Incidence of ABO(H) blood group variants among the Bulgarian population.

The result of a screening of ABO (Hh) variants after investigating the blood group of 106,980 persons are presented. The Ax variant is registered most frequently among the Bulgarian population. As a whole the frequencies of ABO blood group variants Ax, Ael, A3, Aend and Am among the Bulgarian population are similar to that established by other authors among the French population and nearly twice as high as among the population of Bombay. The group of H-deficient phenotypes includes AA1Xh, AHm, OHm variants. Their frequency is significantly lower when compared with the frequency of AHm and OHm variants among the population of Thailand. Variants A1 and Aint with unusually high H-content are classified as A1H, A1Hint, Aint H and integrated as a category of H-excess phenotypes. Their incidence among the Bulgarian population is significantly lower than that registered among Maharastrian, South African Bantu and Indian.     

The Cis AB complex of the ABO system]

Members of six unrelated families from Japan, France, Belgium and Poland were studied in parallel. Major immunological features characteristic of the phenotype produced by the Cis AB complex are the following: 1) The red cell A reactivity is close to normal, is beyond the values of agglutination scores by Helix and by anti-A from B; likewise, with percent agglutination measurements, A reactive appears hiher than that of A2B cells; one sample only is slightly detected by anti-A from Dolichos. 2) The B reactivity, on the contrary, is lower than that of normal AB cells. A single sample is detected by anti-B from A1. All samples are well detected by anti-B from AW, Aend, Ax, Am but none is detected by anti-B from ABx, Cis AB, or by an auto-anti-B. Under standard conditions, percent aggutination is around 80, very close to that of normal AB cells, thus differentiating Cis AB from AB3 (some of which only reach this figure), and from ABx which are very far from this value. 3) An abnormally high reactivity to anti-H antibody is observed, higher than that of normal A2B, similar to that of A2 red cells. 4) Among secretors, A substance is found to be normal or in excess, H substance is in excess, while B substance is only detected by Cis AB red cells inhibition. 5)An anti-B antibody was identified in the samples studied; however, we recently received from Germany a Cis AB samples, the serum of which did not contain anti-B antibody. By these main characteristics, the studied samples seem to be identical; however, agglutination kinetics and thermodynamic methods show that they differ by their reaction with a same anti-B antibody in standard conditions. The reactive structures of the various samples are indeed different from one family to another. The main point is that identical values were observed in all samples within a same family. Thus, the various Cis AB can be considered as different families mutants.     

Quantitative studies of the ABO blood group system. Quantitative changes in agglutinability of antigens A, B and H with regard to the patient's age]

The results of a comparative quantitative investigation about the agglutinability of erythrocyte antigens A, B, and H to be found in fetus (fifth to ninth lunar month), newborns, adults, and old age people are represented. In investigating with anti-H sera it could be found that A-antigens in fetus undergo the same development to be observed in newborns; it amounts to 75% of the agglutinability present in adults. After delivery the agglubinability will essentially increase up to the fifth month; it will only reach the average values of adults, however, after the seventh year of age and will remain unchanged then until the end of life. The agglutinability of B-erythrocytes with anti-B will also change in the same way. In fetus and newborns the agglutinability of A1 erythrocytes with anti-A1 sera is markedly weaker than that determined by anti-A sera. After delivery it will rapidly increase, will be stronger afterwards than the agglutinability with anti-A sera and will have the values to be found in adults after the third year of age. After the 85 year of age, however, there is a tendency of weakening the agglutinability with anti-A1 serum. With growing age the agglutinability of O-erythrocytes with anti-H serum is changed in the same way. Furthermore, it could be detected that the interaction between genes A1 and B to be found in adults will find its expression in a weakening of the agglutinability of A1B-erythrocytes with anti-A1 and anti-B sera, the agglutinability with anti-A serum remaining constant. This behaviour is valid for the whole life of man. In all stages of life an interaction between weak and strong genes can be observed in A2B-erythrocytes.     

Isoimmune anti-A blood group reagents in pigs

Immunization or reimmunization of A-negative pigs with red blood cells (RBC) from A-positive donors yielded anti-A antibodies reacting in high titres with pheno-type A(Ac) RBC and, in some cases, in low dilutions, with phenotype Aw(Ap) RBC also. An attempt to raise the anti-A level by immunization with saliva which contained A substance was likewise successful.
Repeated immunization of A-negative recipients with the RBC of A-positive donors (compatible in all other factors), with the aid of adjuvant, is recommended as the best way of obtaining Aw typing reagents.     

Isoimmune anti-A blood group reagents in pigs.

Immunization or reimmunization of A-negative pigs with red blood cells (RBC) from A-positive donors yielded anti-A antibodies reacting in high titres with phenotype A(Ac) RBC and, in some cases, in low dilutions, with phenotype Aw(Ap) RBC also. An attempt to raise the anti-A level by immunization with saliva which contained A substance was likewise successful. Repeated immunization of A-negative recipients with the RBC of A-positive donors (compatible in all other factors), with the aid of adjuvant, is recommended as the best way of obtaining Aw typing reagents.     

Quantitation of antibody uptake on A group erythrocytes using immunoautoradiography and monoclonal IgM anti-A

The number of antibody molecules on individual erythrocytes was counted in A1, A2, A3 B and A group individuals using immunoautoradiography (IAR) and monoclonal IgM anti-A1. Quantitation was also done for A group pregnant women. The number of antibody molecules on different red cells of an individual varied widely. Gross variations were also noted in cells of different individuals from one and the same group. The mean values of the uptake of the number of antibody molecules showed the following range A1 greater than A2 greater than Ax greater than A3B. When compared to the average for total A1 adults, red cells of pregnant women and newborn infants showed a 10.7% and 19.7% reduction respectively, in antibody uptake. The mean number of antibody molecules per A1 adult red cells was 5.6 +/- 3 X 10(4), while A2 had 0.85 +/- 0.35 X 10(4) molecules, thus showing a significant quantitative variation.     

Membrane characteristics of old and Rauscher leukemia virus infected mouse red blood cells

Some membrane characteristics of normal and Rauscher leukemia virus (RLV)-infected mouse red blood cells (RBC) were compared, both with regard to total populations and young and old groups of cells. Osmotic fragility, density distribution of cells and agglutinability by poly- -lysine (pLys), concanavalin A (ConA), phytohemagglutinin (PHA) and soybean agglutinin (SBA), were examined. RBC from RLV-infected mice were agglutinated at a higher rate and to a higher degree than normal mice RBC by pLys and by the lectins PHA and ConA. These RBC were generally osmotically more resistant and contained a young cell population of unusually high specific gravity. Comparison of RBC from RLV-infected mice with old RBC from normal mice showed some common membrane characteristics. Similarly to old RBC, RBC from RLV-infected mice have a high specific gravity and high agglutinability by pLys. However, they differ in that the RBC from RLV-infected mice are osmotically more resistant and are agglutinated by ConA; they are also agglutinated at a higher rate by PHA.     

The incidence and features of serological ABO subgroups among one million blood donors in Beijing

This study aims to determine the incidence of serological ABO subgroups from a large-scale database, along with the features of blood samples with serological ABO discrepancies. The serological ABO results of one million individuals were randomly sampled from a blood donor database in Beijing between 2009 and 2010. All samples were diagnosed by serological reverse and forward ABO typing using an automatic analyzer. The proportions of the normal ABO types were 27.28%, 31.57%, 30.56%, and 10.16% for blood types A, B, O, and AB, respectively. In samples in which ABO discrepancies or obvious weak agglutinin were identified in the forward or reverse typing, further tests to analyze the ABO subgroup were conducted. The overall incidence of ABO subgroups was 0.047%, with 14 ABO subgroups observed: A2, A3, Ax, Am, Aint, Aend, B2, B3, Bx, Bm, Bel, B(A), cisAB, and AB^h. In conclusion, this study revealed the exact normal ABO and subgroup distributions in the general, healthy population of Beijing using samples from a blood donor database.     

Quantitative flow cytometric analysis of ABO red cell antigens.

A flow cytometry method has been employed to quantitatively compare the expression of A, B and H antigens on various red blood cells (RBC). The H substance was directly labelled by fluorescein-conjugated anti-H lectin and the A and B antigens by indirect staining first with monoclonal anti-A or anti-B antibodies followed by fluorescently, fluorescein (FITC) or phycoerythrin (PE), labelled anti-mouse immunoglobulin (Ig) antibodies. More than a ten-fold difference in cellular fluorescence intensity was found within each sample. Both the percentage and the mean fluorescence of the positive subpopulation for each antigen were determined. Each RBC population was characterized with respect to the expression of A, B or H antigen by a compound mean value that was the calculated product of these two parameters. The results demonstrated a reciprocal relationship between the compound means of A or B and H. The ratio of A/H or B/H was found to be most informative. Homozygotes for A or B had ratios of greater than 200 and greater than 30, respectively, while heterozygotes (AO or BO) had ratios of less than 5. This method could also distinguish between A1 and A2; RBC carrying the A1 phenotype (as determined by agglutination with anti-A1 lectin) showed a higher A/H ratio than those carrying A2. In contrast to the reciprocity in the expression of A (or B) and H found in RBC obtained from different individuals, a direct correlation was found in the expression of these antigens by individual cells within a given population.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity of the cellular distribution of erythrocytic A antigens. Ultramicroscopic study]

A and A1 antigens have been detected on cells of the human erythrocyte series by immunoelectron microscopy. These antigens have been revealed by an indirect method involving various anti-A and anti-A1 antibodies (allo, auto, hetero-antibodies) and peroxidase-conjugated anti-immunoglobulin antibodies. Immunologic labelling has been carried out with erythrocyte or bone marrow cell suspensions which were fixed prior to incubation with reagents. Cells from various A phenotypes were examined. A and A1 antigens were visualized on maturing normoblasts, at every developmental stage. In addition cell to cell variations of the surface labelling of erythrocytes was found in normal phenotypes, suggesting the existence of several populations of cells according to antigenic load.     

Effects of lysolecithin on the surface properties of human erythrocytes

Human red blood cells (RBCs), transformed by incubation with the amphiphatic compound lysolecithin from their normal discocyte shape into echinocytes, have increased rates of agglutination in the presence of either poly- -lysine (PLL) or soybean agglutinin (SBA). Removal of lysolecithin by washing caused a reversal of shape back to the discocyte configuration and a lowering of agglutination rates. Methochlorpromazine, another amphiphatic echinocytogenic substance produced a similar increase in agglutination rates, suggesting that increased agglutinability may be a general property of echinocytes. Lysolecithin treatment of RBCs caused a decrease in the binding of cationized ferritin (CF) particles/μm² of RBC surface. The decrease in CF binding is due to a rearrangement of negative charge bearing molecules on the RBC surface rather than shedding of charged groups. These observations support the hypothesis that integral membrane proteins which bear negative charges and receptors are associated with a cytoskeleton within the red cell. Alterations in cell shape which result in distortion of the cytoskeleton may cause a redistribution of integral membrane proteins which bear charged groups at the RBC surface.     

Red blood cells imaging and antigen-antibody interaction measurement

In the present study the atomic force microscope (AFM) was used to image the surface morphology of red blood cells (RBC) for the first time. The AFM yielded very reproducible images without appreciable modifications of the sample surfaces. In addition to this topographical imaging, we have developed an experimental approach to measure the binding strength between antibody (anti-A), and the RBC antigen A, when reversible bonds between specific molecules such as antigen and antibody mediate the adhesion. The experimental results suggest that the procedure established here may be used for specific antibody detection. This study has also enhanced our understanding under physiological conditions of molecular interaction in particular antigen-antibody.     

An enzyme basis for blood type A intermediate status.

The blood type A is known to be subclassified as A1, A2, and A1-A2 intermediate (Aint), depending upon red cell agglutinability with anti-A1 and anti-H lectins. Approximately 80% of the blood group H-sites remained unglycosylated in type Aint erythrocyte membranes. Plasma from Aint individuals contains a special blood group GalNAc transferase (UDP-GalNAc:2''-fucosylgalactoside-alpha-3-N-acetylgalactosaminyl transferase), which is different from the enzyme in A1 plasma and the enzyme in A2 plasma. A1-enzyme has strong affinity to UDP-GalNAc and 2''-fucosyllactose, A2-enzyme has low affinity to both substrates, and Aint-enzyme has strong affinity to UDP-GalNAc and very low affinity to 2''-fucosyllactose, which is a soluble analog of the H-substances. The low degree of glycosylation of the blood group H-sites due to the low affinity of Aint-enzyme with the H-substances can account for the lower A activity and higher H activity in Aint red cells than in A1 red cells. The blood group A allele can be subdivided into three common alleles, A1, A2, and Aint, each controlling the formation of different types of blood group GalNAc transferases.     

Determination of erythrocyte transit times through micropores. II. Influence of experimental and physicochemical factors

A new red blood cell filtration system, termed the Cell Transit Time Analyzer (CTTA), has been developed in order to measure the individual transit times of a large number of cells through cylindrical micropores in special "oligopore" filters; the system operates on the electrical conductometric principle and employs special computer software to provide several measures of the resulting transit time histogram. Using this system with filters having pore diameters of 4.5 or 5.0 microns and length to diameter ratios of 3.0 to 4.7, we have evaluated the effects of several experimental factors on the flow behavior of normal and modified human RBC. Our results indicate: 1) linear RBC pressure-flow behavior over a driving pressure range of 2 to 10.5 cm H2O with zero velocity intercepts at delta P = 0, thus suggesting the Poiseuille-like nature of the flow; 2) resistance to flow or "apparent viscosities" for normal RBC which are between 3.1 to 3.9 cPoise and are independent of driving pressure and pore geometry; 3) increased flow resistance (i.e., increased transit times) for old versus young RBC and for RBC made less deformable by DNP-induced crenation or by heat treatment at 48 degrees C; 4) increased mean transit time and poorer reproducibility when using EDTA rather than heparin as the anticoagulant agent. Further, using mixtures of heat-treated and normal RBC and various percentile values of the transit time histogram, we have been able to demonstrate the presence of sub-populations of rigid cells and thus the value of measurements which allow statistical analyses of RBC populations.     

Determination of erythrocyte transit times through micropores. II-- Influence of experimental and physicochemical factors

A new red blood cell filtration system, termed the Cell Transit Time Analyzer (CTTA), has been developed in order to measure the individual transit times of a large number of cells through cylindrical micropores in special "oligopore" filters: the system operates on the electrical conductometric principle and employs special computer software to provide several measures of the resulting transit time histogram. Using this system with filters having pore diameters of 4.5 or 5.0 cm and length to diameter ratios of 3.0 to 4.7, we have evaluated the effects of several experimental factors on the flow behavior of normal and modified human RBC. Our results indicate : 1) linear PBC pressure - flow behavior over a driving pressure range of 2 to 10.5 cm H2O with zero velocity intercepts at delta P = 0, thus suggesting the Poiseuille - like nature of the flow; 2) resistance to flow or "apparent viscosities" for normal RBC which are between 3.1 to 3.9 cPoise and are independent of driving pressure and pore geometry; 3) increased flow resistance (i.e., increased transit times) for old versus young RBC and for RBC made less deformable by DNP-induced crenation or by heat treatment at 48 degrees C; 4) increased mean transit time and poorer reproducibility when using EDTA rather than heparin as the anticoagulant agent. Further, using mixtures of heat-treated and normal RBC and various percentile values of the transit time histogram. We have been able to demonstrate the presence of sub-populations of rigid cells and thus the value of measurements which allow statistical analyses of RBC populations.     

Mouse monoclonal antibodies to bovine blood group antigens: additional results

Seven fusions of mouse myeloma cells with spleen cells from mice immunized with bovine red cells yielded 61 clones producing discriminant antibodies out of total of 651 secreting clones. Although antigenic factors of all known bovine blood group systems were present on the donors' cells, the antibodies identified reacted with antigenic factors from only five systems, A, B, F, S and Z. The antibody specificities produced by more than two clones were anti-A1 or -A2 (21 clones), -S (9),- Z(6),-G' (3) and -V1 (3). The absence of clones secreting antibodies to antigens of the other systems, especially the complex C system, remains unexplained. The properties of the antibodies reacting with antigens of the S system (anti-SU", anti-SUU') and of the B system (O-like antibodies) are in accordance with previous interpretations of polyclonal sera and with present knowledge of the genetic map of the B system.     

Direct Antiglobulin Reaction in ABO-Haemolytic Disease of the Newborn

The minimum number of IgG anti-A (or anti-B) molecules detectable on A or B red cells by the antiglobulin reaction was found to be the same—that is, about 150 molecules per red cell—with newborn as with adult cells. Furthermore, the ratio of anti-IgG bound to IgG anti-A (or anti-B) molecules was the same whether the anti-A (or anti-B) molecules were present on newborn or on adult cells and was similar to that found for anti-IgG bound to IgG anti-Rh.In 15 infants (11 group A, 4 group B) with haemolytic disease of the newborn due to ABO-incompatibility the amount of anti-A or anti-B on the red cells ranged from 0·25 to 3·5 μg antibody per ml red cells, corresponding to 90-1,320 antibody molecules per cell; only five infants had more than 0·55 μg antibody per ml of red cells. These amounts are far smaller than those found in most moderate or severe cases of Rh-haemolytic disease.It is concluded that the weak direct antiglobulin reactions observed in ABO-haemolytic disease are due simply to the fact that the number of anti-A (or anti-B) molecules on the infant''s red cells is at the lower limit of sensitivity of the test. Since ABO-haemolytic disease can be quite a severe process it seems probable that IgG anti-A and anti-B molecules are more effective than anti-Rh molecules in bringing about red cell destruction.     

Specificities shared between HLA-A3 and HLA-A11 detected by Fab'2 blocking

Four anti-HLA-A3 sera crossreactive with HLA-A11 were tested with a panel of A3 positive cells pretreated with either Fab'₂ prepared from one of the four anti-A3 sera or Fab'₂ fragments of an anti-A11 serum displaying CYNAP crossreactivity with A3. The anti-A3 Fab'₂ blocked the reactions of all anti-A3 sera with all A3 +, 11– cells; the anti-A11 Fab'₂ blocked the reactions of only some serum-cell combinations. Each of the four anti-A3 sera tested were blocked by the anti-A11 Fab'₂ in a different pattern, indicating that each could have a different anti-A3 specificity. Five different patterns of reactivity were observed among the 21 test lymphocytes. These findings are interpreted as evidence for shared antigenic components between A3 and A11.     

Evaluation by ELISA of anisakis simplex larval antigen purified by affinity chromatography

In order to improve the specificity and sensitivity of the techniques for the human anisakidosis diagnosis, a method of affinity chromatography for the purification of species-specific antigens from Anisakis simplex third-stage larvae (L3) has been developed. New Zealand rabbits were immunized with A. simplex or Ascaris suum antigens or inoculated with Toxocara canis embryonated eggs. The IgG specific antibodies were isolated by means of protein A-Sepharose CL-4B beads columns. IgG anti-A. simplex and -A. suum were coupled to CNBr-activated Sepharose 4B. For the purification of the larval A. simplex antigens, these were loaded into the anti-A. simplex column and bound antigens eluted. For the elimination of the epitopes responsible for the cross-reactions, the A. simplex specific proteins were loaded into the anti-A. suum column. To prove the specificity of the isolated proteins, immunochemical analyses by polyacrylamide gel electrophoresis were carried out. Further, we studied the different responses by ELISA to the different antigenic preparations of A. simplex used, observing their capability of discriminating among the different antisera raised in rabbits (anti-A. simplex, anti-A. suum, anti-T. canis). The discriminatory capability with the anti-T. canis antisera was good using the larval A. simplex crude extract (CE) antigen. When larval A. simplex CE antigen was loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with A. simplex CE antigen, its capability for discriminate between A. simplex and A. suum was improved, increasing in the case of T. canis. The best results were obtained using larval A. simplex CE antigen loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with adult A. suum CE antigen. When we compared the different serum dilution and antigenic concentration, we selected the working serum dilution of (1/4)00 and 1 microg/ml of antigenic concentration.     

Mouse 3T3 cell filtrability correlating with concanavalin A agglutinability

In a previous study of the dextran gel sphere model system, a possible correlation between cell deformability and agglutinability by concanavalin A was indicated. Cell deformability was evaluated as filtrability, using polycarbonate membrane filtration. With 25-mm diameter filters and 5-ml cell suspensions at (0.8–16) · 10⁵ cells/ml, the filtrability at a given filter pore size was highly reproducible and was not affected by variations in cell population, viability, washings of cells retained on filter, or temperature. The filtrability of EDTA-dissociated 3T3 cells through 12-μm pore size filter was 8%, and a suspension of 10⁶ cells/ml was not agglutinated by 600 μg concanavalin A. The filtrability of trypsin-dissociated 3T3 cells was 95%, and these cells were agglutinated by 200 μg of the lectin. EDTA-dissociated SV-3T3 cells had a filtrability of 73% and were also highly agglutinable. Formalin fixation reduced the high filtrability to 6%, and also abolished the agglutinability. As a further test of the correlation, trypsin-dissociated 3T3 cells were admixed with the fixed cells. The agglutinability varied with the proportions of the two cell components, and the admixtures could be separated according to filtrability into the original components with distinctly different agglutinability. Furthermore, 25% of a random population of EDTA-dissociated SV-3T3 cells retained by the filter were found to be non-agglutinable. The separated SV-3T3 cell fractions could also form admixtures of different agglutinability. It is concluded that the agglutinability of mouse 3T3 and SV-3T3 cells by concanavalin A can be correlated with the predicted by cell filtrability.