BIO-FLASH ®
READ HIGHLIGHTED CHANGES
®
BIO-FLASH Syphilis
3000-8602
100 tests
The BIO-FLASH Syphilis is a fully automated chemiluminescent two-step immunoassay for qualitative
measurement of IgG and IgM antibodies to Treponema pallidum in human serum or plasma on the
BIO-FLASH instrument.
Summary
Syphilis is a disease caused by infection with the spirochete bacterium Treponema pallidum, ordinarily
transmitted by sexual contact. However, it can also be transmitted congenitally by transplacental passage of
T. pallidum to the foetus and by blood transfusion. Syphilis disease progresses in stages and may become a
chronic infection if untreated. Syphilis is characterized by episodes of active disease interrupted by periods of
latent infection. The incubation period is estimated to be between 10 and 90 days (three weeks in average).
Early clinical manifestations (primary and secondary stages) primarily involve the skin and mucosal surfaces,
although secondary syphilis is a systemic illness. The serological diagnosis of syphilis is performed
demonstrating the presence of significant levels of specific T. pallidum antibodies in the patient serum/plasma
sample. Syphilis has several clinical manifestations, making laboratory testing a very important aspect of
1
diagnosis.
Blood tests are divided into nontreponemal and treponemal tests. Nontreponemal tests include VDRL (Venereal
Disease Research Laboratory) and RPR (Rapid Plasma Reagin) tests. False positive results on nontreponemal
tests can occur with some viral infections such as varicella and measles, as well as with lymphoma, tuberculosis,
malaria, endocarditis, connective tissue disease, and pregnancy.
Treponemal tests, such as TPHA (Treponema pallidum Haemoagglutination) or FTA-Abs (Fluorescent Treponemal
Antibody Absorption test), ELISA and recently CLIA, are more specific. Treponemal antibody tests usually become
positive two to five weeks after the initial infection.
Besides immunodominant recombinant antigens p15, p17 and p47, BIO-FLASH Syphilis incorporates an additional
recombinant protein (United States Patent, US7700727B2) that enhances sensitivity.
Principle
When BIO-FLASH Syphilis paramagnetic microparticles are mixed and incubated with the sample, if antibodies
specific for T. pallidum are present in the sample they will bind to the specific T. pallidum antigens coated on the
microparticles. A magnetic separation followed by a wash step is done to remove residual sample. Immediately
after, a tracer consisting of isoluminol-labeled anti-human IgG and anti-IgM antibodies is added and may bind to
the antibodies specific for T. pallidum captured by the microparticles. After a second incubation, a magnetic
separation and a wash step, reagents that trigger the chemiluminescent reaction are added. The emitted light is
measured as relative light units (RLU) by the BIO-FLASH luminometer. The RLUs are directly proportional to the
concentration of antibodies specific for T. pallidum in the sample.
The BIO-FLASH utilizes a 4 Parameter Logistic Curve fit data reduction method (4PLC) to generate a Master
Calibration Curve (MCC). The MCC is predefined and lot dependent and it is stored in the instrument through the
cartridge barcode. With the measurement of calibrators (supplied in a separate kit), the predefined MCC is
transformed to a new, instrument specific Working Calibration Curve (WCC). The concentration values of the
calibrators are included in the calibrator tube barcodes.
Reaction Scheme:
BIOKIT, S.A. - Can Malé s/n - 08186 Lliçà d’Amunt - Barcelona - SPAIN
3000-8602_R00_05.2014_eng.docx
BIO-FLASH ®
Components
Reagent cartridge
The BIO-FLASH Syphilis kit contains a reagent cartridge for 100 determinations (REF 3000-8602).
NOTE: Cartridge design is protected under patent (US D565, 741 / EC Design 000762992-0001)
Reagent cartridge composition:
Cartridge has 4 different vials with the following contents:
A.
1 cylindrical vial of microparticle suspension coated
with purified recombinant T. pallidum antigens.
Contains < 0.1% Sodium azide.
B.
1 vial of assay buffer. Contains 0.15 % ProClin 950
and 0.214% Triton X-100.
C.
1 opaque vial of tracer consisting of mouse monoclonal
anti-human IgG and anti-human IgM labeled with
isoluminol. Contains < 0.1% Sodium azide.
D.
1 vial of sample diluent. Contains 0.15% ProClin 950.
Preparation
See the figure below.
Cartridge: Microparticles settle during shipment and storage and require mixing to resuspend.

The first time that the cartridge is used, gently invert the cartridge 30 times avoiding foam formation.
Bubbles may interfere with the instrument liquid sensors.

Check for the complete resuspension of the microparticles. If the microparticles are not totally resuspended
continue to invert the cartridge until the microparticles have been completely resuspended.

If the microparticles do not resuspend or it is detected that the reagent seal is broken, DO NOT USE THE
CARTRIDGE.

Once the microparticles have been resuspended place the cartridge on a solid surface and gently remove the
Red Secure Shipping Tab from the cartridge.

With the cartridge still on a solid surface, press the two tabs placed on the sides of the piercing cap (grey part)
and apply pressure to the top portion of the cartridge until it snaps in a lock position. Once in the locked
position the tabs should not be visible. Do not invert the open cartridge.

Let the cartridge stand for a period of 5 minutes before loading it on the instrument.

Once the cartridge is placed on the instrument additional periodic mixing is automatically performed on board.
BIOKIT, S.A. - Can Malé s/n - 08186 Lliçà d’Amunt - Barcelona - SPAIN
3000-8602_R00_05.2014_eng.docx
BIO-FLASH ®
Precautions
The BIO-FLASH reagents are intended for IN VITRO diagnostic use.
For professional use only.
Sodium azide may react with lead or copper pipes and plumbing creating highly explosive metal azides. Flush
drains with water thoroughly after disposing of the remains of reagents.
WARNING: POTENTIALLY BIOHAZARDOUS MATERIAL.
Dispose all used materials in a suitable biohazardous waste container.
Please do not reuse nor reintroduce any reagent in the cartridges or vials.
WARNING: Component B contains Proclin 950 and Triton X-100. Component D contains Proclin 950.
Hazard statements
H317: May cause an allergic skin reaction.
Precautionary statements
P280: Wear protective gloves/protective clothing/eye protection/face protection.
P302+P352: IF ON SKIN: Wash with plenty of soap and water.
P333+P313: If skin irritation or rash occurs: Get medical advice/attention.
P363: Wash contaminated clothing before reuse.
P501: Dispose of contents/container in accordance with local/regional/national/international regulations.
Sample collection and storage
Use fresh serum (including serum separator tube) or plasma (Li-Heparin, Li-Heparin separator tube, Na-Heparin,
Na-Citrate, EDTA, CPD, CPDA, K-Oxalate and ACD). Other anticoagulants should be evaluated before use. Liquid
anticoagulants as Na-Citrate have a dilution effect and may result in lower concentrations for individual patient
specimens. Samples should not be heat inactivated. Samples showing visible particles should be clarified by
centrifugation. Refer to the CLSI H18-A3 and H21-A5 guidelines for further information on handling, transport,
processing and storage of samples.
Serum
Serum samples can be stored at 2-8°C for 10 days. For longer periods, sera should be frozen at -20°C or colder.
Samples can be freeze/thawed up to 3 times. Mix thoroughly after thawing. Inspect all samples for bubbles and
remove all of them prior to analysis.
Plasma
Plasma samples can be stored at 2-8°C for 10 days. For longer periods, plasma samples should be frozen at -20°C
or colder. Thaw plasma at 37°C. Thaw samples only once. Mix thoroughly after thawing. Inspect all samples for
bubbles and remove all of them prior to analysis.
Sample volume
The sample volume required to perform a single BIO-FLASH Syphilis test varies depending on the type of sample
container used. A test requires at least 50 µL plus dead volume, which is 200 µL for the recommended sample cup
(REF 3000-8209).
Additional materials
The following materials are not supplied with the reagent cartridge and must be purchased separately.
REF 3000-8603
BIO-FLASH Syphilis Calibrators
REF 3000-8604
BIO-FLASH Syphilis Controls
Read carefully their corresponding inserts for more information.
Do not use other calibrators. The information required by the BIO-FLASH instrument to calibrate the BIO-FLASH
Syphilis assay is included in the barcoded vials.
Using controls from other manufacturers may lead to unexpected results.
Ensure that the BIO-FLASH instrument has enough quantity of the following consumables on board before running
samples, calibrators or controls:
REF 3000-8206
BIO-FLASH Cuvettes
Note: Cuvettes design is protected under patent (US D560, 816 / EC Design 000762984-0001)
REF 3000-8204
BIO-FLASH Triggers
REF 3000-8205
BIO-FLASH System Rinse
BIOKIT, S.A. - Can Malé s/n - 08186 Lliçà d’Amunt - Barcelona - SPAIN
3000-8602_R00_05.2014_eng.docx
BIO-FLASH ®
Instrument / test procedure
Refer to the BIO-FLASH Operator’s Manual for the complete assay procedure instructions.
Calibration
Each cartridge barcode contains the Master Calibration Curve (MCC) valid for the reagent lot; however a Working
Calibration Curve (WCC) is required for all tests. The WCC is lot specific and valid throughout the shelf life of the
lot. A new WCC is indicated when controls report outside the acceptable range or when adjustments are made to
the instrument.
Read carefully BIO-FLASH Operator’s Manual for configuring a WCC.
Quality control
Two controls are recommended for a complete quality control program. BIO-FLASH Syphilis Controls Negative
and Positive are designed for this program. Controls should be analyzed at least once every 24 hours each day of
use. Ensure that control results are within the acceptable ranges. A control result not falling within the acceptable
range may indicate invalid test results and corrective measures should be taken by the user. Examine all test
results generated since obtaining the last acceptable quality control check for this analyte. Recalibration may be
indicated. Refer to the instrument’s Operator’s Manual for additional information. Refer to Westgard et al.4 for
identification and resolution of out-of-control situations.
Storage and stability
Unopened reagents are stable until the expiration date shown on the cartridge label when stored at 2-8°C in the
upright position.
Once open the cartridge stability on board the BIO-FLASH instrument or stored at 2-8°C is 5 weeks.
Interpretation of results
The amount of analyte in every sample is determined from the emitted light (RLU) by interpolation in the stored
Working Calibration Curve. BIO-FLASH Syphilis results are reported in S/CO (Signal/Cut-Off). This is a qualitative
test; the numeric value of the result is only indicative of the amount of antibody present. The determination of
anti-Treponema pallidum antibodies, using the BIO-FLASH Syphilis assay, is interpreted as follows:


samples with a result < 1.00 S/CO are considered non reactive (negative).
samples with a result ≥ 1.00 S/CO are considered reactive (positive).
Samples with initially reactive result should be reanalyzed in duplicate. A repeatedly positive result is indicative of
Syphilis infection. Samples with anti-T. pallidum concentration exceeding 200.00 S/CO are reported as >200.00 S/CO.
Limitations
The result of a single sample is not sufficient for the diagnosis of Syphilis disease and therefore the
BIO-FLASH Syphilis results should be used in conjunction with other data; e.g., symptoms, clinical history, results
of other tests and other appropriate information.
A negative result does not exclude the possibility of exposure to or infection with T. pallidum. As in all sensitive
immunoassays, there is the possibility that non-repeatable positive results occur.
Expected results
Syphilis is believed to have infected 12 million people in 1999 with greater than 90% of cases in the developing
2
world. It affects between 700,000 and 1.6 million pregnancies a year resulting in spontaneous abortions, stillbirths,
3
and congenital syphilis. In Sub-Saharan Africa syphilis contributes to approximately 20% of perinatal deaths. In the
developed world, syphilis infections were in decline until the 1980s and 1990s due to widespread use of antibiotics.
Since the year 2000, rates of syphilis have been increasing in the US, UK, Australia and Europe primarily among
2
men who have sex with men. This is attributed to unsafe sexual practices.
2
Increased rates among heterosexuals have occurred in China and Russia since the 1990s. Syphilis increases the
2
risk of HIV transmission by two to five times and co-infection is common (30 - 60% in a number of urban centers).
4
The global prevalence was estimated in around 36.4 million people in 2008.
Performance characteristics
NOTE: The following data are representative; results in individual experiments may vary from these data.
BIOKIT, S.A. - Can Malé s/n - 08186 Lliçà d’Amunt - Barcelona - SPAIN
3000-8602_R00_05.2014_eng.docx
BIO-FLASH ®
Specificity
Specificity assessment was based upon testing a total of 3010 blood donor samples from Banc de Sang i Teixits de
Catalunya (Spain), including serum and EDTA-plasma. In addition, 300 unselected serum samples from routine of
a hospital laboratory in Barcelona (Spain), and 50 samples from pregnant females were also assessed. The
samples repeatedly reactive were retested using other commercial assays. The specificity results are shown in the
following table:
Specimens
tested
n
Positive
Negative
Specificity
n
n
% (95%CI)
Donors from Blood Bank
3010
0
3009*
100.0 (99.9 to 100.0)
Non-selected hospitalized patients
300
7**
293
100.0 (98.7 to 100.0)
Pregnant females (including multiparous)
50
0
50
100.0 (92.9 to 100.0)
*One sample was inconclusive in retesting with BIO-FLASH Syphilis and was not used in calculations
**The 7 samples reactive for BIO-FLASH Syphilis were found also reactive by other commercial assays
Sensitivity
Sensitivity was evaluated by testing specimens that are confirmed positive for antibodies to T. pallidum. The
specimens were obtained from Blood Bank and from Hospital de la Santa Creu i Sant Pau. The sensitivity results
are shown in the following table:
Specimens
tested
n
Confirmed Syphilis positive samples
200
Positive
Negative
Sensitivity
n
n
% (95%CI)
200
0
100.0 (98.2 to 100.0)
External evaluation
An external evaluation was performed at Hospital de la Santa Creu i Sant Pau (Spain) with a total of
500 prospective (non-selected) routine syphilis samples. Samples were characterized by ARCHITECT Syphilis TP
method and were tested with BIO-FLASH Syphilis.
ARCHITECT Syphilis TP
BIO-FLASH
Syphilis
NEG
POS
Total
NEG
POS
Total
431
0
431
3
65
68
434
65
499*
*One sample was inconclusive in retesting with ARCHITECT Syphilis TP
Inconclusive results were not used in calculations. The following results were obtained for relative sensitivity,
specificity and overall agreement:
N
499
Relative Sensitivity
Value
95% CI
95.6% 87.6% to 99.1%
Relative Specificity
Value
95% CI
100.0% 99.1% to 100.0%
Overall Agreement
Value
95% CI
99.4% 98.3% to 99.9%
ARCHITECT Syphilis TP and/or BIO-FLASH Syphilis reactive samples were tested with RPR and TPHA in
accordance to the routine tests algorithm criteria followed in the laboratory. The 3 samples that reported positive for
ARCHITECT Syphilis TP but negative for BIO-FLASH Syphilis were classified by the laboratory as inconclusive
results as RPR and TPHA reported negative results.
BIOKIT, S.A. - Can Malé s/n - 08186 Lliçà d’Amunt - Barcelona - SPAIN
3000-8602_R00_05.2014_eng.docx
BIO-FLASH ®
Syphilis algorithm
consensus
BIO-FLASH
Syphilis
NEG
POS
Total
NEG
POS
Total
431
0
431
0
65
65
431
65
496*
* 4 samples were inconclusive by the algorithm criteria
The following results were obtained for relative sensitivity, specificity and overall agreement:
N
496
Relative Sensitivity
Value
95% CI
100.0% 94.5% to 100.0%
Relative Specificity
Value
95% CI
100.0% 99.1% to 100.0%
Overall Agreement
Value
95% CI
100.0% 99.3% to 100.0%
Precision
Within run and total (run to run and day to day) precision were assessed (following CLSI EP05-A2 Guidelines) over
multiple runs. Results are summarized in the following table:
Mean (S/CO)
0.30
3.10
0.48
0.83
5.41
Negative Control
Positive Control
Negative Sample
Around Cut-Off Level Sample
Positive Sample
Within run
0.009 SD
1.7 % CV
0.018 SD
1.8 % CV
1.7 % CV
Total
0.011 SD
2.8 % CV
0.024 SD
2.6 % CV
2.6 % CV
Reproducibility
Repeatability between lots and between instruments was assessed using 50 samples. Results are shown in the
following table:
Slope
Intercept
R
n
Value
95% CI
Value
95% CI
Value
95% CI
Lot 2 vs. Lot 1
50
1.02
1.00 to 1.04
-0.01
-0.06 to 0.04
0.985
0.974 to 0.991
Instrument 2 vs. Instrument 1
50
0.95
0.93 to 0.97
-0.01
-0.08 to 0.03
0.997
0.994 to 0.998
Interferences
As assessed by the CLSI EP7-A Guidelines, BIO-FLASH Syphilis results are not affected by the following
potentially interfering substances:
Potentially Interfering substance
Haemoglobin
Free bilirubin
Complexed bilirubin
Triglycerides
Rheumatoid factor
Concentration
500 mg/dL
18 mg/dL
18 mg/dL
1300 mg/dL
800 UI/mL
% Interference
≤ 10
≤ 10
≤ 10
≤ 10
≤ 10
Cross-reactivity
A total of 104 positive specimens for a range of potential cross-reactants were analyzed with BIO-FLASH Syphilis.
It is considered that a potential cross-reactant causes cross-reactivity when the diagnostic changes from negative
to positive. The reactive samples were analyzed with commercial assays in order to confirm positivity. Results are
summarized in the following table:
BIOKIT, S.A. - Can Malé s/n - 08186 Lliçà d’Amunt - Barcelona - SPAIN
3000-8602_R00_05.2014_eng.docx
BIO-FLASH ®
Potential Cross-Reaction
SLE (Systemic Lupus Erythematosus)
ANA (Anti-Nuclear antibodies)
RF (Rheumatoid factor)
Drug Users
Heterophile antibodies
Pregnant women (including multiparous)
Flu vaccinated
Paraprotein G and M
anti-EBV (Epstein-Barr virus)
anti-HSV-1 & HSV-2 (Herpes Simplex virus 1 & 2)
anti-VZV (Varicela Zoster virus)
anti-Mumps
anti-Measles
anti-Rubella
anti-Toxo (Toxoplasma gondii)
anti-CMV (Human cytomegalovirus)
anti-HAV (Hepatitis A virus)
HBsAg (Hepatitis B surface antigen)
anti-HCV (Hepatitis C virus)
anti-HEV (Hepatitis E virus)
anti-HIV (Human immunodeficiency virus)
anti-HTLV (Human T-lymphotropic virus)
anti-PV B19 (Parvovirus B19)
Schistosomiasis
E. coli (organism in which vectors were induced)
West Nile fever virus
Chlamydia trachomatis
Neisseria gonorrhoeae
Candida albicans
Lyme disease
Chagas disease
Agreement
3/3
3/3
3/3
3/3
3/3
6/6
3/3
6/6
3/3
6/6
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
5/5
3/3
3/3
3/3
3/3
3/3
3/3
Analytical Sensitivity
Analytical Sensitivity obtained was <0.02 IU/mL. It was determined by interpolating the assay cut-off in the
regression line obtained by testing serial dilutions of the 1st WHO International Standard for human syphilitic
plasma IgG and IgM (NIBSC code: 05/132).
Commercial Panels
The following commercial panels were analyzed with BIO-FLASH Syphilis: Zeptometrix Mixed Titer Syphilis Panel
(K-ZMC002), SeraCare-BBI Diagnostics Syphilis Mixed Titer Performance Panel (PSS202(M)) and SeraCare-BBI
Diagnostics Syphilis Seroconversion Panel (PSS901-1.2). For the two mixed titer panels BIO-FLASH Syphilis
showed 100% agreement with the majority of the methods stated in their respective instructions for use. For the
seroconversion panel, the results obtained were equivalent to other commercial CLIA immunoassays.
BIOKIT, S.A. - Can Malé s/n - 08186 Lliçà d’Amunt - Barcelona - SPAIN
3000-8602_R00_05.2014_eng.docx