Decreased amount of the Rh antigen D in hereditary spherocytosis (HS).

This study was done to determine whether hereditary spherocytosis (HS) red blood cells (RBC) have decreased amounts of Rh antigens. Initially we studied the RBC of five members of one family, two of whom had HS. Using automated quantitative haemagglutination tests, we demonstrated that HS RBC agglutinated less with Rh antisera of four specificities than did normal RBC, indicating that Rh antigens are decreased on HS RBC. In this family, the strength of other blood group antigens on HS RBC was estimated by manual titres and agglutination scores. No appreciable differences in the agglutination of HS and normal RBC were observed with non-Rh antisera. To assess the strength of the D antigen more accurately, the number of D sites was quantitated on the RBC of 19 individuals with HS and 11 of their healthy relatives. HS RBC had 9209 +/- 4084 (mean +/- SD) D sites, whereas the normal RBC had 15 394 +/- 5763 D sites. These two means were significantly different (P less than 0.01). HS RBC were also compared to normal RBC of unrelated individuals who had the same Rh phenotype. These analyses showed that HS RBC had about half of the normal number of D sites. Our data indicate that HS red cells have decreased amount of the Rh antigen D and probably also of other Rh antigens.

Quantitative studies on the D antigen of red cells with the Du phenotype.

Rh-positive red cells (RBCs) that fail to agglutinate with commercial anti-D reagents by the tube method are considered to have the Du phenotype. The quantities of D epitopes on such RBCs have been measured previously in one kindred. The authors report on the number of D epitopes on Du RBCs of 23 unrelated individuals, as calculated by Scatchard's analysis. Cell-bound anti-D was measured by an automated antiglobulin consumption technique. On the average, RBCs of the Rh phenotype CcDue had a mean of 1568 +/-1220 (n = 12) D epitopes per cell. The relatively large range of values in this group implies a heterogeneous genetic background. The lowest number of D epitopes, 285 per cell, was observed on the RBCs of one individual who was apparently homozygous for C. In this case, the D antigen was detected only by adsorption/elution tests. RBCs of the phenotype cDuEe had a mean of 775 +/- 378 epitopes per cell (n = 8), and those from two individuals with phenotype cDue had 2840 and 1560 D epitopes, respectively. Thus, on the average, RBCs with the Du phenotype bear about 10 to 20 times less D antigen than normal Rh-positive RBCs. It is suggested that the low D antigen density of Du red cells may account for their poor immunogenicity.

Quantitative studies on the Rh-antigen D. Effect of the C gene.

Using an automated antiglobulin consumption test and Scatchard's analysis, the authors measured the number of D antigen epitopes on each of 46 samples of washed red cells (RBC) that represented six different Rh phenotypes. A clear D dosage effect was observed only when C was missing from the Rh genome, which confirmed the previously recognized suppressive effect of C. Thus, RBC of the Rh phenotype R2R2 had almost twice as many D epitopes (mean, 30,300/cell) as RBC of the Rh phenotypes R2r and R0r (means, 17,200 and 16,500 respectively). The data indicated that C in trans and cis suppressed similar amounts of D epitopes.

Availability of the inner surface of the red cell membrane to antibodies in digitonin ghosts. Studies on D-negative cells with anti-D.

Studies were done to determine whether antibodies can detect antigens on the inner red cell stromal membrane. When albumin, anti-A, anti-D, or IgG were combined with varying volumes of intact O, Rh-negative red cells (RBCs), these proteins diluted, on the average, to 76 percent of the total volume, which was almost identical to the volume of the supernatant fluid (mean 77%). In contrast, when the protein markers were combined with packed stroma prepared from O, Rh-negative RBCs by digitonin, they diluted, on the average, to 94 percent of the total reaction volume rather than to the volume of the supernatant (mean 70%). Similar dilution was observed in the fluid volume harvested from stromal suspensions by ultracentrifugation, indicating that the protein markers occupied the total fluid volume of the reaction mixture (inside and outside the stroma). Nonspecific adsorption of the protein markers to stroma did not occur since their dilution was unaffected by doubling the stromal volume and since they could be totally recovered in the fluid harvested by ultracentrifugation. These data indicate that antibodies easily traverse the membrane of RBC digitonin stroma but not the membrane of intact RBCs. Therefore, antibodies may be used to detect antigenic determinants on the inner stromal membrane. When anti-D was incubated with Rh-negative stroma, we did not observe consumption of this antibody. Thus, our data did not indicate that the D antigen is present on the cytoplasmic membrane of Rh-negative RBCs.

Modification of acid stromal elution method for complete recovery of bound antibodies.

Erythrocyte-bound antibodies could be eluted from stroma almost completely using a modification of the method of Jenkins and Moore. (1) In the modified method, eight volumes of 0.1M glycine, pH 3.0, were added to one volume of stroma and incubated at 45 degrees C for 30 minutes. This method was optimal for autoantibodies and all alloantibodies studied.

Automatic Marsh method for detection and identification of erythrocyte antibodies in patients and blood donors.

Using a Technicon autoanalyzer method of Marsh 752 sera of patients, Rh-negative subjects in the initial stage of immunization with Rh-positive blood, and healthy blood donors were investigated. In 56 sera (8.7%) positive reactions with standard erythrocytes were found. In 21 of these cases (36%) specificity of antibodies was established. They were mostly alloantibodies or autoantibodies against Rh antigens. In 1 case anti-Ika alloantibody and in 1 case anti-K autoantibody were demonstrated. Antibodies with undetermined specificity belonged to the so called "analyzer antibodies", which are detected only by the automatic methods, and probably have no clinical importance. The possibility of recording by the autoanalyzer of subthreshold levels of alloantibodies undetected by other methods may be of great practical importance of patients receiving multiple transfusions of blood and for immunized blood donors.

[Detection of light chains of warm-type autoantibodies in autoimmune hemolytic anemia with the aid of an autoanalyzer]. / Wykrywanie lancuchów lekkich autoprzeciwcial typu cieplego w niedokrwistosciach autoimmunohemolitycznych przy uzyciu autoanalizatora.

A method was evolved for detection of light chains of erythrocyte autoantibodies using a Technicon II AutoAnalyser with properly selected dilutions of bromelin and methylcellulose and anti-kappa and anti-lambda sera. It was demonstrated that the method is superior to the direct Coombs test because it made possible detection of one or both light chains in warm autoantibodies coating the erythrocytes of 20 patients with autoimmunohaemolytic anaemias in whom they could not have been detected by the manual method. It was found that in warm-type autoantibodies light chains can be detected always and that in some cases only one light chain is found.

Automated screening of red cells for the detection of autoantibodies.

The AutoAnalyser Technicon II system was adapted for screening red cells for the presence of warm-type autoantibodies. A significant decrease in optical density was recorded when the red cells of 38 patients with autoimmune haemolytic anaemia were tested using the bromelin-methylcellulose method. This was never seen with the red cells of 226 patients with other diseases or of healthy blood donors. The method is very simple and more sensitive than the manual direct antiglobulin test.

Sensitivity of automated red cell antibody detection.

The principle of detecting red cell antibodies by automated technique using the two-channel Technicon Auto-Analyser with some modification is described. The sensitivity of automated methods in detecting antibodies of different specificities was determined. The study was carried out with 76 diagnostic human sera produced by the Institute of Hematology, Transfusion Centers and commercial firms: anti-RhD, -C, -E, -CW, -c, -e, anti-K, -k, Kpa, anti-Fya, -Fyb, anti-Jka, -Jkb, anti-M, -S, -s, anti-P1, anti-Lua, anti-Lea, -Leb, anti-H, -A1, anti-I, anti-Lua and anti-Vel. A majority of the antibodies were detected by both automated methods with greater sensitivity than by manual methods. Greatest sensitivity was observed in the detection of anti-S, -Vel and antibodies of the Rh system. Anti-Leb and anti A1 antibodies were not detected. The studies are the basis for introduction of the automated technique into routine use for antibodies in blood donors and recipients.