Clinical relevance of cell-free DNA quantification and qualification during the first month after lung transplantation

BackgroundMany studies have reported the relevance of donor-derived cfDNA (dd-cfDNA) after lung transplantation (LTx) to diagnose and monitor acute rejection (AR) or chronic rejection or infection (INF). However, the analysis of cfDNA fragment size has not been studied. The aim of this study was to determine the clinical relevance of dd-cfDNA and cfDNA size profiles in events (AR and INF) during the first month after LTx.MethodsThis prospective, single-center study includes 62 LTx recipients at the Marseille Nord Hospital, France. Total cfDNA quantification was performed by fluorimetry and digital PCR, dd-cfDNA by NGS (AlloSeq cfDNA-CareDX®), and the size profile by BIABooster (Adelis®). A bronchoalveolar lavage and transbronchial biopsies at D30 established the following groups: not-injured and injured graft (AR, INF, or AR+INF).ResultsQuantification of total cfDNA was not correlated with the patient’s status at D30. The percentage of dd-cfDNA was significantly higher for injured graft patients at D30 (p=0.0004). A threshold of 1.72% of dd-cfDNA correctly classified the not-injured graft patients (negative predictive value of 91.4%). Among recipients with dd-cfDNA >1.72%, the quantification of small sizes (80-120bp) >3.70% identified the INF with high performance (specificity and positive predictive value of 100%).ConclusionWith the aim of considering cfDNA as a polyvalent non-invasive biomarker in transplantation, an algorithm combining the quantification of dd-cfDNA and small sizes of DNA may significantly classify the different types of allograft injuries

Qualification of cfDNA in organ transplantation and blood transfusion : impact on immune tolerance

Outre leurs intérêts dans le diagnostic prénatal non invasif et l’oncologie, les acides nucléiques extracellulaires (ADN cell-free, ADNcf) ont récemment été étudiés dans les transplantations d’organes. En effet, la proportion d’ADNcf issu du donneur (ADNcfdd) permettrait de prédire, diagnostiquer et suivre un rejet du greffon. De même, l’ADNcf semble avoir des capacités inflammatoires et immunomodulatrices, lors des rejets d’organes ou lors de son apport par des transfusions de produits sanguins labiles (PSL).Aussi, l’objectif de cette thèse est de caractériser quantitativement et qualitativement l’ADNcf présent lors des processus inflammatoires après transplantation pulmonaire, par des technologies innovantes.Une première partie de ce travail a permis la mise en place d’un processus préanalytique et analytique robuste pour l’étude de l’ADNcf.La deuxième partie a confirmé, après avoir déterminé des valeurs normales d’ADNcf dans une population saine, que l’ADNcfdd est un marqueur non invasif prometteur du rejet aigu des greffons pulmonaires à partir d’une cohorte prospective.La dernière partie a consisté à analyser le rôle inflammatoire de l’ADNcf dans le contexte de rejet aigu mais aussi lorsque des PSL sont transfusés.En final, ce travail a confirmé que dans des conditions d’analyses maitrisées, l’ADNcf peut être utilisé comme biomarqueur non invasif du suivi de certaines transplantations d’organes. Une meilleure connaissance physiopathologique de l’ADNcf au cours des manifestations de rejet pourrait permettre de nouvelles thérapies ciblées, notamment pour limiter les processus inflammatoires et immunitaires.In addition to their relevance in non-invasive prenatal diagnosis and oncology, extracellular nucleic acids (cell-free DNA, cfDNA) have recently been studied in organ transplantation. In fact, the proportion of donor-derived cfDNA (ddcDNA) could predict, diagnose and monitor graft rejection. Also, cfDNA seems to have inflammatory and immunomodulatory capacities, during organ rejection or during its injection by labile blood products transfusions.The objective of this work is to characterize quantitatively and qualitatively the cfDNA present during inflammatory processes after lung transplantation, using innovative technologies.The first part of this work allowed the implementation of a robust pre-analytical and analytical process for the study of cfDNA.The second part confirmed, after establishing normal values of cfDNA in a healthy population, that cfDNA is a promising non-invasive marker of acute lung graft rejection from a prospective cohort.The last part consisted in the analysis of the inflammatory role of cfDNA in the context of acute rejection but also when labile blood products are transfused.In the end, this work confirmed that under controlled analysis conditions, cfDNA can be used as a non-invasive biomarker for the follow-up of certain organ transplants. A better pathophysiological knowledge of cfDNA during rejection events could allow new targeted therapies, in particular to limit inflammatory and immune processes

Virus de l’hépatite E : implications en transfusion sanguine

Le virus de l’hépatite E (VHE) se transmet principalement par voie féco-orale dans les pays en voie de développement (génotypes 1 et 2) et par voie alimentaire dans les pays développés (génotypes 3 et 4). En 2004, la preuve d’une transmission par l’utilisation de produits sanguins labiles a été établie. Depuis de nombreux cas sont décrits à travers le monde. Les objectifs de cette thèse sont de préciser les avancées concernant le risque transfusionnel du VHE en 2017 et de déterminer la cinétique des anticorps anti-VHE chez des donneurs de sang (population asymptomatique et immunocompétente). Ce travail est le début d’une étude à long terme de suivi de donneurs de sang. Nous disposons d’échantillons des dons de plasma français testés positivement à l’ARN VHE. Grâce à des techniques sérologiques commerciales nous testons les IgM et IgG anti-VHE de chaque donneur. Nous quantifions également les IgG anti-VHE après avoir validé une technique de sérologie quantitative. Nous montrons que les donneurs de plasma testés positivement à l’ARN VHE étaient en début d’infection (IgM et IgG négatives) pour 80% des donneurs. De plus, nous obtenons une hétérogénéité de profil sérologique. En effet, à côté des cinétiques classiques de marqueurs infectieux, nous observons un cas d’absence de réponse immune IgG anti-VHE mais aussi deux cas évocateurs de réinfection par le VHE. Cette étude a également permis de préciser le délai d’apparition des anticorps anti-VHE après un don positif pour l’ARN VHE et la durée de leur persistance

Characterization of the novel HLA‐B*07:482 allele using next‐generation sequencing methods

HLA‐B*07:482 differs from HLA‐B*07:02:01:01 allele by one nucleotide substitution in codon 285 in exon 5

Characterization of the novel HLA‐A*33:246 allele using next‐generation sequencing

HLA‐A*33:246 differs from HLA‐A*33:01:01:01 allele by one nucleotide substitution in codon 135 in exon 3

Characterization of 43 novel HLA-H alleles.

Forty‐three novel HLA‐H alleles, including 32 identified in cohorts of French donors for Hematopoietic Stem Cell Transplantation, have been characterized by Next‐Generation Sequencing (NGS) using a long range PCR approach. A phylogenetic study confirms three main HLA‐H clades

The novel HLA-DQA1*03:01:12 allele identified using next-generation sequencing in a Melanesian individual.

HLA‐DQA1*03:01:12 differs from HLA‐DQA1*03:01:01:01 by one nucleotide substitution in codon 21 in exon 2

Association study between HLA-A, -B, -C, -DRB1 alleles and Psoriatic arthritis in southern France

International audiencePsoriatic arthritis (PsA) is a type of inflammatory arthritis associated with psoriasis. HLA association studies performed in northern Europe, comparing patients with control populations, have shown that the highest risk for PsA is carried by HLA-C*06, HLA-B*57 and HLA-B*27 alleles. This retrospective association study compared HLA-A, -B, -C, and -DR alleles of 500 patients from southern France, who fulfilled the CASPAR criteria for Psoriatic Arthritis (PsA), with 2346 controls from healthy blood donors, using the chi-square test. We classified PsA patients into three different subgroups according to disease: purely axial, purely peripheral and combined axial and peripheral. The 'axial' subgroup was associated with HLA-B*27 (OR = 16.3, p = 2.7 × 10-28) and its haplotypes: HLA- B*27-C*01 (OR = 12.4, p = 1.7 × 10-12) and HLA-B*27-C*02 (OR = 8.7, p = 10 × 10-9). The 'axial and peripheral' and the 'peripheral' subgroups were associated with HLA-C*06 (respectively OR = 1.5, p = 3.6 × 10-10 and OR = 2.4, p = 3.6 × 10-12) and its haplotypes HLA-C*06-B*13 (respectively OR = 2.4, p = 1.2 × 10-6 and OR = 2.8, p = 6.4 × 10-11). This association study on a southern French PsA cohort identifies HLA-C*06 as a marker for peripheral PsA and HLA-B*27 as a marker for purely axial PsA

Therapeutic efficacy of platelet transfusion treated with amotosalen/UVA pathogen inactivation technology (INTERCEPTTM Blood System) in acute myeloid leukemia patients undergoing chemotherapy with curative intent: a single center experience.

The INTERCEPTTM Blood System (Intercept Blood System, Cerus Europe BV, Amersfoort, the Netherlands) has been used to reduce or inactivate pathogen load in platelet concentrates in France for three years

Evaluation of a new complement‐dependent lymphocytotoxicity cross match method using an automated cell counter, the NucleoCounter ® NC ‐3000™

Complement-dependent lymphocytotoxicity cross match (CDC-XM) is the ultimate test of donor/recipient compatibility prior to organ transplantation. This test is based on cell viability, evaluated under fluorescence microscopy by an operator after proper staining. The determination of the positivity threshold may vary depending on the operator. We developed a new method in which the final step of determining cell viability is automated using the NC-3000™ (Chemometec®), an image cytometer able to precisely determine the percentage of dead/live cells in a suspension. After T and B donor cells isolation by negative selection, complement-dependent lysis was performed in macrovolumes in a PCR plate. Then, cell viability was measured by the NC-3000™. The sensitivity and routine CDC-XM results of this new method were compared to those of CDC-XM reference method using Terasaki plates. The sensitivity of CDC-XM expressed in the ASHI scoring system of this method was similar to the reference method results for a dilution range of the positive controls. Similarly, the results of the new method were comparable in a clinical situation to those obtained with the reference method after a study of 10 cross-matches, of which 5 cross-matches with DSA were positive and five cross-matches without DSA were negative. Moreover, ASHI scores were similar to those obtained using the reference method, and the mortality percentage was reproducible (CV < 15%). The assessment of cell viability by the NC-3000™ is easy to perform and highly reproducible but requires CDC-XM to be performed by the macrovolume method. The determination of a precise percentage of viability/mortality by the automation excludes operator variability and allows a better understanding of results close to the decision threshold