THE KAHN TEST FOR SYPHILIS IN THE PUBLIC HEALTH

LABORATORY
C. C. YOUNG.
Director of Laboratories, Michigan State Department of Health,
Lansing, Mich.
Read befdfe the Laboratory Section of the American Public Health Association at the Fifty-first Annual
Meeting, Cleveland, October 17, 1922.

FEW problems give the public health

laboratory director greater concern
than the laboratory diagnosis of
syphilis. On the one side, he deals with
a disease which expresses itself in every
.conceivable clinical forni and on the
other, with a diagnostic test which possesses many inherent possibilities for
error. The Wassermann test is being
utilized more and more by many physicians as a routine examination without
regard to clinical findings. The laboratory is called upon to pass its opinion on the possibility of syphilitic infection on blood specimens received through
the mails without the slightest knowledge
of the condition of the patient. It is
clear tfiat the laboratory carries a heavy
responsibility in furnishing results of the
highest accuracy. This applies particularly to the public health laboratory because there is little opportunity for the
laboratory worker and physician to talk
over the findings as is true, for example,
in the case of the hospital laboratory.
Granting the importance of correct
serum diagnosis of syphilis, it must be
admitted that the Wassermann test does
not entirely supply such a diagnosis. The
many variable elements of this test give
it numerous sources of error, so many
indeed, that it is not uncommon for even
dependable workers to vary in their findings on the same specimen of blood.1 2
In order to help overcome many of the
variable factors of the Wassermann test,
public health laboratory workers have
attempted at various times to standardize
this test. In Great Britain the Medical
Research Committee has done excellent
work along this line3. In this country
also several attempts have been made to
standardize the test. The work of the

New York City Department of Health',

the U. S. Public Health Service5, the
Massachusetts State Board of Health6,
and more recently of Kolmer7, are conspicuous examples. In Michigan also we
have for some time been endeavoring to
help standardize this test. While serving
as chairman of the Laboratory section of
the Michigan Public Health Association
at the 1921 meeting, the writer appointed
a Wassermann standardization committee, the work of which, however, will be
reported in detail in another place.
The standardization of the Wassermann test will bring about greater uniformity in the procedure and wilf undoubtedly enhance the diagnostic value
of the Wassermann test. But it is questionable whether the mere standardization will solve the problem-at least not
until we learn more of the laws which
underlie the Wassermann test.
When Meinicke8, and Sachs and
Georgi9, published their precipitation
tests for syphilis it appeared that at
last we might have a simple test to check
the Wassermann and in time perhaps replace the older and more complicated
test. Although studied to a considerable
degree in Germany these two tests have
not given promising results in this country. The Sachss and Georgi test has been
favorably mentioned by several American workers 10. 11 12.12 Kilduffe" more
recently subjected 430 serums to parallel
Wassermann and Sach-Georgi tests and
concluded that the reaction is neither as
delicate nor as trustworthy as the Wassermann test. To our knowledge no
laboratory worker in this country has'
found it worth while to report the results of either the Meinicke or SachsGeorgi tests to physicians as a check on
96

KAHN TEST

FOR

the Wassermann. Probably the greatest

drawback for the laboratory worker is
the long incubation period (48 hours15)
required for these two tests, permitting

organisms to develop which interfere

with the determination of correct results.
THE KAHN PRECIPITATION TEST

This leads us to the precipitation test

for syphilis which forms the basis of this
paper. This test was evolved about a
year ago by Kahn"' in the laboratories
of the Michigan Department of Health,
and has from the very beginning given
promising results. The main features
are:

(1) The spontaneous reaction in the

case of strongly positive serums. About
80 per cent of the serums giving a four

plus reaction with the Wassermann test,

show spontaneous precipitation after
mixing serum with antigen.
(2) The clumping of the precipitates
after incubation. The four plus and three
plus reactions show clumps instead of
precipitates. This coagulation of the precipitates makes the stronger reactions
unmistakable.
(3) The simplicity of the procedure.
We have not found it necessary to increase the laboratory personnel in adding
this test to our routine procedures because it requires comparatively little time
and effort in its manipulation.
We were particularly encouraged by
the favorable clinical results obtained
with this test by Keim and Wile'7 at the
Dermatological Clinic of the University
of Michigan. The findings of these
workers entirely corroborated our own
in connection with examinations made of
syphilitic inmates of the Michigan penal
institutions where there are 560 cases
under treatment. Our findings also
showed that the combined results of the
Wassermann and Kahn Precipitation
tests approached a higher degree of correct diagnosis as well as a better check on
treatment than the Wassermann test

alone.
On July 1st, 1922, after 15,400 Kahn

SYPHILIS

97

tests had been carried out in this laboratory in conjunction with the Wassermann
test, it was decided to add this test to our
routine procedures. Since that date.,
over 8,000 precipitation tests have been
reported parallel with the Wassermann
test to physicians, with entirely satisfactory results

PROCEDURE OF THE KAHN TEST

In contradistinction to the precipitation tests proposed by Meinicke8, Sachs

and Georgi9, Dreyer and Ward (Sigma
Reaction)'8 and Wang'9 wherein a considerable amount of salt solution is employed for the dilution of the reagents,
the Kahn precipitation test is carried out
in greater concentration and is practically
free from salt solution. The serum is
employed undiluted. The antigen is prepared from dried heart muscle and before extracting with alcohol is first freed
from fat and other nonspecific elements
by several extractions with ether. The
alcoholic extract is cholesterinized by
adding 4 mgm of cholesterin per cubic
centimeter. In diluting the cholesterinized antigen for the tests, approximately
the smallest amount of salt solution is
employed which will keep the lipoids in
solution. This renders the diluted mixture quite unstable and when mixed with
positive serums, shows marked precipitating properties.
Preparation of Antigen: Beef heart is
freed from fat, fiber and blood vessels
in the usual manner and passed several times through a meat grinder. It
is then spread on a platter and dried l*y
means of a revolving fan. The dried
plates are broken up into small particles
and either ground in a mortar or passed
several times through a coffee grinder
until it is mealy and but slightly gritty.
The ground muscle is then extracted
with ether at icebox temperature until
supernatant ether is free from coloring
matter. Between 3 and 4 ether extractions with fresh quantities at 24-hour
intervals are necessary to bring this
about. The ether of the final extraction

98

THE AMERICAN JOURNAL

is filtered off and the ground muscle

dried for several hours at room temperature until free from ether odor.
To 50 gms. of dried material placed in
500 cc. Erlenmyer flask is added 250 cc.
of 95 per cent alcohol and extracted for
nine days in the icebox and one day at
room temperature with occasional shaking. After this period, the alcoholic extract is filtered off and a given amount
cholesterinized by adding 4 mgm. of
cholesterin per 1 cc.
This cholesterinized antigen is diluted
for the tests as follows: Pipette from
0.5 to 1.0 cc. of extract, depending on
the amount needed for the tests, into an
agglutination tube having an inner diam-eter of about three-eighths inch. Pour
three times the amount of salt solution
into this tube from a cylinder and rapidly invert back and forth. The final mixture is opalescent and clear and is ready
for use. The employment of two cholesterinized antigens as a check on one
another is recommended.
Performnce of Test: Measure 0.3 cc.
of clear inactivated (one-half hour at
560 C.) serum into an agglutination
tube and add 0.05 cc. of the diluted antiShake vigorously from
gen mixture.
one to two minutes. The strongly positive serums will show in most cases,
spontaneous precipitation, but the weaker
serums need overnight incubation at
37.5 C. to bring forth the precipitates.
After incubation the strongly positive
serums show precipitation clumps, mak'ing it comparatively easy to read the end
results.
The tests are read in accordance with
the following scale:

OF

PUBLIC HEALTE

terinized antigen. Since we

run

Wasser-

mann tests on all serums, we do not deem

it necessary to further check the cholesterinized antigen test with a noncholes-

terinized one.
The ring or layer modification of the
Kahn test, first reported by Keim and
Wile'7 and later by Herrold20, is not
quite satisfactory. We are dealing, as
Keim and Wile pointed out, with a test
of lesser sensitiveness. Furthermore, the
ring test lacks the definiteness of the
regular test. With any given source of
light one may see from one angle what
appears to be a ring precipitate, but
viewed from another angle nothing is
visible. No test for syphilis can have
permanent value if based on variable
readings; the end result must be obvious.
Indeed, if the Kahn test merely showed
fine precipitates as is true in the case of
the precipitation tests referred to above
where agglutinoscopes have to be employed for reading the results, we would
not have made it a routine test in the
laboratories of the Michigan Department
of Health. It is because the end results
of the Kahn test are unmistakable that
this test is particularly valuable.
COMPARATIVE RESULTS OF WASSERMANN
AND KAHN TESTS

About 93 per cent of serums give the

results with the Wassermann and
Kahn tests. Close to six per cent of
serums give, what we consider a relative
check. These serums give doubtful reactions with the Wassermann test and
either negative or positive reactions with
the Kahn test, or doubtful reactions with
the latter test and negative or positive
1.
A precipitate, consisting of one or more reactions with the Wassermann test.
large clumps =+ + + +.
2. A large flocculent precipitate = + +. Close to one per cent of serums do not
3. Moderate-size floc or granules = + +.
check, giving a negative reaction with
4. Small-size floc or granules =
one test and positive with the other or
5. Fine floc or granules = +.
vice versa.
6. Negative precipitation--.
The results obtained with the WasserA noncholesterinized antigen may also mann and Kahn tests from July to Octobe employed for this test. It is consid- ber, covering 8,070 examinations, are reered of value as a check on the choles- corded in the following table:
same

99

KAHN TEST FOR SYPHILIS

TABLE 1-COMPARISON OF RESULTS
Wassermann Reaction

WASSERMANN AND KAHN REACTIONS ON 8070 SERUMS.

Kahn Reaction
Negative
Positive
Doubtful
1727 positive* tests gave ..................
1672
33
22
332
101
62
495 doubtful tests gave ......
............
5562
50
236
5848 negative tests gave .....
.............
*Positive includes + ++ + + + + and + +; Doubtful includes + and +.
OF

According to this table, of 8070 serums examined

(1) 1672 were positive by both methods
332 were doubtful by both methods
5562 were negative by both methods

(2)

(3)

62 doubtful Wassermann reaction gave positive Kahn reactions

101 doubtful Wassermann reactions gave negative Kahn reactions
236 negative Wassermann reactions gave doubtful Kahn reactions

5.353%S

432 serums showed relative agreement ......................

50 negative Wassermann reactions gave positive Kahn reactions
22 positive Wassermann reactions gave negative Kahn reactions
72 serums showed no agreement.......
RESUME

Experience gained with over 23,000

Kahn tests, 8,000 of which have been
reported to physicians parallel with the
Wassermann test, leads us to the following conclusions:
1. The laboratory diagnosis of syphilis
based on the combined results of the
\Wassermann and Kahn tests possesses a
higher degree of accuracy than that of
the Wassermann test alone.
2. The clinical application of the Kahn
test both in the diagnosis and treatment
of syphilis compares favorably with the
Wassermann

test.

3. The simplicity of the procedure of

the Kahn test makes it readily applicable
as a routine procedure in a public health
laboratory.
4. The employment of the Kahn test
as a check on the Wassermann test will
help reduce the element of skepticism
associated with the older test.
REFERENCES.
Solomon, H. C.: Agreement in Results of the
Wassermann Reaction; A Study of Tests Performed
in 2 Laboratories in 3,000 Successive Hospital Admissions. Jour. Amer. Med. Assn., 74:788, March 20,
1920.
2. Palmer, L. J.: Wassermann Variations; A Study
of the Serums of Seventy-five Patients by Eight
Laboratories. Ibid, 79:724, August 26, 1922.
3. The Wassermann Test: Special Report Series
No. 14 of the Medical Research Committee. H. M.
Stationery Office, London, 1918.
4. Koopman, J.: The Wassermann Reaction as
Carried Out by the Department of Health, Monthly
Bulletin, Department of Health, City of New York,
8:87, 1917. Discussed also in the paper by Ottenberg,
R.: On Reliability of Wassermann Reaction. Arch.
Int. Med. 19: 457, 1917.
1.

93.754%

7566 showed complete agreement .................................

33 positive Wassermann reactions gave doubtful Kahn reactions

........

0,892o%

5. Neil, H. H.: The Complement Fixation Test

for Syphilis. U. S. Public Health Reports, 33:1387,
1918.
6. Hinton, W. A.: A Standardized Method of Performing the Wassermann Reaction. Amer. J. Syph.,
4:598, 1920.
7. Kolmer, J. A.: Studies in the Standardization
of Wassermann Reaction. XXX. A New Complement-Fixation Test for Syphilis Based Upon the Results of Studies in the Standardization of Technic.
Amer. J. Syph., 6:82, 1922.
8. Meinicke E.: Ueber eine neue Method der
serologischen Luesdiagnose. Berl. Klin. Wchnschr.,
54:613, 1917; Ueber die dritte Modifikation meiner
Luesreaktion. Muench. Med. Wchnschr., No. 88,
p. 932, 1919.
9. Sachs, H., and Georgi, W.: Zur Serodiagnostik der Syphilis Mittels Ausflockung durch
cholesterinierte Extrakte. Med. Klinik, No. 33, p.
803, 1918.
10. Hull, T. G., and Faught, E. E.: The SachsGeorgi Precipitation Test for Syphilis. J. of Immunol., 5:521, 1920.
11. Parker, F., Jr., and Haigh, A. V. R.: The
Sachs-Georgi Test for Syphilis. Arch. Derm. and
Syphil., 4:67 (July), 1921.
12. Levinson, S. A.: Floculation Reactions in
Syphilis, with Especial Reference to the Meinicke and
Sachs-Georgi Reactions. Amer. J. Syph., 5:414, 1921.
Comparative Study of the
13. D'Auncy, R.:
Wassermann and Sachs-Georgi Reactions. J. Med.
Research, 42:839, 1921.
14. Kilduffe, R. A.: A Further Note upon a Comparison of the Sachs-Georgi and Wassermann Reactions in the Serologic Diagnosis of Syphilis. Amer.
Jour. of Med. Sciences, 164:523, 1922.
15. Bauer, R., and Nyiri, W. Zur Theorie und
klinischen Verwendbarkeit der Meinicke-Reaktion
(III Modification) Zeitsch. f. Immunitats. Original,
33:325, 1922. These workers suggest the employment
of an incubation period of 72 hours for the Meinicke
reaction.
16. Kahn, R. L.: A Simple Quantitative Precipitation Reaction for Syphilis, Preliminary Communication. Arch. Derm. and Syphil., 5:570, 1922; Second
Communication, ibid, 5:734, 1922; Third Communication, ibid, 6:382, 1922.
17. Keim, H. L., and Wile, U. J.: The Kahn
Precipitation Test in the Diagnosis of Syphilis: A
Preliminary Study. Jour. Amer. Med. Assn., 79:870,
1922.
18. Dryer, G., and Ward, H. K.: A Simple
Quantitative Serum-Reaction for the Diagnosis of
Syphilis. The Lancet, 1:956, 1921.
A Precipitation Test for
19. Wang, C. Y.:
Syphilis. The Lancet, 1:274, 1922.
20. Herrold, R. D.: A Ring or Contact Precipitation Test for Syphilis; A Modification of the Kahn
Test. Jour. Amer. Med. Assn., 79:957, 1922.