Reference values for T, B and NK human lymphocyte subpopulations in adults.

The data presented in this paper are reference ranges for frequencies of thirty-eight subpopulations of T, B and NK lymphocytes, established from a cohort of 253 healthy blood donors aged from 19 to 67. When relevant, the influence of age or sex was taken into account to calculate these reference values. This article is related to the research article entitled "Influence of age, sex and HCMV-serostatus on blood lymphocyte subpopulations in healthy adults" (Apoil et al., 2017) [1]. Immunophenotyping data obtained from each individual is made publicly available for extended analyses.

Influence of age, sex and HCMV-serostatus on blood lymphocyte subpopulations in healthy adults.

Using a standardized immunophenotyping procedure we studied thirty-eight distinct subpopulations of T, B and NK lymphocytes in 253 healthy blood donors aged from 19 to 67. We analysed the influence of age, sex and HCMV seropositivity on each lymphocyte subpopulations and established reference ranges. We observed that aging influences the largest number of lymphocyte subpopulations with a slow increase of CD8+ EMRA T lymphocytes and of the numbers of circulating Tregs. The proportion of HLA-DR+ cells among Tregs increased with age and was correlated to the proportion of HLA-DR+ cells among effector T CD4+ lymphocytes. Sex had a major impact on absolute counts of CD4+ T cells which were higher in females. HCMV-seropositivity was associated with higher frequencies of CD8+ EMRA memory T lymphocytes while a high frequency of terminally differentiated EMRA CD4+ T cells was observed in 80% of HCMV-positive individuals and in none of the HCMV seronegative individuals.

[Feasibility study for Intercept platelets]. / Étude de faisabilité des plaquettes Intercept.

During 3 months, platelet concentrates prepared by "Établissement français du sang Pyrénées-Méditerranée" (Blood bank) were treated with the Intercept process (CERUS©). This study primarily aimed to measure the organizational impact of this technology on transfusion chain. The introduction of Intercept did not raise any major difficulties, but required some adaptations upstream from the deployment. Prior information of health care institutions and physician was essential to anticipate the practical changes, including the prescription of platelet concentrates (CMV negative, irradiation). This study allowed to analyze also the transfusion consequences for patients, in the form of observational studies. The patients transfused with platelet concentrates treated with Intercept received more platelet concentrates (+12.9%), less rich in platelets (-12.8%), the cumulated quantity of platelet being stable.

A simple genotyping procedure without DNA extraction to identify rare blood donors.

BACKGROUND: Transfusion-induced alloimmunization has severe clinical consequences including haemolytic transfusion reactions, impaired transfused RBCs longevity and greater difficulty in finding compatible blood. Molecular analysis of genomic DNA now permits prediction of blood group phenotypes based on identification of single nucleotide polymorphisms. Implementation of molecular technologies in donor centres would be helpful in finding RBC units for special patient populations, but DNA extraction remains an obstacle to donor genotyping. MATERIALS AND METHODS: We propose a simple method compatible with high throughput that allows blood group genotyping using a multiplex commercial kit without the need for DNA extraction. The principle relies on pre-PCR treatment of whole blood using heating/cooling procedure in association with a recombinant hotstart polymerase. RESULTS: In a prospective analysis, we yielded 5628 alleles identification and designated 63 donors with rare blood, that is either negative for a high-frequency antigen or with a rare combination of common antigens. CONCLUSION: The procedure was optimized for simplicity of use in genotyping platform and would allow not only to supply antigen-matched products to recipients but also to find rare phenotypes. This methodology could also be useful for establishing a donor repository for human platelet antigens (HPA)-matched platelets since the same issues are involved for patients with neonatal alloimmune thrombocytopenia or post-transfusion purpura.

[Medical biology accreditation at EFS]. / Accréditation des laboratoires de biologie médicale au sein de l'Établissement français du sang.

The medical biology laboratory accreditation according to the Iso 15189 standard, which main lines are mentioned in the article, is henceforth becoming a statutory obligation in France. All laboratories must apply, at least partly, to the COFRAC, by 31(st) October 2012. The EFS has largely anticipated the necessary steps to reach this objective and has developed an approach based on six basic processes. To date, 24 laboratories of various technical fields are accredited and several other submissions are pending. The Iso 15189 standard requirements match those already implemented at the EFS with the Certification. The trade standard operating procedures are almost included in the risk control management. Through the involvement of all the EFS members following precision action plans based on the sharing of successful experiences and the harmonization of trade practices, this compulsory objective will be reached and the deadline respected.

[Hypotension and adverse transfusion reactions: from the associated clinical signs to the hypotensive transfusion reaction]. / L'hypotension artérielle dans les effets indésirables receveurs: du signe clinique associé à la réaction transfusionnelle hypotensive.

OBJECTIVE: The first aim of this study was to confirm the presence of hypotension blood transfusion reactions and to assess the part of hypotension as a principal event, as defined by the literature but not characterized in French haemovigilance data. As well, recent series of several cases led us to consider a possible incidence increase. STUDY DESIGN: Using a retrospective observation, the haemovigilance data from 2000 to the end of 2007 of two French regions were reviewed. During this period, 1159657 blood units were transfused by nearly 100 hospitals and 3727 adverse reactions observed. RESULTS: One hundred and sixty-eight adverse reactions with hypotension were noticed and analyzed, representing 4.5% of all transfusion reactions and revealing an incidence of 14.5 for 100000 blood units transfused. It turned out to be mostly male recipients, severe reactions and appearing rather in the beginning of transfusions. Although platelets having greater incidence, all types of blood products may be involved. The clinical diagnosis was the following: 40 to 47% were classified as febrile reactions, 13 to 17% were allergic reactions, 8 to 9% were due to immunologic and/or haemolytic reactions, 5 to 7% resulted of cardiologic disorders, 5% resulted of hypovolemic contexts and 22% were unexplained hypotensive transfusion reactions. CONCLUSION: In about three cases out of four, transfusion-induced hypotension was associated with other clinical reactions. Indeed, hypotensive transfusion reactions were identified, having an incidence of 3.2 for 100000 blood units transfused. Furthermore, there was no explanation found for the incidence increase in our region during 2007. A national study was suggested to analyse the national data as well as a prospective study to clear out this type of transfusion reactions.

[Evolution of the blood products distribution in France - detailed analysis in a regional blood transfusion centre]. / Evolution de la distribution des produits sanguins labiles en France - analyse détaillée au sein d'un Etablissement français du sang interrégional.

OBJECTIVE: The present increase of blood products distribution raises some adaptation questions. To understand this evolution, it is necessary to assure patients needs satisfaction. STUDY DESIGN: The study focuses on years 1997 to 2007. All blood products and hospitals are taken into account. The possible impact of size and clinical specialties is analyzed for the main hospitals. RESULTS: The evolution varies according to blood product: continuous drop for autologous red cells and plasmas, drop and rise for homologous red cells and platelets with a turnaround in 2001-2002, reverse and more chaotic movement for homologous plasmas. These movements are the result of public hospitals, with an upsurge of the medium sized ones. Private hospitals go down for all blood products, with a concentration on the larger ones. Surgical hospitals fall from 3 to 4% for all blood products, while medical ones rise of 3% for homologous red cells, 27% for platelets and fall of 10% for homologous plasmas. Private mixed hospitals fall for all blood products, while public ones rise for homologous red cells and platelets. CONCLUSION: Evolution understanding of blood products distribution requires precisions about the kind of products and the hospitals status and specialties: medical, surgical, even obstetrical or urgency-related. The present trend is a rise for public hospitals and medical specialties, which are both the largest. Blood transfusion needs will thus go on rising in the years to come.

[Transfusion protocols in hematopoietic stem cell allogeneic transplantation]. / Consignes transfusionnelles en cas d'allogreffe de cellules souches hématopoïétiques.

Hematopoietic stem cell (HSC) allogeneic transplantation is now commonly used as a therapeutic tool in patients with certain types of hematologic malignancies. Such patients, on account of severe pre-graft conditioning regimens, present with severe marrow aplasia justifying specific transfusion care. Given a complex immunological situation (immediately after transplantation, co-existence of two cell populations with different immunohematological characteristics), transfusion protocols must rest on clear and well-defined recommendations. Recent transfusion recommendations in settings of HSC allogeneic transplantation have defined criteria for the choice of blood products (red blood cell concentrates, plasma and platelet concentrates) depending on recipient and graft immunohematological characteristics (minor/major/mixed ABO compatibility/incompatibility and time of transplantation). Transfusion instructions are summarized in a synthesis document entitled : "Instructions for transfusion following HSC allogeneic transplantation". This document specifies the immunohematological characteristics of blood products and various transfusion protocols (systematic irradiation, negative CMV, etc.). This document is used by the teams who distribute blood products, for selection purposes, as well as by the medical transfusion team when they perform ultimate pre-transfusion control steps.

[Health care units in the French blood banks, why?]. / Les structures de soins dans les établissements de transfusion sanguine, pourquoi faire ?

In France for several years, many patients have been treated in Blood Transfusion Centers belonging to the EFS. This partnership between public hospitals and EFS is appreciated by the patients who find a competent staff in transfusion and apheresis process, in a more pleasant environment than in hospital. There is a total of 93 Health Care Units in Blood Transfusion Centers. Sixty-three of these Health Care Units perform only transfusions and bleeding. In the remaining 30 Health Care Units apheresis, peripheral blood hematopoietic stem, cell harvesting, plasmatic exchanges and extracorporeal photopheresis are also performed. Despite the perfect fit between hospital needs, comfort and easiness for patients, an economical problem remains. At the present time, the reimbursement rate by national health insurance is below the real cost. If unsolved, this discrepancy could force an end to this beneficial partnership.

[Validation of antibody screening by indirect antigloblin test and ABO blood typing by filtration and microplate techniques: assessment of robustness]. / Maîtrise des tolérances métrologiques : application à la recherche des anticorps anti-érythrocytaires (RAI) en test indirect à l'antiglobuline (TIA) et au groupage sanguin ABO par les procédés de filtration et en microplaque.

According to requirements of the French Committee for Accreditation (Cofrac), it is essential to use validated and standardised methods in Immunohematology. This imposes first the knowledge of metrological tolerances for all the technics. Two multicenter studies were carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and tests cells for antibody screening using indirect antiglobulin test on one hand and for reverse grouping on another hand. All equipment used (temperature test chamber, chronometer, pipettes) were calibrated according to Cofrac standards. The antibody screenings were performed manually using 3 different filtration systems: ID Diamed, Biovue Ortho and Scangel Biorad, the same tests cells, a standard 20 ng/mL anti RH1, a positive control anti KEL1 and a negative control; the reverse blood grouping was performed manually using the above mentionned filtration systems and microplate technic with the same A1 and B test cells. These two studies showed that all the tests from the multiples combinations of the above parameters gave the same results and allowed us to define a range of tolerance for 4 critical physical parameters involved in the antibody screening and blood typing.

Molecular polymorphism of O alleles in five populations of different ethnic origins.

Sequences of exons 6 and 7 of the O allele of the ABO gene were studied in 317 individuals of the O phenotype from five different ethnic groups (Basques, Berbers, Akans from the Ivory Coast, and Amerindians: Cayapas from Ecuador and Aymaras from Bolivia). Twenty-one O alleles were characterized, among which 9 differed from all O alleles reported to date. The nine alleles differed from either the O01 allele (four out of nine) or O02 allele (five out of nine) by one to three point mutations. The number of different O alleles in population samples varied greatly: the highest number (13) was observed in Akans, and the lowest (5) in Amerindians. Some rare alleles previously reported by others at low frequencies were found with high frequencies in the Akans. The results also revealed a decreasing frequency of Ov7 alleles from south to north (Akans, Berbers, Basques). Berbers and Basques share two rare alleles, Ov6 and O03, which were not encountered in the other populations studied here.

[Problem-solving in immunohematology: direct compatibility laboratory test ]. / Aide à la décision en immunohématologie: épreuve directe de compatibilité au laboratoire (EDC).

Cross-matching between the serum of a patient and the red blood cells to be transfused is most important for the prevention of hemolytic transfusion reactions in allo-immunized or new-born patients found positive with direct antiglobulin test. Cross-matching is a time-consuming and complex laboratory test. In order to obtain valid results, it is necessary to abide by some technical rules detailed in this article. The choice of the blood units to be cross-matched depends on the patient's clinical story and on the specificity of anti-erythrocyte antibodies present in the serum. The identification and the management of most frequent difficulties met by using the cross-match technique are discussed hereby.

Decision aides in immunohematology: detection of anti-erythrocyte antibodies. / Aide à la décision en immunohématologie: recherche des anticorps anti-érythrocytaires (RAI).

Detection and identification of irregular red-cell antibody in the serum or plasma of a patient is of prime importance for the prevention of hemolytic transfusion reactions and the biological supervision of the hemolytic disease of the foetus or the newborn. Practice in these tests is replete with complex biological problems. Using problem solving strategies, we discuss the recognition and resolution of the most frequent difficulties encountered in red cell antibody identification.

[Analytical validation in erythrocyte immunohematology]. / Validation analytique en immunohématologie érythrocytaire.

In a transfusional or foeto-maternal context, hemolysis by incompatibility due to anti-erythrocyte antibodies (regular or irregular) remains the most frequent and most serious immunological risk in the receiver. In order to prevent this risk, a number of actions must be taken, such as the realization of the immunohematologic analyses for which the methodological practices have been legislated because of their serious clinical consequences. Several elements play a role in the reliability of the analyses and their results: the selection of the reagents and their validation in the routine technique used; the validation of reception; the controls involved in secondary preparations (e.g., blood cells reagent); and the daily internal controls. All this requires the choice of adapted controls and the management of possible anomalies.

[Problem-solving in immunohematology: interpretation of ABO typing and its difficulties]. / Aide à la décision en immunohématologie: interprétation du groupage sanguin ABO et de ses difficultés.

Practice in immunohematology is replete with complex problems that require practitioners' problem-solving performance. In immunohematology, the acquisition of the reasoning process and necessary skills for making clinical decisions is based on teaching problem-solving strategies which potentially reduce errors and improve patient outcome. We discuss the recognition and resolution of the common causes of discrepancies in ABO typing results using problem-solving strategies.

Evolution of alpha 2-fucosyltransferase genes in primates: relation between an intronic Alu-Y element and red cell expression of ABH antigens.

Coding sequences of the paralogous FUT1 (H), FUT2 (Se), and Sec1 alpha 2-fucosyltransferase genes were obtained from different primate species. Analysis of the primate FUT1-like and FUT2-like sequences revealed the absence of the known human inactivating mutations giving rise to the h null alleles of FUT1 and the se null alleles of FUT2. Therefore, most primate FUT1-like and FUT2-like genes potentially code for functional enzymes. The Sec1-like gene encodes for a potentially functional alpha 2-fucosyltransferase enzyme in nonprimate mammals, New World monkeys, and Old World monkeys, but it has been inactivated by a nonsense mutation at codon 325 in the ancestor of humans and African apes (gorillas, chimpanzees). Human and gorilla Sec1's have, in addition, two deletions and one insertion, respectively, 5' of the nonsense mutation leading to proteins shorter than chimpanzee Sec1. Phylogenetic analysis of the available H, Se, and Sec1 mammalian protein sequences demonstrates the existence of three clusters which correspond to the three genes. This suggests that the differentiation of the three genes is rather old and predates the great mammalian radiation. The phylogenetic analysis also suggests that Sec1 has a higher evolutionary rate than FUT2 and FUT1. Finally, we show that an Alu-Y element was inserted in intron 1 of the FUT1 ancestor of humans and apes (chimpanzees, gorillas, orangutans, and gibbons); this Alu-Y element has not been found in monkeys or nonprimate mammals, which lack ABH antigens on red cells. A potential mechanism leading to the red cell expression of the H enzyme in primates, related to the insertion of this Alu-Y sequence, is proposed.

[Prevention of fetal hemolytic disease: it is time to take action]. / Prévention de la maladie hémolytique du foetus et du nouveau-né: il faut agir!

In spite of the progress made since 1970 in specific prevention by anti-rhesus immunoglobulins, and improved management of at-risk pregnancies, allo-immunization due to the erythrocytic Rh 1 antigen (formerly known as Rhesus D or Rh D) remains widespread. In fact, anti-Rh 1 antibodies currently constitute over one-third of the immune antibodies detected after pregnancy. The prevention of allo-immunization against the Rh 1 antigen is therefore still problematical, and concerns approximately one pregnant woman in seven. The etiology and pathology of fetal hemolytic disease have been recalled, and the treatment approach during pregnancy and delivery has been carefully examined. Tests for quantifying the risk of fetomaternal hemorrhage have also been described. This approach aims at improving the methods of preventing allo-immunization (e.g., during pregnancy and delivery) and the efficacy of treatment. It is also stated that if the necessary preventive action is not taken in cases of allo-immunization due to to the Rh 1 antigen, this should be considered a grave medical fault.