Flow cytometric measurements of DNA and other cell components in human tumors: A critical appraisal

doi:10.1016/0046-8177(89)90244-X

Human Pathology

Volume 20, Issue 6, June 1989, Pages 528-548

https://doi.org/10.1016/0046-8177(89)90244-X Get rights and content

Abstract

Fundamental principles' of flow cytometry wity emphasis on DNA measurements and cell cycle analysis in human cells and tissues are summarized. Some of the pitfalls of cell preparation techniques and histogram interpretation are discussed at length. While consensus has been reached for some organs and tumors that DNA quantitation by flow cytometry (or image cytometry) may be of prognostic value, for most cancers studied to date the information remains incomplete. Thoroughly lacking are well-structured prospective studies because retrospective studies, while suggestive, may not necessarily be of the same value. Potential usefulness of other tumor markers is briefly discussed. Many fundamental questions concerning definitions of “diploid” and “aneuploid” tumors have not been satisfactorily settled. While the goal of “objective measurements” is worthy of further pursuit, the interpretation of results is often highly subjective. The biologic reasons for behavioral differences between diploid and aneuploid tumors are still totally obscure.

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Cited by (195)

Evaluation of DNA ploidy with intraoperative flow cytometry may predict long-term survival of patients with supratentorial low-grade gliomas: Analysis of 102 cases
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Citation Excerpt :
Characterization of various cancers, including brain tumors, with FC had been extensively used starting from the end of the 1970s. Multiple studies were mainly directed at cell-cycle analysis, evaluation of proliferative activity, calculation of DNA index, and determination of DNA histogram type, as well as at assessment of their prognostic and predictive values [15–29]. However, reproducibility of these data was low, because applied methods of FC lacked standardization and were highly operator-dependent.
The objective of the present study was evaluation of the prognostic significance of DNA ploidy assessed with intraoperative flow cytometry (iFC) during resection of low-grade gliomas (LGG).
Retrospective analysis of 102 consecutive cases of newly diagnosed WHO grade II supratentorial gliomas, surgical removal of which was accompanied by iFC, was done. There were 46 diffuse astrocytomas (DA) and 56 oligodendrogliomas (OD). According to iFC, 68 tumors (67%) were considered non-aneuploid and 34 (33%) aneuploid. Median extent of resection (EOR) was 95% (mean, 89.0 ± 14.5%). Postoperative FRT with or without adjuvant chemotherapy was administered in 24 cases (24%).
With median follow-up of 29.9 months (range, 9.4–66.9 months), neither median overall survival (OS) nor median progression-free survival (PFS) were reached. The 3-year actuarial OS and PFS rates were 95.8% and 86.3%, respectively. Non-aneuploid DNA histogram type of the tumor was associated with significantly longer OS both in univariate (P = 0.0094) and multivariate (P = 0.0232) analyses, and with longer PFS in univariate analysis (P = 0.0184). Aneuploidy was encountered more frequently in DA, than in OD (43% vs. 25% of cases; P = 0.0488). Tumor progression was noted in 15 DA and 4 patients succumbed to the disease; in this subgroup the main unfavorable prognostic factors for OS and PFS were presence of aneuploidy (P = 0.0510) and MIB-1 index ≧3.9% (P = 0.0141), respectively. Aneuploid DA more frequently progressed to glioblastoma than to anaplastic astrocytoma, but this difference did not reach statistical significance (P = 0.1362). In contrast, only one OD progressed (non-aneuploid neoplasm), and no one patient with such tumor died of the disease.
DNA ploidy assessed with iFC may be effectively used as prognostic indicator in cases of LGG, especially of DA. Aneuploid tumors demonstrate more aggressive clinical course translated into shorter OS of patients. Thus, their detection during surgery may be helpful for decision on the optimal EOR, and for choice of the most appropriate postoperative adjuvant therapy.
Simultaneous paired analysis of numerical chromosomal aberrations and DNA content in osteosarcoma
2001, Modern Pathology
Relatively little is known about the biologic relevance of numerical chromosomal changes in relation to DNA content in osteosarcoma. In this study, by using a series of human osteosarcoma cell lines, we standardized a method for the assessment, on the same nuclei specimen, of both specific chromosome copy numbers by fluorescence in situ hybridization (FISH) and the DNA content by static cytofluorometry or image cytometry. On the same cell lines, we also evaluated the DNA content by using flow cytometry and the chromosome number distribution by metaphase analysis. Comparison between these different methods showed that DNA ploidy level as determined by FISH or metaphase analysis is frequently lower than the ploidy pattern as defined by cytometric methods. By using comparative genomic hybridization, we were able to demonstrate that these discrepancies were due to the presence of several unbalanced chromosome aberrations, specifically gains and high-level amplifications, which affect the total DNA content with less effect on the total chromosome number. Thus, evaluation of DNA ploidy in osteosarcoma cells is needed for a correct interpretation of FISH or cytogenetic data concerning numerical chromosomal changes. Evaluation of tumor ploidy in a series of clinical samples demonstrated that in high-grade osteosarcoma, flow cytometry sometimes may give false results because of the presence of high proportions of contaminating, nonneoplastic cells that cannot be excluded from the flow cytometric assessment but that do not interfere with the evaluation of DNA ploidy by static cytofluorometry or image cytometry, in which only tumor cells are selected for the analysis. The possibility of using this method to evaluate, on the same nuclei sample, both specific chromosomal aberrations and DNA ploidy may allow a better determination of numerical chromosomal changes that may be relevant for the biologic behavior of osteosarcoma.
Analysis of human tumors by laser scanning cytometry
2001, Methods in Cell Biology
The laser scanning cytometer (LSC) is a microscope-based cytofluorometer that combines advantages of flow cytometry and image analysis. Fluorescence of individual cells is measured rapidly by LSC, with sensitivity and accuracy comparable to that of flow cytometry (FC). The unique feature of LSC, which distinguishes it from FC, is that the specimen is interrogated by a single or two laser beams while located on a microscope slide rather than in suspension. Several attributes can be measured and recorded by LSC for each analyzed cell in a given cellular specimen: (1) the integrated fluorescence intensity over the integration contour at a given wavelength band, (2) the value of maximal pixel within the measured area, and (3) the perimeter of the contour. This chapter focuses on the clinical applications of LSC for tumor analysis. Several analytical methods, frequently used in FC for tumor diagnosis and prognosis, have been adapted to LSC and are presented in the chapter. The analytical power of LSC combined with its unique advantages, in particular to correlate measurements with visual examination of the corresponding cell, are of special value for pathologists.
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^☆: Supported by National Institutes of Health grants no. 5R01 CA35745, 5R01 CA34790, and IU0 1CA 41025 to L.G.K.

^∗: Dr Czerniak is a visiting scientist in the Department of Pathology, Montefiore Medical Center. Permanent address: Department of Tumor Pathology, Medical Academy, Szczecin, Poland.

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SymposiumFlow cytometric measurements of DNA and other cell components in human tumors: A critical appraisal☆

Abstract

Pathol Res Pract

Clin Chim Acta

Hum Pathol

Hum Pathol

Cancer Genet Cytogenet

Pharmacol Ther

Cancer Genet Cytogenet

Urology

Comput Biomed Res

Advances in flow cytometry for diagnostic pathology

Lab Invest

Prognostic application of DNA flow cytometry for human solid tumors

Ann NY Acad Sci

Clinical and biological significance of aneuploidy in human tumours

J Clin Pathol

Flow cytometry, cellular DNA content, and prognosis in human malignancy

J Clin Oncol

Analytical and quantitative cytology: A historical perspective

Anal Quant Cytol Histol

Automated cytology and histology: A historical perspective

Anal Quant Cytol Histol

Torbjörn Oskar Caspersson: Curriculum vitae and selected publications

Cytometry

Ultraviolet absorption of epidermoid cancer cells

Science

Spectrophotometric cell sorter

Science

Spectrophotometer: New instrument for ultrarapid cell analysis

Science

Efficacy of a rapid cell spectrophotometer in screening for cervical cancer

Cancer

Flow Cytometry and Sorting

Practical Flow Cytometry

Multistation multiparameter flow cytometry: A critical review and rationale

Cytometry

Laser noise and news

Cytometry

Flow cytometry. Principles and applications. II

Cancer Invest

Comparison of needle aspiration and solid biopsy technics in the flow cytometric study of DNA distributions of surgically resected tumors

Am J Clin Pathol

Flow cytometric evaluation of cell dispersion from human head and neck tumors

Cytometry

Solid tumor preparation for clinical application of flow cytometry

Cytometry

Preparation of tissues for DNA flow cytometric analysis

Cytometry

Nuclei from rat liver; isolation method that combines purity with high yield

Science

The monodisperse cervical smear. Quantitative analysis of cell dispersion and loss with enzymatic and chemical agents

Anal Quant Cytol

A method for dissociation of viable human breast cancer cells that produces flow cytometric kinetic information similar to that obtained by thymidine labeling

Cancer Res

Characterization of cells obtained by mechanical and enzymatic means from human melanoma, sarcoma, and lung tumors

Cancer Res

Comparison of tissue disaggregation techniques of transitional cell bladder carcinomas for flow cytometry and chromosomal analysis

Cytometry

Flow microfluorometric analysis of nuclei isolated from various normal and malignant human epithelial cells

J Histochem Cytochem

DNA distribution in human colon carcinomas and its relationship to clinical behavior

JNCI

Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining

J Cell Biol

A detergenttrypsin method for the preparation of nuclei for flow cytometric DNA analysis

Cytometry

Deoxyribonucleic acid ploidy and cell cycle events in benign colonic epithelium peripheral to carcinoma

Lab Invest

Method for analysis of cellular DNA content of paraffin-embedded pathological material using flow cytometry

J Histochem Cytochem

DNA flow cytometry of paraffin-embedded tissue

Cytometry

Application of DNA flow cytometry to paraffin-embedded archival material for the study of aneuploidy and its clinical significance

Cytometry

Interinstitutional variability in DNA flow cytometric analysis of tumors. The National Cancer Institute's flow cytometry network experience

Cancer

Extraction of nuclei from selected regions in paraffin-embedded tissue

Cytometry

Identification of cell subpopulations by dual-color surface immunofluorescence using biotinylated and unlabeled monoclonal antibodies

Symposium
Flow cytometric measurements of DNA and other cell components in human tumors: A critical appraisal☆