Limits of detection of nuclear DNA abnormalities by flow cytometric DNA analysis. Results obtained by a set of methods for sample‐storage, staining and internal standardization

Abstract

Flow cytometric DNA analysis is an attractive alternative to conventional cytogenetics for diagnosing karyotype changes resulting in abnormal DNA content. We have developed methods for long-term storage of samples and standards, for staining the cells and for standardization of the measurements by two internal standards. In this paper the currently attainable resolution was determined and limiting factors were identified. Normal reference values for male and female leucocytes were determined by analyzing 240 samples from six men and six women, all cytogenetically normal. The DNA content of female cells was 1.5% higher than that of male cells. This finding was used to correct the results for sex related DNA differences. The 95% confidence limits for mononuclear blood cells were 20.79%. The results on granulocytes were exceptionally variable, with 95% confidence limits of k1.798. Reexamination of the normal leucocytes after 1 year showed a long-term drift of up to 2% in the results, indicating a need for regular checks of the reference values. In addition to the sex related differences in DNA content, individual differences of up to 1% were demonstrated. This creates problems as to which reference value to use for a particular sample. Furthermore, tissue related differences in fluorescence were found when six different tissues from one mouse were examined. The means had a range of 0.7%. The tissue related differences add uncertainty to the interpretation of the results. The resolution of heterogeneous populations with slightly different DNA content was examined by analyzing mixtures of cells with known DNA differences ranging from 1.50% to 5.83%. With a coefficient of variation of the peaks of about 2% a DNA difference of approximately 4% was required for accurate determination of the DNA content of the two individual populations. A lower coefficient of variation would increase the resolution, but tissue related differences in fluorescence could then become the limiting factor. To perform reliable analyses on a large-scale routine basis, a number of problems must be solved. We have developed methods for long-term storage of samples and standards (19), for fluorochrome staining of the cells (20) and for standardization of the measurements (21). The methods were designed to make sample collection independent of immediate subsequent analysis, and to make the results directly comparable irrespective of the day of analysis and eventually of the laboratory where the analysis is performed.In this paper the detection limits of these methods were determined and some limiting factors were identified. The strategy for testing the achievable resolution was to exploit the small sex and species related DNA differences in man and mouse. Materials and MethodsHuman leucocytes were separated into granulocytes and mononuclear cells by the method of Boyum (3). Cells from mouse liver, spleen, kidney and thymus were obtained by fine-needle aspiration (18). Mouse bone marrow from the femur and blood from the femoral artery were used. Ce...

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