Determination of homozygote vs heterozygote of Rh blood group antigens via rosette assays.

By sensitizing human red blood cells with antisera specific for corresponding blood group antigens and subsequent rosette formation, we were able to distinguish homozygosity of the antigens from heterozygosity. Three types of rosette assays were utilized: a protein A rosette, an immunoglobulin-coated bead rosette, and an erythrocyte-antibody rosette with human lymphocytes. In the Rh blood group system, we were able to demonstrate the dosage effect of the Rho(D) antigen by rosette assay in addition to determining that the deletion of the C and/or E alleles increased the rosette forming cell count when red blood cells with different genotypes were sensitized with Rh immune globulin (anti-D). The assay proved to be reproducible in distinguishing between homozygous and heterozygous Rh red blood cell antigens, and may be adaptable to study many antigenic markers on cell surfaces.

Improved immunodiagnosis of neutrophil dysfunction in the newborn and infant.

An improved assay for the simultaneous assessment of phagocytic uptake (via Immunobeads) and metabolic integrity (via nitroblue tetrazolium (NBT) dye reduction) was used to evaluate the neutrophil function in neonates, one year olds, and adults. The unique advantage of this assay is that it offers greater standardization since the beads are commercially available with a known quantity of immunoglobulin bound to their surface which allows for considerably less variation than particles opsonized in vivo. Blood samples were collected from 20 full term healthy neonates, 20 healthy one year olds, and 20 healthy adults. The neutrophils were isolated, and phagocytic and killing function compared among the three groups. It was found that the neonates had a small but significant neutrophil dysfunction with respect to phagocytic and intracellular killing ability when compared to the one year olds and adults. Additionally, blood was collected and evaluated from five premature infants with varying degrees of stress. Their neutrophil dysfunction was much more pronounced. Although it was previously thought that full term healthy neonates have no demonstrable neutrophil dysfunction unless stressed in some manner, a subtle dysfunction was identified even in unstressed neonates. It was, however, found to be more pronounced if the neonate was either premature or stressed in some manner. It is hoped that this assay, through its greater sensitivity and ease of standardization, will uncover subtle neutrophil dysfunctions in various disease states that as yet remain undiagnosed. The clinical significance of such subtle neutrophil dysfunctions is not yet known.

HLA-B27, Klebsiella pneumoniae, and the relation to acute anterior uveitis.

Acute anterior uveitis (AAU) is a disease of unknown etiology, although it is frequently associated with various autoimmune diseases. It has recently been shown in patients with ankylosing spondylitis (AS), a disease often associated with AAU, that antiserum raised against a particular isolate of Klebsiella pneumoniae would cross-react with lymphocytes possessing HLA-B27. The present study was performed to evaluate the response of lymphocytes of patients to K. pneumoniae and to determine the correlation with HLA-B27. The circulating immune complex levels in the serum of the patients were also determined for correlation with the presence of HLA-B27 antigen. We were able to demonstrate this cross-reactivity in the samples of AAU patients; however, we did find an increased immune complex level that was independent of HLA-B27 antigen. From our results, we conclude that although AAU is clinically associated with AS, the role of K. pneumoniae in these disease states remains unclear.

Evaluation of neonatal neutrophil function using the combined immunobead/NBT dye reduction assay.

An improved assay for the simultaneous assessment of phagocytic uptake (via Immunobeads) and metabolic integrity (via NBT dye reduction) was used to evaluate the neutrophil function in neonates, one year olds, and adults. The unique advantage of this assay is that it evaluates both phagocytic and killing function simultaneously which appears to make the assay more reliable in the detection of neutrophil dysfunction. This assay allowed us to establish a subtle but significant neutrophil dysfunction in full term unstressed neonates which proved to be more pronounced in both the stressed and the preterm neonates. The one year olds, however, were found to have neutrophil function comparable to that of an adult.

Demonstration of neutrophil dysfunction in the serum of patients with cystic fibrosis.

The neutrophil function of 12 patients with cystic fibrosis was evaluated via the new Immunobead/NBT dye reduction test. While the neutrophils appeared to be hyperfunctional when studied in the standard assay using fetal calf serum, we demonstrated a significant neutrophil dysfunction when studied in their own respective serum. The dysfunction involved both the phagocytic and intracellular killing ability of the cells, which we postulate is due to an inhibitory factor present in the patient's serum. The inhibitory factor did not appear to be related to the level of circulating immune complexes.

Dose response with sensitizing antibody in EA rosette formation.

With the exception of the EA rosette test using SRBC, all other assay systems using BRBC and HRBC showed a significant dose response in rosette-forming cell count when the sensitizing antibody was further diluted. The percent of rosette-forming cell counts of the EA assay with SRBC, however, was unaffected by dilutions of antiserum even at exceedingly high levels. The present results suggest using red cells, which are nonreactive with T lumphocytes for the assay of B lymphocytes, and the use of antibody for sensitization of red blood cells must be adjusted for the optimal dilution necessary for proper EA rosette formation.

Anti-kappa/lambda immunobead for detection of human B lymphocytes.

Several methods were compared for quantitating the B lymphocytes in human peripheral blood. RBC rosette assays including sheep and bovine red blood cells, and immunobead rosette assays including beads coated with anti-kappalambda and trivalent anti-GAM antibodies respectively were investigated. The anti-kappalambda bead assay proved to be simpler, faster, higher in reproducibility, and appeared to be more accurate than any of the other assays.