V.V. Zenin, N.D. Aksenov, A.N. Shatrova, N.V. Klopov, L.S. Cram, A.I. Poletaev
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
Instrumentation for univariate fluorescent flow analysis of chromosome sets has been developed for human cells. A new method for cell preparation and intracellular staining of chromosomes with different dyes was developed. The method includes enzyme treatment (chymotrypsin), incubation with saponin and separation of prestained cells from debris on a sucrose gradient. This procedure makes it possible to get a well stained sample with a minimal amount of contaminates: free chromosomes, cell fragments, etc. A special mixing/stirring device was placed inside the flow chamber of flow cytometer. The rupturing of prestained mitotic cells is performed by means of a small magnetic rod vibrating in an alternating magnetic field. The device works in a stepwise manner: a defined volume of sample is delivered to the breaking chamber for breaking mitotic cells for a defined time period, followed by a buffer wash to move the released chromosomes from the breaking chamber to the point of analysis. The information about the chromosomes appearing at the point of analysis is accumulated in list mode files makes it possible to resolve chromosome sets arising from single cells on the basis of time gating. The concentration of cells in the sample must be kept low to ensure that only one cell at a time enters the breaking device. The developed software classifies chromosome sets according to different criteria: total number of chromosomes, overall DNA content in the set, and the number of chromosomes of certain type. In addition it's possible to determine the presence of extra chromosomes or the loss of chromosome types. Thus this new approach combines the high performance of flow cytometry (quantitation and high throughput) with the advantages of image analysis (cell to cell karyotype analysis and the skills of a trained cytogeneticist). The data analysis capabilities offer extensive flexibility in determining important features of the karyotypes under study. This development offers the potential to duplicate most of what is determined by a clinical cytogeneticist.
Supported by a grant from the DOE-NIS International Partnering Program, Russian State Program of Human Genome Studies, and the NIH (grant RR-01315).
 Institute of Cytology, Russian Academy of Sciences, St-Petersburg, Russia; St-Petersburg Institute of Nuclear Physics, Russian Academy of Sciences, Gatchina, Russia.
 Los Alamos National Laboratory, Los Alamos, NM.
Return to Table of Contents