IMx HBsAg (V2), LN and HBsAg Confirmatory, LN

Transcription

1 IMx HBsAg (V2), LN and HBsAg Confirmatory, General Information The information presented below is an embellishment of parts of Perspectives on Viral Hepatitis by Robert P. Perrillo, M.D. in the Abbott Diagnostics Educational Services Series. HBsAg was first discovered in the blood of Australian aborigines in 1963 and at that time was termed the Australia antigen. 1 The presence of the Australia antigen in various human sera remained a mystery until 1966, when Blumberg and colleagues demonstrated an association between the Australia antigen and viral hepatitis. Since that time researchers have recognized that the presence of the Australia antigen, now referred to as HBsAg, in blood and other body secretions is a marker for hepatitis B viral (HBV) infections. 2 HBV is a DNA containing virus that belongs to a class called the Hepadna viruses (Figure 1). The outside lipoprotein envelope of HBV, the hepatitis B surface antigen (HBsAg), encapsulates the core of the virus and is abundantly produced as spherical and tubular particles. During acute or chronic type B hepatitis as many as to particles may be found per ml of blood, outnumbering virions by as much as 10 6 to 1. The primary antigenic constituent of HBsAg is a protein of 24,000 d and its glycosylated form, 27,000 d, which are coded by the HBV S gene. One hundred or more of these proteins assemble with phospholipid molecules to form the HBsAg particle, 22 nm in diameter, or to form the envelope of the virion. Figure 1. Structure of hepatitis B virus and its antigens. DNA Polymerase HBsAg HBcAg HBeAg DNA Dane Particle HBsAg HBsAg Sphere Tubule November 1999/R2-1- LN and

2 General Information (Cont.) The HBV core contains a distinct antigen, the hepatitis B core antigen (HBcAg). The core also contains a double stranded DNA with a gap in one of the strands, and a DNA polymerase that repairs the gap region. Hepatitis e antigen (HBeAg), a third antigen associated with HBV, is believed to represent an internal component of the core. The HBcAg is not measurable in the serum without detergent treatment to disrupt the complete virion (Dane particle) in order to release the HBcAg. However, the DNA polymerase enzyme, HBeAg and HBV DNA are detectable in serum. Since these markers indicate the presence of replicating virus, they are associated with increased contagiousness. 3 HBV is not cytopathic, and the damaging effects of infection are thought to result from the immune response of the host. 4 Current evidence suggests that an appropriate T lymphocyte response is critical to the clearance of the virus. It has been estimated that there are more than 280 million HBV carriers throughout the world today. Areas of high endemicity include certain parts of Africa, Asia and the Mediterranean basin. In the US, approximately 250,000 cases of acute hepatitis B occur annually, and 0.2% - 0.4% of the population are chronically infected. The virus is largely transmitted either by parenteral exposure (e.g., needlestick or blood transfusion) or by inapparent skin or mucosal exposure (e.g., intimate or sexual contact). Many cases are not associated with an identifiable route of exposure and probably result from hetero- or homosexual person-to-person contact. All major classes of body fluid have been shown to contain the virus with the possible exception of stool that is free of contamination with blood. Experimental studies have demonstrated that HBV is not infectious by the oral, nasal or respiratory routes. 5 Type B hepatitis is frequent among spouses and sexual contacts of persons with acute or chronic HBsAg-positive hepatitis. 6 Infection occurs approximately 10 times more frequently in male homosexuals as compared to the general population. 7 The mechanism of spread related to intimate contact has been clarified to some extent by the finding of HBsAg and HBV DNA in saliva and semen as well as the blood of infected persons. 8 Figure 2 represents the time course of clinical, biochemical and serological events in the typical case of acute hepatitis B. Note the rapid disappearance of HBeAg and HBV DNA. Figure 3 shows the serological profile for a typical chronic HBV infection. LN and -2- November 1999/R2

3 General Information (Cont.) Figure 2. Typical Sequence of HBV Markers in Acute Infection. Duration Incubation (4-12 Weeks) Acute Infection (2-12 Weeks) Recent Acute Infection (2-16 Weeks) Recovery (Years) Relative Concentration HBeAg HBsAg Symptoms Core Window anti-hbe anti-hbc IgM anti-hbs anti-hbc Total HBV DNA Time Figure 3. Hepatitis B Chronic Carrier Serological Profile. Duration Incubation (4-12 Weeks) Acute Infection (6 Months) Chronic Infection (Years) Relative Concentration anti-hbc Total HBeAg HBsAg anti-hbc IgM HBV DNA Time November 1999/R2-3- LN and

4 General Information (Cont.) Sensitive enzyme immunoassays for the detection of HBsAg were first described by Engvall and Perlmann 9-11 and VanWeemen and Schuurs 12 in The first solid phase sandwich enzyme immunoassays were developed in 1976 utilizing polyclonal anti-hbs on a solid phase to capture HBsAg and then detecting with anti-hbs conjugated to enzyme In recent years, immunoassays using monoclonal anti-hbs have been developed for the detection of HBsAg These immunoassays have been used to screen blood and blood products for the presence of HBsAg to prevent transmission of HBV infection to recipients of these products. Clinical Use Tests for the presence of HBsAg are routinely used to diagnose suspected HBV infection and to monitor the status of infected individuals i.e. whether the patient has resolved the infection or has become a chronic carrier of the virus. 22 Approximately 50% of individuals infected with HBV remain asymptomatic. The only method of determining acute or chronic HBV infection in such cases is by the detection of serologic markers such as HBsAg. When symptoms exist, they include malaise, joint aches, anorexia and fever. The length of time between infection and the appearance of symptoms varies depending on the initial dose of virus received. Circulating HBsAg can be detected, and rises to high titers in the acute stage of the disease, before the appearance of symptoms. In the 90-95% of individuals who resolve their infection, HBsAg disappears from circulation and the antibody to HBsAg appears weeks to months later. In 5-10% of patients infected with hepatitis B, circulating HBsAg remains detectable for longer than 6 months and antibody to HBsAg remains undetectable. These individuals are unable to clear the HBV and are classified as hepatitis B chronic carriers. At the start of this stage, symptoms, if present, begin to improve and liver enzymes fall to a lower level. The duration and severity of chronic hepatitis B varies greatly. Healthy carriers continue to demonstrate HBsAg in their serum over a period of years but have no liver abnormalities. Approximately 50% of chronic carriers have chronic active hepatitis, resulting in liver degeneration and potential cirrhosis and increased risk of liver cancer. Positive test results for the presence of HBsAg, HBV DNA and IgM antibody to hepatitis B core antigen will determine that a patient is in the acute stage of HBV infection. The continued presence of HBsAg and HBV DNA and the disappearance of the HBV core IgM marker after 6 months will determine that a patient has entered the chronic stage of hepatitis B. The importance of determining the serologic status of an HBV infected individual is linked to both the need to administer prophylactic measures for close contacts of the patient and to determine the treatment of patients who become chronic carriers. Perinatal transmission in HBV infection from mother to neonate is the major mode of transmission in HBV endemic populations. If the infant is treated with hepatitis B immunoglobulin and HBV vaccine within 12 hours of birth, transmission is prevented in 90% of cases. The Centers for Disease Control have recommended the screening of all pregnant women for the presence of HBsAg before giving birth so that newborns may obtain such prophylactic treatment. 23 The efficacy of anti-viral treatment of chronic HBV carriers has been evaluated by measuring levels of HBsAg and HBV DNA in patients receiving therapy. Circulating levels of these markers are directly related to levels of viral replication. A decrease in HBsAg or HBV DNA during and after therapy indicates the efficacy of the therapeutic regimen. 24 LN and -4- November 1999/R2

5 Assay Summary: IMx HBsAg (V2) (List No ) 1. Configuration Qualitative, third-generation Microparticle Enzyme Immunoassay Anti-HBs sandwich formed in first step producing biotin label; second step binds enzyme label to biotin label. IMx MODE 1 Assay 2. Timing (approximate) 1 Sample: 37 minutes 21 Patient Samples: 51 minutes 3. Sample/Preparation Serum (including serum collected in separator tubes) or plasma collected in sodium heparin, lithium heparin, sodium citrate, potassium oxalate, ACD, CPD, CP2D, CPDA-1, or tripostassium EDTA may be used. Follow the manufacturer s processing instructions for serum and plasma collection tubes. For optimal results, fresh plasma specimens should be centrifuged for 15 minutes at 3,000 x g (RCF). CAUTION: Verify that sample preparation tubes allow for recommended centrifugation conditions. Care must be taken with respect to resistance of sample tubes to centrifugation conditions (see specifications for tubes given by manufacturer). Convert rpm to RCF as follows: RCF = 1.12 r max (rpm/1000) 2 Convert RCF to rpm as follows: rpm = RCF r max RCF rpm r max The relative centrifugal force generated during centrifugation. The rotation per minute of the rotor on which the specimens are being spun (usually the digital readout on the centrifuge will indicate rpm). Radius of the rotor in millimeters. The radius measured is dependent on whether the rotor is a fixed angle rotor or a swinging bucket rotor. This value is typically provided with the rotor, by the manufacturer. * For fixed angle, r max is a measure of the distance from the rotor axis (center) to the bottom of the tube cavity. * For the swinging bucket, r max is a measure of the distance from the rotor axis (center) to the bottom of the tube bucket while it is extended during rotation. 4. Test Results Qualitative test Samples with S/N < are nonreactive and considered negative for HBsAg. Samples with S/N are reactive. Samples with S/CO < are nonreactive and considered negative for HBsAg. Samples with S/CO are reactive. November 1999/R2-5- LN and

6 NOTE: Specimens and controls are determined to be reactive or nonreactive based only upon the S/N or S/CO result. If the ********** flag appears in the S/N or S/CO column, the result is invalid. Refer to the IMx System Operation Manual, Section 10, for additional information. Reactive specimens should be centrifuged (recommended: 8,000 to 10,000 RCF) for 10 minutes and repeat tested. Specimens that repeatedly test S/N or S/CO should be confirmed by a licensed neutralization test. NOTE: The German Federal Public Authority (Paul Ehrlich Institute, Langen, Germany) recommends for Germany that all specimens within a low grayzone option, parameter 58, LOW GRAY, must be edited to "1.600" for result unit S/N or result unit S/CO. Specimens with assay values equal to or higher than the low gray value and less than the cutoff are indicated as grayzone reactive. The letters "GRZ" will appear in the NOTE column on the printout. Refer to the IMx System Operation Manual for a detailed instrument procedure. 5. Dynamic Range This is a qualitative assay. A <1.0 ng/ml to >1.0 mg/ml concentration range of HBsAg is detected by IMx HBsAg. 6. Sensitivity < 1.0 ng/ml HBsAg. Typically sensitivity is in the range ng/ml based upon the Abbott HBsAg Sensitivity Panel. 7. Calibration For Calibration, run MODE 1 Calibrator in duplicate in the first two positions, Positive Control in position 3 and Negative Control in position 4, using the Calibration Mode. The mean rate of the MODE 1 Calibrator is stored. Subsequent carousels are run in MODE 1 and must have a MODE 1 Calibrator, run singly, in the first carousel position, a Positive Control run in position 2 and a Negative Control in position 3. IMx HBsAg calibration should be stable for a minimum of 2 weeks with the same reagent pack or reagent pack lot number. Recalibrate when the controls or the MODE 1 factors are out of range. 8. Carryover Estimated to be less than 1 ppm. Typical performance is 0.3 ppm. At 0.3 ppm carryover, with assay sensitivity at 0.3 ng/ml, 1,000,000 ng/ml HBsAg will cause carryover so that a negative specimen following it in the carousel will test as borderline reactive. Adjacent high level/low level reactive specimens in a carousel should be retested in a different carousel position order. 9. Confirmation of Reactive Results Specimens that test as reactive must be retested after centrifugation (recommended: 8,000 to 10,000 RCF for 10 minutes). If the specimen is repeatedly reactive, then the specimen must be confirmed by neutralization with human anti-hbs. LN and -6- November 1999/R2

7 Step 1 Schematic Reaction Sequence Anti-HBs: Biotin Anti-HBs Microparticles HBsAg Anti-HBs: Biotin HBsAg Antibody-Antigen- Antibody Complex Step 2-3 Alkaline Phosphatase Anti-Biotin Alk Phos Conjugate Enzyme Labeled Complex Step 4 MU P MU Enzyme Labeled Complex 4-Methylumbelliferyl Phosphate (MUP) Methylumbelliferone (MU) is fluorescent. Rate of production of MU is measured. November 1999/R2-7- LN and

8 Instrument Function Step 1 MEIA #2 Diluent Buffer Conjugate (4) Substrate (3) Biotin Probe (2) Microparticles (1) Matrix Blotter Incubation Well Sample Well Predilution Well MEIA #2 Diluent Buffer added to Incubation Well for Probe wash. Transfer Biotin Probe, microparticles and sample to Predilution Well to form sandwich with biotin label. Step 2 Reaction mixture added to matrix. Conjugate added to form enzyme labeled complex. Step 3 MEIA #2 Diluent Buffer Matrix washed with MEIA #2 Diluent Buffer to remove unbound conjugate. Step 4 MUP Substrate added to Matrix; rate of fluorescent product formation measured. LN and -8- November 1999/R2

9 Questions and Answers 1. What types of specimens can be run on IMx HBsAg? Either serum (including serum collected in serum separator tubes) or plasma collected in sodium heparin, lithium heparin, sodium citrate, potassium oxalate, ACD, CPD, CP2D, CPDA-1, or tripotassium EDTA may be used in the IMx HBsAg assay. If the assay will be performed within 14 days after collection, the specimen must be stored at 2 8 C or frozen (-20 C or colder). If the specimen is stored at 2 8 C, it may be stored on or off the clot or red blood cells. If the specimen is stored at -20 C or colder, it must be removed from the clot or red blood cells. Specimens containing particulate matter or red blood cells may give inconsistent results and must be centrifuged (recommended 8,000 10,000 RCF x 10 minutes) before testing. Specimens will be required to pass through the reaction cell glass fiber matrix and should be free of particulate material. For centrifugation follow the manufacturer s processing instructions for serum and plasma collection tubes. For optimal results, fresh plasma specimens should be centrifuged for 15 minutes at 3,000 x g (RCF). CAUTION: Verify that sample preparation tubes allow for recommended centrifugation conditions. Care must be taken with respect to resistance of sample tubes to centrifugation conditions (see specifications for tubes given by manufacturer). Convert rpm to RCF as follows: RCF = 1.12 r max (rpm/1000) 2 Convert RCF to rpm as follows: RCF rpm r max rpm = RCF r max The relative centrifugal force generated during centrifugation. The rotation per minute of the rotor on which the specimens are being spun (usually the digital readout on the centrifuge will indicate rpm). Radius of the rotor in millimeters. The radius measured is dependent on whether the rotor is a fixed angle rotor or a swinging bucket rotor. This value is typically provided with the rotor, by the manufacturer. * For fixed angle, r max is a measure of the distance from the rotor axis (center) to the bottom of the tube cavity. * For the swinging bucket, r max is a measure of the distance from the rotor axis (center) to the bottom of the tube bucket while it is extended during rotation. 2. What types of specimens cannot be run on IMx HBsAg? The quality of the specimen condition will affect the performance of IMx HBsAg. A sample containing bacteria, precipitate, fibrin clots, or cellular debris, etc., will not give consistent results. Old specimens with a history of extensive storage at 2 8 C or multiple freeze-thaw cycles may cause problems even after extensive centrifugation. These specimens and any specimens with noticeable solid contents (fat globules, etc.) should NOT be run on the IMx. Specimens will be required to pass through the reaction cell glass fiber matrix and should be free of particulate material. Do not run heat-inactivated specimens. Heat treatment has been shown to affect the condition of the specimen. Heat November 1999/R2-9- LN and

10 treatment often alters the specimen viscosity, and this may cause inconsistent results in IMx HBsAg due to flow problems. Performance has not been established using cadaver specimens or body fluids other than serum or plasma, such as urine, saliva or pleural fluid. 3. What is the effect of freezing and thawing specimens? Eleven nonreactive and eleven reactive specimens showed no qualitative performance differences when subjected to six freeze-thaw cycles; however, multiple freeze-thaw cycles should be avoided, if possible. Specimens must be mixed thoroughly after thawing and centrifuged prior to use to remove particulate matter and to ensure consistency in the results (recommended 8,000 10,000 RCF x 10 minutes). Frozen specimens that have not been centrifuged can give elevated S/N or S/CO results even elevated enough to be reactive (S/N > 2.0 or S/CO > 1.0). Note: Do not freeze specimens on the clot, red blood cells or serum separator tubes. 4. I see borderline reactive specimens following a relatively high S/N or S/CO specimen. What does this mean? Carryover in the IMx HBsAg assay has been measured to be ppm. Specimen condition can affect the ability of the IMx to control carryover. Specimens that are in poor condition are more difficult to control. Specimens with 1,000,000 ng/ml of HBsAg can carry over enough HBsAg to the following specimen in the process of sample handling so as to cause it to test as a reactive specimen. When a borderline reactive specimen follows a high reactive specimen on initial testing on a carousel, it is a good practice to repeat test both of them in a different carousel order. 5. Will I see an effect of cross contamination between runs with different assays? No. 6. I have obtained a negative rate. What does this mean? Negative rates are invalid results for IMx HBsAg and no S/N or S/CO result will be given. Negative rates arise from: a specimen that blocks flow through the matrix. The specimen should be centrifuged a minimum of 10 minutes (recommended 8,000 10,000 RCF) and retested. an empty MEIA Buffer bottle. Ensure that a minimum of 300 ml is present prior to starting an assay. 7. What is the effect of hemolyzed or lipemic specimens? No qualitative performance differences were observed when Negative and Positive Controls with elevated levels of hemoglobin (up to mg/dl) or lipids (up to 3,000 mg/dl) were tested. Elevated results with hemolyzed specimens arise not from the hemoglobin but from red cell debris or particulate in the sample. Centrifuge prior to testing (recommended 8,000 10,000 RCF x 10 minutes). In general, lipemic specimens do not interfere with the IMx HBsAg results. However, if lipemia is severe enough to affect the specimen viscosity, it may affect the IMx HBsAg result because of flow problems. Any specimen that is difficult to pipette due to lipemia, fat particles or particulate matter should not be tested with the IMx HBsAg assay. LN and -10- November 1999/R2

11 8. Is pretreatment of samples required? The IMx HBsAg assay does not require pretreatment of samples. 9. I forgot to run the MODE 1 Calibrator. What do I do? The results are not valid. The carousel must be rerun with a valid IMx HBsAg MODE 1 Calibrator in the first carousel position. 10. I forgot to run the Negative or Positive Control. What do I do? The results are not valid. The carousel must be rerun with the Positive and Negative Controls in the appropriate positions. 11. I ran the Negative Control in position 2 immediately following the MODE 1 Calibrator and the Positive Control in position 3. What do I do? The results are not valid. The carousel must be rerun with the Positive and Negative Controls in the appropriate positions. 12. How long is the Calibration stable? Curve stability is greater than 2 weeks. However, the calibration can be used as long as the controls test within the control S/N or S/CO range listed in the package insert (NC S/N = , PC S/N = ; NC S/CO = , PC S/CO = ) and the MODE 1 Calibrator is within range. Recalibration should be performed: each time a new reagent lot number is used in an assay, when the reagent pack lot number is not stored as curve 1 or 2, when either of the IMx HBsAg Controls or MODE 1 calibrator is out of range. 13. I have run out of MEIA #2 Diluent Buffer. Can I substitute saline or water? No. You must use IMx MEIA #2 Diluent Buffer (No ). 14. Should I mix or shake my reagents before using them? DO NOT SHAKE. Shaking reagents will cause foaming and possible level sense errors. Reagents that have been shaken must be allowed to sit until all foam or bubbles have disappeared from the reagent bottles. Bubbles will give rise to inaccurate level sensing and reagent aspirations. Gently invert reagents 5 or more times in order to ensure resuspension of the microparticles in bottle #1. Be sure to mix the reagents until the microparticles are completely resuspended, otherwise assay performance may show lower potency. The reagent pack should also be gently inverted between sequential runs. 15. Can I pool reagents or MEIA #2 Diluent Buffer? No, this should not be done due to possible cross contamination or bacterial contamination. November 1999/R2-11- LN and

12 16. I removed my IMx HBsAg Reagent Pack from the refrigerator and noticed a white slurry or paste on the bottom of bottle #1. Can I use it? Yes, bottle #1 contains the microparticles which make up the white slurry you see. Just mix by gently inverting the reagents a minimum of 5 times until the microparticles are resuspended and the white slurry or paste has disappeared. It is important to ensure complete resuspension of the microparticles before placing the reagents in the IMx. Incomplete mixing can affect assay performance by lowering the assay potency. Lower assay potency means decreased positive control S/N or S/CO values. 17. Can I use a reaction cell with a torn matrix? No, it can cause aberrant test results. You must use a new, undamaged reaction cell. 18. I did not invert my reagents between runs. Can I trust my results? As always, if the IMx controls on the carousel are within the specifications listed in the package insert, the assay results are acceptable. 19. I left my reagents out (or in the IMx) overnight. Can I still use them? Leaving the reagents at room temperature or in the IMx for extended periods is not recommended. Mix the reagents by inverting a minimum of 5 times to resuspend the microparticles and run controls. If the controls meet the specifications listed in the package insert, the reagents may continue to be used. 20. The Positive Control S/N or S/CO was out of the package insert required range. Is my run valid? No. The assay is not valid unless the Positive Control S/N or S/CO meets package insert specifications (S/N ; S/CO ). Check expiration dating on the Reagent Kit. If it is expired, replace the kit with a nonexpired kit and reassay. If the kit is not expired, ensure that reagent mixing prior to running the assay was sufficient to resuspend the microparticles. If the reagents continue to give a Positive Control S/N or S/CO out of range, they should not be used. Seek technical assistance to resolve the problem. 21. The Negative Control S/N or S/CO was out of the package insert required range. Is my run valid? No. The assay is not valid unless the Negative Control S/N or S/CO meets package insert specifications (S/N ; S/CO ). Check expiration dating on the Reagent Kit. If it is expired, replace the kit with a nonexpired kit and reassay. If the kit is not expired, ensure that reagent mixing prior to running the assay was sufficient to resuspend the microparticles. If the reagents continue to give a Negative Control S/N or S/CO out of range, they should not be used. Seek technical assistance to resolve the problem. 22. I obtained a CODE 146 CONTROL OUT OF RANGE for the Positive and/or Negative Control. What do I do? If the Positive or Negative Control value does not meet the acceptable range (established by assay parameters XX.21 XX.24), the error message will be printed below each control that is out of range. Refer to the IMx System Operation Manual, Section 10, for appropriate troubleshooting procedures. LN and -12- November 1999/R2

13 23. I obtained a CODE 166 READ ERROR on a specimen. What can I do? CODE 166 READ ERROR means that the sample has a level of fluorescence that exceeds the IMx optics linearity. This usually indicates a very high rate value. If only one specimen (or a few random specimens) gives this result, centrifuge the specimen (recommended 8,000 10,000 RCF x 10 minutes) and repeat the test. If the specimen is reactive, proceed to a confirmatory assay. As this error may be caused by a high analyte concentration, a 1:500 dilution of the sample can be evaluated (see below). However, because levels of HBsAg may become undetectable after sample dilution, a nonreactive result obtained after dilution must not be interpreted as negative. If an entire carousel gives the CODE 166 READ ERROR, the MUP (bottle #3 in the reagent pack) may be contaminated, or there may be an instrument problem. Consult the IMx System Operation Manual, Section 10. The dilution procedure below must be followed by make 2 (serial) dilutions: µl specimen µl Negative Control µl from Step µl Negative Control 24. I am seeing discrepancies between my bead assay and the IMx assay. Discrepant samples can be caused by differences in assay sensitivity, specificity, the ability to detect immune complexes, or the effect of sample conditions on the technology. If a sample is discrepant, retest by both technologies. If the sample is repeatedly reactive, continue with confirmation assay for each technology. Note: Immune complex samples contain complexes of antigen and antibody. Limited technical data indicates IMx HBsAg and Auszyme Monoclonal exhibit equivalent detection of these types of samples, and both of these assays show slightly improved detectability over that of Ausria II. 25. In addition to the microparticles and alkaline phosphatase conjugate, the IMx HBsAg assay also has a biotinylated anti-hbs solution. Why? During assay development, the biotinylated anti-hbs solution was added to enhance the detection of HBsAg at the low end of the dynamic range. This component serves as a bridge between the microparticles and conjugate and enhances the sensitivity of the IMx HBsAg assay. 26. A sample that I tested was repeat reactive in IMx HBsAg, but did not confirm. Why? All highly sensitive immunoassay systems have a potential for nonspecific reactions. Nonspecific interactions in the IMx HBsAg assay may be due to either nonspecific reactions with the reagents of the kit or sample quality issues. Therefore, the specificity of a repeatedly reactive specimen must be confirmed by a licensed neutralization test before reporting a sample as positive. 27. I am seeing ERROR CODE 163 NRMSE TOO LARGE. Poor sample quality is frequently the cause of this error. The customer should centrifuge the sample (recommended 8,000 10,000 RCF x 10 minutes) to remove any particulate matter. Often centrifugation resolves the problem even when the original sample appeared to be clear. The customer should check the MEIA Diluent Buffer volume, as insufficient buffer can cause sporadic results in this assay. 28. I have used the Abbott Proficiency/Sensitivity Panel and am seeing poor precision in the IMx HBsAg assay. The HBsAg sensitivity panel consists of dilutions of HBsAg positive samples in plasma. The panel has a recommended storage of 2 8 C with a 2 year expiration date. Plasma stored for this length of time at 2 8 C may exhibit some shedding of protein similar to what is observed in clinical samples stored for an extended period of November 1999/R2-13- LN and

14 time. The quality of the specimen condition will affect the performance of IMx HBsAg. For optimal product performance, panel members should be centrifuged (recommended 8,000 10,000 RCF x 10 minutes) prior to use to remove particulate matter. 29. Is there a WHO standard for Hepatitis B Surface Antigen? Yes, there is a WHO standard for HBsAg. Abbott has not evaluated this standard and therefore can provide no data on how it performs. 30. Can excessive levels of biotin interfere with the assay? No. Three studies showed no interference from biotin in HBsAg. Study 1 included 15 volunteers to ingest 1.2 mg of biotin a day for 14 days. (The recommended dose is 30 to 100 µg per day). Samples were drawn at the beginning and end of the study and tested in IMx HBsAg and Auszyme monoclonal. All samples were nonreactive. Study 2 spiked high concentrations of biotin into negative plasma. All samples, spiked and unspiked, were nonreactive. Study 3 spiked high concentrations of biotin into positive serum to be sure that no false negatives would occur. All samples, spiked and unspiked, tested positive. 31. Can I use another manufacturer s controls? If a customer wishes to run another external manufacturer s control IN ADDITION TO the controls provided in the masterlot, that is perfectly acceptable. They may not replace our controls with another manufacturer s. If that happens, the run is invalid and must be repeated using Abbott controls in the proper positions. 32. Can I use up the control kits with List Number ? No. The IMx HBsAg Control Kit, List No , is evaluated only for the use with the IMx HBsAg (V2) Kit, List No For this reason, Controls cannot be mixed from kits with different List numbers. 33. If a customer wishes to do a dilution and does not want to use Negative Control, or is out of material, can they use a different diluent? IMx HBsAg does not recommend dilutions of sample. However, dilutions may be required for the confirmatory procedure. In this case, the diluent provided with the HBsAg confirmatory test should be used. LN and -14- November 1999/R2

15 IMx HBsAg Confirmatory Biological Principles of the Procedure The IMx HBsAg Confirmatory assay is based on the Microparticle Enzyme Immunoassay (MEIA) technology. The IMx HBsAg Confirmatory assay (Assay #74) uses the same reagents (IMx HBsAg Reagent Pack, No or IMx HBsAg (V2) Reagent Pack, No ) as the IMx HBsAg assay. For information on the IMx HBsAg assay procedure refer to the appropriate IMx HBsAg assay package insert. The IMx HBsAg Confirmatory assay differs from the IMx HBsAg assay in that the specimen is preincubated with the IMx HBsAg Confirmatory Reagent A [a high titer Anti-HBs (Human) solution] or Reagent B (human plasma nonreactive for HBsAg, Anti-HBs, anti-hcv, HIV-1Ag, and anti-hiv-1/hiv-2) prior to running the assay. Assay Summary: IMx HBsAg Confirmatory (List No ) 1. Configuration Qualitative, third generation Microparticle Enzyme Immunoassay Manually pipetted neutralization prior to assay run Anti-HBs sandwich formed in first step producing biotin label; second step binds enzyme label to biotin label. IMx CALIBRATION MODE ONLY assay 2. Timing (approximate) Confirmation of 1 Sample (8 reaction cells): 34 minutes Confirmation of 5 Samples (24 reaction cells): 44 minutes 3. Sample Serum (including serum collected in serum separator tubes) or plasma collected in sodium heparin, lithium heparin, potassium oxalate, sodium citrate, ACD, CPD, CP2D, CPDA-1, or tripotassium EDTA In order to effectively neutralize specimens across the range of HBsAg concentrations, a sample dilution of 1:500 (or greater in rare cases) is frequently required. All specimens must be run both undiluted and diluted (at 1:500) in the IMx HBsAg Confirmatory assay. This amounts to 4 tests per specimen. 4. Specimen Dilutions Dilutions are made using the IMx HBsAg Confirmatory Dilution Reagent. The recommended dilution is a two step dilution: 10 µl specimen µl Dilution Reagent followed by 20 µl of 1st dilution µl Dilution Reagent 5. Test Results Qualitative test, for use as confirmation only, not reactive screening. Test results depend upon two tests for each specimen: sample + Reagent A (Result A) and sample + Reagent B (Result B) November 1999/R2-15- LN and

16 For data reduction, an undiluted specimen and a 1:500 dilution of that specimen are analyzed as separate samples. Two calculations are reported: S/N or S/CO values (for Result A and for Result B) % Neutralization Two types of flags can be reported: R (reactive) - denotes S/N values or S/CO values POS - indicates that Result B is S/N ( R ) or S/CO ( R ) AND that %Neutralization is %Neutralization = Result B S/N or S/CO - Result A S/N or S/CO X 100 Result B S/N or S/CO (MODE 1 Result) 6. Interpretation of Results and Flags Samples with S/N or S/CO for Result B (undiluted Specimen + Reagent B) less than or respectively are nonreactive and considered negative for HBsAg. In order for a specimen to be considered positive for HBsAg, the following conditions must all be met: The assay must be valid: Positive Control S/N or S/CO and Positive Control %Neutralization The specimen treated with Reagent B must test reactive ( R flag). Calculated sample %Neutralization must be See also the INTERPRETATION OF RESULTS section in the IMx HBsAg Confirmatory package insert. 7. Calibration IMx HBsAg Confirmatory is always run in the CALIBRATION Mode. %Neutralization = Result B S/N or S/CO - Result A S/N or S/CO X 100 Result B S/N or S/CO (MODE 1 Result) LN and -16- November 1999/R2

17 MANUAL STEP Reagent A HBsAg (Specimen) Anti-HBs (Reagent A) Schematic Reaction Sequence Neutralized or partly neutralized HBsAg Step 1 Reduced Complex Formation Anti-HBs Microparticles Neutralized or partly neutralized HBsAg Anti-HBs: Biotin Available HBsAg forms Antibody-Antigen-Antibody Complex Step 2-3 Alkaline Phosphatase Anti-Biotin Alk Phos Conjugate Reduced or No Formation of Enzyme Labeled Complex Step 4 MU P MU Reduced or No Formation of Enzyme Labeled Complex 4-Methylumbelliferyl Phosphate (MUP) Methylumbelliferone (MU) is fluorescent. Rate of production of MU is measured. Reduced fluorescent signal, Neutralization Occurs November 1999/R2-17- LN and

18 MANUAL STEP Reagent B No Anti-HBs HBsAg (Specimen) (Reagent B) Schematic Reaction Sequence Step 1 Anti-HBs Microparticles HBsAg Anti-HBs: Biotin HBsAg Antibody-Antigen-Antibody Complex Step 2-3 Alkaline Phosphatase Anti-Biotin Alk Phos Conjugate Formation of Enzyme Labeled Complex Step 4 MU P MU Enzyme Labeled Complex 4-Methylumbelliferyl Phosphate (MUP) Methylumbelliferone (MU) is fluorescent. Rate of production of MU is measured. LN and -18- November 1999/R2

19 200 µl Sample 50 µl Reagent A or Reagent B Manual Step Pipette samples, 200 µl each; add Confirmatory Reagents; load carousel Step 1 Instrument Function MEIA #2 Diluent Buffer Conjugate (4) Substrate (3) Biotin Probe (2) Microparticles (1) Matrix Blotter Incubation Well Sample Well Predilution Well MEIA #2 Diluent Buffer added to Incubation Well for Probe wash. Transfer Biotin Probe, microparticles and sample to Predilution Well to form sandwich with biotin label. Step 2 Reaction mixture added to matrix. Conjugate added to form enzyme labeled complex. Step 3 MEIA #2 Diluent Buffer Matrix washed with MEIA #2 Diluent Buffer to remove unbound conjugate. Step 4 MUP Substrate added to Matrix; rate of fluorescent product formation measured. November 1999/R2-19- LN and

20 Questions and Answers 1. My MODE 1 Calibrator values are too high in IMx HBsAg Confirmatory, but they run fine on IMx HBsAg (V2). What is the problem? Reagent B can get contaminated with HBsAg if the same pipette tip is used for pipetting Reagent B into all the specimens. A pipette tip that is splashed with an HBsAg positive specimen can easily contaminate the IMx HBsAg Confirmatory reagents. For both Reagents A and B, use a separate pipette tip for each specimen in order to avoid cross-contamination. 2. In the %NEUT result column I got ********** instead of a result. What happened? The ********** flag appears in the %NEUT column if: An error code or invalid S/N or S/CO result was obtained for either Result A or Result B. The specimen should be centrifuged and retested in the IMx HBsAg Confirmatory assay. OR Result B is the same S/N or S/CO value as the MODE 1 Calibrator (1.000). This sample need not be retested, since Result B is nonreactive. 3. I ran a specimen both undiluted and at 1:500. The undiluted specimen had an error code in one of the two reaction cells, however, the 1:500 is flagged POS. Do I need to repeat the undiluted test? Verify that the assay is valid (PC S/N or S/CO and PC %Neut 50%. The undiluted specimen Result B S/N must be or S/CO must be If no result is obtained, the test must be repeated with the undiluted sample. 4. I ran an IMx repeatedly reactive specimen on the IMx HBsAg Confirmatory assay and it did not neutralize. Why? The package insert discusses many of the possible outcomes for confirmation and how to interpret them. Use the package insert as a reference. Additional comments: Specimens must be centrifuged (recommended 8,000 10,000 RCF x 10 minutes) prior to all confirmation testing. The undiluted specimen plus Reagent B is nonreactive (S/N < 1.500; S/CO < 1.000). This indicates that specimen condition (particulate matter or fibrin clots) may have been responsible for the IMx HBsAg reactivity in the initial testing or the wrong sample was retested. A specimen is a strong reactive in the confirmatory assay undiluted and diluted 1:500. The % neutralization is less than The IMx HBsAg Confirmatory test should be repeated with the specimen diluted at 1:500 and 1:5000 to ensure that the dilutions were correct and that the dilution is sufficient to allow neutralization. Also check the carousel to see that Reagent A (violet color in sample well) was added to the correct reaction cells for this specimen. If a specimen is reactive undiluted but does not show 50.00% neutralization, and at 1:500 it is nonreactive, then it may be exhibiting a nonspecific interaction in the IMx HBsAg assay. LN and -20- November 1999/R2

21 5. I forgot to select Assay 74 when running the confirmatory assay. What do I do? Error Code 146 CONTROL OUT OF RANGE will appear. The run is invalid and should be rerun selecting assay I diluted my specimen with buffer (or distilled water). Are my results accurate? Sample dilutions should only be made using human plasma nonreactive for HBsAg, HIV-1 Ag, anti-hbs, anti-hcv and anti-hiv-1/2 as specified in the package insert. IMx HBsAg Negative Control or IMx HBsAg Confirmatory Dilution Reagent is recommended for making dilutions. DO NOT USE DISTILLED WATER OR BUFFER TO MAKE SPECIMEN DILUTIONS. If buffer or distilled water is used, the results are not accurate because the conjugate will nonspecifically bind to the glass matrix on the reaction cell in an unpredictable manner. The results will be inconsistent and generally elevated. 7. What is the minimum number of reaction cells needed to run 1 patient with the IMx HBsAg Confirmatory assay? The total number of reaction cells needed to run 1 patient sample with IMx HBsAg Confirmatory is 8 if controls are included. Two replicates of MODE 1, 2 replicates of Positive Control, 2 replicates of the undiluted sample and 2 replicates of the sample diluted 1:500. November 1999/R2-21- LN and

22 Reproducibility of the IMx HBsAg (V2) Assay Assay reproducibility was determined during the clinical evaluation of the IMx HBsAg (V2) assay. The samples were assayed in replicates of three with three clinical lots in four consecutive runs on three instruments each. Calculations were made with a variance components analysis, using a nested analysis of variance model. Typical results are summarized in Table 1. Table 1 Reproducibility of the IMx HBsAG (V2) Assay Panel Mean S/N Intra-Run Inter-Run Member n Value SD CV(%) SD CV(%) LN and -22- November 1999/R2

23 Sensitivity of the IMx HBsAg (V2) Assay The sensitivity of IMx HBsAg (V2) was evaluated using a 9 member proficiency panel of purified HBsAg (ad and ay) prepared at Abbott Laboratories. Each panel member was assayed in replicates of three, with three clinical lots, three runs each on each of 4 IMx instruments. Typical results are summarized in Tables 1, 2, and 3. Table 1 Detection of Purified HBsAg/ad by IMx HBsAg (V2) Concentration IMx HBsAg (V2) (ng/ml) S/N Value Result Table 2 Detection of Purified HBsAg/ay by IMx HBsAg (V2) Concentration IMx HBsAg (V2) (ng/ml) S/N Value Result Table 3 Sensitivity Calculated by Linear Regression Analysis HBsAg Subtype Sensitivity (ng/ml) ad 0.22 ay 0.17 Typically, the sensitivity results from clinical trials and studies conducted at Abbott Laboratories have ranged from 0.1 to 0.5 ng/ml. November 1999/R2-23- LN and

24 IMx HBsAg (V2)/LN vs IMx HBsAg (V2)/LN Clinical Correlation HBsAg Positives N = 400 IMx HBsAg (V2) LN IMx HBsAg (V2) LN Agreement = 100% IMx HBsAg (V2)/LN vs IMx HBsAg (V2)/LN Clinical Correlation Hospital/Public Health Population N = 600 IMx HBsAg (V2) LN LN IR RR Confirmed (2.17%) (2.0%) (1.17%) (1.33%) (1.50%) (1.17%) LN and -24- November 1999/R2

25 IMx HBsAg (V2)/LN vs IMx HBsAg (V2)/LN Clinical Correlation Specimens Containing Potentially Interfering Substances* N = 132 IMx HBsAg (V2) LN LN IR RR Confirmed 17 4* 3 (12.88%) (3.03%) (2.27%) (2.27%) (2.27%) (2.27%) *Categories tested: Flu Vaccine Recipients (Pre-vaccination) Flu Vaccine Recipients (Post-vaccination) Rheumatoid Arthritis Rheumatoid Factor ANA Positive (SLE) Elevated Liver Enzymes Multiparous Females Pregnant Females 1st Trimester Pregnant Females 2nd Trimester Pregnant Females 3rd Trimester Syphilis Positive (RPR) EBV IgM Positive Toxo IgM Positive CMV IgM Positive HCV HAV IgM Positive Myelloma IgG Myelloma IgM Elevated Alkaline Phosphatase November 1999/R2-25- LN and

26 Bibliography 1. Blumberg BS. Australia Antigen and the Biology of Hepatitis B. Science 1977;197: Tiollais P, Charnay P, and Vyas GN. Biology of Hepatitis B Virus. Science 1981;213: Alter HJ, Seeff LB, Kaplan PM, McAuliffe VJ, Wright EC, Gerin JL, et al. Type B Hepatitis: The Infectivity of Blood Positive for e Antigen and DNA Polymerase after Accidental Needlestick Exposure. N Engl J Med 1976;295: Dienstag JL. Immunologic Mechanisms in Chronic Viral Hepatitis. In: Vyas GN, Dienstag JL, Hoofnagle JH, eds. Viral Hepatitis and Liver Disease. New York: Grune and Stratton, Inc. 1984: Bancroft WH, Snitbhan R, Scott RM, Tingpalapong M, Watson WT, Tantiicharoenyos P, et al. Transmission of Hepatitis B Virus to Gibbons by Exposure to Human Saliva Containing Hepatitis B Surface Antigen. J Infect Dis 1977;135: Perrillo RP, Gelb L, Campbell C, Wellinghoff W, Ellis FR, Overby L, et al. Hepatitis B e Antigen, DNA Polymerase Activity, and Infection of Household Contacts with Hepatitis B Virus. Gastroenterology 1979;76: Szmuness W, Harley EJ, Ikram H, Stevens CE. Sociodemographic Aspects of the Epidemiology of Hepatitis B. In: Vyas GN, Cohen SN, Schmid R, editors. Viral Hepatitis. Philadelphia: Franklin Institute Press 1978: Davidson F, Alexander GJM, Trowbridge R, Fagan EA, Williams R. Detection of Hepatitis B Virus DNA in Spermatozoa, Urine, Saliva, and Leucocytes, of Chronic HBsAg Carriers. A Lack of Relationship With Serum Markers of Replication. J Hepatol 1987;4: Engvall E, Perlmann P. Enzyme-Linked Immunosorbent Assay (ELISA) Quantitative Assay of Immunoglobulin G. Immunochem 1971;8: Engvall E, Perlmann P. Enzyme-Linked Immunosorbent Assay (ELISA) In: Peeters H, editor. Protides of the Biological Fluids. Proceedings of the Nineteenth Colloquium, Burgge. Oxford: Pergamon Press, 1971: Engvall E, Jonsson K, Perlmann P. Enzyme-Linked Immunosorbent Assay II. Quantitative Assay of Protein Antigen, Immunoglobulin G, By Means of Enzyme-Labelled Antigen and Antibody-Coated Tubes. Biochem Biophys Acta 1971;251: VanWeemen BK, Schuurs AHWM. Immunoassay Using Antigen-Enzyme Conjugates. FEBS Letters 1971;15: Wisdom GB. Enzyme-Immunoassay. Clin Chem 1976;22: Wolters G, Kuijpers L, Kacaki J, Schuurs A. Solid-Phase Enzyme-Immunoassay for Detection of Hepatitis B Surface Antigen. J Clin Pathol 1976;29: Wei R, Knight GJ, Zimmerman DH, Bond HE. Solid-Phase Enzyme Immunoassay for Hepatitis B Surface Antigen. Clin Chem 1977;23: David GS, Present W, Martinis J, Wang R, Bartholomew R, Desmond W, et al. Monoclonal Antibodies in the Detection of Hepatitis Infection. Med Lab Sci 1981;38: Drouet J, Couroucé AM, Kalil J, Ropars C, Fellous M. Monoclonal Antibodies to HBsAg Produced by Murine Hybridomas. In: Szmuness W, Alter HJ, Maynard JE, editors. Viral Hepatitis. Philadelphia: Franklin Institute Press, 1982: Goodall AH, Miescher G, Meek FM, Janossy G, Thomas HC. Monoclonal Antibodies in a Solid-Phase Radiometric Assay for HBsAg. Med Lab Sci 1981;38: Kennedy RC, Ionescu-Matiu I, Alder-Storthz K, Henkel RD, Sanchez Y, Dreesman GR. Characterization of Anti- Hepatitis B Surface Antigen Monoclonal Antibodies. Intervirology 1983;19: Shih JW-K, Cote PJ, Dapolito GM, Gerin JL. Production of Monoclonal Antibodies Against Hepatitis B Surface Antigen (HBsAg) by Somatic Cell Hybrids. J Virol Methods 1980;1: Wands JR, Zurawski VR. High Affinity Monoclonal Antibodies to Hepatitis B Surface Antigen (HBsAg) Produced by Somatic Cell Hybrids. Gastroenterology 1981;80: Perrillo R, and Aach R. The Clinical Course and Chronic Sequence of Hepatitis B Virus Infection. Seminars in Liver Disease 1981;1: MMWR June 10, Dusheiko G, Dibisceglie A, Bowyer S, Sachs E, Ritchie M, Schoub B, et al. Recombinant Leukocyte Interferon Treatment of Chronic Hepatitis B. Hepatology 1985;5: LN and -26- November 1999/R2