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.

Responses of artificially reared cat fleas Ctenocephalides felis felis (Bouché, 1835) to different mammalian bloods.

The cat flea, Ctenocephalides felis felis (Bouche, 1835) (Siphonaptera: Pulicidae), which is found worldwide and which parasitizes many species of wild and domestic animal, is a vector and/or reservoir of bacteria, protozoa and helminths. To aid in the study of the physiology and behaviour of fleas and of their transmission of pathogens, it would be of value to improve the laboratory rearing of pathogen-free fleas. The conditions under which artificially reared fleas at the University of Bristol (U.K.) and the Rickettsial Diseases Institute (France) are maintained were studied, with different ratios of male to female fleas per chamber (25 : 50, 50 : 100, 100 : 100, 200 : 200). The fleas were fed with bovine, ovine, caprine, porcine or human blood containing the anticoagulants sodium citrate or EDTA. Egg production was highest when fleas were kept in chambers with a ratio of 25 males to 100 females. In addition, the use of EDTA as an anticoagulant rather than sodium citrate resulted in a large increase in the number of eggs produced per female; however, the low percentage of eggs developing through to adult fleas was lower with EDTA. The modifications described in our rearing methods will improve the rearing of cat fleas for research.

16(th) IHIW: analysis of HLA population data, with updated results for 1996 to 2012 workshop data (AHPD project report).

We present here the results of the Analysis of HLA Population Data (AHPD) project of the 16th International HLA and Immunogenetics Workshop (16IHIW) held in Liverpool in May-June 2012. Thanks to the collaboration of 25 laboratories from 18 different countries, HLA genotypic data for 59 new population samples (either well-defined populations or donor registry samples) were gathered and 55 were analysed statistically following HLA-NET recommendations. The new data included, among others, large sets of well-defined populations from north-east Europe and West Asia, as well as many donor registry data from European countries. The Gene[rate] computer tools were combined to create a Gene[rate] computer pipeline to automatically (i) estimate allele frequencies by an expectation-maximization algorithm accommodating ambiguities, (ii) estimate heterozygosity, (iii) test for Hardy-Weinberg equilibrium (HWE), (iv) test for selective neutrality, (v) generate frequency graphs and summary statistics for each sample at each locus and (vi) plot multidimensional scaling (MDS) analyses comparing the new samples with previous IHIW data. Intrapopulation analyses show that HWE is rarely rejected, while neutrality tests often indicate a significant excess of heterozygotes compared with neutral expectations. The comparison of the 16IHIW AHPD data with data collected during previous workshops (12th-15th) shows that geography is an excellent predictor of HLA genetic differentiations for HLA-A, -B and -DRB1 loci but not for HLA-DQ, whose patterns are probably more influenced by natural selection. In Europe, HLA genetic variation clearly follows a north to south-east axis despite a low level of differentiation between European, North African and West Asian populations. Pacific populations are genetically close to Austronesian-speaking South-East Asian and Taiwanese populations, in agreement with current theories on the peopling of Oceania. Thanks to this project, HLA genetic variation is more clearly defined worldwide and better interpreted in relation to human peopling history and HLA molecular evolution.

Blood group typing in five Afghan populations in the North Hindu-Kush region: implications for blood transfusion practice.

BACKGROUND AND OBJECTIVES: Blood incompatibility arises from individual and ethnic differences in red blood cell (RBC) antigen profiles. This underlines the importance of documenting RBC antigen variability in various ethnic groups. Central Asia is an area with a long and complex migratory history. The purpose of this article is to describe key antigen frequencies of Afghan ethnic groups in the Hindu-Kush region of Afghanistan as a basis for improving blood transfusion practices in that area. MATERIALS AND METHODS: The key ABO, Rh and Kell antigens were investigated in five Afghan populations. In order to depict accurately the blood group gene diversity in the area, DNA from eight additional Pakistani populations were included, and the entire sample set screened using two multiplex polymerase chain reactions sensitive for 17 alleles in 10 blood group genetic systems (MNS, Kell, Duffy, Kidd, Cartwright, Dombrock, Indian, Colton, Diego and Landsteiner-Wiener). RESULTS: Phenotype and allele frequencies fell within the ranges observed in Western European and East Asian populations. Occurrence of DI*01, IN*01, LW*07 and FY*02N.01 and prevalence of ABO*B were consistent with migratory history as well as with putative environmental adaptation in the subtropical environment Hindu-Kush region. CONCLUSION: These findings expand the current knowledge about key antigen frequencies. Regarding occurrence of viral markers, further blood transfusion in the region requires rigorous typing.

Molecular characterization of a new D- - haplotype in a Comorian man.

The D- - phenotype is a genetic variant of the Rh blood group system. It expresses D antigen but lacks C, c, E and e antigens. In D- - phenotype, the RHCE coding region is extensively modified by RHD sequence replacement, nucleotide deletion or splice-site changes. This article reports the identification of a new D- - haplotype in a Comorian man. It exhibits a hybrid gene in which RHCE gene exons 3-8 have been replaced by RHD sequences on the RHCE * C allele background. This allele is associated with no expression of c/C and e/E antigens and overexpression of RhD antigen.

The Arabo-Islamic migrations in Madagascar: first genetic study of the GM system in three Malagasy populations.

The Antemoro are an ethnic group from the southeast coast of Madagascar who claims an Arab origin. Cultural signatures of an Arabo-Islamic influence have been found in this region. Nevertheless, their origins are very contentious. Through this study, we want to determine whether this ethnic group had a particular GM profile that differentiated it from other Malagasy populations, and whether there were detectable genetic traces of the Arabo-Islamic migration. The Gm polymorphisms of IgG immunoglobulins was analysed in a population of Antemoro (N = 85), two other Malagasy populations from northern Fiherena (N = 82) and southern Fiherena (N = 50) and in a Comorian population (N = 171). This last group was used to enlarge the database for genetic comparisons. Results revealed significant contributions from Africa (60%, 0.092 ≤F(ST) ≤ 0.280) and Southeast Asia (40%, 0.043 ≤ F(ST) ≤ 0.590) to the Antemoro genetic pool. No direct genetic relationships with the Middle East. These results bring new insights into the population history of Madagascar.

Linkage disequilibrium between HLA-G*0104 and HLA-E*0103 alleles in Tswa Pygmies.

Nonclassical human leukocyte antigen (HLA)-G and -E loci are separated by approximately 660 kb on the short arm of chromosome 6. Interestingly, some functional and expression characteristics are relatively identical or associated for both molecules. For example, expression of HLA-E on the cell surface has been linked to preferential binding of nonameric leader peptides derived from the signal sequence of HLA-G. It has been suggested that these two molecules act synergistically in modulating susceptibility to infectious or chronic inflammatory diseases. A possible explanation for these observations is that HLA-E and HLA-G are evolving under analogous selective pressures and have functions that place them under selective regimes differing from classical HLA genes. The purpose of this study was to investigate the consistency of this hypothesis based on the characterization of the molecular polymorphism of these two genes and their linkage disequilibrium (LD) in three populations, i.e. Southeastern French (n = 57), Teke Congolese (n = 84) and Tswa Pygmies (n = 74). Allelic frequencies observed for HLA-G and HLA-E and for 14-bp ins/del polymorphism in the three populations were similar to those observed in the literature for populations from corresponding geographic areas. Only one of the recently described HLA-G polymorphisms (HLA-G*01:07-01:16) was found, i.e. HLA-G*01:15 in one individual from Congo. We showed that two haplotypes in Tswa Pygmies, i.e. HLA-G*01:04-E*01:03:01 and G*01:04-E*01:01, exhibited highly significant positive and negative D' values respectively. Although these LD could have functional implications, it is more likely because of the genetic drift as the two other populations did not display any significant LD.

Identification of Frailty in a Population of Former Immigrant Workers in the South of France.

BACKGROUND: Frailty is unevenly distributed across the world but also within different populations in the same country. OBJECTIVES: This study sought to identify frailty in former immigrant workers, known as Chibanis, living in an immigrant hostel in Marseille. The secondary objective was to describe health care access, as well as any chronic diseases reported. DESIGN, PARTICIPANTS AND SETTING: Our descriptive, observational, monocentric study conducted from January to April 2021 included 67 Chibanis, living in an immigrant hostel in Marseille. MEASUREMENTS AND RESULTS: Almost all this population (97%), with a median age of 77 years, presented at least one frailty criterion: 7.5% were malnourished, 55.2% had a grip strength of < 27 kg, and 41.8% were on multiple drugs. Majority of Chibanis (86.6%) had multimorbidity. CONCLUSION: Identifying frailty in this population of Chibanis must be proposed through a specific, adapted care pathway including referral to a specialist.

[Impact of menstrual blood self-representation on contraceptive choice of women]. / Impact des représentations du sang menstruel sur le choix contraceptif des femmes.

OBJECTIVES: To study the psychic self-representations and experiences of menstrual blood in women and their impact on the choice of a contraceptive method, with or without amenorrhea. METHODS: Qualitative study based on semi-structured interviews with French women over age 18, under contraception. RESULTS: Twenty-three interviews were conducted with women of various ages and socio-economic classes. Three themes have been studied: the menarche experience, the representation and experience of menstrual blood, and the representation and experience of amenorrhea induced by contraception. Menarche has been a negative experience for most of them, and menarche is known to influence menstrual self-representation. About menstrual bleeding, two profiles of women could be described. Those with a positive self-representation of menstrual blood considered it necessary for the purification of their bodies as well as for procreation and were reluctant to the idea of amenorrhea induced by their contraception. Those with a negative representation of menstrual blood considered it as a source of physical and mental suffering and accepted the idea of having amenorrhea induced by their contraception, amenorrhea being considered as a treatment or a release. CONCLUSION: The choice of a contraception with or without a induced-amenorrhea seems to be specific to every woman and depends on there self-psychic representation of menstrual blood, independently from their socio-economic class. The results of this study highlighted the effect of women's psychic representations and experience of menstrual blood on their contraceptive choice.

[Structural or functional relationship between immunohematology and distribution - Which elements for controlling risks?] / Lien immunohématologie-délivrance structurel ou fonctionnel ­ Quels éléments pour la maîtrise des risques ?

OBJECTIVE: Transfusion safety is based on the availability of safe and compatible blood products at the right time and to the right patient, and requires close monitoring in order to detect possible incidents. The decree of June 20th 2018, which establishes the national blood transfusion's guiding plan, states that the organization that prevails throughout the national territory is built around an inseparable link between the implementation of erythrocyte immunohematology and the labile blood products delivery by authorised structures. METHOD: The article describes the two types of the link's organization, structural or functional, used to develop the comparative risk-benefit analysis. RESULTS: The structural link, which has fewer interfaces, reduces risk situations that lead to delays in release by default of a compatible product. The cases in which a functional link may have a greater benefit than the risks generated are those related to a geographical distance between the delivery site and the patient's place of care. In these cases, a functional link is possible provided that certain organizational points are mastered. CONCLUSION: The comparative analysis shows that the structural link is to be favoured since that the coherence of the patient's care and his care path is ensured. In certain situations, mainly geographical, the functional link can have a benefit that offsets the risks generated by the new interfaces; provided that the system is secured by a real tripartite collaboration between health care institution, biology laboratory and delivery site.

Relevance and costs of RHD genotyping in women with a weak D phenotype.

OBJECTIVES: For pregnant women, the serologic test results of D antigen will determine the frequency of RBC antibody detection as well as the indication for RhIG prophylaxis. RHD genotyping is the only method that may provide clear guidance on prophylaxis for women with a weak D phenotype. This analysis evaluated the economical implications of using RHD genotyping to guide RhIG prophylaxis among pregnant women with a serological weak D phenotype. METHODS: We compared the costs of 2 strategies in a cohort of 273 women with weak D phenotype. In the first strategy, we did not perform genotyping and all women with weak D phenotypes were treated as if they were D-, thus considered to be a risk of RhD alloimmunization. These women all received the prophylactic follow up. In the second strategy, RHD genotyping was performed on all women with a serologic weak D phenotype. Then, the follow-up will be determined by phenotype deduced from genotype. RESULTS: On the studied cohort, the additional expense occurred by genotyping is 26,536 €. RHD Genotyping has highlighted 162 weak D Type 1, 2 3, that could safely be managed as D+ and 111 partial D to consider as D-. By comparing the 2 strategies, the savings generated by genotyping the patients of our cohort are € 12,046 for the follow up of one pregnancy. Knowing that in France, a woman has on average 2 pregnancies and that the genotyping is carried out only once, the savings generated for the following pregnancies would be € 38,581. CONCLUSIONS: Performing RHD genotyping for pregnant women with a weak D phenotype enables to clearly identify weak D type 1, 2 or 3 from the other variants at risk of alloimmunization. This analysis generates savings in terms of follow-up schedule of pregnant women and RhIG prophylaxis. It also allows saving of D- products for patient with a weak D type 1, 2 or 3 in case of a transfusion need.

Population structure of modern-day Italians reveals patterns of ancient and archaic ancestries in Southern Europe.

European populations display low genetic differentiation as the result of long-term blending of their ancient founding ancestries. However, it is unclear how the combination of ancient ancestries related to early foragers, Neolithic farmers, and Bronze Age nomadic pastoralists can explain the distribution of genetic variation across Europe. Populations in natural crossroads like the Italian peninsula are expected to recapitulate the continental diversity, but have been systematically understudied. Here, we characterize the ancestry profiles of Italian populations using a genome-wide dataset representative of modern and ancient samples from across Italy, Europe, and the rest of the world. Italian genomes capture several ancient signatures, including a non-steppe contribution derived ultimately from the Caucasus. Differences in ancestry composition, as the result of migration and admixture, have generated in Italy the largest degree of population structure detected so far in the continent, as well as shaping the amount of Neanderthal DNA in modern-day populations.

[Microtitration of anti-RH1 antibodies: interest in the follow-up of pregnant women]. / Intérêt de la technique de microtitrage des anticorps anti-RH1 dans le suivi immunohématologique des femmes enceintes.

UNLABELLED: The proposal for a 300 microg anti-RH1 injection at 28 GW by RH:-1 pregnant women complicates the interpretation of the screening for alloantibodies during pregnancy; to distinguish an alloantibody from a passive one is nevertheless important for the care of the patients. Testing a technique allowing this distinction seems thus necessary. MATERIAL AND METHODS: The technique of microtitration of anti- RH1 antibodies is an indirect antiglobulin test. Two hundred specimens were tested in comparison with a standard prepared from a national anti- RH1 standard. If the anti- RH1 concentration measured is lower or equal to the expected concentration, there is a passive antibody. If the concentration is largely higher, we can suspect an allo-immunization. RESULTS: With this technique, 38 alloanti- RH1 and 112 passive anti- RH1 antibodies were confirmed. Twenty-five diagnosis were modified: seven alloanti- RH1 initially labeled passive and 18 passive anti- RH1 previously considered as alloantibodies. 15 cases can not be concluded, because the blood sample was taking away too early after the injection, and 10 cases are on standby, waiting for a control. DISCUSSION: The microtitration is an important exam in the follow-up of the RH:-1 pregnant women when an anti-RH1 antibody is found. This exam should be offered each time we have no information about the anti-D injection, or when an incoherent reaction compared to the presumed date of injection is observed.

Use of the PSA enhancer core element to modulate the expression of prostate- and non-prostate-specific basal promoters in a lentiviral vector context.

Composite promoters combining the prostate-specific antigen (PSA) enhancer core element with promoter elements derived from gene coding for human prostate-specific transglutaminase gene, prostate-specific membrane antigen gene, prostate-specific antigen, rat probasin or phosphoglycerate kinase were characterized for their ability to specifically express the enhanced green fluorescent protein (EGFP) gene in prostate versus non-prostate cancer cell lines when transferred with a human immunodeficiency virus-1-based lentiviral vector. By themselves minimal proximal promoter elements were found to inefficiently promote relevant tissue-specific expression; in all the vectors tested, addition of the PSA enhancer core element markedly improved EGFP expression in LnCaP, a cancer prostate cell line used as a model for prostate cancer. The composite promoter was inactive in HuH7, a hepatocarcinoma cell line used as a model of neighboring non-prostate cancer cells. Among the promoters tested, the combination of the PSA enhancer and the rat probasin promoter showed both high specificity and a strong EGFP expression. Neither a high viral input nor the presence of the cPPT/CTS sequence affected composite promoter behavior. Our data suggest that composite prostate-specific promoters constructed by combining key elements from various promoters can improve and/or confer tissue specific expression in a lentiviral vector context.

[Voluntariness and blood donation: Proceedings of an ethics seminar held at the National Institute for Blood Transfusion]. / Volontariat et don de sang : compte rendu d'un séminaire de l'Institut national de transfusion sanguine.

Voluntariness stands for one of the four pillars of ethics in blood donation; it is, however, more related to tradition than to legislation. Because it seems necessary to apply "marketing" techniques to blood collection in order to meet the needs in blood components, both in terms of quantity and quality, one wonders if this may be at the expense of this principle of voluntariness. This seminar-belonging actually to a series of seminars in Ethics in Transfusion Medicine-aimed at questioning the possible weakness of voluntariness in the field of blood donation. To achieve this goal, specialists of numerous disciplines in medical sciences, law and humanities gathered to discuss all related issues to voluntariness in blood donation.

Sequence family variant loss from the AZFc interval of the human Y chromosome, but not gene copy loss, is strongly associated with male infertility.

BACKGROUND: Complete deletion of the complete AZFc interval of the Y chromosome is the most common known genetic cause of human male infertility. Two partial AZFc deletions (gr/gr and b1/b3) that remove some copies of all AZFc genes have recently been identified in infertile and fertile populations, and an association study indicates that the resulting gene dose reduction represents a risk factor for spermatogenic failure. METHODS: To determine the incidence of various partial AZFc deletions and their effect on fertility, we combined quantitative and qualitative analyses of the AZFc interval at the DAZ and CDY1 loci in 300 infertile men and 399 control men. RESULTS: We detected 34 partial AZFc deletions (32 gr/gr deletions), arising from at least 19 independent deletion events, and found gr/gr deletion in 6% of infertile and 3.5% of control men (p>0.05). Our data provide evidence for two large AZFc inversion polymorphisms, and for relative hot and cold spots of unequal crossing over within the blocks of homology that mediate gr/gr deletion. Using SFVs (sequence family variants), we discriminate DAZ1/2, DAZ3/4, CDY1a (proximal), and CDY1b (distal) and define four types of DAZ-CDY1 gr/gr deletion. CONCLUSIONS: The only deletion type to show an association with infertility was DAZ3/4-CDY1a (p = 0.042), suggesting that most gr/gr deletions are neutral variants. We see a stronger association, however, between loss of the CDY1a SFV and infertility (p = 0.002). Thus, loss of this SFV through deletion or gene conversion could be a major risk factor for male infertility.

Unreliable patient identification warrants ABO typing at admission to check existing records before transfusion.

BACKGROUND AND OBJECTIVES: This study describes patient identification errors leading to transfusional near-misses in blood issued by the Alps Mediterranean French Blood Establishment (EFSAM) to Marseille Public Hospitals (APHM) over an 18-month period. The EFSAM consolidates 14 blood banks in southeast France. It supplies 149 hospitals and maintains a centralized database on ABO types used at all area hospitals. As an added precaution against incompatible transfusion, the APHM requires ABO testing at each admission regardless of whether the patient has an ABO record. The study goal was to determine if admission testing was warranted. MATERIALS AND METHODS: Discrepancies between ABO type determined by admission testing and records in the centralized database were investigated. The root cause for each discrepancy was classified as specimen collection or patient admission error. Causes of patient admission events were further subclassified as namesake (name similarity) or impersonation (identity fraud). RESULTS: The incidence of ABO discrepancies was 1:2334 including a 1:3329 incidence of patient admission events. Impersonation was the main cause of identity events accounting for 90.3% of cases. The APHM's ABO control policy prevented 19 incompatible transfusions. In relation to the 48,593 packed red cell units transfused, this would have corresponded to a risk of 1:2526. CONCLUSION: Collecting and storing ABO typing results in a centralized database is an essential public health tool. It allows crosschecking of current test results with past records and avoids redundant testing. However, as patient identification remains unreliable, ABO typing at each admission is still warranted to prevent transfusion errors.

HLA-DRB1 and DQB1 polymorphisms in southern France and genetic relationships with other Mediterranean populations.

This study presents the results of HLA-DRB1 and DQB1 sequence-specific oligonucleotide probe (SSOP) typing for a population sample of 181 individuals originating from southern France. On the basis of allele and haplotype frequencies, we compared our population with others from the Mediterranean area. Allele frequencies are comparable to those found in other western European populations (France, Portugal, Spain) and indicate neighboring exchanges. The haplotype frequencies showed relationships with North Africans and Jewish populations, as well as the common origin of Moroccan and Lebanese Jews. Therefore, allele frequencies seem to be more able to show recent exchanges while haplotype frequencies might show ancestral relationships. These results may serve as references for future studies of HLA and disease in southern France.