Resolution of RHCE Haplotype Ambiguities in Transfusion Settings.

Red blood cell (RBC) transfusion, limited by patient alloimmunization, demands accurate blood group typing. The Rh system requires specific attention due to the limitations of serological phenotyping methods. Although these have been compensated for by molecular biology solutions, some RhCE ambiguities remain unresolved. The RHCE mRNA length is compatible with full-length analysis and haplotype discrimination, but the RHCE mRNA analyses reported so far are based on reticulocyte isolation and molecular biology protocols that are fastidious to implement in a routine context. We aim to present the most efficient reticulocyte isolation method, combined with an RT-PCR sequencing protocol that embraces the phasing of all haplotype configurations and identification of any allele. Two protocols were tested for reticulocyte isolation based either on their size/density properties or on their specific antigenicity. We show that the reticulocyte sorting method by antigen specificity from EDTA blood samples collected up to 48 h before processing is the most efficient and that the combination of an RHCE-specific RT-PCR followed by RHCE allele-specific sequencing enables analysis of cDNA RHCE haplotypes. All samples analyzed show full concordance between RHCE phenotype and haplotype sequencing. Two samples from the immunohematology laboratory with ambiguous results were successfully analyzed and resolved, one of them displaying a novel RHCE allele (RHCE*03 c.340C>T).

HLA-F and LILRB1 Genetic Polymorphisms Associated with Alloimmunisation in Sickle Cell Disease.

Red blood cell (RBC) transfusion remains a critical component in caring for the acute and chronic complications of sickle cell disease (SCD). Patient alloimmunisation is the main limitation of transfusion, which can worsen anaemia and lead to delayed haemolytic transfusion reaction or transfusion deadlock. Although biological risk factors have been identified for immunisation, patient alloimmunisation remains difficult to predict. We aimed to characterise genetic alloimmunisation factors to optimise the management of blood products compatible with extended antigen matching to ensure the self-sufficiency of labile blood products. Considering alloimmunisation in other clinical settings, like pregnancy and transplantation, many studies have shown that HLA Ib molecules (HLA-G, -E, and -F) are involved in tolerance mechanism; these molecules are ligands of immune effector cell receptors (LILRB1, LILRB2, and KIR3DS1). Genetic polymorphisms of these ligands and receptors have been linked to their expression levels and their influence on inflammatory and immune response modulation. Our hypothesis was that polymorphisms of HLA Ib genes and of their receptors are associated with alloimmunisation susceptibility in SCD patients. The alloimmunisation profile of thirty-seven adult SCD patients was analysed according to these genetic polymorphisms and transfusion history. Our results suggest that the alloimmunisation of SCD patients is linked to both HLA-F and LILRB1 genetic polymorphisms located in their regulatory region and associated with their protein expression level.

Frequency and characterization of RHD and RHCE variants in the Noir Marron population from French Guiana.

BACKGROUND: The RH system is one of the most polymorphic blood group systems due to the proximity and opposite orientation of RHD and RHCE genes. Numerous alleles are described and can affect Rh protein expression. This complexity is especially evident in populations of African origin. We performed RHD and RHCE genotyping of the Noir Marron population in French Guiana. This population belongs to the Maroon community who are direct descendants of African slaves, who escaped from Dutch plantations, in the current day Suriname, during the 17th century. They represent an original ethnic group with highly blended culture. METHODS AND MATERIALS: A total of 89 DNA samples were collected from four different ethnic groups of the Noir Marron population of French Guiana. RHD and RHCE genotyping was performed using DNA microarray and/or sequencing. RESULTS AND DISCUSSION: Significant allelic diversity was shown, with 45% of individuals presenting an RHD gene variant (most common: RHD*DAU, RHD*DIVa, and RHD*DIIIa allele) and 9.4% with a partial D phenotype. Likewise, 85% presenting an RHCE gene variant and 9% a partial RH2 antigen. One original allele was identified in two D+ Noir Marron individuals: a hybrid RHD*DIIIa-CE(9)-D allele, encoding probably a partial D antigen and associated with an RHCE*ce(48C,733G,1006T) allele. The African diversity of RHD and RHCE genes is found in this population with preserved genetic but mixed cultural backgrounds. These data allow us to describe the characteristics of the RH system antigen and highlights a significant number of partial antigens with a risk of alloimmunization.

Qualitative and quantitative comparison of cell-free DNA and cell-free fetal DNA isolation by four (semi-)automated extraction methods: impact in two clinical applications: chimerism quantification and noninvasive prenatal diagnosis.

BACKGROUND: Non-invasive molecular analysis of cell-free DNA (cfDNA) became a sensitive biomarker for monitoring organ transplantation or for detection of fetal DNA (cffDNA) in noninvasive prenatal test. In this study, we compared the efficiencies of four (semi)-automated cfDNA isolation instruments using their respective isolation kit: MagNA Pure 24 (Roche®), IDEAL (IDSolution®), LABTurbo 24 (Taigen®) and Chemagic 360 (Perkin Elmer®). The cfDNA was isolated from 5 plasma samples and the Rhesus D (RhD)-cffDNA from 5 maternal plasmas. The cfDNA were quantified by digital droplet PCR (ddPCR), BIABooster system and QUBIT fluorometer. The cfDNA fragment size profiles were assessed by BIABooster system. Chimerism were quantified by home-made ddPCR and Devyser NGS kit. RhD-cffDNA in maternal plasma were detected between weeks 14 and 24 of amenorrhea using free DNA Fetal RHD Kit® (Biorad®). RESULTS: Statistical tests have shown differences in DNA yield depending on the isolation procedure and quantification method used. Magna Pure isolates smaller cfDNA fragment size than other extraction methods (90% ± 9% vs. 74% ± 8%; p = 0.009). Chimerism was only reliable from LABTurbo 24 extractions using the NGS but not with ddPCR whatever extraction methods. RhD-cffDNA were detected by all isolation methods, although IDEAL and LABTurbo 24 systems seemed more efficient. CONCLUSIONS: This comparative study showed a dependency of cfDNA yield depending on isolation procedure and quantification method used. In total, these results suggest that the choice of pre-analytical isolation systems needs to be carefully validated in routine clinical practice.

[Immuno-hematological follow up of pregnant women]. / Suivi immuno-hématologique des femmes enceintes.

Immuno-hematological follow up of pregnant women. The goal of immuno-hematological follow-up during pregnancy is to organize maternal transfusion safety and optimal management of fetal and neonatal hemolytic disease, that may result from maternal immunization. This follow-up relies on scheduled antibodies detection following regulation. Since May 2017 the refund by social security of the fetal RHD genotyping analysis on maternal plasma, it is possible to limit prophylaxis of allo-immunization to RhD-negative women with a RhD positive fetus. If red blood cell antibody detection is positive, an identification and a quantification of the antibody must be performed. Furthermore, fetomaternal red blood cell incompatibility must be confirmed, in order to adapt the management of allo-immunized pregnancy, for detection of fetal anemia but also to anticipate possible transfusion requirements of the mother and the newborn long before delivery.

Suivi immuno-hématologique des femmes enceintes. L'objectif du suivi immuno-hématologique d'une femme enceinte est d'assurer sa sécurité transfusionnelle mais aussi la prise en charge optimale de la maladie hémolytique foetale et néonatale, pouvant compliquer une immunisation maternelle. Ce suivi repose sur le respect du calendrier de recherche des agglutinines irrégulières (RAI). Le remboursement depuis mai 2017 de l'analyse du génotypage RhD foetal sur sang maternel permet de cibler la prophylaxie anti-D aux seules femmes RhD-négatives avec un foetus RhD-positif. Lorsque la recherche d'agglutinines irrégulières s'avère positive, il est indispensable d'identifier l'anticorps, de le quantifier, de déterminer s'il existe une situation d'incompatibilité foeto-maternelle érythrocytaire. Cela afin d'adapter le suivi de la grossesse et la prise en charge néonatale. Enfin, la connaissance du phénotype de la patiente, allo-immunisée ou pas, est indispensable pour anticiper les éventuels besoins transfusionnels de la mère et du nouveau-né, bien en amont de l'accouchement.

Five-Years Review of RHCE Alleles Detected after Weak and/or Discrepant C Results in Southern France.

Immunohematology laboratories are regularly facing transfusion issues due to serological weaknesses. Altered (partial) RH antigens account for most of them. In some situations, RHCE variant alleles are involved. Herein we present our three-step molecular exploration, with allele frequencies, that has efficiently untangled RH2 phenotype weaknesses and discrepancies in our 2017-2021 cohort. In the last 5 years, the PACA Corse EFS molecular platform received 265 samples from healthy blood donors or patients with C and C/e typing difficulties. The first-intention technique (DNA array and real time PCR for RHCE*CeRN research) detected RHCE variant alleles in 143 cases (54%). The RHCE alleles classically found in African populations were the most frequent, with RHCE*CeRN allele in 40 cases (15%) and (C)ces haplotype type 1 and 2 in 26 cases (10%). A "CE" effect haplotype was suspected in 56 cases, due to the uncommon DCE haplotype that may explain the low C expression. When there were no RHCE*Ce or RHCE*CE alleles, we then searched for RHD polymorphisms by DNA array. We detected the RHD*DAU5 and RHD*DIVa in 18 and 7 cases respectively, suggesting that C ambiguity is related to the presence of these alleles which has never been described with DAU5. If no variant RHCE and RHD alleles were detected, we finally sequenced the 10 exons of both RHCE and RHD genes according to the clinical context and found seven new RHCE alleles. Thus, this molecular strategy would improve the knowledge of RHCE variants' expression and, thus, optimize the transfusion management.

Robinsoniella peoriensis infection following surgery for scoliosis: a case report.

INTRODUCTION: Robinsoniella peoriensis was recently identified as a Gram-positive, spore-forming, anaerobic bacillus originally isolated from swine manure storage pits. Seven isolates have been subsequently reported from human sources. CASE PRESENTATION: We report the case of an infection caused by R. peoriensis in a 45-year-old Caucasian woman after posterior instrumentation correction of idiopathic thoracolumbar scoliosis. The identification was made by culture of samples inoculated onto blood agar and chocolate agar and was confirmed by 16 S ribosomal ribonucleic acid gene sequencing. CONCLUSIONS: We discuss similar cases suggesting that R. peoriensis is responsible for health care-associated infections with the colonic flora as a potential source of infection.