HLA-DRB3*01:01:27 differs HLA-DRB3*01:01:02:01 by a single synonymous substitution in exon 2.

HLA-DQA1*01:02:32 differs HLA-DQA1*01:02:01 by a single synonymous substitution in exon 2.

HLA-C*12:459 differs HLA-C*12:02:02:01 by a single substitution in exon 2.

INTRODUCTION Human amniotic membrane (hAM) is considered to have low immunogenicity since Akle et al. reported no anti-HLA antibodies (Abs) in four volunteers after subcutaneous grafting of fresh hAM in 1981. But the sensitivity of the detection methods has significantly improved since then. Furthermore, hAM graft is a massive local input of allogenic HLA-G whose potential immunogenicity has never been assessed. The objective of this study was to look for the presence of anti-HLA class I, II, and anti HLA-G Abs at 1 and 3 months after hAM transplantation on the ocular surface. MATERIAL AND METHODS Twenty-three patients who required a hAM graft on the ocular surface (cornea and/or conjunctiva) for any indication were enrolled in this prospective clinical study. Sera were collected on the day of transplantation and at 1- and 3-months post-transplantation (M0, M1, M3). Anti-HLA class I, and II Abs were assessed using the Luminex® Single Antigen technique. Anti HLA-G Abs were assessed using an in-house ELISA-based detection assay, derived from the Monoclonal Antibody specific Immobilization of Platelet Antigen (MAIPA) assay. RESULTS Only one patient showed de novo Ab apparition after transplantation. Six patients showed a significant increase of preformed Abs MFI (>50%) during follow up although transient in one out of six. No anti-HLA-G Abs were identified before or after hAM transplantation. CONCLUSION This is the first study to investigate anti-HLA class I, II, and anti HLA-G Abs in a clinical setting after cryopreserved hAM transplantation. The main finding of this study is the minimal humoral immune response in our patients, supporting the immunological safety of the procedure and the excellent clinical tolerance reported across various indications over decades of use.

Post-transplant chimerism monitoring is a cornerstone of hematopoietic stem cell transplantation (HSCT) follow-up, providing early and clinically actionable information on engraftment, graft rejection and disease relapse. Analytical strategies have progressively evolved from STR-based assays towards more sensitive molecular approaches, including qPCR, dPCR and next-generation sequencing (NGS). Although NGS offers high analytical robustness and multiplexing capacity, its routine use remains limited by turnaround time and batch-dependent workflows, restricting its applicability in urgent clinical contexts. Third-generation sequencing (TGS), based on real-time DNA sequencing, emerges as a complementary approach combining molecular sensitivity with enhanced operational flexibility. We evaluated ONtrack (Eurobio/GenDx), the first commercial TGS-based assay for post-transplant chimerism quantification using Oxford Nanopore Technologies, currently available through an early access program. Thirty samples were analysed, including external quality assessment materials, manufacturer controls and clinical specimens from three transplanted patients. Sample types included whole blood, bone marrow and CD3+-sorted cell fractions. ONtrack results were compared with routinely used methods (qPCR, dPCR and NGS). ONtrack demonstrated excellent analytical performance, with strong correlations to qPCR (R2 = 0.955), dPCR (R2 = 0.990) and NGS (R2 = 0.998). Mixed chimerism was reliably detected down to 0.5%, and Bland-Altman analyses confirmed good agreement across methods. Z-score analysis showed satisfactory performance for all evaluated samples. Notably, the complete analytical workflow could be completed in approximately 3 h for a single sample, with fully automated data processing and integrated quality controls. Beyond analytical accuracy, ONtrack offers high operational versatility, enabling both urgent single-sample testing and batch analyses using a standardised workflow. Although the limited number of markers may restrict detection of ultra-low microchimerism, TGS represents a valuable complementary solution for early post-transplant monitoring, particularly in decentralised laboratory settings.

Objectives: The aim of this study was to assess the HLA alloreactivity of cold-stored saphenous vein allografts (CSVAs) by identifying the production of HLA donor-specific antibodies (DSAs) in the recipient. The secondary objective was to evaluate CSVA rejection-related complications, such as CSVA thrombosis and/or aneurysmal degeneration in the recipient. Methods: This was a single-center, prospective, experimental before-and-after study which included participants undergoing CSVA placement, either to create a vascular access (VA) for hemodialysis or to create a lower limb arterial bypass. On Day 1, before CSVA placement, total blood samples were taken for HLA typing by sequence-specific primers (SSPs) and anti-HLA antibody detection using a Luminex assay. One month after CSVA placement, a second blood sample was taken to assess the appearance of donor-specific antibodies or an increase in the level of anti-HLA antibodies. Patency of the CSVA and potential aneurysmal degeneration were evaluated at 3 and 6 months with a Doppler ultrasound checkup. Results: From September 2022 to November 2023, 45 patients were included (30 men, 67%; mean age: 71 ± 12 years). One month after CSVA placement, no appearance of de novo anti-HLA antibody was detected in anti-HLA antibody-negative patients at inclusion (n = 28). Among the patients who already had anti-HLA antibodies at inclusion (n = 17), no increase in anti-HLA antibody levels or appearance of de novo anti-HLA antibodies was detected. Conclusions: This prospective study evaluating the immunogenicity of CSVAs through the appearance of anti-HLA antibodies one month after placement demonstrates that they do not seem to induce any HLA alloreactivity. Therefore, they may be used without the risk of HLA immunization in patients awaiting organ transplantation.

Human amniotic membrane (hAM) is a biocompatible scaffold with suitable mechanical properties (permeability, stability, elasticity, flexibility, resorbability and transparency), that is rich in stem cells and growth factors and is used for tissue repair and regenerative processes. It is routinely used in ocular surgery as it promotes epithelialization, supports cell adhesion and proliferation, and enhances wound healing. It exhibits both immunomodulatory and immunoregulatory properties, contributing to its anti-inflammatory and low immunogenicity risk profile.hAM transplantation is considered unlikely to trigger an immune response against human leukocyte antigens (HLA) and is generally regarded as minimally or non-immunogenic. This conclusion is primarily based on clinical observations, and a single study published 40 years ago that investigated subcutaneous hAM grafting—a procedure that does not reflect current clinical applications.Since then, there has been no laboratory evidence that hAM transplantation is immunologically safe. Notably, the production of anti-HLA antibodies following hAM transplantation has not been investigated, despite the tremendous improvements in the sensitivity of detection techniques. The recent application of hAM to more vascularized areas could raise concerns about potential immune responses.In this perspective paper, we summarize the limited and sometimes contradictory data regarding hAM immunogenicity. Then, we describe the methods we use to assess whether immune responses are triggered after hAM grafting in current clinical indications.

HLA-A*29:198 differs from the HLA-A* 29:02:01:01 allele by one nucleotide substitution in codon 127 in exon 3.