Check samples for laboratory self-assessment in DNA flow cytometry. The national cancer institute's flow cytometry network experience

Abstract

The National Cancer Institute's Flow Cytometry Network (NCI-FCN) is attempting to facilitate the transfer of flow cytometry (FCM) of exfoliated bladder cells from the research laboratory to the clinical laboratory. Demonstrating interinstitutional consistency in FCM analysis of replicate specimens simulating clinical barbotage specimens, fixed to allow easy transportation and storage at room temperature was one specific objective. Simulated barbotage specimens were prepared by mixing cultured aneuploid bladder carcinoma cells with normal or mitogen-stimulated peripheral blood mononuclear cells in different ratios. The samples were fixed in 10% formalin for 30 minutes, stored in buffer, and enucleated with pepsin, pH 1.5, before staining with propidium iodide for FCM DNA analysis. Preservation in ethanol or other common DNA cytochemical reagents was found to be unsatisfactory. In contrast, the formalin-fixed samples showed excellent preservation of quantitative DNA fluorescence and coefficient of variation of histogram peaks for over 2 weeks. Exchange of eight fixed specimens among five network laboratories that analyzed them as "unknowns" showed good overall agreement on histogram data and interpretation, although some noteworthy interlaboratory differences were found. This technique could be used for self-assessment surveys of clinical laboratory performance in DNA FCM of bladder barbotage specimens.

Quality control study by the French Cytometry Association on flow cytometric DNA content and S-phase fraction (S%)

“…In addition, few authors have investigated clinical laboratories with different degrees of experience (3,14,25). Few have involved frozen tumor samples (20,23); the majority used mixtures of normal cells and cell lines (3)(4)(5)14,25), which do not reflect the clinical situation, because the S% of cell lines is generally far higher than the majority of human tumors. Thus, these studies failed to identify prob- lems encountered during the use of mathematic models for low S% values.…”

“…Several quality control studies on FCM DNA analysis have been published (13,20,21,23,25) with the aims of defining the optimal conditions for sample storage with a view to interlaboratory exchanges (4), studying interlaboratory reproducibility during analysis of given samples (3,13,14,16,21,27,28), and determining the prognostic relevance of DNA histogram classification ( 15).…”

Comparative evaluation of fresh, fixed, and cryopreserved solid tumor cells for reliable flow cytometry of DNA and tumor associated antigen

“…We carried out the investigation on fresh tissues obtained from patients in the operating theatre and not on cultured solid tumor cell lines so that the practicability of these methods in a clinical and research setting could be evaluated. Cell suspensions of different tumors and normal tissues (and even subpopulations of cells in such suspensions, such as leucocytes) have a differing susceptibilities to damage by fixation and storage [3,7,491. In addition, to prepare a cell suspension from a tissue block there must be some form of mechanical and/or enzymatic tissue dissociation, and this might change the susceptibility of the cells to injury; a technique might work well for a particular cell line but not be the best method for solid tumor suspensions in routine clinical practice.…”

Significance of DNA quantification in testicular germ cell tumors

“…The nuclear DNA content is considered as a tumor marker (Barlogie et al, 1980(Barlogie et al, , 1983Koss et al, 1989a). The cytophotometric determination of the DNA content in cell nuclei has become a frequent practice in the diagnosis and prognosis of many tu-mors of the male genital system such as tumors of prostate (Zetteberg & Esposti, 1976;Seppelt & Sprenger, 1984;Winkler et al, 1988) kidney (Tanaka et al, 1983;Otto et al, 1984;Frohlander et al, 1989) and bladder (Fossa, 1975;Hoshi et al, 1980;Coon et al, 1989;Koss et al, 1989b).…”