Aims and basic principles of the quantitation server EUROQUANT for DNA image cytometry


 
 

1. Biological background and aims of DNA image cytometry

2. Principles of the method

3. Basic performance standards

4. Diagnostic interpretation of DNA measurements
 
 

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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 tumors. The DNA content cannot be measured directly by cytometry. After 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 cyle 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,

- Occurence 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.

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.

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 2c +/- 0.2c or 4c +/- 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 ( ) 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 G0/1- and 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 the 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.

- 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 non-pathologic 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/G1-phase-fraction.

- DNA-S-Phase-fraction: all nuclei with IOD-values inbetween those of the corresponding G0/1- and 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 occurence of peaks in the duplication (x2, x4, x8, …) regions of euploid or aneuploid DNA-stemlines.
 
 

Figure 1: Descriptors of a DNA histogram peak
 
 
 
 

4.2. Algorithms

Depending on the material under investigation, the clinical or diagnostic question and the sampling strategy DNA measurements can be interpreted for three purposes [12], namely by assisting in: (1) thediagnosis of neoplasia, (2) theprognostication of neoplasia and (3) the monitoring of the therapy.

The interpretation of the measurements should not be based on subjective interpretations but be defined by algorithms. The algorithms for the recognition of histogram peaks and their quantitation are recommended to be applied to the rescaled DNA measurement values directly, not to the binarized DNA histogram.

The following algorithms have been tested to be useful:

(1) Histogram peak: a local maximum statistically different (p< 0.05) from randomly distibuted events in the frequency distribution (for the type of the statistical test, e.g. see [30]).

(2) DNA-index (DI) of a peak: modal value of the peak divided by the modal value of the diploid reference cell peak [33].

(3) DNA stemline ploidy: modal value of a stemline in the "c" units, i.e. DI *2c [13, 40, 51].

(4) DNA-stemline abnormality (aneuploidy): modal value of the peak is outside the 95% confidence region of the modal value of G0/G1 phases of non-pathologic (DNA-euploid) cell populations measured under the same methodological conditions [30], i.e. outside IOD values multiplied by the corrective factor +/- 2*SD of the corrective factor.

(4) Polyploidization: modal value of a measured stemline is not outside the 95% confidence region for the duplicate position, measured in a larger series of non-pathologic, polyploid cell populations under the same methodological conditions [30].

(5) For prognostic purposes a classification of the entire DNA-histogram is proposed, according to the position of the DNA stemlines. The terms to classify the histogram for prognostication should be different from those ones used to identify DNA aneuploidy for diagnostic purposes. Principally the prognostic interpretation must be tumour type specific. Based on previous studies and taken tumour cytogenetics into account [1, 10, 11, 23, 59] the following DNA histogram types are helpful in describing the prognosis of solid tumours:

- peridiploid: a single DNA-stemline has a modal DNA value between 1.8c and 2.2c;

- peritetraploid: a single DNA-stemline or a stemline additional to a peridiploid one has a modal DNA value between 3.6c and 4.4c;

- x-ploid: a single DNA-stemline or a DNA-stemline additional to a peridiploid or peritetraploid one has a modal DNA value outside the thresholds mentioned above. "x" should be substituted by the DNA ploidy value of this stemline (e.g. peritriploid, hyperdiploid etc.).

- multiploid: more than one abnormal DNA-stemline occurs (often called "Manhattan skyline").

For each histogram type the exact position of the stemline should be given. However, one has to take into consideration thatthe prognostic relevance of these classes may be different among the varioustumour types.

(6) Abnormal cells, often called 5c exceeding events or 9c exceeding events, are those cells having nuclear DNA content higher than the duplication or quadruplication region of a normal G0/G1 phase population, i.e. not belonging to G2/M phase fractions. Cave: 5c or 9c are often not approriate thresholds for defining abnormality due to the improper precision of the measurements. The choice of the duplication or quadruplication region depends on the occurence of euploid polyploidization in the tissue under investigation [6, 7, 8, 14, 47].

Those abnormal cells can be given as absolute numbers or as rates (related to the number of all cells measured)

(7) Cell cycle fractions should only be quantitated, if a single DNA stemline has been recognized. In calculation of cell cycle fractions the statistical limitations given by the actual cell numbers must be regarded [30].

The G0/1- as well as the G2/M-phase-fractions comprise all nuclei with DNA values in a region of the modal value +/- 3 times the SD of the corresponding peaks (see fig.1).

(8) Further complex algorithms for interpretation:

- 2c Deviation Index (2cDI) [7]: 

ci is the DNA content of a single nucleus, rescaled by the mean corrective factor of the tissue type under investigation.
- ploidy balance (PB) [46]: 
neu is the number of all cells in euploid regions of the DNA histogram rescaled by the mean corrective factor of the tissue type under investigation (1.8c - 2.2c; 3.6c - 4.4c; 7.2c - 8.8c);

nan is the number of all cells outside the euploid regions of rescaled DNA histogram;

N is the total number of cells.

In conclusion, for the diagnosis of malignancy the DNA-stemline abnormality and/or the occurence of abnormal cells should be regarded. For the prognostication of a known malignant lesion the prognostic histogram types and/or the complex algorithms are recommended.

Before a more detailed and tumour type specific diagnostic interpretation can be proposed, the algorithms above have to be tested in large scale clinical materials in a standardized manner, i.e. regarding the recommendations and basic considerations of this consensus.
 
 

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