NCCLS. Accutest H. pylori WB Test. (Catalog. No. ID-92A225) For Professional and In Vitro Diagnostic Use Only

Transcription

1 NCCLS Accutest H. pylori WB Test (Catalog. No. ID-92A225) For Professional and In Vitro Diagnostic Use Only A Rapid Visual Test for the Qualitative Detection of IgG Antibodies Specific to Helicobacter pylori Accutest H. pylori WB Test 1 of 14

2 TABLE OF CONTENTS Page Number Contents 2 Title 3 Background Summary and Explanation of Test Principle of the Procedure 4 Specimen Collection Fingerstick Whole Blood 5 Materials 6 Test Procedure 8 Interpretation of Results 8 Limitations 9 Quality Control 10 Comments and Technical Assistance Performance Characteristics 14 References Accutest H. pylori WB Test 2 of 14

3 This procedural bulletin is intended to provide a ready outline reference for performance of the assay. These abbreviated directions are not intended to replace the complete package insert. It is the obligation of every manufacturer of medical devices labeled FOR in vitro DIAGNOSTICS USE to provide a complete package insert in accordance with the format and specification recommended by the National Committee for Clinical Laboratory Standards, Villanova, PA 19085; this bulletin is intended for use in conjunction with the Physicians Office Laboratory Procedural Manual, NCCLS Document POL2-T. Accutest H. pylori WB Test For Professional and In Vitro Diagnostic Use Only A Rapid Visual Test for the Qualitative Detection of IgG Antibodies Specific to Helicobacter pylori Accutest H. pylori WB Test 3 of 14

4 INTENDED USE For the visual qualitative detection of IgG antibodies specific to Helicobacter pylori in human whole blood. This test kit is intended as an aid in the diagnosis of H. pylori infection in patients with gastrointestinal symptoms, and is intended for professional laboratory use only. BACKGROUND Summary and Explanation of Test Gastritis and peptic ulcers are one of the most common human diseases. Since the discovery of H. pylori (8), many reports have suggested that this organism is one of the major causes of ulcer diseases (1,3, 4) and stomach cancer. Although the exact role of H. pylori is not fully understood, the eradication of H. pylori has been associated with elimination of ulcer diseases. (3) The human serological responses to infection with H. pylori have been demonstrated. (2) The detection of the IgG antibodies specific to H. pylori has been used as an aid in the diagnosis of symptomatic patients. A serological test may be used either as an adjunct to endoscopy or as an alternative measure in symptomatic patients. (5) The Accutest H. pylori WB test is intended for use in the detection of the IgG antibodies specific to H. pylori in whole blood. It is intended for adult patients only. Principle of the Test The Accutest H. pylori WB Test is a chromatographic immunoassay for the qualitative determination of anti-h. pylori IgG antibodies in human whole blood. The test device contains a membrane strip that is pre-coated with H. pylori antigens on the test band region and H. pylori specific monoclonal antibody on the control band region. The H. pylori antigens-colloidal gold conjugate pad is placed at the end of the membrane. When the H pylori specific IgG antibodies are present in patient samples, the mixture of colloidal gold conjugate, patient sample and developer buffer moves along the membrane chromatographically to the test region (T) and forms a visible line as the antigenantibody-antigen gold particle complex forms. Therefore, the formation of a visible line in the test region (T) indicates a positive result for the detection of H. pylori specific IgG antibodies. When the H. pylori specific IgG antibodies are absent in the patient sample, no visible color band will form in the test region (T). Therefore, the absence of a color band in the test region (T) indicates a negative result for the detection of H. pylori specific IgG antibodies. Accutest H. pylori WB Test 4 of 14

5 SPECIMEN COLLECTION Fingerstick Special patient preparation requirements Method of Collection Volume of specimen required Handling conditions of specimen Clean the area to be lanced with an alcohol swab. Wipe away the first drop of blood with a sterile gauze or cotton. Squeeze the end of the fingertip and pierce with a sterile lancet. Allow the second drop of blood to flow directly into the sample well. Two pipette drops or approximately 40 µl. The specimen obtained from this collection method should be used immediately. All patient samples should be treated as if capable of transmitting disease. Whole Blood Method of collection Volume of specimen required Special patient preparation requirement Handling conditions of specimen Criteria A certified phlebotomist should collect the whole blood into a purple, blue or green top collection tube (containing EDTA, citrate, or heparin respectively) by venipuncture. Two pipette drops or approximately 40 µl. None. The whole blood may be used for testing immediately or may be stored at 2-8 C up to three days. All patient samples should be treated as if capable of transmitting disease. Do not use whole blood stored for more than three days. Accutest H. pylori WB Test 5 of 14

6 Reagents and Materials provided MATERIALS 25 Test devices. Each test cassette contains one test strip with H. pylori antigen coated membrane and colored H. pylori antigen pad. Include disposable transfer pipette. Developer Buffer (8 ml): 50mM Tris-HCI Buffer with 0.1% sodium azide. H. pylori Negative Control (0.4 ml): Normal human serum or plasma diluted in saline solution with 0.1% sodium azide. H. pylori Positive Control (0.4 ml): H. pylori antibody positive human serum or plasma diluted in saline solution with 0.1% sodium azide. One Instruction Insert. Materials Required but not Provided Vacutainer tubes. EDTA, heparin, or citrate for whole blood procedure. Finger lancet for fingerstick blood procedure. Storage and stability The test kit (including the positive and negative controls) should be stored refrigerated (2-8 C) or at room temperature (up to 30 C). The test device should be in the sealed pouch for duration of shelf life. Precautions: FOR In Vitro DIAGNOSTIC USE ONLY. Do not mix reagents from different lots. Do not use test kit beyond expiration date. Do not open the foil pouch until ready to perform the test. Developer buffer, positive and negative controls contain sodium azide which may react with lead or copper plumbing to form potentially explosive metal azides. When disposing of these solutions, always flush with copious amounts of water to prevent azide build-up. The assay performance has not been established for anticoagulants other than those that have been evaluated. Accutest H. pylori WB Test 6 of 14

7 Warning: Potential Biohazardous Material Each donor unit of human plasma or serum used in the preparation of the Positive and Negative Controls was tested by FDA-approved methods for the presence of anti-hiv- 1/HIV-2, HBsAg and anti-hcv, and found to be negative. However, caution should be used when handling and disposing of these items at biosafety level 2, as recommended in the Center for Disease Control and Prevention/National Institutes of Health Manual, Biosafety in Micobiological and Biomedical Laboratories, Assay Procedure TEST PROCEDURE 1. Test device, developer buffer, patient samples, and controls should be brought to room temperature (20 C to 30 C) prior to testing. 2. Remove the test device from its foil pouch when ready to perform the test. Label the device with patient or control identification. 3. Add the specimen to the sample well. For whole blood sample in collection tube: Mix well before using. Hold the provided transfer pipette in a vertical position to draw and add two drops (about 40 µl) of whole blood into the sample well or use a pipette to transfer 40 µl of whole blood into the sample well. Fingerstick blood: Use the provided transfer pipette to collect enough sample (more than 40 µl) of blood. Hold transfer pipette in a vertical position and add two full drops (about 40 µl) of fresh blood into the sample well. 4. Immediately add 3 or 4 drops of developer buffer into the sample well. 5. Start to count the time after adding the developer buffer. Observe the results in 5 minutes. Strong Positive results may be observed sooner. Do not interpret results after 8 minutes. Accutest H. pylori WB Test 7 of 14

8 INTERPRETATION OF RESULTS NEGATIVE: Only one red colored band appears in the control region (C). No apparent faint pink to red colored band appears in the test region (T). A negative result indicates that there is no anti-h. pylori IgG in the patient sample or that IgG concentration is below the detection level. POSITIVE: In addition to the control band, a pink or red colored band also appears in the test region (T). For a very strong positive sample, the control band may be lighter. A positive result indicates that there is anti-h. pylori IgG in the patient sample. INVALID: When no colored band appears in the control region, the test should be voided since an improper test procedure may have been performed or deterioration of reagents may have occurred. Repeat the test with a new test device and if condition persists, contact Jant Pharmacal Corp. at (818) for technical assistance. LIMITATIONS This test kit is to be used for the qualitative detection of IgG antibodies to H. pylori. This test kit should be used for symptomatic individuals with gastrointestinal disorders. It is not intended for use with asymptomatic patients and is not intended for use with pediatric patients. Diagnosis of gastritis and/or peptic ulcer should be made by confirmation with other clinical findings. A positive result suggests the presence of IgG antibodies to H. pylori. It does not distinguish between active infection and colonization by H. pylori. It does not necessarily indicate the presence of gastrointestinal disease. A negative result does not rule out H. pylori infection because the IgG antibodies to H. pylori may be absent or may not be present in sufficient quantity to be detected. The comparison study against biopsy should be performed with serum samples from patients. Accutest H. pylori WB Test 8 of 14

9 QUALITY CONTROL Internal Procedural Control 1. A procedural control is included in the test that is used to indicate proper performance of the test procedure. A colored band appearing in the control region of the membrane indicates proper performance of the test and the device. 2. A clear background in the result window is considered an internal negative control. However, when whole blood samples are tested, the background may appear slightly reddish due to the low level hemolysis of some red blood cells. This is acceptable as long as it does not interfere with the interpretation of test result. The test is invalid if the background fails to clear and obscures the reading of the result. 3. A positive and negative external control must be tested when opening a new test kit. Each operator must test a positive and negative external control once with each 25 count test kit. External Quality Control 1. Positive Control: Add one drop (about 20 µl) of Positive Control in the sample well using the provided transfer pipette by holding it in a vertical position. Immediately add 3 or 4 drops of developer buffer. A positive signal is indicated by two pink to red lines, one in the test region (T) and one in the control region (C). Read results at 5 minutes. 2. Negative Control: Add one drop (about 20 µl) of Negative Control in the sample well using the provided transfer pipette by holding it in a vertical position. Immediately add 3 or 4 drops of developer buffer. A negative signal is indicated by only one pink to red colored line in the control region (C). Read results at 5 minutes. Accutest H. pylori WB Test 9 of 14

10 COMMENTS AND TECHNICAL ASSISTANCE Expected Values The majority of individuals exposed to H. pylori develop IgG antibodies against the organism. It is reported that H. pylori is universally distributed and an estimated 50% of the world s population is colonized by H. pylori. (4) The presence of H. pylori IgG antibodies is a function of age, race geography and clinical conditions. A relatively large proportion of patients who have positive levels of IgG antibodies are asymptomatic. Therefore, the level of IgG antibodies do not necessarily correlate with the severity of clinical symptoms. (6) During the clinical studies of the Accutest H. pylori WB Test, a selected symptomatic population was evaluated. Of the 317 patient samples tested, 217 tested positive for H. pylori IgG, 215 tested biopsy positive, 100 tested anti- H. pylori IgG negative and 102 tested biopsy negative. Because this study was not a random sampling of the population, it does not indicate the prevalence of Helicobacter pylori in symptomatic patients. Technical Service Representative For technical assistance contact: Jant Pharmacal Corporation Ventura Blvd., Suite 505 Encino, CA Phone: (818) Fax: (818) Accutest H. pylori WB Test 10 of 14

11 PERFORMANCE CHARACTERISTICS Accuracy - Comparison with biopsy results The accuracy of the Accutest H. pylori WB Test was evaluated in comparison to biopsy results of human specimens. Out of the three hundred and seventeen (317) samples, two hundred and eighty-three (283) test results agreed with the biopsy results. Thirty-four (34) samples gave discordant results. Out of the thirty four (34) discordant test results, eighteen (18) samples obtained positive results with the Accutest H. pylori WB Test and negative biopsy results. Sixteen (16) samples obtained negative results with Accutest H. pylori WB Test and positive biopsy results. A commercial EIA kit was used to reanalyze the discrepant samples. Out of the eighteen positive Accutest H. pylori WB Test results, fifteen (15) samples were positive, and three (3) samples were negative. Out of the sixteen (16) Accutest H. pylori negative test results, one (1) was negative, four (4) were indeterminate and eleven (11) were positive when tested in comparison to an EIA kit. The biopsy sample comparison results are summarized in Table 1a. Table 1a Accutest Accutest Total H. pylori WB Positive H. pylori WB Negative Biopsy Positive Biopsy Negative Total The comparison study results gave a sensitivity of 92.6% (199/215), a specificity of 82.4% (84/102), and a total agreement of 89.3% (283/317). The relatively low specificity of the serological test results in comparison to the biopsy results may be partially attributed to a sampling error of the biopsy test. Comparison Study with a Commercially Available Rapid H. pylori Test The accuracy of Accutest H. pylori WB Test was also evaluated against a commercially available rapid H. pylori test using serum/plasma specimens. Specimens were tested in a side by side comparison using the Accutest H. pylori WB Test and a commercial rapid H. pylori test. The discrepant specimens were tested with a commercial EIA kit. Out of the one hundred and seventy (170) samples, one hundred and fifty eight (158) gave the same results. Twelve (12) samples gave negative results with Accutest H. pylori WB Test and positive results with the commercial rapid H. pylori test. When tested with an EIA kit, out of these twelve (12) samples, three (3) were positive, seven (7) were negative, and two (2) were indeterminate. The comparison results are summarized in Table 1 b. Accutest H. pylori WB Test 11 of 14

12 Commercially available H. pylori Test Positive Commercially available H. pylori Test Negative Table 1b Accutest H. pylori WB Positive Accutest H. pylori WB Negative Total Total These results gave a relative sensitivity of 84.0% (63/75), a relative specificity of 100% (95/95) and a total agreement of 92.9% (158/170). Test Sensitivity: Since there is no sensitivity standard established for H. pylori IgG antibodies, the following dilution (test sensitivity) studies were performed for comparison purposes. Six (6) H. pylori positive human specimens (serum/plasma) purchased from suppliers were diluted with H. pylori negative human serum. The diluted samples were tested with Accutest H. pylori WB Test and a commercially available rapid H. pylori Test. The results of the test sensitivity study are summarized in Table 2. Table 2 Sample #1 #2 #3 #4 #5 #6 Dilution Factor J C J C J C J C J C J C 1: : : : : : : : J: Accutest H. pylori WB test. C: Commercially available rapid H. pylori test. These results indicated that the sensitivity of the Accutest H. pylori WB Test was comparable to the commercial rapid H. pylori Test. Accutest H. pylori WB Test 12 of 14

13 Specificity: Cross Reactivity The cross reactivity of the Accutest H. pylori WB Test was evaluated by an inhibitory assay. Testing was performed with Campylobacter coli (ATCC 33559), Campylobacter fetus (ATCC 27374), Campylobacter jejuni (ATCC 33560) and Escherichia coli. Different antigens were added to patient samples and assayed by Accutest H. pylori WB Test. All species tested showed no cross reactivity. There were no differences between the control samples and samples with added antigens. Based on these results, it can be concluded that Accutest H. pylori WB Test is specific for the H. pylori IgG antibodies. Non-Specific Interference The Accutest H. pylori WB Test was evaluated for possible interference from visibly hemolyzed, lipemic and icteric samples. Lipemic samples with known level of triglycerides and H. pylori antibody status were tested with the Accutest H. pylori WB Test. Human hemoglobin, bilirubin or albumin was spiked in samples and tested using non-spiked sample as controls. The results indicate that there is no interference in the performance of Accutest H. pylori WB Test by triglycerides up to 2370 mg/dl, hemoglobin up to 10 mg/ml, bilirubin up to 0.5 mg/ml and albumin up to 100 mg/ml. Reproducibility/ Site Study The precision of the Accutest H. pylori WB Test has been evaluated in house and at three independent clinical sites. Three (3) human whole blood specimens with different levels of H. pylori antibodies were diluted and blind labeled into twenty (20) vials each and used for the study. Of the forty (40) positive samples with two levels of H. pylori antibodies, the results were all positive. The results of twenty (20) negative samples rendered 100% agreement with expected results. Fifteen (15) volunteers were also sent to three sites to test for anti-h.pylori with fingerstick blood. Seven (7) people tested positive and eight (8) people tested negative by all three sites. The simplicity of the Accutest H. pylori WB Test has been evaluated at three different sites. Twenty (20) people at each site, most of whom were non-professionals with the equivalent of a high school education, were asked to read the instructions and perform three tests using blind-labeled samples. The results from these studies are summarized in Table 3. For the one hundred and twenty (120) samples with different levels of H. pylori antibodies, results were all positive. For the sixty (60) negative samples, the results were all negative. The results obtained from all sites demonstrate 100% agreement. The filed results demonstrate that the Accutest H. pylori WB Test is easy to perform and can be used to detect visual qualitative results of IgG antibodies specific to H. pylori. The results Accutest H. pylori WB Test 13 of 14

14 are summarized in Table 3. Table 3 Sample Number Field Results Strong Weak Negative Positive Positive Strong Positive Weak Positive Negative Total REFERENCES 1. 1.Anderson, L. P. and Nielsen, H., Peptic ulcer: an infectious disease: Ann. Med. 1993, Evans, D.J., Evans, D.G., Graham, D. Y. and Klein, P.D. A sensitive and specific serologic test for detection of Campylobacter pylori infection, Gastroenterology. 1989, 96: Hunt, R.H. and Mohamed, A.H. The current role of Helicobacter pylori: eradication in clinical practice, Scand. J. Gastroenterol. 1995, 30 suppl 208: Lambert, J.R., Lin, S.K. and Aranda-Michel, J. Helicobacter pylori, Scand. J. Gastroenterol. 1995, 30 suppl. 208: Peura, D.A. The report of digestive health initiative international update conference on Helicobacter pylor, Gastoenterology 1997, 113: B. 6. Tytgat G.N.J. and Rauws, E.A.J. The role of Campylobacter pylori in gastroduodenal diseases: a "believer's" point of view, Gastroenterol. Clin. BioI. 1989, 13: Vaira, D. and Holton, J. Serum immunoglobulin G antibody levels for Campylobacter pylori diagnosis, Gastroenterology. 1989, 97: Warren, J.R. and Marshall, Unidentified curved bacillus on gastric epithelium in active chronic gastritis, LANCET. 1983, 1: Accutest H. pylori WB Test 14 of 14