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).

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.

[Biobanking in a histocompatibility laboratory. Guidelines from the Francophone Society of Histocompatibility and Immunogenetics (SFHI)]. / Critères de conservation des échantillons biologiques dans un laboratoire d'histocompatibilité Les recommandations de la Société francophone d'histocompatibilité et d'immunogénétique (SFHI).

Histocompatibility laboratories perform the biological analyses linked related to organ transplant, hematopoietic stem cells transplant, some immune dysfunction diseases and immuno-allergy after therapeutic treatment. Most of these analyses are prospectively or retrospectively performed on sera and DNA. The Société Francophone d'Histocompatibilité et d'Immunogénétique (SFHI) has made some recommendations in order to define storage conditions and storage lifetime of the samples required in a histocompatibility laboratory. These recommendations have been drawn up by a working group of ten biologists. They have been established on literature review and data from method validation, which has been already performed within French laboratories (collected through a national questionnaire sent to participant laboratories). The recommendations made by the SFHI for the storage of samples for immunogenetics analyses facilitate the harmonization of practices among histocompatibility laboratories.

Prospective Measures of Adherence by Questionnaire, Low Immunosuppression and Graft Outcome in Kidney Transplantation.

BACKGROUND: Non-adherence with immunosuppressant medication (MNA) fosters development of de novo donor-specific antibodies (dnDSA), rejection, and graft failure (GF) in kidney transplant recipients (KTRs). However, there is no simple tool to assess MNA, prospectively. The goal was to monitor MNA and analyze its predictive value for dnDSA generation, acute rejection and GF. METHODS: We enrolled 301 KTRs in a multicentric French study. MNA was assessed prospectively at 3, 6, 12, and 24 months (M) post-KT, using the Morisky scale. We investigated the association between MNA and occurrence of dnDSA at year 2 post transplantation, using logistic regression models and the association between MNA and rejection or graft failure, using Cox multivariable models. RESULTS: The initial percentage of MNA patients was 17.7%, increasing to 34.6% at 24 months. Nineteen patients (8.4%) developed dnDSA 2 to 3 years after KT. After adjustment for recipient age, HLA sensitization, HLA mismatches, and maintenance treatment, MNA was associated neither with dnDSA occurrence, nor acute rejection. Only cyclosporine use and calcineurin inhibitor (CNI) withdrawal were strongly associated with dnDSA and rejection. With a median follow-up of 8.9 years, GF occurred in 87 patients (29.0%). After adjustment for recipient and donor age, CNI trough level, dnDSA, and rejection, MNA was not associated with GF. The only parameters associated with GF were dnDSA occurrence, and acute rejection. CONCLUSIONS: Prospective serial monitoring of MNA using the Morisky scale does not predict dnDSA occurrence, rejection or GF in KTRs. In contrast, cyclosporine and CNI withdrawal induce dnDSA and rejection, which lead to GF.

Characterization of the novel HLA-DQA1*05:13 allele by next-generation sequencing.

DQA1*05:13 differs from DQA1*05:05:01:04 by one nucleotide substitution at position 37 in exon 1.

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*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*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*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.