Consensus report of the task force on standardisation of
DNA flow cytometry in clinical pathology
M. G. Ormeroda*, B. Tribukaitb and Walter
Giarrettic on behalf of the DNA Flow Cytometry Task Force of the
European Society for Analytical Cellular Pathology
a 34 Wray Park Road, Reigate, RH2 ODE, UK;
b Department of Medical Radiobiology, Karolinska Institute,
Box 60212, S-104 01 Stockholm, Sweden;
c Laboratory of Biophsics, Istituto Nazionale per la Ricera sul
Cancro, Viale Bendetto XV, n. 10, 16132 Genova, Italy
Abstract.Guidelines are given to assist the standardisation
of DNA flow cytometry in clinical pathology. They have been agreed by a
group of twelve scientists from nine European countries.
Key words: DNA flow cytometry, standardisation, ploidy, S phase
Published in:Analytical Cellular Pathology17:103-110,(1998)
* Corresponding author:
Michael_Ormerod@compuserve.com ** Members of the group:
A. Böcking, Institut für Cytopatholgie, Medizinische
Einrichtungen, Heinrich Heine Universität, Düsseldorf, Germany;
I. Christensen, Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark;
J.-L. D’Hautcourt, Clinique St. Joseph, Mons, Belgium;
J. Dufer, Institut Jean-Godinot, Reims, France;
W. Giaretti, Laboratory of Biophsics, Istituto Nazionale per la Ricera
sul Cancro, Genova, Italy;
J. Lawry, Institute of Cancer Studies, Medical School, Sheffield, UK;
A. Orfao, Servicio General de Citometria, Laboratorio de Hematologia,
Hospital Clinico Universitario, Salamanca, Spain;
M. G. Ormerod, 34 Wray Park Road, Reigate, UK;
A. Sampredo, Servicio de Proceso de Imagenes, Universidad de Oviedo,
F. Sansonetty, IPATIMUP, Laboratorio Anatomia Patalogica, Hospital de Sao
Joao, Porto, Portugal ;
B. Tribukait, Karolinska Institute, Stockholm, Sweden;
G.K. Valet, Max-Planck-Institut für Biochemie, Cell Biochemistry
Laboratory, Martinsried, Germany.
These guidelines were produced at the request of the council of the
European Society for Cellular Analytical Pathology. Previously, guidelines
have been published by a North American group  who also produced consensus
reviews of the clinical utility of DNA cytometry in bladder cancer ,
carcimoma of the breast , colorectal cancer , neoplastic
haematopathology  and prostate cancer .
Normal resting human cells have 46 chromosomes corresponding to
7.10-12 pg DNA per cell nucleus. During proliferation, the DNA
content doubles. Cells that are replicating DNA (in S phase of the cell
cycle) will have an intermediate content of DNA. In malignancy, structural
and/or chromosomal aberrations are common. Only when the net chromosome
number is changed, can deviations in the DNA content from normal be observed,
giving rise to DNA aneuploidy. Thus lack of abnormality in the DNA content
does not exclude malignancy or the presence of chromosome abnormalities. It
should also be noted that polyploidisation of normal cells during ageing or
after tumour therapy results in an increase in DNA content. Loss of DNA by
apoptosis or necrosis must also be taken into consideration in the
interpretation of DNA histograms.
Measurement of the DNA content of a large number of cells by flow
cytometry gives the DNA histogram from which can be derived the cell ploidy
and the components of the cell cycle, including the S phase fraction. Both
ploidy and S phase fraction may have prognostic significance in certain
2. Nomenclature for nuclear DNA measurements 
The nuclear DNA content can be measured on slides in selected nuclei
by image cytometry (ICM) or non-selectively in a suspension of cells or
nuclei by flow cytometry (FCM). The method used must be defined in the report.
In flow cytometry the following terms are frequently used:
coefficient of variation (CV) which is measured on a peak in
the DNA histogram and is given by 100*standard deviation/mean channel
DNA index (DI) which is the mean channel number of the G1 peak
of the tumour/mean channel number of the G1 peak of normal cells. A DNA
index of 1 corresponds to diploidy (2c).
Ploidy was originally used to refer to the chromosome number. In
cytometry, it is used to describe the overall DNA content. Diploid cells
have a DNA content of normal cells although their chromosomes may be
abnormal. Cells with a DNA index between 1.90 and 2.10 are classified as
DNA tetraploid. Peaks outside the tetraploid and diploid range are referred
to as DNA aneuploid. The DNA index should be corrected to allow for any
variation in the linearity of the amplifier (see section 6.2 below).
3. Sample preparation
Cells from blood, bone marrow, body fluids, irrigations, surgical
biopsies, core biopsies and fine needle aspirates can be used. Fresh
material, specimens stored by freezing or by fixation or material fixed
and embedded for routine histological examination may be used. The best
results are usually obtained by using either fresh or frozen material.
The advantage to using histopathological material is that a region of
interest can be identified by inspection of a conventionally stained
section and then selected. A disadvantage is that antibody staining to
identify the tumour cells, as opposed to normal stromal cells, cannot
Whenever possible, the tumour cells should be identified by use of a
A sample of the material should be checked by conventional staining
and light microscopy to ensure that it contains an adequate number of
tumour cells; 20% has been suggested as the minimum acceptable proportion
if a measurement of S phase is to be made .
Material may be fixed in ethanol or in buffered formaldehyde.
Other fixatives often used in routine histopathology, such as Bouins,
Zenkers, mercuric chloride, should be avoided. If fixed samples are to be
transported to the flow cytometry laboratory care should be taken to fill
the sample tube with fixative so that all of the specimen remains immersed,
even when the sample is shaken.
If an antibody stain is used to identify the tumour cells (see below),
a fixative which preserves the antigen used must be selected. For many cell
surface antigens, fixation can only be performed after antibody labelling.
Several authors have given protocols for cell preparation for
recording DNA histograms from cells [8-14].
The aim is to obtain single cells or nuclei free of clumps with a
minimum of debris. The method of preparation will depend on the way in
which the sample has been stored. Several different methods have been
3.2.1 Fresh or fresh frozen material. For leukaemias or aspirates of, for
example, peritoneal fluid, a single cell suspension can be readily obtained.
Solid tumours can be disassociated mechanically in a buffer containing
detergent (such as 01.% nonidet-P40) which releases the nuclei into
suspension . Sometimes trypsin is included to assist tissue
disaggregation . Vindelov et al. have published details of a
buffer which stabilises the nuclei with spermidine . Prepapration of
nuclei precludes the use of an antibody to a surface or cytoplasmic
antigen to distinguish tumour from normal cells.
3.2.2Freshly fixed material. (a) Samples fixed in ethanol. (b) Formalin-fixed
material. The sample is cut into small pieces (1-2 mm2) and
incubated either with pepsin in 0.1 M HCl or with trypsin to release the
3.2.3Material fixed in formalin and paraffin embedded. The methods used
are all variations on that originally published by Hedley et al. .
A section is cut from a block of paraffin embedded tissue; it should be
at least 50 µm thick, a thinner section will contain too many sliced nuclei
thereby increasing the debris in the DNA histogram. After the section has
been dewaxed and brought through ethanol to water, it is treated with a
proteolytic enzyme (either pepsin or protease) to release the nuclei. It is
advantageous to place the section in a small cassette while carrying out
these procedures; this precaution avoids centrifugation which can clump
The choice of stain is governed by the flow cytometer in use. If the
machine is equipped with a source uv light (mercury arc lamp or uv laser)
and measurement is restricted to DNA, 4’ 6-diamidino-2-phenylindole (DAPI)
is the stain of choice. This dye whose fluorescence is enhanced 200 fold on
binding to DNA is DNA specific. If the flow cytometer is only equipped
with an argon-ion laser producing light at 488 nm, propidium iodide (PI)
should be used. This dye also binds to double stranded RNA and the sample
should be treated with RNase before analysis. DAPI-stained samples can be
analysed immediately after adding the dye. PI stained samples improve if
they are stored at 4°C for a few hours (Ormerod, unpublished data).
It is important that there is sufficient stain to guarantee stoichiometry.
For propidium iodide, at least 20 µg of PI per million cells is recommended.
For DAPI, the dye concentration should be at least 5 µM.
3.4 Cell concentration
The final concentration of cells or nuclei should be about
106/ml. At a lower concentration, the flow rate of the sample
through the cytometer has to be increased which can degrade the CV; if the
concentration is higher, there may be insufficient dye to stain the DNA
3.5 Quality of sample preparation
It is useful if the final preparation is checked under a microscope
(preferably a fluorescent microscope). The following points should be
clumping or excess debris;
that the majority of the nuclei have the appearance of tumour nuclei (for example, are not from granulocytes);
absence of cytoplasmic remnants attached to nuclei.
4. Reference standard for DNA
The ploidy of a sample is calculated by reference to the peak of
diploid cells. In a clinical sample (with the exception of some
lymphomas and leukaemias, see below) there is nearly always some normal,
diploid nuclei present but it can be difficult to identify which peak is
from diploid cells. Identification is helped if a standard reference cell
has been added to the sample (trout or chicken erythrocytes or both ).
Identification of the tumour G1 peak can also be made by using a monoclonal
antibody to identify specific cell types (see below). The reference cells
must be processed identically with the sample and must be added to the sample
at the earliest possible stage. Reference cells cannot be used with
paraffin-embedded material in which it may possible to separate normal and
malignant nuclei using light scatter .
5. Measurement of DNA
(1) A linear amplifier should always be used when measuring DNA. The
linearity of the amplifier should be checked (for example, using standard
fluorescent beads, clumped, fixed lymphocytes, polyploid liver cells, chicken
and trout erythrocytes).
(2) The alignment of the instrument should be checked daily by measuring
the CV of standard beads or fixed, stained lymphocytes. The CV obtainable
will depend on the machine but should be <=2%.
(3) The number of channels in the histogram should be at least 512.
The PMT voltage should be adjusted so that the G1 peak of normal, diploid
cells does not fall below a channel equal to one fifth of the maximum channel
number (that is, 200 in a 1024 channel histogram, 100 in 512 channels).
(4) The electronics should be triggered on the DNA fluorescence.
(5) All signals should be collected, including those from debris, above
a channel number equal to 1/10 diploid G1 peak channel.
(6) The number of events collected should be sufficient to give
10 - 20,000 nuclei in the DNA histogram (excluding debris). The more
complex the DNA histogram, the greater the number events which should be
recorded. If S phase is being recorded, then there should be at least 100
cells in S phase region.
(7) In order to obtain the best CV, the flow rate should be kept low
(typically 100-300 events/sec).
6. Judgement of quality
6.1 Coefficient of variation (CV)
The lower the CV of the peaks in the DNA histogram, the better is
the quality and the greater is the amount of information which can be
derived. A better CV is obtained from fresh than from paraffin-embedded
material. From fresh material, CVs in the order of 3 % or less should
routinely obtained. The quality of paraffin-embedded material depends on
the treatment of the specimen in the histopathology laboratory. CVs of
less than 5% should routinely be obtained. An estimation of the S phase
fraction should not be attempted if the CV of the G1 peak is =>8%.
If the G1 to G2/M ratio does not fall between 1.95 and 2.05, the
linearity of the amplifier should be checked and, if necessary, the
appropriate adjustment made (often it will be found that the offset
on the amplifier is misadjusted).
High debris can interfere with the measurement of the S phase
fraction and could also obscure a small hypodiploid peak. If high debris
routinely causes a problem, the sample collection and preparation should
be re-evaluated. In a small number of cases, an excessive number of
necrotic or apoptotic cells can give excessive debris.
Excessive clumps in the sample is an indication of poor sample
preparation. They should not be a problem in preparations of nuclei. Clumps
will be revealed in the DNA histogram by a peak at a channel number three
times the channel of the diploid G1 peak. Clumps will also be revealed,
and gated out, by pulse shape analysis (see below).
7. Multiparameter analysis
7.1 Pulse shape analysis of the DNA signal
If a laser is used with beam shaping optics, an analysis of the
shape of the signal from the DNA fluorescence is possible and should be
performed (, for example see reference 10). A cytogram of the peak or
width of the DNA signal against area will reveal clumps and abnormally
7.2 Light scatter
When possible, a cytogram of right angle versus forward light scatter
should be displayed. Debris can usually be identified and gated out of the
analysis. In a well prepared sample, the cells from the inflammatory cells
in the tumour can often be identified.
7.3 Antibody stain
An antibody labelled with fluorescein can often be used to
distinguish normal from tumour cells. With epithelial tumours, the
epithelial cells can be identified with a stain for cytokeratin [19-22].
In non-lymphoid malignancies, normal inflammatory cells can be labelled
using an antibody against CD45 (leucocyte common antigen) . In
lymphoid malignancies, an antibody to an appropriate cell surface antigen
needs to selected depending on the classification of the tumour .
8. Evaluation and interpretation of the histogram
The histogram is used to estimate the DNA ploidy of the tumour and
its cell cycle parameters (particularly S phase fraction). If there is
appreciable debris or clumping in the sample and it is not possible to
improve the histogram by multiparametric analysis (see above), a computer
program can be used to correct for these artefacts .
8.1 Measurement of ploidy
(1) A single peak in the DNA histogram corresponding to the channel
of normal human cells is defined as DNA diploid. A peak from G2/M cells
should also be present in the histogram.
(2) In lymphomas and leukaemias, in which a sample may contain few
normal cells, a single peak deviating more than ± 5% from the expected
position for diploid cells should be suspected as aneuploid. In the
presence of added DNA standard cells or selection of the tumour cells by
an antibody (see above), an interpretation of aneuploidy may be confirmed.
In their absence, aneuploidy should be reported with an added caution.
(3) If a peak is observed at a position corresponding to 4c together
with a peak at 8c and S phase cells between these two positions, the tumour
should be reported as tetraploid. In the absence of a additional (peak at 8c
plus S phase), if the percentage of cells at 4c exceeds 3xSD of the
percentage of cells in G2/M in normal tissues, then tetraploidy should be
reported with a cautionary note. Precautions should be taken to ensure that
the excess of cells in G2/M is not an artefact due to cell clumping (see
A tetraploid peak should have a DI within the range 1.90 - 2.10. A
peak falling outside this range should be reported as DNA aneuploid.
(5) Accuracy of determining whether a tumour is DNA diploid will depend
on the CV of the G1 peak. The smaller the CV, the smaller the deviation
from diploid that will be detected. Separation of normal from tumour
cells will improve the measurement of small deviations from diploid.
8.2 Evaluation of cell cycle parameters
A variety of algorithms have been developed to obtain the
percentage of cells in G1, S and G2/M phases of the cell cycle from the
DNA histogram [25-27]. The reliability of the information will depend on
the quality of the DNA histogram. In particular, a low CV, absence of
clumping and low debris are important.
In a histogram with only diploid cells or one clear-cut aneuploid
peak, the rectangle model of Baisch is adequate and can be performed by
setting regions and using a pocket calculator . Commercial software
exists which will attempt to correct background and clumping of nuclei and
will also attempt an estimate of S phase fraction in the presence of
overlapping polyploid peaks. The reliability of such procedures is
9. Reporting results
The following minimum information should be reported:
(1) Ploidy additionally with the DI of all the populations.
(2) CV of the main G1 peak.
(3) S phase fraction.
(4) Brief comment if necessary (for example, inadequate number of cells, high debris, CV too high)
 T. V. Shankey, P. S. Rabinovitch, B. Bagwell, K. D. Bauer,
R. E. Duque, D. W. Hedley, B. H. Mayall and L. L. Wheeless, Guidelines for
implementation of clinical DNA cytometry, Cytometry14 (1993)
 L. L. Wheeless, R. A. Badalament, R. W. deVere White, Y. Fradet
and B. Tribukait, Consensus review of the clinical utility of DNA cytometry
in bladder cancer, Cytometry14 (1993), 478-481.
 D. W. Hedley, G. M. Clark, C. J. Cornelisse, D. Killander, T. Kute
and D. Merkel, Consensus review of the clinical utility of DNA cytometry in
carcinoma of the breast, Cytometry14 (1993), 482-485.
 K. D. Bauer, C. B. Bagwell, W. Giaretti, M. Melamed, R. J. Zarbo,
T. E. Witzig and P. S. Rabinovitch, Consensus review of the clinical utility
of DNA cytometry in colorectal cancer, Cytometry14 (1993),
 R. E. Duque, M. Andreef, R. C. Braylan, L. W. Diamond, and S. C.
Peiper, Consensus review of the clinical utility of DNA cytometry in
neoplastic hematopathology, Cytometry14 (1993), 492-496.
 T. V. Shankey, O.-P. Kallioniemi, J. M. Koslowski, M. L. Lieber,
B. H. Mayall, G. Miller and G. J. Smith, Consensus review of the clinical
utility of DNA cytometry in prostate cancer, Cytometry14
 W. Hiddemann, J. Schumann, M. Andreeff, B. Barthologie, C. J.
Herman, R. C. Leif, B. H. Mayall, R. F. Murphy and A. A. Sandberg,
Convention on nomenclature for DNA cytometry, Cytometry5
 H. A. Crissman and G. T. Hirons, Staining of DNA
in live and fixed cells, in: Methods in Cell Biology. 41,
Z. Darnzynkiewicz, J. P. Robinson and H. A. Crissman, eds, Academic Press,
San Diego (1994) pp. 195-209.
 D. W. Hedley, DNA analysis from paraffin-embedded blocks, in:
Methods in Cell Biology, 41, Z. Darnzynkiewicz, J. P.
Robinson and H. A. Crissman, eds, Academic Press, San Diego (1994)
 M. G. Ormerod, Analysis of DNA –general methods, in Flow
Cytometry: a Practical Approach, M. G. Ormerod, ed. 2nd
edition, IRL at Oxford University Press, Oxford (1994) pp. 119-135.
 F. J. Otto, High-resolution analysis of nuclear DNA employing
the fluorochrome DAPI, in: Methods in Cell Biology,41,
Z. Darnzynkiewicz, J. P. Robinson and H. A. Crissman, eds, Academic Press,
San Diego (1994) pp. 211-217.
 P. S. Rabinovitch, Practical considerations for DNA content and
cell cycle analysis, in: Clinical Flow Cytometry: Principles and
Application, K. D. Bauer, R. E. Duque and T. V. Shankey, eds, Williams
and Wilkins, Baltimore (1993) p. 117-142.
 K. Steck and A. El-Naggar, Solid tumor DNA ploidy analysis, in:
Flow Cytometry Protocols, Methods in Molecular Biology,
91, M. J. Jaroszeski and R. Heller, eds, Humana Press, Totowa, NJ
(1998) pp. 181-195.
 L. L. Vindeløv and I. J. Christensen, Detergent and
proteolytic enzyme-based techniques for nuclear isolation and DNA content
analysis, in: Methods in Cell Biology, 41, Z. Darnzynkiewicz,
J. P. Robinson and H. A. Crissman, eds, Academic Press, San Diego (1994)
 S. E. Petersen, Flow cytometry of human colorectal tumors: nuclear
isolation by detergent technique. Cytometry6 (1985),
 L. L. Vindeløv, I. J. Christensen and N. I. Nissen,
A detergent-trypsin method for the preparation of nuclei for flow cytometric
DNA analysis. Cytometry3 (1983), 323-327.
 D. W. Hedley, M. L. Friedlander, I. W. Taylor, C. A. Rugg, C. A.
Musgrove, Method for analysis of cellular DNA content of paraffin-embedded
pathological material using flow cytometry. J. Histochem. Cytochem.31 (1983), 1333-1335.
 M. G. Ormerod, J. T. Titley and P. R. Imrie, The use of light scatter
when recording a DNA histogram from paraffin-embedded tissue. Cytometry21 (1995), 294-299.
 J. T. Bijman, D. J. T. Wagener, J. M. C. Wessels, P. Van den Broek
and F. C. S. Ramaekers, Cytometry7 (1986) 76-81.
 P. S. Oud, J. B. J. Henderik, H. L. M. Beck, J. A. M. Veldhuizen,
G. P. Voojis, C. J. Herman and F. C. S. Ramaekers, Cytometry6
 J. C. Van der Linden, C. J. Herman, J. G. C. Boenders, M. M. Van de
Sandt and J. Lindeman, Cytometry13 (1992) 163-168.
 S. Wingren, O. Stål and B. Nordenskjöld, Flow cytometric
analysis of S-phase fraction in breast carcinoma using gating on cells
containing cytokeratin. Brit. J. Cancer69 (1994) 546-549.
 R. J. Zarbo, R. D. Brown, M. D. Linden, F. X. Torres, R. E. Nakhleh
and D. S. Schultz, Rapid (one-shot) staining method for two-color
multiparametric DNA flow cytometric analysis of carcinomas using staining
for cytokeratin and leucocyte common antigen, Am. J. Clin. Path.101 (1994), 638-642.
 A. Orfao, R. Garcia-Sanz, M. C. López-Berges, M. B. Vidiales,
M. González, M. D. Cabellero and J. F. San Miguel, A new method for
the analysis of plasma cell DNA content in multiple myeloma samples using a
CD38/propidium iodide double staining technique. Cytometry, 17(1994), 332-339.
 P. S. Rabinovitch, DNA content histogram and cell-cycle analysis,
in: Methods in Cell Biology, 41, Z. Darnzynkiewicz, J. P.
Robinson and H. A. Crissman, eds, Academic Press, San Diego (1994) pp.
 J. V. Watson, S. H. Chambers and P. J. Smith, A pragmatic approach
to the analysis of DNA histograms with a definable G1 peak, Cytometry8 (1987), 1-8.
 M. G. Ormerod, A.W.R. Payne and J.V. Watson, Improved program
for the analysis of DNA histograms, Cytometry8, (1987)