The D typing strategies in several European countries protect carriers of D category VI (DVI) from anti-D immunization but not carriers of other partial D. Besides DVI, one of the clinically most important partial D is D category IV (DIV). A detailed description and direct comparison of the different DIV types was missing.
Study design and methods
RHD nucleotide sequences were determined from genomic DNA. D epitope patterns were established with commercial monoclonal anti-D panels.
DIV comprises several variants of the D antigen with distinct serology, molecular structures, evolutionary origins and ethnic prevalences. The DIV phenotype is determined by 350H shared by all, but not limited to, DIV variants which are further divided into DIVa and DIVb. The DIVa phenotype is expressed by DIV type 1.0 harboring 350H and the dispersed amino acids 62F, 137V and 152T. The DIVb phenotype is expressed by DIV type 3 to type 5 representing RHD-CE-D hybrids. 4 of the 6 postulated DIV variants were encountered among 23 DIV samples analyzed. Of 12 DIV carriers with anti-D, 10 were female and 7 likely immunized by pregnancy. 2 DIV related alleles are newly described: DWN which differs from DIV type 4 by 350D and epitope pattern. DNT carries 152T, known to cause a large D antigen density.
DIV alleles arose from at least 2 independent evolutionary events. DIV type 1.0 with DIVa phenotype belongs to the oldest extant human RHD alleles. DIV type 2 to type 5 with DIVb phenotype arose from more recent gene conversions. Anti-D immunization, especially dreaded in pregnancies, will be avoided not only in carriers of DVI but also in carriers of other D variants like DIV, if our proposed D typing strategy is adopted.
The numerous antigens in the Kell blood group system result from missense nucleotide changes in KEL. Antibodies to antigens in this system can be clinically important. We describe six probands whose plasma contained antibodies to high-prevalence Kell antigens, and discuss their relationship.
Study design and methods
PCR amplification, direct sequencing, RFLP assays, hemagglutination, flow cytometry, and protein modeling were performed by standard methods.
Proband 1 (KUCI), and her serologically-compatible sister, were heterozygous for a nucleotide change in exon 11 (KEL*1271C/T; Ala424Val). Proband 2 (KANT) was heterozygous for KEL*1283G/T (Arg428Leu) and KEL*1216C/T (Arg406Stop) in exon 11. RBCs from Proband 1 and her sister were not agglutinated by plasma from Proband 2; however, RBCs from Proband 2 were agglutinated by plasma from Proband 1. Probands 3, 4, 5, and 6 had the KEL*1391C>T change associated with the previously reported KETI− phenotype. Proband 5 was also homozygous for KEL*905T>C encoding the K11−K17+ phenotype. Hemagglutination studies revealed an association between KUCI, KANT, KETI and K11. Protein modeling indicated that whereas Ala424 and Arg428 are clustered, Val302 and Thr464 are not.
Ala424 in the Kell glycoprotein is associated with the high-prevalence Kell antigen, KUCI (ISBT 006032), which is detected by the antibody of Proband 1. Arg428 is associated with the high-prevalence Kell antigen, KANT (ISBT 006033). The association between KUCI, KANT, KETI, and K11, and the results of protein modeling are discussed.
Genotyping for red blood cell (RBC), platelet and granulocyte antigens is a new tool for clinical pathology, transfusion medicine services and blood banks. Proficiency in laboratory tests can be established by external quality assessments (EQAs), which are required for clinical application in many health care systems. There are few EQAs for molecular immunohematology.
We analyzed the participation and pass rates in an EQA for RBC, platelet and granulocyte antigens. This EQA was distributed by INSTAND, a large non-profit provider of proficiency tests, twice per year since fall 2006 as EQA no. 235 Immunohematology A (molecular diagnostic). The coordinators defined at the outset which alleles are mandatory for detection.
The number of participants steadily increased from 51 to 73 per proficiency by fall 2012. More than 60 institutions utilized this EQA at least once a year. Approximately 80% of them participated in RBC, 68% in platelet and 22% in granulocyte systems. With the exceptions of RHD (82%) and granulocytes (85%), pass rates exceeded 93%. While the pass rate increased for granulocyte and decreased for the ABO system, the pass rates for the other systems changed little over 6 ½ years.
The INSTAND proficiency test program was regularly used for EQA by many institutions, particularly in Central Europe. While the technical standards and pass rates in the participating laboratories were high, there has been little improvement in pass rates since 2006.
The Rhesus Site is a resource for information of the ‘Rhesus’ blood group. It is intended for specialists and non-specialists. The website details research in the field relevant for transfusion medicine, immunohematology, and molecular research. Link areas guide to important publications and to methodological resources for Rhesus. Many data originally presented at The Rhesus Site have been formally published later. The ‘RhesusBase’ section represents the largest database for RHD alleles; the ‘RhesusSurveillance’ section details the results of the largest prospective observational study on anti-D immunization events in D-positive patients. Visitors to the website are encouraged to explore the intricacies of the most complex blood group gene locus.
Rh-Hr blood group system; Antigen D; Rh blood group; Databases as topic; Immunization
transplantation; mismatching; incompatibility; D antigen; haematopoietic progenitor cell
Spray drying techniques are commonly utilized in the pharmaceutical, dairy and animal feed industries for processing liquids into powders but have not been applied to human blood products. Spray dried protein products are known to maintain stability during storage at room temperature.
Study design and methods
Plasma units collected at the donor facility were shipped overnight at room temperature to a processing facility where single-use spray drying occurred. After 48 hours storage at room temperature, the spray dried plasma product was split in two and rehydrated with 1.5% glycine or deionized water and assayed for chemistry analytes and coagulation factors. Matched fresh frozen plasma (FFP) was analyzed in parallel as controls.
Reconstitution was achieved for both rehydration groups within five minutes (n=6). There was no statistically significant intergroup difference in recovery for total protein, albumin, IgG, IgA, and IgM (96% or higher). With the exception of factor VIII (58%), the recovery of clotting factors in the glycine reconstituted products ranged from 72% to 93%. Glycine reconstitution was superior to deionized water.
We documented proteins and coagulation activities were recovered in physiologic quantities in reconstituted spray dried plasma products. Further optimization of the spray drying method and reconstitution fluid may result in even better recoveries. Spray drying is a promising technique for preparing human plasma that can be easily stored at room temperature, shipped, and reconstituted. Rapid reconstitution of the microparticles results in a novel plasma product from single donors.
Red blood cell transfusion is the principal therapy in patients with severe thalassaemias and haemoglobinopathies, which are prevalent in Thailand. Serological red blood cell typing is confounded by chronic transfusion, because of circulating donor red blood cells. We evaluated the concordance of serological phenotypes between a routine and a reference laboratory and with red cell genotyping.
Materials and methods
Ten consecutive Thai patients with β-thalassemia major who received regular transfusions were enrolled in Thailand. Phenotypes were tested serologically at Songklanagarind Hospital and at the National Institutes of Health. Red blood cell genotyping was performed with commercially available kits and a platform.
In only three patients was the red cell genotyping concordant with the serological phenotypes for five antithetical antigen pairs in four blood group systems at the two institutions. At the National Institutes of Health, 32 of the 100 serological tests yielded invalid or discrepant results. The positive predictive value of serology did not reach 1 for any blood group system at either of the two institutions in this set of ten patients.
Within this small study, numerous discrepancies were observed between serological phenotypes at the two institutes; red cell genotyping enabled determination of the blood group when serology failed due to transfused red blood cells. We question the utility of serological tests in regularly transfused paediatric patients and propose relying solely on red cell genotyping, which requires training for laboratory personnel and physicians. Red cell genotyping outperformed red cell serology by an order of magnitude in regularly transfused patients.
blood group; red cell genotyping; dry matching; thalassaemia; Thailand
Although transmission of Plasmodium falciparum (Pf) infection during red blood cell transfusion from an infected donor has been well documented, malaria parasites are not known to infect hematopoietic stem cells. We report a case of Pf infection in a patient 11 days after peripheral blood stem cell transplant for sickle cell disease.
Study Design and Methods
Malaria parasites were detected in thick blood smears by Giemsa staining. Pf HRP2 antigen was measured by ELISA on whole blood and plasma. Pf DNA was detected in whole blood and stem cell retention samples by real-time PCR using Pf species–specific primers and probes. Genotyping of 8 Pf microsatellites was performed on genomic DNA extracted from whole blood.
Pf was not detected by molecular, serologic or parasitologic means in samples from the recipient until day 11 post-transplant, coincident with the onset of symptoms. In contrast, Pf antigen was retrospectively detected in stored plasma collected 3 months prior to transplant from the asymptomatic donor. Pf DNA was detected in whole blood from both the donor and recipient post-transplant, and genotyping confirmed shared markers between donor and recipient Pf strains. Look back analysis of red blood cell donors was negative for Pf infection.
These findings are consistent with transmission by the stem cell product and have profound implications with respect to the screening of potential stem cell donors and recipients from malaria-endemic regions.
Plasmodium falciparum; Sickle Cell Disease; Peripheral Blood Stem Cell Transplant; Real-Time PCR; PfHRP2 Antigen ELISA
Hemolytic reactions (HTRs) can occur from ABO-incompatible platelet transfusions. After a series of cases at our institution, a procedure to screen all plateletpheresis donors for high-titer ABO antibodies was implemented.
Study design and methods
Plasma samples from plateletpheresis donors were screened using pooled 0.8% A1 and 0.8% B RBC in buffered gel. Dilutions of 1 in 150, 1 in 200, and 1 in 250 were sequentially evaluated. A component testing positive for high-titer ABO antibodies is restricted to ABO-identical or group O recipients, or washed.
At the initial dilution of 1 in 150, half of group O components were labeled as high-titer. At the current dilution of 1 in 250, 25% of group O components are labeled as high-titer. No platelet-associated HTR has been reported since screening began.
Universal screening for high-titer ABO antibodies in plateletpheresis donors can be implemented efficiently to reduce the risk of HTRs. The cutoff for classifying a unit as high-titer depends on the serologic method used, and may be customized by the individual facility. Our screening method uses one gel test per donation regardless of blood group, and a plasma dilution of 1 in 250 with pooled A1/B RBCs in buffered gel.
The DARC (Duffy blood group, chemokine receptor) gene encodes for a transmembrane glycoprotein that functions as a chemokine transporter, is a receptor for Plasmodium vivax and knowlesi, and expresses the Duffy blood group antigens (Fy). The Fy(a−b−) phenotype found in people of African descent is typically associated with a −67t>c mutation in the 5′ untranslated region (UTR), which prevents red blood cells being invaded by Plasmodium vivax and knowlesi. The aim of this study was to establish DARC allele frequencies in an African American blood donor cohort, determine a phylogenetic tree for DARC, and compare human and Neandertal DARC genes.
The DARC nucleotide sequence of 54 African American blood donors was determined from genomic DNA. Heterozygous substitutions were resolved by sequencing of haplotype specific amplifications. A phylogenetic tree for DARC was established using the neighbor-joining method with Pan troglodytes as root.
108 haplotypes of the DARC gene could be unambiguously determined from nucleotide position −300 in the 5′ UTR to +300 in the 3′ UTR. 11 different alleles were found, including the clinically relevant FY*A, FY*B, FY*B-67C, FY*B298A, and FY*X alleles. All phenotype predictions based on genotypes matched exactly the serologically determined phenotypes: 52% Fy(a−b−), 28% Fy(a−b+), and 20% Fy(a+b−).
The nucleotide sequencing approach using one amplicon is a practical genotyping method for DARC and allows the determination of haplotypes even in heterozygous constellations. We developed a phylogenetic tree for DARC alleles and postulated a distinct FY*B allele as ancestral for the extant DARC alleles in humans.
The human neutrophil antigen-3 (HNA-3) epitopes reside on the choline transporter-like protein-2 (CTL2). A single-nucleotide substitution (461G>A; Arg154Gln) on the CTL2 gene (SLC44A2) defines the allele SLC44A2*1, which expresses HNA-3a, and SLC44A2*2, which expresses HNA-3b; an additional substitution (457C>T; Leu153Phe) in SLC44A2*1:2 may impact genotyping systems. People who only express HNA-3b may develop anti-HNA-3a. These alloantibodies have been linked to severe transfusion-related acute lung injury (TRALI), which may be a reason to screen blood donors for SLC44A2*2 homozygosity. For Caucasian and Asian populations, SLC44A2 allele frequencies are known. Our primary objective was to determine the SLC44A2 allele frequencies in the African American population.
Study design and methods
Purified DNA from 334 individuals (202 male, 132 female; 241 African American, 93 Caucasian) was collected. Two real-time PCR assays were developed to genotype all samples; results were confirmed by nucleotide sequencing.
In 241 African American donors, the allele frequency of SLC44A2*1 was 93% (85% to <100%; 95% confidence intervals, Poisson distribution) while SLC44A2*2 was 7% (5% to 10%). In 93 Caucasian donors, the allele frequencies of SLC44A2*1 was 83% (71% to 98%) and SLC44A2*2 was 17% (11% to 24%), matching previously reported data for Caucasians but differing from African Americans (p<0.001, Fisher’s exact test).
This study describes the allele frequencies of the 3 known HNA-3 variants in an African American population. We found that African Americans have a significantly lower probability of possessing the SLC44A2*2 allele, and may thus be less likely to form the clinically relevant anti-HNA-3a.
Red blood cell (RBC) preservation is essential to transfusion medicine. Many blood group reference laboratories need a method to preserve rare blood samples for serologic testing at a later date. This study offers a comparison of three common cryoprotective agents and protocols used today: bulk preservation with glycerol and droplet freezing with sucrose/dextrose (S+D) or polyvinyl pyrrolidone (PVP).
Study design and methods
Human blood from 14 volunteers was collected and frozen at set intervals over two weeks with PVP, S+D, or glycerol. The frozen RBCs were later thawed and the percentage of surviving RBCs was determined. Detailed protocols and an instructional video are supplied.
Over a two week period, RBCs preserved with glycerol and thawed with a widely used protocol showed a recovery of 41 ± 16 % (mean ± standard deviation) while those thawed with a modified glycerol protocol showed a recovery of 76 ± 8 %. RBCs preserved by droplet freezing with S+D showed a recovery of 56 ± 11 % while those preserved by droplet freezing with PVP showed a recovery of 85 ± 6 %. Recovery values were similar with ethylenediaminetetraacetic acid (EDTA) or heparin anticoagulants, differing freezing rates, and varying droplet volumes.
Droplet freezing with PVP offered the greatest recovery. While bulk freezing with glycerol can be effective too, droplet freezing may be a more convenient method overall. It requires less effort to thaw, needs much less storage room, and allows blood group laboratories to be frugal with thawing rare samples.
Specialists in Blood Bank (SBB) Technology play important roles in blood banks, transfusion services, regulatory agencies, educational institutions and other facilities where expertise in blood banking, transfusion medicine, cellular therapy, and tissue transplantation is required.
Review of pathways that qualify applicants for a national examination administered by the American Society of Clinical Pathology (ASCP) to become a certified specialist and outcomes of accredited programs. Description of a face-to-face, accredited program including review of management topics included in curriculum.
The first examination was administered in 1954. As of December 2009, the total number certified SBBs was 5,124. There are currently 16 accredited SBB programs in the United States. The programs vary in mode of delivery, length of program, number of students accepted and organization of program officials and faculty but all must follow specific standards and guidelines in order to be accredited.
Students who successfully complete SBB programs have a higher passing rate than those who attempt the certification examination and have not participated in a program. Students can choose among a variety of programs that differ widely in the way they are managed. The role of management in an SBB program ranges from attracting and retaining individuals, to maintaining an accredited program to finally graduating individuals who not only pass the certification examination but who also confidently contribute to the field.
Incompatible blood group antigens are highly immunogenic and can cause graft rejections. Focusing on distinct carbohydrate- and protein-based membrane structures, defined by blood group antigens, we investigated human bone marrow-derived mesenchymal stem cells (MSCs) cultured in human serum. The presence of H (CD173), ABO, RhD, RhCE, RhAG, Kell, urea transporter type B (SLC14A1, previously known as JK), and Duffy antigen receptor of chemokines (DARC) was evaluated at the levels of genome, transcriptome and antigen. Fucosyltransferase-1 (FUT1), RHCE, KEL, SLC14A1 (JK) and DARC mRNA were transcribed in MSCs. FUT1 mRNA transcription was lost during differentiation. The mRNA transcription of SLC14A1 (JK) decreased during chondrogenic differentiation, while that of DARC increased during adipogenic differentiation. All MSCs synthesized SLC14A1 (JK) but no DARC protein. However, none of the protein antigens tested occurred on the surface, indicating a lack of associated protein function in the membrane. As A and B antigens are neither expressed nor adsorbed, concerns of ABO compatibility with human serum supplements during culture are alleviated. The H antigen expression by GD2dim+ MSCs identified two distinct MSC subpopulations and enabled their isolation. We hypothesize that GD2dim+H+ MSCs retain a better “stemness”. Because immunogenic blood group antigens are lacking, they cannot affect MSC engraftment in vivo, which is promising for clinical applications.
stem cell transplantation; mesenchymal cells; blood groups; H antigen; CD173
Rhesus is the clinically most important protein-based blood group system. It represents the largest number of antigens and the most complex genetics of the 30 known blood group systems. The RHD and RHCE genes are strongly homologous. Some genetic complexity is explained by their close chromosomal proximity and unusual orientation, with their tail ends facing each other. The antigens are expressed by the RhD and the RhCE proteins. Rhesus exemplifies the correlation of genotype and phenotype, facilitating the understanding of general genetic mechanisms. For clinical purposes, genetic diagnostics of Rhesus antigens will improve the cost-effective development of transfusion medicine.
Rhesus; blood group; molecular genetics; molecular diagnostic; transfusion
Codon usage in genomes is biased towards specific subsets of codons. Codon usage bias affects translational speed and accuracy, and it is associated with the tRNA levels and the GC content of the genome. Spontaneous mutations drive genomes to a low GC content. Active cellular processes are needed to maintain a high GC content, which influences the codon usage of a species. Loss-of-function mutations, such as nonsense mutations, are the molecular basis of many recessive alleles, which can greatly affect the genome of an organism and are the cause of many genetic diseases in humans.
We developed an event based model to calculate the risk of acquiring nonsense mutations in coding sequences. Complete coding sequences and genomes of 40 eukaryotes were analyzed for GC and CpG content, codon usage, and the associated risk of acquiring nonsense mutations. We included one species per genus for all eukaryotes with available reference sequence.
We discovered that the codon usage bias detected in genomes of high GC content decreases the risk of acquiring nonsense mutations (Pearson's r = -0.95; P < 0.0001). In the genomes of all examined vertebrates, including humans, this risk was lower than expected (0.93 ± 0.02; mean ± SD) and lower than the risk in genomes of non-vertebrates (1.02 ± 0.13; P = 0.019).
While the maintenance of a high GC content is energetically costly, it is associated with a codon usage bias harboring a low risk of acquiring nonsense mutations. The reduced exposure to this risk may contribute to the fitness of vertebrates.
The Bloodgen project was funded by the European Commission between 2003 and 2006, and involved academic blood centres, universities, and Progenika Biopharma S.A., a commercial supplier of genotyping platforms that incorporate glass arrays. The project has led to the development of a commercially available product, BLOODchip, that can be used to comprehensively genotype an individual for all clinically significant blood groups. The intention of making this system available is that blood services and perhaps even hospital blood banks would be able to obtain extended information concerning the blood group of routine blood donors and vulnerable patient groups. This may be of significant use in the current management of multi-transfused patients who become alloimmunised due to incomplete matching of blood groups. In the future it can be envisaged that better matching of donor-patient blood could be achieved by comprehensive genotyping of every blood donor, especially regular ones. This situation could even be extended to genotyping every individual at birth, which may prove to have significant long-term health economic benefits as it may be coupled with detection of inborn errors of metabolism.
BLOODchip; Blood groups; Blood group antigens