Quantitative analysis of chimerism using a short tandem repeat method on a fluorescent automated DNA sequencer.

Stem cell transplantation (SCT) is the treatment of choice for a number of malignant and nonmalignant diseases. Monitoring of SC engraftment or microchimerism (MC) is important for diagnosis of relapse, rejection or graft vs. host disease (GVHD). The goal of this study was to develop a sensitive and relatively simple method for MC lineage analysis using the Visible Genetics fluorescence automated sequencer. Sensitivity of the method was studied by polymerase chain reaction (PCR) amplification of informative short tandem repeats (STR) using donor/recipient DNA mixtures as the templates and DNA extracted from donor and recipient CD3+, CD19+ and CD15+ cells mixed at various ratios. Semi-quantitative analysis was performed using the Visible Genetics software and percent of donor specific signal was calculated. The sensitivity of this method varied from 0.8% to 6.2% for both DNA and cellular MC in CD3+, CD19+ and CD15+ subsets. Regression analysis revealed linearity (r = 0.94) between the number of donor cells in the mixture and intensity of MC fluorescent signal. These data indicate that the Visible Genetics polyacrylamide gel sequencer can be successfully used for MC analysis in SC recipients providing a relatively high level of sensitivity.

An acellular pertussis vaccine in healthy adults: safety and immunogenicity. Pennridge Pediatric Associates.

Recent data indicate that Bordetella pertussis can be an important cause of illness in adolescents and adults. In a randomized observer- and subject-blinded study, adults (> or = 18 years of age) received an acellular pertussis (aP) vaccine containing genetically inactivated pertussis toxin (PT), filamentous hemagglutinin (FHA) and pertactin (PRN), or a saline placebo, and were monitored for safety and immunogenicity. IgG antibodies to PT, FHA, and PRN were measured by enzyme-linked immunosorbent assay (ELISA) and PT neutralization by a Chinese hamster ovary (CHO) cell assay. Local reactions, more common in the aP group, were mild and transient. One month after immunization, geometric mean ELISA antibody concentrations for the aP and placebo groups, respectively, were: anti-PT, 463 and 7.6; anti-FHA, 417 and 18; and anti-PRN, 855 and 14. The anti-PT neutralization titers for the aP and placebo groups were 1:3439 and 1:58 respectively. This aP vaccine is a safe and immunogenic candidate booster vaccine against pertussis for adults.

Household contact study of Bordetella pertussis infections.

Household contacts of primary pertussis cases were evaluated. Infection was determined by culture, direct fluorescent antibody assay, and serological criteria. Agglutinin titers and values of ELISA IgG and IgA antibodies to lymphocytosis-promoting factor, filamentous hemagglutinin, and pertactin were determined. In 39 households 255 subjects were exposed; 114 remained well (group 1), 53 had mild illness (group 2), and 88 had pertussis (group 3). The infection rates were 46% (group 1), 43% (group 2), and 80% (group 3). In a subgroup of subjects seen within 14-28 days of exposure, it was found that none with clinical pertussis had a value of IgG antibody to pertactin in acute-phase sera of > or = 50 ELISA units (EU) per mL or an agglutinin titer of > 256. Of the primary cases, 53% were > or = 13 years of age. These data point out the importance of Bordetella pertussis infections in adolescents and adults as a source of infection in young children. Our subgroup data suggest that high values of antibody to pertactin and high agglutinin titers may be predictive of protection against clinical pertussis.

A rapid flow cytometry assay for HLA antibody detection using a pooled cell panel covering 14 serological crossreacting groups.

Many studies have demonstrated the usefulness of flow cytometry crossmatching (FC-XM) for selection of regraft recipients, and more recently this assay has been shown to correlate with allograft survival in primary cadaveric transplant patients. The need now exists for a practical antibody screening procedure which uses the same methodology. We describe here a simple and sensitive method to screen for HLA antibodies by FC using a pool of 6 cells selected to cover the 14 serological crossreacting groups defined by Rodey. Screenings of 367 sera (255 primary transplant sera, 112 regraft sera) received for monthly antibody testing were performed by both pooled cell FC and complement-dependent cytotoxicity (CDC) assays. Forty of these sera were also FC-screened using a panel of 16 individual cells for comparison with the pooled cell FC screenings. Analysis indicated a strong correlation between the pooled FC-PRA and the individual cell panel FC-PRA (P = .0001) with mean values of 60% and 73%, respectively. Only 2 of the 40 sera screened by both FC methods resulted in PRAs that differed by > 40%. The majority (82%) of the primary patients did not exhibit HLA antibodies by CDC--however, 22% of the CDC negative patients were positive by flow cytometry. Females were more likely to be positive by FC (35%) than males (16%) (P = .0001). Similarly, black patients were more likely to have FC-demonstrable antibodies (28%) than white candidates (14%) (P = .014). The regraft patients who tested positive by either or all methods had a mean PRA for CDC, pooled FC-PRA, and individual cell FC-PRA of 40, 75, and 85, respectively. FC-PRA proved to be a more sensitive technique in both primary and regraft patients.

Outbreak of pertussis in a fully immunized adolescent and adult population.

OBJECTIVE: To evaluate the spread of pertussis in a fully immunized eighth-grade class and the household contacts of two coindex cases of pertussis. DESIGN: Survey of infected subjects and their contacts was performed using culture, direct fluorescent antibody assay, and serological assays to establish the diagnosis of Bordetella pertussis. SETTING: Middle-class parochial school. PARTICIPANTS: A volunteer sample of 15 eighth-grade students and 13 household contacts of two identified cases of B pertussis infection. INTERVENTIONS: All participants had medical histories (including immunization status) and laboratory evaluation for B pertussis infection (including nasopharyngeal specimens and serum samples) obtained initially and 30 days later. After initial evaluation, all subjects received erythromycin ethyl succinate therapy. MAIN OUTCOME MEASURES: Assessment of B pertussis infection as defined by positive nasopharyngeal culture, direct fluorescent antibody, or serological tests. RESULTS: Laboratory evidence of B pertussis infection was found in eight (47%) of 17 immunized eighth-grade classmates and in three (23%) of 13 household contacts, all of whom were 12 years of age or older. CONCLUSIONS: Vaccine-induced immunity wanes by early adolescence. These older age groups may be infected with B pertussis and may serve as reservoirs of infection for other susceptible individuals.