TPHA

200 72503
500 72504
KITS FOR THE QUALITATIVE AND SEMI-QUANTITATIVE
DETECTION OF ANTIBODY TO TREPONEMA
PALLIDUM IN HUMAN SERUM OR PLASMA BY
PASSIVE HAEMAGGLUTINATION

883683 - 2014/12
 TABLE OF CONTENT

1. INTENDED USE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

2. SUMMARY AND EXPLANATION OF THE TEST. . . . . . 5

3. PRINCIPLES OF THE PROCEDURE. . . . . . . . . . . . . . . 5

4. REAGENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

5. WARNING AND PRECAUTIONS. . . . . . . . . . . . . . . . . . 7

6. SPECIMENS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

7. PROCEDURE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

8. TEST LIMITATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

9. PERFORMANCES CHARACTERISTICS . . . . . . . . . . . 13

10. BIBLIOGRAPHY REFERENCES. . . . . . . . . . . . . . . . . . 15

4 [EN]
1. INTENDED USE
TPHA kits are intended for use for the qualitative and semi-
quantitative detection of antibodies to Treponema pallidum in
human serum and plasma. The product may be used for the
screening of blood donors, and to aid in the diagnosis of patients
where syphilis infection is suspected.

2. SUMMARY AND EXPLANATION OF THE TEST

Syphilis is a chronic infection that progresses through distinct
stages of infection: primary, secondary, tertiary, and quaternary.
These stages produce diverse clinical symptoms, typically
producing initial sores known as chancres then syphilitic rash
followed by long periods of dormancy. Untreated infection may
eventually result in cardiovascular problems and neurosyphilis.

The infection is caused by the spirochaete Treponema pallidum, and

is usually acquired by sexual contact, although the disease may be
transmitted by transfusion of infected blood. Intrauterine infection
also occurs. The organism has proved virtually impossible to culture
in artificial media, and diagnosis of the infection usually depends on
the demonstration of antibodies in the blood, which appear soon
after initial infection and may persist for many years.

Tests for Syphilis fall into four categories: direct microscopic

examination, treponemal antibody tests, non - treponemal antibody
tests and direct antigen tests. Because of the long periods of
dormancy and the non - specific nature of non - treponemal tests,
methods that detect specific anti-treponemal antibodies in blood
specimens have become increasingly popular for screening. TPHA
is one such test.

3. PRINCIPLES OF THE PROCEDURE

TPHA kits use preserved avian erythrocytes coated with antigens of
T. pallidum (Nichols strain), which will bind with specific antibody
present in patient’s serum or plasma. The cells are suspended in a
medium containing components to eliminate non - specific reactions.
Positive reactions are shown by agglutination of the cells, negative
reactions by the settling of the cells to a button or small ring.

Although these kits are intended for use primarily as qualitative

tests, antibody levels may be titrated by doubling dilution.

Agglutination patterns may be interpreted by eye or by a plate-

reader capable of reading agglutination patterns. Contact the
concern company for the adaptations and special procedures.
[EN] 5
4. REAGENTS
4.1. Description

Presentation
Identification
Description 72503 72504
on label
200 tests 500 tests
Test Cells
Suspension of Avian
erythrocytes coated with 2 vials 2 vials
R1 Test Cells
antigens of T. pallidum, 7.8 ml 20 ml
containing Bovine Serum
Albumin (BSA)
Control Cells
Suspension of Avian 2 vials 1 vial
R2 Control Cells
erythrocytes, containing 7.8 ml 20 ml
BSA
Diluent
2 vials 1 vial
R3 Diluent Saline solution containing
20 ml 125 ml
Rabbit serum
Positive Control
Human serum containing
antibodies to T .pallidum,
Positive 1 vial 1 vial
R4 negative for HBs Antigen,
Control 1 ml 1 ml
anti-HIV1/2, and anti-
HCV antibodies diluted in
phosphate buffer
Negative Control
Negative 1 vial 1 vial
R5 Rabbit serum in
Control 1 ml 1 ml
phosphate buffer

4.2. Storage and handling requirements

This kit should be stored at +2-8°C. Store bottles up-right. Do not
freeze.
Each item of the kit preserved at +2-8°C can be used up to the
expiry date mentioned on the package. After opening and in the
absence of contamination, the R1, R2, R3, R4 and R5 reagents
preserved at +2-8°C, can be used up to the expiry date shown on
the label.

6 [EN]
5. WARNING AND PRECAUTIONS
For in vitro diagnostic use. For healthcare professional use.

5.1. Health and Safety precautions:

• This test kit should be handled only by qualified personnel trained
in laboratory procedures and familiar with their potential hazards.
Wear appropriate protective clothing, gloves and eye/face
protection and handle appropriately with the requisite Good
Laboratory Practices.

• The control materials supplied are derived from human serum.

They have been tested at donor level and found negative for HBs
Antigen, anti-HIV1/2, and anti-HCV antibodies. No known test
method can offer complete assurance that infectious agents are
absent. Therefore, all human blood derivatives, reagents and
human specimens should be handled as if capable of transmitting
infectious disease, following recommended Universal Precautions
for blood borne pathogens as defined by local, regional and
national regulations.

• Biological spills: Human source material spills should be treated as

potentially infectious.

Spills not containing acid should be immediately decontaminated,

including the spill area, materials and any contaminated surfaces
or equipment, with an appropriate chemical disinfectant that is
effective for the potential biohazards relative to the samples
involved (commonly a 1:10 dilution of household bleach, 70-80%
Ethanol or Isopropanol, an iodophor [such as 0.5% Wescodyne™
Plus, etc.), and wiped dry.

Spills containing acid should be appropriately absorbed (wiped

up) or neutralized, the area flushed with water and wiped dry;
materials used to absorb the spill may require biohazardous waste
disposal. Then the area should be decontaminated with one of the
chemical disinfectants.

Note: Do not place solutions containing bleach into the autoclave !

• Dispose of all specimens and material used to perform the test as

though they contain an infectious agent. Laboratory, chemical or
biohazardous wastes must be handled and discarded in
accordance with all local, regional and national regulations.

• The Safety Data Sheet is available on www.bio-rad.com.

[EN] 7
5.2. Precautions related to the procedure
5.2.1. Preparing
The reliability of the results depends on correct implementation of
the following Good Laboratory Practices:
• Do not mix or associate reagents from different lots within a test
run.
• Do not use expired reagents.
• Before use wait for 30 minutes for the reagents to stabilize at room
temperature (18-30°C).

5.2.2. Processing
• Do not change the assay procedure.
• Use a new distribution tip for each sample.

6. SPECIMENS
Serum or plasma (EDTA, Sodium Citrate, Heparin and ACD)
specimens should be free of blood cells and of obvious microbial
contamination. They may be stored at +2-8°C for up to 7 days
before testing. Specimens needing longer storage should be frozen
at -20°C or lower. Frozen specimens should be thawed and well
mixed before testing. Do not repeat more than 5 freeze/thaw cycles.
Heated samples at 56°C during 3 hours do not impact the results.

Specimens containing up to 100 mg/L of bilirubin, up to 36 g/L of

triglycerid and up to 10 g/L of hemoglobin do not affect the results.
However, it is not recommended to use hyperlipaemic or
hyperhaemolysed sera or plasma.

If the specimens are to be shipped, they must be packaged in

accordance with the regulations in force regarding the transport of
etiological agents and preferably transport frozen.

7. PROCEDURE
REMARK: The kit Positive and Negative Controls (R4 and R5) must
be run with each run of tests.
7.1. Materials required but not provided
• Distilled water
• Sodium hypochlorite (household bleach) and sodium bicarbonate.
• Absorbent paper
• Disposable gloves
• Safety glasses
• Disposable tubes
• Automatic or semi-automatic, adjustable or preset pipettes or
multipipettes to measure and dispense 10 μl, 25 μl, 75 μl and
190 μl.
8 [EN]
• Microplate shaker
• U well plate format (96-well microplates) - Code 83375 (5 plates)
• Container for biohazardous waste

7.2. Assay Procedure (Manual technique)

7.2.1. Qualitative assay

Three wells from the U microplate are needed for each specimen.

The TPHA 500 Kit (Product No. 72504) is intended for screening
large numbers of specimens and contains only a small volume of
Control Cells. It is intended that specimens are screened using only
Test Cells in the first instance, and the Control Cells should be used
when repeating tests on specimens giving a positive result when first
tested.

a. Specimen and Controls Dilution (to 1 in 20)

Add 190 μl of the diluent (R3) to one well.
Add 10 μl of specimen or Positive Control (R4) or Negative Control
(R5) to the same well.
Mix thoroughly.

b. Test
Transfer 25 μl of diluted control or diluted specimen from dilution
step to test well.
Transfer 25 μl of diluted control or diluted specimen dilution step to
control well.
Re-suspend the Test Cells (R1) and the Control Cells (R2) by shaking
the vial Examine for complete suspension.
Add 75 μl of Test Cells (R1) to test well and 75 μl of Control Cells
(R2) to the control well.
Final specimen dilution is 1: 80.
Mix wells thoroughly.
Incubate at room temperature (15-30 °C) on a vibration-free surface
for a minimum of 45 minutes.
Read the settling patterns. Agglutination patterns are stable for at
least three hours if undisturbed.

7.2.2. Semi-Quantitative assay

9 wells of the U microplate are needed for each specimen: One well
for the specimen or control dilution and 8 wells for the titration.

Note: The Positive and Negative Controls (R4 and R5) must be run
with each lot of tests, using the procedure given below.

[EN] 9
a. Specimen and Controls Dilution (to 1 in 20)
Add 190 μl of the diluent (R3) to one well.
Add 10 μl of specimen or Negative Control (R5) or Positive Control
(R4) to the same well.
Mix thoroughly.

b. Titration
Leaving the first well empty, add 25 μl of diluent (R3) to each of the
remaining 7 wells.

0 1 2 3 4 5 6 7 8
190 μl (R3) + 10 μl
25 μl 25 μl 25 μl 25 μl 25 μl 25 μl 25 μl
Specimen or (R4)
of R3 of R3 of R3 of R3 of R3 of R3 of R3
or (R5)
Specimen or
8 wells for titration
control dilutions

Transfer 25 μl of diluted control or specimen to the 1st well and to

the 2nd well, then mix.

Then serially dilute along the well sequence, discarding the excess
25 μl from the final well.
25 μl 25 μl 25 μl 25 μl 25 μl 25 μl

0 1 2 3 4 5 6 7 8 Discarding
excess
25 μl of 25 μl
190 μl (R3) +
25 μl of R3 +
10 μl 25 μl 25 μl 25 μl 25 μl 25 μl 25 μl
diluted 25 μl of
Specimen or of R3 of R3 of R3 of R3 of R3 of R3
control diluted
(R4) or (R5)
control
Specimen
or control 8 wells for titration
dilutions

c. Test
Gently mix the Test Cells (R1) to ensure thorough resuspension.
Add 75 μl of Test Cells (R1) to each well.
Final specimen dilution range after addition of cells is 1: 80 –
1 :10240.
Mix thoroughly.

10 [EN]
 0 1 2 3 4 5 6 7 8

25 μl of
190 μl (R3) + 25 μl of 25 μl of 25 μl of 25 μl of 25 μl of 25 μl of 25 μl of
diluted
10 μl dilution dilution dilution dilution dilution dilution dilution
control
Specimen or + 75 μl + 75 μl + 75 μl + 75 μl + 75 μl + 75 μl + 75 μl
+ 75 μl
(R4) or (R5) of R1 of R1 of R1 of R1 of R1 of R1 of R1
of R1

1:80 1:160 1:320 1:640 1:1280 1:2560 1:5120 1:10240

Specimen or
control 8 wells for titration
dilutions

Incubate at room temperature (15-30°C) on a vibration-free surface

for a minimum of 45 minutes.
Read the settling patterns. Agglutination patterns are stable for at
least three hours if undisturbed.
The titre of the specimen is the reciprocal of the highest dilution
giving agglutination.

7.3. Quality Control

The kit Controls must give the correct result; Negative Control (R5)
is negative and Positive Control (R4) is positive.
When the kit Positive Control (R4) is titrated, the expected end point
is 1:640 - 1:5120.

7.4. Interpretation of the results and Test Validation criteria

Positive Equivocal Negative

Test Cells Control Cells

Full cell pattern covering the bottom
Positive: Strong Negative button
of the well.
Cell pattern covers approx. 1/3 of
Weak Positive Negative button
the well bottom.
Cell pattern shows a distinctly open
Equivocal Negative button
centre.
Cells settled to a compact button,
Negative Negative button
typically with a small clear centre.
Non-specific
Positive reaction. Positive reaction
reaction

[EN] 11
A sample where the Test Cell well is non-reactive should be
considered as negative for T. pallidum.
Reactivity less than equivocal is considered negative.

Any specimen giving equivocal or positive results should be

considered as positive and the test procedure repeated as above, in
duplicate, adding the Control Cells (R2) provided to one set of cells
and Test cells (R1) to the other.
If at least in one of the well with Test cells (R1), the result is equivocal
or positive, the specimen should be considered as positive for
T. pallidum.

For non-specific reaction, if the agglutination is greater in the Test

Cells (R1) than in the Controls Cells (R2), then the sample is
considered positive and should be repeated as above.
When a sample has greater or equal agglutination in the Control
Cells (R2) then the sample should be absorbed using the following
procedure.

7.5. Absorption of non-specific reactions

(Procedure to be used if agglutination is seen in both Test and
Control cells).
1.Add 10 μl of specimen to 190 μl of resuspended Control Cells, mix
well and incubate at room temperature for 30 minutes.
2. Centrifuge to deposit the cells at a minimum of 1500g for 3
minutes.
3. Add 25 μl of supernatant from step 2 to each of 2 wells.
4. Gently mix the Test and Control Cells to ensure thorough
resuspension.
Add 75 μl of Test Cells to the 1st well.
Add 75 μl of Control Cells to the 2nd well.
5. Ensure thorough mixing and incubate at room temperature for a
minimum of 45 minutes.
6. Read and interpret the settling patterns as above.

8. TEST LIMITATION
No single test or definitive reference standard is available for every
stage of the disease. Thus, Syphilis diagnosis relies predominantly
on serological testing, requiring results from both non-treponemal
and treponemal methods.

No diagnostic test provides absolute assurance that a sample does

not contain low levels of antibodies to T. pallidum, such as those
present at a very early stage of infection. Therefore, a negative result
at any time does not preclude the possibility of exposure to infection
with syphilis.

12 [EN]
All treponemal test results tend to remain reactive following
treponemal infection therefore they should not be used to evaluate
response to therapy. Because of the persistence of reactivity,
generally for the life of the patient, the treponemal tests are of no
value in determining relapse or re-infection in a patient who had a
reactive result. In this case, it is recommended to use other assays:
Syphilis Total Ab, Syphilis IgM EIA and RPR.

9. PERFORMANCES CHARACTERISTICS
9.1. Precision Study
Precision study was realized by testing 3 samples (1 negative, 1 low
positive and 1 positive) in 10 replicates in the same run (repeatability)
and in 2 replicates during 5 days on 2 different lots and reading by
2 operators (Intermediate precision).
Repeatability: The 3 samples gave identical results when repeated
10 times.
Intermediate/ inter-lot precision: The 3 samples gave identical
results when tested in the different conditions (40 times).

9.2. Clinical performance

The performances of the TPHA assay have been determined by
testing samples from random blood donors, hospitalized patients,
patients infected with syphilis and on samples positive for markers
unrelated to infection by the T. pallidum.
The studies were carried out on 2 blood donor sites and at the Bio-
Rad site.

9.2.1. Specificity
A total of 5032 samples from blood donors prospectively collected
in 2 different sites were studied.
The samples were either serum (3626), EDTA K2 (539) or Lithium
Heparin plasma (867) tested in a period of less than 7 days after
sampling and were compared to the screening assay used in the
laboratory.

[EN] 13
Table 1 : Blood donor population

Initial Repeated 95%

Specificity
Site Number Reactive Reactive Confidence
(%)
samples (IR) samples (RR) Interval

99.72% 99.43% –
Site # 1 2519 18 7
(2512/ 2519) 99.89%
99.72% 99.43% –
Site # 2 2513* 20 7
(2505 / 2512) 99.89%

38 14
99.72% 99.53% –
Total 5032 (8 positive, (3 positive,
(5017/5031) 99.85%
30 equivocal) 11 equivocal)

*: one sample (true positive) was withdrawn from the calculation.

The total specificity on the blood bank population is equal to

99.72% (5017/5031) with a 95% confidence interval of [99.53% –
99.85%]. Among the 14 false positive samples, 11 were found
repeated equivocal.

A retrospective study was also carried out on 201 frozen samples

from patients from a Sexually Transmitted Disease Center or from
vendors, which were found negative for syphilis.

Specificity on these samples is found at 99.5% (200/201) with a

95% confidence interval at [97.3%- 100.0%].

9.2.2. Sensitivity
The sensitivity study was studied on 435 retrospective frozen serum
samples from the laboratory of Sexually Transmitted Disease
Center, or from frozen samples from vendors. These samples were
characterized positive by immunoassays, FTA assay, RPR/ VDRL
assay or TPHA assays according to their origin.
All the samples were tested first with the CE mark TPHA assay and
then with the Bio-Rad TPHA 500 assay (72504).
Sensitivity on this population is found at 100% (435/435) with a 95%
confidence interval at [99.2%-100.0%].

9.3. Analytical sensitivity

Analytical sensitivity was tested against the WHO International
Standard for Syphilis NIBSC code 05/132. Using the semi-
quantitative protocol, the sensitivity was found at 0.05 IU/mL.

14 [EN]
9.4. Analytical Specificity / Cross Reactivity Study
210 potentially interfering samples containing antibodies against
pathogens that could lead to infectious illnesses (Cytomegalovirus,
Epstein Barr virus, VZV, Rubella virus, Hepatitis C virus, Hepatitis B
virus, HIV 1/2, HTLV 1/2, Toxoplasma gondii, Dengue, Malaria,
Leptospirosis), samples from pregnant women and multiparous
women or samples from patients with immune system disorders
(autoantibodies (SLE), Rheumatoid factors), were tested with the
TPHA assay. Four (4) true positive samples were discarded from the
calculation.
One sample was found repeatable positive with the TPHA test.
The specificity observed on this target population of 99.5 %
(205/206) was similar to the specificity of clinical samples.

9.5. Prozone Effect

The existence of a possible prozone effect was studied by testing 3
samples with high titers (≥ 1:20480) at different dilutions. The
equivalence of results observed among non-diluted and diluted
samples indicates the absence of the prozone effect.

10. BIBLIOGRAPHY REFERENCES

1. Daguet G.L. - Diagnostic Biologique de la Syphilis. Technique et
Biologie, 1995 ; 120 :5-30.
2. Houng H. - Syphilis: new diagnostic directions. Intern. J. STD
and AIDS 1992; 3 : 391- 413.8.
3. Larsen S.A., Hambie E.A., et coll., Specificity, sensitivity and
reproducibility among the fluorescent treponemal antibody
absorption test, the microhemagglutination assay for
Treponema pallidum antibodies, and the hemagglutination
treponemal test for syphilis. J. Clin. Microbiol., 1981 ; 14 : 441 –
445.
4. North MLet Guntz Ph. Serodiagnostic de la syphilis. La Revue
Française des Laboratoires, 1997 ; 294 : 51-58.
5. Paris Hamelin, A., Dreux P. et coll. – Treponematoses : aspects
cliniques et biologiques. Feuill. Biol. 1991a ; 23 : 88-89.
6. Rathlev T. - Haemagglutination tests utilizing antigens from
pathogenic and apathogenic Treponema pallidum
WHO/VDT/RES 1965 ; 77 : 65.
7. Rathlev T. - Haemagglutination test utilizing pathogenic
Treponema pallidum for the serodiagnosis of syphilis. Br J Vener
Dis 1967 ; 43 : 181-5.
8. Sequeira P,J,L. Eldridge A,E. - Treponemal Haemagglutination
test. Br J Vener Dis 1973 ; 49 : 242-8.

[EN] 15
9. Sluis J.J. Van Der. - Laboratory Techniques in the diagnosis of
syphilis: a review. Genitourin Med. 1992 ; 68 : 413-9.
10. Stability of selected serum proteins after long-term storage in
the Janus Serum Bank. Clin Chem Lab Med. 2009. 47:596-606.
11. Tomizawa T, Kasamatsu S. - Haemagglutination tests for
diagnosis of syphilis. A preliminary report. Japan. J. Med. Sci.
Biol. 19, 305-308, 1966.
12. Tomizawa T. Kasamatsu S. Yamaya S. - Usefulness of the
haemagglutination test using Treponema pallidum antigen
(TPHA) for the serodiagnosis of syphilis. Jap J Med Sci Biol
1969 ; 22 : 341-50.

16 [EN]
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Manejar con cuidado.
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31
Bio-Rad
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Tel.: +33 (0)1 47 95 60 00
Fax: +33 (0)1 47 41 91 33 2014/12
www.bio-rad.com 883683