Picogram per cell determination of DNA by flow cytofluorometry

Lee, Greta M.; Thornthwaite, Jerry T.; Rasch, Ellen M.

doi:10.1016/0003-2697(84)90374-9

Cited by 78 publications

(40 citation statements)

References 18 publications

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“…approximately 106 cells ml-' (Vindelov et al, 1983). To increase cell elution, the tissue was mechanically disintegrated with two forceps, after which 1-2 ml of nuclear isolation medium, containing propidium iodide (PI) for the staining of DNA, was added (50 tLg PI ml-', SIGMA P-5264; RNAse 0.1 mg ml-' and SIGMA R5125; Nonidet P 40 0.6% (v/v) in isotonic phosphate buffered saline, GIBCO) (Lee et al, 1984;Thornthwaite et al, 1980). The samples were filtered (140 tsm) and incubated in the dark for 10 min at room temperature, after which they were kept at + 4°C until flow cytometry was performed (within 60 min).…”

Section: Methodsmentioning

confidence: 99%

Flow cytometry in primary breast cancer: improving the prognostic value of the fraction of cells in the S-phase by optimal categorisation of cut-off levels

Sigurðsson

Baldetorp²,

Borg³

et al. 1990

Br J Cancer

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Summary The use of continuous prognostic variables is clinically impractical, and arbitrarily chosen cut-off points can result in a loss of prognostic information. Here we report findings from a study of primary breast cancer, showing how the prognostic value of the fraction of cells in the S-phase of the cell cycle (SPF), as measured by flow cytometry, can be affected by the SPF cut-off level(s) adopted. It was possible to evaluate the SPF in 566 (94%) of 603 consecutive cases where fresh frozen specimens were available in a tumour bank at our department. Clinically, all patients were without distant spread at the time of diagnosis, and the median duration of follow-up was 4 years. Using different survival end-points and x2 values for each cut-off level, two optimal cut-off points, at the 7% and 12% levels, were consistently obtained for the SPF. The aim of the current study was to elicit optimal cut-off levels for the SPF in primary breast cancer, as determined with FCM.Correspondence: H. Sigurdsson. Received 3 January 1990; and in revised form 9 July 1990. Patients and methodsIn the health care region of southern Sweden hormone receptor analyses are routinely performed on specimens from patients with primary breast cancer, any residual tumour specimens being stored in a tumour bank at the Oncology Department at University Hospital in Lund.The study included 603 consecutive cases where specimens were available in the tumour bank, and which fulfilled the following inclusion criteria: (1) tumour sample from primary breast cancer (cancer in situ excluded); (2) diagnosed during the period between September 1982 and January 1986; (3) clinically without signs of distant metastasis at the time of diagnosis; (4) sufficient tumour specimen to yield a DNA histogram; and (5) tumour cells microscopically identified in a cytopathologic investigation of all imprints used for making cell suspensions for DNA analysis.The median age of the patient population was 63 years (range 26-97). Tumour size was taken from the pathological report and usually determined on a unfixed specimen. A median of ten axillary lymph nodes was investigated. The distribution of cases by tumour size was as follows: 0-20mm, n=250 (41%); 21-50mm, n=317 (53%); and > 51 mm, n = 36 (6%). Distribution by axillary lymph node status was: node-negative, n = 303 (50%); node-positive, n = 277 (46%); and no axillary staging performed, n = 23 (4%).Laboratory methods Tumour samples were taken from biopsies originally obtained for steroid receptor analysis. Fresh specimens from the macroscopic mammary tumours were taken by the examining pathologist, except in a few cases where it was done during surgery. The specimens were stored frozen (-70°C), and later analysed (in a single laboratory) at the Oncology Department in Lund. Flow

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Section: Methodsmentioning

confidence: 99%

Flow cytometry in primary breast cancer: improving the prognostic value of the fraction of cells in the S-phase by optimal categorisation of cut-off levels

Sigurðsson

Baldetorp²,

Borg³

et al. 1990

Br J Cancer

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“…Lee et al [40] and Silvestrini et al [38] later studied the relationship of DNA ploidy and ovarian cancer with similar results. Brescia et al [41] examined the DNA content of 99 ovarian carcinomas by flow cytometric analysis of nuclei obtained from paraffin-embedded tissue.…”

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confidence: 67%

“…The data for 22 diploid, normal tonsil samples resulted in an average G0 coefficient of variation of 2.18 ± 0.46 SD, an average DNA index (peak channel of normal tonsil divided by the peak of DNA standard) of 1.42 ± .033 SD, and an average S-phase fraction of 4.37 ± 1.37 SD. The DNA content of TRBC is 5.0 pg/nucleus [40] . The DNA content of the diploid populations was about 7.4 pg/nucleus, which more closely resembled non-paraffinized tissue G0 populations of 7.9 pg/nucleus [39,40,45,46] .…”

Section: Resultsmentioning

confidence: 98%

“…The DNA content of TRBC is 5.0 pg/nucleus [40] . The DNA content of the diploid populations was about 7.4 pg/nucleus, which more closely resembled non-paraffinized tissue G0 populations of 7.9 pg/nucleus [39,40,45,46] . The fixation and paraffination affected the ability of DAPI to bind to the same extent to A-T regions as in fresh tissues.…”

Section: Resultsmentioning

confidence: 98%

“…We have been pioneers in developing better ways to measure DNA in tissues [39,40,[43][44][45][46] . The purpose of the research presented here is to show the best procedures for measuring DNA in nuclei derived from paraffinated tissues.…”

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confidence: 99%

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Comparison of clinical staging of benign and malignant ovarian tissues with DNA flow cytometry

Thornthwaite

Stanfill²,

Ahmed³

2013

JST

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The prevention, diagnosis and treatment of ovarian cancer are major issues. The outcome of patients with advanced ovarian cancer is poor despite aggressive therapy including surgery, combination drug chemotherapy and radiation treatments. From the literature, the direct correlation between DNA ploidy and survival is greatly enhanced using high resolution DNA measurements, which results in a coefficient of variation (CV) range between 1%-2% (1.42 ± 0.19, n=66) for trout red blood cells (TRBC) and 2%-3% (2.18 ± 0.46 SD, n=22) for tonsil nuclei derived from formalin-fixed, paraffin-embedded tissues (deparaffinated). DNA nuclear determinations from 50 ovarian cancer and 21 benign patients is presented. This was accomplished by measuring the DNA content of nuclei simultaneously isolated from deparaffinated tissues, stained with the fluorescent DNA specific dye, 4', 6-diamidino-1-phenylindole (DAPI) and analyzed on a high resolution flow cytometer. A high percentage of aneuploidy (92.0%) was determined from the ovarian cancer patients, especially of the aneuploid DNA histogram types, such as hypodiploid, multiploid and hypertetraploid (64.0%), which have shown poor prognosis in a variety of cancers including ovarian. Furthermore, aneuploidy was detected in 23.8% of the benign patients. DNA flow cytometry may complement pathological assessment of ovarian cancer to better determine malignancy, thus justifying a closer follow-up with more specialized approaches to treatment in clinical trials that involve new therapies including the use of chemically defined, natural products, which may help offset the overall poor prognosis seen in ovarian cancer.

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Correlation between p53, c-erbB-2, and topoisomerase II? expression, DNA ploidy, hormonal receptor status and proliferation in 356 node-negative breast carcinomas: prognostic implications

Olsson²,

et al. 1999

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Picogram per cell determination of DNA by flow cytofluorometry

Cited by 78 publications

References 18 publications

Flow cytometry in primary breast cancer: improving the prognostic value of the fraction of cells in the S-phase by optimal categorisation of cut-off levels

Flow cytometry in primary breast cancer: improving the prognostic value of the fraction of cells in the S-phase by optimal categorisation of cut-off levels

Comparison of clinical staging of benign and malignant ovarian tissues with DNA flow cytometry

Correlation between p53, c-erbB-2, and topoisomerase II? expression, DNA ploidy, hormonal receptor status and proliferation in 356 node-negative breast carcinomas: prognostic implications

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