FORSCells: 40-days fixed prepared reagent for detection of anti-Forssman in humans.

In 2012, the FORS system was accepted by the International Society of Blood Transfusion as the 31st blood group system. Forssman (Fs) antigen (Ag) expression is most commonly found on sheep red blood cells (RBC) but rare in human RBC. Anti-Fs antibodies (Ab) are naturally occurring in human sera and are predominantly IgM but they can also be IgG. To this day, the global prevalence of the FORS system is unknown. Currently, there is a lack of natural FORS1-positive RBC available to use for anti-Fs screening in large populations. This study was designed to produce FORS1-positive cells viable for 40 days use in the anti-Fs screening. Three to 5% FORS1-positive cells were produced using sheep's blood and CellStab stabilizer solution. The quality of the FORS1-positive cells was investigated in more than three independent experiments of ABO titration, osmotic fragility test and supernatant haemolysis. For each batch of FORS1-positive cells produced, an extended antibody panel was performed. To demonstrate that the FORS1-positive cells can be used for up to 40 days, anti-Fs screening and classification were carried out in a patient and donor population. Antigenic expression and membrane integrity of FORS1-positive cells remained stable for 40 days. Good FORS1 Ag preservation was established, and minimal haemolysis was observed. In conclusion, a novel and easy-to-produce reagent has been developed and submitted to a patent with stable FORS1 Ag expression. With this FORS1-positive cell suspension, it is now possible to screen and classify anti-Fs Ab in large populations.

A plate-based histo-blood group antigen binding assay for evaluation of human norovirus receptor binding ability.

Human norovirus is a leading cause of gastroenteritis worldwide. Although two in vitro cultivation methods have been reported, they cannot provide mechanistic insights into viral inactivation. Receptor-binding assays supplement these assays and give insight into capsid integrity. We present a streamlined version of a receptor-binding assay with minimal time-to-result while maintaining accuracy and high throughput. We validate assay performance for physical and chemical inactivation treatments of a norovirus GII.4 capsid. The assay produces a high positive/negative ratio (25.3 ± 4.9) in <2.5 h and has a limit of detection of 0.1 µg/ml capsid. This method is a valuable additional tool for understanding human norovirus inactivation.

Evolution of technology for molecular genotyping in blood group systems.

The molecular basis of the blood group antigens was identified first in the 1980s and 1990s. Since then the importance of molecular biology in transfusion medicine has been described extensively by several investigators. Molecular genotyping of blood group antigens is one of the important aspects and is successfully making its way into transfusion medicine. Low-, medium- and high-throughput techniques have been developed for this purpose. Depending on the requirement of the centre like screening for high- or low-prevalence antigens where antisera are not available, correct typing of multiple transfused patients, screening for antigen-negative donor units to reduce the rate of alloimmunization, etc. a suitable technique can be selected. The present review discusses the evolution of different techniques to detect molecular genotypes of blood group systems and how these approaches can be used in transfusion medicine where haemagglutination is of limited value. Currently, this technology is being used in only a few blood banks in India. Hence, there is a need for understanding this technology with all its variations.

Selective capture of human red blood cells based on blood group using long-range surface plasmon waveguides.

An optical biosensor based on long-range surface plasmon-polariton waveguides is applied to the detection of blood group antigen A on whole erythrocytes. The biosensor consists of straight gold waveguides embedded in CYTOP with an etched fluidic channel. The gold waveguides were functionalized with immunoglobulin G against blood group A (anti-A IgG) by forming a self-assembled monolayer (SAM) of 16-mercaptohexadecanoic acid (16-MHA) and then conjugating the anti-A IgG through carbodiimide chemistry. In order to demonstrate anti-A surface selectivity, solutions of O-type, B-type, A-type and AB-type red blood cells (RBCs) were sequentially injected over an anti-A functionalized waveguide. Surfaces were regenerated by lysing attached cells with distilled/deionized water (DDI H2O). The efficiency of surface regeneration with DDI H2O was very high as determined by performing six sequential binding/regeneration cycles of A RBC capture on the same anti-A surface. Also, five solutions of different A RBC concentrations, ranging from 1.14 × 10(5)cells/ml to 1.83 × 10(6)cells/ml, were injected over an anti-A surface to determine the limit of detection (LOD), which was found to be less than 3 × 10(5)cells/ml. Finally, the response produced by a single cell bound to a waveguide was determined by relating the number of bound cells to the response produced, from which the signal-to-noise ratio for single cell detection was determined to be ~95. The waveguides are promising as simple, low-cost and compact transducers, functionalized using standard thiol-based chemistries, for the selective detection of cells.

Prevalence of Rh, Duffy, Kell, Kidd & MNSs blood group antigens in the Indian blood donor population.

BACKGROUND & OBJECTIVES: Little data are available regarding the frequencies of the blood group antigens other than ABO and RhD in the Indian population. Knowledge of the antigen frequencies is important to assess risk of antibody formation and to guide the probability of finding antigen-negative donor blood, which is especially useful when blood is required for a patient who has multiple red cell alloantibodies. This study was carried out to determine the frequencies of the D, C, c, E, e, K, k, Fy(a), Fy(b), Jk(a), Jk(b), M, N, S and s antigens in over 3,000 blood donors. METHODS: Samples from randomly selected blood donors from Delhi and nearby areas (both voluntary and replacement) were collected for extended antigen typing during the period January 2009 to January 2010. Antigens were typed via automated testing on the Galileo instrument using commercial antisera. RESULTS: A total of 3073 blood samples from donors were phenotyped. The prevalence of these antigens was found to be as follows in %: D: 93.6, C: 87, c: 58, E: 20, e: 98, K: 3.5, k: 99.97, F(a) : 87.4, Fy(b) : 57.6, Jk(a) : 81.5, Jk(b) : 67.4, M: 88.7, N: 65.4, S: 54.8 and s: 88.7. INTERPRETATION & CONCLUSIONS: This study found the prevalence of the typed antigens among Indian blood donors to be statistically different to those in the Caucasian, Black and Chinese populations, but more similar to Caucasians than to the other racial groups.

Bifidobacterial α-galactosidase with unique carbohydrate-binding module specifically acts on blood group B antigen.

Bifidobacterium bifidum is one of the most frequently found bifidobacteria in the intestines of newborn infants. We previously reported that B. bifidum possesses unique metabolic pathways for O-linked glycans on gastrointestinal mucin (Yoshida E, Sakurama H, Kiyohara M, Nakajima M, Kitaoka M, Ashida H, Hirose J, Katayama T, Yamamoto K, Kumagai H. 2012. Bifidobacterium longum subsp. infantis uses two different ß-galactosidases for selectively degrading type-1 and type-2 human milk oligosaccharides. Glycobiology. 22:361-368). The nonreducing termini of O-linked glycans on mucin are frequently covered with histo-blood group antigens. Here, we identified a gene agabb from B. bifidum JCM 1254, which encodes glycoside hydrolase (GH) family 110 α-galactosidase. AgaBb is a 1289-amino acid polypeptide containing an N-terminal signal sequence, a GH110 domain, a carbohydrate-binding module (CBM) 51 domain, a bacterial Ig-like (Big) 2 domain and a C-terminal transmembrane region, in this order. The recombinant enzyme expressed in Escherichia coli hydrolyzed α1,3-linked Gal in branched blood group B antigen [Galα1-3(Fucα1-2)Galß1-R], but not in a linear xenotransplantation antigen (Galα1-3Galß1-R). The enzyme also acted on group B human salivary mucin and erythrocytes. We also revealed that CBM51 specifically bound blood group B antigen using both isothermal titration calorimetry and a solid-phase binding assay, and it enhanced the affinity of the enzyme toward substrates with multivalent B antigens. We suggest that this enzyme plays an important role in degrading B antigens to acquire nutrients from mucin oligosaccharides in the gastrointestinal tracts.

Massive fetomaternal hemorrhage secondary to intrauterine intravascular transfusion.

BACKGROUND: Small-volume fetomaternal hemorrhage is frequently observed after intrauterine transfusion. The Kleihauer-Betke test, the reference method for identifying fetomaternal hemorrhage, cannot be used after intrauterine transfusion, because the adult red blood cells used for transfusion cannot be distinguished from maternal red blood cells. CASE: Massive fetomaternal hemorrhage secondary to intrauterine transfusion led to fetal hemorrhagic stroke. We used a method based on blood group identification in the maternal blood to confirm and to quantify fetomaternal hemorrhage. CONCLUSION: Fetal stroke may result from severe hypovolemia and low cerebral blood flow caused by fetomaternal hemorrhage, rather than from fetal anemia itself.

Isolation and purification of blood group antigens using immuno-affinity chromatography on short monolithic columns.

Monolithic columns have gained increasing attention as stationary phases for the separation of biomolecules and biopharmaceuticals. In the present work the performance of monolithic convective interaction media (CIM(®)) chromatography for the purification of blood group antigens was established. The proteins employed in this study are derived from blood group antigens Knops, JMH and Scianna, equipped both with a His-tag and with a V5-tag by which they can be purified. In a first step a monoclonal antibody directed against the V5-tag was immobilized on a CIM(®) Disk with epoxy chemistry. After this, the immobilized CIM(®) Disk was used in immuno-affinity chromatography to purify the three blood group antigens from cell culture supernatant. Up-scaling of the applied technology was carried out using CIM(®) Tubes. In comparison to conventional affinity chromatography, blood group antigens were also purified via His-tag using a HiTrap(®) metal-affinity column. The two purifications have been compared regarding purity, yield and purification speed. Using the monolithic support, it was possible to isolate the blood group antigens with a higher flow rate than using the conventional bed-packed column.

Human blood group genes 2010: chromosomal locations and cloning strategies revisited.

Thirty human blood group systems are now recognized. Corresponding genes have been cloned and characterized for all of the systems and localized to single cytogenetic bands on 14 autosomes and the X chromosome. In this review, we summarize this information, highlighting the most recently defined blood group system (Rh-associated glycoprotein) and the developing understanding of the P1 system and the complex molecular basis for its phenotypes.

A review of the Chido/Rodgers blood group.

The C4 protein plays an important role in maintaining health and, in some situations complicated by poor expression of the C4 protein, may lead to or exacerbate certain diseases. The blood groups Chido and Rodgers are epitopes on the C4 protein, and polymorphisms associated with these epitopes may lead to the formation of antibodies to the Chido or Rodgers antigens in transfused patients. Identification of anti-Ch or anti-Rg is still based on the antibody neutralization with plasma from Ch-positive or Rg-positive individuals and lack of reactivity with qualified Ch-negative or Rg-negative RBCs. These antibodies may be useful in genetic studies of C4 polymorphisms or, in the case of C4-deficient patients, a signal of the potential for serious illnesses. The recognition of the extreme polymorphism of the C4 gene and the gene complex RCCX should lead to more insights in the understanding of disease risk and potential treatment.

Blood group genotyping by high-throughput DNA analysis: application to the French panel of RBC reagents.

BACKGROUND: The use of blood group genotyping for the prediction of antigen expression has been discussed in clinical transfusion settings, but much less for reagent red blood cells selection. In France, the Centre National de Référence pour les Groupes Sanguins (CNRGS) produces a reference panel of reagent red blood cells, mainly used for red cell antibody identification. The use of high-throughput DNA analysis has never been applied to blood donors whose red blood cells are used as reagents. The aim of this study was to compare the serological phenotype and that predicted from DNA analysis in such donors, and to determine the benefit of DNA analysis in reagent red blood cells selection strategy. STUDY DESIGN AND METHOD: Red blood cells of 346 blood donors were typed with two different reagents for each antigen. The genotyping was performed by using HEA v1.2 BeadChips, BioArray Solutions, Immucor. The comparison between the serologically determined phenotype and that predicted from DNA analysis held on 8876 paired results obtained from 10 blood group systems and 25 antigens. RESULTS: A 99.95% concordance was observed. Four cases of discrepancy for RH, KEL, LU and DO blood group systems were analyzed. Genotyping precisions were of special interest for the Duffy blood group system. CONCLUSION: Systematic DNA analysis brings important information on reagent red blood cells selection. It can be used at a routine level. Especially, the notion of "antigen of double dose" which is specified in several countries by government bodies should evolve regarding data obtained from DNA analysis. This should improve the quality of reagent red blood cells as first step for antibody identification.

High molecular weight blood group A trisaccharide-polyacrylamide glycoconjugates as synthetic blood group A antigens for anti-A antibody removal devices.

Specific immunoadsorption of blood group antibodies by synthetic antigens immobilized on support matrices in the peri-transplantation period provides a promising solution to hyperacute rejection risk following ABO-incompatible transplantation. In this study, we investigated binding interactions between anti-A antibodies and synthetic blood group A trisaccharide conjugated with polyacrylamide of different molecular weights (30 and 1000 kDa). The glycopolymers were equipped with biotin tags and deposited on streptavidin-coated sensor chips. The affinity and kinetics of anti-A antibodies binding to glycoconjugates were studied using surface plasmon resonance (SPR). The high molecular weight conjugate (Atri-PAA(1000)-biotin) enhanced antibody binding capacity by two to three fold compared with the low molecular weight conjugate (Atri-PAA(30)-biotin), whereas varying the carbohydrate content in Atri-PAA(1000)-biotin (20 mol % or 50 mol %) did not affect antibody binding capacity of the glycoconjugate. The obtained results suggest that immunoadsorption devices, especially hollow fiber-based antibody filters which are limited in available surface area for antigen immobilization, may greatly benefit from the new synthetic high molecular weight polyacrylamide glycoconjugates.

Identification and characterization of major proteins carrying ABO blood group antigens in the human kidney.

BACKGROUND: It is generally admitted that ABO(H) blood group antigens are linked to lipids and proteins. Although glycolipids carrying ABO antigens have been well characterized in human kidneys, glycoproteins carrying ABO antigens are largely unknown, and their molecular properties remain to be elucidated. METHODS: All the blood group A antigen-linked proteins in human kidney could be solubilized and captured on immobilized Helix pomatia lectin that recognizes A antigens. These proteins were separated on SDS-PAGE gels. The gel pieces containing protein bands immunoreactive with anti-A antibody were excised, in-gel digested with trypsin, and analyzed by nanoLC tandem mass spectrometer. Protein candidates that carry ABO antigens were confirmed by immunoprecipitation and double-labeled immunofluorescense microscopy. RESULTS: All the glycoproteins carrying ABO antigens were found to be Asn-linked glycoproteins, and presented as multiple bands on SDS-PAGE with molecular masses ranging from 60 to 270 kDa. The protein bands were subjected for mass spectrometric analysis, which identified 121 distinct proteins with high confidence. Of the identified proteins, 55 N-glycosylated, membrane proteins were selected as glycoprotein candidates that carry ABO antigens. Among them, most abundantly expressed proteins as estimated by the number of peptide matches in the MS spectrometric analysis, such as platelet endothelial cell adhesion molecule 1, plasmalemmal vesicle-associated protein, and von Willebrand factor, were further characterized. CONCLUSIONS: Several glycoproteins were identified that represented major glycoproteins carrying ABO antigens in the human kidney, which exhibited distinct features in localization to most of vascular endothelial cells.

Prevalência dos tipos sangüíneos A, B e AB em gatos domésticos mestiços da cidade de Porto Alegre, Rio Grande do Sul, Brasil / Prevalence of blood types A, B and AB in mixed breed domestic cats of Porto Alegre, Rio Grande do Sul, Brazil

O presente estudo teve como objetivo determinar a prevalência dos tipos sangüíneos em felinos domésticos, mestiços, da cidade de Porto Alegre, Rio Grande do Sul, Brasil. Foram selecionados aleatoriamente 100 gatos, clinicamente saudáveis, mestiços e sem parentesco entre si. Amostras de sangue foram coletadas da veia jugular e a tipagem sangüínea foi realizada através do teste Rapid Vet H Feline (DMS Laboratories, Flemington, USA) e do teste hemaglutinação em tubo de ensaio. A tipagem reversa foi realizada para confirmar o tipo e a presença de aloanticorpos naturais. No presente estudo encontrou-seprevalência de 97% e 3% de gatos do tipo A e B, respectivamente. Não foram encontrados gatos do tipo AB. Os resultados indicam que no sul do Brasil há uma alta prevalência de gatos domésticos mestiços do tipo A, entretanto a prevalência de gatos do tipo B encontrada no trabalho é mais alta do que aquelas relatadas em alguns países. O conhecimento da prevalência dos tipos sangüíneos da população de gatos de uma região pode auxiliar na determinação dos riscos de reações transfusionais e de ocorrência de isoeritrólise neonatal, e estes podem ser prevenidos através de tipagem sangüínea e teste de compatibilidade sangüínea.

The present study was carried out to determine the prevalence of the blood types in mixed breed domestic cats of Porto Alegre, Rio Grande do Sul, Brasil. A hundred of clinically healthy mixed breed and non related cats were selected. Blood samples were collected from the jugular vein and the blood typing was performed by Rapid Vet H Feline test(DMS Laboratories, Flemington, USA) and by hemagglutination tube test. Reverse blood typing was done to confirm the blood types and the presence of alloantibodies. In the present study the prevalence of blood type A and B in cats were 97% and 3%, respectively. No AB blood type cats were found. These results indicate that there is a high prevalence of blood type A mixed breed domestic cats in the south of Brazil, although the prevalence o B cats found is higher than reported in some countries. The knowledge of feline blood types prevalence in cat population can help to determine the risks of transfusion reactions and neonatal isoerythrolysis, and these can beavoid by blood typing and cross-matching test.

[Contribution of the genetic fingerprintings compared to grouping ABO/Rhesus technique in the expertise of filiation]. / Apport des empreintes génétiques par rapport à la technique de groupage ABO/Rhésus dans l'expertise de filiation.

Paternity is based on biological analyzes that have drastically developed during the past 20 years. According to scientific developments, paternity testing was based on red blood groups studies, the analysis of red cell enzymes and plasma proteins polymorphisms, the typing of the HLA antigens, and the DNA polymorphism in its various forms. This study aims at comparing two analyses: red blood groups and DNA polymorphism. The performance of each test is analyzed in this report, based on a study of 142 cases. Indeed, the numbers of case of paternity exclusion are respectively 6 and 45 by the classic method and the genetic one. Thanks to studies based on the gene amplification of microsatellites, the efficiency of this reference technique has been proved, however, the classic one makes it possible in the cases of exclusion to lead to a certain decision without recourse to other systems. Of these facts, beyond the most efficient biological analysis, it is very important to think about paternity testing as a process in which biological tests are only one step.

Blood transfusionist extraordinaire: Marie Cutbush Crookston.

After receiving her BSc from the University of Melbourne, Australia, Marie set sail for England to pursue a career. In London, she worked with Dr P.L. Mollison for 10 years, and together they published many articles in the areas of hemolytic disease of the newborn, red cell survival, red cell preservation, and the identification of new antibodies. In 1957, she married Dr John Crookston and moved to Toronto. In Toronto, she directed and participated in various research projects while acting as a consultant to the Blood Transfusion Laboratory at Toronto General Hospital. Her enthusiasm for the field of Transfusion Medicine, her keen eye, and intellect resulted in many discoveries, both on her own or in collaboration with others. Marie is now retired but is fondly remembered by Blood Transfusionists in Canada and elsewhere.

Back to the beginnings: an autobiography.

After receiving BS and MS degrees from the University of Washington in Seattle, I entered its new medical school in 1947, receiving an MD degree in 1951. After internship and residency, I obtained a 2-year postdoctoral fellowship in hematology under the guidance of Dr Clement Finch. The last 6 months of the fellowship were spent in London, England, at Dr Patrick Mollison's Blood Transfusion Research Unit. There I met and worked with Marie Cutbush (later Crookston) who has been a long-term friend. On returning to Seattle, I joined the faculty of the medical school and became the associate director of the Puget Sound Blood Center. There, I supervised the blood typing and cross-matching laboratory, introducing methods I had learned in London and measuring the effectiveness of various cross-matching procedures. My own research was largely directed toward human genetic polymorphism, and I wrote a textbook published in 1969, describing the biochemical structure, function, inheritance, and geographic distribution of the genetic markers. Subsequently, I discovered that 2 forms of inherited immunodeficiency disease were due to deficiencies of the enzymes adenosine deaminase and purine nucleoside phosphorylase. In 1979, I became the director of the blood center and was shortly afterwards elected to the National Academy of Sciences. I retired in 1987 and have spent most of the intervening years relearning to play the violin and exploring the wonders of chamber music.

LU21: a new high-frequency antigen in the Lutheran blood group system.

BACKGROUND AND OBJECTIVES: The Lutheran blood group system comprises 18 antigens numbered LU1 to LU20, with two numbers obsolete. Thirteen antigens are of high frequency. MATERIALS AND METHODS: Serological tests were performed by conventional methods. The monoclonal antibody-specific immobilization of erythrocyte antigens (MAIEA) assay was carried out with monoclonal antibodies to Lutheran glycoprotein. All exons of the LU gene were amplified by the polymerase chain reaction (PCR) and directly sequenced from genomic DNA. RESULTS: A patient was found to have an antibody to a high-frequency red cell antigen during her second pregnancy. The antibody was shown to be Lutheran-related and was distinguished from all reported Lutheran antibodies. MAIEA suggested the antibody was defining a novel epitope in domain 1 of the Lu-glycoprotein. Sequencing of the LU gene revealed a new homozygous single-point mutation, C282G, in exon 3, encoding an Asp94Glu change in the first domain of the Lu-glycoprotein. CONCLUSIONS: The antibody detected a new high-frequency Lutheran antigen, numbered LU21, that appears to result from an Asp94Glu substitution in the first, N-terminal domain of the Lu-glycoprotein.