A new modification of our detergent technique for the preparation of nuclei for flow cytometric DNA analysis is described. The attainment of low coefficients of variation of the peaks and of quantitative staining of nuclei from different tissues was a problem with the original method. This was solved in the new modification by trypsinization of the unfixed nuclei. The nuclei were stabilized by spermine. A simple procedure for long-term storage of samples at -80°C was integrated into the method. The fluorescence of the nuclei was stable for at least 3 hours after staining. Light exposure protection of the samples was essential. No cell loss was caused by storage or staining. The method was successfully applied on samples including: (a) Normal tissues-human lymphocytes, granulocytes and spleen. Mouse lymphocytes, bone marrow, spleen, liver, kidney and thymus. (b) Human neoplasms-lung cancer, breast cancer, lymphoma, leukemia, bladder cancer and cancer of the oral cavity. Key terms: Flow cytometry, DNA, propidium iodide, solid tissues, preparation, storage A previously published method for the preparation of fineneedle aspirates from solid tissues for flow cytometric DNA analysis (9) has been useful for examination of human solid tumors (1,13,14). The method is based on detergent lysis of cell membranes and produces stained nuclei in monodisperse suspension in a single step. However, the low salt procedure of the technique, which has been used most extensively, has two major drawbacks: (a) Tissues rich in cytoplasm, for instance liver cells, are not prepared optimally because residual cytoplasm contaminates the nuclei, causing clumping and nonspecific staining. In the DNA distribution this produces asymmetrical peaks with high coefficients of variation (cv). ( b ) Staining of different types of cells is not perfectly quantitative. Thus a comparison of human lymphocytes and granulocytes for example showed a 6% difference in fluorescence.The original method was therefore modified with the aim of obtaining a preparation technique that would produce optimal results in a broad spectrum of tissues in regard to

In this trial, ABVD therapy for 6 to 8 months was as effective as 12 months of MOPP alternating with ABVD, and both were superior to MOPP alone in the treatment of advanced Hodgkin's disease. ABVD was less myelotoxic than MOPP or ABVD alternating with MOPP.

Determination of nuclear DNA content by flow cytometry requires comparison with a reference standard. The use of external standards such as lymphocytes or granulocytes is time-consuming and inaccurate.Chicken red blood cells (CRBC) have a DNA content of 35% of the human diploid value and have been widely used as internal standard. The ratio calculated on the basis of the peak channel numbers of the standard and

A simple procedure for long-term storage of cells for flow cytometric DNA analysis was developed and tested. The cells were stored as single cells or fine-needle aspirates suspended in a citrate buffer with dimethylsulfoxide (DMSO), or as small blocks of tissue from solid tumors. The cells were stored for up to one y e a r b y freezing at -80°C. Statistical analysis of the results showed no change in the fractions of cells in the cell cycle phases as determined by deconvolution of the DNA-histograms. It was found that in addition to the intrinsic sample variation from the parameter estimation by deconvolution, there was significant intrad a y and interday variation. Hence the most accurate results are obtained if different aliquots of a sample are measured on different d a y s rather than on the s a m e day. Use of the storage method thus has the potential of increasing the accuracy of t h e analysis. The storage method m a k e s sample collection independent of immediate subsequent analysis. This has enabled us to perform large internally controled experiments, involving more samples than can be analyzed in one day, to examine t u m o r samples f r o m different hospitals and to utilize fully the capacity of our flow cytometer. The method was a prerequisite f o r developing an accurate standardization procedure for DNA content determination.Key terms: Flow cytometry, DNA analysis, sample storage, fixation procedures The development of techniques for rapid staining of unfixed cells for flow cytometric DNA analysis (3,7,8) has greatly simplified the application of this method by shortening the preparation time from hours to minutes. However, the fact that unfixed samples must be analyzed in the flow cytometer within a few hours after sampling has created practical problems: (a) In large experiments more samples are produced than can be analyzed in one day. cation of flow cytometry and increase the accuracy of the results.Methods for poststaining fixation have been reported to solve this problem (1, 4). A number of fixation procedures have been tested in our laboratory in combination with our staining method (8). An important feature of this method is that no centrifugation steps are involved and the cell loss is therefore minimal. T o preserve this feature the added fixative could not be removed again. It was invariably found that clumping, or changes in fluorescence, or both, were induced by fixation. The changes were progressive with time and therefore not compatible with quantitative measurements.Cryopreservation before staining of the cells was subsequently found to be useful. This paper describes the effect on the DNA distribution of storage of cells up to 1 year, with particular emphasis on the identification of sources of systematic and random variation. Materials and MethodsThe tumors used were the hypotetraploid JB-1 ascites tumor and a hyperdiploid human malignant melanoma transplanted to nude mice (6). The tumors were stored either suspended in buffer or as small blocks of tissue (approx. 5 x 5 x 5 mm)...

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...