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.

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.

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.

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.

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.

Identification of an erythrocyte component carrying the Duffy blood group Fya antigen.

The erythrocyte component carrying the Duffy blood group antigen Fya has been identified as a 35- to 43-kilodalton protein. The protein is degraded by proteases, chymotrypsin, and Pronase, which destroy its antigenicity on intact erythrocytes. Its unusual property of aggregating on being boiled in 5 percent sodium dodecyl sulfate with 5 percent 2-mercaptoethanol distinguishes it from other erythrocyte membrane proteins described to date.

[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.

Exposure of cryptantigens on red blood cell membranes in patients with acquired immune deficiency syndrome or AIDS-related complex.

Patients with acquired immune deficiency syndrome (AIDS) or AIDS-related complex (ARC) are subject to recurrent and severe infections due to organisms known to cause red blood cell membrane modifications. These red cell modifications include the exposure of novel carbohydrate cryptantigens that can react with naturally occurring antibodies and potentially result in hemolysis. We examined the frequency of cryptantigen exposure on the surface of red cells from AIDS/ARC patients. Blood samples from 108 patients with AIDS/ARC and from 65 non-AIDS/ARC patients were tested for most common forms of cryptantigens. The lectin Arachis hypogaea agglutinated red cells from 7% (8/108) of the AIDS/ARC patients and 3% (2/65) of non-AIDS/ARC patients, indicating the presence of T, Tk, or Th cryptantigen exposure. One sample from an AIDS patient with E. coli sepsis had T activation with polyagglutinable red cells. None of the samples showed evidence of exposed Tn or acquired B antigens. These results show that red cell cryptantigen exposure does occur in AIDS patients with a prevalence similar to that previously reported in patients with sepsis or malignancy. For this reason, and because polyagglutination has been associated with in vivo hemolysis, cryptantigen exposure should be considered in the differential diagnosis in AIDS patients with suspected immune hemolysis; it can be tested for by performing a minor crossmatch with ABO compatible serum.

Freeze-thaw effects on the detection of blood group substances in detergent extracts by enzyme-linked immunosorbent assays (ELISA).

The sensitivity of an enzyme-linked immunosorbent assay (ELISA) for human blood group antigens extracted from blood stains with the surfactant, n-octyl-beta-D-glucopyranoside (OBG), at concentrations below the critical micelle concentration can be increased by the introduction of a single freeze-thaw step. The ELISA signals increase from 3- to 4-fold for OBG extracts of 80 nl bloodstains. The ELISA signal enhancement occurs irrespective of the age of the bloodstains, at least for bloodstains up to 1 year old. The origin of the effect has been investigated and its is demonstrated that the freeze-thawing cycle increases the extent of adsorption of the blood group determinants in OBG-solubilized complexes onto microtitre plates. Gel filtration has been used to analyse the composition of OBG extracts of bloodstains in terms of the carriers of the blood group substances, protein and phospholipid in fresh and freeze-thawed extracts. It was found that freeze-thawing alters the distribution of blood group active material in the lipid-protein OBG complexes leading to a greater proportion of blood group active material in higher molecular weight complexes. The freeze-thaw effect is eliminated on the addition of a cryoprotectant, such as glycerol, and the factors which contribute to changes in the microstructure of OBG extracts on freeze-thawing are discussed.

Immuno- and histochemical studies on galactan and human blood group-related receptors in the bovine lung.

Using a monoclonal anti-galactan antibody and streptococcus B type II antibody, the distribution of lung galactan could be demonstrated for the first time in a vertebrate organ. In addition to the immunochemical demonstration of the bovine lung galactan, a human blood group A-like glycoprotein is detectable by lectinological methods in the bovine lung tissue. Various other lectin-receptors, for instance those of the peanut lectin (PNA) or for lectins with L-fucose (UEA) and N-acetyl-lactosamine (ECA) specificity show a typical staining pattern in bovine lung.

Recombinant forms of Gerbich blood group antigens: expression and purification.

Recombinant forms of normal glycophorin C (GPC), carrying the high frequency Gerbich blood group antigens, and its natural deletion mutants of Yus and Ge type (all combined with oligohistidyl tag) were expressed in CHO and COS 7 cells. The stable expression of all recombinant forms of GPC in CHO cells was obtained, but the level of expression was low and detectable only by flow cytometry. The high level of transient expression of GPC recombinant forms in COS 7 cells allowed their purification on Ni-NTA-agarose. The purified recombinant GPC and mutants of Yus and Ge type behaved in SDS-PAGE similarly to normal GPC forms from RBC membranes. The recombinant GPC.Yus and GPC.Ge mutants appeared as diffuse bands, suggesting the similar heterogeneity of glycosylation that was observed in natural GPC.Yus and GPC.Ge glycoproteins. The flow cytometry analysis of the transfected CHO and COS 7 cells showed that binding of anti-GPC monoclonal antibodies to GPC variants was accordant with the known fine specificity of these antibodies. The obtained recombinant forms of GPC carrying common Gerbich antigens may be useful in serology, and also as model molecules for structure-function studies.

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.

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.

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.

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.