P0069 ViraQ HBV Trend 25 P0069 The kit insert contains a detailed protocol and should be read carefully before testing the run control to ensure optimal performance
Table of contents Intended Use... 3 Key to Symbols Used... 3 Principle of method... 3 Traceability of HBV-DNA concentration in IU/mL and copies/ml... 4 Kit Contents... 5 Storage Instructions... 5 Warning and precautions... 5 Test Procedure... 6 Expected assay response values... 6 Interpretation of results... 6 Performance characteristics... 6 Limitations... 8 References... 9 P0069 ViraQ HBV Trend 25 2
Intended Use P0069 ViraQ HBV Trend 25 is intended to be used as external run control for monitoring consistency in analytical sensitivity of multiplex nucleic acid test (NAT) blood screening assays for detection of HBV-DNA as defined in Table 1. P0069 ViraQ HBV Trend 25 is not a replacement for the internal kit controls or calibrators required for the release of test results. The product is intended for performance evaluation only. Table 1. Nucleic acid blood screening kits covered by this run control Manufacturer Equipment Agent Test kits Hologic/Grifols Diagnostic Solutions TIGRIS PANTHER Hepatitis B virus Procleix ULTRIO Plus Procleix ULTRIO Elite Key to Symbols Used Manufacturer Lot number Catalogue number Store below -30 C Biological substance Category B Do not re-use Device for performance evaluation Expiry date Contents Caution Read instructions for use Principle of method P0069 ViraQ HBV Trend 25 enables blood screening laboratories to monitor the analytical sensitivity of transcription mediated amplification (TMA) assays for the qualitative detection of hepatitis B virus (HBV)-DNA in plasma or serum samples. The external run control is designed to mimic naturally occurring plasma specimens with a low concentration of HBV-DNA. The concentration of HBV-DNA in P0069 ViraQ HBV Trend 25 control samples is set at 25 copies (cps)/ml as measured in replicate Siemens bdna 3.0 assays (equivalent to 4.7 International Units (IU)/mL). This level is chosen close to the 95% lower limit of detection (LOD) of the Ultrio Plus and Ultrio Elite 1-4 assays. The external run control generates sample to cut-off (S/CO) ratios in the saturation, dynamic and negative ranges of the Ultrio assay versions. Changes in frequency of non-reactive, dynamic and saturated reactive results on the run control over time may indicate suboptimal performance of the automated NAT screening systems or deterioration of assay reagents. The external run control tubes are barcoded and comparable in size to blood donor collection tubes. The thawed tubes are ready for use and can be placed at random positions in laboratory equipment sample racks. P0069 ViraQ HBV Trend 25 3
Traceability of HBV-DNA concentration in IU/mL and copies/ml Traceability and securing continuous and stable performance of P0069 ViraQ HBV Trend 25 is based on four viral standards (figure 1) described below: Historically the EUROHEP genotype A 5 was the first HBV-DNA standard used for standardisation purposes. The 1 st, 2 nd and 3 rd WHO HBV-DNA standards 6,7,8 are 1:500 dilutions of the EUROHEP genotype A standard. The primary standard is the 1 st WHO HBV-DNA international standard (97/746) with an assigned value in IU/ml. The secondary VQC-Sanquin HBV-DNA genotype A standard (S0011) was prepared by mixing plasma units from one donor. A homogeneous pool was aliquoted, snap frozen in liquid nitrogen and stored at -70 C 9. The tertiary inactivated BQC HBV-DNA genotype A standard (S0043) was prepared by heat inactivation of a 1:2500 dilution of the native VQC-Sanquin HBV-DNA genotype A standard (S0011) 9,10,11,12. Subsequently P0069 ViraQ HBV Trend 125 is manufactured by gravimetrically recorded dilution steps from S0043. Figure 1: Traceability chain of P0069 HBV Trend control to the EUROHEP genotype A standard, 1 st WHO IS (97/746), the VQC-Sanquin HBV-DNA genotype A standard and BQC inactivated HBV-DNA genotype A standard. The closed arrows indicate a calibration experiment, while the open arrows depict manufacturing steps. BQC has quantified its viral standards in copies/ml using the branched (b) DNA 3.0 test as the reference assay 12. Expression in nucleic acid copies/ml (which is a SI unit) allows comparison between different viruses, genotypes, limiting dilution analysis for estimating NAT efficiency and relation to 50% minimum infectious dose for understanding residual transfusion transmission risk (the MID50 is estimated at 3.16 (1-10) copy) 13,14,15,16. The HBV DNA concentration in copies/ml (and 95% confidence interval (C.I.)) was originally established in suitable dilutions of the S0011 VQC-Sanquin standard in 28 Siemens bdna P0069 ViraQ HBV Trend 25 4
3.0 assays at 2.15 (2.11-2.20).10 8 copies/ml as was estimated from the results reported in proficiency studies and in house testing at Sanquin. The secondary S0011 VQC Sanquin standard was calibrated against the International Standard in the WHO collaborative study 6 and when analysing the results of bdna 3.0 assay separately 1 IU was found to be equivalent to 5.33 (5.11-5.55) copies/ml. The S0043 BQC standard has been quantified against the S0011 VQC Sanquin HBV standard in 2 x 6 bdna 3.0 assays on suitable dilutions at a concentration (95%CI) of 7.23(4.82-10.9).10 6 copies/ml. The calibration of the S0043 BQC inactivated standard against the S0011 VQC-Sanquin standard was also compared by Ct analysis (13 x 2 parallel assays) on 2000 copies/ml samples. Samples of the two preparations tested in TaqScreen 2.0 assay showed a potency (95%C.I.) of the latter standard of 1.22 (1.09-1.38) 17. The production and quality control methods are designed such that the traceability to the primary and secondary standards as described above is guaranteed and that the virus concentration in consecutive batches of P0069 HBV Trend 25 run control (95%CI) is maintained consistently at 25 (18-38) copies/ml or 3.4 (3.4-7.1) IU/mL. Kit Contents 10 Tubes, each containing 1.5 ml run control without preservatives in polypropylene tubes with screw caps. Storage Instructions Store the run controls at or below -30 C for a maximum of two years (reference actual expiry date). Stability experiments showed less than 10% degradation of HBV-DNA after storing samples in liquid format at 2-8 C and at room temperature for 8 hours. Warning and precautions P0069 ViraQ HBV Trend 25 contains HBV particles that are inactivated by a pasteurization procedure guaranteeing sufficient reduction of infectivity. The plasma matrix is prepared from human plasma tested negative for blood borne virus markers (HBV-DNA, HCV-RNA, HIV-RNA, HBsAg, anti-hbc, anti-hiv, anti-hcv and anti- Treponema pallidum). No test method can offer complete assurance that products derived from human blood cannot transmit (unknown) infectious agents. Observe the universal precautions for prevention of transmission of infectious agents when handling these materials 18,19. Do not pipette by mouth. Use personal protective equipment, including lab coats, gloves and safety glasses. Do not eat, drink or smoke in areas where HBV run controls are handled. Disinfect spills using a 0.5% hypochlorite solution (1:10 v/v household bleach) or equivalent disinfectant. Dispose unused or spilled materials according to the normal practices for biological waste disposal in your institution. If precipitates are visible, mix the run controls for 2 minutes thoroughly. P0069 ViraQ HBV Trend 25 5
Test Procedure Thaw the run control quickly in a water bath at 37 C, by gently mixing until frozen contents are just thawed. Remove the run control tube from the water bath immediately. Vortex the run control. Give a short spin in a centrifuge before releasing screw cap from vial. Minimise the time period from thawing until usage of the run control. When not used immediately place in refrigerator. The duration is limited to 8 hours storage until use. The controls should be handled and tested in a manner identical to that of clinical specimens in the assay procedure being evaluated. The external run control tubes are barcoded for automated data-processing. The run controls can be placed at random positions in sample racks of the equipment. After testing discard the remaining run control. Note: To minimize degradation of HBV-DNA in the run control do not leave the run control longer than 8 hours on the NAT equipment before the actual processing of the sample in the assay. Do not refreeze! Expected assay response values Qualitative detection of HBV DNA in Grifols Procleix Ultrio Plus and Elite The expected distribution of assay response values on P0069 ViraQ HBV Trend 25 Control per 1000 test runs in the Ultrio assay versions is presented in Table 2. Table 2. Expected distribution of S/CO response values in Ultrio Plus and Elite assays. Range of assay response values Expected frequency per 1000 runs Test runs Interpretation Saturated: S/CO 12 870 Positive Dynamic: 1 S/CO<12 57 Positive Non-reactive S/CO <1.0 73 Negative Interpretation of results For daily monitoring, the Westgard 20 rules can be applied on non-reactive results which equals the event being outside the 95% confidence interval. To monitor consistency in analytical sensitivity, the frequencies of non-reactive, dynamic and saturated results obtained should be compared to the expected frequencies (Table 2) and/or peer groups. The chi-square test can be used to identify abberant trends. An aberrant trend is defined by p<0.05 and should be investigated to identify the cause. Performance characteristics Quantitation HBV-DNA in run control by viral load assay. The viral load (and 95% CI) results on P0069 ViraQ HBV Trend 25 in quantitative HBV DNA assay are shown in Table 3. Table 3. Measured viral load in P0069 ViraQ Trend 25 (see also limitations) Unitage Average 95 % confidence interval Copies/mL 1 IU/mL 1 25 4.7 17-38 3.4-7.1 1 Siemens Versant bdna 3.0 assay, only (Figure 1) P0069 ViraQ HBV Trend 25 6
Analytical sensitivity of the Ultrio Plus and Elite assay. The analytical sensitivity of the Ultrio Plus and Elite assay versions has been evaluated in different validation studies 1-4,17,19. Table 4 shows the 95% LOD of the Ultrio Plus and Ultrio Elite on the 1 st and 2 nd WHO (97/746 and 97/750), S0011 HBV genotype A and S0043 HBV genotype A inactivated standard (S0043 is used for manufacturing of the run control). From this table it is concluded that the Ultrio Plus and Ultrio Elite assays have the same analytical sensitivity. Thus, P0069 ViraQ Trend 25 is suitable for both assays. Table 4. Analytical sensitivity of Procleix Ultrio versions on Sanquin and BQC HBV-DNA standards and the 2 nd WHO HBV subtype A standard HBV-DNA standard Assay 50% LOD (CI) 95% LOD (CI) n version copies/ml copies/ml Ref EUROHEP genotype A Ultrio Plus 96 3.6 (2.7-4.7) 26 (19-36) 1 st WHO (97/746) Ultrio Plus 20 4.0 (2.1-7.8) 29 (15-59) 17 2 nd WHO (97/750) Ultrio Plus 791 3.7 (3.3-4.1) 27 (23-32) S0011 Sanquin HBV Ultrio Plus 48 4.9 (3.7-6.4) 44 (31-70) genotype A Ultrio Elite 24 3.8 (2.5-5.6) 35 (22-58) 1 S0043 BQC HBV genotype Ultrio Plus 24 6.6 (3.6-12) 48 (27-90) A inactivated Ultrio Elite 25 5.7 (4.0-8.2) 41 (24-90) 17 # IU/mL LOD values converted to copies per ml using factor of 5.33 copies per IU. The HBV DNA concentration in P0069 ViraQ HBV Trend 25 Control is set at approximately the 90% LOD of the Ultrio versions. Commutability of the inactivated standard to the native standard The results on standard dilution series of S0043 BQC HBV genotype A inactivated standard were used to design the P0069 ViraQ Trend HBV 25 control at approximately the 90% LOD (or 0.6 x 95% LOD). The test results of Ultrio Plus, Elite on the inactivated S0043 BQC standard differ from the native S0011 VQC-Sanquin standard. It is assumed this (not significant) difference is caused by lack of commutability of the inactivated BQC standard between testing in the bdna assay (used for calibration) and the target Ultrio Plus and Elite. Distribution characteristics of the S/CO results. Figure 2 shows the S/CO values in 193 Ultrio Elite Assay runs on P0069 ViraQ HBV Trend 25 Control in a histogram which confirm the expected reactivity. Obviously, the S/CO values are not normally distributed. A transformation of S/CO values to obtain normally distributed S/CO values appears not possible. Therefore monitoring S/CO response values in Levey-Jennings charts to identify responses outside confidence limits cannot be applied in a statistically valid manner. Only the proportions of S/CO results in the dynamic and negative range can be used to identify lack of performance. Figure 2. S/CO Distribution on P0069 ViraQ HBV Trend 25 in Ultrio Plus Assay (n=193) P0069 ViraQ HBV Trend 25 7
Predicted and observed reactivity rate in Ultrio versions. The observed non-reactivity levels in two different ViraQ HBV DNA run controls of 125 and 25 copies/ml in Ultrio Plus (P0065 and P0069) are decreased when compared to probit analysis data on BQC standard dilution series (P0031) in Ultrio Plus and Elite; combined (Table 5). When compared to the claimed reactivity by the manufacturer the results on the run control confirm consistency in reactivity of Ultrio Plus and Elite, Ultrio batches and run control batches. BQC product HBV-DNA level Copies/mL Table 5. Predicted and observed number of non-reactive response values in Ultrio assays of non-reactive non-reactive results Predicted number Observed number of results per 1000 per 1000 P0031 BQC HBV DNA standard dilution panel 1 x 95% LOD 43 50 N.A. P0065 ViraQ Check 125 2.90 x 95% LOD 125 6 0 (0/339) P0069 ViraQ Trend 25 0.60 x 95% LOD 25 121 70 (16/193) & 5.33 copies/ml is one IU/ml is applied Limitations P0069 ViraQ HBV Trend 25 Control is not intended to be used for evaluation of the analytical or diagnostic sensitivity of NAT blood screening assays. P0069 ViraQ HBV Trend 25 Control must not be substituted for the mandatory controls or calibrators provided with IVD test kits for calculating the cut off and/or criteria for releasing test results. The Poisson distribution in samples with low HBV concentrations will imply around 92 % reactive results will be found on P0069 ViraQ HBV Trend 25 Control in NAT blood screening assays. Therefore non-reactive, or low reactive response values on the run control should not be used for a decision to reject the test run. The expected distributions of assay response values on P0069 ViraQ HBV Trend 25 that are presented in this package insert are based on evaluation studies involving a limited number of runs and reagent batches of the Ultrio and Ultrio Plus assays. It cannot be guaranteed that future reagent batches of the Ultrio Plus and Elite assay versions will generate the same reactivity. It cannot be guaranteed that the inactivated BQC standard from which P0069 ViraQ HBV Trend 25 is prepared is commutable across different NAT methods. Commutability of lyophilized or heat-inactivated virus in WHO and BQC standards with regard to reported concentrations in IU/mL and copies/ml by different quantitative HBV DNA assays has not been investigated. Therefore establishing accuracy of viral load measurements using the run control is not appropriate The manufacturers of the quantitative HBV DNA assays have prepared their internal calibrators based on different standards and this may cause systematic differences in quantitative results between methods. The accuracy of different quantitative assays in reporting in IU or copies/ml values could have been affected by the differences in calibration of the NAT systems and by heat inactivation of the BQC standard. Therefore laboratories should define their own geometric mean and 95% CI in the applied test system and interpret results according to the Westguard rules 18. P0069 ViraQ HBV Trend 25 8
References 1. Grabarczyk P, van Drimmelen H, Kopacz A, Gdowska J, Liszewski G, Piotrowski D, Górska J, Kuśmierczyk J, Candotti D, Lętowska M, Lelie N, Brojer E. Head-to-head comparison of two transcription-mediated amplification assay versions for detection of hepatitis B virus, hepatitis C virus, and human immunodeficiency virus Type 1 in blood donors. Transfusion. 2013;53:2512-2524 2. Assal A, Barlet V, Deschaseaux M, Dupont I, Gallian P, Guitton C, Morel P, David B, and De Micco P. Comparison of the analytical and operational performance of two viral nucleic acid test blood screening systems: Procleix Tigris and cobas s 201. Transfusion 2009;49:289-300 3. Koppelman M, Assal A, Chudy M, Torres P, de Villaescusa RG, Reesink HW, Lelie PN, Cuypers HT. Multi-center performance evaluation of a transcription-mediated amplification assay for screening of human immunodeficiency virus-1 RNA, hepatitis C virus RNA, and hepatitis B virus DNA in blood donations Transfusion 2005;45:1258-66 4. Grabarczyk P, Koppelman M, Boland F, Sauleda S, Fabra C, Cambie G, O Riordan K, Van Drimmelen H, O Riordan J, Lelie N. Inclusion of human immunodeficiency virus type 2 (HIV-2) in a multiplex transcription mediated amplification assay does not affect detection of HBV and hepatitis B and C virus genotypes: A Multi-center performance evaluation study. Transfusion 2015 [Epub ahead of press]. 5. Heermann KH, Gerlich WH, Chudy M, Schaefer S, Thomssen R.Quantitative detection of hepatitis B virus DNA in two international reference plasma preparations. Eurohep Pathobiology Group.J Clin Microbiol. 1999 Jan;37(1):68-73. 6. Saldanha J, Gerlich W, Lelie N, Dawson P, Heermann K, Heath A; WHO Collaborative Study Group.An international collaborative study to establish a World Health Organization international standard for hepatitis B virus DNA nucleic acid amplification techniques.vox Sang. 2001 Jan;80(1):63-71. 7. Baylis SA, Heath AB, Chudy M, Pisani G, Klotz A, Kerby S, Gerlich W.An international collaborative study to establish the 2nd World Health Organization International Standard for hepatitis B virus DNA nucleic acid amplification technology-based assays.vox Sang. 2008 May;94(4):358-62 8. Fryer JF, Heath AB, Wilkinson DE, Minor PD and the collaborative study group. Collaborative study to evaluate the proposed 3rd WHO International Standard for hepatitis B virus (HBV) for nucleic acid amplification technology (NAT)-based assays. WHO ECBS Report 2011; WHO/BS/2011.2170 9. Van Drimmelen A.A.J., Lelie PN. Preparation of inactivated secondary viral standards: Safety assessment of quality control samples for viral serology and NAT assays in blood screening laboratories. BQC document number CE4006. 10. Lelie PN, Reesink HW, Lucas CJ. Inactivation of 12 viruses by heating steps applied during manufacture of a hepatitis B vaccine. J Med Virol. 1987;23:297-301. 11. Lelie PN, Reesink HW, Niessen J, Brotman B, Prince AM. Inactivation of 10(15) chimpanzee-infectious doses of hepatitis B virus during preparation of a heatinactivated hepatitis B vaccine.j Med Virol. 1987 Nov;23(3):289-95. 12. Collins ML, Zayati C, Detmer JJ, Daly B, Kolberg JA, Cha TA, Irvine BD, Tucker J, Urdea MS. Preparation and characterization of RNA standards for use in quantitative branched DNA hybridization assays. Anal Biochem. 1995 20;226:120-9. 13. Weusten J, Vemeulen M, Van Drimmelen H, Lelie PN. Refinement of a viral transmission risk model for blood donations in serconversion window phase screened by nucleic acid testing in different pool sizes and repeat test algorithms. Transfusion 2011;51:203-15 P0069 ViraQ HBV Trend 25 9
14. Komiya Y, Katayama K, Yugi H, Mizui M, Matsukura H, Tomoguri T, Miyakawa Y, Tabuchi A, Tanaka J, Yoshizawa H. Minimum infectious dose of hepatitis B virus in chimpanzees and difference in the dynamics of viremia between genotype A and genotype C.Transfusion. 2008 Feb;48(2):286-94 15. Hsia CC, Purcell RH, Farshid M, Lachenbruch PA, Yu MY. Quantification of hepatitis B virus genomes and infectivity in human serum samples.transfusion. 2006 Oct;46(10):1829-35 16. Kleinman SH, Lelie N, Busch MP. Infectivity of human immunodeficiency virus-1, hepatitis C virus, and hepatitis B virus and risk of transmission by transfusion.transfusion. 2009 Nov;49(11):2454-89. 17. Lelie N., Van Drimmelen H. and the International NAT Study Group. Calibration and stability of WHO and secondary viral standards. http://www.nibsc.org/spotlight/sogat/blood_virology/past_meetings.aspx SoGAT XXIV 8-9 May 2013, Ljubljana, Slovenija 18. Centers for Disease Control (CDC). Update: Universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis B virus, and other blood borne pathogens in health-care settings. MMWR 1988; 37:377-388. 19. Centers for Disease Control (CDC). Guidelines for prevention of transmission of human immunodeficiency virus and hepatitis B virus to health-care and public-safety workers. MMWR 1989; 38(S-6): 1-36. 20. Westgard rules. www.westgard.com. P0069 ViraQ HBV Trend 25 10
P0069 ViraQ HBV Trend 25 11
BioQControl B.V. Visseringlaan 25 2288 ER Rijswijk The Netherlands Tel: +31 (0)88 235 33 33 Fax: +31 (0)88 235 33 00 Internet: www.bioqcontrol.com KI4065 v1.0 June 2016 P0069 ViraQ HBV Trend 25 12