Specimens

This study is conducted upon ten thousands of blood donors over one
year time period (January to December 2009) of both sexes (8720 males
and 1280 female) and age group ranging from 18 to 42 years at blood
bank unit of Sohag University Hospital.
Blood is collected from blood donors who are apparently normal and give
no history of risk factors. Samples were collected and examined within 24
hours.
Blood donors were carefully selected after complete history taking and
physical examination to assess eligibility and to ensure that the donor
would not be negatively affected, e.g. become anaemic. The donated units
of blood were screened and were discarded if positive for any test

Method
We compared data obtained by the Treponema Pallidum Particle
Agglutination (TPPA; PaGIA method, Diamed) to those obtained by:
1. Venereal Disease Research Laboratory test (VDRL Carbon
Antigen, Biotec).
2. Chemiluminescent microparticle immunoassay (CMIA; Abbott
Architect) as standard test.

1) TPPA (DiaMed AG, PaGIA)

New particle gel immunoassay (DiaMed AG, Cressier sur Morat,
Switzerland) with three recombinant Treponema pallidum antigens was
evaluated with serum samples from the blood donors. It proved to be a
simple, rapid (20 min).
The PaGIA consists of a microtube containing a gel matrix and red
polymer particles sensitized with recombinant antigens TpN15, TpN17,
and TpN47 in a ready-to-use suspension.
Principles
Red coloured polymer particles are sensitised with the recombinant
antigens TpN15, TpN17, TpN47. When these particles are mixed with
patients serum, the specific antibodies react with the syphilis antigens on
the bead surface, causing agglutination of the particles. This simple and
specific method gives a clear result in only 15 minutes
To obtain a spatial separation of agglutinated and non-agglutinated
particles, the reaction mixture is centrifuged through a gel filtration
matrix. Agglutinated particles are trapped on top of the gel or dispersed
within the gel (= positive reaction), while free non-agglutinated particles
form a pellet at the bottom of the microtube (= negative reaction). Due to
the red colour of these particles, the result can be read visually with ease.

Material required to perform the test

ID-Card PaGIA-Syphilis Antibody Test

ID-PaGIA Syphilis Antibody Test polymer particles
coated with T. pallidum recombinant antigens TpN15, TpN17 and TpN47, red
coloured, ready-to-use suspension.
DiaMed Syphilis positive + negative control

Instrument required
Incubator.
Pipettes or pipette tips (optional) to deliver the volumes specified.
Centrifuge for DiaMed Microplate Technique
Test procedure
ID-Cards which show signs of drying, have bubbles, damaged seals,
drops of gel or supernatant in the upper part of the microtubes or on the
underside of the aluminum foil are excluded.
Clearing samples by centrifugation at 1500 g for 10 minutes before use.
We allow samples and reagents to reach room temperature before use.
1. Identify the ID-Card with the patient name.
2. Remove the aluminium foil from as many microtubes as required
by holding the ID-Card in the upright position.
3. Pipette 10 L of sample into the funnel of the appropriate
microtube, such that it does not come into contact with the gel
supernatant. Discard used tips.
4. Vortex the ID-PaGIA Syphilis antibody test Particles tube for 5
seconds before use. Add 50 L of the particle suspension to the
sample in the microtube funnel taking care to not touch the sample
or wall of the microtube with the pipette tip.
5. Re-vortex the particles at least every 5 minutes to ensure that the
suspension remains homogenous.
6. Incubate the ID-Card for 5 minutes at room temperature.
7. Centrifuge the ID-Card for 10 minutes in the ID-Centrifuge.
8. Read and record the results.

Interpretation of the results:

1. Strong positive reaction (++) corresponds to a complete

agglutination, seen as a red line on top of the gel or just below the
surface of the gel (1), or by agglutinates distributed only within the
upper part of the gel (2).
2. Weak positive reaction (+) can be distinguished when some particles
reach the bottom of the microtube with agglutinates still visible in the
upper part of the gel (3), or throughout the gel (4).
A positive result indicates the presence of specific anti-T.P.antibodies.
3. A negative reaction (-) corresponds to a complete sedimentation of
the particles as a pellet at the bottom of the microtube, and no
agglutinated particles visible within the gel (5).
A negative result indicates the absence of detectable anti-T.
pallidum antibodies.
4. Doubtful reaction (?): fibrin residues in the sample may trap nonagglutinated particles, presenting a fine red line on the surface of the
gel while the rest of the particles are at the bottom of the microtube
and no agglutinates in-between (6). Repeat test after clearing the
sample by centrifugation at 1500 g for 10 minutes.
If the doubtful reaction persists, the result should not be considered
valid.

2) Venereal Disease Research Laboratory test (VDRL)

BIOTEC VDRL Carbon Antigen is used as a non-Treponemal test for the
qualitative and semi-quantitative detection of syphilis using serum
(heated or unheated) or plasma.
Principle of the Method
The VDRL test is non-treponemal in that the antibodies detected are
not specific for T.P., although their presence in patients serum or plasma
is strongly associated with infection by the organism. This test measures
antibody (IgG and IgM) produced in response to lipoidal material
released from damaged host cells as well as to lipoprotein-like material
released from the spirochaetes.
These antibodies tend to disappear after successful treatment of the
infection.
BIOTEC VDRL Carbon Antigen consists of modified VDRL antigen
containing microparticulate carbon, which aggregates in the presence of
reagin type antibodies in serum or plasma, indicating a positive result.
The aggregation can be read macroscopically.
Non-reactive samples typically appear as a smooth non-aggregated
pattern, which may form buttons in the centre of the test area.
Sample used should not be contaminated, haemolysed, turbid or lipaemic
Requirement
1. VDRL Carbon Antigen (0.1% sodium azide) in glass vials.
The reagent is ready to use.
It must be well shaken to ensure homogeneity.
Bring to room temperature before use.
2. Controls: Positive and negative.
3. Timer
4. 0.85% physiological saline (semi quantitative test only).
5. Test tubes for sample dilution (semi quantitative test only).
6. Test Slides (white background).
7. Pipettes or droppers for dispensing 15 and 50l.
8. Disposable stirrers.

Procedure:
Qualitative Test
1 Allow all reagents, controls and samples to reach room temperature
before use.
2 Draw the sample into a dispenser (50l) taking care not to transfer
any cellular elements.
3 Hold the dispenser over a tile / test card circle and allow the
specimen to fall onto the test card. It is important to hold the
dispenser in a vertical position whilst dispensing the sample.
4 Spread the specimen evenly over the entire test circle using stirrer.
5 Shake the vial of carbon antigen reagent to ensure even mixing.
6 Into another dispenser withdraw 15l of carbon antigen.
7 Keeping the dispenser in a vertical position allow the antigen to fall
onto the specimen. Do not mix.
8 Rotate the tile or card for 8 minutes.
9 Read and interpret results macroscopically in good light.
Semi Quantitative Test
1 Prepare doubling dilutions of the sample from the undiluted
specimen to 1:32 using physiological saline. Mix well.
2 Place one drop (50l) of each dilution onto a separate test card
circle.
3 Using a stirrer spread each dilution evenly over the test circle,
starting at the highest dilution (1:32), proceeding to the lowest
(1:2).
4 Continue as from 1.5 in the Qualitative Test.
After 8 minutes rotation, read the test and note the last circle in the
dilution series that has a positive result.
If the highest dilution tested (1:32) is reactive, proceed with a further
dilution series by preparing doubling dilutions of the sample from 1:32 to
1:512 using physiological saline.
Mix well and continue as from 2nd step in the semi quantitative Test.

INTERPRETATION
Qualitative Test
1 Positive result
Reactive (positive) samples display characteristic agglutination
ranging from slight (weak-reactive) to intense (reactive). A strong
positive reaction is seen as large aggregates in the centre of the test
circle. Weakly positive reactions are seen as small aggregates around
the edge of the test circle.
2 Negative result
Negative results show no aggregates. The carbon antigen either
remains in a smooth suspension or forms a distinct button.

Semi Quantitative Test

Results may be graded from strong to non-reactive and the titre
expressed as the reciprocal of the last dilution showing a positive
reaction.
1. Strong Reactive (SR): Large clumps of carbon particles with a
clear background.
2. Reactive (R): Large clumps of carbon particles, more dispersed
than strong reactive.
3. Weak Reactive (WR): Small clumps of carbon particles with
light grey background.
4. Trace Reactive (TR): Slight clumping of carbon particles,
typically seen as a button of aggregates in the centre of the test
circle or dispersed around the edge of the test circle.
5. Non-Reactive (NR): A smooth grey pattern or a button of
nonaggregated carbon particles in the centre of the test circle.
6. Reactive samples should be recorded as antibody positive and
must be subjected to further tests to determine the presence or
absence of specific anti-Treponemal antibody.

3) Chemiluminescent Microparticle Immunoassay:

The chemiluminescent microparticle immunoassay (Abbott Architect) is a
fully automated treponemal antibody test.
Principles of the procedure
It's a two-step immunoassay for the qualitative detection of antibody to
TP in human serum or plasma using CMIA technology with flexible assay
protocols, referred to as Chemiflex.
In the first step, sample, microparticles coated with recombinant TP
antigens (TpN15, TpN17 and TpN47) and Assay Diluent are combined.
Anti-TP antibodies present in the sample bind to the TP coated
microparticles. After washing, the acridinium-labeled anti-human IgG
and IgM conjugate is added in the second step. Following another wash
cycle, Pre-Trigger and Trigger Solutions are added to the reaction
mixture. The resulting chemiluminescent reaction is measured as relative
light units (RLUs). A direct relationship exists between the amount of
anti-TP antibodies in the sample and the RLUs detected by the
ARCHITECT i* optical system.
The presence or absence of anti-TP antibodies in the specimen is
determined by comparing the chemiluminescent signal in the reaction to
the cutoff signal determined from a previous Syphilis TP calibration. If
the chemiluminescent signal in the specimen is greater than or equal to
the cutoff signal, the specimen is considered reactive for anti-TP

System used:
ARCHITECT i2000SR

Material required
1. ARCHITECT Syphilis TP Reagent Kit (8D06)
Microparticles: TP (E.coli, recombinant) antigen coated
microparticles in MES buffer.
Conjugate: Murine anti-IgG/anti-IgM acridiniumlabeled
conjugate in MES buffer with protein (bovine) stabilizer.
Syphilis TP Assay Diluent containing MES buffer.
2. Other Reagents :
Pre-Trigger Solution: Pre-Trigger Solution containing 1.32%
(w/v) hydrogen peroxide.
Trigger Solution: Trigger Solution containing 0.35 N sodium
hydroxide.
Wash Buffer: Wash Buffer containing phosphate buffered
saline solution. Preservatives: antimicrobial agents.
3. Controls and Calibrators :
8D06-01 ARCHITECT Syphilis TP Calibrator
8D06-10 ARCHITECT Syphilis TP Controls
4. Other materials :
Reaction vessel.
Septum.
Sample cups.
Replacement cups.
Instrument required
Centrifuge.
Pipettes or pipette tips (optional) to deliver the volumes
specified.

PROCEDURE
1. Calibration
Single sample of each Control must be tested to evaluate the
assay calibration. Ensure that assay control values are within the
S/CO ranges specified in the control package insert. Calibrator
1 should be priority loaded. Once calibration is accepted and
stored, all subsequent samples may be tested

2. Quality control
Single sample of each control can be tested once every 24 hours
each day of use for each reagent lot.
Ensure that assay control values are within the acceptable
ranges specified in the control package insert. If a control is out
of its specified range, the associated test results are invalid and
must be retested. And recalibration may be indicated.
Before loading the reagent kit on the system for the first time,
the microparticle bottle requires mixing to resuspend
microparticles that have settled during shipment or storage:

3. Test procedure

Load samples then press RUN.

The ARCHITECT i System performs the following functions:

1. Moves the sample carrier to the sample processing queue.

2. Loads a reaction vessel (RV) into the process path.
3. Aspirates and transfers sample into the RV.

4. Advances the RV one position and transfers microparticles and

assay diluent into the RV.
5. Mixes, incubates and washes the reaction mixture.
6. Adds conjugate to the RV.
7. Mixes, incubates and washes the reaction mixture.
8. Adds Pre-Trigger and Trigger Solutions.
9. Measures chemiluminescent emission to detect the presence of
anti-TP antibodies in the sample.
10.Aspirates contents of RV to liquid waste and unloads RV to
solid waste.
11.Calculates the result.
Calculation
The ARCHITECT i System calculates the cutoff (CO) using the mean
chemiluminescent signal (RLU) from three replicates of the Calibrator 1
and stores the result.
Cutoff (CO) = Calibrator 1 Mean RLU x 0.20
The ARCHITECT Syphilis TP assay calculates a result based on a cutoff
determined by the following calculation.
S/CO = Sample RLU / Cutoff RLU
The cutoff RLU is stored for each reagent lot calibration.

Interpretation of Results
Specimens with S/CO values < 1.0 are considered nonreactive
Specimens with S/CO values > 1.0 are considered reactive.