Comparative studies on the detection of benzodiazepines in serum by means of immunoassays (FPIA).

Serum was tested for benzodiazepines by fluorescence polarization immunoassay (FPIA) on Abbott's ADx system using the benzodiazepine serum reagents (Benzo S) and the benzodiazepine urine reagents (Benzo U) after pretreatment of specimens by means of acetone precipitation. The following sera were included for comparing the two methods: negative sera spiked with various benzodiazepines; 80 sera randomly selected out of a total of 8654 serum specimens from impaired drivers; and blood specimens from individuals who stated that they had taken benzodiazepines. The different benzodiazepines were added to serum at concentrations of 25, 75, and 300 ng/mL. The low-dose benzodiazepines flunitrazepam and triazolam were additionally tested at serum concentrations of 10 ng/mL. Because of the better reproducibility and the shift of the dynamic range to lower concentrations, the Benzo S assay was found to be more sensitive than the Benzo U assay after acetone precipitation. The direct ADx benzodiazepine serum assay has clear advantages over the acetone precipitation method, especially with regard to therapeutic concentrations and for the detection of the highly potent and low-dose benzodiazepines flunitrazepam and triazolam.

Simultaneous detection of X- and Y-bearing human sperm by double fluorescence in situ hybridization.

Double fluorescence in situ hybridization (FISH) was used to detect sex chromosomes in decondensed human sperm nuclei. Biotinylated X chromosome specific (TRX) and digoxigenin-labeled Y chromosome specific (HRY) probes were simultaneously hybridized to sperm preparations from 12 normal healthy donors. After the hybridization, the probes were detected immunocytochemically, using two different and independent affinity systems. Ninety-six percent of the 12,636 sperm showed fluorescent labeling, of which 47.4% were haploid X and 46.8% were haploid Y. A frequency of 0.46% of XX-bearing sperm (0.28% disomic, 0.18% diploid) and 0.38% YY-bearing sperm (0.21% disomic, 0.17% diploid) was found. The overall proportions of X- and Y-bearing sperm in the ejaculates were 47.9% and 47.2%, respectively, which was not significantly different from the expected 50:50 ratio. In addition 0.21% of cells appeared to be haploid XY-bearing sperm, 0.62% were diploid XY-bearing cells, and 0.05% of cells were considered to be tetraploid cells. The application of double FISH to human sperm using X-chromosome and Y-chromosome probes has allowed a more accurate assessment of the sex chromosal complements in sperm than single FISH method and quinacrine staining for Y-bodies.

Detection of chromosome 17- and X-bearing human spermatozoa using fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) with DNA probes specific to chromosomes 17 and the X has been applied to human ejaculated sperm. After sperm nuclei were decondensed with EDTA and DTT, biotinylated alpha satellite DNA probes TR17 and TRX were separately used on preparations from thirteen healthy donors. After hybridization 96% of sperm were labelled with the TR17 probe and 48% of sperm were labelled with the TRX probe. Frequencies of 0.33% disomic 17 and 0.29% disomic X sperm were found. The frequencies of diploid sperm were assessed as 0.37% using the TR17 probe and 0.20% using the TRX probe which labelled only one half of the sperm; after correcting the result from the X-probe to 0.40% the two frequencies are very similar.

Chromosome errors at mitotic anaphase.

Errors in mitotic divisions were assayed using various satellite DNAs as probes, hybridized in situ, to show that they included nondisjunction, chromosome and chromatid lagging, chromatid malsegregation, and monopolar segregations. The total rates of error were 1.7, 1.1, and 0.6% for chromosomes X, 17, and 18, respectively. Lagging was the most common error for all chromosomes and chromatid malsegregation, a source of 3:1 segregations occurred at about the same frequency as nondisjunction. In some cells, lagging of both X chromatids occurred and there were several cells where both X chromosomes showed errors in segregation. The disjunction of chromosomes was shown to be independent of their segregation and is speculated to involve a different mechanism.

Localization by in situ hybridization of a type I keratin intermediate filament gene (Krt-1.14) to band D of mouse chromosome 11.

A probe from the 3' noncoding region of a murine type I keratin intermediate filament (IF) gene (Krt-1.14) localizes to band D of murine Chromosome 11 using in situ hybridization. This localization provides a physical confirmation of the assignment of the type I keratin genes by linkage analysis in the mouse. It also demonstrates that the Krt-1.14 genes are at a single locality in the mouse in contrast to the two locations on the short and long arms of chromosome 17 in humans.

Chromosome elimination in micronuclei: a common cause of hypoploidy.

An excess of hypoploid cells has repeatedly been reported in studies of aneuploidy and has often been attributed to technical artifact. We have examined at least 200 anaphase or early-telophase cells from each of 28 normal women and found that chromosome or chromatid lagging occurs in an average of 2.43% of cells. In a separate study, we have examined the frequency of micronuclei in cytochalasin B-arrested, binucleate cells and shown that a similar frequency of cells (1.6%) contain one or more micronuclei. Using in situ hybridization of an alpha centromeric probe (alpha R1), which hybridizes to 9 of the 22 human autosomes, we were able to infer that most, if not all, of the micronuclei contain whole chromosomes or chromatids. Since the loss of a chromosome by lagging will induce hypoploid daughter nuclei (two where a chromosome is lost and one where a chromatid is lost), we conclude that lagging is a major mechanism for chromosome loss in human lymphocyte cultures. This loss occurs in the cells of normal individuals under control conditions.

Chromosome flexion: potential for assessing the state of spindle assembly.

A novel measurement, that of chromosome flexion, has been used to assess the degree of spindle polymerization at metaphase in human lymphocytes. It was found that this measurement showed a highly repeatable quantitative response to nocodazole exposure. Thus this measurement could be used to assess the potential for chemicals to depolymerize spindles. Under controlled conditions, individual differences were observed between the subjects which might be related to their age. However, the response to nocodazole-induced spindle depolymerization, as measured by flexion, was uniform for all subjects. 4 chemicals reported to induce aneuploidy in mammalian cells were used in a flexion assay. Only substances known to depolymerize microtubules reduced chromosome flexion.

A perspective of chromosome displacement as a primary step in the induction of aneuploidy.

This paper considers whether the model proposing chromosome displacement as a primary step in the induction of mitotic aneuploidy (Ford and Roberts, 1983a) is consistent with experimental data relating to spindle structure, chromosomal attachment to spindles, the promotion of aneuploidy and the relative involvement of the different human chromosomes in aneuploidy. It is concluded that the model is consistent with all the parameters thus far tested for autosomal chromosomes.