P0154 ViraQ HBV Trend 50 P0154

P0154 ViraQ HBV Trend 50 P0154 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... 3 Kit Contents... 4 Storage Instructions... 4 Warning and precautions... 4 Test Procedure... 5 Expected assay response values... 5 Intepretation of results... 5 Performance characteristics... 5 Limitations... 7 References... 8 2 P0154 ViraQ HBV Trend 50

Intended Use P0154 ViraQ HBV Trend 50 is intended to be used as external run control for monitoring consistent performance and analytical sensitivity of multiplex nucleic acid test (NAT) blood screening assays for detection of HBV-DNA (see Table 1). P0154 ViraQ HBV Trend 50 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 Test kits covered by this run control Equipment Agent Test kits TIGRIS PANTHER Hepatitis B virus Procleix ULTRIO Plus Procleix ULTRIO Elite Key to Symbols Used Manufacturer Lot number Catalogue number Store between -15 C and -40 C Biological substance Category B Device for performance evaluation Expiry date Contents Caution Read instructions for use Principle of method P0154 ViraQ HBV Trend 50 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 P0154 ViraQ HBV Trend 50 control samples is set at 50 copies (cps)/ml as measured in replicate Siemens bdna 3.0 assays (equivalent to 9.4 International Units (IU)/mL). This level is chosen close at the 95 % lower limit of detection (LOD) of the Ultrio Plus, Ultrio Elite 1-4 assays. The external run control generates sample to cut-off (S/CO) ratios in the saturation, dynamic and negative range of the Ultrio assay versions. Changes in frequency of nonreactive, 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 donor blood collection tubes. The thawed tubes are ready for use and can be placed at random positions in laboratory equipment sample racks. Traceability of HBV-DNA concentration in IU/ml and copies/ml A HBV-DNA genotype A standard has been prepared by heat inactivation of HBV-DNA reactive plasma units pool from one donor in the chronic phase. The HBV-DNA standard is inactivated by heat treatment 5-8. The inactivated standard has been quantified in the Siemens Versant bdna 3.0 assay 9 against similar concentrations of the historically established secondary Sanquin HBV-DNA genotype A standard 1,10. The Sanquin HBV-DNA P0154 ViraQ HBV Trend 50 3

genotype A standard has been calibrated at 5.33 copies per IU against the first WHO HBV- DNA (97/746) standard 1,11,12. The accurate calibration of the secondary Sanquin HBV genotype A and inactivated BQC genotype A standards in copies/ml and IU/ml has been confirmed in analytical sensitivity studies of the Procleix TMA Ultrio plus and Ultrio Elite assays in which the WHO and BQC standards have been compared 13,14. The inactivated HBV-DNA genotype A standard being the source material for manufacturing of the run control has therefore been adequately quantified in IU/ml and copies/ml. Both the secondary Sanquin HBV genotype A standard and inactivated BQC HBV genotype A standards are available at long term and stored at -80 C under secured conditions in sufficient quantities. The viral concentration in the run control samples is targeted at 50 cps/ml (9 IU/mL). The production and quality control methods guarantee a consistent virus concentration in consecutive ViraQ Trend Control batches. Kit Contents 10 Tubes, each containing 1.5 ml P0154 ViraQ HBV Trend 50 in polypropylene tubes with screw caps and comparable in size to Vacutainer tubes used for donor sample collection. The run control contains HBV-DNA genotype A standard diluted in human plasma without preservatives. Storage Instructions The run controls should be stored at or below -30 C to ensure minimal degradation of HBV- DNA. Once thawed the run control samples should be used immediately. Do not refreeze the controls after thawing. Any control sample that appears cloudy or contains precipitates after thawing and mixing should be discarded of. Warning and precautions Although P0154 ViraQ HBV Trend 50 contains inactivated HBV particles 5, the plasma may still be potentially bio-hazardous (see MSDS). The matrix is prepared from human blood plasma that tested negative for blood borne viruses (HBV-DNA, HCV-RNA, HIV-RNA, HBsAg, 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 transmission prevention of infectious agents when handling these materials 15,16. 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. Once thawed, do not re-freeze and thaw the run control samples to avoid formation of cryoprecipitates that could alter reactivity or cause pipetting errors in the automated sampling systems. Store run controls in an upright position. 4 P0154 ViraQ HBV Trend 50

Test Procedure Thaw the run control quickly in a water bath at 37 C. Mix gently during thawing until contents are just thawed. Immediately after thawing remove the run control tube from the water bath. 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 control samples. The controls should be handled and tested in a manner identical to that of clinical specimens in the test procedure being evaluated. Expected assay response values The expected distribution of assay response values on P0154 ViraQ HBV Trend 50 Control per 1000 test runs in the Ultrio plus and elite versions is presented in Table 1 Table 1 shows the measured proportion of non-reactive, dynamic and saturated S/CO response values in 1311 Ultrio Plus assay runs on P0154 ViraQ HBV Trend 50 run control. The dynamic and saturated response values proportions in Table 1 were established assuming Poisson distribution and were based on S/CO ratios obtained from multiple P0154 ViraQ Trend 50 Control and Ultrio reagent batches. Table 1. Expected proportion of assay response values on P0154 ViraQ HBV Trend 50 Control S/CO ratio Response Expected range 12.0 saturated 91 100% 1.0 12.0 dynamic 2.2 4.1% <1.0 nonreactive 0.2 1.0% * Ultrio Plus and Ultrio Elite. Intepretation of results P0154 ViraQ HBV Trend 50 Control should react as described in tables 1 and 4. Users could apply the chi-square test for comparison of the frequency of nonreactive, dynamic and saturated results obtained with different reagent batches and compare those with the frequencies given in Table 1. Statistical significant differences should be investigated to identify the cause. The Westgard rules can be applied on nonreactive results, which equals being outside the 99 % confidence interval. Performance characteristics Analytical sensitivity The analytical sensitivity of the Ultrio plus and Ultrio Elite assays is evaluated in different validation studies 1-4,10,13,14. Table 2 compares the 50% and 95% LODs in these studies showing consistent analytical sensitivity on the different HBV standards as well on different versions of the Procleix assays. P0154 ViraQ HBV Trend 50 5

Table 2. Analytical sensitivity of Procleix Ultrio versions on Sanquin and BQC HBV-DNA standards and three consecutive WHO HBV genotype 1a standards Assay 50 % LOD (CI) 95 % LOD (CI) HBV-DNA standard n Ref version copies/ml copies/ml Sanquin secondary Ultrio Plus 48 4.9 (3.7-6.4) 44.4 (30.6-70.3) HBV genotype A # 4 Ultrio Elite 24 3.8 (2.5-5.6) 34.5 (22.1-58.0) BQC Inactivated HBV genotype A Ultrio plus 30 6.7 (3.1-15.1) 65.4 (25.3-584) 1 1 st WHO HBV Ultrio plus 303 4.9 (4.3-5.6) 25.1 (20.8-31.4) 14 (97/750)* Ultrio Elite 229 4.8 (4.1-5.6) 24.5 (20.3-31.4) # calibrated on 1 st HBV WHO (96/790) standard at 5.33 copies per IU 1,12 calibrated on Sanquin HBV genotype A standard in multiple bdna 3.0 assays * IU/mL LOD values converted to copies per ml using factor of 5.33 copies per IU 1,12. Observed and predicted response on P0154 ViraQ HBV Trend 50 run control in Ultrio plus and Ultrio Elite Table 3 compares the observed and predicted test results on P0154 ViraQ HBV Trend 50 run control in the Ultrio versions. Table 3. Observed and predicted number (Ultrio plus) of non-reactive results on P0154 ViraQ HBV Trend 50 based on probit analysis of percentage reactive results on inactivated HBV standard dilutions. HBV run control level Cps/mL Predicted number of nonreactive results per 1000 1 x 95 % LOD 65 (25-584) 50 3 x 95 % LOD 195 (75-1752) 7.5 5 x 95 % LOD 325 (125-11680) 2.5 Observed number of nonreactive results per 1000 P0154 ViraQ HBV Trend 50 50 73 5.5 Figure 1 shows the S/CO values in 1311 Ultrio Assay runs on P0154 ViraQ HBV Trend 50 Control. When assessing the S/CO distribution on the P0154 ViraQ HBV Trend 50 Control, there are three distinct populations of Ultrio response values, i.e. nonreactive, dynamic and saturated. A transformation of S/CO values to obtain normal distributed S/CO values is not available. Therefore monitoring S/CO response values in Levey-Jennings charts to identify responses outside confidence limits cannot be applied in a statistically valid manner and is not meaningful. 6 P0154 ViraQ HBV Trend 50

Figure 1. Distribution of S/CO values on P0154 ViraQ HBV Trend 50 in 1311 Ultrio Plus runs (see also table 4). This figure shows the results ordered on S/CO values. There are two distinct groups. The first small group present dynamic results whereas the second large group present saturated results. Table 4. Measured response rates of different Ultrio Plus and Ultrio Elite batches on P0154 ViraQ HBV Trend 50 run control Assay Batch n % Neg. % Dyn. Pos. %Sat. Pos. % Positive 1 103 1.0% 0.0% 99.0% 99.0% 2 299 0.7% 2.0% 97.3% 99.3% 3 67 1.5% 1.5% 97.0% 98.5% Ultrio Plus 3 180 0.0% 0.6% 99.4% 100.0% 3 204 0.5% 1.0% 98.5% 99.5% 3 228 0.4% 3.9% 95.6% 99.6% 3 90 0.0% 2.2% 97.8% 100.0% 3 91 2.2% 1.1% 96.7% 97.8% Ultrio Elite 4 49 0.0% 38.8% 61.2% 100.0% All all 1311 0.6% 3.2% 96.3% 99.4% Limitations P0154 ViraQ HBV Trend 50 Control cannot be used to evaluate the diagnostic sensitivity of NAT blood screening assays. P0154 ViraQ HBV Trend 50 Control is not a substitution for the mandatory controls or calibrators provided with IVD test kits for calculating the cut off and/or criteria for releasing test results. The expected distributions of assay response values on P0154 ViraQ HBV Trend 50 Control presented in this package insert were based on evaluation studies involving a limited number of assays and reagent batches. P0154 ViraQ HBV Trend 50 7

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;53:5012-5024. 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;45:1508-66. 4. Grabarczyk P, Koppelman M, Boland F, Sauleda S, Fabra C, Cambie G, O Rioedan K, Van Drimmelen H, Vermeulen M, O Riordan J, Lelie N. Multi-center performance evaluation of the Procleix Ultrio Elite Assay on the panther System. Submitted. 5. 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 (manuscript in preparation). 6. 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 Nov;23(3):297-301. 7. Mauler R, Merkle W, Hilfenhaus J. Inactivation of HTLV-III/LAV, hepatitis B and non- A/non-B viruses by pasteurization in human plasma protein preparations. Dev Biol Stand. 1987;67:337-51. 8. Nowak T, Niedrig M, Bernhardt D, Hilfenhaus J Inactivation of HIV, HBV, HCV related viruses and other viruses in human plasma derivatives by pasteurisation. Dev Biol Stand. 1993;81:169-76. 9. 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. 10. Lelie PN, Van Drimmelen AAJ, Cuypers HTM, Best SJ, Stramer Hyland SL C,.Allain J- P, Moncharmont P, Defer C, Nubling CM, Glauser A, da Silva Cardoso M, Viret J-F, Lankinen M, Grillner L, Wirthmuller U, Coste J, Schottstedt V, Masecar B. and E.M. Dax.Sensitivity of HCV-RNA and HIV-RNA blood screening assays. Transfusion. 2002,42:527-36. 11. 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. 12. 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;37:68-73. 8 P0154 ViraQ HBV Trend 50

13. Van Drimmelen H., Bremer C, Gerlich W, Quint W, Ullum H, O Riordan J, Gdowska J, Grabarczyk P, Brojer E, Lelie N, Commutability of inactivated or lyophilised virus in external quality control (EQC) samples for NAT. http://www.nibsc.org/spotlight/sogat/blood_virology/past_meetings.aspx SoGAT XXII 14-15 April 2011, Rome, Italy. 14. Nico Lelie, Harry van Drimmelen 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. 15. 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. 16. 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. 17. Westgard rules. www.westgard.com. P0154 ViraQ HBV Trend 50 9

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BioQControl B.V. Visseringlaan 50 2288 ER Rijswijk The Netherlands Tel: +31 (0)88 235 33 33 Fax: +31 (0)88 235 33 00 Internet: www.bioqcontrol.com KI4154 v1.1 January 2016 12 P0154 ViraQ HBV Trend 50