The fine specificity of Lewis blood group antibodies. Evidence for maturation of the immune response.

Eight human Lewis blood group antibodies were characterized for their fine specificity by the use of specific immunoadsorbents and a kinetic enzyme-linked immunosorbent assay technique. Examination of sera following immunoglobulin fractionation showed IgM anti-Le(a) exhibiting broad cross-reactivity with structures biochemically related to the Lewis antigens. IgG anti-Le(a) binding was restricted to Le(a) and Le(b) These findings are consistent with the concept of affinity maturation of the immune response, which has been previously demonstrated only in animal model systems.

Correction of cellular autofluorescence in flow cytometry by mathematical modeling of cellular fluorescence.

A method for the correction of background fluorescence in flow cytometry with special relevance to the quantitation of low levels of cellular surface membrane antigens is presented. The method is based on the mathematical modeling of cellular fluorescence distributions of background fluorescence (autofluorescence control or irrelevant antibody control) and total fluorescence (positively stained cells). Algorithms based on two models and utilizing only the routinely available background and total fluorescence histograms are developed and implemented in computer programs. These allow estimation of the fluorescence histogram corresponding exclusively to immunofluorescence staining of the cell surface antigen of interest. Thus, the correction of background fluorescence is effected solely with software processing of routinely available data; no additional hardware or parameter determinations are necessary. Two models were chosen to be physically plausible and to represent extremes in correlation between background and probe fluorescence. Extremes were chosen to assess the solution dependence on model and to provide bounds to the actual solution when no information on correlation is available. Results are presented for both computer simulations and for an actual assay of the CR1 complement receptor on human erythrocytes to test and illustrate the technique. Alternatively, data can be tested assuming a particular model to explore the relationship, if any, between specific and nonspecific fluorescence.

A rapid and accurate single-drop modification of the acid-elution technique for detecting fetomaternal hemorrhage.

A single-drop modification of the acid-elution technique (Kleihauer-Betke) for quantitating fetomaternal hemorrhage is described. It obviates the need for the tedious and time-consuming manual counting of background adult cells. Rather, this is achieved by automated red-blood cell counting of the initial specimen and delivery of a standard volume (1 microliter) of a standard dilution (1:1,000) in the form of a droplet to a microscope slide. The droplet is left to dry undisturbed at room temperature and then stained. The fetal cells are manually counted while the total number of cells is calculated from the initial red-blood cell count, standard volume, and standard dilution. Determinations on 4 different concentrations of fetal/adult red cell mixtures are performed. Results indicate improved accuracy and precision relative to the standard technique in significantly less time for volumes of fetomaternal hemorrhage requiring more than the standard dose of Rho(D)-immune globulin.

Neutralization of P blood group antibodies by synthetic solid-phase antigens.

P blood group system antibodies are encountered frequently in clinical transfusion practice. The authors describe an improved method for removing P antibodies from patient specimens without dilution. Solid-phase synthetic P antigens were used to neutralize serums containing P blood group system antibodies; 26 alloantibodies with specificity other than P were not inhibited. The synthetic P antigens are also used to characterize the heterogeneous immunochemical fine specificity of antibodies to P1 and P + P1 + Pk.

Comparison of monoclonal antisera with conventional antisera for Lewis blood group antigen determination.

Monoclonal blood grouping sera provide a means of overcoming difficulties inherent in the use of conventional polyclonal reagents. Two murine monoclonal anti-Lewis reagents were tested and found to be comparable to conventional reagents for use in red cell phenotyping. These reagents may detect Le(a) antigen on Le(a-b+) red cells with greater sensitivity than previously available polyclonal anti-Lewis reagents.

Detection of anti-Lea in Le(a-b+) individuals by kinetic ELISA.

Utilizing a kinetic enzyme-linked antiglobulin binding assay, eight examples of anti-Lea were detected in individuals whose Lewis phenotype is Le(a-b+). These antibodies were not detectable by routine hemagglutination techniques, but were indistinguishable from anti-Lea made by Le(a-b-) individuals in terms of their immunologic fine specificity. However, unlike most anti-Lea antibodies from Le(a-b-) individuals, none of the anti-Lea antibodies had an IgG component. Anti-Lea antibodies in Le(a-b+) individuals may represent immune responses on the continuum between autoimmunity and alloimmunity.

Neutralization of Lewis blood group antibodies by synthetic immunoadsorbents.

The neutralization of antibodies to the Lewis blood group systems is important in confirming the presence of these antibodies and in investigating complex alloantibody problems. An improved method for neutralizing Lewis antibodies is described. Insoluble immunoadsorbents containing synthetic Lewis antigens are used to physically remove the antibody from serum. Thirty-five sera containing Lewis antibodies were neutralized completely using this technic; 23 non-Lewis alloantibodies were not inhibited. In contrast with current methods, this technic does not dilute the patient serum and results in an affinity purified serum sample.