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A number of models have been proposed for the analysis of flow cytometric DNA histogram data (1, 2, 4-9,
11-18, 25, 28-29) and a comparative review of the various methods has been published (3). .As expected, some
models performed better than others, not only with simulated but also with experimentally derived data; however, none was ideal. The models that performed best
tended to be those requiring a large mainframe computer, as they generally contained a large number of
variables for which a solution had to be found. These
include the position of the mean of the G1 and G2+M
peaks, the standard deviations of these peaks, the age
distribution of the population (22), the rate of DNA
synthesis, and the relative durations of the G1, S, and
G2 +M phase times with their standard deviations. Thus
far, 12 variables have been defined, all of which may
have to be considered simultaneously, which is not a
trivial task, even with a large computer. Herein lies one
of the central problems. The experimental DNA histogram contains, at best, two peaks and a trough corresponding to G1, G2 +M, and S-phase, respectively. This
is a very simple data set with which to compute 12
variables, and this must be totally inadequate compared
with the complexity of the biology.
The work presented in this paper was undertaken to
simplify the analysis of DNA histograms and to produce
THEORY
Assumptions
In our attempt to produce a minimum assumption
and computing model (MAC) we have only assumed
that the data are normally distributed.
The Computer Program
The method of analysis will be illustrated by a description of the program flow diagram and the components of
its various steps.
Step 1. Eliminate high-frequency noise, if this exists,
by spreading the data with a constant standard deviation of 0.75 channels and performing the appropriate
summation (21).
Step 2. Input the start and end channels over which
the analysis is to take place.
Step 3. Compute a first approximation for the mean
and standard deviation (SD) of the G1 peak. These are
achieved by finding the channel containing the maxi-
ERF(Gl(x) - kG1)
Ps(x) =
-m
ERF(G2(x) + kG2)
-m
ERF(kG1)
S/(G1*2)
(2)
When convergence has been obtained, the S-phase distribution is calculated and subtracted from the experimental data set to give the discrete G1 and G2+M
distributions from which the proportions in each phase
and the G2 +M:G1 ratio are calculated.
0 2 mean
GI mean
v = C [t(n~.fin)lC fin)-[C[t(n).fin)l/C
fin)].
(3)
(4)
RESULTS
Simulated Data
A selection of eight histograms is shown in Figure 3.
These were chosen for illustration, as they exhibit widely
different forms and fairly marked differences in the proportions in the cell cycle phases. Figure 4 shows the
model-predictd proportions in each phase plotted against
the known proportions from the analyses in Figure 3.
The solid and open circles represent G1 and S, respectively, and the squares represent G2+M. The line has
been drawn with unit slope. Ex2 for the 24 comparisons
was 5.19 with 15 degrees of freedom, p < 0.0001 that
the
of
the oredicted from the true could have
____ deviations
_ _ _.
.~
~~
~~~
60 -
0)
EXPERIMENTAL DATA
Z 40-
0*
CB/
/
n
/
0
sets were scaled individually to the maximum height of each histogram. For display purposes, these data have been reduced from 10-bit
to &bit resolution.
20
40
60
True Percentage
FIG. 4. Model-predicted percentages plotted against known percentages for the data analysed in Figure 3. 0 ,G1; 0,
S-phase, 0,
G2+M.
GZ+M:Gl ratio. In a series of over 350 DNA histograms from five different cell lines in both control and
drug- or radiation-perturbed populations, the G2 +M:G1
ratio was 2.009 with 95%confidence limits of 0.015. This
overall mean value does not differ significantly from the
expected ratio of 2.0, p > 0.05. There was somewhat
greater variability in the subgroup of drug- and radiation-perturbed data, where the mean was 2.014 with
95% confidence limits of 0.021. The computed proportions in G1, S, and G2+M varied within the ranges of
10 to 90%, 7 to 90% and 5 to 80%, respectively, in a
multiplicity of combinations. A selection of six histogram analyses is shown in Figure 5 to illustrate the
wide variety of forms that can be analysed by the model.
Duplicate samples. In a number of experiments, duplicate flasks were set up by one of the authors P.J.S.).
These were then stained and run on the flow cytometer
by S.H.C. and analysed by J.V.W. The latter two authors
did not know which were duplicates. Figure 6 shows the
proportions in G1, S,and G2+M from the first sample
plotted against the comparable values from the second
sample. The closed and open circles depict G1 and S,
respectively, and the squares represent G2+M. A total
FIG.5. A selection of histograms derived from experimental data. Display directly analagous to Figure 3.
6ol
f 40
DISCUSSION
Q)
In the work presented in this paper, we have attempted to produce a method for analysing DNA histograms that is easy to use, robust, and fast on a
microprocessor with only 28K addressable memory. As
far as user friendliness is concerned, the program
could hardly be easier. Only two numbers are required,
the start and end channels over which the analysis is to
take place. The procedure has been proven to be robust,
with only two failures in over 350 analyses. In each of
these two cases, there was no definable G1 peak, with
the whole population being arrested in late S/G2+M
after drug and radiation perturbation. In both cases, the
progam assumed that the first peak it defined was the
G1 compartment when this was in fact the G2 +M peak.
It then attempted to find a G2+M peak in a region of
the histogram where there were no data. This can be
corrected by inserting a program queued request for
-F
OO
20
40
60
ADDENDUM
The algorithms described in this paper were written
in Fortran IV to run on the DEC LSI 11/23 microcomputer. We have now upgraded one of our dedicated microprocessors with the 11/73 CPU and the programs
execute in about 7-10 s with this processor. They have
also been compiled with Fortran 77 to execute on a VAX
8600. A version has been written in Pascal in conjunction with Dr. M. Ormerod (Institute of Cancer Research,
Sutton, UK) to run on the Ortho 2150 computer system.
Similar versions are being prepared for both the BectonDickenson (FACS) and Coulter computer systems.
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