T H E AMERICAN JOURNAL OF CLINICAL PATHOLOGY
Vol. 35, No. 6, pp. 479-487
June, 1961
Copyright © 1961 by The Williams & Wilkins Co.
Printed in U.S.A.
A CRITICAL RE-EVALUATION OF THE DIPHENYLAMINE TEST FOR
MALIGNANCY
ROBERT E. ZIPF, M. D., JAMES P. P. MURPHY, B. S., AND BERNARD J. KATCHMAN, P H . D .
Department of Research, Miami Valley Hospital, Dayton, Ohio
Received, November 2, 1960; revision received,
December 20; accepted for publication February
28, 1961.
Dr. Zipf is Director of Research and Associate
Pathologist, Department of Pathology; Mr.
Murphy is Research Biochemist; and Dr. Katchman is Assistant Director of Research.
This work was supported in part by the Charles
F. Kettering Foundation, Yellow Springs, Ohio;
the Montgomery County Society for Cancer Control, Dayton, Ohio; and the Ella Lowe Gunckle
Fund, Dayton, Ohio.
gated concurrently with the DPA studies in
order to correlate these 2 tests.
This paper is a report of the results of our
investigation of the DPA and of the "no
DPA" reactions and an evaluation of their
usefulness as diagnostic aids in diseased
states. The results of a study of (1) the
experimental conditions of the tests, and of
(2) added chemicals (to serum from normal
persons) that affect color intensity in both
color reactions are detailed separately.9
METHOD
The method used in the clinical studies
was basically that of Waldo and Zipf.12 A
slight modification in the procedure was
necessary to perform studies of concentration
of serum and to use the 1-cm. cell of the
Beckman DK recording spectrophotometer.
These modifications gave values in normal
and pathologic serum comparable to those
obtained by the original method. To serum
(0.2 to 2.4 ml.) was added 5.6 ml. of 5 per
cent trichloroacetic acid and water to make
8 ml. This mixture was heated for 5 min.
in a boiling water bath, cooled to room
temperature, and filtered. A 2-ml. aliquot
of the filtrate was added to 6 ml. of the
DPA reagent (180 ml. of glacial acetic acid
plus 20 ml. of concentrated sulfuric acid and
1 Gm. of diphenylamine). This mixture was
heated for 30 min. in a boiling water bath,
cooled to room temperature in an ice-bath,
and then read in the Beckman DK recording
spectrophotometer between 400 to 900 m/*,
using a water blank.
Characteristic spectral curves were obtained for normal and pathologic serums
over the ranges of serum concentration
studied. These spectral curves have 2 peaks,
1 at 675 niM and the other at 530 myu. A
plot of optical density vs. serum concentration for either peak usually resulted in a
straight line that passed through the origin.
In some instances it was noted that straight
479
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For the past several years, workers concerned with the development of diagnostic
tests have observed that a more intense
color is obtained when diphenylamine reacts
with serum from patients with rheumatic
fever,1, 2- 7 nonspecific inflammations,4
malignant neoplasms,11 rheumatoid arthritis, 3, 6 typhoid fever, hepatic lesions,10
and leukemia,12 than with serum from
healthy persons. These reports indicate that
the diphenylamine (DPA) reaction is more
likely to be a response to generalized insult
than to any specific disease state. In view
of the report12 that the DPA reaction could
be used as a diagnostic test for malignancy,
it became necessary to re-evaluate the reaction in order to ascertain conditions for
specificity, if any exist.
The DPA test, as described in the literature, involved measurement of the developed
color intensity at a single wave length and
at a convenient concentration of serum.
Our studies of the DPA reaction to ascertain its specificity were broadened to
include the effect of serum concentration
and of chemicals added to serum on the
visible absorption spectrum of the developed
color. In the course of these studies, it was
noted that a characteristic color was obtained in serum even when diphenylamine
was omitted from the standard colordeveloping reagent. A similar finding has
been described by Hess and co-workers.8
This "no DPA" color reaction was investi-
480
ZIPF ET
lines that intersect the optical density axis
were obtained, as well as curvilinear plots
that intersect the origin. These noncharacteristic plots, observed in about 10 per
cent of the data, were randomly distributed
and could not be ascribed to any particular
pathologic condition. The fact that about
90 per cent of the serums examined gave
linear plots over the whole range of serum
concentration (2.4 ml. maximal concentration) was a good indication that the test
conditions were maximal for color development.
Vol. 35
AL.
Conditions for study of the second color
reaction ("no DPA") were identical to
those of the DPA reaction except that
diphenylamine was omitted from the
standard color-developing reagent. The peak
of 470 nut, thus obtained, was very broad,
but nevertheless readily recognized. Plots
of the optical density at 470 nui vs. concentration of serum gave straight lines passing
through the origin; there was a smaller frequency of noncharacteristic plots at this
wave length than in the DPA reaction. For
clinical evaluation, however, both reagents
gave comparable results.
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04
Oi
PATH0L08ICAL
FIG.
normal
FIG.
optical
12
l<«
20
14
CONCENTRATION CURVE
OS
l«
NORMAL
14
S«
3 «
4 0
CONCENTRATION
CURVE
1 (upper). Characteristic spectral curves revealing peaks at 675 imj and 530 nip for
and pathologic serums when used in the DPA reaction.
2 (lower). From the characteristic spectral curves in Figure 1, corresponding plots of
density vs. concentration of serum are indicated.
June 1961
481
D I P H E N Y L A M I N E T E S T F O B MALIGNANCY
RESULTS
Figure 1 represents examples of characteristic spectral curves obtained in the
DPA reaction for normal and pathologic
serums; the corresponding plots of optical
density vs. concentration of serum reveal
linearity of the developed color at 675 nut
and 530 nuj (Fig. 2). Similarly, the data
for the "no DPA" reaction are illustrated
in Figures 3 and 4.
The linear plots of optical density vs.
concentration (Figs. 2 and 4) pass through
the origin and thereby substantiate the
validity of the use of a water blank in the
spectrophotometric analysis.
10
08
06
04
^r\l
/
02
^"
"^^_
/ / / ^ ~ ~ ~ ~
01
/
•*^
/
<
^S*
JS
,-r
S
—
—
normal
pathological
470INIWAVELENGTH
0-6
normal
pathologica
2-4
32
CONCENTRATION
CURVE
FIG. 3 (upper). Characteristic spectral curves indicating a peak of 470 nut for normal and
pathologic serums when used in the "no DPA" reaction.
FIG. 4 (lower). From the characteristic spectral curves in Figure 3, corresponding plots of
optical density vs. concentration of serum are indicated.
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Serums designated as normal were obtained from healthy blood bank donors;
individual as well as pooled serum was used.
Pathologic serums were obtained from
persons with a recorded clinical diagnosis.
All chemicals used were of highest purity
commercially available. Nevertheless, during
the course of this study, it was noted that
some batches of acid reagents produced
distorted curves. The impurity responsible
for these shifts in the spectrums is unknown,
and it should be noted that only by examination of the absorption spectrums of the
reaction mixture can this effect be ascertained.
482
ZIPF ET
Vol 35
AL.
TABLE 1
DPA
I N D E X I N NORMAL AND PATHOLOGIC S E R U M S
675 m/i
Standard
Mean value deviation*
530 mp
Standard
95% Tolerance limits Mean value deviation*
95% Tolerance limits
Single Valuesf
Normal
Malignant
Nonmalignant
Leukemic
53
107
107
127
316
450
437
403
316
450
437
403
±
±
±
±
104
216
218
259
253
367
363
333
40
91
95
104
253
367
363
333
±
±
±
±
79
183
193
212
Smoothed Values!
81
117
118
458
428
423
458 ± 175
428 ± 247
423 ± 255
372
350
347
64
98 '
99
372 ± 138
350 ± 208
347 ± 214
* Standard deviation of individual observations.
f D P A index at indicated wave length for 0.8 ml. of serum.
| D P A index for 0.8 ml. of serum obtained from linear plot of serum concentration curve.
TABLE 2
COLOH I N D E X * I N NOEMAL AND PATHOLOGIC
S E R U M S AT 470 M,U
Mean
Value
Standard 95% Tolerance
Limits
Deviation!
Single V a l u e s !
Normal
Malignant
Nonmalignant
Leukemic
104
155
160
140
21
57
46
57
104
155
160
140
± 41
± 114
± 94
± 117
Smoothed Values§
Malignant
Nonmalignant
Leukemic
159 ± 74
154 ± 100
159 ± 119
* Color developed without DPA in reagent.
f Standard deviation of individual observations.
J D P A index at indicated wave length for
0.8 ml. serum.
§ D P A index for 0.8 ml. serum obtained from
linear plot of serum concentration curve.
Table 1 is an analysis of the data obtained
from the study of serums (0.8 ml.) from 240
healthy persons and 109 patients with
various disease states by means of the DPA
reaction. The concentration of serum chosen
for analysis is comparable to that used by
Waldo and Zipf.12 The mean value for normal
persons of 253 units (optical density X
1000) at 530 m^i compares favorably with
that of 248 units described by Waldo and
Zipf12 at this wave length. For purposes of
distinguishing between the serums of healthy
and diseased persons, 95 per cent limits of
tolerance for individual observations have
been calculated.
The DPA index at 675 mM is 316 ± 104
units and at 530 m/x, 253 ± 79 units for
healthy persons. The mean DPA index for
diseased persons is greater at either wave
length than the mean indices for healthy
persons at the corresponding wave length.
Thus, there is good discrimination at either
wave length between healthy and diseased
persons. Similarly, the indices at 470 va/j.
(no DPA in the reagent) also distinguish
readily between the serums of healthy and
diseased persons (Table 2). In serum from
diseased patients, however, neither the
DPA nor the "no DPA" reaction manifests
distinct enough differences in the indices to
permit distinction of a specific disease state
among pathologic serums.
The agreement between corresponding
pairs of data (Tables 1 and 2) indicates that
determination of a single value is just as
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Malignant
Nonmalignant
Leukemic
June 1961
D I P H E N Y L A M I N E T E S T FOR
483
MALIGNANCY
TABLE 3
RELATIVE
FREQUENCY
OF
OCCURRENCE
OF
DPA
INDICES
SERUMS
AMONG
NORMAL
AND
675 mix
Number of persons
Percentage of persons detectable as
nonnormal at the 5 % probability
level*
95th percentile D P A index
Malignant
530 irnt
Nonmalignant
Leukemic Normal
Malignant
Iff
Normal
PATHOLOGIC
Leukemic
240
41
71
35
57
33
42
240
41
71
35
57
33
46
400
658
678
638
318
550
577
523
reliable an index as a calculated value from
a concentration curve for either of the color
reactions.
It should be noted that this lack of specificity by the DPA test at 530 nu* is not in
agreement with the selectivity of this test
reported by Waldo and Zipf.12 For this
reason, we have recorded in Table 3 and
Figure 5 the relative frequency of occurrence
of ranges of DPA indices at 675 nu* and 530
nijU in serums from healthy and diseased
persons. For completeness of data presentation, similar treatment for the "no DPA"
reaction (peak at 470 nui) is recorded in
Table 4 and Figure 6.
The data in Tables 3 and 4 are arranged
to show the number of indices that fall in a
particular range for each of the test methods
and for the 4 categories of individuals. In
each of these tables is summarized the
percentage of patients detectable as diseased;
i.e., that percentage of patients whose indices
fall outside the 95th percentile index of the
healthy persons.
Thus, at 530 mji, 57 per cent of nonmalignant, 71 per cent of malignant, and 46
per cent of leukemic persons have indices
greater than the 95th percentile of the
normal DPA index. The uniformity of these
percentages at the 3 wave lengths is a good
indication of the reliability of the test
methods. It should be noted, however, that
these data illustrate the wide distribution
of indices and overlap among the serums
from patients with malignant, nonmalignant,
and leukemic conditions (Figs. 5 and 6),
TABLE 4
R E L A T I V E FREQUENCY OF OCCURRENCE OF C O L O R
I N D I C E S * AT 470 M^U AMONG NORMAL AND
PATHOLOGIC SERUMS
Number of persons
Percentage of persons detectable as
non -normal at the
5%
probability
levelf
95th percentile (no
DPA) color index
Normal
Malignant
Nonmalignant
224
41
61
32
59
27
41
137
280
288
296
Leukemic
* Color developed without D P A in reagent.
t Percentage of persons with color indices
exceeding t h e 95th percentile index of t h e normal
group where t h e 95th percentile color index is
t h a t index which exceeds 95 per cent of t h e persons' indices in a group.
and because of this overlap, the DPA and
"no DPA" tests are not capable of distinguishing between various diseases. The tests
may be used to distinguish between healthy
and diseased persons, and, in this sense, may
be a reliable measure of the extent of
humoral reaction to a generalized insult of
the body.
DISCUSSION
The DPA reagent of Waldo and Zipf,12 as
well as that used in our own studies, is
constituted of 1 part of sulfuric acid and 9
parts of glacial acetic acid (containing 0.5
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* Percentage of persons with D P A index exceeding the 95th percentile index of t h e normal group
where t h e 95th percentile DPA index is t h a t index which exceeds 95 per cent of the persons' indices
in a group.
484
ZIPF ET
Vol. 35
AL.
675mu
530mu
NORMAL
NORMAL
MALIGNANT
MALIGNANT
NONMALIGNANT
NONMALIGNANT
40
30
20
10
_^d
40
30
10
II
Ul
u
Id
0.
LLn
40
30
20
10
CL
XX
LEUKEMIC
in ML
LEUKEMIC
40
30
20
10
XI0*3
^d
X
0*5 0*6 0*7
01 0 2 0 3 0-4 0 3-Q,
0*6 0 7
OPTICAL DENSITY
F I G . 5. The relative frequency of occurrence of ranges of D P A indices at 675 imt and
530 m/i among normal and pathologic serum.
*~51
0*2
0*4
jr
per cent diphenylamine). Niazi and State, 11
as well as Ayala and co-workers,1 used the
Dische reagent per se, which is constituted
of 1 part of sulfuric acid and 39 parts of
glacial acetic acid (containing 1 per cent
diphenylamine). Dische6 has already indicated that with these reagents the most
important variable with respect to sensitivity and selectivity or specificity is the
sulfuric acid-glacial acetic acid ratio. Thus,
the Dische reagent does not produce color
when reacted with hexoses, and its absorption spectrum reveals a strong peak at 530
m/i and a very weak one at 675 imi; this
is in contrast to the DPA reagent that
reacts with a wide variety of substances
including hexoses, and results in strong
peaks at 530 m/i and 675 m,u. Therefore,
although these reagents are similary constituted, they do have different ranges of
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20
June 1961
DIPHENYLAMINE
TEST FOR
485
MALIGNANCY
4 7 0 muMALIG N A N T
OPTICAL
0-1
0 3
LEUKEMIC
0 2
DENSITY
FIG. 6. The relative frequency of occurrence of ranges of "DPA" indices at 470 nn< among normal
and pathologic serums.
reaction response, and in this sense may be
regarded as different reagents. Nevertheless,
our results are in agreement with those
reported by Niazi and State. 11 They, too,
could distinguish only between healthy and
diseased serums; high values were observed
in serum from postpartum patients and in
persons with pulmonary tuberculosis, rheumatic fever, inflammations, and so on.
On the other hand, our results do not
confirm the specificity claimed by Waldo
and Zipf12 for the DPA reagent in its response
to leukemic serum. In reviewing the Waldo
data, 2 discordant facts are evident; the
data reveal no high values for serums other
than that derived from leukoproliferative
disorders. Our experience, as well as that
of Niazi and State11 and others, has shown
high values for many malignancies and
nonmalignancies, as well. In addition, we
have observed low values in serums of
patients with leukoproliferative disorders,
while under clinical control, and have received no indication that the color index
rises precipitously while the patients are
under therapy. In order to demonstrate the
latter point, we have prepared Table 5.
It can be seen from Table 5 that many
patients with leukoproliferative diseases,
controlled by therapy, manifest low normal
DPA indices at 530 m/i over a long period
of time; even at death there was no unusually
high DPA index.
It is more likely that the DPA reaction
measures a general response to insult of the
body, as evidenced by the elevated DPA
indices that are observed in serums from
patients diagnosed as having polycythemia,
malignancies, leukoproliferative disorders,
and nonmalignancies, as described here, and
rheumatic fever, inflammations, rheumatoid
arthritis, typhoid fever, and hepatic lesions,
as described by others.
We have reported that the "no D P A "
reactions give a distribution of indices comparable to those of the DPA reagent observed at 530 niju and 675 niju. This correlation between the DPA and "no DPA" test
would seemingly implicate a single substance or blood component as the responsible
factor in both tests. In a subsequent paper, 9
evidence is described which would indicate
that the material in serum from healthy
persons reponsible for the color produced in
the DPA and "no DPA" tests is related to
the neuraminic acids. It may be convenient
to ascribe the increase in color-forming
material observed in the serum of diseased
persons to neuraminic acid containing
glycoprotein; however, the chemical studies
at least leave room for the possibility that
under the conditions of these serum tests,
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02
NONMALIGNANT
486
Z I P F ET
AL.
TABLE 5
DPA
Patient
T . R.
I N D E X AT 530 M/X DURING T H E R A P Y
Diagnosis
Monoblastic leukemia
W. R.
Lymphosarcoma with infiltration of bone marrow
W. R.
Polycythemia
Polycythemia
M. E .
Polycythemia
A. C.
Gangrene of leg with leukocytic infiltration
W. H .
Chronic granulocytic leukemia
M. N.
J . J.
S. G.
Chronic lymphatic leukemia
Chronic lymphatic leukemia
Lymphosarcoma
May 6, 1955
May 11, 1955
June 5, 1955
J u n e 7, 1955
June 29, 1955*
June 17, 1955
J u l y 20, 1955
August 4, 1955
August 21, 1952
October 23, 1952
November 20, 1952
July 23, 1953
September 19, 1953
1954
August 2, 1955
September 8, 1955
June 21, 1955
July 19, 1955
June 23, 1955
July 23, 1955
J u l y 6, 1955
June 7, 1955
July 15, 1955
March 15, 1956
M a r c h 20, 1956
M a r c h 26, 1956
April 3, 1956
April 18, 1956
July 10, 1956*
March 22, 1955
M a y 6, 1955
June 17, 1955
October 1, 1955
December 10, 1955
June 1, 1954
J a n u a r y 4, 1955
M a y 2, 1955
July 30, 1955f
September 3, 1953
September 14, 1953
September 25, 1953
December 9, 1953
1954
M a y 4, 1955
October 29, 1955f
* Patient died.
t Patient still under treatment.
Therapy
Triethylene melanine
6-Mercaptopurine
DPA Index
410
410
410
P-32
Triethylene melanine
260
280
P-32
P-32
P-32
P-32
P-32
P-32
P-32
P-32
Postamputation
X-ray
Myleran
Myleran
Myleran
Myleran
Myleran
P-32
P-32
P-32
P-32
P-32
X-ray
P-32
P-32
P-32
P-32
P-32
P-32
Triethylene melanine
Triethylene melanine,
P-32
Triethylene melanine,
P-32
Triethylene melanine,
P-32
285
215
390
380
295
308
337
509
347
516
577
495
300
265
265
295
295
208
201
182
255
225
215
332
350
296
350
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S. L.
Date
June 1961
487
D I P H E N Y L A M I N E T E S T F O R MALIGNANCY
certain normally occurring metabolites simple a executar, es usate pro sequer le
(fructose, sedoheptulose, and so on) may be curso clinic de un therapia.
responsible for producing enhanced serum
Acknowledgments. The authors wish to acknowlDPA indices.
edge t h e technical assistance of M r s . Lois Chiles
Neither the DPA nor the "no D P A " who performed chemical tests, M r . K e n n e t h Busch
reagent manifests specificity, when reacted for the statistical analysis, and D r . L. E . Loveless
with serum, to warrant their utilization as a for his efforts on this paper.
specific screening test for leukemia or any
REFERENCES
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1. Es describite datos additional pertinente con respecto al test a diphenylamina
(DPA) pro malignitate, insimul con datos
pertinente con respecto al test a "nulle
DPA."
2. Ambe tests manifesta intensificate indices de color in specimens de sero ab subjected morbide.
3. Le analyse statistic del datos indica que
—ben que ambe tests pote esser usate pro
distinguer inter subjectos malade e subjectos
normal—ni le un ni le altere pote esser usate
pro distinguer inequivocamente inter subjectos con diverse morbos.
4. Es proponite que iste tests, que es
A.:
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im
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10. K U M A T E , J., VIVAR, G. M., AND B E N A V I D E S ,
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10:468, 1954.
Experientia,
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1. Additional data pertaining to the DPA
test for malignancy are described, as well as
data on the "no DPA" test.
2. Both tests manifest enhanced color
indices in specimens of serum from diseased
persons.
3. Statistical analysis of the data indicates
that, although either test may be used to
distinguish between healthy and diseased
persons, neither test can be used to distinguish unequivocally between persons with
various diseases.
4. It is suggested that these tests that are
simple to perform may be used to follow the
clinical course of therapy.
LABIO, C. R.: Comparative studies of serum
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