Part I: Basic Considerations and Recommendations for Preparation, Measurement and Interpretation

A task force of invited experts in the field of diagnostic DNA image cytometry, especially consisting of participants from the PRESS (Prototype Reference Standard Slides) and EUROPATH (European Pathology Assisted by Telematics for Healthcare) projects, but open to any other scientist or physician revealing experience in that new diagnostic procedure (names are given in the Annex A) agreed upon the following updated consensus report during the 5th International Congress of the ESACP 1997 in Oslo. This report is based on the preceeding one [9] and on results of the above mentioned European research projects. It deals with the following items: – Biological background and aims of DNA image cytometry, – Principles of the method, – Basic performance standards, – Diagnostic interpretation of DNA measurements, – Recommendations for practical use. Readers are invited to contribute remarks and comments on DNA ploidy through: http://www.esacp.org/esacimag.html 1. Biological background and aims of DNA image cytometry Quantitation of nuclear DNA content by cytometry has come into practice for assistance in the diagnosis and grading of malignant tumours. The DNA content cannot be measured directly by cytometry. After 0921-8912/98/$8.00  1998 – IOS Press. All rights reserved 190 G. Haroske et al. / 1997 ESACP consensus report. Part I quantitative DNA-staining, the nuclear IOD (Integrated Optical Density) is the cytometric equivalent of its DNA content. The quantitation of nuclear DNA requires a rescaling of the IOD values by comparison with those from cells with known DNA content. Therefore the DNA content is expressed in a “c” scale in which 1c is half the mean nuclear DNA content of cells from a normal (non-pathological) diploid population in G0/G1 cell cycle phase. For practical reasons as a term being accepted and used throughout the literature “DNA ploidy” will be further used. However, we want to point out that in practice the cytometric evaluation of nuclear DNA content is often improperly called “DNA ploidy” which is assumed to be the quantitative cytometric equivalent of “chromosomal ploidy”. Both terms are not identical. Whereas “chromosomal ploidy” is theoretically detectable by cytogenetic methods in each single cell, its DNA content cannot be equated with a certain chromosomal outfit [56,57,60]. The term “DNA ploidy” should therefore preserved for the description of DNA stemlines, but not for single cells. Indeed, the quantity of nuclear DNA may be changed by the following mechanisms: replication, polyploidization, gain or deletion. Each affects the size or the number of chromatids. Furthermore viral infections may change the nuclear DNA content detectable by flow and image cytometry. Among others, the unspecific effects of cytostatic or radiation therapy, vitamin B12 deficiency, apoptosis, autolysis and necrosis on nuclear DNA content play also a role [5,13,49,58,61,64]. All these effects have to be taken into consideration when a diagnostic interpretation of DNA histograms is performed. At present the basic aim of diagnostic DNA cytometry is to identify DNA stemlines outside the euploid regions as abnormal (or aneuploid) at a defined statistic level of significance. Furthermore DNA image cytometry should give information about – Number of abnormal (sive aneuploid) DNA stemlines, – Polyploidization of euploid or aneuploid DNA stemlines, – Cell cycle fractions, – Occurrence of rare cells with an abnormally high DNA content. During the past few years a huge body of methodological experience has been gathered allowing ICM-DNA users to perform their DNA measurements at a high level of quality. Recommendations for the entire process of preparation and measurement are given in Annex B. 2. Principles of the method Because DNA image cytometry results in nuclear IOD values, equivalent but not identical with nuclear DNA content, the quantitation of nuclear DNA requires a rescaling of IOD values by comparison with those from cells with known DNA content, so-called reference cells. By means of reference cells the arbitrary unit scale (a.u.) will be transformed in a reference unit scale (2c, 4c, 8c, for example) [16,56]. In general, there are two types of reference cell systems: external and internal ones, respectively. Whereas the external reference cells are very easily to identify by the investigator, but often not to prepare in parallel with the clinical sample, the internal reference cells have the advantage of sharing all preparatory steps with the anlysis cells in the clinical specimens. The nuclear IOD values of reference cells own the same methodological limitations in terms of precision of the measurements as the appropriate IOD values of the analysis cells. G. Haroske et al. / 1997 ESACP consensus report. Part I 191 The mean ratio between the modal IOD values of the non-pathologic cells of the tissue under study and the reference cells used is called corrective factor. This corrective factor must be applied to DNA measurements from the clinical sample before any DNA histogram interpretation [56]. Due to the methodological variability, mentioned above, the corrective factor is not constant. The accuracy of each diagnostic DNA evaluation depends decisively on the standard deviation (SD) of the corrective factor used during the rescaling procedure [30]. Because most of the interpretations of DNA measurements are population-based, the results are usually displayed as DNA histograms. The bin size of such histograms should be adapted to the precision of the actual measurements, i.e., the lower the variability in the reference cell peak, the smaller the bin size of histogram classes could be. The grammalogues “ICM-DNA” (image cytometric DNA) and “FCM-DNA” (flow cytometric DNA) are good descriptors used to designate the type of nuclear DNA measurement. 3. Basic performance standards The usual precision of recent DNA image cytometric measurements should at least allow DNA stemlines to be identified as abnormal (or aneuploid), if they deviate more than 10% from the diploid (2c) or tetraploid region (4c), i.e., if they are outside 2± 0.2c or 4± 0.4c. To achieve this goal with an error probability p < 0.05 the test statistics [30] require a measurement performance described by: – the CV of the ratios between modal IOD-values of reference cells and non-pathologic G0/1 cells in a series of measurements is <5% (comp. Fig. 1: SD of the peak position); – the relative standard error (rSEM = CV/ √ n) of reference cells in each sample is <1.5%. Furthermore, a DNA-stemline should be identified as polyploid within the duplication position of a G0/1-phase-fraction ±0.2c (at 4c), and ±0.4c (at 8c), respectively, with an error probability p < 0.05 if – the CV of the ratios between modal IOD-values of non-pathologic G0/1and G2/M-phase-fractions in a series of measurements is <2.5%. Every scientist and physician who applies DNA image cytometry is free to choose his appropriate methodological specification, if he only meets the performance standards above. The different aspects of the measuring process and of the interpretation should be regularly subjected to quality control measures in order to warrant a steadily high level of quality of the diagnostic procedure. Appropriate protocols for such a quality assurance guide are described in Part II of this paper. 4. Diagnostic interpretation of DNA measurements 4.1. Definition of basic terms of ICM-DNA assessment (for illustration see Fig. 1) – DNA histogram: frequency distribution of IOD values obtained by quantitative DNA stains and rescaled by reference cells in “c” units. The class width should be twice the standard deviation of the IOD of the G0/1-phase-fraction of reference cells. – Histogram peak: a significant local maximum in the DNA histogram. 192 G. Haroske et al. / 1997 ESACP consensus report. Part I Fig. 1. Descriptors of a DNA histogram peak. – Modal value of a histogram peak: the most frequent value, i.e., the mean value of the histogram class containing the highest number of nuclei. – Corrective factor: mean ratio between the modal IOD values of the G0/1-phase-fractions of nonpathologic cells and of reference cells in a series of measurements under the same methodological conditions. – DNA-stemline: G0/G1-phase-fraction of a proliferating cell population. – DNA-G0/1-phase-fraction: all nuclei belonging to a peak which does not represent a duplication of a lower peak. – DNA-G2/M-phase-fraction: all nuclei belonging to a peak in the duplication region of a G0/G1phase-fraction. – DNA-S-phase-fraction: all nuclei with IOD-values in between those of the corresponding G0/1and its G2/M-phase-fraction counterpart, and not belonging to other stemlines. – DNA-euploidy: that type of DNA distributions which cannot be differentiated from those of normal (resting, proliferating, or polyploidizing) cell populations. – DNA-aneuploidy: those types of DNA distributions which are statistically different from those of normal (resting, proliferating, or polyploidizing) cell populations. – DNA-polyploidy: the occurrence of peaks in the duplication (×2,×4,×8, . . .) regions of euploid or aneuploid DNA-stemlines.