e~/8 er of Drawing Blood:
For proper performance of all serologic tests the blood
SEROLOGIC DIAGNOSTIC METHODS
Basis for Serologic Reactions:
When certain substances are introduced into the animal
body the tissues respond with the formation of special gamma
globulins. These gamma globulins are called" antibodies"
and are capable of combining in a specific manner with the
substances called "antigens" that evoked their formation.
Complete antigens are usually proteins. The specific chem-
ical group in a complex antigen responsible for antibody
formation is called' 'hapten. "
Antigen- antibody reactions are highly specific. An
antibody will react only with the species of antigen which
evoked its formation. This can be illustrated as the combin-
ation of a given chemical configuration with its complementary
image:
Serologic and immunologic methods utilize the specificity
of antigen-antibody reactions for the diagnosis of disease.
In particular, diseases due to infectious agents are character-
ized by the development of specific antibodies against the
responsible microorganism. The presence of a specific
antibody in the serum thus suggests past or present infection
of the person with the microorganism. Detection in the test
tube of specific antibodies is, therefore, a valuable tool in
the diagnosis of diseases suspected to be of infectious origin,
particularly if the etiologic agent cannot be recovered.
While antigen-antibody reactions "are specific, cross re-
actions will occur in serologic procedures and immunologic
tests between antigens possessing closely related chemical
groups and antibodies. Knowledge of this possibility will avoic.
confusion.
Time for Taking Specimens:
For most conclusive results, always obtain one specimen
of blood serum as early as possible in the disease, another
about ten days later, and a third about four weeks after onset
of the illness.
5-e...-.un must be obtained properly. Venous blood is drawn
~±aseptic precautions (see p. 132) and permitted to clot in
~ 5:erile test tube. The clot is separated from the test tube
z::..:. and the serum left at refrigerator temperature overnight
.::;>erroIt clot retractIon. The tube is then centrifuged and
- c.ear serum, free from hemolysis, is transferred by pipet
:::> another lIghtly stoppered, sterile tube. The serum
~- -':d be kept cold and brought to the laboratory as quickly as
- -s:ble.
==-- :-pretation of Results:
1: antibodies are present in the same concentration (titer)
~ a.:: :hree specimens obtained as described on p. 286, they
-e. ~ost certamly acquired in the past and have nothing to
.:n the present Illness. If, on the other hand, their con-
_-:::-a"on (titer) rises very markedly in the course of the
;.....::.ESS (L e., from the first to the third specimens) they help
es:a.blish the definite diagnosis.
:-x::-_-\ntitoxin Neutralization:
er-ain bacteria, e. g., Corynebacterium diphtheriae or
s=-:dium botulinum, produce soluble toxins. Antibodies to
- _~e: xins, i. e., antitoXins, are able to neutralize them
-- =:xed in proper proportions. This reaction takes place
~~. a.Dsence of complement (see p. 299). This principle is
- -_ :n the typIng of botulinus toxin and in the virulence test
_ ::"?!:ilieria bacilli.
- _::::::a 'on Tests:
-e:-..ain organisms evoke the production of antibodies in
-- and these antibodies can agglutinate the causative
~m5 in vitro.
--~cations:
.c.entification of isolated bacteria by slide or tube test
see p. 279) by mixing with known sera.
easurement of antibody concentration in patient's
senun by mixing serum dilutions with diagnostic anti-
gens, especially the following:
li. almonella species (typhoid, paratyphoid fevers,
etc. )
Brucella (undulant fever)
c. Proteus OX19, OX2, OKX (rickettsial diseases;
see p. 295)
c. Pasteurella pestis (plague)
e. Pasteurella tularensis (tularemia)
Leptospira species (leptospirosis)
 3. Other diseases - In some cases of "primary atypical b. The flagellar "H" antigen (= Hauch) is prepared by
pneumonia, " a nonspecific agglutination reaction with growing the bacteria on agar in Blake bottles and
streptococcus MG is positive (see p. 297); cold agglu- washing the growth off with saline containing O. 50/0
tination tests are also frequently positive (see p. 293). formalin. Shake well, and store in the refrigerator.
Sera from patients with rheumatoid arthritis often In "H" antigens the formalin treatment preserves
agglutinate sensitized sheep cells (see p. 294). the flagellar structures on the surface while in '.'0"
B. Technic of Agglutination Tests: antigens the treatment removes them. A third
1. Tube method - Make serial twofold dilutions of serum group of somatic"K" antigens are thermolabile,
with saline in the following manner. Place O. 5 ml. and occur as envelopes surrounding the cell body.
saline in each of a series of small test tubes. Add They may interfere with "0" agglutination and must
O. 5 ml. serum to first tube, mix well by drawing up sometimes (e. g., in E. coli serotypes) be destroyed
·and expelling repeatedly from pipet, and transfer 0.5 by heating before the "0" antigen can be determined.
ml. to second tube. Proceed similarly until last tube One important "K" antigen is the Vi antigen of viru-
of the row is reached, then discard 0.5 ml. To each lent typhoid bacilli.
tube add 0.5 ml. of a bacterial suspension suitably 2. Factors in interpretation - For the diagnosis of infec-
standardized for density. Shake the rack of tubes, tion by serologic tests the following are important:
then incubate overnight at 37 C. (98. 6 F.) or 50 C.
Q Q Q
a. The best proof of active disease (e. g., typhoid)
(122 F. ).
Q
other than identification of the organism is the find-
A positive reaction (++++) consists in complete ing of a rising agglutination titer when serum is
clumping of the bacteria with complete clearing of the secured from the patient at intervals during the
supernatant fluid. A negative reaction (-) consists of second and third weeks of the illness.
uniform turbidity in the suspension without clumped b. Significant agglutination titers may persist in the
sediment. blood for years after clinical or subclinical infec-
2. Slide agglutinations are often useful for rapid identi- tion or after vaccination. The finding of a single
fication of bacteria. A suspension of unknown organ- positive agglutination test therefore does not prove
isms from a fresh culture is mixed with a drop of the existence of active disease.
specific antiserum on a glass slide. On another area c. There are some cross-reactions between various
of the same slide the organisms are mixed with a drop enteric organisms encountered in agglutination
of saline. After standing or gentle rotation of the slide ests. The organism agglutinated by the patient's
for a few minutes, the slide is observed grossly and serum in the highest dilution is ordinarily con-
under the microscope. A positive result is indicated sidered the most significant.
by rapid clumping of bacteria and loss of motility in c. The following antigenic patterns are often encoun-
the serum drop but not in the saline drop (high dry tered in salmonella infections:
objective, subdued light) (see p. 279).
A similar rapid slide agglutination test may be per-
Titers observed with antigens
formed with commercial salmonella or brucella anti-
"0" "H" "Vi"
gens for the identification of antibodies in unknown sera.
and 3rd weeks 1:80 < 1:40 Low
C. Interpretation of Agglutination Tests for Salmonella Infec-
. ac ive typhoid and
tions (Typhoid, Paratyphoid, Etc.):
:erer risin
1. "0" and "H" antigens and antibodies - Many motile
-=-cination in 1:80 or more, Low
bacteria have two principal antigenic components: :.:.e past
a. The somatic "0" antigen (= Ohne Hauch) is obtain- not rising
_:L~:er < 1:40 1:40 or
ed when thick suspensions of bacteria are treated
--:e more
with an equal quantity of absolute alcohol with
vigorous stirring and shaking. Incubate the mixture Similar" receptor analysis" can occasionally be
for 12 to 24 hours at 37 C. (98. 6 F.), then shake
Q Q
carried out in enteric fevers other than typhoid,
well. Add a quantity of saline equal to the amount e. g., dysentery.
of alcohol added, to reduce alcohol concentration
to 330/0,and store in refrigerator. Adequate "0" =-. _-_:::~ Tests:
antigens are obtained from E. coli by heating the .--=:ibodies against soluble antigens (' 'precipitins' ') are
culture for one hour in boiling water bath. ~onstrable by their formation of a visible precipitate
as the result of antigen-antibody reaction. As the precipitate
is often soluble in excess of either antibody or antigen, the
two have to be mixed in accurate proportions. Precipitin
tests are used clinically for the diagnosis of syphilis (see p.
298), for the typing of ,B-hemolytic streptococci, for the
serologic identification of proteins (e. g., identification of
blood stains), and in trich~nella and echinococcus infections.
Opsonocytophagic Tests:
Antibodies which enhance the ability of leukocytes to in-
gest bacteria are called "opsonins." They can be demonstrated
by mixing patient's fresh citrated blood with a suspension of
This technic can also be used for indirect staining:
antigen + its antibody A + anti-A-antibody-fluorescent
e. g., + (e. g., antivirus + (e. g., antihuman globulin
'-;:rus) human antibody) from rabbit)
Such indirect technics permit the localization of some
a--"gens or antibodies which cannot otherwise be established
_:-..present methods.
-::::::::> ement Fixation Tests:
7he reaction consists of two parts:
•..•..Part I - Mechanism of Reaction: Specific antibody com-

00
bacteria and comparing the degree of phagocytosis with that bines with ("fixes'~ complement only in the presence of
of controls. Such tests are occasionally employed in brucel- corresponding specific antigen:
losis but are of doubtful significance for diagnosis.

:J
Bacteriolysin Test: iliC
-.?eC Complement
Antibodies can occasionally be demonstrated by their bound
ability to dissolve bacteria. While the antigen-antibody re- --.. ("fixed")
actions described above take place in the absence of com-
plement, the latter is an essent.al component in this and the

0
folloWing reactions. Complement is a substance present in
most fresh mammalian sera and destroyed by heating at 56°C.
(138° F. ) for 30 minutes. This procedure is called' 'inacti-
vation" of a serum. In most tests complement is used in the
form of pooled guinea pig serum of established satisfactory ,,-- ~:;c [] Complement
potency (fresh or reconstituted from the dehydrated state). _ not bound
In a test for bacteriOlysis, suspensions of the organism (' 'not fixed")
are mixed with serum which is being tested for antibodies. ~
Complement is added and the mixture incubated at 37°C.
(98.6° F.) for one hour, or (in the case of cholera) injected
intraperitoneally into guinea pigs. Control mixture s con-
taining normal serum are included in the test. At the end of :=. Part 2 - Test for Presence of Free (Not Bound) Com-
the incubation period the organisms in contact with specific :Lement: "Hemolytic system" is added to above mixture
antibodies will have dissolved, while those mixed with normal . ~antigen + antibody + complement. •'Hemolytic system"
serum should be unchanged. The test is used mainly in chol- onsists of sheep cells + anti-Sheep cell hemolysin (i. e.,
era and leptospirosis (as agglutination-lysis). serum from rabbits repeatedly injected with sheep cells).
-: free complement is present, it will combine with and
Fluorescent Antibody Technics: ::emolyze the sheep cells (i. e., if the original antigen did
A new serologic technic is based on the conjugation of anti- • fit the antibody, the two did not combine, hence no
body molecules with fluorescent dyes (e. g., fluorescein, omplement was bound).
rhodamine). Such" lab'eled" antibody may be used to locate
antigen microscopically because of the high specificity of the
antigen-antibody bond. Fluorescent-Iabeled
rapidly identifY specific microorganisms
antibody can
in smears from
cultures, pus, tissue, or exudates, and can establish the
localization of antigens in tissue section. This technic is
used for bacteriologic and virologic diagnosis and for the
8U[]~LYSIS
study of "hypersensitivity" diseases.
 This complement fixation test is negative. Hemolysis
is present.
If free complement is not present (because it was all
bound by original antigen-antibody combination), the
sheep cells will remain intact:
2. After the injection of animal serum (e. g., antitoxin).
individuals often develop high titers of agglutinins
(which will agglutinate sheep cells).
3. The agglutinins mentioned under 1 and 2 can be re'
moved from the serum by absorption with guinea pig
kidney.
4. The agglutinins developing in mononucleosis are not

Ou
No absorbed with guinea pig kidney but are absorbed by
complement washed and boiled beef red cells.
available _ NO
LYSIS .::be ~o. Saline Serum Sheep cells Final serum
ml. m!. m!. dilution (titer)
l 0.4 0.1 O. 1 1:7
Sheep cells + Hemolysin 2 0.25 0.25 of Tube 1 O. 1 1:14
3 0.25 0.25 of Tube 2 O. 1 1:28
This complement fixation test is positive. No 4 0.25 0.25 of Tube 3 0.1 1:56
hemolysis. 5 0.25 0.25 of Tube 4 O. 1 1:112
Between parts 1 and 2, the antigen-antibody-com-
plement mixture is incubated for varying periods· at 37°C.
(98. 6°F.) or kept at refrigerator temperature for
5

3
. 0.25
0.25
0.25
0.25
0.25
0.25
of Tube
of Tube
of Tube
5
6
7
0.1
O. 1
O. 1
1:225
1:450
1:900
••fixation" of complement prior to addition of the indicator 9 0.25 0.25 of Tube 8 O. 1 1:1800
"hemolytic" system. 10 0.25 0.25 of Tube 9 0.1 1:3600
C. Complement fixation tests are used commonly in the 1: 0.25 0.25 of Tube 10 O. 1 1:7200
diagnosis of the following diseases: .2 0.25 - - - - - O. 1 Control
1. Syphilis (see p. 298).
2. Viral diseases (see p. 296). Keep tubes at room temperature for two hours or at
3. Rickettsial diseases (see p. 295). -·C. (98. 6°F.) for one hour, and place in refrigerator
4. Fungous diseases (especially coccidioidomycosis, .·ernight. Shake gently and note clumping of cells.
histoplasmosis, blastomycosis).
They are occasionally employed in the diagnosis of '.' .' gg utination Test:
brucellosis, parasitic diseases (cysticercosis, trichin- '- some cases of "primary atypical pneumonia" (PAP)
osis, echinococcosis) and gonorrheal arthritis. "'--ces appear in the serum which are capable of agglu-
--- -" human group 0 red cells in the cold, but not at room
Sheep Cell Agglutination Test for Infectious Mononucleosis --::.;ature or at 37°C. (98.6· F.). The test is performed by
(Paul-Bunnell Test, "Heterophil Agglutination Test"): .- serial twofold dilutions of the patient's serum in saline
This is a nonspecific reaction based on the finding that ~ .. g an equal amount of a 1"/0suspension of washed
persons with infectious mononucleosis develop a high titer of '. group 0 red cells. The mixtures are shaken well and
sheep cell agglutinating antibodies. - :he refrigerator overnight. A positive result is indi-
A. Technic: Sheep blood is obtained fresh and the cells _~ .•. :inding clumped cells immediately on removing the
washed three times with saline. A 2% suspension of cel .:;:;om the refrigerator which do not remain clumped after
is then prepared. Sera from patients are inactivated at ~5 have been at room temperature for three to four
56°C. (133° F. ) for 30 minutes. Whenever possible ser~ -.:;. Control tubes with normal serum and with saline-red
should be obtained as early as possible in the illness and _ ::::ix1ures are included.
ten days later, to observe the rise in antibodies. A 7-p degree of positivity of this test depends on the etiology
series of small test tubes is set up as shown in chart on .• some extent the severity of the illness. PAP caused by
next page (Davidsohn, JAMA 108:289, 1937). -~_ ses is never associated with a positive cold agglu-
B. Problems of Interpretation: _ - test. PAP due to the Eaton-Meiklejohn pneumonitis
1. Normal persons may have agglutination titers up to --5 and perhaps other agents gives cold agglutination tests
1: 112. - _ :x>rtion to the severity of the illness. Early treatment
::.cases with tetracycline drugs seems to interfere with
~ elopment of a positive test.
Antistreptolysin Titer:
Persons infected with l3-hemolytic streptococci often
develop antibodies against the hemolysin 0 produced by the
streptococcus. This antibody can be tested for by its ability
to inhibit hemolysis of red cells by a standardized strepto-
coccus hemolysin. The test should be carried out only in a
laboratory having considerable experience and standardized
reagents at its disposal. A reliable technic is indicated by
Rantz, L. A., et al. , Am. J. Med. 5: 3, 1948.
Finding persistently high antistreptolysin titers (in excess
of 125 units) suggests recurrent or persistent infection with
hemolytic streptococci and reaction to such infection. NOTE:
Adequate penicillin therapy of hemolytic streptococcus infec-
tions often interferes with the development of antistreptolysin
in high ti ter.

Serologic Tests for Rheumatoid Arthritis:

Most patients with active rheumatoid arthritis possess an
unusual serum protein which is capable of reacting with gamma
globulin. This substance (' 'rheumatoid factor") can be demon-
strated by several serologic tests. Most of these tests con-
sist in the agglutination of particles (latex, bentonite, tannic-
acid-treated red blood cells, etc.) onto which human gamma
globulin has been adsorbed by diluted serum from rheumatoid
patients. The interaction of rheumatoid factor and gamma
globulin can also be demonstrated as a precipitin reaction and
may represent an antigen-antibody reaction.

Virus Neutralization Tests:

In many viral infections it is not practical to attempt to
isolate the etiologic agent. The diagnosis usually rests on
the demonstration that antibodies capable of neutralizing the
infectious agent appeared in the course of the illness. It is
essential, therefore, to obtain at least two and preferably
three specimens of the patient's serum during the illness: the
first as early as possible after onset of the illness; the second
ten to 14 days later; and the third about four weeks after onset
of the illness.
The sera must be obtained in an aseptic manner (see p.
132) and must be kept refrigerated. The laboratory test con-
sists of mixing suitable proportions of serum with infectious
material, inoculating it into susceptible laboratory animals,
fertile eggs, or tissue cultures, and observing the presence
or absence of protection from viral effects.
The viral diseases in which neutralization tests are com-
monly available are indicated in the table on p. 296.

Virus Hemagglutination-Inhibition Test:

A number of viruses (e. g., influenza, Newcastle, mumps,
encephalitides) agglutinate erythrocytes, and this reaction
[Cont'd. on p. 298.]
 - _. -
:"0111' III
'",' Il:ItClllI I< A Pllt Itu h Dc ('UI'J'I~IH'( V(~l'tm W.<I1 I' 1~llx
OX-19 OX-2 OX-K
Typhus Epidemic typhus R. prowazeki Central World-wide Lice ++++ + 0
Murine typhus R. mooseri Short duration World-wide Rat flea ++++ + 0
Brill's disease* R. prowazeki Central East Coast U.S. None ++++ + 0
Spotted Rocky Mountain R. rickettsi Peripheral North and South Ticks +++ ++ 0
Fevers spotted fever America
Mediterranean R. conori Pnmaryand Mediterranean Dog ticks ++++ ++ 0
(boutonneuse) fever palms and soles
South African tick R. rickettsi Primary lesion South Africa Ticks +++ +++ ++
fever
Other spotted Various Various Australia .. India, Ticks +++ ++ Qcc.
fevers Russia
Rickettsialpox R. akari Primary lesion, New York, Mites 0 0 0
central rash Boston
Scrub typhus R. orientalis Primary lesion, Japan, Burma. Mites 0 0 +++
Tsutsugamushi fever central rash Malaya. New
Guinea
Q fever Coxie Ha burneti None World-wide (Air-borne 0 0 0
Diagnosis Based On:
1. Exposure to known vectors in endemic areas. 4. Results of Weil-Felix test on early and later serum
2. Clinic.al appearance. specimens; rise in titer is diagnostic.
3. Isolation of rickettsiae (infre.quently done; see p. 393). 5. Results of complement fixation tests witb acute and
convalescent sera; rise in titer is diagnostic.
6. Response to chemotherapy is often suggestive.
Chloramph<micol (Chloromycetin®) or the tetracyclines are the drugs of choice.
 Disease Etiologic Agent Specimen Submitted for Examination Test Remarks
Croup Hemadsorption, Throat swab; blood serum, Virus isolation, Different viruses in
(infantile) croup-assoc. acute and convalescent. hemagglutination- different years.
viruses inhibition.
Denflue Denllue virus Blood serum, acute and convalescent. * Virus neutralization. Not often available.
Encephalitis Specific viruses Blood serum, acute and convalescent. * Virus neutralization, Only rise in titer diagnostic.
(Japanese B, St. Louis, complement fixation.
Western and Eastern
Equine, Venezuelan) Brain (autopsy). Virus isolation.
Gingivostomatitis, Herpes simplex Vesicle fluid, saliva, spinal fluid. t Virus isolation, In eggs, mlce, raoolts, tissue cuJ.-
herpes labialis, cold virus Blood serum, acute and convalescent. * virus neutralization, ture. Only rise in titer signifi-
sores, encephalitis complement fixation. cant. Most adults have antibodies.
Herpangina, pleurodynia, Coxsackie A Throat washings, stools. t
Spinal fluid. Virus isolation, Many different virus types. Tis-
aseptic meningitis and B Blood serum acu~e and convalescent. * neutralization, C-F. sue culture. Suckling mice.
Inclusion blennorrhea Large virus Epithelial scrapings of conjunctiva of Stained smear, egg Elementary bodies seen.
lower lid. inoculation. Occurs especially in newborn.
Infectious Unidentified Blood for white blood cell count. WBC and differential, Atypicallymphocytes.
mononucleosis Blood serum, acute and convalescent. * Paul-Bunnel test. Rising heterophil agglutination
titer.
Influenza Influenza viruses Throat washings. t Virus isolation, ]n eggs, mice, or tissue culture.
A, B, C (several Blood serum, acute and convalescent. * hernagglutination- Only greater than four-fold in-
sublypes) inhibition, C-F. crease in titer diagnostic.
Ker atoconjW1ctivi tis, Adenovirus, Swabs or scrapings from conjunctiva. Virus isolation, Tissue culture.
epidemic type B Blood serum, acute and convalescent. :\< virus neutralization.
Lymphocytic LCM virus Blood serum, acute and convalescent. :\< Virus neutralization. Only rise in titer diagnostic.
choriomenin~tis
Lymphopathia LGV virus Pus o:r: tissue biopsy. t Skin test (Frei), Elementary bodies.
venereum Blood serum, acute and convalescent. * virus isolation. In eggs and mice.
Mumps Mumps virus Saliva, spinal fluid. t Virus isolation, In eggs.
Blood serum, acute and convalescent. * hemagglutination- Only rise in titer significant.
inhibition, C-F.
Meningitis. aseptic Coxsackie, ECHO, Spinal fluid. blood serum, acute and Virus isolation, Tissue culture. eggs. suckling
(viral) H. simplex, polio, convalescent. * neutralization, C-F. mice.
others

_ Co_=: n.e:::.a.gg~.
=m::.a PAP; aee::e :ac::.'";e a=:.c. ~escen:...* ~ isolation. C-F.
respiratory disease virus neutralization.
lARD)
Psittacosis Sputum, blood. t Virus isolation,
Blood serwu, acute and convalescent. * com lement fixation.
Brain tissue (autopsy). Smears for Negri
bodies.
Virus isolation. In mice.
Whole blood. Rickettsia isolation, In guinea pigs.
Blood serum. acute and convalescent. * complement fixation, Only 'rise in titer significant.
Weil-Felix test. For results see chart p. 295.
Spinal, ventricular fluid, lymph node. Toxoplasma isolation, In mice.
Blood .!?erum, acute and convalescent. :0:: neutralization test. Often not available. Special
corn lement fixation. detest.
Epithelial scrapings from conjunctiva Stained smear, Elementary bodies seen.
of upper tarsus. Virus isol. In fertile eggs.
Scrapings from base of fresh lesion. Stained smear, Special fixation and stain.
Vesicle fluid. virus isolation, In eggs and rabbit cornea.
Dried crusts from lesions. complement fixation. Crusts serve as anti en.
Blood serumt in first week. Virus isolation, In mice or monkeys.
Blood serum, acute and convalescent. * virus neutralization, Only rising titer diagnostic.
Liver tissue (autopsy). histologic Midzonal necrosis and
diagnosis. . 'Councilman" bodies.

*Collect sterile blood as early as possible in disease and again two tCollect materials as early as possible in disease, ship to
and four weeks after onset of illness. Let blood clot, send sterile laboratory frozen, without thawing. Maintain aseptic
serum to laboratory, keep refrigerated, label adequately. precautions, label adequately.
may be specifically inhibited by antiserum. This reaction
forms the basis of a number of diagnostic tests for viral in-
fections.
To standardized amounts of virus, serial dilutions of the
serum under test are added (after heating the serum for 30
minutes at 56°C.), and the mixture is shaken well. An equal
amount of 0.5% suspension of human Type 0 or chicken eryth-
rocytes is added and the mixture incubated at room temper-
ature for 45 to 60 minutes. Care must be taken not to disturb
the test tubes or racks. The tubes are then read for agglu-
tination of the red blood cells. The titer of a given serum is
defined as the highest dilution of serum which effects com-
plete inhibition of red cell agglutination.
Commonly Used Serologic Tests for Syphilis (S. T. S. ):
The tests below are accepted in the 1955 Manual of the
Public Health Service, U. S. Department of Health, Education,
and Welfare (Serologic Tests for SyphiliS, Public Health
Service Pub!. No. 411, U. S. Government Printing Office,
Washington 1955). Others are occasionally used. Adherence
to the published standard method is essential in obtaining re-
liable results.
If more than one test is desired it is customary to per-
form one complement-fixation and one flocculation test. The
greater the agreement between different tests and the results
of different laboratories, the more confidence may be placed
in reports of these serologic findings. The different technics
estimate the presence of reagin, not of specific antibody
directed against T. pallidum.
A. Complement-Fixation Test: Kolmer-Wasserman.
B. Flocculation Tests: Hinton, Kahn, Kline, Mazzini,
APHA Reference Test.
C. VDRL (Ven.Dis.Ref.Lab.).
See the above manual for the standard technic of these
tests.
Antigens for S. T.S.:
The causative agent of syphilis, T. pallidum, has not yet
been grown in vitro. Thus antigens prepared from virulent T.
pallidum are not available. Experimentally, antigens from
the Reiter strain of treponemes appear to have significant
specificity in complement -fixation test s, but they are not yet
widely used.
It has been found empirically that sera from syphilitic in-
dividuals will flocculate or fix complement in the presence of
alcoholic extracts of beef heart muscle to which cholesterol
and lecithin have been added. Such standardized extracts
~rdiolipin-Iecithin antigens) are employed in most S. T. S.
...sed for the assay of reagin. Living treponemes from animal
:es'ons are employed in the TPI (see p. 300).
?:'J.ids to Be Tested by S. T. S.:
A. Patient's Serum: Draw 5 to 10 m!. of sterile venous blood
and permit to clot in dry sterile test tube. Blood samples
should not be taken during high fever or soon after an-
esthesia or alcohol intoxication, for these and other con-
ditions (see below) tend to give false-positive reactions.
Centrifuge the clotted blood and remove the cell-free
serum. Store in refrigerator until test can be performed.
Just prior to test the serum is inactivated at 56°C.
(133°F.) for 30 minutes to destroy complement.
3. pinal Fluid: Collect 3 m!. spinal fluid in sterile test
tube, free from blood cells (see p. 247).
:::Zerpretation of Results of S. T. S.:
_. In untreated adults the S. T. S. will become positive in 60
to 80% of cases in the primary stage (four to eight weeks
after exposure), whether primary lesions are present or
not. If manifest secondary lesions develop, the blood
:est will be positive in 90 to 100% of the cases and the
spinal fluid in 30 to 70%. In late (tertiary) lues, the blood
~s positive - if visceral lesions are developing - in 60 to
80% of cases. If there are no visceral lesions but C. N. S.
involvement, the spinal fluid is a better guide. Note,
:"owever, that in tabes dorsalis, both blood and C.S.F.
!:lay be negative in the presence of progressive symptoms
and signs. In general paresis both blood and C. S. F. are
almost always positive.
_. :n the newborn, and in infants up to ten weeks, the blood
:est may reflect the mother's reaction and does not
:1ecessarily indicate infection of the infant. It is
:,nportant, therefore, to follow titers of infants born of
mothers with positive serology but without evidence of
activity of the disease (e. g., adequately treated).
:. Antiluetic therapy usually results in gradual reversion
0: positive serologic tests to negativity. This, however,
::lay take many months. A positive blood test in an ade-
".lately treated individual is therefore no justification for
ex:ending or repeating therapy unless there is evidence of
activity of the disease or rise in complement fixation
:::er. Antiluetic therapy, particularly with penicillin,
administered to darkfield-positive, seronegative individ-
:zals often interferes with the development of a positive
complement fixation test.
=. Anticomplementary Specimens: Occasionally blood sam-
:l2.esare encountered which fix complement in the absence
.. beef heart antigen. Such specimens cannot satisfactor-
 :.::ese preparations microscopic<llly in the darkfield and de-
ily be examined by the complement fixation test. This
:::-n::ine the highest dilution of the test serum which rapidly
occurs particularly in hemolysed or contaminated speci-
=obilizes the organism. This dilution gives the titer of
mens. Such samples can be submitted to precipitation or 7:-eponema-immobilizing a.ntibody.
flocculation tests. "Anticomplementary" meanS neither . nother reaetion, called Treponema pallidum immune
positive nor negative result. ~--e:-ence phenomenon (TPIA), may also be used as a specific
E. Biologic false-positive serologic reactions for syphilis E.::-o:ogic test for syp.hilis. In the pres.ence of specific anti-
occur in 0.01 to 0.1% of all positive tests. Commonly .:<:. and complement, killed spirochetes adhere to human red
they are weakly positive (except in yaws, pinta, and - .. cells. The adherence does not take place in the absence
bejel, where they are the same as in syphilis). . :;>ecific antibody.
Such biologic false-positive reactions may occur In
yaws, pinta (mal del pinto), bejel (all closely related
spirochetal infections), 50 to 100% of cases; leprosy, 40
to 70% of cases; infectious mononucleosis, 10 to 50% of
cases; malaria, 10 to 40% of cases; rat-bite fever and
C-reactive protein is a substance in the serum of certain
relapsing fever, 20 to 50% of cases; all protozoan and
=-eI:ts which reacts with the somatic C polysaccharide of
viral infectious diseases, occasionally; all diseases
ococci. It can be detected by a precipitin reaction with
associated with abnormally high globulin levels; all im-
c;.:::nococcus
polysaccharide, but is more commonly meas-
munization and vaccination procedures; any fever of long
-,:-::
by precipitation with specific antiserum prepared in
duration; all" collagen diseases." .
:·s.
Unless supported by clinical findings, never accept the
reactive protein is a 13- globulin, perhaps bound to
result of a single serologic test for syphilis as proving the
diagnosis. Always request at least two examinations. Con-
=C- lipid. It is not found in the serum of normal individuals,
.: appears within 14 to 26 hours follOWinginflammation or
sider conditions commonly responsible for biologic false- "":;;E.einjury. It is consistently found in the serum of patients
positive reactions. _~ bacterial infections, active rheumatic fever, acute myo-
==.a: infarction, and widespread malignant disease. It is
Quantitative S. T. S.:
commonly present in rheumatoid arthritis, virus infec-
Any serologic test for syphiliS may be carried out with
" and active tuberculosis, but only rarely in chronic
dilutions of serum, and the highest dilution giving a ++++ re-
~=..;:a, multiple myeloma, and limited primary neoplasm.
action may be reported. This permits evaluation of trends in
TI:e aboratory determination for C-reactive protein thus
serologic response (result of therapy, indication of relapse,
follow-up of infants).
-=s a nonspecific test for the presence of inflammation,
::55.e injury, or necrosis.
Number of units = serum dilution giving ++++ reaction
x 4.

Treponema Pallidum Immobilization Test (TPI):

Serum from individuals with syphilis contains an antibody
which results in the rapid immobilization of actively motile
T. pallidum. This test has the greatest diagnostic specificity
because it measures a true antibody against the etiologic
organism of syphilis rather than the development of reagin
against a lipid extract of mammalian tissue which shows only
accidental relationship to the presence of syphilis. There-
fore, TPI can conclusively differentiate between biologic
false-positive tests and true syphilitic positives. Unfortu-
nately the test is not easy to perform and is available only in
a few central laboratories.
Technic: To dilutions of the test serum add a suspension
of actively motile T. pallidum freshly extracted from the =: ::etails see Roantree, R. J., and Rantz, L. A.: Arch.
testicular chancre of an infected rabl:>it. A similar mixture ed. 96:674, 1955.
containing normal human serum is used as a control. Observe