Separating the Wheat from the Chaff among HLA-DQ Eplets.

In transplantation, anti-HLA Abs, especially targeting the DQ locus, are well-known to lead to rejection. These Abs identified by Luminex single Ag assays recognize polymorphic amino acids on HLA, named eplets. The HLA Eplet Registry included 83 DQ eplets, mainly deduced from amino acid sequence alignments, among which 66 have not been experimentally verified. Because eplet mismatch load may improve organ allocation and transplant outcomes, it is imperative to confirm the genuine reactivity of eplets to validate this approach. Our study aimed to confirm 29 nonverified eplets, using adsorption of eplet-positive patients' sera on human spleen mononuclear cells and on transfected murine cell clones expressing a unique DQα- and DQß-chain combination. In addition, we compared the positive beads patterns obtained in the two commercially available Luminex single Ag assays. Among the 29 nonverified DQ eplets studied, 24 were confirmed by this strategy, including the 7 DQα eplets 40E, 40ERV, 75I, 76 V, 129H, 129QS, and 130A and the 17 DQß eplets 3P, 23L, 45G, 56L, 57 V, 66DR, 66ER, 67VG, 70GT, 74EL, 86A, 87F, 125G, 130R, 135D, 167R, and 185I. However, adsorption results did not allow us to conclude for the five eplets 66IT, 75S, 160D, 175E, and 185T.

Improving HLA typing imputation accuracy and eplet identification with local next-generation sequencing training data.

Assessing donor/recipient HLA compatibility at the eplet level requires second field DNA typings but these are not always available. These can be estimated from lower-resolution data either manually or with computational tools currently relying, at best, on data containing typing ambiguities. We gathered NGS typing data from 61,393 individuals in 17 French laboratories, for loci A, B, and C (100% of typings), DRB1 and DQB1 (95.5%), DQA1 (39.6%), DRB3/4/5, DPB1, and DPA1 (10.5%). We developed HaploSFHI, a modified iterative maximum likelihood algorithm, to impute second field HLA typings from low- or intermediate-resolution ones. Compared with the reference tools HaploStats, HLA-EMMA, and HLA-Upgrade, HaploSFHI provided more accurate predictions across all loci on two French test sets and four European-independent test sets. Only HaploSFHI could impute DQA1, and solely HaploSFHI and HaploStats provided DRB3/4/5 imputations. The improved performance of HaploSFHI was due to our local and nonambiguous data. We provided explanations for the most common imputation errors and pinpointed the variability of a low number of low-resolution haplotypes. We thus provided guidance to select individuals for whom sequencing would optimize incompatibility assessment and cost-effectiveness of HLA typing, considering not only well-imputed second field typing(s) but also well-imputed eplets.

Two-field resolution on-call HLA typing for deceased donors using nanopore sequencing.

The current practice of HLA genotyping in deceased donors poses challenges due to limited resolution within time constraints. Nevertheless, the assessment of compatibility between anti-HLA sensitized recipients and mismatched donors remains a critical medical need, particularly when dealing with allele-specific (second field genotyping level) donor-specific antibodies. In this study, we present a customized protocol based on the NanoTYPE® HLA typing kit, employing the MinION® sequencer, which enables rapid HLA typing of deceased donors within a short timeframe of 3.75 h on average at a three-field resolution with almost no residual ambiguities. Through a prospective real-time analysis of HLA typing in 18 donors, we demonstrated the efficacy and precision of our nanopore-based method in comparison to the conventional approach and without delaying organ allocation. Indeed, this duration was consistent with the deceased donor organ donation procedure leading to organ allocation via the French Biomedicine Agency. The improved resolution achieved with our protocol enhances the security of organ allocation, particularly benefiting highly sensitized recipients who often present intricate HLA antibody profiles. By overcoming technical challenges and providing comprehensive genotyping data, this approach holds the potential to significantly impact deceased donor HLA genotyping, thereby facilitating optimal organ allocation strategies.

Identification of a novel HLA-A null allele, HLA-A*01:420N allele.

The novel HLA-A*01:420N allele has two changes in exon 4 leading to premature stop codon.

Identification of a novel HLA-B allele, HLA-B*08:304.

The novel allele B*08:304 differs from B*08:01:01:01 by one nucleotide substitution in exon 2.

Identification of a novel HLA-C allele, HLA-C*03:618N.

The novel HLA-C*03:618N allele has one change in exon 1 leading to a premature stop codon.

Characterization of the novel HLA-C*08:258 and -C*12:374 alleles by two different sequencing-based typing techniques.

The novel HLA-C*08:258 and -C*12:374 alleles differ from HLA-C*08:02:01:01 and -C*12:03:01:01 by one nucleotide substitution.

Identification of the new HLA-DRB1*15:213 allele in a French cardiac transplant recipient.

The new HLA-DRB1*15:213 allele results from one nucleotide substitution in exon 3 of HLA-DRB1*15:02:01.

Assessing the allogenic realness of the Cw1/12/15 pattern occurring in the LABScreen single antigen assay.

Several technical limitations of Luminex single antigen (LSA) assays have been described so far. This study focused on a reactivity pattern observed in many sera that cannot be explained by eplets described in the Epitope Registry database and sometimes appearing against a self-HLA allele or antigen. In most cases, this pattern is revealed by a discrepant result when compared with other assays (Luminex PRA, cell-binding assays such as flow cytometry cross match, LSA from another manufacturer…). We focus here on the Cw1/12/15 pattern appearing on the LABScreen class I LSA provided by One Lambda. We documented its behavior using this LSA after acid denaturation of the beads, using Lifecodes LSA from Immucor, and adsorption of sera either on spleen mononuclear cells from deceased donors or on single HLA transfected cell clones. We studied 33 sera from different patients positive for the three Cw beads, selected from our routine patients' LSA database. Nine patients had transplants from a Cw12 or Cw15 donor without any pejorative evolution of the graft, nor post-transplant MFI (mean fluorescence intensity) increase of the Cw1/12/15 beads. A significant increase of MFI was observed after acid denaturation of the LABScreen beads. All sera tested by Lifecodes LSA were negative for these Cw beads. Finally, we found no significant difference of MFI after adsorption on cells from either origin. Therefore, the Cw1/12/15 pattern appears to be a false positive reactivity of the LABScreen single antigen assay.

The novel HLA-C*17:64Q allele characterized by two different sequencing-based typing techniques.

The novel allele HLA-C*17:64Q differs from HLA-C*17:01:01:02 by insertion of a Lysine in exon 2.

Deciphering the role of the conjugate's phycoerythrin label in complement-mediated interference occurring in HLA single antigen Luminex bead assays.

Complement-mediated interference is a well described phenomenon in single antigen bead (SAB) Luminex assay that leads to falsely low or negative results for anti-HLA antibody (Ab). In a context of high amount of Ab, the enrichment of the Ab around the bead can lead to complement cascade activation and deposition, thereafter impairing Ab detection. EDTA is now routinely used to circumvent this interference. In this report, we attempted to decipher the role of the phycoerythrin (PE) label conjugated to the secondary Ab in this interference. Indeed, PE is a huge molecule (240 kDa) that could participate to limiting access of the conjugate to its Ab target on the bead. To this purpose, 22 sera displaying complement interference without pre-treatment with EDTA were compared on SAB assay with three detection strategies: the recommended PE-conjugated secondary Ab (IgGPE), an Alexa Fluor 532-conjugated Ab (IgGAF) bearing a tiny 724 Da fluorochrome, and a biotinylated Ab followed by PE-conjugated streptavidin (IgGBiot). Complement interference occurred with the three detection methods, but its depth, defined by the percentage of MFI loss with neat serum, was the highest for IgGPE. Our study highlighted the partial role of the PE fluorochrome in complement interference in SAB assays.

HLA graph, a free and ready-to-use bioinformatics tool to explore anti-HLA eplets reactivity pattern.

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.

Utility of assessing CD3+ cell chimerism within the first months after allogeneic hematopoietic stem-cell transplantation for acute myeloid leukemia.

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.

Characterization of the novel HLA-C*04:450 allele by next-generation sequencing.

HLA-C*04:450 differs from HLA-C*04:01:01 by one nucleotide substitution in codon 328 in exon 7.

Characterization of the novel HLA-DRB1*04:326 allele by next generation sequencing.

HLA-DRB1*04:326 differs from HLA-DRB1*04:01:01 by one nucleotide substitution in codon 23 in exon 2.

A one-step assay for sorted CD3+ cell purity and chimerism after hematopoietic stem cell transplantation.

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 (R2 = 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.

Antibodies against HLA cross-reactivity groups: From single antigen bead assay to immunoinformatics interpretation of epitopes.

Identification of anti-human leukocyte antigen (HLA) antibodies (Abs) is based on Luminex™ technology. We used bioinformatics to (i) study the correlations of mean fluorescence intensities (MFIs) for all the possible allele pairs, and (ii) determine the degree of epitope homology between HLA antigens. Using MFI data on anti-HLA Abs from 6000 Luminex™ assays, we provide an updated overview of class I and II HLA antigen cross-reactivity in which each node corresponded to an allele and each link corresponded to a strong correlation between two alleles (Spearman's ρ > 0.8). We compared these correlations with the serological groups and the results of an epitope analysis. The strongest correlations concerned allele-specific Abs directed against the same antigen. For the HLA-A locus, the highest values of Spearman's ρ reflected broad specificity. For the HLA-B locus, graphs defined the HLA-Bw4 public epitope, and correlations between HLA-A and -B alleles were only present for beads with the same Bw4 public epitope. For the HLA-C locus, we identified two groups that differed with regard to their KIR ligand subclassification. Lastly, the HLA-DRB1 subgroups were part of a network. In the epitope analysis, Spearman's ρ was related to the number of matched epitopes within pairs of alleles. The combination of Spearman's ρ with simple, undirected graphing constitutes an effective tool for understanding routinely encountered cross-reactivity profiles. Based on this model, we have implemented an online data visualization tool available at http://cusureau.pythonanywhere.com/.

Flow cytometry crossmatching to investigate kidney-biopsy-proven, antibody-mediated rejection in patients who develop de novo donor-specific antibodies.

INTRODUCTION: The appearance of de novo donor-specific anti-human leukocyte antigen antibodies (dnDSAs) after kidney transplantation is independently associated with poor long-term allograft outcomes. The objective of the present study was to evaluate the predictive value of a flow cytometry crossmatching (FC-XM) assay after the appearance of dnDSAs related to antibody-mediated allograft rejection (ABMR) after kidney transplantation. MATERIALS AND METHODS: A total of 89 recipients with dnDSAs after transplantation were included. The crossmatching results were compared with the dnDSA profile (the mean fluorescence intensity (MFI), the complement-binding activity, and the IgG subclass profile) and the biopsy's morphological features. RESULTS: Of the 89 patients, 59 (66%) were positive in an FC-XM assay, 17 (19%) had complement-binding DSAs, 55 (62%) were positive for IgG1 and/or IgG3 in a solid phase assay, and 45 (51%) had morphological biopsy features linked to ABMR. CONCLUSION: An FC-XM assay was unable to discriminate between cases with or without ABMR on biopsy findings; it had a low positive predictive value (<70%) and a low negative positive predictive value (<42.9%), taking into account the sensitivity of our assay (limit of detection: DSAs with an MFI >3000). In this context, the height of the MFI of the dnDSAs might be enough for a high positive predictive value for ABMR and additional testing for complement binding activity can remain optional.

Analytical performance evaluation and enhancement of the ADVIA Centaur® HIV Ag/Ab Combo assay.

BACKGROUND: Fourth-generation immunoassays (such as the ADVIA Centaur® HIV Ag/Ab Combo (CHIV) assay) have improved the early diagnosis of human immunodeficiency virus (HIV), and their sensitivity and specificity usually exceed 99%. In regions with a low prevalence of HIV infection, however, the regular occurrence of false positives interferes with a medical laboratory's workflow. The additional reagent and staff costs associated with false positives can nevertheless be avoided or reduced by gaining a better knowledge of the CHIV assay's performance. OBJECTIVES/STUDY DESIGN: To improve our HIV diagnosis strategy, we retrospectively analyzed all the Centaur® CHIV assays and confirmatory tests performed at Amiens University Medical Center between 2012 and 2018. We used open-source machine learning software to process this large database, develop a predictive model, and identify a new cut-off for Centaur® CHIV index interpretation. RESULTS: A total of 56,682 HIV serological assay results were analyzed. The results of the CHIV assay were initially reactive or indeterminate for 449 samples. After p24 antigen and/or immunoblotting, there were 171 (38%) false positives and 278 (62%) confirmed true positives. The application of a cut-off of 2.12 led to reclassification of 130 of the 171 false positives as true negatives. Combining our predictive model with medical record analysis reduced the number of false positive CHIV assay results from 171 to 12. CONCLUSIONS: The efficiency of the Centaur® CHIV assay can be increased by adjusting its cut-off for positivity. This adjustment may reduce the number of unnecessary confirmatory tests and accelerate the delivery of HIV test results.

Protein biosynthesis, a target of sorafenib, interferes with the unfolded protein response (UPR) and ferroptosis in hepatocellular carcinoma cells.

Sorafenib is the first line treatment for advanced hepatocellular carcinoma (HCC). We explored its impact on the proteostasis of cancer cells, i.e. the processes that regulate the synthesis, maturation and turn-over of cellular proteins. We observed that sorafenib inhibits the production of the tumour marker alpha-foetoprotein (AFP) in two different HCC cell lines, an effect that correlated with a radical inhibition of protein biosynthesis. This effect was observed at clinically relevant concentrations of sorafenib and was not related to the effect of sorafenib on the transport of amino acids across the plasma membrane or the induction of the unfolded protein response (UPR). Instead, we observed that sorafenib inhibits translation initiation and the mechanistic target of rapamycin (mTOR) signaling cascade, as shown by the analysis of phosphorylation levels of the protein 4EBP1 (eukaryotic translation initiation factor 4E binding protein 1). We explored the consequences of this inhibition in HCC cells. We observed that overall sorafenib is a weak inducer of the UPR that can paradoxically prevent the UPR induced by tunicamycin. We also found no direct synergistic anticancer effect between sorafenib and various strategies that inhibit the UPR. In agreement with the possibility that translation inhibition might be an adaptive stress response in HCC cells, we noted that it protects cancer cell from ferroptosis, a form of oxidative necrosis. Our findings point to the modulation of protein biosynthesis and mTOR signaling as being important, yet complex determinants of the response of HCC cells to sorafenib.