SYPHILIS TPHA

Qualitative and semiquantitative determination, by passive hemagglutination, of anti-Treponema pallidum antibodies

TEST SUMMARY In case of positive result repeat the analysis using • If the results are incompatibles with clinical
Anti-Treponema Pallidum antibodies, contained in the even the control cells as described below. presentation, they have to be evaluated within a total
serum or plasma, produce agglutination of erythrocytes clinical study.
coated with antigens of Treponema Pallidum. Reagents Well 1 Well 2 Well 3 • For in vitro diagnostic use only.
SAMPLES Diluent 40 µl 25 µl 25 µl CALIBRATION
Serum or plasma citrate specimens should be free of Positive and Negative control sera should be always used
blood cells. Stability 7 day at 2-8°C. Specimens needing Samples 10 µl 25 µl 25 µl to distinguish an eventual background’s agglutination of
longer storage should be frozen at -20°C. Frozen from well 1 from well 2 reactive.
specimens should be thawed and well mixed before
(reject 25 µl) TEST PERFORMANCE
testing.
Precision
REAGENTS Test cells -- -- 75 µl
n = 10 assays of a positive sample
Test cells CV = 8.1%
Chicken’s tannate erythrocytes and coated with total Control cells -- 75 µl -- Accuracy = -2.5%
extractive antigen of Treponema Pallidum cultivated in Specificity
rabbit’s testicles; conservative and stabilizer. Shake sweetly the microplate to obtain a mix of reagents Two independent studies on 2900 donor sera have
into the wells. Incubation sheltered from the vibrations for shown 100 % consensus with existing test methods.
Control cells 60 minutes.
Preserved chicken erythrocytes; conservative and An independent study on 200 antenatal sera has shown
stabilizer. RESULTS INTERPRETATION 100 % specificity.

Diluent Positive (strong): Full cell pattern covering the bottom of Sensibility
Saline solution; conservative and stabilizer. the well. A study on 110 characterised positive samples gave
Positive (weak): Cell pattern covers approx. 1/3 of the 100% positive results. This included 2 samples tested
Positive control well bottom. negative by other commercial TPHA tests but confirmed
Human base stabilized solution of anti-Treponema Negative: Cells settled to a compact button, typically with FTA positive and IgM EIA positive.
Pallidum antibodies with a titre that gives a clear a small clear centre. Interferences
agglutination. Indeterminate: Cells pattern shows a distinctly open False positivity has been verified in case of Leprosy,
Negative control centre. Infectious Mononucleosis and connective’s disease.
Proteicl solution not reactive with suspension. Non specific: Positive reaction.
WASTE DISPOSAL
REAGENTS PREPARATION AND STORAGE Intermediate results, for ex. a ring of hemagglutination
Product is intended for professional laboratories. Waste
with bottom in centre, indicate uncertain results. The well
Reagents are ready to be use. products must be handled as per relevant security cards
2 (control well) has to give negative result to indicate
Erythrocytes suspension must be resuspended with much and local regulations.
sample fitness to be tested, in fact eventual agglutinates
care. Make the suspension homogeneous by sweet
depend on a-specific agglutinines that invalidate the test, PACKAGEING
inversion.
therefore they must be removed by incubation of 25 µl of CODE AK00601 (100 TESTS)
Stability: The reagents are stable until expiration date on
the label to 4°C. sample with 500 µl of erythrocytes control for 30 minutes Test cells 1 x 8 ml
Do not freeze. and following centrifugation. Control cells 1 x 5 ml
Use the supernatant during the test. Diluent 1 x 25 ml
MATERIAL REQUIRED BUT NOT SUPPLIED Positive control 1 x 0.5 ml
SEMIQUANTITATIVE PROCEDURE
Normal equipment of laboratory. Negative control 1 x 0.5 ml
Microplate with U bottom, micropipette, centrifuge, test- Follow the scheme of qualitative procedure continuing
dilutions until 10° well. CODE AK00600 (200 TESTS)
tube for centrifuge.
Test cells 1 x 16 ml
PRECAUTION Reagents Well 1 Well 2 Well 3 … Well 10 Control cells 1 x 10 ml
All reagents contain 0.095% of sodium azide. Diluent 1 x 25 ml
Reagent may contain some non-reactive and preservative Diluent 40 µl 25 µl 25 µl … 25 µl Positive control 1 x 0.5 ml
components. It is suggested to handle carefully it, Negative control 1 x 0.5 ml
Sample 10 µl 25 µl 25 µl … 25 µl
avoiding contact with skin and swallow. REFERENCES
from from from well 9
Perform the test according to the general “Good well 1 well 2 Tomizawa T. e coll. – Jap. J. Sci. Biol. 19; 305 (1966).
Laboratory Practice” (GLP) guidelines. (reject 25 µl) Sequiera P.J.L. e coll. – Brit. J. Vener. Dis. 49; 242
QUALITATIVE PROCEDURE (1972).
Predispose dilutions of serum, into a microplate with “U” Erythro- -- -- 75 µl … 75 µl MANUFACTURER
bottom, following scheme below, pipetting diluent and cytes test
serum. LTA s.r.l.
Erythro- -- 75 µl -- … --
Using the same pipette (inspiring and discharging many cytes Via Milano 15/F
times) mix with care contents of well before to transfer control 20060 Bussero (Milan) ITALY
into the following well. Discharge 25 µl from last well of Tel: ++39 02 95409034
every series. Execute test using positive and negative Titre … … 1 : 80 … 1 : 10240 Fax: ++39 02 95334185
control instead of sample. e-mail: info@ltaonline.it
The TPHA kit is intended for screening large numbers of Website: http://www.ltaonline.it
specimens and contains only a small volume of Control Final specimen dilution range after addition of cells is 1:80 –
1:10240. SYMBOLS
Cells. It is intended that specimens are screened using
only Test Cells in the first instance, and the Control Cells
be used when repeating tests on specimens giving a Shake sweetly the microplate to obtain a mix of reagents F Only for IVD use
positive result when first tested. into the wells. Incubation sheltered from the vibrations for
60 minutes. C Lot of manufacturing
Reagents Well 1 Well 2 Well 3
RESULTS INTERPRETATION B Code number
According to illustrated modalities, value the last well that
Diluent 40 µl 25 µl 25 µl
shows a clear agglutination. I Storage temperature interval
Samples 10 µl 25 µl
from well 1
25 µl
from well 2
For the results to be valid the negative control must give a K Expiration date
negative result and the positive control must give a titre of
640-2560. J Warning, read enclosed documents
(reject 25 µl)
NOTE L Read the directions
Test cells -- -- 75 µl • Test gives positive results in case of treated syphilis
too. A Biological risk
Shake sweetly the microplate to obtain a mix of reagents • Human sera used in controls have been found Mod. 01.06 (ver. 3.8 - 15/10/2010)
into the wells. Incubation sheltered from the vibrations for negative in the reaction with HIV and HbsAg.

H
60 minutes. However, they should be handled with care.