An optimized procedure for Luminex-based human platelet antigen-specific antibody screening and identification (PakLx assay) with a cost-effective approach and improved sensitivity.

BACKGROUND AND OBJECTIVES: Detection of anti-platelet antibodies is required for the diagnosis of foetal/neonatal alloimmune thrombocytopaenia. The most commonly used methods for anti-platelet antibody detection are the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) and the Luminex bead assay (PakLx). However, for economic reasons, the use of the PakLx assay is limited. MATERIALS AND METHODS: In the present study, we evaluated the performance of an optimized protocol based on a half-volume of PakLx reagents. We compared two alternative procedures: one with a half-volume of all components including patient samples, and another based on a half-volume of reagents but a standard volume of patient sample. RESULTS: Our results obtained with a panel of 67 samples demonstrate improved sensitivity when using a standard sample volume. CONCLUSION: In the event of an inconclusive result with this optimized protocol (e.g., incomplete panel of positive Luminex beads), we recommend testing the sample with an alternative protocol (e.g., MAIPA or the original PakLx protocol).

Donor-targeted serotherapy as a rescue therapy for steroid-resistant acute GVHD after HLA-mismatched kidney transplantation.

Acute graft-versus-host disease (GVHD) is a rare but frequently lethal complication after solid organ transplantation. GVHD occurs in unduly immunocompromised hosts but requires the escalation of immunosuppression, which does not discriminate between host and donor cells. In contrast, donor-targeted therapy would ideally mitigate graft-versus-host reactivity while sparing recipient immune functions. We report two children with end-stage renal disease and severe primary immune deficiency (Schimke syndrome) who developed severe steroid-resistant acute GVHD along with full and sustained donor T cell chimerism after isolated kidney transplantation. Facing a therapeutic dead end, we used a novel strategy based on the adoptive transfer of anti-HLA donor-specific antibodies (DSAs) through the transfusion of highly selected plasma. After approval by the appropriate regulatory authority, an urgent nationwide search was launched among more than 3800 registered blood donors with known anti-HLA sensitization. Adoptively transferred DSAs bound to and selectively depleted circulating donor T cells. The administration of DSA-rich plasma was well tolerated and notably did not induce antibody-mediated rejection of the renal allografts. Acute GVHD symptoms promptly resolved in one child. This report provides a proof of concept for a highly targeted novel therapeutic approach for solid organ transplantation-associated GVHD.

HLA-DRB3/4/5 mismatches are associated with increased risk of acute GVHD in 10/10 matched unrelated donor hematopoietic cell transplantation.

Matching for HLA-A, -B, -C, and -DRB1 loci (8/8 match) is currently the gold standard for unrelated donor hematopoietic cell transplantation (HCT). In Europe, patients are also matched at the HLA-DQB1 loci (10/10 match). However, there is increasing evidence that matching at HLA-DRB3/4/5 loci may help to lower transplant-related morbidity and mortality. We therefore investigated the impact of HLA-DRB3/4/5 mismatches on outcomes in 1975 patients who received a first 10/10 matched unrelated donor (MUD) HCT in France from 2000 to 2012 for a hematological malignancy. High-resolution typing was performed at HLA-A, -B, -C, -DRB1, -DQB1, -DPB1, and -DRB3/4/5 loci for all donor/recipient pairs. Compared with DRB3/4/5-matched pairs, patients who received a MUD HCT from a DRB3/4/5 mismatched donor had a significantly increased risk of grade II-IV acute graft-versus-host disease (aGVHD) (Adjusted Hazard Ratio (HR) 1.43 (1.07 to 1.90)) associated with lower graft-versus-host disease-free and relapse-free survival (GRFS) (Adjusted HR 1.20 (1.02 to 1.42)). Conversely, we observed no differences in terms of chronic GVHD, nonrelapse mortality, relapse and overall survival. However, we believe that patients stand to benefit from DRB3/4/5 loci being considered for unrelated donor selection to improve GRFS and then quality of life after unrelated HCT.

Involvement of circulating soluble HLA-G after liver transplantation in the low immunogenicity of hepatic allograft.

Graft rejection is a critical risk in solid-organ transplantation. To decrease such risk, an understanding of the factors involved in low immunogenicity of liver allografts could potentially make it possible to transfer this tolerogenic property to other transplanted organs. HLA-G, a natural physiological molecule belonging to the Human Leukocyte Antigen class (HLA) Ib family that induces tolerance, is associated with fewer rejections in solid-organ transplantation. In contrast to HLA-G, HLA antigen incompatibilities between donor and recipient can lead to rejection, except in liver transplantation. We compared HLA-G plasma levels and the presence of anti-HLA antibodies before and after LT to understand the low immunogenicity of the liver. We conducted a large prospective study that included 118 patients on HLA-G plasma levels during a 12-month follow-up and compared them to the status of anti-HLA antibodies. HLA-G plasma levels were evaluated by ELISA at seven defined pre- and post-LT time points. HLA-G plasma levels were stable over time pre-LT and were not associated with patient characteristics. The level increased until the third month post-LT, before decreasing to a level comparable to that of the pre-LT period at one year of follow-up. Such evolution was independent of biological markers and immunosuppressive treatment, except with glucocorticoids. An HLA-G plasma level ≤ 50 ng/ml on day 8 after LT was significantly associated with a higher rejection risk. We also observed a higher percentage of rejection in the presence of donor specific anti-HLA antibodies (DSA) and an association between the increase in HLA-G plasma levels at three months and the absence of DSA. The low immunogenicity of liver allografts could be related to early elevated levels of HLA-G, which lead, in turn, to a decrease in anti-HLA antibodies, opening potential new therapeutic strategies using synthetic HLA-G proteins.

Characterization of the novel HLA-DQA1*01:39 allele by next-generation sequencing.

DQA1*01:39 differs from DQA1*01:02:01:01 by one nucleotide substitution at position 49 in exon 1.

Characterization of the novel HLA-B*08:67:02N allele by next-generation sequencing.

HLA-B*08:67:02N differs from B*08:01:01:01 by one nucleotide substitution at position 224 in exon 2.

Characterization of the novel HLA-DQA1*03:11 allele by next-generation sequencing.

DQA1*03:11 differs from DQA1*03:03:01:01 by one nucleotide substitution at position 664 in exon 4.

Characterization of the novel HLA-DQA1*05:18 allele by next-generation sequencing.

DQA1*05:18 differs from DQA1*05:01:01:03 by one nucleotide substitution at position 94 in exon 2.

Characterization of the novel HLA-B*18:181 allele by next-generation sequencing.

B*18:181 differs from B*18:01:01:02 by one nucleotide substitution at position 1043 in exon 6.

Characterization of the novel HLA-B*51:296 allele by next-generation sequencing.

B*51:296 differs from B*51:01:01:01 by one nucleotide substitution at position 1030 in exon 6.

Characterization of the novel HLA-B*40:450 allele by next-generation sequencing.

B*40:450 differs from B*40:01:02:01 by one nucleotide substitution at position 5 in exon 1.

Characterization of the novel HLA-DRB1*08:97 allele by next-generation sequencing.

DRB1*08:97 differs from DRB1*08:03:02:01 by one nucleotide substitution at position 485 in exon 3.

Characterization of the novel HLA-B*44:452 allele by next-generation sequencing.

B*44:452 differs from B*44:02:01:01 by one nucleotide substitution at position 527 in exon 3.

Characterization of the novel HLA-DQB1*02:162N allele by next-generation sequencing.

DQB1*02:162N differs from DQB1*02:02:01:01 by one nucleotide substitution at position 276 in exon 2.

Characterization of the novel HLA-DQB1*03:417 allele by next-generation sequencing.

DQB1*03:417 differs from DQB1*03:01:01:01 by one nucleotide substitution at position 179 in exon 2.

Characterization of the novel HLA-B*44:476 allele by next-generation sequencing.

B*44:476 differs from B*44:03:01:01 by one nucleotide substitution at position 934 in exon 5.