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Tpha syphilis assay haemagglutination test - Newmarket BiomedicalTpha syphilis assay haemagglutination test - Newmarket Biomedical
Instructions for Almedica models including: NB007, NB008, NB009, NB007 New Bio Tpha Syphilis Test, NB007, New Bio Tpha Syphilis Test, Tpha Syphilis Test, Syphilis Test
NewBio TPHA syphilis test – Almedica
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DocumentDocumentNewBio TPHA NB007 REF NB008 NB009 2797 1. Intended Use Intended for the qualitative detection of Treponema pallidum IgG and IgM antibodies to syphilis in human serum, EDTA plasma or CSF and to determine the titre level of the samples. The intended use population is patients with a suspected syphilis infection or at elevated risk of syphilis infection who attend STI clinics or other healthcare settings. This assay is not intended for automated use. This assay is not intended for blood screening or as a confirmatory assay on donor samples. 2. Principle of assay Syphilis 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 infection is a chronic condition that typically progresses through distinct primary, secondary, tertiary, and quaternary stages of infection. 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 organism cannot be routinely cultured in artificial media, and diagnosis of the infection usually depends on the demonstration of antibodies in the blood, which appear soon after initial infection. NewBio TPHA uses preserved avian erythrocytes coated with extracted antigens of T.pallidum (Nichols strain). Specific antibodies present in a sample of plasma or serum bind to these antigens when the sample is incubated with the erythrocytes. This causes the erythrocytes to agglutinate, then settle to form a characteristic pattern in the test well. Non-specific reactions are eliminated by the use of absorbents. 3. Components Name Description 100 tests NB007 200 tests NB008 1000 tests NB009 Test Cells Avian erythrocytes coated with antigens of T. pallidum 8.5 mL 2 x 8.5 mL 2 x 40 mL Control Cells Avian erythrocytes 8.5 mL 2 x 8.5 mL 1 x 40 mL Sample Saline solution Diluent containing absorbents 20 mL 2 x 20mL 4 x 50mL Positive Rabbit antiserum Control Titre 1/1280 1.5 mL 1.5 mL 1.5 mL Negative Normal Rabbit Serum Control Instructions for use. 1.5 mL 1.5 mL 1.5 mL 4. Additional required materials Micro-pipettes capable of delivering: 10, 25, 75 and 190L 96-well U well micro-plates 5. Reagent preparation Bring all reagents and samples to room temperature before use. Kit controls must be run with each assay. Ensure Test and Control Cells are thoroughly re-suspended. Newmarket Biomedical Ltd. Unit 1, Lanwades Business Park, Kentford, Suffolk CB8 7PN UK 19005 v2.2 EN 2024/04 Page 1 of 8 6. Storage and shelf life after first opening 1. Test cells and Control cells must be stored in an upright position at 28°C. Do not freeze. 2. After opening, Test cells, Control cells, Sample Diluent and Controls are stable for up to 3 months when stored upright at 28°C. 3. Do not use after the expiration date. 7. Warnings and precautions 1. NewBio TPHA is for in vitro diagnostic use only. For professional use only. 2. Test cells, Control cells, Sample Diluent and Controls contain sodium azide (< 0.1% w/v) as a preservative, which can accumulate in lead or copper pipes to form potentially explosive azides. To prevent azide buildup, flush with large volumes of water after disposing of solutions containing azide into the drains. 3. Reagents and Controls contain material of animal origin. Any bovine albumin used in the manufacture of this product is sourced from donor animals that have been inspected and certified by Veterinary Service inspectors to be disease free. 4. Do not freeze Test cells, Control cells, Sample Diluent and Controls. 5. Test cells and Control cells must be thoroughly re-suspended prior to use. Failure to do so could result in an inadequate dilution and erroneous results. 6. Test cell and Control cell erythrocytes should be covered by suspension medium during storage, where this has not been the case then erythrocytes should be re-suspended. Failure to do so could result in clumping in the test well. 7. Test cells, Control cells and Sample Diluent from the same lot may be pooled using good laboratory practices. 8. Reagents showing visible signs of microbial growth or gross turbidity may indicate degradation and should be discarded according to local rules. 9. The effects of microbial contamination in specimens cannot be predicted. 10. Do not use Test cells, Control cells, Sample Diluent, or Controls after the expiration date. 11. Do not interchange caps between the Positive and Negative Control vials. Controls are differentiated by colour coded caps and the vial label. If caps are inadvertently switched, the Control tubes should be discarded. 12. Samples exhibiting gross lipemia, haemolysis or icterus may be compromised and may require alternative testing. 13. Deviations from the NewBio TPHA Instructions for Use can lead to erroneous results. 14. Dispose of leftover reagents in a safe manner, in accordance with local regulations. 8. Sample collection, handling, and storage NewBio TPHA may be used for testing with either human serum or EDTA plasma specimens for up to 7 days after collection. Specimens should be free of particulate matter to prevent interference with the assay result. If erythrocytes or other visible components are present in the specimen, remove by centrifugation to prevent interference with the test results. Store EDTA plasma and serum specimens at 2-8°C for up to 7 days. EDTA plasma and serum specimens can be frozen at less than -20°C for up to one month, thawed and mixed thoroughly prior to testing. Specimens may be frozen and thawed up to 5 times. NewBio TPHA may be used for testing with CSF for up to 5 days after collection. Store CSF samples at 2-8°C for up to 5 days. For longer periods store at less than -20°C. Allow all specimens to equilibrate to room temperature before use. 9. Assay procedure Serum and plasma testing Each sample requires 3 wells plus 2 additional wells for Positive and Negative Controls. Note: For NewBio TPHA 1000 only run Control Cells on retest. Newmarket Biomedical Ltd. Unit 1, Lanwades Business Park, Kentford, Suffolk CB8 7PN UK 19005 v2.2 EN 2024/04 Page 2 of 8 1. Sample Dilution (to 1 in 20) Add 190L of sample diluent to the first well. Add 10L of sample to the same well. Mix thoroughly. Note: Kit controls are pre-diluted (i.e., diluted 1 in 20) 2. Test Add 25L of Positive Control and Negative Control to designated test wells. Transfer 25L of diluted sample from step 1 to a test well. Transfer 25L of diluted sample from step 1 to a control well. Re-suspend the Test and Control Cells thoroughly. Add 75L of Test Cells to Positive Control and Negative Control wells. For diluted samples add 75L of Test Cells to test wells, and 75L Control Cells to control wells. (Final sample or Control dilution is 1 in 80) Mix wells thoroughly. Incubate at 15-30°C on a vibration-free surface for 45 - 60 minutes. Read the agglutination patterns. Patterns are stable if undisturbed. Sample titration assay procedure (optional) 9 wells are needed for each sample from 1 in 80 to 1 in 10240 dilution. 2 additional wells for Positive and Negative Controls (if run at 1 in 80 only) 1 additional well is needed if Controls Cells are run 1. Sample Dilution (to 1 in 20) Add 190L of sample diluent to the first well. Add 10L of sample to the same well. Mix thoroughly. Note: Kit controls are pre-diluted (i.e. diluted 1 in 20) 2. Titration Leave the second and third wells empty, add 25L of diluent to well 4 to well 10 in the sequence. Transfer 25L from step 1 to the second and third wells. Transfer 25L from step 1 to the fourth well and mix, then serially dilute along the well sequence, discard the excess 25L from the final well. Note: Care must be taken to avoid carryover of sample between serial dilution steps Kit Positive Control can be titrated if required 3. Test Re-suspend the Test Cells and Control Cells thoroughly Add 75L of Control Cells to well 2 Add 75L of Test Cells to wells 3 to 10. (Final sample dilution for Test Cells is 1 in 80 1 in 10,240) Mix wells thoroughly. Incubate at 15-30°C on a vibration-free surface for 45 - 60 minutes. Read the agglutination patterns. Patterns are stable if undisturbed. The titre of the sample is the reciprocal of the final positive sample dilution. CSF testing 1 Dilute CSF sample 1 in 5 with TPHA sample diluent. 2 Place 25µL of diluted sample in to 2 `U' wells. 3 Add 75 µL of Test Cells to well 1 4 Add 75 µL of Control Cells to well 2 5 Incubate on a vibration free surface for 1 hour at room temperature. 6 Interpret result according to pack insert. CSF Titration 1. Add 25µl CSF to 100 µL TPHA Diluent (1 in 5 dilution) 2. Prepare a row of 9 wells, leaving the first 2 empty, and adding 25 µL TPHA Diluent to each remaining 7 wells. 3. Add 25µL diluted CSF to each of the first 3 wells of the prepared row 4. Mix the contents of well 3 and transfer 25 µL to well 4, mix and repeat for the remainder of the series, and discard 25µL from the last well. 5. Add 75µL TPHA Control cells to the 1st (control) well. 6. Add 75µL TPHA Test cells to the remaining wells. The dilution series in wells 2-9 is now 1 in 20 to 1 in 2560. 7. Mix, incubate and interpret as above Newmarket Biomedical Ltd. Unit 1, Lanwades Business Park, Kentford, Suffolk CB8 7PN UK 19005 v2.2 EN 2024/04 Page 3 of 8 10. Control procedure The Positive and Negative Controls must be run with each assay. If required, the Kit Positive can be titrated, and the expected end point is 1/640 1/2560. Additional QC testing may be performed by the operator by the inclusion of other characterised specimens or reference material. The Positive Control should produce a positive result and the Negative Control should produce a negative result with the test. If the appropriate results are not obtained with the controls, the assay is considered invalid and all samples within that assay should be retested. TPHA Controls are pre-diluted. They should be added directly to the reaction well without being diluted in TPHA Sample Diluent. Test Cells are added directly to the Controls. 11. Interpretation of results A sample where the Test Cell well is non-reactive should be considered as negative for T.pallidum antibodies. Reactivity less than equivocal is considered negative. A sample where the Test Cell well is reactive or equivocal indicates antibodies to T.pallidum resulting from a syphilis infection. The sample should be repeated in duplicate. Where either repeat duplicate result is reactive or equivocal the sample should be considered as positive for T.pallidum antibodies. Where both duplicate repeat results are non-reactive then the samples are determined as non-reactive. Where a sample is reactive in both Test and Control Cells, if the agglutination is greater in the Test Cells, then the sample is considered positive and should be repeated as above. When running the sample titration procedure, a titre of 1/80 is considered reactive and the sample should be repeated in duplicate. Reactive results may indicate active, past, or successfully treated syphilis infections. Examples of result interpretation are shown in the figure below. Positive Positive Positive Equivocal Negative Negative Positive Positive Positive Positive Equivocal Negative Positive Positive Positive Positive Negative Negative Positive Positive Positive Equivocal Negative Negative Newmarket Biomedical Ltd. Unit 1, Lanwades Business Park, Kentford, Suffolk CB8 7PN UK 19005 v2.2 EN 2024/04 Page 4 of 8 Test cells + (strong) + (equal to CC) + (weak) + - Control cells + (weak) + (equal to TC) + (strong) + Repeat Y Y Y Y N Y Absorption N Y Y N N N Interpretation TP positive TP positive TP positive TP positive TP negative TP negative Absorption of Non-specific Reactions (only to be performed where a sample has greater or equal agglutination in the Control cells than the Test Cells) 1. Add 10L of sample to 190L of re-suspended Control Cells, mix thoroughly and leave for 30 minutes. 2. Centrifuge to deposit the cells at a minimum of 1500g for 3 minutes. 3. Add 25L of supernatant from step 2 to each of 2 wells. 4. Ensure Test and Control Cells are re-suspended. · Add 75L of Test Cells to the first well. · Add 75L of Control Cells to the second well. 5. Mix wells thoroughly and incubate at 15-30°C on a vibration-free surface for 45 - 60 minutes 6.Read and interpret patterns as above. During absorption of Non-Specific reactions, the supernatant is added directly to the reaction well without dilution in Sample Diluent. Performing this step incorrectly may result in false negative results. 12. Performance characteristics Limit of detection NewBio TPHA has an expected limit of detection of 0.1 IU/mL against the WHO 1st IS for human syphilitic plasma IgG NIBSC code:05/122. Reproducibility Assay reproducibility in serum and plasma was assessed using a characterised, mixed titre panel comprising 25 syphilis positive and 5 syphilis negative samples. The panel was tested using multiple lots of NewBio TPHA on 5 testing days over a 7-day period, in duplicate, with two separate runs on each testing day. Reproducibility Study rate of agreement Samples Agreement N= Total N= Rate of Agreement 95% CI Syphilis positive 250 250 100.00% 98.54 100% Syphilis negative 50 50 100.00% 92.89 100% Overall 300 300 100.00% 98.78 100% Cross reactivity and interference 140 syphilis negative serum and plasma samples containing antibodies to infectious diseases (Rubella, Toxoplasma, Borrelia, EBV, HCV, HBV, HAV, HIV, HTLV, Herpes, Chlamydia), ANA antibodies, Rheumatoid Factor antibodies and samples from pregnant (multiparous) subjects were tested in NewBio TPHA. All samples gave the expected negative result. 151 syphilis positive serum and plasma samples containing these antibodies and samples from pregnant (multiparous) subjects were tested in NewBio TPHA. All samples gave the expected positive result. Prozone Prozone effects may be seen at very high antibody levels for haemagglutination assays. In studies for NewBio TPHA with serum and plasma, no negative results were obtained at high levels of TP antibodies up to 100 IU/mL. Diagnostic sensitivity A panel of 205 commercially sourced, well characterised TP positive samples (157 serum and 48 EDTA plasma) were tested using the NewBio TPHA in comparison with NewBio PK TPHA 2000. The true clinical status for the commercially obtained syphilis positive samples was presumed to be that defined by the vendor assay results. Initial testing for NewBio TPHA v PK TPHA 2000 Sample Agreement Agreement measure N= Serum PPA 157 EDTA plasma PPA 48 Combined PPA 205 Total N= 157 48 205 ROA 100.0% 100.0% 100.0% 95% CI 97.68-100.0% 92.60-100.0% 98.22-100.0% Newmarket Biomedical Ltd. Unit 1, Lanwades Business Park, Kentford, Suffolk CB8 7PN UK 19005 v2.2 EN 2024/04 Page 5 of 8 Statistical summary against clinical status Sample Agreement Agreement measure N= All samples Sensitivity 205 Total N= 205 ROA 100.0% 95% CI 98.22-100.0% Diagnostic specificity A panel of 1248 known TP negative EDTA plasma samples were tested using the NewBio TPHA in comparison with NewBio PK TPHA 2000. Initial reactive samples were retested in duplicate with the relevant method. Initial testing for NewBio TPHA v PK TPHA 2000 Sample Agreement Agreement measure N= EDTA plasma NPA 1236 Total N= 1238 ROA (%) 99.84 95% CI (%) 99.42-99.98 Repeat testing for NewBio TPHA v PK TPHA 2000 Sample Agreement Agreement measure N= EDTA plasma NPA 1245 Total N= 1246 ROA 99.92 95% CI 99.55-100.0 Statistical summary by sample type against clinical status -- after repeat testing Sample Agreement Agreement Total ROA measure N= N= EDTA plasma Specificity 1247 1248 99.92 95% CI 99.55-100.0 CSF sample testing CSF samples collected for routine screening for neurosyphilis in clinical laboratories in Europe were tested using NewBio TPHA in comparison with Serodia TPPA. Although TPPA does not have a published claim for CSF samples, this method has been widely considered as state of the art as an aid to diagnosis of neurosyphilis. Serodia TPPA Positive Negative NewBio Positive 34 0 TPHA Negative 0 46 Agreement measure Diagnostic sensitivity Diagnostic specificity OPA Agreement N= 34 46 80 Total N= 34 46 80 ROA (%) 100 100 100 95% CI (%) 89.72 - 100 92.29 - 100 95.49 - 100 13. Limitations NewBio TPHA may be used for serum, EDTA plasma and CSF samples. No interfering substances have been identified, however TPHA can cross react with other treponemal infections such as T.pertenue and T.carateum so positive results should be confirmed by another method. In early primary syphilis, occasionally, specific antibodies may not be detected. Newmarket Biomedical Ltd. Unit 1, Lanwades Business Park, Kentford, Suffolk CB8 7PN UK 19005 v2.2 EN 2024/04 Page 6 of 8 14. Key to symbols REF Catalogue number IVD IVD In Vitro Diagnostic Medical Device Manufactured by EU Authorised Representative EU importer Distributor Temperature limitation Use by LOT Batch code Consult instructions for use 15. Post Market Surveillance Should this IVD be implicated in any serious incident a report shall be made to the manufacturer and competent authority of the Member State in which the user and/or the patient is established. www.new-bio.com info@new-bio.com 16. Summary of Safety and Performance SSP can be obtained from the EUDAMED website https://ec.europa.eu/tools/eudamed. Newmarket Biomedical Ltd. Unit 1, Lanwades Business Park, Kentford, Suffolk CB8 7PN UK 19005 v2.2 EN 2024/04 Page 7 of 8 17. Literature references 1. Rathlev T. - Haemagglutination tests utilizing antigens from pathogenic and apathogenic Treponema pallidum WHO/VDT/RES 1965 ; 77 : 65. 2. Tomizawa T, Kasamatsu S. - Haemagglutination tests for diagnosis of syphilis. A preliminary report. Japan. J. Med. Sci. Biol. 19, 305-308, 1966. 3. Rathlev T. - Haemagglutination test utilizing pathogenic Treponema pallidum for the serodiagnosis of syphilis. Br J Vener Dis 1967 ; 43 : 181-5 4. 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. 5. Sequeira P,J,L. Eldridge A,E. - Treponemal Haemagglutination test. Br J Vener Dis 1973 ; 49 : 242-8. 6. 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. 7. Wasley G.D. & Wong H.H.Y. Syphilis Serology Principles and Practice. Oxford Medical Publications 104 105 8. Manual of Test for Syphilis, U.S. Department of Health, Education, And Welfare, 1969 For instructions in other languages, please visit our website http://www.new-bio.com or contact your distributor. Other languages are available on request. Newmarket Biomedical Ltd. Unit 1, Lanwades Business Park, Kentford, Suffolk CB8 7PN UK 19005 v2.2 EN 2024/04 Page 8 of 8