Cytometry of the cell cycle: In search for perfect methodology for DNA content analysis in tissue specimens

Since the dawn of cytometry DNA has been the most frequently measured cellular constituent.1,2 Its measurement provides information on DNA ploidy and cell position in the acell cycle. It may also alert to the presence of apoptotic cells that are characterized by fractional DNA content.3,4 DNA content measured by cytometry is defined as “DNA index” (DI) and is also referred as “DNA ploidy”; for normal (euploid) cells in G0/G1 phase DI = 1.0. Cells in G2/M phase have DI = 2.0 and the S-phase cells are characterized by 1.0 < DI < 2.0. Note that “DNA ploidy” should not be confused with the term “ploidy” that refers to the number of complete sets of chromosomes in a cell where somatic cells containing two complete sets of chromosomes, each derived from the parent, are diploid and the mature gametes, either sperm- or egg-cells, haploid. Progress in developing DNA content assays by cytometry, primarily utilizing flow cytometry (FC) instrumentation, has been driven by introduction of new fluorochromes with the increasing specificity and stoichiometry of binding to DNA and by modifications of the cell preparation and staining procedures.5 There were two parallel but intertwined directions (goals) in method development. One was to make the DNA quantification as accurate as possible. High accuracy enables one to detect minor variation in DNA ploidy and thereby identify aneuploid cells (e.g., for tumor diagnosis) with minimal alteration in DNA content. High accuracy is also of importance in detecting the cell cycle-specific effects of antitumor drugs as well as in assessing the fraction of cells in S-phase in human tumors, considered to be an important clinically prognostic biomarker.6 The coefficient of variation (CV) of the mean value of DNA content of population of cells with uniform content, such as G1 cells is a generally accepted indicator of accuracy of DNA content analysis by FC. Formally undeclared but distinctive competition between different laboratories to outdo each other and report the data with the most accurate DNA content measurement, reflected by minimal CV, has been performed through years. It is now possible to measure DNA content with an accuracy that allows one to discriminate lymphocytes of different gender persons differing by ~1% in number of base pairs (XX vs XY chromosomes).7 While analysis of DNA content in cells maintained in cultures or hematopoietic cells is relatively straightforward there are often problems in isolating cells from normal or diseased tissues and measuring their DNA with good accuracy. The second direction in method development thus was to design convenient approach of tissue dispersal that can lead to isolation of individual cells or cell nuclei amenable to DNA staining and accurate analysis by FC. The tissues differ from each other by the quantity and composition of the intercellular matrix, and by adherence and fragility of the cells embedded in the matrix. It was difficult therefore to develop the methodology that can be universally applied to different tissues, including the cryo-preserved samples of the archival material. Several published methods either were not universally applicable and/or were unable to provide good accuracy in DNA content analysis. Up to date, the most commonly used protocol is the one developed by Vindelov and his colleagues, which is based on a combined treatment of tissues with nonionic detergent and trypsin.8 It allows for high resolution of DNA quantification and also provides internal controls. Their original paper introducing the methodology has been already cited over 1,600 times (Thomson Reuters ISI) and is among the most popular articles ever published in field of FC. In the current issue of Cell Cycle Heinlein et al. describe novel approach for analysis of DNA content of cells isolated from different tissues.9 The method of cell isolation described by these authors is based on the mechanical dispersion of cells by pressing the tissue through mesh with 100 μm openings followed by filtering of the cell suspension through 35 μm cell strainers. The tissues and the cells are suspended in a solution of PBS and EDTA at all times during the isolation procedure and then fixed in ethanol. Thus, there is no cell exposure to detergent and/or proteolytic enzymes. The major virtue of this technique is simplicity. The method also appears to be universally applicable to tissues of different types and generates data of high resolution. Because of these virtues it is likely that it may find wide applications. Caution should be exercised when interpreting the presence of objects with the “sub-G1” DNA content on the histograms as the evidence of apoptotic cells. In the methods that rely on use of detergents,10 the lysis of apoptotic cells results on release of individual chromatin fragments, often several from a single apoptotic cell. Also, individual chromosomes or chromosome aggregates having the sub-G1 DNA content are being released from mitotic cells upon their lysis. The frequency of the “sub-G1 objects” cannot be therefore considered to represent the frequency of apoptotic cells, and DNA content analysis cannot be used as a single parameter identifier.4 Since no detergents are used in the method of Heinlein et al.,9 it is less likely that fragmentation of apoptotic cells can contribute to the bias in estimation of the “sub-G1 objects.” One has to be aware, however, that some cells may become mechanically damaged by disruption of tissue and the enforced passage through the mesh and their fragments may be recorded as the as “sub-G1 objects.” Each method of cell isolation from tissues and their DNA content analysis has its own bias in terms of some degree of selectivity in representing euploid vs aneuploid cells, mitotic vs non-mitotic cells or quantification of the “sub-G1” population. Since there is already vast literature presenting the data obtained by the detergent-trypsin method, it would be of importance to compare side-by-side, using the very same tissues samples, the classic method of Vindelov et al.,8 with the approach described by Heinlein et al.9