BIO-FLASH. BIOKIT, S.A. - Can Malé s/n Lliçà d Amunt - Barcelona - SPAIN

Transcription

1 READ HIGHLIGHTED CHANGES BIO-FLASH anti-hbc tests The BIO-FLASH anti-hbc is a fully automated chemiluminescent two-step immunoassay for qualitative measurement of total antibodies to hepatitis B virus core antigen (anti-hbc) in human serum or plasma on the BIO-FLASH instrument. Summary Hepatitis B is a liver infection produced by the Hepatitis B Virus (HBV) that represents a major global health concern. It causes a number of liver diseases, ranging from acute and chronic hepatitis to cirrhosis and primary liver cancer. Diseases caused by HBV have a global distribution. It is estimated that approximately one-third of the world population has been infected with HBV. Of these, approximately 350 million individuals are chronically infected and at risk of serious illness and death, mainly from liver cirrhosis and hepatocellular carcinoma. HBV infection varies markedly in different geographic areas, and chronic HBV prevalence can range from 0.2 to 15%. The main modes of HBV transmission are perinatal exposure to an infected mother, horizontal, parenteral and sexual, and the relative rates of these vary throughout the world. Parenteral and sexual transmission predominates in industrialized countries, whereas horizontal and perinatal transmission predominates in developing countries. 1,2 During the viral infection many serological markers appear. One of these markers is the anti-hbc. The hepatitis B core antigen (HBcAg) is an internal component of hepatitis B virus which is antigenically distinct from hepatitis B surface antigen (HBsAg). HBcAg/anti-HBc antigen-antibody system was described by Almeida et al. in Anti-HBc can usually be detected in the blood just after the detection of HBsAg and before the onset of clinically apparent hepatitis. In acute hepatitis B with recovery, anti-hbc is the only serological marker of infection in the period between HBsAg clearance and the appearance of anti-hbs. The anti-hbc antibodies may persist for life while the antibodies anti-hbs in some patients can became negative. In chronic hepatitis B the anti-hbc is detected with HBsAg, but in some individuals this antigen can become undetectable. Blood donations taken when anti-hbc is the only serological marker of HBV may be infectious 4 and it has been suggested that screening for anti-hbc as well as for HBsAg might further reduce the incidence of post transfusion hepatitis B. 5 In fact, screening for anti-hbc of blood donations is compulsory in several countries. The measurement of anti-hbc, HBsAg and anti-hbs is recommended in the case of immunodeficient patients, such as HIV positive and immunosuppressed individuals due to the increased risk of hepatitis B reactivation. 6 On the other hand, the screening for anti-hbc should also be performed on any individual before the administration of hepatitis B vaccine to know the immune status in order to vaccinate only the anti-hbc negative individuals. Principle When BIO-FLASH anti-hbc paramagnetic microparticles and the assay buffer are mixed and incubated with the sample, if specific anti-hbc antibodies are present in the sample, they will bind to the HBc antigen coated on the microparticles. A magnetic separation followed by a wash step is done to remove residual sample. Immediately after, a tracer consisting of an isoluminol-labeled rabbit IgG antibody anti-hbc is added and may bind to the HBc antigen coated on the microparticles. After a second incubation, a magnetic separation and a wash step, reagents that trigger the chemiluminescent reaction are added. The emitted light is measured as relative light units (RLU) by the BIO-FLASH luminometer. The RLUs are inversely proportional to the anti-hbc concentration in the sample. The BIO-FLASH utilizes a 4 Parameter Logistic Curve fit data reduction method (4PLC) to generate a Master Calibration Curve (MCC). The MCC is predefined and lot dependent and it is stored in the instrument through the cartridge barcode. With the measurement of calibrators (supplied in a separate kit), the predefined MCC is transformed to a new, instrument specific Working Calibration Curve (WCC). The concentration values of the calibrators are included in the calibrator tube barcodes.

2 Reaction Scheme: Triggers URL Components Reagent cartridge The BIO-FLASH anti-hbc kit contains a reagent cartridge for 100 determinations (REF ). Note: Cartridge design is protected under patent (US D565,741 / EC Design ) Reagent cartridge composition: Cartridge has 4 different vials with the following contents: A. 1 cylindrical vial of microparticle suspension coated with recombinant HBc antigen. Contains <0.1% Sodium azide. B. 1 vial of assay buffer. C. 1 opaque vial of tracer consisting of anti-hbc rabbit IgG labeled with isoluminol. Contains <0.1% Sodium azide. D. 1 empty vial. Preparation See the figure below. Cartridge: Microparticles settle during shipment and storage and require mixing to resuspend. The first time that the cartridge is used, gently invert the cartridge 30 times avoiding foam formation. Bubbles may interfere with the instrument liquid sensors. Check for the complete resuspension of the microparticles. If the microparticles are not totally resuspended continue to invert the cartridge until the microparticles have been completely resuspended. If the microparticles do not resuspend or it is detected that the reagent seal is broken, DO NOT USE THE CARTRIDGE. Once the microparticles have been resuspended place the cartridge on a solid surface and gently remove the Red Secure Shipping Tab from the cartridge. With the cartridge still on a solid surface, press the two tabs placed on the sides of the piercing cap (grey part) and apply pressure to the top portion of the cartridge until it snaps into a lock position. Once in the locked position the tabs should not be visible. Do not invert the open cartridge. Let the cartridge stand for a period of 5 minutes before loading it onto the instrument. Once the cartridge is placed on the instrument additional periodic mixing is automatically performed on board.

3 Precautions The BIO-FLASH reagents are intended for IN VITRO diagnostic use. For professional use only. Sodium azide may react with lead or copper pipes and plumbing creating highly explosive metal azides. Flush drains with water thoroughly after disposing of the remains of reagents. WARNING: POTENTIALLY BIOHAZARDOUS MATERIAL. Dispose of all used materials in a suitable biohazardous waste container. Please do not reuse nor reintroduce any reagent in the cartridges or vials. Sample collection and storage Use fresh serum (including serum separator tube) or plasma (EDTA, Li-Heparin, Na-Heparin, Na-citrate and ACD). Other anticoagulants should be evaluated before use. Samples should not be heat inactivated. Samples showing visible particles should be clarified by centrifugation. Refer to the CLSI H18-A3 and H21-A5 guidelines for further information on handling, transport, processing and storage of samples. Serum Serum samples can be stored at 2-8 C for 8 days. For longer periods, sera should be frozen at -20 C or colder. Samples can be freeze/thawed up to 3 times. Mix thoroughly after thawing. Inspect all samples for bubbles and remove all of them prior to analysis. Plasma Plasma samples can be stored at 2-8 C for 8 days. For longer periods, plasma samples should be frozen at -20 C or colder. Thaw plasma at 37 C. Thaw samples only once. Mix thoroughly after thawing. Inspect all samples for bubbles and remove all of them prior to analysis. Sample volume The sample volume required to perform a single BIO-FLASH anti-hbc test varies depending on the type of sample container used. A test requires at least 100 µl plus dead volume, which is 200 µl for the recommended sample cup (REF ). Additional materials The following materials are not supplied with the reagent cartridge and must be purchased separately. REF REF BIO-FLASH anti-hbc Calibrators BIO-FLASH anti-hbc Controls Read carefully their corresponding inserts for more information. Do not use other calibrators. The information required by the BIO-FLASH instrument to calibrate the BIO-FLASH anti-hbc assay is included in the barcoded vials. Using controls from other manufacturers may lead to unexpected results.

4 Ensure that the BIO-FLASH instrument has enough quantity of the following consumables on board before running samples, calibrators or controls: REF BIO-FLASH Cuvettes Note: Cuvette design is protected under patent (US D560,816 / EC Design ) REF BIO-FLASH Triggers REF BIO-FLASH System Rinse Instrument / test procedure Refer to the BIO-FLASH Operator s Manual for the complete assay procedure instructions. Calibration Each cartridge barcode contains the Master Calibration Curve (MCC) valid for the reagent lot; however a Working Calibration Curve (WCC) is required for all tests. The WCC is lot specific and valid throughout the shelf life of the lot. A new WCC is indicated when controls report outside the acceptable range or when adjustments are made to the instrument. Read carefully BIO-FLASH Operator s Manual for configuring a WCC. Quality control Two levels of control are recommended for a complete quality control program. BIO-FLASH anti-hbc Controls Negative and Positive are designed for this program. Controls should be analyzed at least once every 24 hours each day of use. Ensure that control results are within the acceptable ranges. A control result not falling within the acceptable range may indicate invalid test results and corrective measures should be taken by the user. Examine all test results generated since obtaining the last acceptable quality control check for this analyte. Recalibration may be indicated. Refer to the instrument s Operator s Manual for additional information. Refer to Westgard et al. for identification and resolution of out-of-control situations. 7 Storage and stability Unopened reagents are stable until the expiration date shown on the cartridge label when stored at 2-8 C in the upright position. Once open the cartridge stability on board the BIO-FLASH instrument or stored at 2-8 C is 8 weeks. Traceability of calibrators and controls The values are reported in AU/mL and were determined over multiple runs on the BIO-FLASH instrument using specific lots of reagents and against an internal standard referenced to the Paul-Ehrlich-Institute anti-hbc IgG (HBc Reference Material 82, IgG-anti-HBc). Interpretation of results The amount of analyte in every sample is determined from the emitted light (RLU) by interpolation in the stored Working Calibration Curve. BIO-FLASH anti-hbc results are reported in AU/mL (Arbitrary units/ml). This is a qualitative test; the numeric value of the result is only indicative of the amount of antibody present. The determination of anti-hbc, using the BIO-FLASH anti-hbc assay, is interpreted as follows: samples with a result < 0.90 AU/mL are considered non reactive (negative). samples with a result 0.90 and < 1.00 AU/mL are considered indeterminate (grey zone). samples with a result 1.00 AU/mL are considered reactive (positive). Samples with initially reactive or indeterminate result must be reanalyzed in duplicate. If both retest values are lower than 0.9, the final interpretation of the test is negative for anti-hbc antibodies. If either duplicate retest result is positive or indeterminate, the sample should be further investigated with supplemental test and the results used in conjunction with other data; e.g., symptoms, clinical history. A repeatedly positive result is indicative of HBV infection. Samples with an anti-hbc concentration exceeding AU/mL are reported as > Limitations The result of a single sample is not sufficient for the diagnosis of infection with HBV and therefore the BIO-FLASH anti-hbc results should be used in conjunction with other data; e.g., symptoms, clinical history, results of other tests and other appropriate information. A negative result does not exclude the possibility of exposure or infection with HBV. As in all sensitive immunoassays, there is the possibility that non-repeatable positive results occur.

5 Expected results The anti-hbc antibody is the most frequent marker of HBV virus infection because it is found in acute, chronic or past infection. The prevalence of anti-hbc and other markers of Hepatitis B infection vary widely in the world, from high (70-95%, Africa, Asia and Western Pacific) to intermediate (20-55%, Southern and Eastern Europe) and low (4-6%, Western Europe, North America and parts of South America). 2 The implementation of immunization programs should decrease the incidence of HBV infection. Performance characteristics NOTE: The following data are representative; results in individual experiments may vary from these data. Specificity Specificity assessment was based upon testing unselected blood donor serum samples, including first time donors, and hospitalized patients. A total of 5097 non-selected serum samples from two Blood Banks were tested fresh, 3536 from EFS Centre Atlantique site de Tours (France) and 1561 from EFS Nord de France Lille. The samples Repeatedly Reactive were retested using another commercial test. Then specificity was calculated, with the results shown in the following table: Specimens tested Initially reactive (IR) Repeatedly reactive (RR) RR that were indeterminate RR that were confirmed positive Specificity n n n n n % Blood donors Hospitalized patients Sensitivity A panel of samples from different sources with verified anti-hbc positivity was tested with BIO-FLASH anti-hbc in comparison with a commercially available EIA and CLIA anti-hbc methods. This panel includes 28 fresh same day samples ( 1 day after sampling). The results and sensitivity are shown in the following table: Specimens Reference Method tested Reactive Sensitivity n n % EIA method CLIA method In the case of the 403 samples evaluated versus EIA-method, two samples were in the grey zone for BIO-FLASH and another one for the reference method. For this reason, those samples were not taken into account for the sensitivity calculation. Seroconversion sensitivity Additional sensitivity studies were carried out by testing 3 Hepatitis B seroconversion panels. The BIO-FLASH anti-hbc results were in agreement with the results of other commercial methods for all samples. Precision Within run and total (run to run and day to day) precisions were assessed (following CLSI EP05-A2 Guidelines) over multiple runs. Results are summarized in the following table: Mean (AU/mL) Within run Total Negative Control SD SD Positive Control % CV 5.8% CV Around Cut-Off Level % CV 7.3% CV Reproducibility Repeatability between replicates, between lots and between instruments was assessed using 50 samples. Results are shown in the following table:

6 n Co-negativity Co-positivity Replicate 2 vs. Replicate % 100% Lot 2 vs. Lot % 100% Instrument 2 vs. Instrument % 100% Interferences As assessed by the CLSI EP7-A2 Guidelines, BIO-FLASH anti-hbc results are not affected by the following potentially interfering substances: Potentially Interfering substance Concentration % Interference Hemoglobin 500 mg/dl 10 Free bilirubin 18 mg/dl 10 Complexed bilirubin 18 mg/dl 10 Triglycerides 1300 mg/dl 10 Rheumatoid factor 800 IU/mL 10 HAMA 1 µg/ml 10 Cross reactivity A total of 111 positive specimens for a range of potential cross-reactants and samples from pregnant women were analyzed with BIO-FLASH anti-hbc. It is considered that a potential cross-reactant causes cross-reactivity when the diagnostic changes from negative to positive or vice versa. The reactive samples were compared to a reference assay in order to evaluate the agreement between assays. Results are summarized in the following table: Potential Cross-Reaction Agreement ANA (Anti-Nuclear Antibodies) 5/5 anti-cmv (Cytomegalovirus) 6/6 anti-e. coli 12/12 anti-ebv (Epstein-Barr Virus) 4/4 anti-hav-igg (Hepatitis A Virus) 6/6 anti-hav-igm (Hepatitis A Virus) 5/5 anti-hdv IgG (Hepatitis D Virus) 5/5 anti-hev (Hepatitis E Virus) 4/4 anti-hiv (Human Immunodeficiency Virus) 7/7 anti-hsv (Herpes Simplex Virus) 5/5 anti-rubella 10/10 anti-toxoplasma 4/4 anti-vzv (Varicela Zoster Virus) 5/5 Elevated IgG 5/5 Elevated IgM 4/4 Mononucleosis 4/4 Pregnant 7/7 Pregnant (multiparous) 5/5 RF (Rheumatoid factor) 5/5 RPR (Rapid Plasma Reagin) 3/3 Limit of Detection and Analytical Sensitivity Assessed by the CLSI EP17- A Guidelines, the Limit of Detection (LoD) is set at 0.39 AU/mL. Analytical Sensitivity obtained was 0.74 PEI-U/mL. It was determined by interpolating the assay cut-off in the regression line obtained by testing serial dilutions of anti-hbc standard of the Paul-Ehrlich-Institute (HBc Reference Material 82, IgG-anti-HBc). According to internal studies with the BIO-FLASH anti-hbc, 1.00 PEI-U/mL is equivalent to approximately 1.4 IU/mL of the WHO First International Standard for anti-hbc (NIBSC code 95/522). Prozone effect The BIO-FLASH anti-hbc is a two-step inhibition assay. The test protocol includes a washing step after the sample incubation that precludes the prozone effect. Experimentally no prozone effect was observed with a sample as high as AU/mL.