DIMDI Reg.-Nr.: DE/CA22/1116-135-IVD
Rapid immunochromatographic test for qualitative detection of tetanus antitoxin antibodies in whole blood, serum or plasma samples
a) General information
Tetanus is a serious disease, leading to prolonged hospitalisation with sequelae, and that may lead to death (20% of the cases). This infectious disease is due to the exotoxin produced by Clostridium tetani. This ubiquitous Gram-positive anaerobic bacillus is occasionally present in animal digestive tract, and persists in animal droppings and in the ground in a very resisting sporulated form. In case of wound, it penetrates within the body. Then, the tetanus toxin blocks neurotransmitter release, and leads to muscular contractures and spasms.
Since the disease confers no immunity, vaccination is the only prevention. In France, there still are about twenty cases of tetanus per year; immunisation coverage is insufficient in adults.
Immunoprotection is proven by measuring anti-tetanus antibody concentration. As part of emergency medicine, knowing the real-time immune status in subjects at risk (deep injuries) enhances better management with appropriate anti-tetanus prophylaxis.
Keul-o-test Tetanus test is a rapid immunoassay for qualitative detection of tetanus antitoxin antibodies in serum, plasma, or whole blood samples.
The assay is intended for helping anti-tetanus prophylaxis for patients at risk of tetanus.
The assay uses an immunochromatographic technique, which uses an association of tetanus anatoxin bound on the membrane (test area (T)), anti-tetanus antibodies possibly present, and tetanus anatoxin bound to colloidal gold particles. During migration of a positive sample, anti-tetanus antibodies are firstly caught by the anatoxin bound to the particles. Under the influence of the diluent, this complex migrates along the membrane, then is secondly immobilised by the anatoxin bound to the test area (T), producing a pink test line. If there is an internal control line in the control area (C) indicates the test correctly works.
II. Keul-o-test Tetanus KIT COMPONENTS
Each kit contains everything needed to perform the tests.
III. STORAGE AND STABILITY
- All Keul-o-test Tetanus kit components should be stored between +4°C and +30°C within its original packaging.
- Do not freeze the kit.
- Keul-o-test Tetanus is stable until the expiry date mentioned on the packaging label.
- For "in vitro" diagnostic use and professional use only.
- Read carefully instructions before performing the test.
- Do not use after the expiry date indicated on the packaging.
- Do not use a test from a damaged protective wrapper.
- Handle all the samples as if they contained infectious agents. When the assay is finished, carefully discard the samples after autoclaving them for at least one hour. It is also possible to treat them with a 0.5% to 1% sodium hypochlorite solution for one hour before disposal.
- Wear protective clothing such as laboratory coats and disposable gloves while assaying samples.
- Do not eat, drink or smoke in the area where specimens and kit reagents are handled.
- Avoid any contact between hands and eyes or nose during specimen collection and testing.
V. SPECIMEN COLLECTION
may be performed with serum, plasma, or whole blood samples.
The samples should be collected under standard laboratory conditions (aseptically to avoid any haemolysis).
It is recommended to immediately analyse the blood samples (< 4 hours).
If the test is performed within 48 hours after collection, the sample should be stored in a fridge (+2°C to +8°C). After 48 hours, it should be frozen. The frozen samples should be wholely thawed, carefully mixed, and brought to room temperature before assaying. Avoid repetitive freezing and thawing.
If the serum sample is cloudy, highly viscous, or contains particles, it should be diluted before the test within a similar volume (V/V) of dilution buffer (not provided, but available on request).
VI- TEST PROCEDURE
- Bring samples and Keul-o-test Tetanus components to room temperature prior to testing.
- Remove the reaction device from its protective wrapper by tearing along the split.
- Label the device by indicating the patient's name or control number.
- Collect the sample (serum or plasma) with the pipette and, by holding it vertically, dispense one drop of sample (25 µL) in the well (w). If whole blood is used, dispense two drops (50 µL) in the well (w).
- Add exactly 3 or 4 drops of diluent (150 µL) in the well (w).
- Read the results after 20 minutes.
DO NOT READ AFTER 30 MINUTES.
VII- TEST RESULT READING
Only one coloured line appears in the control area (C).
The sample is considered as negative. The antibody concentration is lower than 0.1 IU/mL.
Besides the control line, a clearly visible line appears in the test area (T). The sample is considered as positive. The antibody concentration is higher than 0.1 IU/mL.
If no clear coloured line appears in the control area (C), the result is not valid. In this case, it is recommended to perform a new test.
VII- PERFORMANCES CHARACTERISTICS
1) Detection threshold
The Keul-o-test Tetanus detection threshold study was performed on both whole blood and serum, whose tetanus antitoxin IgG titrations were determined through ELISA (tetanus antitoxin IgG ELISA reagent Virotech). The detection threshold was also calibrated against the international standard (NIBSC 76/589).
Thanks to this study, the detection threshold of the test was defined to 0.1 IU/mL on serum and whole blood.
2) Sensitivity and specificity
A clinical study was conducted in an emergency unit on 159 patients suffering from cutaneous-mucous wounds. This study enabled to obtain 89% sensitivity on whole blood, and 94% sensitivity on serum. The obtained specificity is equal to 100%.
3) Internal validation
The test internal validation was performed on 129 samples (74% of them were positive): 109 serum samples and 20 whole blood samples. The obtained values, compared with the reference test (Virotech) indicate 91% sensitivity, and 97% specificity.
The intra-assay precision study was performed on 3 replicates of 3 serum samples with 0 IU/mL concentration; 0.17 IU/mL, and > 6 IU/mL. The values were correctly identified in 100% of the cases.
The inter-assay precision study was performed from 10 serum samples: 4 negative samples, 4 limit serum samples (0.10; 0.12; 0.15; 0.17 IU/mL), and 2 positive serum samples (1.6, and >6 IU/mL). The samples were analysed under the same conditions 3 times in a row on 3 different lots.
The negative and positive values were correctly identified in 100% of the cases.
IX- TEST LIMITATIONS
- Keul-o-test Tetanus test is intended to qualitative determination of tetanus antitoxin IgG antibody concentration in serum, plasma or whole blood.
- The absence of line in the test area after 20 minutes may indicate a residual and non-protective tetanus antitoxin antibody concentration.
- The anti-tetanus prophylaxis selection should also be based on clinical criteria (wound depth and soiling).
- As for any diagnostic method, the doctor should assess the test result by taking into account other clinical data.
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