Evaluation Report

January 2001

Evaluation Report

NUMBER

MDA 01001

Monolisa® anti-HCV PLUS versions 1 and 2

© Crown Copyright

£60

© Crown Copyright 2001 Apart from any fair dealing for the purposes of research or private study, or criticism, or review, as permitted under the Copyright, Designs & 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 Colgate, NORWICH, NR3 1BQ.

An evaluation of Monolisa® anti-HCV PLUS version 1 (product code 72311 / 72312) and version 2 (product code 72317 / 72318) Keith R. Perry Christine S. Burgess Olapeju F. Ogunade John V. Parry Microbiological Diagnostics Assessment Service (MiDAS) Hepatitis and Retrovirus Laboratory Virus Reference Division Central Public Health Laboratory 61 Colindale Avenue LONDON NW9 5HT Pauline Rogers Statistics Unit PHLS Communicable Disease Surveillance Centre 61 Colindale Avenue LONDON NW9 5EQ

Contents

Summary

3

Introduction

5

Description of the assay

9

Method

13

Specimen panel

15

Results

17

Technical appraisal

37

Conclusions

39

Acknowledgements

41

References

43

Appendix

47

MDA Evaluation Report: Monolisa® anti-HCV PLUS versions 1 and 2

1

2

MDA Evaluation Report: Monolisa ® anti-HCV PLUS versions 1 and 2

Summary

Background

This report focuses on two versions of the Monolisa® anti-HCV PLUS enzyme immunoassay. The two versions have different substrate and stopping solution concentrations. North London Blood Centre (NLBC) and Southampton Blood Centres (SBC) undertook specificity studies. MiDAS undertook the sensitivity evaluation and small specificity assessments. Throughout the report results are compared with evaluations of other anti-HCV kits. Evaluation panel

Version 1 of the assay was assessed against a panel of 2885 specimens. Of these, 2090 were blood donors’ specimens, 199 were other anti-HCV negative specimens and 148 were ‘screen reactive and RIBA 3.0 negative’ specimens. One-hundred and one anti-HCV positive and 22 indeterminate samples were tested. A further 222 specimens were from 28 commercial seroconversion panels and 100 from five commercial low and mixed titre panels. Reactivity with three quality control samples was also assessed. Version 2 was assessed against a panel of 2459 specimens. Of these, 2328 were blood donors’ specimens. A small panel of 131 specimens was tested against two lots of the kits to establish whether the sensitivity was similar to that found for version 1. Of the 131 specimens, 40 were randomly selected anti-HCV positive, 64 were specimens from seven seroconversion panels and 15 were from a commercial low titre panel. Two quality control samples and 10 genotyped samples from a pre-batch acceptance panel were also employed. Specificity findings

The repeat reactive rate for Monolisa® anti-HCV PLUS version 1 was 0.29% (95% confidence interval 0.1 – 0.6%) based on testing 2090 specimens from blood donors, and 0% (95% confidence interval 0 – 1.8%) based on testing 199 other anti-HCV negative specimens. Of 148 anti-HCV ‘screen reactive / RIBA HCV 3.0 negative’ specimens, sixteen were repeatedly reactive by Monolisa ® anti-HCV PLUS version 1. The repeat reactive rate for each lot of the version 2 assay was 0% (95% confidence interval 0 – 0.3%) Sensitivity findings (randomly selected positives)

The sensitivity of Monolisa ® anti-HCV PLUS version 1 was 100% (95% confidence interval 96.4 – 100%) based on initial reactions with 101 anti-HCV positive specimens. The OD/CO ratios were between 1.2 and 10.7 (mean 8.50, median 8.73). Five specimens gave OD/CO ratios less than 5.0 (range 1.2 – 4.6). The sensitivity of both lots of the version 2 assay was 100% (95% confidence interval 91.2 – 100%) based on testing 40 positive specimens. The OD/CO ratios were all above 5.0, though the specimens were not the same as


3

Summary

those used to evaluate version 1. Sensitivity findings (seroconversion / performance panels)

The ability of the Monolisa® anti-HCV PLUS version 1 assay to detect early antibody was compared with four other anti-HCV kits. The sensitivity scores for 24 seroconversion panels and five performance panels were combined to give a total sensitivity score. For each kit the aggregate sensitivity score was obtained by summing the total number of reactive samples. An analysis of the number of days later each kit detected primary infection compared with the most sensitive anti-HCV kit was also performed. The Ortho HCV ELISA Test System with Enhanced SAVe was the most sensitive assay by seven points with a total score of 125 (mean delay 0.58 days) , followed by PRISM anti-HCV assay with a score of 118 (mean delay 1.38 days). Monolisa® anti-HCV PLUS v1 was the next most sensitive assay with a score of 110 (mean delay 2.88 days). The median delay for these three kits and Abbott HCV EIA 3rd generation was zero. Eight commercial panels were used to assess the sensitivity of the version 2 assay. The two lots of the version 2 assay gave scores of 40 and 41 compared with 37 for the version 1 assay, suggesting that there had been an improvement in sensitivity. Quality control samples and lot variation

Three quality control samples gave values in a range suitable for statistical assay control. The version 2 assay was also tested against a PHLS HCV prebatch acceptance panel consisting of 10 genotyped samples. All were detected except one sample (genotype 1a) with an indeterminate RIBA HCV 3.0. Onehundred and fifty-nine specimens were tested by two lots of the version 2 kit. All gave the same qualitative result, except for one seroconversion panel member (PHV904-04). Overall, samples that were negative or had low OD/CO ratios had good agreement. Strongly positive samples had a statistically significant difference in OD/CO ratios. Technical appraisal

Both versions of the kit were presented as standard microplate-based enzymeimmunoassays, and with good packaging and labelling of the reagents. The assays were simple to perform and took approximately 2½ hours to complete. Conclusions

The Monolisa® anti-HCV PLUS assays were in the same order of sensitivity as other anti-HCV kits. The two versions were of similar sensitivity and both had acceptable specificity. On the basis of the studies reported here, these kits appear to be suitable as first line screening assays in the Blood Service and in clinical diagnostic laboratories.

4


Introduction

This report describes results obtained from assessments of two versions of the Monolisa® anti-HCV PLUS enzyme–immunoassay. Results from this evaluation were compared with those obtained from the evaluation of other HCV screening assays published in previous MDA reports1-4. The assay is based on the ‘indirect’ or anti-globulin format and utilises three recombinant proteins in the non-structural (NS3 and NS4) and structural areas of the hepatitis C virus genome. Hepatitis C virus (HCV) is the most frequent cause of parenterally transmitted and sporadic chronic non–A, non–B hepatitis (NANBH)5,6. Since anti-HCV tests have been available it has become apparent that the majority of all posttransfusion hepatitis (PTH) occurring since the introduction of sensitive HBsAg screening of blood donations had been due to HCV infection. Where anti-HCV screening of blood donors has become routine the incidence of PTH has fallen to very low levels 7. HCV induces an antibody response in infected individuals, most of whom continue to be seropositive and remain persistently infected. They are therefore potentially infectious 8,9. The genetic cloning and expression of a non-structural HCV antigen (c100–3) from the plasma of a chimpanzee in which infection had been induced by inoculation with serum from a human case of putative parenterally-transmitted NANBH made it possible to develop the assays for anti–HCV, even though the virus had not been visualised or grown in cell culture10. The c100–3 antigen (coded by the NS4 region of the HCV genome) was used in an early anti-HCV immunoassay to demonstrate the association with NANB hepatitis and then to develop the first generation of anti–HCV screening assays5. In the next stage of development, assays were designed to incorporate other HCV antigens encoded by the core and NS3 genes. Some of the assays now available incorporate an additional antigen from the NS5 region of the HCV genome, which codes for an RNA polymerase, and accurately identify 90–95% of patients with chronic HCV infection11. Immunoassays using both recombinant and synthetic peptide antigens have now been developed and commercialised. Figure 1 is a diagrammatic representation of the HCV genome and some of the antigens that have been employed in the development of diagnostic anti–HCV assays. Theoretical models of the appearance and clearance of viral antigens and antiviral antibody markers have been developed for various viruses. In the case of HCV, it can be shown by reverse transcription of HCV RNA and its amplification by the polymerase chain reaction (RT-PCR) that the virus is often persistent and that individuals' immune responses to it are diverse. Assay methods that permit immune responses to be recognised as antibodies against individual viral proteins (eg RIBA, LIA, WB, Deciscan, Matrix) reveal differences between individuals, and over time, in the range and intensity of antibody responses to


5

Introduction

specific HCV gene products in the same individual12. Reactivity in EIAs of specimens from anti–HCV positive individuals may also be influenced by viraemia and genotype13 ie the sera from patients with viraemia and those infected with type 1 have shown greater EIA reactivity. Recent exposure and an evolving immune response may sometimes explain absent or feeble serological reactivity, but in many cases these explanations seem inadequate. Nor is it clear that particular anti–HCV profiles correlate with the severity of infection or the prognosis. In view of the above considerations, it is not surprising that no serological method that might be considered a ‘gold standard’ has been devised to confirm infection with HCV. Moreover, some supplemental anti–HCV assays employ antigens so similar to those incorporated in screening kits made by the same or other manufacturers that they could lead to false confirmation of positive reactions in the screening test. Another difficulty is that the supplemental serological assays lead to a proportion of screen reactive specimens being classified, on the basis of single band reactivity, as anti–HCV indeterminate. On the basis of attempts to amplify HCV RNA by PCR from these specimens and other evidence some of these sera contain HCV but most, particularly in a low–risk population, do not14,15. Figure 1: Diagrammatic representation of the HCV genome 5’

Structural

3010-3033aa

Non-structural

3’

~9.6 kb

core

C

envelope

E1

E2

NS2

NS3

. p21 gp36 gp70 p7 p23

p70

p56/p58

p68

}

4

p27

NS5b

}

3

p8

NS5a

}

2

} }

} } 1

4a NS4b

5

6

7

Features of the viral proteins 1. RNA binding, nucleocapsid protein, LTßR binding 2. Virion glycoproteins, E1-E2 heterodimers , neutralisation targets 3. NS2-3 autoprotease 4. N-terminal Ser protease, C-terminal NTPase/RNA helicase 5. Ser protease cofactor 6. Ser phosphoprotein, Interferon sensitivity determining region (ISDR). 7. RNA-dependent RNA polymerase , terminal transferase

Notes: The spectrum of diagnostic antigens derived from the genome of HCV has evolved since the first immunoassays were developed. The first assays employed a single polypeptide (c100–3) antigen produced by recombinant DNA expression. Currently available anti–HCV assays incorporate several ‘recombinant’ antigens and synthetic oligopeptide antigens derived mostly from the core/E1, NS3, NS4 and NS5 genes. Diagram adapted from Reed KE and Rice CM (1998): Molecular characterisation of Hepatitis C virus in Reesink HW (Ed), Hepatitis C Virus, 2nd edition, Karger, Switzerland. p4

6


Introduction

Development of HCV RT-PCR assays enabled early detection of hepatitis C infection and these assays have since been introduced into routine blood bank screening in the UK. Their introduction offers potential benefits because of the higher HCV incidence in blood donors relative to other transfusion transmitted infections, as well as the longer potentially infectious seronegative window period. The high genome copy number during seroconversion also made HCV a suitable candidate for detection in pools of samples 16. Financial and practical considerations influenced the current size of the pools (96 samples), but it is expected that pool sizes will gradually reduce, eventually leading to single sample testing17. More recently an HCV antigen EIA has been developed that claims similar seroconversion sensitivity to commercial HCV RNA tests18. This may prove to be a more economic and robust approach than RT-PCR to closing the seronegativity window. At present serological and supplemental serological assays still have a valuable role in the diagnosis of HCV infection. For example, it is possible that antiHCV positive blood, which is RT-PCR negative, could transmit HCV, as has been demonstrated in a chimpanzee experimental model system19. It is also known that diagnostic PCR testing is technically exacting and errors can arise, either as false positives (usually as a result of contamination of a specimen with extraneous HCV genetic material) or as false negatives if there has been loss of viral RNA and inadequate transcription to cDNA or because of the presence of inhibitors of PCR. In a PCR performance assessment exercise only five of 31 participating laboratories obtained faultless results20 suggesting that, in routine use, PCR is likely to be less robust as a confirmatory procedure than a serological test. In any case, antibody tests and RT-PCR detect different facets of HCV infection and cannot be expected to give identical results on all occasions. Nevertheless RT-PCR is an increasingly useful diagnostic tool and it is helpful to check serological confirmations by detection of HCV RNA 21. Specimens that the supplemental serological assays indicate are anti-HCV positive very often contain viral RNA by RT-PCR 8,22. Conversely, there have been reports of antiHCV negative or indeterminate blood specimens that are RT-PCR positive and which might therefore transmit HCV23. The specimens employed in this evaluation had previously been characterised. In the main, HCV status was based on complete agreement between all EIAs employed. When not all EIAs were in accord, status was determined on the basis of results in three or four supplementary immunoblot assays. RT-PCR tests were not done, particularly as the specimens in the panel had not all been stored optimally for that investigation.


7

8



A summary of the characteristics of Monolisa ® anti–HCV PLUS version 1 and 2 kits is given in Table 1. This includes details such as the product number, specimen volume required and antigens used in the assay. We show the current registration status of the kit with European and American agencies where regulatory controls are in place. Quotes from the instructions that accompany the kit, indicating the manufacturer's claims for the performance of the kit and applications for which it is recommended, are also given in Table 2. Both versions of Monolisa® anti–HCV PLUS are indirect enzyme–immunoassays which use microwells as the solid phase. The antigens coated on the microwells are purified recombinant proteins from the non–structural (NS3 and NS4) and structural regions of the hepatitis C genome. During incubation of the samples and controls antibodies to HCV, if present, bind to the solid phase antigens. The residue of the specimen is then washed away. In the next step, peroxidase–labelled goat anti–human IgG binds to the specific antibodies already bound to the antigens on the solid phase. Samples that did not contain specific antibody do not bind the conjugate to the well. After unbound conjugate has been removed by washing, the presence of enzyme immobilised in complexes is detected by the addition to the wells of substrate, comprising a solution of tetramethylbenzidine (TMB) and dimethyl sulphoxide (DMSO). The enzyme reaction is stopped by the addition of sulphuric acid and absorbance values are read using a spectrophotometer at 450nm, with a 620nm reference filter. The amount of conjugate, and hence colour, in the wells is, over a small range, proportional to the concentration of antibody to HCV in the sample. The version 2 assay includes a change in the substrate (kit reagents R8 and R9) and stopping solution (R10) concentrations (Table 1 and 21).


9


Table 1: Assay information General: ®

Assay name Manufacturer Product number Number of tests in one pack Test volume ISO9000 series certification FDA registered PEI registered AFSSaPS registered CE Mark UK launch date

Monolisa anti-HCV PLUS Sanofi Diagnostics Pasteur Ltd 72311 / 72312 96 / 480 20µl 9001 No Yes Yes NA 1996

®

Monolisa anti-HCV PLUS version 2 BioRad Laboratories 72317 / 72318 96 / 480 20µl 9001 No Yes Yes NA January 2000

Presentation: Assay type Antigens on solid phase Conjugate Chromogen Peroxidase substrate buffer

Indirect NS3, NS4 and structural recombinant proteins peroxidase-labelled goat antibody against human IgG tetramethylbenzidine (TMB) / DMSO tetramethylbenzidine (TMB)

Stopping solution Sample colour change Positive control wells per run Negative control wells per run Reading wavelength Cut-off calculation

Sodium citrate and sodium acetate solution pH5.2 containing H 2O2 (0.009%) and DMSO (4%)

Citric acid and sodium acetate solution pH4.0 containing H 2O2 (0.015%) and DMSO (4%)

1.5N Sulphuric acid

1N Sulphuric acid

Purple to blue 3 2 450 / 620nm Mean positive control OD / 5

Stages: Preparation / loading time (90 tests) Sample incubation Wash step Conjugate incubation Wash step Substrate incubation Assay completion time Total time to completion Number of optional procedures

20 minutes 37°C or 40°C / 60 minutes 3x 37°C or 40°C / 30 minutes 4x 18 - 30°C / 30 minutes 2 hours 2 hours 20 minutes 2

Additional equipment required Absorbent paper, Water bath or dry incubator Micropipette to deliver, 20µl, 80µl, 100µl, 200µl, 1ml, Disposable tips Graduated cylinders - 10ml, 200ml, 1l ; Latex gloves and protective safety glasses Microplate washing system, Microplate reading device

Notes: FDA = Food and Drug Administration, USA; PEI = Paul-Ehrlich-Institut, Germany AFSSaPS = Agence Française de Sécurité Sanitaire des Produits de Santé, France.

10



Table 2: Claims and limitations of the assay Claims for the assay (from the kit insert) Monolisa® anti-HCV PLUS allows the detection of the antibodies associated with an infection by Hepatitis C virus in patient serum or plasma.

Limitations of the assay (from the kit insert) -Given the diversity of the immunological reactions of infected patients by the hepatitis C virus (especially during seroconversions), some differences of detection between tests can be observed depending o n the kind of antigenic proteins used. A negative result with a screening test does not exclude the possibility of exposition or infection by hepatitis C virus -Results just below the cutoff value (Vs -10% = 10

0.8

0.6

-0 .3 9 0.5 9 0.4 -

0. 2

30 days). This is particularly evident for IMx HCV (mean delay 9.6 days). This kit showed poor performance in one particular panel (PHV907) that had a large interval between the last negative and first positive bleed. When this panel is removed from the analysis IMx has a mean delay of 3.7 days. The effect of outlying results is indicated by the large difference between the mean and median delays. The median value may therefore represent a more robust indication of differences in sensitivity. A small analysis of six seroconversion panels adjusted to include the version 2 kit, showed that version 1 had a range of 0 to -20 days delay (mean 5.67 days, median 3 days) and version 2 had a 0 to -20 days delay (mean 4.67 days, median 1 day). Ortho® HCV 3.0 Test System with enhanced SAVe was the most sensitive with a mean delay of 0 days (median 0 days), followed by PRISM™ anti–HCV assay with a mean delay of 0.5 days (median 0 days).


29

Results

Table 15: Comparative timing of the detection of seroconversion Anti-HCV assay

Product number

Ortho HCV enhanced SAVe (Ortho Diagnostics)

PRISM™ anti-HCV (Abbott Laboratories Ltd)

Monolisa ® anti-HCV PLUS v1 (Sanofi Diagnostics Pasteur)

Abbott HCV EIA 3rd generation (Abbott Laboratories Ltd)

Delay in detecting seroconversion in each

MDA

panel compared with the most sensitive assay reference

Range (Days)

Mean (Days)

Median (Days)

9307401

0 to 7

0.58

0

00051

6A52-48

0 to 10

1.38

0

97/72

72312

0 to 20

2.88

-2

01001

7A16-23

0 to 41

4.83

-2

95/03

3A99-20

0 to 146

9.63

0

-

®

IMx HCV (Abbott Laboratories Ltd)

Notes: The upper limit of the range is, to some extent, influenced by the intervals between bleeds for any individual panel The mean and median values provide a better general guide to each assay's ability to detect seroconversion. When any assay failed to detect a seroconversion in a panel it was given an arbitrary extra 3 days delay for that panel.

Figure 5: Comparative timing of detection of primary HCV infection (mean value) based on 24 seroconversion panels.

Earliest anti-HCV detection

Ortho HCV 3.0 enhanced SAVe

30

Monolisa anti-HCV Plus EIA v1 Abbott HCV EIA 3rd gen.

5

0

IMx HCV

10

15 days

PRISM anti-HCV


Results

n

Comparison of reactivities for two lots of the version 2 assay. Two lots of the version 2 assay were tested against 171 specimens (40 anti-HCV negative, 40 anti-HCV positive, 64 seroconversion panel specimens, 15 low titre specimens and 12 quality control samples). Results for 159 of the specimens are shown in this section and results for the 12 QC samples are shown in the follow ing section (Table 17b and 18). The 40 anti–HCV negative specimens were unreactive by both lots with little discernible difference in reactivity between lot 9M512T (mean OD/CO 0.15, SD 0.11) compared with lot 9M513U (mean OD/CO 0.16, SD 0.09). The 40 anti–HCV positive specimens gave slightly higher reactivities in lot 9M513U (mean OD/CO 11.06, SD 1.37) compared with lot 9M512T (mean OD/CO 9.95, SD 1.43). (Table 16, Appendix Table 26). A plot of the reactive samples from the two lots is given in Figure 6a. From this it can be seen that the major ity of the points lie to one side of the line of equality, also indicating that lot 9M513U tends to give higher values than 9M512T. Of the 79 specimens represented by seven seroconversion panels and one low titre panel (Table 16), 41 were reactive. These and the 40 anti-HCV positive specimens accounted for a total of 81 reactive specimens (detected by lot 9M513U). However one specimen was not detected by lot 9M512T. The fourth member of panel PHV904 was detected by lot 9M513U (OD/CO 1.64) but not by lot 9M512T (OD/CO 0.81) (Table 16, Appendix Table 27). The Pearson correlation coefficient for the 81 reactive samples was 0.97 indicating a high degree of linearity between the two batches (this tests whether the results are linearly related, not whether the values agree). Using the analysis recommended by Altman and Bland25, the difference between the two test results and the mean of the two test results were then calculated and plotted against each other (Figure 6b) to investigate any relationship between measurement error and the true value (estimated by the mean). The mean difference was shown to be significantly different from zero (t = -7.25, degrees of freedom = 80, p < 0.0001). Overall the results did not produce equivalent results on the two lots. The data were further analysed for three subgroups (ie specimens from long standing infections, seroconversion and low titre panels). The mean difference was shown to be significantly different from zero for the 40 long-standing infection specimens (t = -11.96 d.f = 39 and p < 0.0001), but not significantly different from zero for the seroconverter samples (t = -0.74, df=26, p=0.4647) and the low titre samples (t = -2.95,df=13, p=0.0114). On the basis of these small numbers, the two lots appear to be equivalent at low values but not equivalent at high values.


31

Results

Table 16: Comparison of reactivities for two lots of Monolisa® anti–HCV PLUS version 2 Specimens

Number of specimens

Anti-HCV negative Anti-HCV positive BBI - PHV904 BBI - PHV907 BBI - PHV908 BBI - PHV910 BCP - 6211 BCP - 6212 BCP - 6214 BBI - PHV104

Number reactive Lot Lot 9M513U 9M512T 0 0 40 40 4 3 3 3 7 7 3 3 2 2 4 4 4 4 14 14

40 40 7 7 13 5 10 9 13 15

TOTAL 159 Note: Refer to tables 26 - 27 for details of S/CO ratios

81

80

Figure 6a: Comparison of reactivities for 81 reactive specimens tested by two lots of the Monolisa® anti–HCV PLUS version 2 kit. 14 13 12

Lot 9M513U: OD/CO ratio

11 10 9 8

Line of equivalence

7 6 5 4 3 2 1 0 0

1

2

3

4

5

6

7

8

9

10

11

12 13

14

Lot 9M512T: OD/CO ratio

32


Results

Figure 6b: The mean and difference between reactivities for 81 specimens tested by two lots of the Monolisa ® anti–HCV PLUS version 2 kit. 3

Difference between two tests

2

1

+2SD

0 Mean

-1

-2 -2SD

-3 0

2

4

6

8

10

12

14

Mean of two tests

MDA Evaluation Report: Monolisa® anti–HCV PLUS versions 1 and 2

33

Results

n

Quality control samples Replicates of three quality control (QC) samples were all detected by version 1 of the assay. The QC sera comprised one control from the PHLS and two from the National Institute for Biological Standards and Control (Table 17a). Two QC samples were tested by two lots of the version 2 assay. Both were detected and would be suitable anti–HCV controls (Table 17b). A QC specimen / statistical assay control should be chosen to have a reactivity within the linear dynamic range of the assay, ideally 2–3 times the cut–off. The version 2 assay was also tested by a PHLS HCV pre–batch acceptance panel 1 (PBAP) consisting of 10 genotyped samples. All were detected except sample 2 (genotype 1a) which had an indeterminate RIBA HCV 3.0 (Table 18). Table 17a: Quality control samples tested by version 1 of the assay Kit lot

PHLS HCV QC1

6C501.U

Mean

OD/CO 3.26 3.15 3.08 3.07 3.07 2.80

NIBSC anti-HCV (95/604) OD/CO 3.30 3.31 3.55 NT NT NT

NIBSC anti-HCV (94/732) OD/CO 2.98 3.13 2.70 NT NT NT

3.08

3.39

2.94

Table 17b: Quality control samples tested by version 2 of the assay Kit lot

PHLS HCV QC1

NIBSC anti-HCV British working standard

9M513U

Mean

9M512T

Mean

34

(99/B201-01) OD/CO 2.21 2.13 2.17 1.88 1.97 1.82

(99/588-001-W1) OD/CO 3.28 3.15 3.02 2.59 3.59 3.33

2.03

3.16

2.04 2.06 2.12 1.89 1.94 1.93

3.07 3.04 2.91 2.82 3.15 3.10

2.00

3.02


Results

Table 18: PHLS HCV pre–batch acceptance panel 1 (PBAP) tested by Monolisa® anti–HCV PLUS version 2 Sample no. batch 98/B120

Genotype ('in-house')

Serotyping (Murex)

1 2 3 4 5 6 7 8 9 10

2a 1a 1a 1b 3a (a) 3b (ß) 1a 2b 1b 1a

2 1 1 1 3 2 or 3 1 2 1 1

RIBA Monolisa anti-HCV Plus v2 HCV 3.0 lot 9M513U lot 9M512T OD/CO OD/CO + ind + + + + + + + +


3.74 0.53 1.30 1.90 3.03 1.88 1.49 6.96 2.17 2.68

3.56 0.53 1.18 1.77 2.87 1.90 1.35 6.88 2.18 2.57

35

Results

n

Manufacturer’s kit control results The results for the controls provided in both versions of the Monolisa ® anti–HCV PLUS kit are shown in Tables 19a and 19b. All results were within the limits specified by the kit insert. Although The coefficient of variation (CV) was better in version 2 there were insufficient test runs to draw any conclusions.

Table 19a: Manufacturer’s kit control (Monolisa® anti–HCV PLUS v1). Batch (and number of assay runs)

Negative control OD

Positive control OD

Cutoff

6C001.U (5 test runs) 1-plate kit

Range Mean Median SD %CV

0.026 - 0.052 0.034 0.031 0.01 29.412

1.180 - 1.351 1.267 1.266 0.061 4.815

0.236 - 0.27 0.253 0.253 0.012 4.743

6C501.U (5 test runs) 5-plate kit


0.016 - 0.028 0.024 0.026 0.005 20.833

1.205 - 1.413 1.322 1.356 0.081 6.127

0.241 - 0.283 0.265 0.271 0.016 6.038

Table 19b: Manufacturer’s kit control (Monolisa® anti–HCV PLUS v2). Batch (and number of assay runs)

36

Negative control OD

Positive control OD

Cutoff

9M513.U (2 test runs)


0.026 - 0.032 0.029 0.029 0.004 13.793

1.146 - 1.154 1.150 1.150 0.006 0.522

0.229 - 0.231 0.230 0.230 0.001 0.435

9M512.T (2 test runs)


0.019 - 0.021 0.020 0.020 0.001 5.000

1.161 - 1.172 1.167 1.167 0.008 0.686

0.232 - 0.234 0.233 0.233 0.001 0.429


Technical appraisal

Monolisa® anti–HCV EIA versions 1 and 2 both employ microtitre plates comprising 12 x 8 U-well strips. The instructions provided with the kits were clear and easy to follow. The kit and reagent packaging and labelling were good and the reagents were very easy to prepare and use. Two different kit sizes are available from the manufacturer (96 and 480 tests). For the evaluation, tests were processed manually and each 96 well assay run took approximately 2.5 hours to complete. The sample deposition indicator in the sample diluent had a clear colour change from purple to blue when samples or controls were added. This colour change occurred for all samples used in the evaluation. Sample diluent (80µl) was added to the required number of wells before addition of sample (20µl) and was supplied in ready–to–use liquid form. Adequate volumes of preserved anti–HCV negative control serum and anti–HCV positive control serum were each provided in ready–to–use liquid form. Two negative controls wells and three positive control wells are used for each assay run. The conjugate (peroxidase labelled goat antibody directed against human IgG heavy chain) was also provided in ready–to–use liquid form. The working substrate was prepared by diluting reagent R9 with reagent R8 in a ratio of 1:10 (eg 1ml R9 + 10mls R8). A volume of 10 mls is sufficient for use in up to 12 strips. Table 20: Reagents supplied in the kit version 2 reagents

Product code 72317

Product code 72318

1

5

1 vial (100 mls)

2 vials (2 x 250 mls)

R3: Negative control

1 vial (1 ml)

1 vial (3 mls)

R4: Positive control

1 vial (1 ml)

1 vial (3 mls)

R6: Sample diluent

1 vial (15 mls)


R7: Conjugate

1 vial (15 mls)


R8: Peroxidase substrate buffer

1 vial (60mls)


Reagent R1: Coated microplate (12 x 8 well strips) R2: Washing solution

R9: Chromogen (TMB)

1 vial (5 mls)

2 vials (2x 5 mls)

R10: Stop solution

1 vial (28 mls)


4

12

Product code 72311

Product code 72312

1 vial (100 mls) 1 vial (60mls) 1 vial (1 ml)

2 vials (2 x 240 mls) 3 vials (3 x 60 mls) 1 vial (3 mls)

Adhesive microplate covers

version 1 (differences from v2) Washing solution Peroxidase substrate buffer Chromogen (TMB)


37

38


Conclusions

Readers are encouraged to study carefully the results presented and to draw their own conclusions from them. We offer the following comments: Specificity l

The Monolisa® anti–HCV PLUS assay was found, in routine blood screening, to have acceptable specificity (repeat reactive rate 0.29% version 1 ; 0% version 2). In testing stored anti–HCV negative blood donor specimens, potentially false positive and IDU samples the initial and repeat reactive rate for the version 1 assay was 0%.

l

A proportion of selected ‘screen reactive / RIBA 3.0 negative’ specimens were reactive by Monolisa ® anti–HCV PLUS v1. The reactive specimens (16 of 148) were previously screen reactive by Monolisa® HCV ‘new antigens’, Abbott HCV 2nd generation or Ortho HCV 3.0 ELISA.

l

All anti–HCV positive specimens were reactive in both versions, giving a sensitivity of 100%

l

The total seroconversion score and calculation of timing of detection of primary infection indicate that Monolisa® anti–HCV PLUS was marginally less sensitive than Ortho HCV ELISA and the PRISM anti– HCV assay. Both versions of Monolisa® anti–HCV PLUS were of similar sensitivity.

l

Monolisa® anti–HCV PLUS v1 and v2 were both more sensitive than the previous version Monolisa® anti–HCV ‘new antigens’.

Sensitivity

Quality control and kit lot variation l

Three anti–HCV quality control materials were identified as suitable for statistical assay control. Statistical assay controls need to be chosen so that they give reactivities in the linear part of the dynamic range of each assay (neither too weak nor too strong), and therby permit sensitive monitoring of changes in the performance of each run.

l

A comparison of two version 2 kit production lots was undertaken for randomly selected anti-HCV negative and positive specimens, seroconversion panels and a performance panel. The more critical antiHCV positive samples that had low OD/CO ratios showed good agreement. However, strongly positive samples had a statistically


39

Conclusions

significant difference in OD/CO values, although this difference may not be consequential in the clinical setting. Presentation l

Monolisa® anti–HCV PLUS was well presented and easy to perform. Including specimen addition, 96 specimens and controls could be test ed manually within 2½ hours.

Applications l

40

Monolisa® anti–HCV PLUS appears to be suitable as a first–line screening assay in clinical laboratories and blood centres.


Acknowledgements

We thank colleagues at the following blood centres for providing their specificity data: l

North London Blood Centre, Colindale Ave, London. NW9 5BG.

l

Southampton Blood Centre, Coxford Rd, SO16 5AF

We thank the following for providing serum samples: l

Dr John Barbara, David Wenham and Alan Bendall, North London Blood Centre, Colindale NW9.

l

Colleagues in the PHLS Virus Reference Division.


41

42


References

1.

Perry KR, Palmer DR, McAlpine L, Mortimer PP, Parry JV (1995): An evaluation of ten anti–HCV screening assays Medical Devices Agency Evaluation Report: MDA/95/03

2.

Perry KR, Parry JV (1996): An evaluation of the ACCESS® HCV immunoassay and an update on five other anti–HCV assays Medical Devices Agency Evaluation Report: MDA/96/26

3.

Giles RE, Perry KR, Parry JV, Wenham D, Reeves I (1997): Abbott PRISM™ automated anti–HCV assay system. Medical Devices Agency Evaluation Report: MDA/97/72

4.

Burgess C, Perry K, Parry J (2000): Ortho anti–HCV enhanced SAVe . Medical Devices Agency Evaluation Report: MDA 00051

5.

Kuo G, Choo QL, Alter HJ et al (1989): An assay for circulating antibodies to a major etiologic virus of human non–A, non–B hepatitis. Science 244: 362–364

6.

Alter MJ, Hadler SC, Judson FN, Mares A, Alexander WJ, Hu PY, Miller K, Moyer LA, Fields HA, Bradley DW, et al (1990): Risk fac tors for acute non–A, non–B hepatitis in theUnited States and association with hepatitis C infection. JAMA 264: 2231–2235

7.

Donahue JG, Munoz A, Ness PM, Brown DE Jr, Yawn DH, McAllister HA, Reitz BA, Nelson KE (1992): The declining risk of post–transfusion hepatitis C infection. New England Journal of Medicine. 327: 369–373

8.

Choo QL, Weiner AJ, Overby LR, Kuo G, Houghton M, Bradley DW (1990): Hepatitis C virus: the major causative agent of viral non–A, non– B hepatitis. British Medical Bulletin 46: 423–441

9.

Garson JA, Tedder RS, Briggs M, Tuke P, Glazebrook JA, Trute A, Parker D, Barbara JA, Contreras M, Aloysius S (1990): Detection of hepatitis C viral sequences in blood donations by ‘nested’ polymerase chain reaction and prediction of infectivity Lancet 335: 1419–1422


43

References

44

10

Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M (1989): Isolation of a cDNA clone derived from a blood–borne non–A, non–B viral hepatitis genome. Science 244: 359–362

11.

Younossi ZM, McHutchison JG (1996): Serological tests for HCV infection. Viral Hepatitis Reviews 2: 161–173

12

Cuypers HT, Winkel IN, van der Poel CL, Reesink HW, Lelie PN, Houghton M, Weiner A (1991): Analysis of genomic variability of hepatitis C virus Journal of Hepatology 13: suppl 4: S15–19

13.

Dhaliwal SK, Prescott LE, Dow BC, Davidson F, Brown H, Yap PL, Follett EA, Simmonds P (1996): Influence of viraemia and genotype upon serological reactivity in screening assays for antibody to hepatitis C virus. Journal of Medical Virology 48: 184–190

14

Pawlotsky JM, Fleury A, Choukroun V, Deforges L, Roudot– Thoraval F, Aumont P, Duval J, Dhumeaux D (1994): Significance of highly positive c22–3 ‘indeterminate’ second–generation hepatitis C virus (HCV) recombinant immunoblot assay (RIBA) and resolution by third– generation HCV RIBA. Journal of Clinical Microbiology 32: 1357–1359

15.

Tobler LH, Busch MP, Wilber J, Dinello R, Quan S, Polito A, Kochesky R, Bahl C, Nelles M, Lee SR (1994): Evaluation of indeterminate c22–3 reactivity in volunteer blood donors. Transfusion 34: 130–134

16.

Flanagan P, Barbara J (1999): PCR testing of plasma pools: from concept to reality. Transfusion Medicine Review 13 (3): 164-76

17.

Legler TJ, Köhler M, Heerman K–H. (1999): High throughput extraction, amplification and detection (HEAD) of HCV–RNA in individual blood donations. Journal of Clinical Virology 13: 95–103

18.

Aoyagi K, Ohue C, Iida K, Kimura T, Tanaka E, Kiyosawa K, Yagi S (1999): Development of a simple and highly sensitive enzyme immunoassay for hepatitis C virus core antigen. Journal of Clinical Microbiology 37: 1802–1808.20.

MDA Evaluation Report: Monolisa ® anti–HCV PLUS versions 1 and 2

References

19.

Beach MJ, Meeks EL, Mimms LT, Vallari D, DuCharme L, Spelbring J, Taskar S, Schleicher JB, Krawczynski K, Bradley DW (1992) Temporal relationships of hepatitis C virus RNA and antibody responses following experimental infection of chimpanzees. Journal of Medical Virology 36: 226–237

20

Zaaijer HL, Cuypers HT, Reesink HW, Winkel IN, Gerken G, Lelie PN (1993): Reliability of polymerase chain reaction for detection of hepatitis C virus Lancet 341: 722–724

21

Dow BC, Coote I, Munro H, McOmish F, Yap PL, Simmonds P, Follett EA (1993): Confirmation of hepatitis C virus antibody in blood donors. Journal of Medical Virology 41: 215–220

22

Weiner AJ, Kuo G, Bradley DW, Bonino F, Saracco G, Lee C, Rosenblatt J, Choo QL, Houghton M (1990): Detection of hepatitis C viral sequences in non–A, non–B hepatitis Lancet 335: 1–3

23.

Watanabe M, Ohkoshi S, Tawaraya H, Miyajima T, Shimotohno K, Kamimura T, Asakura H (1994): Hepatitis C viral markers in patients who received blood that was positive for hepatitis C virus core antibody, with genetic evidence of hepatitis C virus transmission. Transfusion 34: 125–129

24.

Perry KR, Giles RE, Burgess C, Kenny K, Sallett K, Parry J (1999): Anti–HCV commercial panel data: comparative performance data for anti–HCV assays using commercial seroconversion and other panels. Medical Devices Agency Evaluation Report: MDA/99/49

25.

Bland JM, Altman DG (1986): Statistical methods for assessing agreement between two methods of clinical measurement. Lancet: Feb 8th 1986; 307-310


45

46


Appendix

n

Key to the appendix The appendix contains the following information. Tables of data have been presented in summary or graphic form in earlier parts of the report: l

protocol

l

seroconversion panel scores and S/CO ratios (Table 21, Table 22, Table 23)

l

performance panel scores and S/CO ratios (Table 24, Table 25)

l

comparison of two production batches of the version 2 assay (Table 26, Table 27)

l

details of potential false positive specimens

l

contact details for participating manufacturer and UK agent

l

manufacturer’s comments

l

how to obtain MDA evaluation reports


47

Appendix

n

Evaluation protocol Procurement of product for evaluation, duration of evaluation and training of evaluator The evaluator will require for evaluation a package which contains sufficient assays to test the panel of sera, together with any ancillary reagents and consumables. The kits will be used in conjunction with the equipment that is either provided by the manufacturer or available at MiDAS and has been agreed to meet the requirements of the manufacturer’s representative. It is anticipated that, assuming satisfactory performance, the laboratory work will be completed within four weeks of the date of commencement. Another 1216 weeks will be required to analyse the data and produce an MDA evaluation report. Before evaluation starts, the manufacturer will be invited, if they so wish, to train the evaluator(s) in the use of the kits and equipment and to satisfy themselves that the evaluator(s) 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 described during the training period or any subsequent changes must be confirmed in writing. All assay data will be stored on the MiDAS database for the duration of the evaluation. The data may subsequently be downloaded to floppy disc, tape or other long-term back-up system. In addition the evaluators will keep clear records of the practical work. All original print-outs from the plate reader will be retained, together with any reader print-outs from confirmatory assays. Content of the evaluation The object of this evaluation is to assess the ability of the Monolisa® anti-HCV PLUS assay to detect, with a high degree of sensitivity and specificity, antiHCV in human serum and plasma. To do this, the kits will be tested against a panel of sera/plasma samples comprising material found to be either reactive or unreactive by anti-HCV screening assays currently in use in the UK. Those samples selected as 'screening test positive' are further classified according to their reactivity in the RIBA confirmatory test. Seroconversion panels, low and mixed titre panels obtained from Boston Biomedica Inc, USA and Bioclinical Partners Inc, USA will also be included in the evaluation panel.

48


Appendix

Storage of samples Aliquots of each serum specimen will be distributed into plastic tubes with screw-cap lids with sealers. Each sample will be coded so that the evaluators will not know the expected results. 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 l l

l

the packaging and labelling of the materials the clarity of the operating instructions the ease of use and reliability of the products, including equipment supplied for the evaluation health and safety considerations.

Discordant results A discrepancy will arise when a result by the kit under evaluation disagrees with the consensus result obtained by other kits under evaluation and/or those obtained at the Virus Reference Division. If this occurs tests will be repeated in duplicate on the same aliquot of the serum. Should the discrepancy remain, the specimen will be tested in confirmatory assays ie. RIBA HCV 3.0, LIA–HCV–3 (Innogenetics) and PCR. If, in the view of the evaluators, the kit manufacturer makes a justifiable case, aliquots of specimens giving discrepant results may be made available, where practically possible, to the manufacturer. 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, in the usual style, will be prepared for the Medical Devices Agency. BioRad Diagnostics will be given the opportunity to comment on the results of the evaluation of their product before the MDA evaluation report is published. Results obtained in other tests undergoing evaluation will not be disclosed at this time. Manufacturer's written comments, where relevant, will be appended to the report.


49

Appendix

n

S/CO ratios for seroconversion panels Twenty–eight seroconversion panels were included in the evaluation to assess the sensitivity of the Monolisa® anti–HCV PLUS version 1 assay at the time of seroconversion. Results from 24 of these could be compared with other anti–HCV kits (Table 21). Details of OD/CO ratios for each panel are given in Tables 22a–c and scores compared with Monolisa anti–HCV new antigens kit are given in Table 23. The OD/CO ratios for version 2 of the assay are given in the Appendix section entitled ‘Comparison of two production batches of the version 2 assay’ (Table 27). Details of S/CO ratios for other assays previously evaluated can be found in a separate report entitled Anti–HCV Commercial Panel Data24. This report also includes other supplementary information eg RIBA HCV 3.0, b–DNA and polymerase chain reaction results, supplied by Boston Biomedica Inc, USA and by BioClinical Partners Inc, USA.

50

MDA Evaluation Report: Monolisa ® anti–HCV PLUS versions 1 and 2

Appendix


51

Appendix

Table 22a: Monolisa® anti-HCV PLUS v1 : OD/CO ratios for BBI seroconversion panels PHV901 – PHV911. Panel

Days since first bleed

OD/CO

0 PHV901-01 65 PHV901-02 97 PHV901-03 99 PHV901-04 104 PHV901-05 106 PHV901-06 131 PHV901-07 139 PHV901-08 159 PHV901-09 166 PHV901-10 203 PHV901-11 Cutoff = 0.27; kit lot 6C501.U

0.16 0.12 6.36 6.58 6.92 6.97 8.47 8.30 8.68 8.70 9.25

0 PHV902-01 2 PHV902-02 7 PHV902-03 9 PHV902-04 14 PHV902-05 16 PHV902-06 21 PHV902-07 Cutoff = 0.26; kit lot 6C501.U

0.11 0.10 0.89 2.57 8.05 8.45 9.19

0 PHV903-01 5 PHV903-02 7 PHV903-03 12 PHV903-04 14 PHV903-05 19 PHV903-06 21 PHV903-07 26 PHV903-08 Cutoff = 0.26; kit lot 6C501.U

0.11 0.17 0.22 0.65 0.80 4.25 5.25 6.47

PHV904-01 0 PHV904-02 2 PHV904-03 7 PHV904-04 9 PHV904-05 14 PHV904-06 21 PHV904-07 23 Cutoff = 0.26; kit lot 6C501.U

0.07 0.08 0.33 1.24 3.30 5.19 6.66

0 PHV905-01 4 PHV905-02 7 PHV905-03 11 PHV905-04 14 PHV905-05 18 PHV905-06 21 PHV905-07 25 PHV905-08 28 PHV905-09 Cutoff = 0.27; kit lot 6C501.U

0.04 0.04 0.07 0.35 0.66 1.42 2.99 6.03 6.30

52

Panel


OD/CO

0 PHV906-01 2 PHV906-02 7 PHV906-03 10 PHV906-04 14 PHV906-05 17 PHV906-06 21 PHV906-07 Cutoff = 0.26; kit lot 6C001.U

1.98 3.90 6.01 6.59 6.97 8.81 8.00

0 PHM907-01 4 PHM907-02 7 PHM907-03 13 PHM907-04 18 PHM907-05 21 PHM907-06 164 PHM907-07 Cutoff = 0.28; kit lot 8H018Q

0.05 0.08 0.04 0.15 0.88 1.29 7.83

0 PHV908-01 3 PHV908-02 5 PHV908-03 11 PHV908-04 13 PHV908-05 19 PHV908-06 25 PHV908-07 27 PHV908-08 32 PHV908-09 35 PHV908-10 41 PHV908-11 45 PHV908-12 48 PHV908-13 Cutoff = 0.28; kit lot 8H018Q

0.05 0.04 0.06 0.11 0.17 0.84 4.50 5.57 7.31 7.42 8.15 8.48 8.57

0 PHV909-01 28 PHV909-02 30 PHV909-03 Cutoff = 0.28; kit lot 8H018Q

0.12 2.42 2.43

0 PHV910-01 4 PHV910-02 8 PHV910-03 11 PHV910-04 15 PHV910-05 Cutoff = 0.23; kit lot 8H018Q

0.21 0.40 0.47 2.19 3.12

0 PHV911-01 3 PHV911-02 14 PHV911-03 21 PHV911-04 24 PHV911-05 Cutoff = 0.21; kit lot 8H018Q

0.04 0.05 1.04 6.94 8.94


Appendix

Table 22b: Monolisa® anti-HCV PLUS v1: OD/CO ratios for BBI seroconversion panels PHV912 – 915 and BCP seroconversion panels 6211 – 6216 Panel


OD/CO

0 PHV912-01 4 PHV912-02 7 PHV912-03 Cutoff = 0.28; kit lot 8H018Q

0.30 0.28 8.05

0 PHV913-01 2 PHV913-02 7 PHV913-03 9 PHV913-04 Cutoff = 0.28; kit lot 8H018Q

0.10 0.34 2.99 2.90

0 PHV914-01 5 PHV914-02 9 PHV914-03 12 PHV914-04 16 PHV914-05 19 PHV914-06 24 PHV914-07 30 PHV914-08 33 PHV914-09 Cutoff = 0.28; kit lot 8H018Q

0.03 0.04 0.04 0.08 0.75 1.56 4.48 5.40 5.30

0 PHV915-01 5 PHV915-02 12 PHV915-03 14 PHV915-04 Cutoff = 0.28; kit lot 8H018Q

0.14 0.47 0.86 1.44

150 6211-31 154 6211-32 157 6211-33 161 6211-34 164 6211-35 168 6211-36 171 6211-37 182 6211-38 186 6211-39 189 6211-40 Cutoff = 0.21; kit lot 8H018Q

0.08 0.07 0.04 0.05 0.06 0.02 0.02 0.72 2.53 4.08

0 6212-01 12 6212-02 14 6212-03 23 6212-04 26 6212-05 32 6212-06 37 6212-07 53 6212-08 55 6212-09 Cutoff = 0.23; kit lot 8H018Q

0.05 0.06 0.08 0.36 0.53 1.29 1.48 7.00 7.45

Panel


OD/CO

0 6213-01 2 6213-02 8 6213-03 11 6213-04 15 6213-05 18 6213-06 28 6213-07 30 6213-08 35 6213-09 37 6213-10 43 6213-11 46 6213-12 Cutoff = 0.23; kit lot 8H018Q

0.07 0.04 0.03 0.03 0.04 0.06 0.07 0.08 0.09 0.26 3.84 3.79

0 6214-01 2 6214-02 8 6214-03 10 6214-04 16 6214-05 18 6214-06 23 6214-07 25 6214-08 30 6214-09 32 6214-10 49 6214-11 53 6214-12 56 6214-13 Cutoff = 0.23; kit lot 8H018Q

0.05 0.06 0.04 0.06 0.06 0.02 0.03 0.05 0.28 1.13 7.05 7.77 8.22

0 6215-01 3 6215-02 10 6215-03 20 6215-04 Cutoff = 0.21; kit lot 8H018Q

0.04 0.05 0.05 1.90

0 6216-01 3 6216-02 8 6216-03 10 6216-04 15 6216-05 17 6216-06 23 6216-07 Cutoff = 0.23; kit lot 8H018Q

0.08 0.09 0.06 0.04 0.06 0.06 0.49


53

Appendix

Table 22c: Monolisa® anti-HCV PLUS v1: OD/CO ratios for BCP seroconversion panels 6222, 9041-9047 and SD976. Panel


OD/CO

6222-01 0 6222-02 2 6222-03 17 6222-04 19 6222-05 24 6222-06 26 6222-07 36 6222-08 40 Cutoff = 0.23; kit lot 8H018Q

0.02 0.03 0.03 0.02 0.04 0.06 0.14 1.15

9041-01 0 9041-02 24 9041-03 27 9041-04 31 9041-05 62 9041-06 64 9041-07 69 9041-08 71 Cutoff = 0.41; kit lot 8D523X

0.09 0.05 0.05 0.04 5.68 6.07 6.16 6.26

9044-01 0 9044-02 4 9044-03 17 9044-04 21 9044-05 25 9044-06 29 Cutoff = 0.41; kit lot 8D523X

0.08 0.05 0.06 0.22 1.13 1.87

9045-01 0 9045-02 2 9045-03 7 9045-04 9 9045-05 26 9045-06 32 9045-07 37 9045-08 41 Cutoff = 0.38; kit lot 8D523X

0.03 0.04 0.05 0.04 0.03 0.07 0.73 2.40

9046-01 0 9046-02 69 9046-03 72 9046-04 76 9046-05 79 Cutoff = 0.38; kit lot 8D523X

0.06 5.68 5.33 4.95 5.25

54

Panel


OD/CO

9047-01 0 9047-02 2 9047-03 10 9047-04 12 9047-05 19 9047-06 21 9047-07 28 9047-08 30 9047-09 35 9047-10 37 Cutoff = 0.38; kit lot 8D523X

0.09 0.12 0.10 0.07 0.12 0.16 0.73 4.12 5.89 6.45

SD976-01 0 SD976-02 58 SD976-03 71 SD976-04 154 SD976-05 164 SD976-06 225 SD976-07 229 SD976-08 238 SD976-09 310 SD976-10 313 SD976-11 319 SD976-12 376 SD976-13 380 SD976-14 383 SD976-15 454 SD976-16 458 SD976-17 462 SD976-18 524 SD976-19 526 SD976-20 531 Cutoff = 0.26; kit lot 6C501.U

0.08 0.07 0.08 0.05 0.07 0.06 0.07 0.07 0.10 0.07 0.08 0.06 0.07 0.06 2.20 2.15 4.21 8.10 8.32 7.84


Appendix

Table 23: Detection of anti-HCV in seroconversion panels by Monolisa® antiHCV ‘version 1’ and ‘new antigens’ versions Panel

Number of specimens in panel

The number of positive samples and the number of days from initial bleed to first reactive sample (shown in parenthesis) Monolisa® anti-HCV Monolisa® anti-HCV new PLUS v1 antigens (72311/2) (72306/7)

PHV901 PHV902 PHV903 PHV904 PHV905 PHV906 PHV907 PHV908 PHV909 PHV910 PHV911 PHV912 PHV913 PHV914 6211 6212 6213 6214 6215 6216 SD976

11 7 8 7 9 7 7 13 3 5 5 3 4 9 10 9 12 13 4 7 20

9 (97) 4 (9) 3 (19) 4 (9) 4(18) 7 (0) 2 (21) 7 (25) 2 (28) 2 (11) 3 (14) 1 (7) 2 (7) 4 (19) 2 (186) 4(32) 2 (43) 4 (32) 1 (20) 0 (>23) 6 (454)

6 (106) 4 (9) 5 (12) 3 (14) 3 (21) 5 (7) 3 (18) 5 (32) 2 (28) 3 (8) 3 (14) 1 (7) 2 (7) 5 (16) 0 (>189) 4 (32) 2 (43) 3 (49) 1 (20) 0 (>23) 6 (454)

TOTAL

173

73

66


55

Appendix

n

S/CO ratios for performance panels Five performance panels (worldwide, low and mixed titre) were included in the evaluation in order to help establish the sensitivity of the Monolisa ® anti–HCV PLUS v1 assay. Comparative scores for each panel are given in Table 24 and details of OD/CO ratios for each panel are given in Table 25. Panels PHV103 and PHV203 were used for the calculation of performance panel scores (Table 12). The OD/CO ratios for one low titre panel tested by version 2 of the assay are given in the Appendix section entitled ‘Comparison of two production lots of the version 2 assay’ (Table 27) Details of S/CO ratios for other assays previously evaluated can be found in a separate report entitled Anti–HCV Commercial Panel Data24. This report also includes other supplementary information eg RIBA HCV 3.0, b–DNA and polymerase chain reaction results, supplied by Boston Biomedica Inc, USA and by BioClinical Partners Inc, USA.

56


Appendix

Table 24a: Detection of anti–HCV in performance panels (positive specimens) Number of positive reactions

Panel

Number of Monolisa® positive Monolisa® antianti-HCV specimens HCV PLUS EIA new in panel v1 antigens

Ortho HCV 3.0 ELISA enhanced SAVe (short incubation)

IMx® HCV

Abbott HCV EIA 3rd generation

PRISM™ HCV

ACCESS® HCV

72312

72306

9307401/601

3A99-20

7A16-23

6A52-48

34310

PHV103

14

14

13

14

14

13

14

14

PHV104

14

12

14

14

13

NT

NT

NT

PHV203

23

21

21

21

21

23

20

21

PHV204

23

23

NT

23

23

NT

NT

NT

WWHV301 Notes:

18

18

18

18

NT

18

NT

NT

The score was calculated by summing the number of positive samples for each of the seroconversion panels. A higher score suggests higher sensitivity.

Table 24b: Detection of anti–HCV in performance panels (negative specimens) Number of positive reactions Panel

Number of negative specimens in panel

Monolisa® anti-HCV PLUS EIA

Monolisa® Ortho HCV 3.0 anti-HCV ELISA enhanced new SAVe (short antigens incubation)

IMx® HCV

Abbott HCV EIA 3rd generation

PRISM® HCV

ACCESS® HCV

72312

72306

9307401/601

3A99-20

7A16-23

6A52-48

34310

PHV103

1

0

0

0

0

0

0

0

PHV104

1

0

0

0

0

NT

NT

NT

PHV203

2

0

0

0

0

0

0

0

PHV204

2

0

NT

0

0

NT

NT

NT

WWHV301

2

0

0

0

NT

0

NT

NT


57

Appendix

Table 25a: Monolisa ® anti-HCV PLUS v1: OD/CO ratios for performance panels Panel

Anti-HCV status

Monolisa® antiHCVPLUSv1 (OD/CO)

PHV103-01 Positive PHV103-02 Positive PHV103-03 Positive PHV103-04 Positive PHV103-05 Positive PHV103-06 Positive PHV103-07 Positive PHV103-08 Positive PHV103-09 Positive PHV103-10 negative PHV103-11 Positive PHV103-12 Positive PHV103-13 Positive PHV103-14 Positive PHV103-15 Positive Cutoff = 0.27; kit lot 6C501.U

4.87 5.65 4.15 9.32 9.95 5.60 1.32 3.87 6.55 0.32 5.86 5.12 4.19 4.14 6.61

PHV104-01 Positive PHV104-02 Positive PHV104-03 Positive PHV104-04 Positive PHV104-05 Positive PHV104-06 Positive PHV104-07 Positive PHV104-08 Positive PHV104-09 Positive PHV104-10 Positive PHV104-11 Positive PHV104-12 negative PHV104-13 Positive PHV104-14 Positive PHV104-15 Positive Cutoff = 0.21; kit lot 8H018Q

0.62 12.27 3.66 10.71 0.73 10.98 8.44 2.82 3.48 4.52 2.23 0.03 2.00 3.24 8.24

58

Panel

Anti-HCV status

PHV203-01 Positive PHV203-02 Positive PHV203-03 Positive PHV203-04 Positive PHV203-05 negative PHV203-06 Positive PHV203-07 Positive PHV203-08 Positive PHV203-09 Positive PHV203-10 Positive PHV203-11 Positive PHV203-12 Positive PHV203-13 Positive PHV203-14 Positive PHV203-15 negative PHV203-16 Positive PHV203-17 Positive PHV203-18 Positive PHV203-19 Positive PHV203-20 Positive PHV203-21 Positive PHV203-22 Positive PHV203-23 Positive PHV203-24 Positive PHV203-25 Positive Cutoff = 0.26; kit lot 6C001.U

Monolisa® antiHCVPLUSv1 (OD/CO)

3.00 2.99 0.99 3.73 0.18 9.69 8.46 7.09 3.86 2.82 4.35 2.68 2.72 6.28 0.09 10.58 5.64 11.41 8.45 4.66 7.18 10.71 5.53 1.65 0.11


Appendix

Table 25b: Monolisa® anti-HCV PLUS v1: OD/CO ratios for performance panels Panel

Anti-HCV status

PHV204-01 Positive PHV204-02 Positive PHV204-03 Positive PHV204-04 Positive PHV204-05 Positive PHV204-06 Positive PHV204-07 Positive PHV204-08 Positive PHV204-09 Positive PHV204-10 negative PHV204-11 Positive PHV204-12 Positive PHV204-13 Positive PHV204-14 Positive PHV204-15 Positive PHV204-16 Positive PHV204-17 Positive PHV204-18 Positive PHV204-19 Positive PHV204-20 Positive PHV204-21 Positive PHV204-22 Positive PHV204-23 negative PHV204-24 Positive PHV204-25 Positive Cutoff = 0.21; kit lot 8H018Q

Monolisa® antiHCV PLUS v1 (OD/CO)

9.26 6.90 7.67 9.64 10.67 4.97 5.20 11.21 5.23 0.05 11.94 9.72 4.51 11.09 10.14 2.25 9.93 10.79 6.66 8.30 7.01 10.12 0.13 4.84 5.59

Panel

Murex

Innogenetics

serotype

INNO-LiPA

WWHV301-01 1 1b WWHV301-02 not typable 1b WWHV301-03 3 3a/b WWHV301-04 2 2a/c WWHV301-05 negative not tested WWHV301-06 4 4c/d not typable WWHV301-07 2 WWHV301-08 negative not tested WWHV301-09 1 1b, 2a/c WWHV301-10 1 2 WWHV301-11 1 1b WWHV301-12 2 2 WWHV301-13 1,2 1a/b, 2a/c WWHV301-14 (3) 3a WWHV301-15 (4) 4 WWHV301-16 (4) 4h not typable WWHV301-17 (4) WWHV301-18 1 1b WWHV301-19 not typable 1a WWHV301-20 1 1a Cutoff = 0.21; kit lot 8H018Q

Monolisa® antiHCV PLUS v1 (OD/CO)

9.27 9.49 10.44 10.37 0.00 11.77 8.28 0.23 9.80 11.36 7.34 10.18 10.96 9.54 10.47 11.53 7.38 9.41 5.94 8.48

Notes: ( ) = stronger reactivity with the peptide indicated but does not meet all of the manufacturer's criteria; type confirmed by research amplication method


59

Appendix

n

Comparison of two production lots of the version 2 assay Forty anti–HCV negative, forty anti–HCV positive and eight commercial panels were tested by Monolisa® anti–HCV PLUS version 2 assay (Table 13). Details of comparative OD/CO ratios are given in Tables 26 and 27. Table 26: OD/CO ratios for positive and negative specimens tested by Monolisa® anti–HCV PLUS version 2. Anti-HCV negatives (blood donor sera) VRD No Lot: 9M512T Lot: 9M513U OD/CO OD/CO 00-02001 0.12 0.16 00-02002 0.06 0.07 00-02003 0.08 0.10 00-02004 0.19 0.23 00-02005 0.09 0.11 00-02006 0.10 0.12 00-02007 0.08 0.10 00-02008 0.06 0.08 00-02009 0.07 0.08 00-02010 0.08 0.09 00-02011 0.22 0.25 00-02012 0.07 0.09 00-02013 0.11 0.14 00-02014 0.12 0.22 00-02015 0.11 0.12 00-02016 0.17 0.17 00-02017 0.06 0.15 00-02018 0.09 0.11 00-02019 0.09 0.11 00-02020 0.24 0.29 00-02021 0.12 0.20 00-02022 0.20 0.26 00-02023 0.12 0.14 00-02024 0.09 0.14 00-02025 0.12 0.13 00-02026 0.16 0.22 00-02027 0.30 0.27 00-02028 0.22 0.17 00-02029 0.49 0.51 00-02030 0.21 0.21 00-02031 0.21 0.10 00-02032 0.29 0.18 00-02033 0.10 0.11 00-02034 0.57 0.45 00-02035 0.12 0.09 00-02036 0.15 0.16 00-02037 0.14 0.13 00-02038 0.12 0.13 00-02039 0.10 0.12 00-02040 0.06 0.07 Count Minimum Maximum Mean Median SD

60

40 0.06 0.57 0.15 0.12 0.11

40 0.07 0.51 0.16 0.13 0.09

VRD No 00-10313 00-10314 00-10315 00-10316 00-10317 00-10318 00-10319 00-10320 00-10321 00-10322 00-10323 00-10324 00-10325 00-10326 00-10327 00-10328 00-10329 00-10330 00-10331 00-10332 98-41795 98-41796 98-41797 98-41798 98-41799 98-41801 98-41802 98-41803 98-41804 98-41805 98-41806 98-41807 98-41808 98-41809 98-41810 98-41811 98-41812 98-41813 98-41814 98-41815 Count Minimum Maximum Mean Median SD

Anti-HCV positives Lot: 9M512T Lot: 9M513U OD/CO OD/CO 10.65 12.07 8.19 8.35 10.68 11.98 12.24 12.70 11.16 12.45 11.61 12.53 12.43 12.53 9.10 10.34 10.00 11.36 12.38 12.29 10.32 11.14 7.86 8.00 12.23 13.10 8.14 9.18 9.41 10.69 11.34 11.48 10.03 11.09 11.10 11.86 9.33 10.45 10.29 11.80 9.68 11.39 7.09 9.17 9.62 10.60 10.34 11.33 8.82 10.41 10.13 11.17 7.59 8.72 9.50 10.97 9.74 12.15 11.73 12.28 10.18 11.73 8.57 8.97 9.89 12.09 11.00 12.00 8.38 9.73 7.62 9.07 10.92 13.10 11.28 12.23 8.19 9.32 9.26 10.47 40 7.09 12.43 9.95 10.01 1.43

40 8.00 13.10 11.06 11.35 1.37


Appendix

Table 27: Monolisa ® anti–HCV PLUS version 2 OD/CO ratios for seven seroconversion panels and one low titre panel. Panel


PHV904-01 PHV904-02 PHV904-03 PHV904-04 PHV904-05 PHV904-06 PHV904-07

0 2 7 9 14 21 23

PHV907-01 PHV907-02 PHV907-03 PHV907-04 PHV907-05 PHV907-06 PHV907-07

lot 9M513U OD/CO

lot 9M512T OD/CO

cutoff = 0.231

cutoff = 0.232

0.15 0.20 0.44 1.64 4.81 6.86 5.92

0.14 0.19 0.44 0.81 2.22 5.06 6.87

0 4 7 13 18 21 164

0.12 0.12 0.10 0.43 1.33 1.88 9.99

0.10 0.13 0.12 0.53 1.20 2.04 9.35

PHV908-01 PHV908-02 PHV908-03 PHV908-04 PHV908-05 PHV908-06 PHV908-07 PHV908-08 PHV908-09 PHV908-10 PHV908-11 PHV908-12 PHV908-13

0 3 5 11 13 19 25 27 32 35 41 45 48

0.10 0.08 0.09 0.20 0.41 0.37 4.95 5.25 6.31 6.67 6.94 6.24 6.78

0.11 0.06 0.07 0.21 0.38 0.43 4.70 5.27 6.88 7.80 6.44 8.24 6.04

PHV910-01 PHV910-02 PHV910-03 PHV910-04 PHV910-05

0 4 8 11 15

0.23 0.20 1.39 2.74 4.31

0.26 0.22 1.22 3.19 4.20

6211-31 6211-32 6211-33 6211-34 6211-35 6211-36 6211-37 6211-38 6211-39 6211-40

150 154 157 161 164 168 171 182 186 189

0.11 0.08 0.09 0.11 0.07 0.07 0.12 0.97 2.44 2.94

0.10 0.08 0.10 0.08 0.07 0.08 0.11 0.77 1.13 3.07

Panel


6212-01 6212-02 6212-03 6212-04 6212-05 6212-06 6212-07 6212-08 6212-09

0 12 14 23 26 32 37 53 55

6214-01 6214-02 6214-03 6214-04 6214-05 6214-06 6214-07 6214-08 6214-09 6214-10 6214-11 6214-12 6214-13 PHV104-01 PHV104-02 PHV104-03 PHV104-04 PHV104-05 PHV104-06 PHV104-07 PHV104-08 PHV104-09 PHV104-10 PHV104-11 PHV104-12 PHV104-13 PHV104-14 PHV104-15


lot 9M513U OD/CO

lot 9M512T OD/CO

cutoff = 0.231

cutoff = 0.232

0.07 0.18 0.10 0.41 0.72 1.38 1.96 8.60 8.68

0.05 0.14 0.14 0.39 0.76 1.16 1.82 8.79 9.97

0 2 8 10 16 18 23 25 30 32 49 53 56

0.11 0.10 0.08 0.09 0.04 0.09 0.06 0.12 0.87 1.98 8.66 7.66 6.95

0.10 0.10 0.07 0.08 0.07 0.08 0.07 0.12 0.84 1.90 7.59 7.21 7.44

Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive negative Positive Positive Positive

3.20 12.40 5.23 12.35 2.17 10.73 9.64 4.23 4.97 6.53 3.23 0.15 3.72 5.76 7.99

3.39 >=12.93 5.29 10.93 1.94 9.38 8.60 3.69 4.39 6.68 2.99 0.13 3.01 3.39 6.91

61

Appendix

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Details of potential positive specimens Details of the 45 potential positive samples used in the evaluation of Monolisa anti-HCV PLUS version 1 (Table 4) are listed below.

62

Random number

Comment

26 28 31 39 54 68 77 88 103 110 115 121 125 145 153 161 163 185 191 192 200 214 216 234 250 286 289 290 302 305 339 344 348 354 363 396 462 466

Anti-nuclear antibody Thyroid microsomal / Thyroglobulin Tonsilitis, Glandular fever Tonsilitis, Cervical adenitis DNA antibody Primary biliary cirrhosis Thyroid microsomal / Thyroglobulin Rheumatoid factor positive Thyroid microsomal / Thyroglobulin Cervical lymphadenopathy, Glandular fever Thyroid microsomal / Thyroglobulin Alcohol related liver disease Hepatocellular carcinoma Glandular fever Alcohol-related liver disease No data Primary biliary cirrhosis Thyroid microsomal / Thyroglobulin Thyroid microsomal / Thyroglobulin Alcohol related liver disease Rheumatoid factor positive Primary sclerosing cholangitis Hepatocellular carcinoma Rheumatoid factor positive Parietal cell antibodies Sore throat Thyroid microsomal / Thyroglobulin 3/7 malaise, headache, chest pains Thyroid microsomal / Thyroglobulin Acute Parvovirus B19 Hepatocellular carcinoma Thyroid microsomal / Thyroglobulin Acute Parvovirus B19 Hepatocellular carcinoma Double stranded DNA antibodies Auto-immune chronic active hepatitis Alcohol related liver disease Auto-immune chronic active hepatitis


Appendix

Random number

Comment

483 521 576 670 671 676 729

Primary biliary cirrhosis Rheumatoid factor positive Double-stranded DNA antibodies Rheumatoid factor positive Alcohol-related liver disease Primary sclerosing cholangitis Parietal cell antibodies

Random numbers are taken from the MDA/95/03 evaluation report on antiHCV screening assays.


63

Appendix

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Contact details for participating manufacturer and UK agent Product: Monolisa® anti–HCV PLUS Product code: 72311 / 72312 Product: Monolisa® anti–HCV PLUS version 2 Product code: 72317/72318

Manufacturer Bio–Rad Laboratories 3 boulevard Raymond Poincaré 92430 Marnes-la-Coquette France Tel : +33 (0) 1 47 95 60 00 Fax: +33 (0) 1 47 41 91 33

UK agent Bio–Rad Laboratories (UK) Ltd Bio–Rad House Maylands Avenue Hemel Hempstead Hertfordshire HP2 7TD Tel : 020 8328 2209 Fax: 020 8328 2550

64


Appendix

n

Manufacturer’s comments


65

Appendix

n

How to obtain MDA evaluation reports MDA evaluation reports are published by the Medical Devices Agency, an Executive Agency of the Department of Health. They are available free of charge to the UK National Health Service (NHS), and are for sale to commercial organisations and other interested parties. A free catalogue of available reports can be obtained from the Orders Department, or downloaded from the MDA web site: www.medical–devices.gov.uk Ordering Send your order to the address given below, stating the number, title and quantity of each report required. Your reports will be dispatched by second class post the following working day. If you are not a representative of the NHS, you will be invoiced separately. Non–NHS customers are reminded that it is not possible to offer refunds for reports ordered in error. Orders Department Room 1207 Medical Devices Agency Hannibal House Elephant and Castle London SE1 6TQ Tel: Fax: E–mail:

020-7972 8181 020-7972 8105 dep@medical–devices.gov.uk

Enquiries General enquiries should be directed to the MDA Orders Department, as above. Technical enquiries should be addressed to Dr. Keith R. Perry at MiDAS: Tel: Fax: E–mail:

66

020–8200–4400 ext 3949 020–8200–1569 [email protected]


ISBN 1 84182 306 6 36-22960-1