Flow cytometric enumeration of micronucleated reticulocytes: high transferability among 14 laboratories.

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.

Immune response of infants and children to disseminated infections with Neisseria meningitidis.

Acute- and convalescent-phase sera from 34 children and 10 young adults were studied to determine if, at what age, and to which antigens of Neisseria meningitidis they respond during disseminated disease. Seven children older than two years of age who were infected with group C or Y strains developed significant increases in both binding and bactericidal antibody. Children infected with group B strains infrequently (eight [31%] of 26) had measurable increases in serum antibody to this capsular polysaccharide; response was meager when it did occur, was unrelated to age, and was considerably poorer than that of young adults, of whom 80% responded. Convalescent-phase sera from all children contained bactericidal antibody. Binding capacity for group B polysaccharide accounted for only 35% of the bactericidal activity in convalescent-phase sera of children infected with group B strains. Bactericidal antibody in the sera of children who did not respond to capsular polysaccharides was often to a lipooligosaccharide antigen.

Human immune response to various doses of group Y and W135 meningococcal polysaccharide vaccines.

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.

Heterogenity of serotypes of Neisseria meningitidis that cause endemic disease.

Three collections of strains of Neisseria meningitidis that caused meningococcal disease during nonepidemic periods were serotyped to determine whether serotypes that cause endemic disease are more heterogeneous than those responsible for epidemic disease. Thirty-four strains isolated from pediatric patients in Houston, Texas, from February 1977 to March 1978 were of three separate serogroups and 11 serotypes; 27 contemporary (1977-1978) strains from predominantly military populations, obtained nationwide, were of six serogroups and six serotypes, while 11 strains isolated at military posts in the southwest United States from 1970 through 1976 were of four serogroups and five serotypes. Between 9% and 20% of the strains were nontypable, while type II strains which were responsible for the epidemics in the Northern and Western Hemispheres earlier in the 1970's, accounted for only 20%-44% of the strains. In contast to epidemics, which appear to be caused by a single serotype, endemic meningococcal disease appears to be caused by a broad, heterogeneous distribution of serotypes. Thus, development of a serotype-specific vaccine may have limited application to the prevention of endemic meningococcal disease.

Separation and purification of immunoglobulins M,A and G from small volumes of human sera by a continuous, inline chromatographic process.

A continuous, in-line chromatographic process for the separation of immunoglobulins M, A and G from small volumes of human sera is described. The process involves the sequential and uninterrupted application of molecular sieve, ion-exchange, immunoadsorbent-affinity and de-saltin-concentrating chromatographic methodologies. From 1-3 ml of starting serum it yields 45-55%, by weight, of the three classes of immunoglobulins in 36 h. Each immunoglobulin fraction is free of contamination by immunoglobulins of the other two classes and retains full biological activity as measured by a sensitive immune lysis assay.

Immunoepidemiology of meningococcal disease in military recruits. I. A model for serogroup independency of epidemic potential as determined by serotyping.

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.

Response to antigenic determinants of Neisseria meningitidis lipopolysaccharide investigated with a new radioactive antigen-binding assay.

Adaptations of the Farr technique have resulted in a specific and reproducible radioactive antigen-binding assay for antibodies directed against the lipopolysaccharide (LPS) of Neisseria meningitidis. LPS was intrinsically labeled with 14C acetate during 16-hr growth in a modified Frantz media, extracted by hot phenol-H2O, and purified by dialysis, ultracentrifugation, and ethanol precipitation. LPS, which aggregates in aqueous solutions, was maintained in a monomeric form in 3% sodium deoxycholate (NaD) as determined by gel filtration on Sephadex G-75. Since NaD is insoluble in (NH4)2SO4, polyethylene glycol, 20%, was used to precipitate immunoglobulins of all three major classes.