Design, cohort profile and comparison of the KTD-Innov study: a prospective multidimensional biomarker validation study in kidney allograft rejection.

There is an unmet need for robust and clinically validated biomarkers of kidney allograft rejection. Here we present the KTD-Innov study (ClinicalTrials.gov, NCT03582436), an unselected deeply phenotyped cohort of kidney transplant recipients with a holistic approach to validate the clinical utility of precision diagnostic biomarkers. In 2018-2019, we prospectively enrolled consecutive adult patients who received a kidney allograft at seven French centers and followed them for a year. We performed multimodal phenotyping at follow-up visits, by collecting clinical, biological, immunological, and histological parameters, and analyzing a panel of 147 blood, urinary and kidney tissue biomarkers. The primary outcome was allograft rejection, assessed at each visit according to the international Banff 2019 classification. We evaluated the representativeness of participants by comparing them with patients from French, European, and American transplant programs transplanted during the same period. A total of 733 kidney transplant recipients (64.1% male and 35.9% female) were included during the study. The median follow-up after transplantation was 12.3 months (interquartile range, 11.9-13.1 months). The cumulative incidence of rejection was 9.7% at one year post-transplant. We developed a distributed and secured data repository in compliance with the general data protection regulation. We established a multimodal biomarker biobank of 16,736 samples, including 9331 blood, 4425 urinary and 2980 kidney tissue samples, managed and secured in a collaborative network involving 7 clinical centers, 4 analytical platforms and 2 industrial partners. Patients' characteristics, immune profiles and treatments closely resembled those of 41,238 French, European and American kidney transplant recipients. The KTD-Innov study is a unique holistic and multidimensional biomarker validation cohort of kidney transplant recipients representative of the real-world transplant population. Future findings from this cohort are likely to be robust and generalizable.

Preclinical evaluation of a semi-automated and rapid commercial electrophoresis assay for von Willebrand factor multimers.

BACKGROUND: The von Willebrand factor (VWF) multimer test is required to correctly subtype qualitative type 2 von Willebrand disease (VWD). The current VWF multimer assays are difficult, nonstandardized, and time-consuming. The purpose of this study was to evaluate the clinical utility of the commercial VWF multimer kit by Sebia (Lisses, France), an electrophoresis technique yielding same-day results. METHODS: Ten healthy volunteer plasma samples, in-house reference plasma (IRP) and commercial normal plasma (CNP) samples, 10 plasma samples from patients with a known VWD type, 1 hemophilia A plasma sample, and 7 external quality assurance (EQA) samples were analyzed using the commercial VWF multimer kit. Additional coagulation testing included measurements of VWF antigen (VWF:Ag), VWF activity (VWF:Ac), and FVIII activity (FVIII:C). RESULTS: The CNP results revealed a relative loss of the highest molecular weight multimers; therefore, IRP was preferred as the reference sample. The interpretations of 10 patients with a known VWD type could be successfully reproduced and agreed with previous VWF multimer results. In all EQA surveys, the multimer results and final VWD diagnosis agreed with expert opinion. CONCLUSIONS: The VWF multimer assay by Sebia is easy to perform and can be successfully implemented in any clinical laboratory for second-stage evaluation of VWD. The resolution power of multimer distribution is adequate to correctly classify VWD types 1, 2A, 2B, and 3.

Isatuximab-Specific Immunofixation Electrophoresis Assay to Remove Interference in Serum M-Protein Measurement in Patients with Multiple Myeloma.

BACKGROUND: Isatuximab, an IgG-kappa (IgGκ) anti-cluster of differentiation 38 (CD38) monoclonal antibody approved for use in patients with relapsed or refractory multiple myeloma (MM), can potentially interfere with the visualization of endogenous monoclonal protein (M-protein) on standard immunofixation electrophoresis (IFE) and lead to inaccurate classification of a patient's response to therapy. The Hydrashift 2/4 isatuximab IFE assay (Hydrashift isatuximab assay) removes isatuximab interference from IFE. Using samples from patients enrolled in clinical trials of isatuximab-based therapy for MM, we demonstrate how the Hydrashift isatuximab assay improves the ability to detect residual M-protein and offer recommendations for when the assay is most useful. METHODS: Samples from 141 patients with a variety of known M-protein isotypes were selected and analyzed by standard IFE and the Hydrashift isatuximab assay. A positive control containing isatuximab was run on every standard IFE and Hydrashift gel. RESULTS: The Hydrashift isatuximab assay reliably shifted the migration of isatuximab in patient samples. Standard IFE was adequate for determining 104 patients' M-protein status, and the Hydrashift isatuximab assay confirmed these results. In samples from 37 patients with a history of IgGκ MM and a single IgGκ band visible on standard IFE near the isatuximab migration site, the Hydrashift isatuximab assay was able to separate isatuximab from endogenous M-protein, identifying residual M-protein in 17 samples and preventing false-positive interpretations of standard IFE in 20 samples. CONCLUSIONS: The Hydrashift isatuximab assay is most useful in patients with known IgGκ MM when a single IgGκ band appears near the isatuximab migration site on standard IFE during isatuximab-based therapy. CLINICALTRIALS.GOV REGISTRATION NUMBERS: NCT03275285 and NCT03319667.

Evaluation of a semi-automatic isoelectric focusing method for apolipoprotein E phenotyping.

A qualitative, semi-automatized method for apolipoprotein E (apoE) phenotyping by isoelectric focusing method has been evaluated on 40 serum samples from patients previously genotyped for apoE, especially as regards concordance with genotyping, but also repeatability and reproducibility of the method, and sample storage. Total concordance with genotyping and good precision criteria, together with its practicability and requirement of a little sample volume, lead to conclude to the usefulness of this method to help clinicians in the diagnosis of dyslipidemic and neurodegenerative diseases.

Development of new quantitative mass spectrometry and semi-automatic isofocusing methods for the determination of Apolipoprotein E typing.

BACKGROUND: Apolipoprotein E (Apo E) is a 36 Kda glycoprotein involved in lipid transport. It exists in 3 major isoforms: E2, E3 and E4. ApoE status is known to be a major risk factor for late-onset Alzheimer's and cardiovascular diseases. Genotyping is commonly used to obtain ApoE status but can show technical issues with ambiguous determinations. Phenotyping can be an alternative, not requiring genetic material. We evaluated the ability to accurately type ApoE isoforms by 2 phenotyping tests in comparison with genotyping. METHODS: Two phenotyping techniques were used: (1) LC-MS/MS detection of 4 ApoE specific peptides (6490 Agilent triple quadripole): After its denaturation, serum was either reduced and alkylated, or only diluted, and then trypsin digested. Before analysis, desalting, evaporation and resuspension were performed. (2) Isoelectric focusing and immunoprecipitation: serum samples were neuraminidase digested, delipidated and electrophoresed on Hydragel ApoE (Sebia agarose gel) using Hydrasys 2 Scan instrument (Sebia, Lisses, France). ApoE isoforms bands were directly immunofixed in the gel using a polyclonal anti human ApoE antibody. Then, incubation of the gel with HRP secondary antibody followed by TTF1/TTF2 substrate allowed the visualization of ApoE bands. The results of the two techniques were compared to genotyping. RESULTS: Sera from 35 patients previously genotyped were analyzed with the 2 phenotyping techniques. 100% concordance between both phenotyping assays was obtained for the tested phenotypes (E2/E2, E2/E3, E2/E4, E3/E3, E3/E4, E4/E4). When compared to genotyping, 3 samples were discordant. After reanalyzing them by both phenotyping tests and DNA sequencing, 2/3 discrepancies were confirmed. Those can be explained by variants or rare ApoE alleles or by unidentified technical issues. 102 additional samples were then tested on LC-MS/MS only and compared to genotyping. The data showed 100% concordance. CONCLUSION: Our 2 phenotyping methods represent a valuable alternative to genotyping. LC-MS/MS has the advantage of being fully specific, with identification of the different isoforms and can be considered as a reference method. Sebia isofocusing technique was concordant with LC-MS/MS. Plus, it is a rapid, semi-automated assay that can be easily implemented in clinical laboratories.

[Development of a laboratory test on dried blood spots for facilitating early diagnosis of alpha-1-antitrypsin deficiency]. / Diagnostic biologique du déficit en alpha-1-antitrypsine : développement d'un test simplifié sur goutte de sang recueillie sur papier-filtre.

Alpha- 1-antitrypsin (A1AT) deficiency is a hereditary autosomal codominant genetic disorder resulting in low circulating levels of A1AT and leading to lung and/or liver disease. It remains underdiagnosed and only 5 to 10% of PIZZ patients, the most common form of severe A1AT deficiency, would be actually identified in France. Facilitating early diagnosis of A1AT deficiency would allow a better management of this disease; therefore we have developed and standardized in three laboratories involved in this study, a diagnostic test on dried blood spots (DBS) including quantitative A1AT measurement, phenotyping by IEF electrophoresis and, if necessary, genotyping by SERPINA1 gene sequencing. We performed a quantitative assay on 90 DBS samples by immunoturbidimetric or immunonephelometric methods. We demonstrated that both methods were suitable for this type of sampling and the results obtained were highly correlated (R(2)>0.9) between the three laboratories: for a target value of 1.00 g/L, the results obtained from the three laboratories were between 1.00 and 1.02 g/L. Phenotyping and genotyping were performed under redefined operating conditions and adapted to the analysis of DBS samples. The results were comparable with those obtained for venous blood samples. Following this work, it becomes possible to provide pulmonologists with a reliable kit to perform a capillary blood sampling on filter paper which would allow a large-scale screening of A1AT deficiency in the population particularly affected by this genetic condition.