Evaluation of Next-Generation Sequencing and Crystal Digital PCR for Chimerism Monitoring of Post-Allogeneic Hematopoietic Stem Cell Transplantation.

Hematopoietic stem cell transplantation (HSCT) is a curative treatment for most hematologic diseases. To evaluate the level of donor engraftment, chimerism must be carefully monitored after HSCT. Short tandem repeats, quantitative PCR (qPCR), and, more recently, digital PCR (dPCR) are widely used to determine the proportions of donor and recipient cells after HSCT. The screening and quantification of chimerism have been evaluated by 2 new methods: a ready-to-use next-generation sequencing (NGS)-based method using the Devyser ChimerismNGS kit and an original combination of the Stilla crystal digital PCR (cdPCR) platform with 3-color multiplexing capacity using GenDX KMRtrack reagents. The genotyping of 4 HSCT pairs by cdPCR using 11 triplex mixes of the GenDX KMRtype kit was consistent at 98.8% with qPCR. Informative samples (n = 20) from 6 donor-recipient pairs and 1 external proficiency test demonstrated the reliability of the results (0.1% to 50%) for the 2 methods. The methods are also highly sensitive (0.1%) and accurate. The chimerism values of the 2 methods are correlated and concordant with those of the reference methods. In addition, the ADVYSER software (Devyser) is user-friendly and well adapted to chimerism monitoring. In conclusion, these 2 innovative methods are easy to perform and user-friendly in all molecular, hematology, and immunogenetic laboratories and allow the genotyping and monitoring of chimerism with high performance and sensitivity.

Pulmonary Alveolar Microlithiasis in Children Less than 5 Years of Age.

OBJECTIVE: To collect all published cases up to January 2019 of pulmonary alveolar microlithiasis (PAM) in patients age 5 years and under and to compare their characteristics with those of the 1022 cases in the most recent all-age cohort published in 2015. STUDY DESIGN: We identified 28 cases of PAM worldwide in children age 5 years and under, accounting for only 2%-3% of all cases. RESULTS: Children seem more frequently symptomatic, notably with more cough and severe acute respiratory failure, but had no reported extrapulmonary manifestation. Children with PAM evidenced less typical radiologic findings, with frequent ground glass opacities not reported in adult cases and milder calcifications as less frequent, smaller, and mainly restricted to the lower lobes. CONCLUSIONS: PAM remains an uncommon diagnosis in young children, as symptoms and radiologic findings are less specific. Physicians should be aware to look for calcifications in chest computed tomography at mediastinal window and avoid elution of the bronchoalveolar lavage to find microliths. Collecting longitudinal data through an international registry would help in characterizing PAM to predict disease progression and plan lung transplantation.

[Haploidentical hematopoietic stem cell transplantation: Guidelines from the Francophone society of marrow transplantation and cellular therapy (SFGM-TC)]. / Greffe de cellules-souches hématopoïétiques haplo-identiques : recommandations de la Société francophone de greffe de moelle et de thérapie cellulaire (SFGM-TC).

Haploidentical hematopoietic stem cell transplantation (HSCT) is being increasingly used due to improvement of the transplantation procedures allowing a reduction of graft-versus-host-disease (GVHD) and of transplant-related mortality (TRM). Such improvements have been particularly observed after administration of T-replete HSCT graft associated to an in vivo T cell depletion by the administration of high-doses of cyclophosphamide (HD-Cy) after transplantation. Here, we have analyzed the results of haplo-identical T replete HSC transplants, in particular, when performed with post-transplant HD-Cy in order to provide recommendations for the clinical practice. Criteria of choice for a haploidentical donor by priority order are absence of donor-specific antibodies (DSA) and to prioritize: CMV seronegative recipient/donor couples, ABO matching in case of deserythrocytation, male donor for a male recipient, the youngest donor. There is no clear argument in favor of the use of bone marrow versus peripheral blood stem cells (PBSC) after non myeloablative conditioning regimen, while after ablative conditioning PBSC seem to be associated with higher risks of GVHD without obvious impact on survival. Results of haploidentical HSCT, confirmed by several groups, are interesting in lymphomas (in particular Hodgkin disease) and for acute leukemia. Outcomes of patients rely on age, disease status at transplant and conditioning intensity. At equivalent disease risk, results of haploidentical HSCT seem comparable to those of HLA matched HSCT, raising the question of the classification of such transplants as alternatives. In all cases, we recommend to include patients in prospective clinical trials.

RHCE*cE734C allele encodes an altered c antigen and a suppressed E antigen not detected with standard reagents.

BACKGROUND: The RH blood group system has many RHCE variant alleles that have arisen through gene conversion or nucleotide changes. Two probands, with red blood cells (RBCs) that were D+C+E-c+(w) e+ were sent to our laboratories to resolve the weak c expression. STUDY DESIGN AND METHODS: Hemagglutination tests were performed by automated and manual procedures. Genomic DNA analysis was performed by sequencing of Exons 1 to 10 of RHCE and RHD. RESULTS: The probands' RBCs did not react with standard monoclonal anti-E reagents from Bio-Rad, Diagast, DiaMed, Immucor, Ortho, and Quotient. The RBCs reacted variably with anti-c reagents from Diagast, DiaMed, Immucor, or Ortho and did not react with the Quotient anti-c reagent. Surprisingly, sequencing results of RHCE showed the presence of C/G at Position 676 (E/e polymorphism) and the association of the E polymorphism with a 734T>C transition in Exon 5 of the RHCE, encoding a Leu245Pro amino acid substitution in the mature RhcE polypeptide. Replacement of leucine 245 by proline in the eighth transmembrane domain of the RhcE protein may have a steric effect on the protein such that most anti-E reagents do not bind and the interaction between anti-c and c antigen is also affected. CONCLUSION: We report a novel RHCE*cE allele, RHCE*cE734C, which was assigned the provisional ISBT allele name RHCE*cE.14 or RHCE*03.14. It was found in two probands whose RBCs had weakened c expression and typed E- with conventional anti-E reagents. These data, once again, highlight the fact that the genotype does not always reflect the phenotype.