DiaSorin S.p.A. Strada per Crescentino - 13040 Saluggia (Vercelli) - Italy Tel. 39.0161.487.093 - Fax 39.0161.487.

628

Pay attention to changes!

HBsAg Confirmatory Test (310110) 1. INTENDED USE The HBsAg Confirmatory Test is an in vitro neutralization assay for confirmation of the presence of hepatitis B surface antigen (HBsAg) in human serum and plasma samples found repeatedly reactive for HBsAg by LIAISON HBsAg (310100) or ETI-MAK-4 (N0019). 2. SUMMARY AND EXPLANATION OF THE TEST With the discovery and correlation of the hepatitis B surface antigen (HBsAg) to type B viral hepatitis, the problem of detecting potentially infectious blood units became critically important. Since that time, test methods with increasing sensitivity and specificity have been developed in order to allow for HBsAg detection in donor and patient populations. In spite of the high specificity achieved with these test methods, the possibility exists that falsely reactive results may be encountered due to the presence of non-specific interfering substances, artifacts in the reagents, or the type of methodology used. To reduce this possibility and avoid falsely reactive results in diagnosing HBV infection as well as discarding of useful blood units, neutralization tests were developed to ensure that HBsAg-reactive results are caused by the presence of the surface antigen and not by non-specific interference. 3. PRINCIPLE OF THE PROCEDURE The HBsAg Confirmatory Test is based on the principle of binding inhibition or neutralization of binding activity. A neutralizing reagent containing human antibodies to HBsAg is added to one aliquot of each specimen found repeatedly reactive (neutralized aliquot). As a control procedure, anti-HBs negative human serum is added to the other aliquot (non-neutralized aliquot). If the neutralizing reagent has been added to a sample containing HBsAg, the antibodies in the neutralizing reagent will bind to the HBsAg, forming an antigen-antibody complex. If the neutralizing reagent has been added to a sample containing an interfering substance, the antibodies in the neutralizing reagent will not bind to the interfering substance. Dilution may be used to ensure neutralization of samples with elevated concentrations of HBsAg that may exceed the potency of the HBsAg Confirmatory Test neutralizing reagent. In the confirmation procedure, each reactive sample is incubated in the presence of a solid matrix coated with mouse monoclonal antibodies to HBsAg, whereupon either a solid-matrix antibody-antigen-antibody complex or a solid-matrix antibodyinterfering substance complex is formed. Next, the antibody conjugate from the screening kit, which contains sheep antibodies to HBsAg, is added. If an antibody-antigen-antibody complex is present, the antibody conjugate will bind to the complex only partially. If an antibody-interfering substance complex is present, the antibody conjugate will bind non-specifically to the interfering substance. Therefore, if the repeatedly HBsAg-reactive result was caused by the presence of HBsAg in the sample, the antibodies in the neutralizing reagent will neutralize the reactivity of HBsAg by inhibiting the binding of the antibody conjugate. The resulting signal will be lower than the signal obtained in the aliquot to which anti-HBs negative human serum was added. Conversely, if the repeatedly HBsAg-reactive result was caused by the presence of an interfering substance, the antibodies in the neutralizing reagent will not neutralize the interfering substance, and the antibody conjugate will bind to it non-specifically. The resulting signal will be similar to that of the aliquot to which anti-HBs negative human serum was added. If the signal of the neutralized aliquot is significantly lower than the signal of the non-neutralized aliquot, the presence of HBsAg in the sample is confirmed. 4. MATERIALS PROVIDED
Anti-HBs (1 vial, 0.7 mL) Specimen diluent (1 vial, 16 mL) Number of tests Human serum/plasma containing at least 20,000 mIU/mL anti-HBs antibodies referenced to WHO Anti-Hepatitis B Immunoglobulin 1st International Reference Preparation (1977), preservatives, an inert red dye (ready to use). Human serum/plasma negative for HBsAg and anti-HBs, preservatives (ready to use). 20 specimens including controls (LIAISON HBsAg). 40 specimens including controls (ETI-MAK-4).

5. EQUIPMENT AND MATERIALS REQUIRED BUT NOT PROVIDED For LIAISON HBsAg repeatedly reactive results LIAISON HBsAg (code 310100). LIAISON HBsAg negative and positive controls (code 310101). Equipment and materials required to run the assay with LIAISON HBsAg. For ETI-MAK-4 repeatedly reactive results ETI-MAK-4 (code N0019) provided with necessary accessories. Equipment and materials required to run the assay with ETI-MAK-4.

ConfirmtestA4-en.fm

November 16, 2006 2:03 pm

6. WARNINGS AND PRECAUTIONS For in vitro diagnostic use. Do not mix reagents or exchange components from kits with different lot numbers. LIAISON HBsAg controls are not kit lot specific and may be safely interchanged even from different lots. All serum and plasma units used to produce the components provided in this kit have been tested for the presence of HBsAg, anti-HCV, anti-HIV-1, anti-HIV-2 and found to be non-reactive. As, however, no test method can offer absolute assurance that pathogens are absent, all specimens of human origin should be considered potentially infectious and handled with care. 7. SAFETY PRECAUTIONS Do not eat, drink, smoke or apply cosmetics in the assay laboratory. Do not pipette solutions by mouth. Avoid direct contact with all potentially infectious materials by using protective clothing such as lab coats, protective glasses and disposable gloves. Wash hands thoroughly at the end of each assay. Avoid splashing or forming an aerosol. Any reagent spills should be washed with a 5% sodium hypochlorite solution and disposed of as though potentially infectious. All samples, biological reagents and materials used in the assay must be considered potentially able to transmit infectious agents. They should therefore be disposed of in accordance with the prevailing regulations and guidelines of the agencies holding jurisdiction over the laboratory, and the regulations of each Country. Disposable materials must be incinerated; liquid waste must be decontaminated with sodium hypochlorite at a final concentration of 5% for at least half an hour. Liquid waste containing acid must be neutralized before treatment. Any materials to be reused must be autoclaved using an overkill approach (USP 24, 2000, p. 2143). A minimum of one hour at 121C is usually considered adequate, though the users must check the effectiveness of their decontamination cycle by initially validating it and routinely using biological indicators. 8. STORAGE AND STABILITY OF REAGENTS Upon receipt, the reagents must be stored at 2-8C. Do not freeze. When the reagents are stored sealed, they are stable at 2-8C up to the expiry date. Once opened the reagents are stable for eight weeks when properly stored at 2-8C between two successive uses. Avoid bacterial contamination. The reagents should not be used past the expiry date indicated on the vial labels. 9. SPECIMEN COLLECTION AND PREPARATION Either human serum or plasma may be used. The anticoagulants citrate, EDTA and heparin have been tested and may be used with this assay. Blood should be collected aseptically by venipuncture, allowed to clot, and the serum separated from the clot as soon as possible. Samples having particulate matter, turbidity, lipaemia, or erythrocyte debris may require clarification by filtration or centrifugation before testing. Grossly haemolyzed or lipaemic samples as well as samples containing particulate matter or exhibiting obvious microbial contamination should not be tested. Check for and remove air bubbles before assaying. If the assay is performed within seven days of sample collection, the samples may be kept at 2-8C; otherwise they should be aliquoted and stored deep-frozen (20C or below). If samples are stored frozen, mix thawed samples well before testing. Five samples with different reactivity were stored for seven days at 2-8C and underwent five freeze-thaw cycles. The results showed no significant differences. LIAISON HBsAg - The minimum volume of specimen required is 700 L for slightly reactive samples (e.g., index value < 50) and 20 L for highly reactive samples (e.g., index value > 50). ETI-MAK-4 - The minimum volume of specimen required is 300 L for slightly reactive samples (e.g., absorbance below 3.000) and 20 L for highly reactive samples (e.g., absorbance above 3.000). 10. ASSAY PROCEDURE FOR LIAISON HBsAg (310100) Specimen treatment 1. POSITIVE CONTROL AND SLIGHTLY REACTIVE SAMPLES (index value < 50): Mix 330 L specimen and 33 L anti-HBs antibodies in one tube (neutralized aliquot) as well as 330 L specimen and 33 L specimen diluent in another tube (non-neutralized aliquot). Incubate the specimens for one hour 5 minutes at room temperature (20-25C). Treat controls and specimens in parallel. 2. HIGHLY REACTIVE SAMPLES (index value > 50): Dilute specimens 1:10,000 with specimen diluent before testing. Operate as follows: Dispense 4 L of each specimen and 36 L specimen diluent into test tubes and thoroughly mix with a Vortex to ensure adequate mixing (1:10 intermediate predilution). Dispense 4 L of the 1:10 intermediate predilution and 36 L specimen diluent into clean test tubes and thoroughly mix with a Vortex to ensure adequate mixing (1:100 intermediate predilution). Dispense 7 L of the 1:100 intermediate predilution and 693 L specimen diluent into clean test tubes and thoroughly mix with a Vortex to ensure adequate mixing (1:10,000 predilution). The predilutions may be stored at 2-8C for 24 hours. Mix 330 L diluted specimen and 33 L anti-HBs antibodies in one tube (neutralized aliquot) as well as 330 L diluted specimen and 33 L specimen diluent in another tube (non-neutralized aliquot). Incubate the specimens for one hour 5 minutes at room temperature (20-25C). If the index value of the 1:10,000 predilution is still greater than 100 for the non-neutralized aliquot, repeat the test after further diluting the specimen 1:20 (e.g., 35 L of the 1:10,000 predilution + 665 L specimen diluent). If the index value of the 1:10,000 predilution is less than 0.9 for the non-neutralized aliquot, repeat the test on the 1:100 intermediate predilution of the specimen. Operate as follows: Dispense 7 L of each specimen and 693 L specimen diluent into test tubes and thoroughly mix with a Vortex to ensure adequate mixing.

ConfirmtestA4-en.fm

November 16, 2006 2:03 pm

Assay procedure Assay LIAISON HBsAg negative and positive controls with each specimen run. Strict adherence to the analyzer operators manual ensures proper assay performance. Each test parameter is identified via the bar codes on the reagent integral label. In case of malfunction of the bar code reader, the data can be entered manually. For details, refer to the analyzer operators manual. The analyzer operations are as follows: 1. Dispense calibrators of LIAISON HBsAg test into the reaction module (as required). 2. Dispense one non-treated negative control, one non-treated positive control, and two positive controls (neutralized and non-neutralized aliquots) of LIAISON HBsAg control set into the reaction module. 3. Dispense specimens in duplicate (i.e., neutralized and non-neutralized aliquots) into the reaction module. 4. Dispense buffer C. 5. Dispense coated magnetic particles. 6. Incubate. 7. Dispense conjugate into the reaction module. 8. Incubate. 9. Wash with Wash/System liquid. 10. Add the Starter Kit and measure the light emitted. 11. QUALITY CONTROL FOR LIAISON HBsAg Quality control is suggested once per day of use, or according to guidelines or requirements of local regulations or accredited organizations. Non-treated negative and positive controls may not be run whenever a LIAISON HBsAg test has been already performed on that day. LIAISON controls are available for internal quality control. Whenever controls lie outside the expected ranges, calibration should be repeated and controls retested. The performance of other controls should be evaluated for compatibility with this assay before they are used. Appropriate value ranges should then be established for quality control materials used. 12. CALCULATION OF RESULTS FOR LIAISON HBsAg Percent neutralization is given by the following formula: RLU for non-neutralized aliquot RLU for neutralized aliquot x 100 RLU for non-neutralized aliquot RLU for negative control of HBsAg test RLU = relative light units. 13. INTERPRETATION OF RESULTS FOR LIAISON HBsAg The test is valid if neutralization of the LIAISON HBsAg positive control (code 310101) is at least 50%. A specimen is not confirmed positive when the index value of the non-neutralized aliquot (mixed with specimen diluent) is less than 0.9 irrespective of the outcome of percent neutralization (= negative specimen). A specimen is not confirmed positive when the index value of the non-neutralized aliquot (mixed with specimen diluent) is greater than or equal to 0.9 and percent neutralization is less than 50% (= presence of an interfering substance). A specimen is confirmed positive when the index value of the non-neutralized aliquot (mixed with specimen diluent) is greater than or equal to 0.9 and percent neutralization is greater than or equal to 50%. A specimen should be further diluted with specimen diluent (up to 1:200,000) and neutralization repeated when the index value of the non-neutralized aliquot (mixed with specimen diluent) is greater than 50 (neat specimen) or 100 (diluted specimen) and percent neutralization is less than 50%.
Specimen dilution Neat Index value (non-neutralized aliquot) < 0.9 0.9-50 0.9 > 50 < 0.9 1:10,000 0.9-100 0.9 > 100 1:100 1:200,000 0.9-100 0.9 0.9-100 0.9 % Neutralization Any value < 50% 50% < 50% Any value < 50% 50% < 50% < 50% 50% < 50% 50% Interpretation of results Not confirmed (HBsAg-negative specimen). Not confirmed (interfering substance). Confirmed (true HBsAg-positive specimen). Further dilute (1:10,000) and retest. Retest 1:100 dilution. Not confirmed (interfering substance). Confirmed (true HBsAg-positive specimen). Further dilute (1:200,000) and retest. Not confirmed (interfering substance). Confirmed (true HBsAg-positive specimen). Not confirmed (interfering substance). Confirmed (true HBsAg-positive specimen).

ConfirmtestA4-en.fm

November 16, 2006 2:03 pm

Calculation example Example 1 RLU for non-neutralized aliquot RLU for neutralized aliquot RLU for negative control of HBsAg test 1925 418 x 100 1925 366 Example 2 RLU for non-neutralized aliquot RLU for neutralized aliquot RLU for negative control of HBsAg test 1881 1744 x 100 1881 351 = 9% Non-confirmed specimen. = 1881 = 1744 = 351 Index value = 3.8 = 97% Positive specimen. = 1925 = 418 = 366 Index value = 4.0

14. SPECIFIC PERFORMANCE CHARACTERISTICS FOR LIAISON HBsAg 14.1. Analytical specificity Analytical specificity may be defined as the assay ability to correctly classify the specimens, that is to neutralize HBsAg in the presence of potentially interfering factors in the sample matrix (e.g., anticoagulants, haemolysis, effects of sample treatment), or to fail to neutralize cross-reactive substances. Interference . Controlled studies of potentially interfering substances or conditions showed that the assay performance was not affected by anticoagulants (citrate, EDTA, heparin), haemolysis (up to 100 mg/dL haemoglobin), lipaemia (up to 3000 mg/dL triglycerides), bilirubinaemia (up to 20 mg/dL bilirubin), or by freeze-thaw cycles of samples. 14.2. Repeatability Four samples, containing different HBsAg concentrations, were treated separately and assayed in replicates of 22 to determine repeatability of neutralization. Percent neutralization was then calculated for each specimen replicate. The variability shown in the table below did not result in sample misclassification.
Repeatability Number of determinations Mean neutralization (%) Standard deviation Coefficient of variation (%) A 22 87.8 1.0 1.1 B 22 89.9 1.1 1.2 C 22 87.8 6.9 7.8 D 22 88.1 4.5 5.1

14.3. Diagnostic specificity and sensitivity Diagnostic specificity was assessed by testing 20 specimens giving repeatedly reactive results by LIAISON HBsAg test, but known to be false positive samples. HBsAg Confirmatory Test correctly did not confirm those specimens. Diagnostic sensitivity was assessed by testing 462 specimens giving repeatedly reactive results by LIAISON HBsAg test and known to be true positive samples (healthy HBsAg carriers, acute and chronic hepatitis B patients). Out of those, 388 were single specimens and 74 were seroconversion or follow-up specimens from 19 patients. HBsAg Confirmatory Test correctly confirmed those specimens as positive (diagnostic sensitivity: 100% - 95% confidence interval: 99.2-100%).

ConfirmtestA4-en.fm

November 16, 2006 2:03 pm

15. ASSAY PROCEDURE FOR ETI-MAK-4 (N0019) Specimen treatment 1. POSITIVE KIT CONTROL AND REPEATEDLY REACTIVE SAMPLES with absorbance below 3.000: Mix 150 L specimen and 15 L anti-HBs antibodies in one tube (neutralized aliquot) as well as 150 L specimen and 15 L specimen diluent in another tube (non-neutralized aliquot). Incubate the specimens for one hour 5 minutes at room temperature (20-25C). Treat specimens and controls in parallel. 2. REPEATEDLY REACTIVE SAMPLES with absorbance above 3.000: Dilute specimens 1:10,000 with specimen diluent before testing. Operate as follows: Dispense 4 L of each specimen and 36 L specimen diluent into test tubes and thoroughly mix with a Vortex to ensure adequate mixing (1:10 intermediate predilution). Dispense 4 L of the 1:10 intermediate predilution and 36 L specimen diluent into clean test tubes and thoroughly mix with a Vortex to ensure adequate mixing (1:100 intermediate predilution). Dispense 3 L of the 1:100 intermediate predilution and 297 L specimen diluent into clean test tubes and thoroughly mix with a Vortex to ensure adequate mixing (1:10,000 predilution). The predilutions may be stored at 2-8C for 24 hours. Mix 150 L diluted specimen and 15 L anti-HBs antibodies in one tube (neutralized aliquot) as well as 150 L diluted specimen and 15 L specimen diluent in another tube (non-neutralized aliquot). Incubate the specimens for one hour 5 minutes at room temperature (20-25C). If the absorbance value of the 1:10,000 predilution is still greater than 3.000 for the non-neutralized aliquot, repeat the test after further diluting the specimen 1:20 (e.g., 15 L of the 1:10,000 predilution + 285 L specimen diluent). If the absorbance value of the 1:10,000 predilution is less than the cut-off multiplied by 0.9 for the non-neutralized aliquot, repeat the test on the 1:100 intermediate predilution of the specimen. Operate as follows: Dispense 3 L of each specimen and 297 L specimen diluent into test tubes and thoroughly mix with a Vortex to ensure adequate mixing. Assay procedure Bring all reagents to room temperature (20-25C) before assaying. Perform all assay steps in the order given and without any appreciable delays between the steps. Three negative and two positive controls (supplied in the ETI-MAK-4 kit) should be assayed with each run of samples to be confirmed. The samples submitted to the confirmatory test must be the same which resulted repeatedly reactive at screening. Controls and samples should be submitted to the same process and incubation time. A disposable tip should be used for dispensing each control and sample. Adjust the thermostatically-controlled humid chamber to 37 1C. Identify the wells as follows: 1, 2, 3 negative control (non-treated) 4, 5 positive control (non-treated) 6 positive control (neutralized aliquot) 7 positive control (non-neutralized aliquot) 8 first specimen to be confirmed (neutralized aliquot) 9 first specimen to be confirmed (non-neutralized aliquot) 10 second specimen to be confirmed (neutralized aliquot) 11 second specimen to be confirmed (non-neutralized aliquot) etc. 1. Dispense 100 L negative control to the bottom of 1, 2, 3 wells and 100 L positive control to the bottom of 4, 5 wells . 2. Dispense 100 L positive control to the bottom of 6 (neutralized aliquot) and 7 (non-neutralized aliquot) wells . 3. Dispense 100 L first sample to be confirmed to the bottom of 8 (neutralized aliquot) and 9 (non-neutralized aliquot) wells . 4. Dispense 100 L second sample to be confirmed to the bottom of 10 (neutralized aliquot) and 11 (non-neutralized aliquot) wells . Operate in the same way for all samples to be confirmed. 5. Apply a cover sealer in order to prevent evaporation. 6. Incubate for one hour 5 min at 37 1C in a thermostatically-controlled humid chamber. 7. At the end of the incubation, remove and discard the cover sealer. Aspirate the liquid and rinse each well five times with 0.3 mL wash buffer. Avoid overflows from the reaction wells. 8. Dispense 100 L tracer (supplied in the ETI-MAK-4 kit) into all wells. Repeat step 5. 9. Incubate for one hour 5 min at 37 1C in a thermostatically-controlled humid chamber. Repeat step 7. 10. Dispense 100 L chromogen/substrate (supplied in the ETI-MAK-4 kit) into all wells. 11. Incubate for 30 2 min at room temperature , away from intense light. 12. Dispense 100 L stop solution (supplied in the ETI-MAK-4 kit) into all wells in the same order as for chromogen/substrate and taking the same time intervals. 13. Read the absorbance of samples with a spectrophotometer. Absorbance may be read at 450 nm or 450/630 nm in the bichromatic mode.

ConfirmtestA4-en.fm

November 16, 2006 2:03 pm

16. CALCULATION OF RESULTS FOR ETI-MAK-4 Calculation of cut-off value The cut-off value is determined by adding 0.030 to the mean absorbance for the negative control values (NC x ). Cut-off value = NC x + 0.030. Run validation criteria The following criteria should be used to validate quality control when evaluating results. Always calculate and evaluate mean control values for each plate, even if plates are combined to form a single batch. The absorbance value for the blank well must range between 0.000 and 0.150. 0.000 BLK 0.150. The mean absorbance for the negative control must be greater than 0.010 and less than 0.050. 0.010 < NC x < 0.050. Negative samples and controls generate very low absorbance values, which may lead to negative results after subtracting the blank. If this occurs with the negative control, the cut-off value should be computed algebraically. Each negative control absorbance value must be greater than 0.010 and less than 0.055. 0.010 < NC < 0.055. If any one of the negative control absorbance values does not meet these criteria, it should be discarded and the mean value recalculated using the remaining two values. If more than one negative control absorbance value does not meet these criteria, the run is invalid and must be repeated. The difference between the mean positive control absorbance value and the mean negative control absorbance value must be greater than or equal to 0.500. PC x NC x 0.500. If not, the run is invalid and must be repeated. Calculation example The following data must only be considered an example and should not be employed instead of the data obtained by the user. Absorbance for the negative control
Negative control sample No. 1 2 3 Absorbance at 450/630 nm 0.009 0.011 0.007

Mean absorbance, NC x = 0.009. Absorbance for the positive control

Positive control sample No. 1 2 Absorbance at 450/630 nm 1.538 1.416

Mean absorbance, PC x = 1.477. Cut-off value (NC x + 0.030) = 0.009 + 0.030 = 0.039. 0.9 x cut-off value = 0.9 x 0.039 = 0.035. Positive Negative control difference (P N) = 1.477 0.009 = 1.468. In the given example, the P N difference meets the run validation criteria because it is greater than 0.500; thus the technique is acceptable and data should be considered valid. Calculation of percent neutralization Percent neutralization is given by the following formula: Absorbance for non-neutralized aliquot Absorbance for neutralized aliquot Absorbance for non-neutralized aliquot Absorbance for negative control of ETI-MAK-4 test The test is valid if neutralization of the ETI-MAK-4 positive control is at least 35%. Example 3 Absorbance for non-neutralized aliquot Absorbance for neutralized aliquot Absorbance for ETI-MAK-4 negative control 1.182 0.034 x 100 1.182 0.009 In the given example, the positive control percent neutralization meets the run validation criteria because it is greater than 35%; thus the technique is acceptable and data should be considered valid. = 98% Valid test. = 1.182 = 0.034 = 0.009 x 100

ConfirmtestA4-en.fm

November 16, 2006 2:03 pm

17. INTERPRETATION OF RESULTS FOR ETI-MAK-4 Specimens with absorbance below 3.000 A specimen is not confirmed positive when the absorbance of the non-neutralized aliquot (mixed with specimen diluent) is less than the cut-off multiplied by 0.9, irrespective of the outcome of percent neutralization (= negative specimen). A specimen is not confirmed positive when the absorbance of the non-neutralized aliquot (mixed with specimen diluent) is greater than or equal to the cut-off multiplied by 0.9 and percent neutralization is less than 35% (= presence of an interfering substance). A specimen is confirmed positive when the absorbance of the non-neutralized aliquot (mixed with specimen diluent) is greater than or equal to the cut-off multiplied by 0.9 and percent neutralization is greater than or equal to 35%.
Absorbance (non-neutralized aliquot) < 0.9 x cut-off 0.9 x cut-off 0.9 x cut-off % Neutralization Any value < 35% 35% Interpretation of results Not confirmed (HBsAg-negative specimen). Not confirmed (interfering substance). Confirmed (true HBsAg-positive specimen).

Specimens with absorbance above 3.000 A specimen is not confirmed positive when the absorbance value of the non-neutralized aliquot (mixed with specimen diluent) is less than the cut-off multiplied by 0.9 irrespective of the outcome of percent neutralization (= negative specimen). A specimen is not confirmed positive when the absorbance value of the non-neutralized aliquot (mixed with specimen diluent) is greater than or equal to the cut-off multiplied by 0.9 and percent neutralization is less than 35% (= presence of an interfering substance). A specimen is confirmed positive when the absorbance value of the non-neutralized aliquot (mixed with specimen diluent) is greater than or equal to the cut-off multiplied by 0.9 and percent neutralization is greater than or equal to 35%. A specimen should be further diluted with specimen diluent (up to 1:200,000) and neutralization repeated when the absorbance value of the non-neutralized aliquot (mixed with specimen diluent) is greater than 3.000 and percent neutralization is less than 35%.
Specimen dilution Neat Absorbance (non-neutralized aliquot) < 0.9 x cut-off 0.9 x cut-off to 3.000 0.9 x cut-off > 3.000 < 0.9 x cut-off 1:10,000 0.9 x cut-off to 3.000 0.9 x cut-off > 3.000 1:100 1:200,000 0.9 x cut-off to 3.000 0.9 x cut-off 0.9 x cut-off to 3.000 0.9 x cut-off % Neutralization Any value < 35% 35% < 35% Any value < 35% 35% < 35% < 35% 35% < 35% 35% Interpretation of results Not confirmed (HBsAg-negative specimen). Not confirmed (interfering substance). Confirmed (true HBsAg-positive specimen). Further dilute (1:10,000) and retest. Retest 1:100 dilution. Not confirmed (interfering substance). Confirmed (true HBsAg-positive specimen). Further dilute (1:200,000) and retest. Not confirmed (interfering substance). Confirmed (true HBsAg-positive specimen). Not confirmed (interfering substance). Confirmed (true HBsAg-positive specimen).

Calculation example Example 4 Absorbance for non-neutralized aliquot Absorbance for neutralized aliquot Absorbance for ETI-MAK-4 negative control 1.925 0.418 x 100 1.925 0.009 Because the percent neutralization is greater than 35%, the patient specimen is confirmed positive for the presence of HBsAg. Example 5 Absorbance for non-neutralized aliquot Absorbance for neutralized aliquot Absorbance for ETI-MAK-4 negative control 1.881 1.744 x 100 1.881 0.009 Because the percent neutralization is less than 35%, the patient specimen is not confirmed positive for the presence of HBsAg. = 7% Non-confirmed specimen. = 1.881 = 1.744 = 0.009 = 79% Positive specimen. = 1.925 = 0.418 = 0.009

ConfirmtestA4-en.fm

November 16, 2006 2:03 pm

18. SPECIFIC PERFORMANCE CHARACTERISTICS FOR ETI-MAK-4 18.1. Analytical specificity Analytical specificity may be defined as the assay ability to correctly classify the specimens, that is to neutralize HBsAg in the presence of potentially interfering factors in the sample matrix (e.g., anticoagulants, haemolysis, effects of sample treatment), or to fail to neutralize cross-reactive substances. Interference . Controlled studies of potentially interfering substances or conditions showed that the assay performance was not affected by anticoagulants (citrate, EDTA, heparin), haemolysis (up to 50 mg/dL haemoglobin), lipaemia (up to 3000 mg/ dL triglycerides), bilirubinaemia (up to 20 mg/dL bilirubin), or by freeze-thaw cycles of samples. 18.2. Repeatability Four samples, containing different HBsAg concentrations, were treated separately and assayed in replicates of 20 to determine repeatability of neutralization. Percent neutralization was then calculated for each specimen replicate. The variability shown in the table below did not result in sample misclassification.
Repeatability Number of determinations Mean neutralization (%) Standard deviation Coefficient of variation (%) A 20 58.9 7.0 11.8 B 20 59.2 7.9 13.3 C 20 62.6 6.0 9.6 D 20 62.8 9.2 14.6

18.3. Diagnostic specificity and sensitivity Diagnostic specificity was assessed by testing four specimens giving repeatedly reactive results by HBsAg test, but known to be false positive samples. HBsAg Confirmatory Test correctly did not confirm those specimens. Diagnostic sensitivity was assessed by testing 100 specimens giving repeatedly reactive results by HBsAg test and known to be true positive samples (acute and chronic hepatitis B patients). HBsAg Confirmatory Test correctly confirmed those specimens as positive (diagnostic sensitivity: 100% - 95% confidence interval: 96.4-100%).

200/007-842, G - 09/2006

ConfirmtestA4-en.fm

November 16, 2006 2:03 pm