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HLA-B*58:02:04 differs from HLA-B*58:02:01 by one synonymous nucleotide in codon 215 in exon 4., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

In patients awaiting an allogeneic haematopoietic stem cell transplantation, platelet transfusion is a risk factor for anti-HLA class I immunization because the resulting donor-specific antibodies complicate the allograft process. The objective of the present study was to determine the feasibility of a novel eplet-based strategy for identifying HLA class I mismatches between potential donors and the recipient when pre-allograft platelet transfusions were required. We included 114 recipient/haploidentical relative pairs. For each pair, we entered HLA-class I typing data into the HLA Eplet Mismatch calculator, defined the list of mismatched eplets (for the recipient versus donor direction) and thus identified the shared HLAs to be avoided. Using this list of HLAs, we defined the theoretical availability of platelet components (PCs) by calculating the virtual panel-reactive antibody (vPRA). We also determined the number of PCs actually available in France by querying the regional transfusion centre's database. The mean ± standard deviation number of highly/moderately exposed eplets to be avoided in platelet transfusions was 5.8 ± 3.3, which led to the prohibition of 38.5 ± 2 HLAs-A and -B. Taking into account the mismatched antigens and the eplet load, the mean ± standard deviation theoretical availability of PCs (according to the vPRA) was respectively 34.49% ± 1.95% for HLA-A and 80% ± 2.3% for HLA-B. A vPRA value below 94.9% for highly or moderately exposed eplets would predict that 10 PCs were actually available nationally. Although epitope protection of HLA molecules is feasible, it significantly restricts the choice of PCs., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-B*07:491 differs from HLA-B*07:02:01 by one nucleotide substitution in codon 218 in exon 4., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-C*05:282Q differs from HLA-C*05:01:01 by one nucleotide substitution in codon -24 in exon 1., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-DRB1*14:255 differs from HLA-DRB1*14:54:01 by one nucleotide substitution in codon 205 in exon 4., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-DQB1*06:03:47 differs from HLA-DQB1*06:03:01 by one synonymous nucleotide substitution in codon 158 in exon 3., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-DPB1*1359:01 differs from HLA-DPB1*03:01:01 by one nucleotide substitution in codon 77 in exon 2., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-C*03:613 differs from HLA-C*03:02:02 by one nucleotide substitution in codon 190 in exon 4., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Introduction: HLA antigens are highly polymorphic, and their immunogenicity is dependent on the configurations of polymorphic amino acids. Monitoring anti-HLA immunization is essential in organ transplantation, as antibodies directed against HLA molecules are a major cause of rejection. Anti-HLA antibodies are not specific for HLA antigens, but recognize B-cell epitopes present on HLA molecules., Methods: To better understand antibody reactivity patterns, we calculated the Spearman correlation of the mean fluorescence intensity (MFI) of anti-HLA antibodies identified by a single-antigen assay performed using a Luminex® immunobeads assay on a large number of samples. Then, we built a computer tool analyzing antibody reactivity patterns with an accessibility by a web browser linked to the International Epitope Registry. We also extended our model to Onelambda® and Lifecodes® single-antigen class I and class II assays., Results and Discussion: The resulting MFI correlations reflect HLA antibody cross-reactivity and eplets similarity. We built HLA Graph, a computer tool that analyzes the eplets involved in antibody reactivity profiles. HLA Graph is usable with Onelambda® and Lifecodes® single-antigen class I and class II assays. The interpretation of reactivity against alleles not tested by the antibody assays and against the alpha and beta chains of HLA-DQ and HLA-DP loci were also developed., Conclusion: HLA Graph is a free and ready-to-use bioinformatics tool that can be used by all laboratories performing anti-HLA antibody identification by immunobead assay., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

After allogeneic hematopoietic stem-cell transplantation (alloHSCT), the chimerism assay is used to monitor cell engraftment and quantify the respective proportions of donor/recipient cells in blood or bone-marrow samples. Here, we aimed to better assess the utility of determining CD3 + cell chimerism within the first 6 months post alloHSCT. One hundred and thirty five patients diagnosed with acute myeloid leukemia were enrolled in this study. We observed significantly lower overall survival and relapse free survival for patients without full donor chimerism (<95%, <98%, <99%) in whole blood at Day 30, as well as at Day 90 after alloHSCT, than for patients with full donor chimerism. This outcome was not observed when assessing selected CD3 + cells. However, at Day 90, patients with discordant whole blood versus selected CD3 + cell chimerism showed both significantly lower overall survival and relapse free survival, giving an interest to assess selected cells chimerism., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-C*04:438 differs from HLA-C*04:01:01 by one nucleotide substitution in codon 237 in exon 4., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-DQA1*01:80 differs from HLA-DQA1*01:04:01 by one nucleotide substitution in codon -18 in exon 1., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-C*04:450 differs from HLA-C*04:01:01 by one nucleotide substitution in codon 328 in exon 7., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-DRB1*04:326 differs from HLA-DRB1*04:01:01 by one nucleotide substitution in codon 23 in exon 2., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-C*01:214 differs from HLA-C*01:02:01:01 by one nucleotide substitution in codon -10 in exon 1., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

A hematopoietic chimerism assay is the laboratory test for monitoring engraftment and quantifying the proportions of donor and recipient cells after hematopoietic stem cell transplantation recipients. Flow cytometry is the reference method for determining the purity of CD3 + cells on the chimerism of selected CD3 + cells. In the present study, we developed a single-step procedure that combines the CD3 + purity assay (using the PCR-based Non-T Genomic Detection Kit from Accumol, Calgary, Canada) and the qPCR chimerism monitoring assay (the QTRACE qPCR assay from Jeta Molecular, Utrecht, the Netherlands). First, for the CD3 + purity assay, we used a PCR-friendly protocol by changing the composition of the ready-to-use reaction tubes (buffer and taq polymerase) and obtained a satisfactory calibration plot (R 2 = 0.8924) with a DNA reference scale of 2 ng/μl. Next, 29 samples (before and after CD3 positive selection) were analyzed, the mean cell purity was, respectively, 19.6% ± 6.45 and 98.9% ± 1.07 in the flow cytometry assay; 26.8% ± 7.63 and 98.5% ± 1.79 in the PCR-based non-T genomic detection assay. Our results showed that the CD3 + purity assay using a qPCR kit is a robust alternative to the flow cytometry assay and is associated with time savings when combined with a qPCR chimerism assay., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Flow cytometry crossmatching (FC-XM) assay is the most sensitive cell-based method for detecting donor-specific antibodies (DSAs). However, the use of FC-XM remains limited by methodological and clinical variations. This basic assay cannot discriminate between complement-fixing and noncomplement-fixing antibodies. FC-XM also detects patient all antibodies bound to donor cells and not only DSAs against to HLA molecules. Pretest factors associated with a donor's medical care can affect test results by changing the number, viability and target on lymphocytes (such as rituximab on CD20 + B-cells). Assay adjustment can be performed to improve the sensitivity and specificity of FC-XM. Pronase treatment (0.5-1 mg/mL) prevents false-positive B-cell FC-XM due to nonspecific immunoglobulin binding by Fc receptors and binding of surface immunoglobulins onto the surface of B-cells. Pronase treatment (2 mg/mL) or a serum incubation step with an anti-rituximab monoclonal antibody (Ab) prevents the interference induced by rituximab therapy. The use of 7 aminoactinomycin-D (7-AAD) or fluorochrome-conjugated C4d Ab, after complement incubation, allows complement-fixing antibodies to be distinguished from noncomplement-fixing antibodies. The use of donor endothelial precursor cells as target cells allows the detection of nonmajor histocompatibility complex Ab-binding endothelial cells. However, lymphocyte crossmatches still had some limits in specificity and sensitivity. This implies that this assay must be interpreted with the virtual crossmatch., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

In clinical organ transplantation, flow cytometry crossmatching can be performed on total blood with a hemolysis step or after a preliminary mononuclear cell separation step using a standard Ficoll-Hypaque protocol. Here, we compared the Ficoll-Hypaque step with a faster technique for isolating mononuclear cells (the SepMate tube), using the same samples (collected and stored at room temperature for 0, 24, 48 or 72 hours). We found that the SepMate separation protocol is easily applied to flow cytometry crossmatching (with or without pronase treatment), provided that the samples have been stored at room temperature for 48 hours or less. Conversely, the Ficoll-Hypaque protocol should be used if the samples have been stored for more than 48 hours at room temperature., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Flow cytometry crossmatching (FC-XM) is the most sensitive cell-based method for detecting donor-specific antibodies in clinical organ transplantation. Unfortunately, background FC-XM reactivity is elevated in assays with B lymphocytes-partly because of nonspecific immunoglobulin binding by Fc receptors and B-cell surface immunoglobulins. To reduce the background reactivity in a B-cell FC-XM assay, we treated lymphocytes with pronase (1 mg/mL for 30 minutes). This treatment drastically reduced the presence of kappa light chains and Fc receptors (CD32b), while the concomitant decrease in CD19, CD20 and major histocompatibility complex (MHC) I and II expression on B-cells was acceptable. Higher pronase concentrations (>2 mg/mL) started to significantly affect CD19, CD20, MHC-I and -II expression on B-cells. In subsequent prospective experiments (on 42 donor cells tested with 102 sera), we found that pronase treatment was associated with a relative increase of the sensitivity and specificity in our B-cell FC-XM assay., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Before kidney transplantation, recipients are routinely screened for preformed antibodies and prospective crossmatches. In this study, we compared prospective Luminex donor-specific crossmatches (LumXm) with the levels of identified, donor-specific antibodies (DSAs). LumXm was performed for 108 patient sera, 84 of which were positive for preformed antibodies and 24 of which were negative. Although LumXm can detect class I DSAs (anti-A and anti-B) with a mean fluorescence intensity (MFI) as low as 2300, the assay has a 'grey zone' for MFIs up to 4000 with a sensitivity of 54% and a specificity of 100%. LumXm can detect a class II DSA (anti-DRB1) with an MFI as low as 1300 and a sensitivity of 93% and a specificity of 99%. However, these correlations were obtained with two precautions: autocrossmatching and single-antigen bead assay with denaturing buffer were performed in discordant cases. Moreover, LumXm failed to detect anti-Cw and anti-DP in the 10 cases studied. LumXm, therefore, displays certain discrepancies with respect to single-bead assays--especially for antibodies with a low MFI. Unfortunately, LumXm has a low sensitivity for anti-A and anti-B class I antibodies., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)