the same combination of HLA sequences. While advanced technologies for HLA typing and direct sequencing can precisely capture any mismatches in DNA sequence between a donor's and recipient's HLA, the SAB assay, due to its limited variety in sequence representation, is unable to precisely detect alloantibodies specifically against the donor HLA mismatches. We sought to develop a complementary method using a different technology to detect and characterize anti-donor HLA antibodies on a personalized basis. The screening tool is a custom peptide array of donor HLA-derived sequences for probing post-transplant sera of the organ recipient to assess the risk for antibody-mediated rejection. On a single array for one donor-recipient pair, up to 600 unique peptides are made based on the donor's HLA protein sequences, each peptide carrying at least one mismatched residue in a 15-amino acid sequence. In our pilot experiments to compare antigen patterns for pre- and post-transplant sera on these arrays, we were able to detect anti-HLA signals with the resolution that also allowed us to pinpoint the immune epitopes involved. These personalized antigen arrays allow high-resolution detection of donor-specific HLA epitopes in organ transplantation.

Background: Alloimmunization to red blood cells (RBCs) can cause serious transfusion reactions and complicate the search for compatible blood products. Alloantibodies can be detected for periods ranging from a few days to several years, yet the mechanisms controlling the duration of detectability remain unknown. We studied the detection durations in patients forming multiple antibodies to investigate whether the duration is more strongly determined by conditions present at the time of each transfusion (peritransfusion factors) or by more stable patient-specific factors likely to persist across transfusions., Study Design and Methods: We studied retrospective medical records for alloimmunized patients at Massachusetts General Hospital and Brigham and Women's Hospital (1461 patients; 2187 antibodies)., Results: Antibodies discovered simultaneously in a patient shared similar fates: 76% persisted through the last screen or first became undetectable during the same screen. Simultaneously identified antibodies were also more persistent than sequentially identified antibodies (mean, 9.2 months vs. 4.9 months; p < 10 -3 ). Within a patient, antibodies discovered simultaneously tended to be detected for similar periods of time (mean difference, 25 days), compared to the detection period for sequentially discovered antibodies (107 days, p < 10 -3 )., Conclusions: The similarity in detection duration of simultaneously identified antibodies suggests that peritransfusion factors are important determinants of alloantibody detectability and duration. We also find some evidence that detection durations for sequentially identified antibodies are also more highly correlated than those for randomly selected antibodies across all patients, suggesting that patient-specific factors also play a role in determining alloantibody persistence., (© 2017 AABB.)

Antibody-mediated rejection is associated with heterogeneous kidney allograft outcomes. Accurate evaluation of risk for graft loss at time of diagnosis is necessary to offer personalized treatment. In contrast with serological and molecular assessment, morpho-histological evaluation of antibody-mediated rejection lesions has not significantly evolved. This relies on Banff classifications designed to be of diagnostic discriminatory power rather than prognostic and face quantitative and qualitative limitations. Here we developed a method of Computer-assisted Analysis of Graft Inflammation (CAGI) to improve the classification of allograft inflammation. Digitization of immunostained biopsy sections, image processing and algorithm-driven analysis allowed quantification of macrophages, T cells, B cells, and granulocytes per unit surface of interstitium, capillaries or glomeruli. CAGI was performed on biopsy specimens of 52 patients with extensively phenotyped antibody-mediated rejection. Macrophage numbers in capillaries and interstitium, but not Banff scores or the amount of other immune cell subsets, correlated with donor-specific antibody (DSA) mean fluorescence intensity and DSA-C3d status. The quantity of macrophages in the interstitium and DSA-C3d status were the only independent predictors for significant allograft loss at the time of antibody-mediated rejection diagnosis (hazard ratio 3.71 and 2.34, respectively). A significant strategy integrating the DSA-C3d assay and the quantification of interstitial macrophages allowed identification of three groups with distinct renal prognosis: DSA-C3d - , DSA-C3d + /macrophages-low and DSAC3d + /macrophages-high. Thus, CAGI brings a missing piece to the antibody-mediated rejection puzzle by identifying morpho-histological processes that bridge in vitro parameters of DSA pathogenicity and graft loss. Hence, this approach could be useful in future integrated strategies of risk evaluation., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Background: Several publications have reported an increase in nonspecific reactions when automated technologies such as solid phase are used for the detection of red blood cell alloantibodies. However, there is little known about patient-specific factors associated with these reactions and the clinical importance of these nonspecific reactions., Study Design and Methods: We performed a 6-year retrospective review of our blood bank records and all newly reported unidentified (UID) reactivity using a test tube polyethylene glycol (t-PEG) and solid-phase method for the detection and identification of alloantibodies was recorded. Patient factors, such as underlying diagnosis, age, sex, ABO, Rh type, ethnicity, and subsequent antibody formation were recorded in each case., Results: We determined that there was a significant increase in new UID reactions recorded in solid phase (20 per 10,000 tests) when compared to the t-PEG (1.8 per 10,000 tests) method for the detection of antibodies (p ≤ 0.0001). Solid-phase UID reactions were significantly associated with female sex (p = 0.04) and certain diagnoses, such as chronic or autoimmune disease, cancer, pregnancy, surgery, and trauma. Approximately 16% of patients developed a new auto- or alloantibody subsequent to their detected UID using solid phase., Conclusions: When solid phase is used for antibody identification, there is greater sensitivity toward nonspecific reactivity when compared to the t-PEG method. Patient sex and underlying diagnosis may explain the increased incidence of new UID reactivity in the solid-phase technology. Finally, UID reactivity should not be overlooked due to a notable percentage of subsequent clinically significant antibodies after UID detection., (© 2017 AABB.)

Aim: To study the association of serum levels of anti-myeloperoxidase (MPO) antibody with retinal photoreceptor ellipsoid zone (EZ) disruption in diabetic retinopathy., Methods: Consecutive patients with type 2 DM [diabetes mellitus with no retinopathy (NODR; n=20); non-proliferative diabetic retinopathy (NPDR; n=18); proliferative diabetic retinopathy (PDR; n=16)] and healthy controls (n=20) between the ages of 40 and 65years were included. Disruption of EZ was graded by spectral domain optical coherence tomography as no disruption of EZ and disrupted EZ. The serum levels of anti-MPO antibody was analyzed using standard protocol. Association between the variables was evaluated using multiple regression analysis., Results: A significant difference was found between the serum levels of anti-MPO antibody in various study groups (p<0.001). A positive association was found between EZ disruption and levels of anti-MPO antibody [adjusted odd's ratio (AOR)=1.079, CI 1.010-1.124, p=0.04]. A significant positive correlation was found between logMAR visual acuity and grade of disruption (AOR=1.008, CI 1.006-5.688, p=0.04)., Conclusions: An increased serum anti-MPO antibody levels is associated with retinal photoreceptor EZ disruption and decreased visual acuity in diabetic retinopathy., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: This is a cross-sectional study designed to evaluate the histologic characteristics of graft injury in the presence of anti-angiotensin II type 1 receptor antibody (AT1R-Ab) and anti-endothelial cell antibody (AECA)., Methods: Non-HLA antibody testing was included in the posttransplant evaluation for 70 kidney recipients. Biopsies were performed for cause for 47 patients and as protocol for the remaining 23 patients. Biopsy-proven rejection was defined according to the Banff 2009-2013 criteria. AT1R-Ab was measured on an ELISA platform. Patients were divided into 3 groups based on AT1R-Ab levels (>17, 10-17, and <10 U/ml). AECA was evaluated using an endothelial cell crossmatch (ECXM) in patients whose HLA antibody level was insufficient to cause a positive flow cytometric crossmatch., Results: AT1R-Ab levels were higher in patients diagnosed with antibody mediated rejection compared to those with no rejection (P = 0.004). Glomerulitis (g) and peritubular capillaritis (ptc) scores were independently correlated with increased AT1R-Ab concentrations in the presence or absence of HLA-DSA (P = 0.007 and 0.03 for g scores; p = 0.005 and 0.03 for ptc scores). Patients with a positive ECXM had higher AT1R-Ab levels compared to those with a negative ECXM (P = 0.005). Microcirculation inflammation (MCI = g + ptc score) was higher in patients with a positive ECXM and with AT1R-Ab >17 U/ml, although this did not reach statistical significance (P = 0.07)., Conclusions: The data show an association between non-HLA antibodies detected in the ECXM and AT1R ELISA and microvascular injury observed in antibody mediated rejection.

Introduction: chronic urticaria (CU) is a skin disorder characterized by transient, pruritic wheals persisting for longer than 6 weeks. The etiopathogenesis of the disease is still unclear, but there is evidence that autoimmunity and endocrine dysfunction may be involved., Aim: the aim of this study was to determine whether chronic urticaria is statistically associated with thyroid autoimmunity., Patients and Methods: in a prospective case-control study, we compared the frequency of thyroid auto-antibodies (thyroglobulin antibody, anti-Tg and thyroid peroxidase antibody, anti-TPO) in 70 patients with chronic urticaria and in 70 healthy volunteers. Thyroid auto-antibodies and thyroid hormones (thyroxine (T4), triiodthyronine (T3) and thyroid stimulating hormone (TSH) were measured in all subjects., Results: thyroid functional abnormalities were found in 8 (11.43%) patients. Anti-Tg and anti-TPO were positive in 16 (23%) and 21 (30%) patients, respectively. In control group, only one subject (1.42%) had abnormalities in thyroid hormonal status, and two subjects (2.86%) had positive thyroid auto-antibodies. Compared with the control group, the frequency of both anti-Tg and anti-TPO was significantly higher in those with chronic urticaria (P < 0.05)., Conclusion: this study shows a significant association between chronic urticaria and thyroid autoimmunity, and that tests to detect thyroid auto-antibodies are relevant in patients with chronic urticaria., Competing Interests: • Conflict of interest: none declared.

Introduction: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a rare condition that may lead to intracerebral haemorrhage (ICH) in the fetus or neonate. Platelet alloimmunisation causing FNAIT has been described in association with fetal cerebral ventriculomegaly (VM), presumably due to subclinical ICH. The objective of this study was to assess the association between fetal VM and platelet alloimmunisation., Methods: This is a case series of pregnancies with fetal VM screened for platelet alloantibodies from 2003 to 2012. Cases of multiple pregnancies, structural anomalies, aneuploidies, or congenital infection were excluded., Results: Of 45 pregnancies with fetal VM that were screened for platelet alloantibodies, 5 (11%) were positive. There was only one antenatal ICH, with confirmed fetal severe thrombocytopenia before termination of pregnancy. The other cases were treated with intravenous immunoglobulins without prior fetal blood sampling. No other case of neonatal thrombocytopenia was confirmed., Conclusions: The prevalence of platelet alloimmunisation was high in this series of fetal VM. Prospective large studies are needed to confirm the role of platelet alloimmunisation in fetal VM., (© 2016 S. Karger AG, Basel.)

Conclusions: When an antibody is detected, its specificity should be determined and its likely clinical significance should be assessed. When one antibody has been identified, it becomes necessary to confirm the presence of additional significant antibodies to ensure that compatible blood is provided to the patient. To perform this confirmation, specific reagent red blood cells (RBCs) are selected; these are called selected cells. Though the most common use of selected cells is for antibody confirmation, they can also be used for several other immunohematologic applications. In a developing country like India, the performance of antibody screening for unexpected antibodies on a routine basis is a comparatively new phenomenon, and those laboratories performing advanced immunohematologic testing would need to use selected cells to arrive at an accurate conclusion. This report defines selected cells and enumerates sources of these RBCs. Detailed immunohematologic applications are discussed with applicable case studies.

Background: RhIG prophylaxis for D- pregnant women prevents hemolytic disease of the newborn and typically depends on results of serologic D typing. Interpretation and follow-up of weak D serology is variable. Recent recommendations promote genotyping for RHD status determination in those with weak D serology. Canadian Blood Services performs comprehensive serologic prenatal testing in four provinces. Genotyping is used to determine D typing in patients with weak D., Study Design and Methods: A serologic algorithm identified which patients require genotyping for RHD determination. Genotyping was performed on one of two commercially available platforms., Results: Only 0.4% of D- patients met criteria for genotyping. Sixty-one percent were weak D Type 1, 2, or 3. Thirty percent had a partial or weak D other than Type 1, 2, or 3. Eleven had variants which remained unresolved. Seventeen were D+ and four were D-., Conclusions: Genotyping of patients with weak D serology led to an identified genotype in most patients. RhIG administration was avoided in 66% who were weak D Type 1, 2, or 3 or were D+. The use of a serologic algorithm to select patients for RHD genotyping identifies a majority of patients with weak D types not at risk for alloimmunization. This approach limits the number of genotyping investigations and the cost of providing classification for weak D types., (© 2016 AABB.)

Screening for de novo donor-specific antibodies (dnDSA) in stable kidney transplant recipients is routine practice in some centers. Patients with DSA are at increased risk of graft loss and early intervention may improve outcomes. However, the costs and benefits of dnDSA surveillance are unknown. A medical decision analysis to examine a screening strategy was developed for kidney transplant recipients who had stable graft function and were DSA negative 1 year posttransplant. In the base case, a modest 25% reduction in graft loss in dnDSA-positive patients treated with increased immunosuppression resulted in 0.04618 quality-adjusted years (QALYs) gained. However, benefits from reduced graft loss were eliminated if there was a small increased risk of death from added therapy. The incremental cost effectiveness was marginal at approximately $120 000-250 000 per QALY, but could be more or less favorable depending on several key variables such as efficacy of treatment, screening costs, incidence rate of subclinical dnDSA, and patient survival. Screening performed the best in patients with lower mortality rates and higher baseline incidence rates of dnDSA. Further study is warranted to gather the necessary high-quality evidence to justify screening., (© Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Anecdotal evidence has suggested an association of intravenous drug abuse with alloimmunization; however, published data are limited to case reports., Objective: The purpose of this study was to determine whether women with a history of intravenous drug abuse have an increased risk of alloimmunization., Study Design: A retrospective cohort study was performed with the use of data from a single-center blood bank and perinatal database from 2008-2014. Blood bank data were used to identify women with alloimmunization, which was defined as a positive antibody screen in pregnancy not due to naturally occurring antibodies, agglutinins, autoantibodies, or Rh immunoglobulin administration. Intravenous drug abuse was ascertained from a comprehensive database that has captured all drug abuse in pregnancy since 2008. For women who contributed >1 pregnancy to the database, only the most recent pregnancy was included. The rates of alloimmunization among women with a history of intravenous drug abuse and general obstetric populations were calculated and compared. The distribution of alloantibody types, proportion of Rh-group alloantibodies, and patient Rh status were assessed for intravenous and non-intravenous drug abuse-associated alloimmunization. Characteristics and outcomes between intravenous and non-intravenous drug abuse-associated alloimmunization were assessed for women with clinically significant alloantibodies., Results: Alloimmunization was more common in women with a history of intravenous drug abuse (11/305 women; 3.6%) compared to women without a history of intravenous drug abuse (288/16,022 women; 1.8%; relative risk, 2.00; 95% confidence interval, 1.11-3.62). Needle-sharing was present in 7 and suspected in 4 women with an intravenous drug abuse history. Among women with a history of intravenous drug abuse, none had a history of transfusion or traditional risk factor for alloimmunization. The distribution of alloantibodies was different between intravenous drug abuse- and non-intravenous drug abuse-associated alloimmunization. Rh group alloantibodies and Rh-negative status were more common in women with a history of intravenous drug abuse. Among Rh-negative women with a history of intravenous drug abuse, 50% of RhD alloimmunization cases occurred in nulliparous women. The rate of multiple alloantibodies was not different between intravenous drug abuse- and non-intravenous drug abuse-associated alloimmunization., Conclusion: Maternal history of intravenous drug abuse is associated with an increased risk of alloimmunization. Approximately 1 in 30 intravenous drug abuse women may be diagnosed with an alloantibody in pregnancy. Given the current US opioid epidemic, increased vigilance in screening is required. Needle-sharing represents a possible mechanism for intravenous drug abuse-associated alloimmunization; however, limited obstetric care, failure to obtain Rh immunoglobulin, or failure to identify early pregnancy loss cannot be excluded., (Copyright © 2016 Elsevier Inc. All rights reserved.)