A comparative study of two routinely used protocols for ex vivo erythroid differentiation.

BACKGROUND: Erythropoiesis is a complex developmental process in which a hematopoietic stem cell undergoes serial divisions and differentiates through well-defined stages to give rise to red blood cells. Over the last decades, several protocols have been developed to perform ex vivo erythroid differentiation, allowing investigation into erythropoiesis and red cell production in health and disease. RESULTS: In the current study, we compared the two commonly used protocols by assessing the differentiation kinetics, synchronisation, and cellular yield, using molecular and cellular approaches. Peripheral blood CD34+ cells were cultured in a two-phase (2P) or a four-phase (4P) liquid culture (LC) and monitored for 20 days. Both protocols could recapitulate all stages of erythropoiesis and generate reticulocytes, although to different extents. Higher proliferation and viability rates were achieved in the 4P-LC, with a higher degree of terminal differentiation and enucleation, associated with higher levels of the erythroid-specific transcription factors GATA-1, KLF-1, and TAL-1. Although the 2P-LC protocol was less efficient regarding terminal erythroid differentiation and maturation, it showed a higher yield of erythroid progenitors in the erythropoietin (EPO)-free expansion phase. CONCLUSIONS: We provide data supporting the use of one protocol or the other to study the biological processes occurring in the early or late stages of erythroid differentiation, depending on the physiological process or pathological defect under investigation in a given study.

First description of a D-CE-D hybrid gene on a weak D Type 2 molecular background.

BACKGROUND: RhD phenotypes that express a significantly reduced amount of RhD antigen per red blood cell may be mistyped as RhD-negative by standard serologic methods. The molecular identification of weak D Type 1, 2, or 3 carriers allows managing them as RhD-positive and, thus, rationalizes the use of RhD-negative stock units and the administration of Rh-immunoglobulin prophylaxis, avoiding unnecessary costs and possible side effects. STUDY DESIGN AND METHODS: One sample was investigated for confirming a D-C-E+c+e- phenotype. Rh phenotyping was performed with the microplate direct hemagglutination test. DNA array analysis was performed using the BeadChip wRhD kit, and the RHD gene was explored by sequencing to determine the molecular background associated with RhD-negative phenotype. RESULTS: Molecular investigations showed a lack of amplification of Exons 3 through 7 and c.1154G>T transversion in Exon 9, suggesting an RHD-CE-D composite on a weak D Type 2 background. We attempted to precisely identify the two recombination sites generating this hybrid allele. The 5' and 3' breakpoints were located in Introns 2 and 7, which showed concentration of mobile Alu sequences most likely involved in the RHD-cE(3-7)-weak D Type 2 allele. CONCLUSION: Altogether, we identified the first example of an RHD-CE-D large hybrid allele on a weak D Type 2 background associated with an RhD-negative phenotype. By investigating the RHCE-D breakpoint zones, we suggest a mobile element-mediated recombination.

New KEL*01M and KEL*02M alleles: structural modeling to assess the impact of amino acid changes.

BACKGROUND: The KELL antigens are carried by the well-folded and highly polymorphic glycoprotein KELL, belonging to the M13 family of metalloproteases. Anti-KEL, particularly anti-KEL1, are clinically significant. We retrospectively investigated genomic DNA from samples with uncertain KEL1 or KEL2 phenotype and identified six novel Kmod alleles. We then considered a model of the protein three-dimensional (3D) structure to assess the impacts of the amino acid changes. STUDY DESIGN AND METHODS: The 19 exons of the KEL gene were polymerase chain reaction amplified and sequenced. Modeling was performed using the experimental 3D structure of human endothelin-converting enzyme-1 in the presence of the metabolite phosphoramidon. RESULTS: We identified four novel KEL*01M alleles with amino acid substitutions p.Arg447Trp, p.Gly641Arg, p.Ala645Val, and p.Gly703Arg found buried within helices of the ectodomain catalytic lobe. We also revealed one new KEL*02M allele with p.Gly263Glu in contact with solvent (water) located within the second lobe of the ectodomain. One sample with c.575G>C transversion (p.Arg192Pro) on a KEL*02 background showed a weakened reactivity for KEL1. According to our 3D modeling, these amino acid substitutions may have a profound impact on the protein structure. CONCLUSION: This study is especially interesting with regard to the description of four new KEL*01M alleles. Indeed, to date only two KEL*01M alleles have been described and our data suggest a nonnegligible incidence of KEL1 variants. Serologic KEL2-negative results as well as any ambiguity implying either KEL1 or KEL2 in donors should always be confirmed by means of genotyping analysis and discrepancies between these methods require sequencing of KEL gene.

Heterogeneity of alleles encoding high- and low-prevalence red blood cell antigens across Africa: useful data to facilitate transfusion in African patients.

Ethnic variations in red blood cell (RBC) antigens can be a source of alloimmunization, especially in migrant populations. To improve transfusion safety in continental Africa and countries with African migrants, we performed RBC genotyping to determine allele frequencies coding for high- and low-prevalence antigens. A total of 481 blood samples were collected in ethnic groups from West, Central and East Africa. Molecular typing was performed using a polymerase chain reaction - reverse sequence specific oligonucleotide method. Results demonstrated no DI*1, DI*3, YT*2, SC*2, LW*7, KN*2 alleles in any sample and the CO*2 allele was rare. The frequency of LU*1 was comparable to that of European-Caucasians (2%) except in Biaka pygmies (8%). The frequency of CROM*-1 was high in Mbuti pygmies (13%). High frequency of KN*7 and KN*6 may reflect selection pressure in the countries investigated. Analysis of Dombrock allele patterns confirmed uneven distribution of the DO*1 and DO*2 alleles with high frequencies of DO*-4 and DO*-5 in all groups. Altogether, findings demonstrated extensive allele-frequency heterogeneity across Africa and suggested that knowledge of patient ethnicity gives information about the high-prevalence antigens that may be lacking. These data are medically useful to support transfusion care of African migrants living in countries where the majority of the population is from a different ethnical background.

A comprehensive survey of both RHD and RHCE allele frequencies in sub-Saharan Africa.

BACKGROUND: The RH system is one of the most polymorphic blood group systems with numerous allele variants affecting Rh polypeptides expression. This complexity is at the origin of difficulties for transfusion of African patients especially sickle cell disease patients requiring chronic transfusion therapy with high risk of immunization. As a complete survey of RH variants is lacking in African populations, we performed red blood cell genotyping to determine the type and frequency of RHD and RHCE alleles in sub-Saharan African populations. STUDY DESIGN AND METHODS: A total of 347 blood samples were collected from individuals of six nonpygmoid and three pygmoid populations. RH typing was performed using two single-tube multiplex polymerase chain reaction amplifications (BioArray Solutions, Immucor). RESULTS: All six sub-Saharan nonpygmoid populations exhibited constant variety in both type and frequency of aberrant RHD and RHCE alleles. Predicted partial RH1 (1.8%) and RH5 (0.9%) phenotypes were less than expected. Conversely, predicted partial phenotype RH2 (5.5%) was frequent. Data confirmed the high frequency of samples positive for the non-clinically significant RH10/RH20 antigens (39.5%) and revealed a high frequency of RH54 (DAK, 8.1%). The pygmoid groups showed higher percentages of predicted partial RH antigens and greater heterogeneity reflecting wide genetic differentiation. CONCLUSION: Our data show that frequencies of aberrant RHD and RHCE alleles were similar, irrespective of location and ethnicity. In view of the predicted frequencies and relative clinical significance of both private antigens and high-prevalence antigens absent, the most relevant assays for individuals of African descent in a transfusion setting are for 1) partial RH2 in the patient and 2) RH54 (DAK) in the donor.

Molecular analysis of inactive and active RHD alleles in native Congolese cohorts.

BACKGROUND: In Africa, RHD alleles have not been fully characterized. The purpose of this study was to identify inactive and active RHD alleles at the molecular level in Congolese cohorts. STUDY DESIGN AND METHODS: Blood samples were collected from people living in central Congo populated by Teke ethnic group. A total of 110 D- and 40 D+ samples from Congo-Brazzaville and Teke groups, respectively, were selected for RHD genotyping using allele-specific primer polymerase chain reaction and sequencing. RESULTS: In the 110 D- samples, RHD exon amplifications were observed in 7 samples that were subsequently identified by sequencing as weak D type 4 variants. In the remaining 103 D- samples, the frequencies of RHD gene deletion, RHDpsi pseudogene, and RHD-CE-D(s) hybrid gene were 0.75685, 0.20560, and 0.04468, respectively. In the D+ samples, 26 individuals carried at least a regular RHD gene; 9 carried aberrant RHD alleles belonging to the African D clusters, that is, DAU, DIVa, and weak D type 4; 3 carried RHDpsi in trans with a DAU allele including one novel RHD allele (V279M, S333N, T379M) named DAU-7; and 2 others were partially determined. CONCLUSION: This study revealed a high frequency of weak D type 4 alleles that confirmed the need to use indirect antiglobulin test to improve transfusion safety in the Congo and in countries hosting Congolese people. Findings also indicated that there is a geographic variation in RHD allele distribution and showed that RHD gene deletion is the most prevalent cause of the D- phenotype in the Congolese population.

DNA-based typing of Kell, Kidd, MNS, Dombrock, Colton, and Yt blood group systems in the French Basques.

The Basques demonstrate peculiar characteristics regarding blood group systems. Although ABO, Rhesus, and Duffy have been extensively studied in this population, the distribution of other groups remains largely unknown. Therefore, we evaluated the frequency of less-explored- or still noninvestigated blood groups using DNA-based assays and interpreted these data in the view of population genetics. Polymorphisms of KEL (Kell), SLCA14A1 (Kidd), GYPA/GYPB (MNS), ART4 (Dombrock), AQP1 (Colton), and ACHE (Yt) blood group genes were determined from a sample of more than 100 autochthonous French Basques using allele-specific primer PCR (PCR-ASP) methods. Our results were compared with those previously obtained by the use of serology from both Basque and non-Basque European populations. MNS*1 and JK*1 allele frequencies were comparable with those reported from Basque samples. Conversely, the KEL*1 allele frequency differed significantly. To our knowledge, this is the first time that the other three systems are studied in the Basque population. DO*1 and CO*1 allele frequencies, being respectively 0.35 and 0.96, were significantly inferior to those published from various European populations. There were some discrepancies regarding these six blood systems when comparing molecular typing with serology. These findings may be explained by differences in either criteria for individual selection or technical assays. Nevertheless, these results constitute additional data to be included in the chapter of Basque biological anthropology.