A two-centre validation study of sterility test of corneal storage media with elimination of interfering antimicrobials in compliance with the European Pharmacopoeia.

The purpose of this study was to validate the sterility test of corneal culture and deswelling/transport media using a device for removal of antimicrobials before incubation in BACTEC™ automated system in two Italian Eye Banks. Corneal culture medium, TISSUE-C, and deswelling/transport medium, CARRY-C, were inoculated with 10-100 cfu of six European Pharmacopoeia (EP) reference strains and either treated with medical device RESEP for removal of antimicrobials (RESEP+ group) or left untreated (RESEP- group) before injection into the BACTEC Plus bottles. The same steps were repeated in the absence of inocula with tryptone soy broth samples as negative controls, and the inocula were also directly injected in the BACTEC™ bottles as growth controls. All the samples were incubated in BACTEC™ automated system for 7 days, and the time to detection of microbial growth was recorded automatically. At both the Eye Banks, in the RESEP+ groups, microbial growth was detected in 100% of samples. In the RESEP- group, the method sensitivity ranged from 66.7 ± 21.1 to 88.9 ± 6.4% for TISSUE-C samples while for CARRY-C samples the method sensitivity ranged from 94.5 ± 5.1 to 100%. The method specificity corresponded to 100% for all the groups at both Eye Banks. This two-centre validation study showed that the use of RESEP increased the sensitivity of sterility test using BACTEC™ automated system up to 100% and, consequently, allowed validation of the method for sterility testing of corneal storage and deswelling/transport media according to the EP requirements. The test could not be validated without the use of RESEP.

Comparison of serological and molecular typing for HLA-A and -B on cord blood lymphocytes.

HLA class I typing by standard microcytotoxicity testing has been unsatisfactory for 14.5% of 1644 cord blood samples. In this study, we evaluated the capacity of PCR-SSP in solving problems in HLA-A,B typing with serological methods. With this aim we have compared serology with PCR-SSP in 100 cord blood samples with doubtful or unreliable HLA-A,B typing. PCR-SSP was successful in amplifying HLA-A,B alleles in all 100 cord blood samples. Forty-six typings gave discrepant results with the 2 methods (serology and PCR-SSP). Typings were considered discrepant also in the case of inability to define a split. For 19 specimens, no serological conclusion was drawn due to high mortality of the cell suspension, while PCR-SSP allowed the definition of a clear typing. In 6 cases it was necessary to infer information from serology to define the current typing. Finally, in 3 other cases it was impossible to exclude or attribute the antigen/allele B67 or B4802. PCR-SSP for HLA-A,B can improve the overall reliability of HLA-A,B typing requiring a small amount of blood although, with the set of sequence specific primers adopted, a number of alleles are still poorly defined.

ABO genotyping in Italian blood donors.

BACKGROUND: Traditional ABO blood group serology is based on the immunoreactivity of antisera with the carbohydrate A, B and H antigens. Progress in the molecular biology of the ABO system has recognized the molecular basis of the red cell (RBC) antigens and has provided a genetic model for ABO polymorphism at the molecular level. Recently, this genetic model was tested in a large number of individuals. MATERIALS AND METHODS: In this study we applied DNA analysis to determine the frequency of ABO genotypes in a group of blood donors for whom the ABO type was known. Two hundred and fifty healthy Italian blood donors were analyzed using polymerase chain reaction (PCR) to amplify two different regions of genomic DNA, each of which contained a different nucleotide polymorphism. The amplified product was digested with 4 restriction enzymes that revealed differences among A, B and O individuals. To analyze the genes at polymorphic sites 261 and 703 we used the restriction enzymes BstE II and Kpn I, and Hpa II and Alu I and compared the PCR determined genotypes to serologically determined phenotypes. RESULTS AND CONCLUSIONS: The results were consistent for all unrelated individuals; however, 2 of 100 individuals with the 0 phenotype carried one allele that differed from the proposed genetic model. This novel O allele, termed 0(2) by Yamamoto et al., was found in our series with a frequency of 1%. The blood group AB0 genotype of 250 healthy Italian blood donors was: 13 AA/AO(2), 37 AO(1), 11 BB, 39 B0(1), 50 AB, 98 0(1)0(1) and 2 0(1)0(2). This method should be applicable not only in forensic medicine but also in immunohematology when serology fails.

Congenital brain damage spares the basic patterns of parental behavior in affected mice.

Pregnant albino mice were treated with 5-azacytidine so that the embryonic brains were affected late in their morphological ontogeny. The offspring showed retarded body growth and a conspicuous reduction in the size of the cerebral hemispheres as measured at the end of development. Histological alterations were found in the hippocampus and the cingulate cortex. No behavioral alterations were detected during development, with the exception of the hyperactivity which probably caused the better performance of treated offspring observed in a self-feeding test. This functional abnormality, attributed by previous authors to retardation in telencephalic development, persisted into adulthood. The parental behavior of virgin females towards a weak stimulus-object was robust. Treated subjects were non-neophobic, seldom aggressive and showed clearcut parental responses. In addition, although the frequency of overall parental tendency was lower in the treated subjects, it gradually approached that of the controls across repeated trials. The brain structures affected by this treatment seem influential on behavioral organization and habituation to novelty, not on basic patterns of behavior, which are probably rooted in phylogenetically more ancient structures.