This laboratory previously described a single-laser flow cytometric method, which effectively resolves micronucleated erythrocyte populations in rodent peripheral blood samples. Even so, the rarity and variable size of micronuclei make it difficult to configure instrument settings consistently and define analysis regions rationally to enumerate the cell populations of interest. Murine erythrocytes from animals infected with the malaria parasite Plasmodium berghei contain a high prevalence of erythrocytes with a uniform DNA content. This biological model for micronucleated erythrocytes offers a means by which the micronucleus analysis regions can be rationally defined, and a means for controlling interexperimental variation. The experiments described herein were performed to extend these studies by testing whether malaria-infected erythrocytes could also be used to enhance the transferability of the method, as well as control intra- and interlaboratory variation. For these studies, blood samples from mice infected with malaria, or treated with vehicle or the clastogen methyl methanesulfonate, were fixed and shipped to collaborating laboratories for analysis. After configuring instrumentation parameters and guiding the position of analysis regions with the malaria-infected blood samples, micronucleated reticulocyte frequencies were measured (20,000 reticulocytes per sample). To evaluate both intra- and interlaboratory variation, five replicates were analyzed per day, and these analyses were repeated on up to five separate days. The data of 14 laboratories presented herein indicate that transferability of this flow cytometric technique is high when instrumentation is guided by the biological standard Plasmodium berghei.
Repeated phenotypic analysis of mouse peripheral blood leukocytes over short periods of time (2 weeks) has been difficult because of the very limited volumes of blood available under guidelines of the Institutional Animal Care and Use Committee. The loss of leukocytes and variations among laboratories during conventional flow cytometry sample preparation based on lysing and repeated washing have been limiting factors when measuring multiple parameters in small samples. We describe a method of phenotypic analysis using a no-lyse, no-wash staining technique combined with fluorescent triggering for data collection that can be performed on volumes of 20 muL or less of whole blood per set of markers in one tube. This method allows repeated phenotypic analysis of peripheral whole blood from mice. Fluorescent triggering with anti-CD45-PE/Cy5 antibody allows high-quality phenotypic data to be collected for CD4, CD8, TcR- beta, CD45R (B220), CD11b, and Gr-1 epitopes on leukocytes from mouse peripheral blood without lysis. The markers selected cover the major populations in peripheral mouse blood. Reproducibility and time-course data are presented for sampling periods as long as 4 weeks. Data produced by flow cytometers manufactured by two different companies show well-correlated results. An instrument equipped with a gated amplifier or a photomultiplier tube suitable for Cy7 conjugates could measure additional parameters. Because of interference from unlysed erythrocytes, scatter parameters are not useful for identifying cell populations with this method.
One hundred twenty strains of Neisseria meningitidis were serotyped with use of cross-absorbed rabbit antisera in a bactericidal test. Fifty-eight epidemic strains of serogroup B, C, and Y that occurred simultaneously among military recruits at two basic training centers during a period of epidemic meningococcal disease were compared with 62 strains of serogroups A, B, C, and Y isolated worldwide. Antisera to the six original antigenic factors of the Gold serotyping schema were adequate for typing 94% of strains, including all of the epidemic strains. The array of serotyping factors in the epidemic strains differed from those in the nonepidemic strains. Epidemic strains were almost exclusively of two serotypes, with type CII predominant among strains of groups B and C. Concurrent strains of groups B and C were invariably of the same serotype. A model for the epidemic potential of meningococcal strains, which is based on their serotype and serogroup antigens, and a modification of the original Gold typing schema are presented.
A divalent vaccine containing equal weights of Neisseria meningitidis group Y and group W135 capsular polysaccharides was inoculated subcutaneously into groups of 32 military recruit volunteers at doses of 10, 25, 50, and 100 micrograms in 10-microliter/microgram volumes. At 4 weeks, the two higher doses induced significantly greater binding antibody responses than did the two lower doses. Differences in response were not found between the two higher doses or between the two lower doses. An additional 32 volunteers received a dose of 25 micrograms in a 20-microliter/microgram volume. Binding antibody response to this vaccine did not differ from the response to doses of 10 and 25 microgram in 10-microliter/microgram volumes. In contrast, bactericidal antibody responses did not differ among doses. Bactericidal antibody was induced in 100% of individuals with low (greater than 4 log2) preexisting serum bactericidal activity, regardless of dose. Bactericidal antibody nonresponse was restricted to individuals with high preexisting serum bactericidal titers. The discrepant dose response between binding and bactericidal antibody resulted from the induction of nonlytic antibody by the higher doses. We conclude that there are no advantages to doses in excess of 5 micrograms of these two chemically similar polysaccharides for the target population of young adult military recruits.
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