Evaluation Report MHRA Four anti-hbc assays. 60 (Free to NHS) MHRA Report number MHRA Evaluation Report. Crown Copyright.

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1 November 2003 NUMBER Evaluation Report MHRA Four anti-hbc assays MHRA Evaluation Report MHRA Report number Crown Copyright 60 (Free to NHS)

2 WHAT YOU CAN EXPECT FROM MHRA EVALUATION REPORTS The Device Evaluation Service (DES) aims to provide independent and objective evaluations of medical devices available on the UK market. Specialist centres, mainly in NHS Trusts, do the evaluations under long-term contract to, and in accordance with, protocols approved by the MHRA. The evaluations are usually of a unit supplied by the manufacturer. We would expect this unit to be representative of the product on the market but cannot guarantee this. Prospective purchasers should satisfy themselves with respect to any modifications that might be made to the product type after MHRA s evaluation. The reports are intended to supplement, not replace, information already available to prospective purchasers. Medicines and Healthcare products Regulatory Agency (MHRA) The Medical Devices Agency has merged with the Medicines Control Agency to form the Medicines and Healthcare products Regulatory Agency. Crown Copyright 2003 Apart from any fair dealing for the purposes of research or private study, or criticism, or review, as permitted under the Copyright, Designs and Patents Act, 1988, this publication may only be reproduced, stored or transmitted in any form or by any means with the prior permission, in writing, of the Controller of Her Majesty s Stationery Office (HMSO). Enquiries concerning reproduction outside those terms should be sent to HMSO at the undermentioned address: The Copyright Unit, Her Majesty s Stationery Office, St. Clements House, 2-16 Colegate, NORWICH, NR3 1BQ.

3 Four anti-hbc assays Rachel White Emma Delieu Keith R Perry Microbiological Diagnostics Assessment Service Evaluations and Standards Laboratory Health Protection Agency - Colindale 61 Colindale Ave London NW9 5HT John V Parry Sexually Transmitted and Blood Borne Virus Laboratory Health Protection Agency - Colindale

4 Contents Summary Introduction Description of the assays Method Specimen panel Results Technical appraisals Conclusions Acknowledgements References Appendix Evaluation protocol Description of assays (tables 27-28) Claims for the assays (tables 29-32) CO/S ratios for seroconversion panels (tables 33-36) CO/S ratios for dilution series (table 37) CO/S ratios for two production batches (tables 38-43) Contact details for manufacturer and UK agent...66 Manufacturer s comments How to obtain MHRA evaluation reports

5 Summary Background Four commercial anti-hbc assays, AxSYM CORE, Bioelisa anti-hbc, Murex anti-hbc (total) and Vitros anti-hbc, were evaluated to determine their ability to detect antibody to hepatitis B virus core antigen (anti-hbc). This investigation was undertaken by the Microbiological Diagnostics Assessment Service (MiDAS). Evaluation panel The evaluation panel consisted of 1,405 specimens. Of these, 500 specimens were from blood donors, 500 from injecting drug users (IDUs), 85 HBsAg positive/ IgM anti-hbc negative serum specimens, 50 antenatal serum specimens, 63 specimens from four commercial anti-hbc seroconversion panels, 15 specimens from one commercial anti-hbc performance panel, 59 specimens that were dilutions of ten positive anti-hbc specimens and replicates of a single 'total' anti-hbc quality control sample. A subset of the main panel was used to assess a second production lot of each assay. Specificity findings Specificity was determined for each assay by testing approximately 500 specimens from blood donors and 180 anti-hbc negative specimens from IDUs. Murex anti-hbc (total) had no repeatedly reactive specimens in either specimen category, giving 100% specificity. For AxSYM CORE one specimen was repeatedly false reactive, giving a 99.9% specificity. The Bioelisa anti- HBc and Vitros anti-hbc each had three repeatedly false reactive specimens, a specificity of 99.6%. Sensitivity findings Randomly selected positives Sensitivity was determined by testing 316 anti-hbc positive specimens from IDUs. Of these, Murex anti-hbc (total) detected 314 giving a sensitivity of 99.4%, AxSYM CORE detected 312 (98.7%), Vitros anti-hbc detected 301 (95.3%) and Bioelisa anti-hbc detected 295 (93.4%). Seroconversion panels Four anti-hbc seroconversion panels were tested and the seroconversion sensitivity of the anti- HBc assays were compared by employing two scoring systems. In the first system the number of specimens found to be reactive for each of the seroconversion panels was added to obtain an aggregate score. With this system the AxSYM CORE, Bioelisa anti-hbc and Vitros anti-hbc assays each had scores of 30, and the Murex anti-hbc (total) had a score of 26. To provide better discrimination between the performance of each assay a second approach was employed in which the highest score was awarded to the assay that first became positive with the highest level of activity, and the lowest score to the assay that became positive last and with the lowest reactivity. With this system the scores were 16 for Bioelisa and Vitros anti-hbc, 13 for AxSYM CORE and 10 for Murex anti-hbc (total). Dilution series Ten anti-hbc positive specimens were diluted from 1/50 to 1/500,000 in anti-hbc negative human serum and the analytical sensitivity of each of the anti-hbc assays was compared by 2

6 Summary employing two scoring systems as with seroconversion panels. By adding the number of positives found in each dilution series, the most sensitive anti-hbc assays were AxSYM CORE with 37 positives, Murex anti-hbc (total) with 36 and Bioelisa anti-hbc and Vitros anti-hbc with 34. The second scoring system awarded the highest score to the assay that remained positive to the highest titre and with the highest level of activity. The scores obtained were 41 for AxSYM CORE, 39 for Murex anti-hbc (total), 25 for Vitros anti-hbc and 23 for Bioelisa anti-hbc. Evaluation of assay cut-off The delta values for Murex anti-hbc (total) were evenly distributed (overall positive delta 3.63, overall negative delta -4.43), indicating that its cut-off has been set to optimise both sensitivity and specificity. The delta values for the AxSYM CORE, Bioelisa anti-hbc and Vitros anti-hbc assays show the negative delta value is substantially higher than the positive delta value, indicating a bias in the assays performance that favours specificity over sensitivity. The sensitivity and specificity data on the specimens from IDUs and blood donors bear this out, with the Murex anti-hbc assay providing the best overall accuracy. Comparison of two production lots A subset of the main evaluation panel, consisting of 167 specimens, was used to compare two production lots of each anti-hbc assay. There was generally close agreement between results for the two lots. The only qualitative differences between lots were observed for the Vitros anti- HBc assay and the AxSYM CORE. In the first production lot evaluated Vitros anti-hbc detected 34 anti-hbc positive specimens from IDU's and the second detected 36 anti-hbc positive specimens from IDU's. For the AxSYM CORE assay one less reactive sample was detected from the dilution series in the second production lot compared with the first. Conclusion The sensitivity of the anti-hbc assays varied from % for the anti-hbc IDU specimens, but the most sensitive kits in this analysis were not necessarily the most sensitive in terms of seroconversion sensitivity or detection of anti-hbc positive dilutions. Despite the variability, it is likely that all of the anti-hbc kits examined could have a role in HBV confirmatory testing algorithm, further to characterise HBsAg positive specimens and to assist in determining whether HBV infection is past or current. In many countries anti-hbc assays are used to screen blood donations. In this evaluation all anti-hbc assays had specificities of more than 99% and would therefore be good candidates for blood screening. 3

7 Introduction Antibody to Hepatitis B virus core antigen (anti-hbc) is a marker of past and current Hepatitis B virus (HBV) infection (1). It is the first HBV antibody marker to appear, and persists for many years following initial infection. During HBV infection, a complex and evolving array of antigens and antibodies may be detected in serum and other biological fluids. Uncomplicated acute HBV infection is characterised by the presence of HBV surface antigen (HBsAg) in serum and the development of IgM anti-hbc (2). During convalescence HBsAg is cleared and IgG anti-hbc, followed by anti-hbs (antibody to HBV surface antigen) develop. In chronic HBV infection HBsAg remains present and anti-hbc is present, but not anti-hbs. Occasionally anti-hbc may be the only HBV-specific marker detected. The finding of anti-hbc reactivity alone most often reflects a waning antibody response in a historic infection but, particularly when at high titre, it may be due to resolution of acute or chronic HBsAg carriage prior to the appearance of anti-hbs. As such, a donor whose specimen has such a profile may still be infectious (3,4). In the clinical laboratory anti-hbc tests are employed as part of a HBV testing algorithm, used to identify past and current HBV infection, and further to characterise HBsAg positive specimens. Currently, UK blood centres do not screen for anti-hbc and rely on highly sensitive HBsAg assays alone to identify donations that may transmit HBV. A number of other countries, including several European countries and the USA, screen blood donors for anti-hbc. Anti-HBc only positive blood donations, with no detectable HBsAg and anti-hbs, may be the source of posttransfusion HBV infection, which anti-hbc screening could prevent (5). Radioimmunoassays (RIA) for anti-hbc have largely been replaced by enzyme immunoassays (EIA) which have been shown to have similar sensitivity and specificity (6). However, further studies have shown greater variation in the sensitivity and specificity between anti-hbc EIAs (7,8). MiDAS has evaluated four of the currently available commercial anti-hbc total (IgM and IgG anti- HBc) kits. This follows on from the evaluation of HBsAg kits since They include two automated immunoassays, AxSYM CORE and Vitros anti-hbc and two microplate EIAs, the Bioelisa anti-hbc and Murex anti-hbc (total). All four assays are based on a competitive EIA format. Each assay was evaluated against the same serum specimen panel, which had been constructed to minimise potential bias. The panel included serum samples from blood donors to determine each assay s specificity. Serum specimens from injecting drug users and antenatal patients consisted of anti-hbc positive and negative sera to determine sensitivity and specificity. Seroconversion panels were used to test the kits ability to detect anti-hbc at the time of seroconversion. Anti-HBc dilution series were included to test the analytical sensitivity of the kits. HBsAg positive/ IgM anti-hbc negative sera were added as a further challenge of the kits ability to detect anti-hbc. Quality control sera were used throughout the evaluation to assess inter-run variation. A smaller panel of specimens was tested in a second lot of each kit to assess lot-tolot reproducibility. 4

8 Description of the assays A summary of the different assays, which includes details of the manufacturer and product number, is provided in table 1. A comparison of the main characteristics of the assays such as specimen volumes and the main components of the assays is shown in table 2. Information on the different assay stages and any additional equipment required can be found in the Appendix. Quotations from the instructions supplied with the kit, including details about the performance of the assays and their limitations can also be found in the Appendix. Figure 1 shows the basic principle of the competitive immunoassay format used in the anti-hbc assays. Each specimen is added to a HBcAg coated solid phase and any anti-hbc will bind to the HBcAg. An anti-hbc conjugate is then added to the solid phase and will bind to any free HBcAg sites on the well. If anti-hbc is present in the specimen there will be little or no reaction from the conjugate as it will not have been able to bind to the well. If no anti-hbc is present in the specimen there will be a strong reaction as conjugate is able to bind to the well. Table 1: Assays evaluated Assay name Manufacturer/ UK agent Product number Tests per kit UK launch date AxSYM CORE Abbott Diagnostics Ltd 7A tests Bioelisa anti-hbc Biokit, S.A tests 1990 Murex anti-hbc (total) Abbott-Murex Diagnostics 8G21-01/ 8G tests/ 480 tests Vitros anti-hbc Ortho-Clinical Diagnostics tests December 1999 Figure 1: Principle of competitive assay format Anti-HBc +ve * * Competitive Assay Anti-HBc -ve * * * * * * HBc antigen Anti-HBc Non-specific antibodies * Conjugate (enzyme-labelled IgG anti-hbc) 5

9 Description of the assays Table 2: Assay characteristics Assay name AxSYM CORE Bioelisa anti-hbc Murex anti-hbc (total) Vitros anti-hbc Assay type competitive microparticle competitive microplate competitive microplate competitive enzyme enzyme immunoassay enzyme immunoassay enzyme immunoassay immunoassay Sample volume 186µl + 50µl void volume = 50µl + 70µl void volume 50µl 50µl 236µl = 120µl Solid phase rhbcag coated microparticle rhbcag coated wells rhbcag coated wells rhbcag coated wells Conjugate mouse monoclonal antianti-hbc (human) conjugated rabbit IgG anti-hbc monoclonal anti-hbc HBc conjugated to to alkaline phosphatase conjugated to HRP conjugated to HRP HRP Substrate Controls per run Cut-off Computation 4-Methylumbelliferyl phosphate (MUP) 1 anti-hbc positive 1 anti-hbc negative index calibrator (mean rate) / 2 (reactive = S/CO < 1.00) Chromogen (TMB) 2 anti-hbc positive 4 anti-hbc negative 2 blank wells (mean negative control + mean positive control)x0.4 (reactive <1.00) Chromogen (TMB) 2 anti-hbc positive 2 anti-hbc negative (mean negative control + mean positive control)/2 (reactive < 1.00) Luminogenic substrates 1 anti-hbc positive 1 anti-hbc negative Lot specific calibration determines cut-off. (reactive <1.00) Equivocal zone none +/- 10% of cut-off none >1.00 and <1.2 Notes: rhbcag = Recombinant Hepatitis B virus core antigen TMB = 3, 3', 5, 5' - tetramethylbenzidine HRP = Horseradish peroxidase 6

10 Description of the assays Abbott AxSYM CORE AxSYM CORE kit components AxSYM CORE is a Microparticle Enzyme Immunoassay for the qualitative detection of anti-hbc in human serum and plasma. The assay uses microparticles coated with recombinant Hepatitis B virus core antigen (rhbcag) as the solid phase. The conjugate is antibody to Hepatitis B virus core antigen (human) conjugated to alkaline phosphatase. Both of these are contained within the reagent pack, which has sufficient volume for 100 tests, and must be stored at 2-8 C (figure 2). Once the reagent pack is loaded onto the AxSYM analyser it can be used for a maximum of 112 cumulative hours, after which time it must be discarded. The 4-methylumbelliferyl phosphate (MUP) substrate should be stored at 2-8 C when not in use. Once loaded onto the analyser the substrate may be used for 14 days, after which time it must be discarded. The single index calibrator is supplied in ready to use (liquid) form with each reagent pack. A new calibration is required for each new lot of reagent and if any of the AxSYM CORE controls give a result outside the specified range. Once a calibration has been accepted, all the subsequent samples using this lot of reagents may be tested without further calibration. The AxSYM CORE controls are supplied as a pack containing one anti-hbc positive control and one anti-hbc negative control, both supplied in liquid ready to use form. The controls may be stored at 2-8 C until the expiry date on the packaging. Each of the positive and negative controls should be tested at least once every 24 hours for every day of use. The AxSYM CORE control values must be within the acceptable ranges specified in the package insert. If a control value is out of range, the test results are invalid and the specimens must be retested. AxSYM CORE procedure The basic procedure of the AxSYM CORE assay involves incubating in one well, sample, sample diluent and the microparticles coated with recombinant Hepatitis B virus core antigen (rhbcag). Should anti-hbc be present it will bind to the rhbcag coated microparticles and form an antigenantibody complex. A portion of this reaction mixture is transferred to the matrix cell, where the microparticles bind irreversibly to the glass fibre matrix. The anti-hbc (human) alkaline phosphatase conjugate is dispensed onto the matrix cell and binds with the rhbcag antigenic sites on the microparticles which are not already occupied by anti-hbc from the sample. The matrix cell is washed to remove materials not bound to the microparticles and the substrate, 4- methylumbelliferyl phosphate is added. The alkaline phosphatase conjugate will produce a fluorescent product, which is measured by the AxSYM analyser MEIA optical assembly. The AxSYM analyser The AxSYM analyser is an automated, random access, closed system, ie only dedicated assays may be run on it (figure 3). The analyser has four main areas: sampling centre, processing centre, waste and supply centre and system control area. In the sampling centre, samples and reagents are pipetted into reaction vessels that are subsequently transferred into the processing centre. The processing centre is a temperature controlled environment whose function is to mix and transport the samples and reagents, incubate the reaction mixtures, perform optical measurements and unload used consumables to the waste centre. The waste and supply centre stores and delivers bulk solutions and collects 7

11 Description of the assays and stores liquid and consumable waste. The system control centre consists of a touch screen monitor, keyboard, printer and software that is used to control the analyser for imputing sample details, viewing results, monitoring QC parameters and performing maintenance procedures. Figure 2: Axsym CORE reagents Figure 3 : AxSYM analyser 8

12 Description of the assays Bioelisa anti-hbc Bioelisa anti-hbc is a competitive immunoenzymatic method for the determination of anti-hbc in human serum. The Bioelisa anti-hbc kit components are shown in figure 4 and should be stored at 2-8 C. The microtitre plate should be brought to room temperature inside the bag before opening and any unused strips stored in a tightly sealed bag at 2-8 C. Washing solution must be diluted 1/10 in distilled water and is stable for 2 weeks if stored at 2-8 C. The substrate-3, 3, 5, 5 -tetramethyl benzidine (TMB) solution is prepared 5-10 minutes before use, is not stable once prepared, and should be stored protected from light. The procedure for the Bioelisa anti-hbc involves a competitive reaction between antibodies, if present, in the sample and conjugate. An aliquot of sample is added to the appropriate well in the plate, the rabbit IgG anti-hbc conjugated to horseradish peroxidase is added to each well and left to incubate. The plate is washed to remove unbound material. Substrate-TMB solution is added to each well and will react with any conjugate bound to the well. The reaction is stopped by adding sulphuric acid and read at 450nm with 630nm reference filter within 30 minutes. Figure 4: Bioelisa anti-hbc kit 9

13 Description of the assays Murex anti-hbc (total) Murex anti-hbc (total) is a competitive enzyme immunoassay for the detection of anti-hbc in human serum or plasma. The Murex anti-hbc (total) kit components should all be stored at 2-8 C (figure 5). The microtitre plate should be brought to room temperature inside the bag before opening and any unused strips should be stored in a tightly sealed bag at 2-8 C. The sample diluent, once opened, is stable for 1 week at 2-8 C and must not be opened for use more than 3 times. Substrate concentrate is diluted in an equal volume of substrate diluent and is stable at 2-8 C or C for 2 days. The wash fluid is diluted 1 in 20 with distilled water and may be stored at C for one month. The basic procedure of the Murex anti-hbc (total) assay involves adding sample diluent to every well, then adding samples and controls to the appropriate wells. The plate is left to incubate to allow any anti-hbc in the sample to bind to the rhbcag coated wells. The plate is washed to remove any unbound material, then monoclonal anti-hbc conjugated to horseradish peroxidase is added to every well and incubated to allow the conjugate to react with rhbcag sites that are not already occupied. The plate is again washed and substrate-tmb solution is added to every well, which will react with any conjugate present. The reaction is stopped by adding sulphuric acid and read at 450nm with 630nm reference filter within 15 minutes. Figure 5: Murex anti-hbc (total) kit 10

14 Description of the assays Vitros anti-hbc Vitros anti-hbc kit components The Vitros anti-hbc is an automated immunoassay for the qualitative detection of anti-hbc in human serum or plasma. The assay is performed on the Vitros ECi Immunodiagnostic system using the anti-hbc reagent pack, signal reagent, calibrator and controls supplied (figure 6). All assay components must be stored at 2-8 C. The reagent pack consists of 100 wells coated with rhbcag, assay reagent and horse radish peroxidase (HRP)-mouse monoclonal anti-hbc conjugate. Once opened the reagent pack may be stored at 2-8 C for up to 13 weeks. The signal reagent consists of luminogenic substrates and once loaded onto the system must be used within 7 days. The calibrator is lot specific and must be used with reagent packs of the same lot. Each calibrator can be used for 6 calibrations and a calibration is performed with every new lot and every 28 days thereafter. After opening the calibrator it may be stored 2-8 C for up to 13 weeks. The positive and negative controls are tested with every calibration and a minimum of once every 24 hours. Once opened they may be stored at 2-8 C for up to 5 days. Vitros anti-hbc procedure The principle of the Vitros anti-hbc assay involves incubating an aliquot of sample in a rhbcag coated well. Any anti-hbc in the sample will bind to the rhbcag on the wells surface and unbound material is removed by washing. HRP-mouse monoclonal anti-hbc conjugate is added to the well and will react with any available rhbcag. Conjugate that does not bind to the well is removed by washing. The luminogenic substrate solution is then added to the wells and will react with HRP in any bound conjugate. Any light emitted is read by the Vitros system. Vitros ECi Immunodiagnostic system The Vitros ECi Immunodiagnostic system, illustrated in figure 7, is an automated, random access, closed system, which means only dedicated assays may be run on it. The system is controlled via a computer keyboard and touch screen monitor. The icon driven touch screen monitor allows the user to enter patient samples, order tests, monitor the status of the sample and perform routine maintenance. The operational unit of the system consists of the sample supply centre, the reagent management centre and the processing centre. In the sample supply centre the sample metering probe aliquots the sample from the sample cup to the well in the processing centre. The reagent management centre stores the reagent packs in a temperature controlled environment, dispenses the wells to the processing centre and the reagent metering probe will dispense reagent into the well. Inside the processing centre the well contents will be mixed and incubated, wells washed with universal wash reagent, signal reagent added, and the luminometer will perform the readings of the chemiluminescent reaction in the wells. 11

15 Description of the assays Figure 6: Vitros anti-hbc assay Figure 7: Vitros ECi Immunodiagnostic system 12

16 Description of the assays Supplementary assays In house radioimmunoassay (RIA) for total anti-hbc (COMPRIA) The in house RIA for anti-hbc total (COMPRIA) is based on the competitive format. The RIA was used for the initial characterisation of the anti-hbc specimen panel and as an independent reference assay. The RIA HBcAg coated wells, controls and radiolabel diluent are stored at -30 C and brought to room temperature before use. The working dilution of radiolabelled anti-hbc conjugate is prepared on the day of use. The procedure involves adding 50µl of each patient s serum specimen and assay controls to the appropriate HBcAg coated well and incubating for 30 minutes at room temperature to allow any anti-hbc to bind to the wells. A sufficient volume of 125I labelled anti-hbc concentrate is added to the label diluent to give a solution of 80, ,000 counts per minute (CPM) per 50µl. After incubation 50µl of radiolabelled anti-hbc is added to the wells and left to incubate overnight at room temperature in a moist chamber and will compete for binding to the HBcAg coated wells with any antibody in the specimen. The next day the plates are washed to remove any unbound radiolabel, and the amount of radiolabel bound is measured with a gamma counter. Results from the COMPRIA are normally expressed as % inhibition but were converted to CO/S to compare with the other anti-hbc assays in this evaluation. On a few occasions the raw count was not available when conducting the data analysis, and the results were only given as % inhibition. HBsAg and anti-hbs assays The Murex HBsAg version 3 (product number GE34/36, lot H682910) and Murex anti-hbs (product number GE20/23, lot H666011) were used as supplementary HBV assays. If the commercial anti-hbc assays gave repeatedly discordant results the samples were tested by these assays to assist with the interpretation of the results. This manufacturer was chosen for the supplementary HBV assays as the Murex HBsAg version 3 was one of the most sensitive and specific assays in previous MHRA HBsAg evaluations (9). No MHRA evaluations have been performed on anti-hbs kits. 13

17 Method The evaluation was carried out according to a protocol agreed by the manufacturers (see Appendix). Each manufacturer supplied 2 lots of their anti-hbc assays for evaluation, details of which are listed in table 3. The kits were tested against a panel of serum specimens designed to be minimally biased and comprised 500 specimens from injecting drug users and 500 specimens from blood donors to determine assay sensitivity and specificity. The panel also included commercially available anti-hbc seroconversion panels to challenge the ability of the anti-hbc assays to detect anti-hbc at the time of seroconversion. Anti-HBc dilution series were included to challenge the ability of the anti-hbc assay to detect increasing titres of anti-hbc. Quality control sera were added to each plate or run to monitor intraplate and interplate variation. Antenatal samples and HBsAg positive/ IgM anti-hbc negative samples were a final set of specimens added to challenge the ability of the assays to detect anti-hbc (table 4a). A smaller panel of specimens were tested on a second lot of the assay to assess lot-to-lot variation (table 4b). Each test was performed according to the manufacturer s instructions. Prior to the start of the evaluation training was provided on the use of the AxSYM analyser and the Vitros ECi Immunodiagnostic system by the relevant companies. For the Bioelisa and Murex assays the plates were washed with a Tecan Columbus washer. The optical densities were read by a Bio- Tek EL808 ultra microplate reader linked to a computer with KC4 software. Before testing the specimen panel a performance/ washer efficiency test was conducted to check for carryover. This involved testing multiple replicates of a strong anti-hbc positive specimen interspersed with replicates of an anti-hbc negative specimen. The results of the anti-hbc assays were compared and any specimen that had given discordant results were retested in duplicate. If the results were repeatedly discordant the specimens were tested on the HBsAg and anti-hbs supplementary assays to help determine the anti-hbc status of each specimen. The results of the anti-hbc tests were converted from S/CO to CO/S to allow us to compare the results with other kits. Employing this convention, a CO/S greater than 1.0 is reactive, and less than 1.0 is negative. A draft of this report was sent out to each manufacturer in November 2003, allowing them the opportunity to comment prior to the publication of the final report. The response from the manufacturer s who replied are reproduced in the Appendix. Table 3: Batch numbers for anti-hbc assays Assay name Lot 1 Expiry date Lot 2 Expiry date AxSYM CORE 90064LU02 05/12/ LU02 20/01/2003 Bioelisa anti-hbc I /08/2003 A /01/2004 Murex anti-hbc (total) H /04/2003 H /04/2003 Vitros anti-hbc /07/ /10/

18 Specimen panel Table 4a: Main specimen panel for the evaluation of anti-hbc assays Sample category 1. Blood donors'/healthy adults' sera Number Negative human serum (NHS) 2x 1 3. Injecting drug users (IDUs) HBsAg 'resolving carrier' specimens (NLBC and BPL) 2x 3 5. HBsAg positive, IgM anti-hbc negative Seroconversion panels: total anti-hbc BBI - PHM935A 20 BCP BCP Profile - RP Performance panels: total anti-hbc BBI - PHE102 (IgM anti-hbc low titre) Dilution of anti-hbc positive (9 specimens x 6 dilutions - 1 specimen x 5 dilutions) 2x Quality control samples HPA - total anti-hbc QC serum: sample 1 6x 1 Three replicates of suitable controls on each plate (estimate) Ante-natal specimens 50 TOTAL (number of tests) 1405 Notes: BBI = Boston Biomedica Inc, USA; BPL = BioProducts Laboratory, UK BCP = BioClinical Partners, USA Profile = Pyramid Profile Diagnostics, USA NLBC = North London Blood Centre, UK HPA = Health Protection Agency, UK 15

19 Specimen panel Table 4b: Specimen panel for the assessment of a second batch of the anti-hbc assays Sample category 1. Blood donors'/healthy adults' sera Number Negative human serum (NHS) 2x 1 3. Injecting drug users (IDUs) HBsAg 'resolving carrier' specimens (NLBC and BPL) 2x 3 5. HBsAg postive, IgM anti-hbc negative Dilution of anti-hbc positive (3 specimens x 6 dilutions) 2x Quality control samples PHLS - total anti-hbc QC serum: sample 1 3x 1 Three replicates of suitable controls on each plate (estimate) 10 TOTAL (number of tests) 167 Notes: BPL = BioProducts Laboratory, UK NLBC = North London Blood Centre, UK HPA = Health Protection Agency, UK 16

20 Results Key to the presentation of results The presentation of results is intended to allow readers to draw their own conclusions from the data on the assay. The data are presented as tables are figures summarising: Blood donor specimen results (tables 5-7 & figures 8-11) Anti-HBc reactivities for specimens from injecting drug users (tables 8-12 & figures 12-15) Assay cut-off evaluation (table 13) Antenatal specimen anti-hbc reactivities (tables 14-16) Detection of anti-hbc in HBsAg positive/ IgM anti-hbc negative specimens (tables 17-18) Detection of anti-hbc in seroconversion panels (tables & figures 16-19) Detection of anti-hbc in dilution series (table 21) Summary of specificity findings (table 22) Summary of sensitivity findings (table 23) Quality control sera (table 24 & figures 20-23) Manufacturers kit controls (table 25) Comparison of two production lots (table 26 & figures 24-27) 17

21 Results Blood donor specimen results The four commercial anti-hbc assays were evaluated against 500 blood donors specimens to determine their specificities (table 5). AxSYM CORE and Murex anti-hbc were tested on one set of blood donors specimens and the Bioelisa anti-hbc and Vitros anti-hbc on a second set. Any of these specimens found to be reactive by an assay were retested in duplicate. If the specimen was repeatedly reactive it was tested by HBsAg and anti-hbs supplementary assays to determine more precisely the HBV status. Specimens that were confirmed to be reactive for another HBV marker were removed from the specificity calculation and the results can be seen in table 6. A summary of the findings on falsely reactive specimens is shown in table 7. Four specimens were found to be repeatedly reactive with the AxSYM CORE assay, three were confirmed anti-hbc reactive specimens and one could not be tested by supplementary assays, and were removed from the specificity calculation. This gave the AxSYM CORE assay a specificity of 100% (95% confidence interval %). Of the four specimens removed from the from the AxSYM CORE specificity calculation two samples did not contain enough volume to be tested by Murex anti-hbc assay and replaced by new blood donors. The Murex anti-hbc assay had the two repeatedly reactive specimens, which were confirmed to be anti-hbc positive, also giving the Murex anti-hbc assay a specificity of 100% (95% confidence interval %). The Bioelisa anti-hbc assay had thirteen initially reactive and six repeatedly reactive specimens, four of which were confirmed anti-hbc reactive specimens. The Vitros assay found the same six repeatedly reactive specimens, giving both assays a specificity of 99.6%. Specimens that were confirmed as anti-hbc reactive were also anti-hbs reactive, indicating a recovered infection. The distributions of the CO/S values given by the anti-hbc negative blood donor specimens for each assay are shown in figures Assays with good discrimination have few, or no, specimens wrongly classified and few reactions in close proximity to the cutoff. The AxSYM CORE had no falsely reactive specimens and the majority of the values were far from the cutoff. The Bioelisa anti-hbc assay had two falsely reactive specimens and the majority of the values were far from the cut-off. The Murex anti-hbc (total) assay had no falsely reactive specimens and and the majority of the values were far from the cut-off. The Vitros anti-hbc assay had two falsely reactive specimens and the majority of the values were also far from the cut-off. The density of anti-hbc positive specimens used in the MiDAS evaluation, where the anti-hbc negative blood donors were interspersed among anti-hbc positive sera, is unlikely to be encountered elsewhere, even in reference centres. The potential for carryover or cross contamination at centres other than MiDAS is therefore less. 18

22 Results Table 5: Specificity findings for the blood donors specimens Total number Bioelisa anti-hbc Vitros anti-hbc Supplementary testing initial repeat initial repeat COMPRIA HBsAg anti-hbs result result result result result result result Assigned Sample ID CO/S CO/S CO/S CO/S CO/S S/CO S/CO status positive 02N positive 02N positive 02N positive AxSYM CORE Murex anti-hbc Supplementary testing initial result Sample ID CO/S CO/S CO/S CO/S inhibition* S/CO S/CO status NT NT 6.50 NT NT unknown** NT NT positive positive positive Notes: NT = Not tested, as not enough specimen remaining ** Sample status unknow n as specimen could not be tested by supplementary assays Bioelisa anti-hbc Vitros anti-hbc Supplementary testing initial result Number initially repeat result repeat result Number repeatedly Assay tested* negative negative (%)** interval (%)** CO/S** CO/S** CO/S** AxSYM Murex Bioelisa Vitros Notes: * Confirmed positive sera w ere removed from total w hen calculating specificity ** Specificity and statisitics are based on the repeat reactive rate initial result initial result Specificity repeat result repeat result 95% Confidence COMPRIA % COMPRIA result HBsAg result HBsAg result Range anti-hbs result anti-hbs result Mean Table 6: Summary of results for the confirmed reactive blood donors specimens. Assigned * COMPRIA % inhibition > 70% is reactive. Data could not be converted to CO/S as raw count data w as not available. Table 7: Summary of results for the repeatedly false reactive blood donors specimens. Median Assigned Sample ID CO/S CO/S CO/S CO/S CO/S S/CO S/CO status negative 02N negative 19

23 Results Figure 8: Distribution of CO/S values for blood donors specimens tested by AxSYM CORE number of samples cut-off = CO/S ranges Figure 9: Distribution of CO/S values for blood donors specimens tested by Bioelisa anti- HBc number of samples cut-off = CO/S range

24 Results Figure 10: Distribution of CO/S values for blood donors specimens tested by Murex anti- HBc (total) number of samples cut-off = CO/S range Figure 11: Distribution of CO/S values for blood donor specimens tested by Vitros anti-hbc number of samples cut-off = CO/S range

25 Results Anti-HBc reactivities for specimens from injecting drug users Specimens from 500 injecting drug users (IDU) consisted of 316 anti-hbc positive and 180 anti- HBc negative specimens and four samples that were removed from the evaluation. Of the four specimens that were removed, no clear anti-hbc status could be established on two specimens, and two negative specimens were not included as no result was obtained on the AxSYM CORE. The procedure for determining the anti-hbc status of each specimen is described at the end of this section. The sensitivity findings for all four commercial anti-hbc assays and the COMPRIA, based on the 316 anti-hbc positive specimens, are shown in table 8. The sensitivity figures quote the repeat reactive rate as retesting was conducted to confirm HBV status, however specimens whose anti- HBc status changed from negative to positive would not normally be detected in a HBV testing algorithm. Murex anti-hbc (total) was the most sensitive assay at 99.4% ( % 95% confidence interval) and the lowest sensitivity was achieved by the Bioelisa anti-hbc at 93.4% (95% confidence interval %). Based on the 180 anti-hbc negative IDU specimens the Murex anti-hbc (total) and the COMPRIA were the most specific kits, both with 100% specificity (98-100% 95% confidence interval) on initial and repeat testing (table 9). The three other kits all had one repeatedly false positive result, giving a specificity of 99.4%. The distribution of the reactivities for IDU specimens tested against the four anti-hbc assays can be seen in figures When specimens were retested because of discordant initial results, the retest result was used for the analysis. Assays that discriminate well between positives and negatives have few, or no, wrongly classified specimens and few reactions in close proximity to the cut-off. AxSYM CORE gave four reproducible false negative results, all just below the cutoff, and one false positive result. Bioelisa anti-hbc gave 21 reproducible false negatives, three of which are classified as borderline by the assay's interpretive criteria, and one false positive. Murex anti-hbc (total) gave three reproducible false negative results and no false positive reactions. Vitros anti-hbc produced 15 false negative results, four of which are classified as borderline by the assay's interpretive criteria, and 1 false positive. The 498 IDU samples were tested on four anti-hbc kits. On initial testing results on 456 (91.2%) specimens were concordant between the four assays and 44 (8.8%) results were discordant. The discordant specimens were retested in duplicate by the discordant assay(s). If only one assay was discordant then only that assay was retested, if more than one assay was discordant then the specimen was retested by all anti-hbc assays. Table 10 shows the 14 specimens that were confirmed anti-hbc positive or negative following repeat testing. The remaining 30 specimens were also tested for HBsAg and anti-hbs. Twenty specimens were positive by one or both of the supplementary assays and they were designated to be anti-hbc positive (table 11). The AxSYM and Murex assays found the majority of these specimens positive and the Bioelisa and Vitros assays only found a few positive specimens. The remaining 10 specimens were negative by the supplementary assays and their anti-hbc status was determined on the basis of the anti-hbc assays (table 12). Four specimens were majority positive and two were majority negative and were assigned that status. Two were assigned positive as the two most sensitive and specific anti-hbc assays, AxSYM CORE and Murex anti-hbc, agreed on the status of the specimen. There were 2 specimens whose status was undetermined as no clear consensus could be decided upon, as the two most sensitive assays did not agree, and the results were not included in the specificity and sensitivity calculation. Overall the IDU specimens were composed of 316 (63.7%) positive specimens and 180 (36.3%) negative specimens. 22

26 Results Table 8: Sensitivity findings for 316 anti-hbc positive IDU specimens Total number Number initially Number repeatedly Sensitivity 95% confidence Range Mean Median Assay tested positive positive (%)* interval (%)* CO/S* CO/S* CO/S* AxSYM Bioelisa Murex Vitros COMPRIA Notes: * figures based on the repeat reactive rate Table 9: Specificity findings for 180 anti-hbc negative IDU specimens Total number Number initially Number repeatedly Specificity 95% confidence Range Mean Median Assay tested negative negative (%)* interval (%)* CO/S* CO/S* CO/S* AxSYM Bioelisa Murex Vitros COMPRIA Notes: * figures based on the repeat reactive rate 23

27 Results Table 10: CO/S values for 14 IDU specimens designated positive or negative following retesting AxSYM CORE Bioelisa anti-bhc Murex anti-hbc Vitros anti-hbc COMPRIA initial repeat initial repeat initial repeat initial repeat initial repeat assigned Sample ID CO/S CO/S CO/S CO/S CO/S CO/S CO/S CO/S CO/S CO/S status NT 0.37 NT 0.59 NT 0.32 NT negative NT 0.37 NT 0.79 NT 0.37 NT negative NT 0.46 NT 0.27 NT 0.39 NT negative NT 0.64 NT 0.29 NT 0.26 NT negative NT NT 0.65 NT 0.37 NT negative NT NT 0.36 NT 0.33 NT negative NT 0.73 NT 0.68 NT 0.61 NT negative NT NT 1.11 NT 1.49 NT positive NT 9.77 NT NT NT positive NT NT 9.09 NT 2.67 NT positive NT NT NT NT positive NT 1.49 NT 6.15 NT NT positive NT 1.30 NT 3.79 NT NT positive NT NT 1.25 NT 1.99 NT positive Note: Positive specimens are shaded grey NT = Not tested 24

28 Results Table 11: CO/S values for 20 IDU specimens assigned positive by supplementary assays AxSYM CORE Bioelisa anti-bhc Murex anti-hbc Vitros anti-hbc COMPRIA Supplementary tests initial repeat initial repeat initial repeat initial repeat initial repeat HBsAg anti-hbs Assigned Sample ID CO/S CO/S CO/S CO/S CO/S CO/S CO/S CO/S CO/S CO/S CO/S CO/S status positive NT 2.79 NT positive positive NT 1.16 NT positive positive positive positive positive positive positive positive positive positive positive NT 1.22 NT 0.45 NT positive NT NT positive positive positive NT 5.22 NT positive NT 4.19 NT positive Note: Positive specimens are shaded grey NT = Not tested 25

29 Results Table 12: CO/S values for 10 IDU specimens whose status was decided by the four anti-hbc assays alone AxSYM CORE Bioelisa anti-hbc Murex anti-hbc Vitros anti-hbc COMPRIA Supplementary tests initial repeat initial repeat initial repeat initial repeat initial repeat HBsAg ahbs Assigned Sample ID CO/S CO/S CO/S CO/S CO/S CO/S CO/S CO/S CO/S CO/S CO/S CO/S status positive NT 3.28 NT positive NT 1.91 NT positive NT 2.09 NT positive NT NT 0.33 NT negative negative NT NT 0.53 NT positive positive undetermined undetermined Note: Positive specimens are shaded grey NT = Not tested 26

30 Results Figure 12: Distribution of reactivities for IDU specimens tested by AxSYM CORE number detected cut-off = CO/S ranges negative samples positive samples Figure 13: Distribution of reactivities for IDU specimens tested by Bioelisa anti-hbc number detected cut-off = CO/S range negative samples positive samples

31 Results Figure 14: Distribution of reactivities for IDU specimens tested by Murex anti-hbc (total) number detected cut-off = CO/S range negative samples positive samples Figure 15: Distribution of reactivities IDU specimens tested by Vitros anti-hbc number detected cut-off = CO/S range negative samples positive samples

32 Results Assay cut-off evaluation Delta values are a statistical measure, for a particular assay, of the distribution of the population of reactivities given by specimens containing the marker sought (delta positive) and those lacking it (delta negative), benchmarked against the assay's cut-off. They provide an insight into whether the assay's threshold has been set at a level that provides a suitable balance of sensitivity and specificity. The delta values for the positive and negative anti-hbc specimens are shown in table 13. The overall positive delta values for the anti-hbc assays were between 1.65 and The overall negative delta values were between and The delta analysis demonstrated a good balance between sensitivity and specificity only for the Murex assay. The delta values for the Murex anti-hbc (total) are more evenly distributed (mean positive delta 3.63, mean negative delta -4.43), suggesting that this cut-off has been set to optimise both sensitivity and specificity. The sensitivity and specificity data on the specimens from IDUs and blood donors bear this out, with the Murex anti-hbc (total) assay providing the best accuracy. The delta values for the AxSYM CORE, Bioelisa ati-hbc assay, Vitros anti-hbc assay and COMPRIA show the negative delta value is further from the cut-off than the positive delta value. The implication of this is that for these assays performance is biased towards high specificity, at some potential cost to sensitivity. Table 13: Delta values for 369 anti-hbc positive specimens and approximately 680 anti-hbc negative specimens Anti-HBc positive specimens delta values Anti-HBc negative specimens delta values Injecting drug users HBsAg positive/ IgM anti-hbc Overall positive delta Injecting drug users Blood donor specimens Overall negative delta value (n~680)* Assay (n=316) negative (n=53) value (n=369) (n=180) (n~500)* AxSYM Bioelisa Murex Vitros COMPRIA 2.17 N/A NT Notes: N/A = Not available NT = Not tested * The number of blood donor specimens and the total number of anti-hbc negative specimens for each assay are: AxSYM CORE 497 and 677, Bioelisa anti-hbc 496 and 676, Murex anti-hbc (total) 498 and 678 and Vitros anti-hbc 496 and

33 Results Antenatal specimen reactivities Fifty antenatal specimens, obtained from an area of South London with a relatively high HBV prevalence, were tested against all four anti-hbc assays and the COMPRIA assay. Due to limited specimen volume the AxSYM CORE and Murex anti-hbc assays were tested on one set of 50 antenatal specimens and the Bioelisa and Vitros anti-hbc assays on a second set of specimens. Both sets of antenatal specimens were tested by COMPRIA. Any specimens that gave discrepant results between the assays were retested and were also tested for HBsAg and anti-hbs. The AxSYM CORE and Murex anti-hbc assays identified 14 (28%) positive specimens and 36 (72%) negative specimens. All assays were 100% sensitive against the positive antenatal specimens (table 14). The Murex anti-hbc and COMPRIA were 100% specific with the negative antenatal specimens (table 15). The AxSYM CORE assay had 1 repeatedly false positive specimen that was negative by the other assays and the supplementary tests (table 16), giving a specificity of 97.2% with the negative antenatal specimens. The Bioelisa anti-hbc and Vitros anti-hbc assays identified 8 (16%) positive specimens and 42 (84%) negative specimens. Overall the three assays were 100% sensitive with the positive specimens and 100% specific with the negative specimens. The Bioelisa assay had 1 false positive specimen on initial testing, that was negative upon retesting and negative by the supplementary assays. However, it should be noted that the sample size for antenatal specimens is too low to make strong statements about sensitivity and specificity. 30

34 Results Assay Number of positive samples tested Total positive 95% Sensitivity confidence (%) interval (%) Range CO/S Mean CO/S Median CO/S Antenatal specimens - set 1 AxSYM Murex COMPRIA Antenatal specimens - set 2 Bioelisa Vitros COMPRIA Assay Table 14: Sensitivity findings for antenatal specimens Table 15: Specificity findings for antenatal specimens Number of negative samples tested Total Specificity negative (%) 95% confidence interval (%) Range CO/S Mean CO/S Median CO/S Antenatal specimens - set 1 AxSYM Murex COMPRIA Antenatal specimens - set 2 Bioelisa Vitros COMPRIA Table 16: CO/S ratios for 2 discordant specimens anti-hbc testing anti-hbc testing Supplementary tests Bioelisa Vitros AxSYM Murex COMPRIA HBsAg Anti-HBs Sample ID initial CO/OD repeat CO/OD CO/S initial CO/S repeat CO/S CO/OD CO/S OD/CO OD/CO 02K0239a b

35 Results Detection of total anti-hbc in HBsAg positive/anti-hbc IgM negative specimens Eighty-five HBsAg positive/ anti-hbc IgM negative specimens were tested against all four anti- HBc total assays and the COMPRIA assay. Of these, 53 were anti-hbc positive by at least one anti-hbc assay. The Murex and Vitros anti-hbc assays detected the highest number of anti- HBc positive specimens at 52 (98.1%) (table 17). The AxSYM assay detected the lowest number of anti-hbc positive specimens among the commercial assays evaluated at 50 (94.3%). Table 18 shows the specimens that gave discordant results between the assays. The Vitros anti-hbc assay detected 1 positive result that was not detected by the other assays. Two specimens were negative by AxSYM CORE but positive in all other assays and one specimen was positive only by the Murex anti-hbc and AxSYM CORE assays. Table 17: Anti-HBc reactivity of 85 HBsAg positive/ anti-hbc IgM negative specimens Number samples Number anti-hbc Number positives Sensitivity 95% confidence Range Mean Median Assay tested positives detected (%) interval (%) CO/S CO/S CO/S AxSYM Bioelisa Murex Vitros COMPRIA* Notes: * Statistics are not given for RIA as results are only expressed as % inhibition Table 18: CO/S ratios for 5 discordant specimens Initial data COMPRIA testing anti-hbc testing Supplementary test HBsAg Anti-HBc initial retest retest AxSYM Bioelisa Murex Vitros Anti-HBs Sample IgM % % ID S/CO (Units) inhibition inhibition CO/S CO/S CO/OD CO/S CO/S S/CO

36 Results Detection of anti-hbc in seroconversion panels Four seroconversion panels were included in the sensitivity evaluation of the anti-hbc assays to assess its ability to detect anti-hbc at the time of seroconversion One of the seroconversion panels was from Boston Biomedica Inc, two were from BioClinical Partners Inc and one was from Profile Diagnostics Inc. The number of positive specimens found for each of the seroconversion panels was added to obtain an aggregate score (table 19a). The most sensitive assay detects the highest number of positive specimens. AxSYM CORE, Bioelisa anti-hbc and Vitros anti-hbc all detected the same number of positive specimens and have the highest seroconversion sensitivity. Since the assays all gave similar scores with the seroconversion panels a second scoring system was used to show more discrimination in the assay sensitivity. For each panel every assay was given a score. The highest score (5 points since 5 kits have been evaluated) was awarded to the assay that first became positive and with the highest level of activity. The lowest score (1) was awarded to the assay that became positive last and with the lowest level of activity. For comparative purposes an aggregate score was obtained for each kit by adding the scores for each of the seroconversion panels (table 19b). The most sensitive assay is deemed to be the one that overall detected anti-hbc in the panels earlier than the other assays, as indicated by obtaining the highest score. Vitros anti-hbc and Bioelisa anti-hbc assays were the two most sensitive assays at detecting seroconversion with a score of 16, separating these two assays away from the AxSYM CORE. The least sensitive commercial assay was the Murex anti-hbc (total) with a score of 10 and the COMPRIA had the lowest overall score of 5. Figures illustrate the detection of anti-hbc in the four seroconversion panels. Details of the detection of anti-hbc and the CO/S ratios can be found in the Appendix. PHE102 is an anti-hbc IgM low titre performance panel containing 15 undiluted serum or plasma specimens from Boston Biomedica Inc. The panel has 14 specimens with reactivities near the sensitivity limits of four anti-hbc IgM tests currently approved by the FDA and one specimen with negative reactivity. Table 20 shows the CO/S ratios for the four anti-hbc assays and COMPRIA. All assays detected all 14 anti-hbc IgM positive specimens and were unreactive with the negative specimen. Table 19a: Comparative detection of anti-hbc in four seroconversion panels (based on the number of reactive samples) Assay Seroconversion panels - number of positive samples and days since first bleed in brackets Aggregate score BBI- PHM935A BCP-6278 BCP-6281 PROFILE-RP009 AxSYM 8 (66) 2 (37) 4 (41) 16 (29) 30 Bioelisa 8 (66) 3 (33) 4 (41) 15 (31) 30 Murex 6 (85) 1 (41) 3 (43) 16 (29) 26 Vitros 8 (66) 2 (37) 4 (41) 16 (29) 30 COMPRIA 8 (66) 1 (41) 3 (43) 14 (36) 26 Table 19b: Comparative detection of anti-hbc in four seroconversion panels (based on the first reactive sample with the highest level of activity) Assay Seroconversion panels - highest points given to first positive sample with the highest level of activity Aggregate score BBI- PHM935A BCP-6278 BCP-6281 PROFILE-RP009 AxSYM Bioelisa Murex Vitros COMPRIA

37 Results Figure 16: Detection of PHM935A seroconversion panel by anti-hbc assays log10 CO/S cut-off = days since first bleed axsym bioelisa murex vitros COMPRIA Figure 17: Detection of BCP-6278 seroconversion panel by anti-hbc assays log10 CO/S 1.00 cut-off = days since first bleed axsym bioelisa murex vitros COMPRIA 34

38 Results Figure 18: Detection of BCP-6281 seroconversion panel by anti-hbc assays log10 CO/S 1.00 cut-off = days since first bleed axsym bioelisa murex vitros COMPRIA Figure 19: Detection of Profile-RP009 seroconversion panel by anti-hbc assays log10 CO/S cut-off = days since first bleed axsym bioelisa murex vitros COMPRIA 35

39 Results Table 20: CO/S ratios for the PHE102 performance panel tested by five anti-hbc assays Anti-HBc evaluation testing BBI information sheet AxSYM Bioelisa Murex Vitros Anti-HBc IgM Anti-HBc IgM CORE anti-hbc anti-hbc anti-hbc COMPRIA Abbott EIA* Abbott RIA* Panel ID CO/S CO/S CO/S CO/S CO/S S/CO S/CO PHE PHE PHE PHE PHE PHE PHE PHE PHE PHE PHE PHE PHE PHE PHE Notes: Results are expressed as cut-off to specimen absorbance ratios. Ratios equal to or greater than 1.0 are considered reactive. * Data for both assays are taken from Procedure B in the BBI information sheet 36

40 Results Detection of anti-hbc in dilution series Ten anti-hbc dilution series were included as part of the sensitivity rating to determine the analytical sensitivity of the assays. The dilution series were prepared by using ten total anti-hbc positive specimens that were diluted in ten-fold steps from 1/50 to 1/500,000 in negative human serum. All ten dilution series were tested on the four anti-hbc assays and the COMPRIA. The number of positive specimens found by each assay in each dilution series was added to give an aggregate score (Table 21a). The most sensitive assay detects the highest number of positive specimens. AxSYM CORE was the most sensitive assay with the dilution series, followed by the Murex anti-hbc (total) assay. Since the last three assays all gave the same score a second scoring system was used to show more discrimination in the sensitivity. For each dilution series every assay was given a score. The highest score (5 points since 5 kits have been evaluated) was awarded to the assay which remained positive to the highest titre and with the highest level of activity. The lowest score (1) was awarded to the assay that remained positive to the lowest titre and with the lowest level of activity. An aggregate score was determined for each kit by adding the scores obtained in each dilution series (table 21b). With this scoring system AxSYM CORE and Murex anti-hbc (total) were still the most sensitive kits and had significantly higher scores than the other three assays. The Vitros anti-hbc was the third most sensitive assay with a slightly higher dilutions score than the other two assays. Further details on the detection of anti-hbc and the CO/S ratios can be found in the Appendix. Table 21a: Comparative detection of anti-hbc in 10 dilution series (based on the number of reactive samples) Assay Dilution series - one point given for every positive sample Aggregate score AxSYM Bioelisa Murex Vitros COMPRIA Table 21b: Comparative detection of anti-hbc in 10 dilution series (based on the highest titre) Assay Dilution series - Highest points given to the assay which remained positive to the highest Aggregate titre and lowest points to the assay which remained positive to the lowest titre score AxSYM Bioelisa Murex Vitros COMPRIA

41 Results Summary of specificity findings An overall specificity rating was obtained by adding the number of anti-hbc negative blood donor and IDU specimens that were correctly identified by the anti-hbc kits. In table 22 the kits are ranked according to the overall specificity, with the most specific at the top. The Murex anti-hbc (total) assay was the most specific with 100% specificity and no false positive specimens detected in either sample group. AxSYM CORE was the second most specific assay, closely followed by the Bioelisa and Vitros anti-hbc assays. Table 22: Summary of specificity findings for 4 anti-hbc assays Assay Number blood donors Number IDU number negative Number initially negative Number repeatedly negative Specificity (%) samples Murex AxSYM Bioelisa Vitros Rank Summary of sensitivity findings Assay An overall sensitivity rating was obtained by adding the number of anti-hbc positives for the IDU, HBsAg positive/ anti-hbc IgM negative specimens, seroconversion panels, performance panel and the dilution series. In table 23 the kits have been ranked according to the overall sensitivity, with the most sensitive at the top. AxSYM CORE was the most sensitive assay, closely followed by the Murex anti-hbc (total) assay. Table 23: Summary of the sensitivity findings for 5 anti-hbc assays IDU HBsAg positive/ IgM Serconversion panels PHE102 Performance Dilution series Overall sensitivity anti-hbc panel score AxSYM Murex Vitros COMPRIA Bioelisa Rank 38

42 Results Quality control sera Replicates of a HPA total anti-hbc quality control sera were tested by all four anti-hbc assays and the COMPRIA to determine intra-plate reproducibility. Replicates of this QC serum were also tested against a second kit lot (table 24). A QC specimen/ statistical assay control should have a reactivity within the linear dynamic range of the assay. When the evaluation began only one anti-hbc QC serum was available for testing, which had not been optimised to detect all anti-hbc assays. The AxSYM CORE, Murex anti-hbc (total) and COMPRIA all detected the QC sera within this range. The QC values for the Bioelisa anti-hbc assay were negative and the Vitros anti-hbc assay were within the assays borderline range. Table 24: CO/S ratios for the HPA anti-hbc quality control sera tested by 5 anti-hbc assays LOT 1** - ANTI-HBc QC SERA Assay CO/S 1 CO/S 2 CO/S 3 CO/S 4 CO/S 5 CO/S 6 mean AxSYM Bioelisa Murex Vitros COMPRIA* LOT 2** - ANTI-HBc QC SERA Assay CO/S 1 CO/S 2 CO/S 3 CO/S 4 CO/S 5 CO/S 6 mean AxSYM Bioelisa Murex Vitros Notes: * a second lot was not tested for the COMPRIA ** lot numbers for each assay can be found in table 26 The inter-run reproducibility of all four anti-hbc assays was determined using the HPA anti-hbc total quality control sera. The QC sera was tested with each run of the AxSYM CORE and Vitros anti-hbc automated assays. The Bioelisa anti-hbc had a QC sera on every plate The Murex anti-hbc (total) had a QC sera on three different locations on each plate and the mean value was calculated. The range of QC sera values obtained with each assay during lot 1 testing is shown in figures 20 to 23. The AxSYM CORE, Bioelisa anti-hbc and Murex anti-hbc (total) values were within 2 standard deviations of the mean except for one value. The Vitros anti-hbc CO/S values were all within 2 standard deviations of the mean. 39

43 Results Figure 20: Inter-run variation of the HPA anti-hbc quality control sera with AxSYM CORE sd sd CO/S 1.6 mean -1 sd sd number of runs Figure 21: Inter-run variation of the HPA anti-hbc quality control sera with Bioelisa anti-hbc assay sd sd CO/S 0.7 mean sd sd number of runs 40

44 Results Figure 22: Inter-run variation of the HPA anti-hbc quality control sera with Murex anti-hbc (total) sd sd 3.2 mean CO/S sd sd Number of runs Figure 23: Inter-run variation of the HPA anti-hbc quality control sera with Vitros anti-hbc assay sd sd CO/S 0.80 mean -1 sd sd number of runs 41

45 Results Manufacturers kit controls The results for the positive and negative controls provided by the manufacturer of the four anti- HBc assays are shown in table 25. All results for the four assays were within the specified limits described in the kit inserts. Table 25: Results for manufacturers kit controls Number of assay runs used to Positive control Range CO/S Mean CO/S Median CO/S % CV Assay calculate ranges AxSYM Bioelisa Murex Vitros Number of assay runs used to Negative control Range CO/S Mean CO/S Median CO/S % CV Assay calculate ranges AxSYM Bioelisa Murex Vitros

46 Results Comparison of two production lots The full specimen panel (table 3a) was used to evaluate one production lot of each anti-hbc assay and a subset of the specimen panel (table 3b) was used to evaluate a second production lot. Table 26 shows a comparison of the number of specimens detected by each production in all four anti-hbc assays. The CO/S values for each specimen panel in the two production lots can be found in the Appendix. The 50 blood donor specimens were unreactive by both production lots of the four anti-hbc assays. Additionally 2 replicates of a known negative human plasma were unreactive by both lots. The overall specificity for each assay does not show any variation between production lots. Fifty IDU sera were tested on both production lots of each assay and the number of positive IDU specimens detected by each assay is shown in table 26. The AxSYM CORE assay was tested on one set of IDU specimens and detected the same number of specimens in both lots. The remaining three assays were tested on a different set of IDU specimens. Bioelisa and Murex anti-hbc assays showed no variation between lots, although the Murex assay did detect a higher number of positive specimens. The Vitros anti-hbc assay detected 34 positive specimens in lot 1 and 36 in lot 2, showing some variation between the production lots. Two specimens that were positive in lot 2 were borderline in lot 1 by the Bioelisa criteria and on retesting one specimen gave a positive result. The dilution panel data shows the number of positive specimens detected in each production lot for all four assays. Dilution panels and detected the same number of specimens in each lot of all four assays. For the panel the AxSYM CORE assay detected four positive specimens in lot 1 and three in lot 2, showing a slight reduction in the sensitivity of the second lot. All other assays detected four positive specimens in each production lot. Three HBsAg resolving carrier specimens were tested on all assays in duplicate and were detected by each production lot for all four assays. Ten HBsAg positive/anti-hbc IgM negative specimens were tested on the assays. Four samples were positive by all assays with each production lot. No variation in sensitivity was found with this group of specimens. All anti-hbc positive specimens from both production lots were compared in a Bland Altman graph for each assay (figures 24-27). At lower CO/S ratios there is less difference than can be seen at higher CO/S ratios. 43

47 Results Table 26: Comparison of 2 production lots for each anti-hbc assay Number of AxSYM CORE Bioelisa anti-hbc Murex anti-hbc Vitros anti-hbc Category samples lot: LU02 lot: 91285LU02 lot: I-1202 lot: A-3903 lot: H lot: H lot: 0530 lot: 0540 Blood donor sera Negative human plasma 2* Total negative samples: Injecting drug users (IDU) Dilution panel Dilution panel Dilution panel HBsAg resolving carrier - ' * HBsAg resolving carrier - ' * HBsAg resolving carrier - ' * HBsAg +/anti-hbc IgM negative 10 NT NT ** 4 Total positive samples: Notes: NT - Not tested as the AxSYM CORE evaluation was complete before these samples were added to the specimen panel * samples were tested in duplicate ** production lot '0550 used as these samples were added after initial lot 1 testing was complete 44

48 Results Figure 24: Bland Altman comparison of mean and difference between the reactivites of 38 positive anti-hbc specimens tested by 2 production lots of AxSYM CORE 20.0 CO/S difference for each positive sample tested by both lots sd sd 5.0 mean sd sd mean CO/S for each positive sample tested by both lots Figure 25: Bland Altman comparison of mean and difference between the reactivites of 53 positive anti-hbc specimens tested by 2 production lots of Bioelisa anti-hbc CO/S difference for each positive sample tested by both lots sd sd mean sd sd mean CO/S for each positive sample tested by both lots 45

49 Results Figure 26: Bland Altman comparison of mean and difference between the reactivites of 58 positive anti-hbc specimens tested by 2 production lots of Murex anti-hbc (total) 15.0 CO/S difference for each positive sample tested by both lots +2 sd sd 5.0 mean sd -2 sd mean CO/S for each positive sample tested by both lots Figure 27: Bland Altman comparison of mean and difference between the reactivites of 57 positive anti-hbc specimens tested by 2 production lots of Vitros anti-hbc 20.0 CO/S difference for each positive sample tested by both lots sd +1 sd mean -1 sd -2 sd mean CO/S for each positive sample tested by both lots 46

50 Technical appraisal AxSYM CORE assay Installation and initial calibration of the AxSYM analyser were carried out by an engineer from Abbott Diagnostic laboratories. Three days training in the use and basic maintenance of the system was given to the MiDAS staff responsible for performing the evaluation. Training comprised basic instruction and practice in using the touch screen monitor and system software; system initialisation and fault diagnosis; loading calibration and testing reagents and specimens; and daily, weekly and monthly maintenance procedures. After initial familiarisation, the system was straightforward to operate. The touch-screen monitor and on-board software were user friendly for sample testing and maintenance. The daily and weekly maintenance checks were easy to perform and not overly time consuming. Very few problems were encountered during the evaluation. A large sample volume of 236µl is required for each test. This is a problem if you need to repeat a test, as this doubles the volume of sample required. The AxSYM analyser gives an exception message for samples that were insufficient or inappropriate for testing. These samples were aliquoted a second time and retested. All samples were centrifuged before use. Two IDU negative samples gave exceptions on repeat testing and no result was obtained. Approximate processing times are 5 minutes for pipetting one sample tray (15 samples), 20 minutes until the first result and 75 minutes to complete 90 samples (6 trays). Bioelisa anti-hbc assay The Bioelisa anti-hbc kit instructions for using the reagents and running an assay were clear and easy to follow. The kit and reagent packaging were clearly identified and it was easy to prepare reagents requiring reconstitution (wash buffer and substrate solution). The sample volume was 50µl and required no dilution. The washing stage was performed using a Tecan Columbus washer and the initial washer efficiency test showed no carryover when applying the wash cycle specified in the kit instructions. Additional equipment used was a Grant Waterbath and BioTek Ultramicroplate reader. For the evaluation, tests were processed manually and each 96 well plate took approximately 2.5 hours to complete. Murex anti-hbc (total) The Murex anti-hbc (total) kit instructions for using the reagents and running an assay were clear and easy to follow. The kit and reagent packaging were clearly identified and it was easy to prepare reagents that required reconstitution (wash buffer and substrate solution). 50µl of sample diluent was added before the addition of 50µl of sample, causing a colour change in the diluent and making it easier to identify the wells where sample had been added. The washing stages were performed using a Tecan Columbus washer and the washer efficiency preevaluation test showed no carryover when applying the wash cycle specified in the kit instructions. Additional equipment used was an Abbott-Murex dry incubator and BioTek Ultramicroplate reader. For the evaluation, tests were processed manually and each 96 well plate took approximately 2.5 hours to complete. 47

51 Technical appraisal Vitros anti-hbc assay Installation and initial calibration of the Vitros ECi Immunodiagnostic system were carried out by an equipment support specialist from Ortho-Clinical Diagnostics. Two days training in the use and basic maintenance of the system was given to the MiDAS staff responsible for performing the evaluation. Training comprised basic instruction and practice in using the touch screen monitor and system software; system initialisation and fault diagnosis; loading, calibration and testing reagents and specimens; and daily, weekly and monthly maintenance procedures. After initial familiarisation, the system was straightforward to operate. The touch-screen monitor and on-board software were user friendly for sample testing and maintenance. The daily, weekly and maintenance checks were easy to perform and not overly time consuming. The main problem encountered during the evaluation were sampling errors. The error messages indicated that the samples contained clots or bubbles, or that there was inadequate sample volume. In this case the specimen was sampled again after a visual inspection to check that no clots or bubbles were present, or aliquoted a second time. All samples were centrifuged before use. The only other problem encountered was a malfunction in the incubator that contained 50 samples. All 50 samples were rejected and no results could be obtained. The sample incubation stage is the only stage in the process where all results will be lost if a malfunction occurs. Approximate processing time for pipetting and sampling one sample tray (10 samples) is 10 minutes. It takes 65 minutes until the first result becomes available and 95 minutes until the results for 60 samples (6 trays) becomes available. 48

52 Conclusions Readers are encouraged to study carefully the results presented and to draw their own conclusions from them. We offer the following comments: Specificity The Murex anti-hbc assay gave no false positive results for the 498 anti-hbc negative specimens from blood donors and 180 anti-hbc negative specimens from IDUs. The Murex anti- HBc was therefore the most specific anti-hbc assay evaluated, with an overall specificity of 100%. The AxSYM CORE assay generated a reproducible false positive reaction with one specimen, giving an overall specificity of 99.9%. The Vitros anti-hbc and Bioelisa anti-hbc assays each gave rise to reproducible false positive reactions on three specimens, giving overall specificities of 99.6%. Sensitivity The overall sensitivity rankings were obtained by adding the number of confirmed anti-hbc positive specimens that were detected by the anti-hbc assays. The anti-hbc positive specimens were from IDUs, commercial seroconversion and performance panels, dilutions series, and 'HBsAg positive/ IgM anti-hbc negative' specimens. Overall the AxSYM CORE assay was the most sensitive anti-hbc assay, detecting 443 of the positive specimens, followed by Murex anti-hbc detecting 442. Vitros anti-hbc and Bioelisa anti- HBc detected 431 and 424 respectively. No kit was consistently the most sensitive for all categories of specimen. For example, the Murex anti-hbc detected the most anti-hbc IDU positive specimens and those that were 'HBsAg positive/ IgM anti-hbc negative'; it was the second most sensitive kit for detecting anti-hbc in the dilution series, but it was the fourth most sensitive kit for detecting anti-hbc seroconversion. The Bioelisa anti-hbc and Vitros anti-hbc assays were the most sensitive kits for detecting anti-hbc seroconversion but ranked as the least sensitive kits against the IDU's and the dilution series. Evaluation of assay cut-off determination The delta values for the Murex anti-hbc kit are evenly distributed (overall positive delta 3.63, overall negative delta -4.43), indicating that its cut-off has been set to optimise both sensitivity and specificity. The delta values for the AxSYM CORE, Bioelisa anti-hbc and Vitros anti-hbc assays show the negative delta value is substantially further from the cut-off than the positive delta value, indicating a bias in assay performance that would favour specificity over sensitivity. The implication of this is that these assays' performance is biased towards high specificity, at some potential cost to sensitivity. This may be a suitable compromise for screening blood donations as the residual HBV risk is mostly from carriers who are HBsAg negative, yet still potentially infectious, and these typically have high titre anti-hbc. 49

53 Conclusions Quality control A single HPA anti-hbc (total) quality control specimen was used throughout the evaluation. Unlike evaluations for other markers (e.g. HIV, HBsAg) for which more quality control specimens have been available this was the only anti-hbc control available when the evaluation began. The AxSYM CORE and Murex anti-hbc (total) were able to consistently detect the quality control sample. However, it was unreactive in the Bioelisa anti-hbc assay and for the Vitros anti-hbc its reactivity was within the assay's borderline range. For the four anti-hbc assays under evaluation the quality control reactions were nearly all within 2 standard deviations of the CO/S mean. Presentation All assays were well presented and easy to perform. The AxSYM CORE took 75 minutes to complete a run of 90 samples. Vitros anti-hbc took 95 minutes to complete a run of 60 samples. The Bioelisa anti-hbc and Murex anti-hbc (total) took 2.5 hours to complete one 96 well plate. Applications The sensitivity of the anti-hbc assays varied from % for the anti-hbc IDU specimens, but the most sensitive kits in this analysis were not necessarily the most sensitive in terms of seroconversion sensitivity or detection of anti-hbc positive dilutions. Despite the variability, it is likely that all of the anti-hbc kits examined could have a role in HBV confirmatory testing algorithms, further to characterise HBsAg positive specimens and to assist in determining whether HBV infection is past or current. In many countries anti-hbc assays are used to screen blood donations. In this evaluation all anti-hbc assays had specificities of more than 99% and would therefore be good candidates for blood screening. 50

54 Acknowledgements We thank the HPA Colindale - SRMD, Virus Reference Division and the following for providing serum samples: North London Blood Centre, Colindale NW9 HPA London, Department of Virology, Kings College Hospital (Dulwich site), London SE22. 51

55 References 1. Hoofnagle JH, Gerety RJ, Ni LJ, et al (1974): Antibody to Hepatitis B core antigen. The New England Journal of Medicine. 290: Mahoney FJ (1999): Update on Diagnosis, Management and Prevention of Hepatitis B virus infection. Clinical Microbiology Reviews. 12: Grob P, Jilg W, Bornhak H et al (2000): Serological pattern Anti-HBc alone : Report on a workshop. Journal of Medical Virology. 62: Alhababi F, Sallam TA, Tong CYW (2003): The significance of anti-hbc only in the clinical virology laboratory. Journal of Clinical Virology. 27: Mosley JW, Stevens CE, Aach RD, et al (1995): Donor screening for anti-body to Hepatitis B core antigen and Hepatitis B virus infection in transfusion recipients. Transfusion. 1: Troisi CL, Hollinger FB (1987): Current tests for antibody to Hepatitis B core antigen used to screen donors for non-a, non-b hepatitis are comparable to the original radioimmunoassay for hepatitis B core antigen. Transfusion. 27: Parkinson AJ, McMahon J, Hall D et al (1990): Comparison of enzyme immunoassay for the detection of anti-body to hepatitis B core antigen as the only marher of Hepatitis B infection in a population with a high prevalence of Hepatitis B. Journal of Medical Virology. 30: Caspari G, Beyer H-J, Elbert G et al (1989): Unsatisfactory specificities and sensitivities of six enzyme immunoassays for antibodies to Hepatitis B core antigen. Journal of Clinical Microbiology. 27: O Connor T, Delieu E, Cole M, Perry KP (2003): Enzygnost HBsAg 5.0 EIA. Medicines and Healthcare products Regulatory Agency: MHRA

56 Appendix Evaluation protocol Protocol for evaluation of anti-hbc kits Procurement of product for evaluation, duration of evaluation and training of evaluator The evaluator will require for evaluation a package that contains sufficient anti-hbc kits to test a panel of specimens, together with ancillary reagents and consumables that are recommended by the manufacturer. Two batches of the kit are required to investigate lot-to-lot reproducibility. The kits will be used in conjunction with equipment, such as spectrophotometer and plate washers, or automated analyser, that is either provided by the manufacturer or is available at MiDAS and has been agreed to meet the requirements of the manufacturer's representative. Before evaluation starts, the manufacturer will be invited to train the evaluator in the use of the kits and equipment and to satisfy themselves that the evaluator is properly trained. Conduct of the evaluation The product will be used in exactly the manner laid down in the manufacturer's instructions. Any modifications to the instructions provided with the kit that are described during the training period, or any subsequent changes must be confirmed in writing. All microtitre plates will be read on a plate reader eg Bio-Tek EL808 linked to a computer with KC4 software. All data will be stored on the anti-hbc kit database for the duration of the evaluation. The data may subsequently be down-loaded to tape or other long-term back-up system. In addition the evaluators will keep clear records of the practical work. All original printouts from the plate reader or automated systems will be retained, together with any reader printouts from confirmatory assays. Content of the evaluation / specimen panel The object of this evaluation is to assess the ability of total anti-hbc kits to detect, with a high degree of sensitivity and specificity, anti-hbc in human serum and plasma. To do this, the kits will be tested against an unbiased panel of plasma specimens obtained from injecting drug users and blood donors. In addition, this evaluation will incorporate commercially available anti-hbc performance panels and quality control samples and standards (Table 2). Specimens previously tested because of false positive screening reactions will not be included in this study because of the potential bias that could be introduced against particular assays. Storage of samples Aliquots of each serum specimen will be distributed into plastic tubes with screw-cap lids with sealers. The aliquots will be stored at -20 C or below until required and at 4 C for the duration of the evaluation. Thawing will be carried out at room temperature. Other aspects of the evaluation The following features of the kits will be noted and may be remarked on in the report: l packaging and labelling of the materials l clarity of the operating instructions l ease of use and reliability of the products, including equipment supplied for the evaluation l health and safety considerations. 53

57 Appendix Table 2a: Panel for the evaluation of anti-hbc kits (lot 1). Sample category Number 1. Blood donors'/healthy adults' sera Negative human serum (NHS) 2x 1 3. Injecting drug users (IDUs) HBsAg 'resolving carrier' specimens (NLBC and BPL) 2x 3 5. HBsAg positive, IgM anti-hbc negative Seroconversion panels: total anti-hbc BBI - PHM935A 20 BCP BCP Profile - RP Performance panels: total anti-hbc BBI - PHE102 (IgM anti-hbc low titre) Dilution of anti-hbc positive (9 specimens x 6 dilutions - 1 specimen x 5 dilutions) 2x Quality control samples HPA - total anti-hbc QC serum: sample 1 6x 1 Three replicates of suitable controls on each plate (estimate) Ante-natal specimens 50 TOTAL (number of tests) 1405 Notes: BBI = Boston Biomedica Inc, USA; BPL = BioProducts Laboratory, UK NLBC = North London Blood Centre HPA = Health Protection Agency, UK; Table 2b: Panel for the evaluation of anti-hbc kits (lot 2). 1. Blood donors'/healthy adults' sera Negative human serum (NHS) 2x 1 3. Injecting drug users (IDUs) HBsAg 'resolving carrier' specimens (NLBC and BPL) 2x 3 5. HBsAg postive, IgM anti-hbc negative Dilution of anti-hbc positive (3 specimens x 6 dilutions) 2x Quality control samples HPA - total anti-hbc QC serum: sample 1 3x 1 Three replicates of suitable controls on each plate (estimate) 10 TOTAL (number of tests) 167 Notes: BBI = Boston Biomedica Inc, USA; BPL = BioProducts Laboratory, UK NLBC = North London Blood Centre HPA = Health Protection Agency, UK Discordant results A discordant result will arise when the kit under evaluation gives a result that disagrees with the observed consensus. If this occurs tests will be repeated in duplicate on the same aliquot of the serum. If the result is still discordant further investigations will be undertaken at the Sexually Transmitted and Blood Borne Virus Laboratory. This will include testing by other commercial or 'in-house' anti-hbc kits, other hepatitis B serological markers (eg anti-hbs, HBsAg, HBeAg, anti- HBe, IgM anti-hbc, as appropriate) and HBV DNA assays. Analysis of results and evaluation report Raw data will be transferred from the laboratory computer onto a database specifically prepared for these evaluations. The data entry will be checked by a second person. A detailed report will be prepared for publication by the MHRA. The manufacturers' will be given the opportunity to comment on the results of the evaluation of their product before the MHRA evaluation report is published. Results obtained in other manufacturers' tests undergoing evaluation will not be disclosed at this time. Manufacturers' written comments, where relevant, will be appended to the report. 54

58 Appendix Description of the assays This section provides further details of the assays under evaluation. Table 27 gives details of the stages of the procedure for each assay. Table 28 describes any additional equipment used. Table 27: Stages of the assays Stages of assays AxSYM CORE Bioelisa anti-hbc Murex anti-hbc (total) Vitros anti-hbc Preparation/sample loading 30 minutes/ 90 samples 30 minutes/ 96 samples 30 minutes/ 96 samples 20 minutes / 60 samples Incubation status Waterbath Dry incubator Sample incubation incubated together 30 minutes at 37 C Number of washes for 60 minutes at 5 Conjugate incubation 37 C 30 minutes at 37 C Number of washes Automated 4 5 Automated Substrate incubation 30 minutes at room temperature 30 minutes at 37 C Reading 450nm with 630nm reference filter 450nm with 630nm reference filter Total incubation times 75 minutes 90 minutes 90 minutes 55 minutes Table 28: Additional equipment required Additional equipment required AxSYM CORE Bioelisa anti-hbc Abbott AxSYM system analyser Waterbath/Dry incubator Micropipettes: µl Plate washer Disposable tips Plate reader Micropipettes: µl Multichannel micropipettes: µl Disposable tips Distilled water Disposable reagent troughs Murex anti-hbc (total) Vitros anti-hbc Dry incubator Vitros Eci analyser Plate washer Micropipettes: µl Plate reader Micropipettes: µl Multichannel micropipettes: µl Disposable tips Distilled water Disposable reagent troughs 0.5M to 2M Sulphuric acid 55

59 Appendix Claims for the assays and their limitations The kit insert for each assay details any claims made on the performance of the assay and any limitations. This information is reproduced in tables Table 29: AxSYM CORE - Claims for the assay and its limitations (quoted from the kit insert) Claims DETECTABILITY: The detectability of anti-hbc in a population of 212 samples previously identified as positive for HBsAg was determined by AxSYM CORE and IMx CORE procedures. Anti-HBc was detected in 212/212 (100%) of the samples by AxSYM CORE and 209/212 (98.6%) by the IMx CORE assay. SPECIFICITY: Samples from 1370 random blood donors and 674 random hospital patients were tested by AxSYM CORE and IMx CORE. AxSYM CORE and IMx CORE showed 99.9% agreement (1368/1370) for random blood donors and 100% agreement (674/674) for the random hospital patients. SENSITIVITY: The sensitivity of the AxSYM CORE assay is an anti-hbc concentration of less than 1 PEI unit/ml, standardised against the Reference Standard of the Paul-Ehrlich-Institute, Langen, Germany. Limitations For diagnostic purposes and in order to differentiate acute HBV infection from chronic HBV infection, the detection of anti-hbc must be correlated with patient symptoms and other hepatitis B viral serological markers. The AxSYM CORE assay is limited to the detection of anti-hbc in human serum or plasma. It is recognised that presently available methods for anti-hbc detection may not detect all potentially infectious units of blood or possible cases of Hepatitis B. False reactive results may be obtained with any diagnostic test. Table 30: Bioelisa anti-hbc - Claims for the assay and its limitations (quoted from the kit insert) Claims Evaluations have been done to check the performance of the Bioelisa anti-hbc compared to a licensed RIA assay. Sera from patients in different stages of Hepatitis B, vaccinated against HB, Delta hepatitis (hepatitis D), post-transfusion non-a, non-b hepatitis (hepatitis C), acute hepatitis A, hepatitis produced by Epstein-Barr virus and cytomegalovirus, and rheumatoid factor have been tested. The overall agreement was 99.4%. Limitations The Bioelisa anti-hbc kit detects specimens with a concentration of anti-hbc antibodies equal to or higher than PEI/ml. Specimens with an anti-hbc concentration lower than 1.5 PEI/ml are not detected. 56

60 Appendix Table 31: Murex anti-hbc (total)- Claims for the assay and its limitations (quoted from the kit insert) Claims A total of 447 specimens from patients with known antibody to hepatitis B core antigen were tested and found to be reactive with Murex anti-hbc (total). The diagnostic sensitivity of Murex anti-hbc (total) on this population of specimens is therefore estimated to be 100% (447/447) with a lower 95% confidence limit of 99.18% (443/447) by the binomial distribution. A total of 360 specimens from patients with conditions unrelated to HBV infection and confirmed as negative for antibody to hepatitis B core antigen were also tested with Murex anti-hbc (total). The diagnostic specificity of Murex anti-hbc (total) on this population of clinical specimens is estimated to be 100% (360/360) with a lower 95% confidence limit of 98.98% (356/360). A total of 5344 routine donor specimens from two European and two South American blood transfusion centres were screened with Murex anti-hbc (total). In the study, 99.14% (5298/5344) of specimens were non-reactive and 0.86% (46/5344) were repeatedly reactive. The screening specificity of Murex anti-hbc (total) on this population of negative routine donor specimens is estimated to be 99.74% (5298/5312) with 95% confidence limits of 99.84% (5304/5312) to 99.56% (5289/5312) by the binomial distribution. Limitations This test is to be used only with individual (unpooled) serum, EDTA plasma or citrate plasma samples. Murex anti-hbc (total) has not been evaluated for any other purpose. Use of highly haemolysed samples, incompletely clotted sera, plasma samples containing fibrin or samples with microbial contamination may give rise to erroneous results. A negative result with an antibody detection test does not preclude the possibility of infection. It is recommended that repeatedly reactive specimens be investigated by supplemental testing. Table 32: Vitros anti-hbc - Claims for the assay and its limitations (quoted from the kit insert) Claims for the assay Sensitivity was determined by testing serial dilutions of the Paul-Ehrlich-Institute (PEI) anti-hbc reference serum, in 6 determinations using 2 reagent lots. The overall sensitivity of the Vitros anti-hbc assay for the PEI reference serum was <1Units/mL. 244 patient samples previously determined as reactive for anti-hbc in another commercially available assay were tested in the Vitros anti-hbc assay. The sensitivity for this population of samples in the Vitros anti-hbc assay was calculated as 100% (244/244). These data suggest that sensitivity may vary between 98.5 and 100% depending on the sample population studied. Samples from 2000 presumably healthy blood donors, and 201 clinical samples were tested in the Vitros anti-hbc assay. The specificity for the Vitros anti-hbc assay for the donor samples was calculated as 99.6% (1987/1995). The specificity for the Vitros anti-hbc assay for the clinical samples was calculated as 100% (200/200). Limitations The results from this or any other diagnostic kit should be used and interpreted only in the context of the overall clinical picture. A negative test does not exclude the possibility of exposure to hepatitis B virus. Levels of anti-hbc may be undetectable both in early infection and late after infection. Heterophilic antibodies in serum or plasma samples may cause interference in immunoassays. Results which are inconsistent with clinical observations indicate the need for additional testing. Samples containing triolein (<33.9mmol/L), haemoglobin (<5 g/l) or bilirubin (<0.342mmol/L) do not interfere with the clinical interpretation of these results. Do not use turbid samples. 57

61 Appendix CO/S ratios for 4 seroconversion panels Days since first Four seroconversion panels were included in the sensitivity testing to assess the ability of an assay to detect anti-hbc at the time of seroconversion. Details of the CO/S ratios for each panel are given in tables Each table shows comparative information taken from the seroconversion panels data sheets on other relevant HBV markers tested for. Each seroconversion panel is a set of undiluted plasma samples from serial bleeds from a single plasma donor collected during a period of Hepatitis B seroconverison. Table 33: BBI - PHM935A panel Axsym anti-hbc evaluation testing Bioelisa Murex Vitros COMPRIA Data from panel information sheets HBV DNA Roche HBsAg Abbott IgM Abbott IgM bleed CO/S CO/S CO/S CO/S CO/S PCR Imx IMx Corzyme-M BLD BLD BLD x x x x x x x x x x >4x >4x x x x x > x >6.6 Note: BLD = Below limit of detection Table 34: BCP panel Days since first Axsym anti-hbc evaluation testing Bioelisa Murex Vitros COMPRIA Data from panel information sheets Research use HBsAg Abbott HBsAg ahbc IgM bleed CO/S CO/S CO/S CO/S CO/S PCR Imx VIDAS* Abbott >50,000, >50,000, Notes: * > 0.13 reactive 58

62 Appendix Days since first Table 35: BCP panel Axsym anti-hbc evaluation testing Bioelisa Murex Vitros COMPRIA Data from panel information sheets Research use HBsAg Roche Cobas Abbott bleed CO/S CO/S CO/S CO/S CO/S PCR VIDAS HBsAgII CORE-M < < Table 36: Profile - RP009 panel Data from panel anti-hbc evaluation testing information sheets Days since Axsym Bioelisa Murex Vitros COMPRIA HBsAg Abbott IgM Abbott first bleed CO/S CO/S CO/S CO/S CO/S Auszyme Corzyme-M

63 Appendix CO/S ratios for 10 dilution series Ten anti-hbc dilution series were included as part of the sensitivity rating to determine the ability of the assays to detect anti-hbc at lower concentrations. The dilution series were prepared using ten anti-hbc total positive specimens that were diluted in ten-fold steps from 1/50 to 1/500,000 in negative human serum. Tables 37a-b show the CO/S ratios for each dilution series. Table 37a: Dilution series Axsym CO/S Bioelisa CO/S Murex CO/S Vitros CO/S COMPRIA CO/S Sample ID Dilution a neat b 1/ c 1/ d 1/5, e 1/50, a neat b 1/ c 1/ d 1/5, e 1/50, f 1/500, a neat b 1/ c 1/ d 1/5, e 1/50, f 1/500, a neat b 1/ c 1/ d 1/5, e 1/50, f 1/500, a neat b 1/ c 1/ d 1/5, e 1/50, f 1/500,

64 Appendix Table 37b: Dilution series Axsym CO/S Bioelisa CO/S Murex CO/S Vitros CO/S COMPRIA CO/S Sample ID Dilution a neat b 1/ c 1/ d 1/5, e 1/50, f 1/500, a neat b 1/ c 1/ d 1/5, e 1/50, f 1/500, a neat b 1/ c 1/ d 1/5, e 1/50, f 1/500, a neat b 1/ c 1/ d 1/5, e 1/50, f 1/500, a neat b 1/ c 1/ d 1/5, e 1/50, f 1/500,

65 Appendix CO/S ratios for two production lots A smaller specimen panel, including 50 blood donor specimens, 50 injecting drug user specimens and 3 dilution series were tested on a second production lot. Tables show the CO/S ratios for specimens tested in both lots. Table 38: Blood donor specimens AxSYM CORE Bioelisa anti-hbc Murex anti-hbc Vitros anti-hbc Sample Lot 1 Lot 2 Sample Lot 1 Lot 2 Sample Lot 1 Lot 2 Sample Lot 1 Lot 2 ID CO/S CO/S ID CO/S CO/S ID CO/S CO/S ID CO/S CO/S N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

66 Appendix Table 39: Injecting drug user specimens AxSYM CORE Bioelisa anti-hbc Murex anti-hbc Vitros anti-hbc Sample Lot 1 Lot 2 Sample Lot 1 Lot 2 Sample Lot 1 Lot 2 Sample Lot 1 Lot 2 ID CO/S CO/S ID CO/S CO/S ID CO/S CO/S ID CO/S CO/S

67 Appendix Table 40: 10 Dilution series AxSYM CORE* Bioelisa anti-hbc Murex anti-hbc Vitros anti-hbc Sample Lot 1 Lot 2 Sample Lot 1 Lot 2 Sample Lot 1 LOt 2 Sample Lot 1 Lot 2 Dilution ID CO/S CO/S ID CO/S1 CO/S2 CO/S1 CO/S2 ID CO/S1 CO/S2 CO/S1 CO/S2 ID CO/S1 CO/S2 CO/S1 CO/S2 neat A A A A / B B B B / C C C C /5, D D D D /50, E E E E /500, F F F F neat A A A A / B B B B / C C C C /5, D D D D /50, E E E E /500, F F F F neat A A A A / B B B B / C C C C /5, D 0.93 NK D D D /50, E 0.59 NK E E E /500, F F F F Notes: NK = not known, as an 'Exception result' was given by AxSYM * Each sample was tested once due to large sample volume 64

68 Appendix Table 41: Negative human plasma Negative human plasma LOT 1 LOT 2 Assay CO/S 1 CO/S 2 CO/S 1 CO/S 2 AxSYM Bioelisa Murex Vitros Table 42: HBsAg resolving carriers LOT 1 LOT 2 LOT 1 LOT 2 LOT 1 LOT 2 Assay CO/S1 CO/S2 CO/S1 CO/S2 CO/S1 CO/S2 CO/S1 CO/S2 CO/S1 CO/S2 CO/S1 CO/S2 AxSYM Bioelisa Murex Vitros Table 43: HBsAg positive/ IgM CORE negative specimens AxSYM CORE Bioelisa anti-hbc Murex anti-hbc Vitros anti-hbc Sample Lot 1 Lot 2 Lot 1 Lot 2 Lot 1 Lot 2 Lot 1 Lot 2 ID CO/S CO/S CO/S CO/S CO/S CO/S CO/S CO/S NT NT NT NT NT NT NT NT NT NT Notes: NT = Not tested 65

69 Appendix Contact details for manufacturer and UK agent AxSYM CORE and Murex anti-hbc (total) Abbott Diagnostics Ltd Abbott House Norden Road Maidenhead Berkshire SL6 4XF Tel: Fax: Bioelisa anti-hbc Launch-Biokit Diagnostics Ash House Ash Road New Ash Green Longfield Kent DA3 8JD Tel: Fax: Vitros anti-hbc Ortho-Clinical Diagnostics Johnson & Johnson Holmers Farn Way High Wycombe Bucks HP12 4DP Tel: Fax:

70 Appendix Manufacturer s comments Bioelisa anti-hbc: 67

71 Appendix Vitros anti-hbc 68

72 69 Appendix