# L_2022178EN.01000301.xml

| 5.7.2022 | EN | Official Journal of the European Union | L 178/3 |
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5.7.2022

EN

Official Journal of the European Union

L 178/3

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COMMISSION IMPLEMENTING REGULATION (EU) 2022/1107

of 4 July 2022

laying down common specifications for certain class D in vitro diagnostic medical devices in accordance with Regulation (EU) 2017/746 of the European Parliament and of the Council

(Text with EEA relevance)

THE EUROPEAN COMMISSION,

Having regard to the Treaty on the Functioning of the European Union,

Having regard to Regulation (EU) 2017/746 of the European Parliament and of the Council of 5 April 2017 on in vitro diagnostic medical devices and repealing Directive 98/79/EC and Commission Decision 2010/227/EU (1), and in particular Article 9(1) thereof,

Whereas:

| (1) | For certain class D in vitro diagnostic medical devices falling within the scope of Regulation (EU) 2017/746, harmonised standards do not exist as regards certain requirements of Annex I to that Regulation, and there is a need to address public health concerns as the risk associated with the use of those devices is significant for public health and patient safety. It is therefore appropriate to adopt common specifications for those devices in respect of those requirements. |
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(1)

For certain class D in vitro diagnostic medical devices falling within the scope of Regulation (EU) 2017/746, harmonised standards do not exist as regards certain requirements of Annex I to that Regulation, and there is a need to address public health concerns as the risk associated with the use of those devices is significant for public health and patient safety. It is therefore appropriate to adopt common specifications for those devices in respect of those requirements.

| (2) | Regulation (EU) 2017/746 replaces Directive 98/79/EC of the European Parliament and of the Council (2). The common technical specifications set out in Commission Decision 2002/364/EC (3) for certain devices covered by Directive 98/79/EC remain relevant. Those common technical specifications have therefore been taken into account and where necessary updated to reflect the state of the art. |
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(2)

Regulation (EU) 2017/746 replaces Directive 98/79/EC of the European Parliament and of the Council (2). The common technical specifications set out in Commission Decision 2002/364/EC (3) for certain devices covered by Directive 98/79/EC remain relevant. Those common technical specifications have therefore been taken into account and where necessary updated to reflect the state of the art.

| (3) | To allow manufacturers, other economic operators, notified bodies and other actors to adapt to this Regulation, and to ensure its proper application, it is appropriate to defer its application. However, in the interest of public health and patient safety, manufacturers should be allowed to comply with the common specifications laid down in this Regulation on a voluntary basis before its date of application. |
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(3)

To allow manufacturers, other economic operators, notified bodies and other actors to adapt to this Regulation, and to ensure its proper application, it is appropriate to defer its application. However, in the interest of public health and patient safety, manufacturers should be allowed to comply with the common specifications laid down in this Regulation on a voluntary basis before its date of application.

| (4) | To ensure a continuous high level of safety and performance of devices, as a transitional measure it should be provided that devices that are in conformity with Decision 2002/364/EC are to be presumed to be in conformity with the requirements for certain performance characteristics set out in Annex I to Regulation (EU) 2017/746 until the date of application of this Regulation. |
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(4)

To ensure a continuous high level of safety and performance of devices, as a transitional measure it should be provided that devices that are in conformity with Decision 2002/364/EC are to be presumed to be in conformity with the requirements for certain performance characteristics set out in Annex I to Regulation (EU) 2017/746 until the date of application of this Regulation.

| (5) | The Medical Device Coordination Group has been consulted. |
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(5)

The Medical Device Coordination Group has been consulted.

| (6) | The measures provided for in this Regulation are in accordance with the opinion of the Committee on Medical Devices, |
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(6)

The measures provided for in this Regulation are in accordance with the opinion of the Committee on Medical Devices,

HAS ADOPTED THIS REGULATION:

Article 1

Common specifications

This Regulation lays down common specifications for certain class D in vitro diagnostic medical devices in respect of the requirements regarding the performance characteristics set out in Section 9.1, points (a) and (b), Section 9.3 and Section 9.4, point (a), of Annex I to Regulation (EU) 2017/746.

in vitro

Annex I lays down common specifications for devices covered by Annexes II to XIII, as specified in that Annex.

Annex II lays down common specifications for devices intended for detection of blood group antigens in the ABO, Rh, Kell, Duffy and Kidd blood group systems.

Annex III lays down common specifications for devices intended for detection or quantification of markers of human immunodeficiency virus (HIV) infection.

Annex IV lays down common specifications for devices intended for detection or quantification of markers of human T-cell lymphotropic virus (HTLV) infection.

Annex V lays down common specifications for devices intended for detection or quantification of markers of hepatitis C virus (HCV) infection.

Annex VI lays down common specifications for devices intended for detection or quantification of markers of hepatitis B virus (HBV) infection.

Annex VII lays down common specifications for devices intended for detection or quantification of markers of hepatitis D virus (HDV) infection.

Annex VIII lays down common specifications for devices intended for detection of markers of variant Creutzfeldt-Jakob disease (vCJD).

Annex IX lays down common specifications for devices intended for detection or quantification of markers of cytomegalovirus (CMV) infection.

Annex X lays down common specifications for devices intended for detection or quantification of markers of Epstein-Barr virus infection (EBV).

Annex XI lays down common specifications for devices intended for detection of markers of Treponema pallidum infection.

Treponema pallidum

Annex XII lays down common specifications for devices intended for detection or quantification of markers of Trypanosoma cruzi infection.

Trypanosoma cruzi

Annex XIII lays down common specifications for devices intended for detection or quantification of markers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

Article 2

Definitions

For the purposes of this Regulation, the following definitions apply:

| (1) | ‘true positive’ means a specimen known to be positive for the target marker and correctly classified by the device; |
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(1)

‘true positive’ means a specimen known to be positive for the target marker and correctly classified by the device;

| (2) | ‘false negative’ means a specimen known to be positive for the target marker and misclassified by the device; |
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(2)

‘false negative’ means a specimen known to be positive for the target marker and misclassified by the device;

| (3) | ‘false positive’ means a specimen known to be negative for the target marker and misclassified by the device; |
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(3)

‘false positive’ means a specimen known to be negative for the target marker and misclassified by the device;

| (4) | ‘the limit of detection’ (‘LOD’) means the smallest amount of the target marker that can be detected; |
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(4)

‘the limit of detection’ (‘LOD’) means the smallest amount of the target marker that can be detected;

| (5) | ‘nucleic acid amplification techniques’ (‘NAT’) means methods of detection and/or quantification of nucleic acids by either amplification of a target sequence, by amplification of a signal or by hybridisation; |
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(5)

‘nucleic acid amplification techniques’ (‘NAT’) means methods of detection and/or quantification of nucleic acids by either amplification of a target sequence, by amplification of a signal or by hybridisation;

| (6) | ‘NAT system’ means the combination of devices used for extraction, amplification and detection of nucleic acids; |
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(6)

‘NAT system’ means the combination of devices used for extraction, amplification and detection of nucleic acids;

| (7) | ‘rapid test’ means a qualitative or semi-quantitative in vitro diagnostic medical device, used singly or in a small series, which involves non-automated procedures (except the reading of results) and has been designed to give a fast result; |
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(7)

‘rapid test’ means a qualitative or semi-quantitative in vitro diagnostic medical device, used singly or in a small series, which involves non-automated procedures (except the reading of results) and has been designed to give a fast result;

in vitro

| (8) | ‘robustness’ means the capacity of an analytical procedure to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage; |
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(8)

‘robustness’ means the capacity of an analytical procedure to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage;

| (9) | ‘cross-reactivity’ means the ability of non-target analytes or markers to cause false positive results in an assay because of similarity, e.g. the ability of non-specific antibodies binding to a test antigen of an antibody assay, or the ability of non-target nucleic acids to be reactive in a NAT assay; |
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(9)

‘cross-reactivity’ means the ability of non-target analytes or markers to cause false positive results in an assay because of similarity, e.g. the ability of non-specific antibodies binding to a test antigen of an antibody assay, or the ability of non-target nucleic acids to be reactive in a NAT assay;

| (10) | ‘interference’ means the ability of unrelated substances to affect the results in an assay; |
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(10)

‘interference’ means the ability of unrelated substances to affect the results in an assay;

| (11) | ‘whole system failure rate’ means the frequency of failures when the entire process is performed as prescribed by the manufacturer; |
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(11)

‘whole system failure rate’ means the frequency of failures when the entire process is performed as prescribed by the manufacturer;

| (12) | ‘first-line assay’ means a device used to detect a marker or analyte, and the use of which may be followed by the use of a confirmatory assay; devices intended solely to be used to monitor a previously determined marker or analyte are not considered first-line assays; |
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(12)

‘first-line assay’ means a device used to detect a marker or analyte, and the use of which may be followed by the use of a confirmatory assay; devices intended solely to be used to monitor a previously determined marker or analyte are not considered first-line assays;

| (13) | ‘confirmatory assay’ means a device used for the confirmation of a reactive result from a first line assay; |
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(13)

‘confirmatory assay’ means a device used for the confirmation of a reactive result from a first line assay;

| (14) | ‘supplemental assay’ means a device that is used to provide further information for the interpretation of the test result of another assay; |
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(14)

‘supplemental assay’ means a device that is used to provide further information for the interpretation of the test result of another assay;

| (15) | ‘virus typing device’ means a device used for typing with already known positive samples, not used for primary diagnosis of infection or for screening; |
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(15)

‘virus typing device’ means a device used for typing with already known positive samples, not used for primary diagnosis of infection or for screening;

| (16) | ‘95 % positive cut-off value’ means the analyte concentration where 95 % of test runs give positive results following serial dilutions of an international reference material, where available, e.g. a World Health Organisation (WHO) International Standard or reference material calibrated against the WHO International Standard. |
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(16)

‘95 % positive cut-off value’ means the analyte concentration where 95 % of test runs give positive results following serial dilutions of an international reference material, where available, e.g. a World Health Organisation (WHO) International Standard or reference material calibrated against the WHO International Standard.

Article 3

Transitional provisions

1.   From 25 July 2022 until 25 July 2024, devices that are in conformity with the common technical specifications set out in Decision 2002/364/EC shall be presumed to be in conformity with the requirements regarding the performance characteristics set out in Section 9.1, points (a) and (b), Section 9.3 and Section 9.4, point (a), of Annex I to Regulation (EU) 2017/746.

During that period manufacturers of devices that are not in conformity with the common technical specifications set out in Decision 2002/364/EC shall duly justify that they have adopted solutions that ensure a level of safety and performance that is at least equivalent thereto.

2.   From 25 July 2022 until 25 July 2024 devices that are in conformity with the common specifications set out in this Regulation shall be presumed to be in conformity with the requirements regarding the performance characteristics set out in Section 9.1, points (a) and (b), Section 9.3 and Section 9.4, point (a), of Annex I to Regulation (EU) 2017/746.

Article 4

Entry into force and date of application

This Regulation shall enter into force on the twentieth day following that of its publication in the Official Journal of the European Union.

Official Journal of the European Union

It shall apply from 25 July 2024.

However, Article 3 shall apply from 25 July 2022.

This Regulation shall be binding in its entirety and directly applicable in all Member States.

Done at Brussels, 4 July 2022.

For the Commission

The President

Ursula VON DER LEYEN

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(1)  
  OJ L 117, 5.5.2017, p. 176.

1

(2)  Directive 98/79/EC of the European Parliament and of the Council of 27 October 1998 on in vitro diagnostic medical devices (OJ L 331, 7.12.1998, p. 1).

2

(3)  Commission Decision 2002/364/EC of 7 May 2002 on common technical specifications for in vitro-diagnostic medical devices (OJ L 131, 16.5.2002, p. 17).

3

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ANNEX I

GENERAL COMMON SPECIFICATIONS

Part I – Requirements for performance characteristics of devices covered by Annexes II to XIII

| Performance characteristics | Requirement |
| --- | --- |
| All performance characteristics set out in Section 9.1, points (a) and (b), Section 9.3 and Section 9.4, point (a), of Annex I to Regulation (EU) 2017/746 | 1. | The determination of performance characteristics shall be carried out in direct comparison with a state-of-the-art device. The device used for comparison shall be one bearing CE marking, if on the market at the time of the performance evaluation. |

Performance characteristics

Requirement

All performance characteristics set out in Section 9.1, points (a) and (b), Section 9.3 and Section 9.4, point (a), of Annex I to Regulation (EU) 2017/746

1.

The determination of performance characteristics shall be carried out in direct comparison with a state-of-the-art device. The device used for comparison shall be one bearing CE marking, if on the market at the time of the performance evaluation.

| 2. | Devices used for determination of status of specimens used in determination of performance characteristics shall be state-of-the-art devices bearing CE marking. |
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2.

Devices used for determination of status of specimens used in determination of performance characteristics shall be state-of-the-art devices bearing CE marking.

| 3. | If discrepant results are identified as part of determination of performance characteristics, these results shall be resolved as far as possible, by one or more of the following:






  — | by evaluation of the discrepant specimen in further devices, |
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3.

If discrepant results are identified as part of determination of performance characteristics, these results shall be resolved as far as possible, by one or more of the following:

—

by evaluation of the discrepant specimen in further devices,

| — | by use of an alternative method or marker, |
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—

by use of an alternative method or marker,

| — | by a review of the clinical status and diagnosis of the patient, |
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—

by a review of the clinical status and diagnosis of the patient,

| — | by the testing of follow-up specimens. |
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—

by the testing of follow-up specimens.

| 4. | The determination of performance characteristics shall be performed on a population equivalent to the European population. |
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4.

The determination of performance characteristics shall be performed on a population equivalent to the European population.

Whole system failure rate

| 5. | As part of the required risk analysis the whole system failure rate leading to false negative results shall be determined in repeat assays on low-positive specimens. |
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5.

As part of the required risk analysis the whole system failure rate leading to false negative results shall be determined in repeat assays on low-positive specimens.

Analytical sensitivity and analytical specificity, interference

| 6. | For devices intended for use with plasma the manufacturer shall verify the performance of the device using all anticoagulants which the manufacturer indicates for use with the device, for at least 50 plasma specimens (for devices intended for detection and/or quantification of infectious agents, 25 positive and 25 negative). |
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6.

For devices intended for use with plasma the manufacturer shall verify the performance of the device using all anticoagulants which the manufacturer indicates for use with the device, for at least 50 plasma specimens (for devices intended for detection and/or quantification of infectious agents, 25 positive and 25 negative).

Analytical and diagnostic specificity, interference and cross-reactivity

| 7. | The manufacturer shall select the potential interfering substances to be evaluated taking account of the composition of the reagents and configuration of the device. |
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7.

The manufacturer shall select the potential interfering substances to be evaluated taking account of the composition of the reagents and configuration of the device.

Batch-to-batch consistency

| 8. | For devices intended to detect antigens and antibodies, the manufacturer’s batch testing criteria shall ensure that every batch consistently identifies the relevant antigens, epitopes, and antibodies and is suitable for the claimed specimen types. |
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8.

For devices intended to detect antigens and antibodies, the manufacturer’s batch testing criteria shall ensure that every batch consistently identifies the relevant antigens, epitopes, and antibodies and is suitable for the claimed specimen types.

| 9. | The manufacturer’s batch release testing for first-line assays shall include at least 100 specimens negative for the relevant analyte (1). |
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9.

The manufacturer’s batch release testing for first-line assays shall include at least 100 specimens negative for the relevant analyte (1).

Part II – Requirements for performance characteristics of devices referred to in Annexes III to XIII

| Performance characteristic | Requirement |
| --- | --- |
| Analytical and diagnostic sensitivity | 10. | Devices intended by the manufacturer for testing body fluids other than serum or plasma, e.g. urine, saliva, etc., shall meet the same requirements as serum or plasma devices. The manufacturer shall test specimens from the same individuals in both the devices to be approved and in a respective serum or plasma device. (2) |

Performance characteristic

Requirement

Analytical and diagnostic sensitivity

10.

Devices intended by the manufacturer for testing body fluids other than serum or plasma, e.g. urine, saliva, etc., shall meet the same requirements as serum or plasma devices. The manufacturer shall test specimens from the same individuals in both the devices to be approved and in a respective serum or plasma device. (2)

| 11. | Devices for self-testing shall meet the same requirements as respective devices for professional use. |
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11.

Devices for self-testing shall meet the same requirements as respective devices for professional use.

| 12. | Positive specimens used in the performance evaluation shall be selected to reflect different stages of the respective disease(s), different antibody patterns, different genotypes, different subtypes, mutants, etc. |
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12.

Positive specimens used in the performance evaluation shall be selected to reflect different stages of the respective disease(s), different antibody patterns, different genotypes, different subtypes, mutants, etc.

| 13. | Seroconversion panels shall start with a negative bleed(s) and shall have narrow bleeding intervals as far as possible. Where this is not possible, manufacturers shall provide a justification in the performance evaluation report. |
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13.

Seroconversion panels shall start with a negative bleed(s) and shall have narrow bleeding intervals as far as possible. Where this is not possible, manufacturers shall provide a justification in the performance evaluation report.

| 14. | For devices intended by the manufacturer to be used with serum and plasma the performance evaluation must demonstrate serum to plasma equivalency. This shall be demonstrated for at least 25 positive donations. |
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14.

For devices intended by the manufacturer to be used with serum and plasma the performance evaluation must demonstrate serum to plasma equivalency. This shall be demonstrated for at least 25 positive donations.

| 15. | For devices detecting or quantifying antigens or nucleic acids, the target antigen(s) or target nucleic acid region(s) respectively shall be specified in the instructions for use. |
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15.

For devices detecting or quantifying antigens or nucleic acids, the target antigen(s) or target nucleic acid region(s) respectively shall be specified in the instructions for use.

| 16. | For devices detecting or quantifying antibodies against an infectious agent, the target antigen(s) of those antibodies shall be specified in the instructions for use. |
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16.

For devices detecting or quantifying antibodies against an infectious agent, the target antigen(s) of those antibodies shall be specified in the instructions for use.

Analytical and diagnostic specificity

| 17. | Devices intended by the manufacturer for testing body fluids other than serum or plasma, e.g. urine, saliva, etc., shall meet the same requirements as serum or plasma devices. The performance evaluation shall test specimens from the same individuals in both the devices to be approved and in a respective serum or plasma device. (2) |
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17.

Devices intended by the manufacturer for testing body fluids other than serum or plasma, e.g. urine, saliva, etc., shall meet the same requirements as serum or plasma devices. The performance evaluation shall test specimens from the same individuals in both the devices to be approved and in a respective serum or plasma device. (2)

| 18. | Devices for self-testing shall meet the same requirements as respective devices for professional use. |
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18.

Devices for self-testing shall meet the same requirements as respective devices for professional use.

| 19. | Negative specimens used in a performance evaluation shall be defined so as to reflect the target population for which the device is intended, such as blood donors, hospitalised patients, pregnant women, etc. |
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19.

Negative specimens used in a performance evaluation shall be defined so as to reflect the target population for which the device is intended, such as blood donors, hospitalised patients, pregnant women, etc.

| 20. | Specificity shall be based on repeatedly reactive false positive results in specimens negative for the target marker. |
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20.

Specificity shall be based on repeatedly reactive false positive results in specimens negative for the target marker.

| 21. | For devices intended by the manufacturer to be used with serum and plasma the performance evaluation must demonstrate serum to plasma equivalency. This shall be demonstrated for at least 25 negative donations. |
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21.

For devices intended by the manufacturer to be used with serum and plasma the performance evaluation must demonstrate serum to plasma equivalency. This shall be demonstrated for at least 25 negative donations.

Analytical and diagnostic specificity, interference and cross-reactivity

| 22. | The manufacturer shall include specimens such as, where applicable:






  — | specimens representing related infections, |
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22.

The manufacturer shall include specimens such as, where applicable:

—

specimens representing related infections,

| — | specimens from multigravida, i.e. women who have had more than one pregnancy, or rheumatoid factor (RF) positive patients, |
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—

specimens from multigravida, i.e. women who have had more than one pregnancy, or rheumatoid factor (RF) positive patients,

| — | specimens containing human antibodies to components of the expression system, for example anti-E. coli, or anti-yeast. |
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—

specimens containing human antibodies to components of the expression system, for example anti-E. coli, or anti-yeast.

Performances obtained by lay persons

| 23. | Relevant parts of the performance evaluation shall be carried out (or repeated) by appropriate lay persons to validate the operation of the device and the instructions for use. The lay persons selected for the performance evaluation shall be representative of the intended users groups. |
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23.

Relevant parts of the performance evaluation shall be carried out (or repeated) by appropriate lay persons to validate the operation of the device and the instructions for use. The lay persons selected for the performance evaluation shall be representative of the intended users groups.

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(1)  This requirement shall not apply to devices covered by Tables 1 and 2 of Annex XIII.

(2)  This requirement shall not apply to devices referred to in Tables 4, 5 and 6 of Annex XIII.

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ANNEX II

COMMON SPECIFICATIONS FOR DEVICES INTENDED FOR DETECTION OF BLOOD GROUP ANTIGENS IN THE ABO, RH, KELL, DUFFY AND KIDD BLOOD GROUP SYSTEMS

Scope

This Annex applies to devices intended for detection of blood group antigens in the ABO, Rh, Kell, Duffy and Kidd blood group systems.

Table 1 applies to performance evaluation of devices detecting blood group antigens in the ABO, Rh, Kell, Duffy and Kidd blood group systems.

Table 2 applies to manufacturer’s batch-to-batch consistency testing of reagents and reagent products to determine blood group antigens in the ABO, Rh, Kell, Duffy and Kidd blood group systems (test reagents, control materials).

Table 1. Performance evaluation of devices detecting blood group antigens in the ABO, Rh, Kell, Duffy and Kidd blood group systems

| Reagent specificity | Number of tests per method claimed by the manufacturer | Total number of specimens to be tested for a launch device | Total number of specimens to be tested for a new formulation, or use of well-characterised reagents | General qualification criteria | Specific qualification criteria | Acceptance criteria |
| --- | --- | --- | --- | --- | --- | --- |
| Anti-ABO1 (Anti-A), Anti-ABO2 (Anti-B), Anti-ABO3 (Anti-A,B) | ≥500 | ≥3 000 | ≥1 000 | Clinical specimens: 10 % of the test population
  Neonatal specimens: > 2 % of the test population | ABO specimens shall include > 40 % A and B antigen positive specimens which may include specimens from group A, group B and group AB | All of the reagents shall show comparable performance to state-of-the-art CE marked devices with regard to claimed reactivity of the device.
  For CE marked devices where the application or use has been changed or extended, further testing shall be carried out in accordance with the requirements outlined in column 2 above (“Number of tests per method claimed by the manufacturer”). |
| Anti-RH1 (Anti-D) | ≥500 | ≥3 000 | ≥1 000 | Performance evaluation of Anti-D reagents shall include tests against a range of weak RH1 (D) and partial RH1 (D) specimens, depending on the intended use of the product.
  Weak and/or partial D cells shall account for > 2 % of RH1 (D) positive specimens. |
| Anti-RH2 (Anti-C), Anti-RH4 (Anti-c), Anti- RH3 (Anti-E) | ≥100 | ≥1 000 | ≥200 | |
| Anti-RH5 (Anti-e) | ≥100 | ≥500 | ≥200 | |
| Anti-KEL1 (Anti-K) | ≥100 | ≥500 | ≥200 | |
| Anti-JK1 (Jka), Anti-JK2 (Jkb) | ≥100 | ≥500 | ≥200 | |
| Anti-FY1 (Fya), Anti-FY2 (Fyb) | ≥100 | ≥500 | ≥200 | |
| Note: Positive specimens used in the performance evaluation shall be selected to reflect variant and weak antigen expression. |

Reagent specificity

Number of tests per method claimed by the manufacturer

Total number of specimens to be tested for a launch device

Total number of specimens to be tested for a new formulation, or use of well-characterised reagents

General qualification criteria

Specific qualification criteria

Acceptance criteria

Anti-ABO1 (Anti-A), Anti-ABO2 (Anti-B), Anti-ABO3 (Anti-A,B)

≥500

≥3 000

≥1 000

Clinical specimens: 10 % of the test population

Neonatal specimens: > 2 % of the test population

ABO specimens shall include > 40 % A and B antigen positive specimens which may include specimens from group A, group B and group AB

All of the reagents shall show comparable performance to state-of-the-art CE marked devices with regard to claimed reactivity of the device.

For CE marked devices where the application or use has been changed or extended, further testing shall be carried out in accordance with the requirements outlined in column 2 above (“Number of tests per method claimed by the manufacturer”).

Anti-RH1 (Anti-D)

≥500

≥3 000

≥1 000

Performance evaluation of Anti-D reagents shall include tests against a range of weak RH1 (D) and partial RH1 (D) specimens, depending on the intended use of the product.

Weak and/or partial D cells shall account for > 2 % of RH1 (D) positive specimens.

Anti-RH2 (Anti-C), Anti-RH4 (Anti-c), Anti- RH3 (Anti-E)

≥100

≥1 000

≥200

Anti-RH5 (Anti-e)

≥100

≥500

≥200

Anti-KEL1 (Anti-K)

≥100

≥500

≥200

Anti-JK1 (Jka), Anti-JK2 (Jkb)

≥100

≥500

≥200

Anti-FY1 (Fya), Anti-FY2 (Fyb)

≥100

≥500

≥200

Note: Positive specimens used in the performance evaluation shall be selected to reflect variant and weak antigen expression.

Table 2. Manufacturer’s batch-to-batch consistency testing of reagents and reagent products to determine blood group antigens in the ABO, Rh, Kell, Duffy and Kidd blood group systems

1.   Test reagents

| Blood group reagents | Minimum number of control cells to be tested as part of specificity testing | Acceptance criteria |
| --- | --- | --- |
| | Positive reactions | | Negative reactions | Each batch of reagent shall exhibit unequivocal positive or negative results by all techniques claimed by the manufacturer in accordance with the results obtained from the performance evaluation data. |
| | A1 | A2B | Ax | | B | O | |
| Anti-ABO1(Anti-A) | 2 | 2 | 2 (1) | | 2 | 2 | |
| | B | A1B | | | A1 | O | |
| Anti-ABO2(Anti-B) | 2 | 2 | | | 2 | 2 | |
| | A1 | A2 | Ax | B | O | | |
| Anti-ABO3(Anti-A,B) | 2 | 2 | 2 (1) | 2 | 4 | | |
| | R1r | R2r | WeakD | | r’r | r”r | rr |
| Anti-RH1 (Anti-D) | 2 | 2 | 2 (1) | | 1 | 1 | 1 |
| | R1R2 | R1r | r’r | | R2R2 | r”r | rr |
| Anti-RH2 (Anti-C) | 2 | 1 | 1 | | 1 | 1 | 1 |
| | R1R2 | R1r | r’r | | R1R1 | | |
| Anti-RH4 (Anti-c) | 1 | 2 | 1 | | 3 | | |
| | R1R2 | R2r | r”r | | R1R1 | r’r | rr |
| Anti-RH3 (Anti-E) | 2 | 1 | 1 | | 1 | 1 | 1 |
| | R1R2 | R2r | r”r | | R2R2 | | |
| Anti-RH5 (Anti-e) | 2 | 1 | 1 | | 3 | | |
| | Kk | | | | kk | | |
| Anti-KEL1 (Anti-K) | 4 | | | | 3 | | |
| | Jk(a+b+) | | | | | Jk(a–b+) | | |
| Anti-JK1 (Anti-Jka) | 4 | | | | | 3 | | |
| | Jk(a+b+) | | | | | Jk(a+b–) | | |
| Anti-JK2 (Anti-Jkb) | 4 | | | | | 3 | | |
| | Fy(a+b+) | | | | | Fy(a–b+) | | |
| Anti-FY1 (Anti-Fya) | 4 | | | | | 3 | | |
| | Fy(a+b+) | | | | | Fy(a+b–) | | |
| Anti-FY2 (Anti-Fyb) | 4 | | | | | 3 | | |
| Note: Polyclonal reagents shall be tested against a wider panel of cells to confirm specificity and exclude presence of unwanted contaminating antibodies. |

Blood group reagents

Minimum number of control cells to be tested as part of specificity testing

Acceptance criteria

Positive reactions

Negative reactions

Each batch of reagent shall exhibit unequivocal positive or negative results by all techniques claimed by the manufacturer in accordance with the results obtained from the performance evaluation data.

A1

A2B

Ax

B

O

Anti-ABO1(Anti-A)

2

2

2 (1)

2

2

B

A1B

A1

O

Anti-ABO2(Anti-B)

2

2

2

2

A1

A2

Ax

B

O

Anti-ABO3(Anti-A,B)

2

2

2 (1)

2

4

R1r

R2r

WeakD

r’r

r”r

rr

Anti-RH1 (Anti-D)

2

2

2 (1)

1

1

1

R1R2

R1r

r’r

R2R2

r”r

rr

Anti-RH2 (Anti-C)

2

1

1

1

1

1

R1R2

R1r

r’r

R1R1

Anti-RH4 (Anti-c)

1

2

1

3

R1R2

R2r

r”r

R1R1

r’r

rr

Anti-RH3 (Anti-E)

2

1

1

1

1

1

R1R2

R2r

r”r

R2R2

Anti-RH5 (Anti-e)

2

1

1

3

Kk

kk

Anti-KEL1 (Anti-K)

4

3

Jk(a+b+)

Jk(a–b+)

Anti-JK1 (Anti-Jka)

4

3

Jk(a+b+)

Jk(a+b–)

Anti-JK2 (Anti-Jkb)

4

3

Fy(a+b+)

Fy(a–b+)

Anti-FY1 (Anti-Fya)

4

3

Fy(a+b+)

Fy(a+b–)

Anti-FY2 (Anti-Fyb)

4

3

Note: Polyclonal reagents shall be tested against a wider panel of cells to confirm specificity and exclude presence of unwanted contaminating antibodies.

2.   Control materials (red cells)

The phenotype of red cells used in the control of blood typing reagents listed above shall be confirmed using an established device(s).

---

(1)  Only where reactivity against these antigens is claimed.

---

ANNEX III

COMMON SPECIFICATIONS FOR DEVICES INTENDED FOR DETECTION OR QUANTIFICATION OF MARKERS OF HUMAN IMMUNODEFICIENCY VIRUS (HIV) INFECTION

Scope

| 1. | This Annex applies to devices intended for detection or quantification of markers of human immunodeficiency virus (HIV) infection.
  Table 1 applies to first-line assays for HIV-1/2 antibody (anti-HIV-1/2) and first-line combined antigen/antibody assays for HIV-1/2 (HIV-1/2 Ag/Ab) which are not rapid tests.
  Table 2 applies to first-line assays for anti-HIV-1/2 and HIV-1/2 Ag/Ab which are rapid tests.
  Table 3 applies to confirmatory assays for anti-HIV-1/2.
  Table 4 applies to antigen tests for HIV-1 and HIV Ag/Ab assays.
  Table 5 applies to qualitative and quantitative NAT devices for HIV ribonucleic acid (RNA).
  Table 6 applies to HIV-1/2 self-tests. |
| --- | --- |

1.

Table 1 applies to first-line assays for HIV-1/2 antibody (anti-HIV-1/2) and first-line combined antigen/antibody assays for HIV-1/2 (HIV-1/2 Ag/Ab) which are not rapid tests.

Table 2 applies to first-line assays for anti-HIV-1/2 and HIV-1/2 Ag/Ab which are rapid tests.

Table 3 applies to confirmatory assays for anti-HIV-1/2.

Table 4 applies to antigen tests for HIV-1 and HIV Ag/Ab assays.

Table 5 applies to qualitative and quantitative NAT devices for HIV ribonucleic acid (RNA).

Table 6 applies to HIV-1/2 self-tests.

Definitions

| 2. | For the purposes of this Annex, the following definitions apply:






  (1) | ‘seroconversion HIV specimen’ means:






  — | p24 antigen and/or HIV RNA positive, and |
| --- | --- | --- | --- |

2.

(1)

‘seroconversion HIV specimen’ means:

—

p24 antigen and/or HIV RNA positive, and

| — | recognised by the antibody first-line assays, and |
| --- | --- |

—

recognised by the antibody first-line assays, and

| — | positive or indeterminate in confirmatory assays. |
| --- | --- |

—

positive or indeterminate in confirmatory assays.

| (2) | ‘early seroconversion HIV specimen’ means:






  — | p24 antigen and/or HIV RNA positive, and |
| --- | --- | --- |

(2)

‘early seroconversion HIV specimen’ means:

—

p24 antigen and/or HIV RNA positive, and

| — | not recognised by the antibody first-line assays, and |
| --- | --- |

—

not recognised by the antibody first-line assays, and

| — | indeterminate or negative in confirmatory assays. |
| --- | --- |

—

indeterminate or negative in confirmatory assays.

Table 1. First-line assays: anti-HIV-1/2, HIV-1/2 Ag/Ab (requirements for antibody detection)

| Performance characteristic | Specimen | Specimen numbers, features, use | Acceptance criteria |
| --- | --- | --- | --- |
| Diagnostic sensitivity | Positive specimens | ≥400 HIV-1
  ≥100 HIV-2
  including 40 non-B-subtypes
  including 25 positive ‘same day’ fresh serum specimens (≤ 1 day after specimen taking)
  all available HIV/1 subtypes shall be represented by at least 3 specimens per subtype | all true positive specimens shall be identified as positive |
| | Seroconversion panels | ≥30 panels
  at least 40 early seroconversion HIV specimens shall be tested | diagnostic sensitivity during seroconversion shall represent the state of the art
  all seroconversion HIV specimens shall be identified as positive |
| Diagnostic specificity | Unselected blood donors (including first-time donors) (1) | ≥5 000 | ≥99,5% |
| Hospitalised patients | ≥200 | Potential limitations for specificity, if any, shall be identified |
| Cross-reactivity | Potentially cross-reacting specimens | ≥100 in total
  (such as RF+, from related virus infections, from pregnant women, subjects recently vaccinated against any infectious agent) |

Performance characteristic

Specimen

Specimen numbers, features, use

Acceptance criteria

Diagnostic sensitivity

Positive specimens

≥400 HIV-1

≥100 HIV-2

including 40 non-B-subtypes

including 25 positive ‘same day’ fresh serum specimens (≤ 1 day after specimen taking)

all available HIV/1 subtypes shall be represented by at least 3 specimens per subtype

all true positive specimens shall be identified as positive

Seroconversion panels

≥30 panels

at least 40 early seroconversion HIV specimens shall be tested

diagnostic sensitivity during seroconversion shall represent the state of the art

all seroconversion HIV specimens shall be identified as positive

Diagnostic specificity

Unselected blood donors (including first-time donors) (1)

≥5 000

≥99,5%

Hospitalised patients

≥200

Potential limitations for specificity, if any, shall be identified

Cross-reactivity

Potentially cross-reacting specimens

≥100 in total

(such as RF+, from related virus infections, from pregnant women, subjects recently vaccinated against any infectious agent)

Table 2. Rapid tests: anti-HIV-1/2, HIV-1/2 Ag/Ab (requirements for antibody detection)

| Performance characteristic | Specimen | Specimen numbers, features, use | Acceptance criteria |
| --- | --- | --- | --- |
| Diagnostic sensitivity | Positive specimens | ≥400 HIV-1
  ≥100 HIV-2
  including 40 non-B-subtypes
  all available HIV/1 subtypes shall be represented by at least 3 specimens per subtype | all true positive specimens shall be identified as positive |
| Seroconversion panels | ≥30 panels
  at least 40 early seroconversion HIV specimens shall be tested | diagnostic sensitivity during seroconversion shall represent the state of the art
  all seroconversion HIV specimens shall be identified as positive |
| Diagnostic specificity | Unselected blood donors (including first-time donors) | ≥1 000 | ≥ 99 % |
| Hospitalised patients | ≥200 | Potential limitations for specificity, if any, shall be identified |
| Cross-reactivity | Potentially cross-reacting specimens | ≥200 specimens from pregnant women
  ≥100 other potentially cross-reacting specimens in total (e.g. RF+, from related infections) |

Performance characteristic

Specimen

Specimen numbers, features, use

Acceptance criteria

Diagnostic sensitivity

Positive specimens

≥400 HIV-1

≥100 HIV-2

including 40 non-B-subtypes

all available HIV/1 subtypes shall be represented by at least 3 specimens per subtype

all true positive specimens shall be identified as positive

Seroconversion panels

≥30 panels

at least 40 early seroconversion HIV specimens shall be tested

diagnostic sensitivity during seroconversion shall represent the state of the art

all seroconversion HIV specimens shall be identified as positive

Diagnostic specificity

Unselected blood donors (including first-time donors)

≥1 000

≥ 99 %

Hospitalised patients

≥200

Potential limitations for specificity, if any, shall be identified

Cross-reactivity

Potentially cross-reacting specimens

≥200 specimens from pregnant women

≥100 other potentially cross-reacting specimens in total (e.g. RF+, from related infections)

Table 3. Confirmatory assays: anti-HIV-1/2

| Performance characteristic | Specimen | Specimen numbers, features, use | Acceptance criteria |
| --- | --- | --- | --- |
| Diagnostic sensitivity | Positive specimens | ≥200 HIV-1
  ≥100 HIV-2
  Including different stages of infection and reflecting different antibody patterns | Identification as “confirmed positive” or “indeterminate”, not as “negative” |
| Seroconversion panels | ≥15 seroconversion panels/low titre panels
  ≥40 early seroconversion HIV specimens | Diagnostic sensitivity during seroconversion shall represent the state of the art
  All seroconversion HIV specimens shall be identified as positive |
| Diagnostic specificity | Blood donors | ≥200 | No false positive results / no neutralisation |
| Hospitalised patients | ≥200 |
| Cross-reactivity | Potentially cross-reacting specimens | ≥50 in total (including specimens from pregnant women, specimens with indeterminate results in other confirmatory assays) |

Performance characteristic

Specimen

Specimen numbers, features, use

Acceptance criteria

Diagnostic sensitivity

Positive specimens

≥200 HIV-1

≥100 HIV-2

Including different stages of infection and reflecting different antibody patterns

Identification as “confirmed positive” or “indeterminate”, not as “negative”

Seroconversion panels

≥15 seroconversion panels/low titre panels

≥40 early seroconversion HIV specimens

Diagnostic sensitivity during seroconversion shall represent the state of the art

All seroconversion HIV specimens shall be identified as positive

Diagnostic specificity

Blood donors

≥200

No false positive results / no neutralisation

Hospitalised patients

≥200

Cross-reactivity

Potentially cross-reacting specimens

≥50 in total (including specimens from pregnant women, specimens with indeterminate results in other confirmatory assays)

Table 4. Antigen tests: HIV-1, HIV Ag/Ab (requirements for antigen detection)

| Performance characteristic | Specimen | Specimen numbers, features, use | Acceptance criteria |
| --- | --- | --- | --- |
| Diagnostic sensitivity | Positive specimens | ≥50 HIV-1 antigen positive
  ≥50 cell culture supernatants including different HIV-1 subtypes and HIV-2 | all true positive specimens shall be identified as positive (after neutralisation if applicable) |
| Seroconversion panels | ≥20 seroconversion panels/low titre panels
  ≥40 early seroconversion HIV specimens | diagnostic sensitivity during seroconversion shall represent the state of the art
  all seroconversion HIV specimens shall be identified as positive |
| Analytical sensitivity | First International Reference Reagent HIV-1 p24 Antigen, NIBSC code: 90/636 | | ≤ 2 IU/ml |
| Diagnostic specificity | Blood donors | ≥200 | ≥ 99,5 % after neutralisation or, if no neutralisation test available, after resolution of the specimen status |
| Hospitalised patients | ≥200 | Potential limitations for specificity, if any, shall be identified |
| Cross-reactivity | Potentially cross-reacting specimens | ≥50 |

Performance characteristic

Specimen

Specimen numbers, features, use

Acceptance criteria

Diagnostic sensitivity

Positive specimens

≥50 HIV-1 antigen positive

≥50 cell culture supernatants including different HIV-1 subtypes and HIV-2

all true positive specimens shall be identified as positive (after neutralisation if applicable)

Seroconversion panels

≥20 seroconversion panels/low titre panels

≥40 early seroconversion HIV specimens

diagnostic sensitivity during seroconversion shall represent the state of the art

all seroconversion HIV specimens shall be identified as positive

Analytical sensitivity

First International Reference Reagent HIV-1 p24 Antigen, NIBSC code: 90/636

≤ 2 IU/ml

Diagnostic specificity

Blood donors

≥200

≥ 99,5 % after neutralisation or, if no neutralisation test available, after resolution of the specimen status

Hospitalised patients

≥200

Potential limitations for specificity, if any, shall be identified

Cross-reactivity

Potentially cross-reacting specimens

≥50

Table 5. Qualitative and quantitative NAT devices for HIV RNA

| 1. | For target sequence amplification devices, a functionality control for each specimen (internal control) shall reflect the state of the art. This control shall as far as possible be used throughout the whole process, i.e. extraction, amplification/hybridisation, detection. |
| --- | --- |

1.

| 2. | Genotype and/or subtype detection shall be demonstrated by appropriate primer or probe design validation and shall also be validated by testing characterised genotyped specimens. |
| --- | --- |

2.

| 3. | Potential cross-reactivity of non-target nucleic acid sequences shall be analysed by appropriate primer or probe design validation and shall also be validated by testing selected specimens. |
| --- | --- |

3.

| 4. | Results of quantitative NAT devices shall be traceable to international standards or calibrated reference materials, if available, and be expressed in international units utilised in the specific field of application. |
| --- | --- |

4.

| 5. | Qualitative HIV NAT devices intended to be used to detect the presence of HIV in blood, blood components, cells, tissues or organs, or in any of their derivatives, in order to assess their suitability for transfusion, transplantation or cell administration shall be designed to detect both HIV-1 and HIV-2. |
| --- | --- |

5.

| 6. | Qualitative HIV NAT devices, other than virus typing devices, shall be designed to compensate for the potential failure of a HIV-1 NAT target region by using two independent target regions.








  Performance characteristic | Specimen | Specimen numbers, features, use | Acceptance criteria |
| --- | --- | --- | --- | --- |
| Analytical sensitivity | WHO International Standard HIV-1 RNA; WHO International Standard HIV-2 RNA; or calibrated reference materials | NAT sensitivity and NAT LOD shall be validated by dilution series of reference materials, testing of replicates (minimum 24) at different analyte concentrations, including those with transition from positive to negative results with the respective NAT device.
  LOD shall be expressed as 95% positive cut-off value (IU/ml) after statistical analysis (e.g. Probit). (2)

  quantitative NAT: definition of lower, upper quantification limit, precision, accuracy,
  ‘linear’ measuring range, ‘dynamic range’.
  Reproducibility at different concentration levels | According to the state of the art |
| HIV geno-/subtype sensitivity | all relevant genotypes/subtypes, preferably from international reference materials
  potential substitutes for rare HIV subtypes (to be quantified by appropriate methods): cell culture supernatants; in vitro transcripts; plasmids | Qualitative NAT: at least 10 specimens/genotype or subtype
  Quantitative NAT: dilution series for demonstration of quantification efficiencies | According to the state of the art |
| Diagnostic sensitivity | Positive specimens reflecting the routine conditions of users (e.g. no pre-selection of specimens) | Quantitative NAT: ≥100
  Comparative results with another NAT system shall be generated in parallel | According to the state of the art |
| Seroconversion panels | Qualitative NAT: ≥10 panels
  Comparative results with another NAT system shall be generated in parallel | According to the state of the art |
| Diagnostic specificity | Blood donor specimens | Qualitative NAT: ≥500
  Quantitative NAT: ≥100 | According to the state of the art |
| Cross-reactivity | Potentially cross-reacting specimens | ≥10 human retrovirus positive specimens (e.g. HTLV) | According to the state of the art |
| Carry-over | High HIV RNA positive;
  HIV RNA negative | At least five runs with alternating high-positive and negative specimens shall be performed during robustness studies. The virus titres of the high positive specimens shall be representative of high virus titres occurring naturally. | According to the state of the art |
| Detection in relation to antibody status | HIV-RNA positives: anti-HIV negative, anti-HIV positive | Pre-seroconversion (anti-HIV negative) and post-seroconversion (anti-HIV positive) specimens | According to the state of the art |
| Whole system failure rate | HIV RNA low-positive | ≥100 HIV RNA low-positive specimens shall be tested. These specimens shall contain a virus concentration equivalent to three times the 95 % positive cut-off virus concentration. | ≥99% positive |

6.

Performance characteristic

Specimen

Specimen numbers, features, use

Acceptance criteria

Analytical sensitivity

WHO International Standard HIV-1 RNA; WHO International Standard HIV-2 RNA; or calibrated reference materials

NAT sensitivity and NAT LOD shall be validated by dilution series of reference materials, testing of replicates (minimum 24) at different analyte concentrations, including those with transition from positive to negative results with the respective NAT device.

LOD shall be expressed as 95% positive cut-off value (IU/ml) after statistical analysis (e.g. Probit). (2)

quantitative NAT: definition of lower, upper quantification limit, precision, accuracy,

‘linear’ measuring range, ‘dynamic range’.

Reproducibility at different concentration levels

According to the state of the art

HIV geno-/subtype sensitivity

all relevant genotypes/subtypes, preferably from international reference materials

potential substitutes for rare HIV subtypes (to be quantified by appropriate methods): cell culture supernatants; in vitro transcripts; plasmids

Qualitative NAT: at least 10 specimens/genotype or subtype

Quantitative NAT: dilution series for demonstration of quantification efficiencies

According to the state of the art

Diagnostic sensitivity

Positive specimens reflecting the routine conditions of users (e.g. no pre-selection of specimens)

Quantitative NAT: ≥100

Comparative results with another NAT system shall be generated in parallel

According to the state of the art

Seroconversion panels

Qualitative NAT: ≥10 panels

Comparative results with another NAT system shall be generated in parallel

According to the state of the art

Diagnostic specificity

Blood donor specimens

Qualitative NAT: ≥500

Quantitative NAT: ≥100

According to the state of the art

Cross-reactivity

Potentially cross-reacting specimens

≥10 human retrovirus positive specimens (e.g. HTLV)

According to the state of the art

Carry-over

High HIV RNA positive;

HIV RNA negative

At least five runs with alternating high-positive and negative specimens shall be performed during robustness studies. The virus titres of the high positive specimens shall be representative of high virus titres occurring naturally.

According to the state of the art

Detection in relation to antibody status

HIV-RNA positives: anti-HIV negative, anti-HIV positive

Pre-seroconversion (anti-HIV negative) and post-seroconversion (anti-HIV positive) specimens

According to the state of the art

Whole system failure rate

HIV RNA low-positive

≥100 HIV RNA low-positive specimens shall be tested. These specimens shall contain a virus concentration equivalent to three times the 95 % positive cut-off virus concentration.

≥99% positive

Table 6. Additional requirements for HIV-1/2 self-tests

| Performance characteristic | Specimens (3) | Number of lay persons |
| --- | --- | --- |
| Result interpretation (4) | Interpretation of results (5) by lay persons reflecting the following range of reactivity levels:






  — | non-reactive |

Performance characteristic

Specimens (3)

Number of lay persons

Result interpretation (4)

Interpretation of results (5) by lay persons reflecting the following range of reactivity levels:

—

non-reactive

| — | reactive |
| --- | --- |

—

reactive

| — | weak reactive (6) |
| --- | --- |

—

weak reactive (6)

| — | invalid |
| --- | --- |

—

invalid

≥ 100

Diagnostic sensitivity

lay persons that are known positive

≥ 200

Diagnostic specificity

lay persons that do not know their status

≥ 400

Lay persons that are at high risk of acquiring the infection

≥ 200

---

(1)  Blood donor populations shall be investigated from at least two blood donation centres and consist of consecutive blood donations, which have not been selected to exclude first-time donors.

(2)  Reference: European Pharmacopeia 9.0, 2.6.21 Nucleic acid amplification techniques, Validation.

(3)  For each body fluid claimed for use with the device, e.g. whole blood, urine, saliva, etc., the sensitivity and specificity of the device for self-testing in the hands of lay persons shall be defined against the confirmed patient infectious status.

(4)  The result interpretation study shall include reading and interpretation of the test results by at least 100 lay persons, with each lay person subjected to reading results covering the specified range of result reactivity levels. The manufacturer shall determine the concordance between lay person reading and professional user reading.

(5)  Tests shall be performed prior to the result interpretation study using whenever possible the specimen type intended by the manufacturer. The tests may be performed on contrived specimens based on the natural matrix of the respective specimen type.

(6)  A higher proportion of the specimens shall be in the low-positive range close to the cut-off or LOD of the test.

---

ANNEX IV

COMMON SPECIFICATIONS FOR DEVICES INTENDED FOR DETECTION OR QUANTIFICATION OF MARKERS OF HUMAN T-CELL LYMPHOTROPIC VIRUS (HTLV) INFECTION

Scope

This Annex applies to devices intended for detection or quantification of markers of human T-cell lymphotropic virus (HTLV) infection.

Table 1 applies to first-line assays for antibodies against HTLV I or II (anti-HTLV I/II) which are not rapid tests.

Table 2 applies to first-line assays for anti-HTLV I/II which are rapid tests.

Table 3 applies to confirmatory assays for anti-HTLV I/II.

Table 4 applies to NAT devices for HTLV I/II.

Table 1. First-line assays: anti-HTLV I/II

| Performance characteristic | Specimen | Specimen numbers, features, use | Acceptance criteria |
| --- | --- | --- | --- |
| |
| Diagnostic sensitivity | Positive specimens | ≥300 HTLV-I
  ≥100 HTLV-II
  including 25 positive ‘same day’ fresh serum specimens (≤ 1 day after specimen taking) | all true positive specimens shall be identified as positive |
| Seroconversion panels | To be defined when available | diagnostic sensitivity during seroconversion shall represent the state of the art, if applicable |
| Diagnostic specificity | Unselected blood donors (including first-time donors) (1) | ≥5 000 | ≥ 99,5% |
| Hospitalised patients | ≥200 | Potential limitations for specificity, if any, shall be identified |
| Cross-reactivity | Potentially cross-reacting specimens | ≥100 in total
  (e.g. RF+, from related virus infections, from pregnant women) |

Performance characteristic

Specimen

Specimen numbers, features, use

Acceptance criteria

Diagnostic sensitivity

Positive specimens

≥300 HTLV-I

≥100 HTLV-II

including 25 positive ‘same day’ fresh serum specimens (≤ 1 day after specimen taking)

all true positive specimens shall be identified as positive

Seroconversion panels

To be defined when available

diagnostic sensitivity during seroconversion shall represent the state of the art, if applicable

Diagnostic specificity

Unselected blood donors (including first-time donors) (1)

≥5 000

≥ 99,5%

Hospitalised patients

≥200

Potential limitations for specificity, if any, shall be identified

Cross-reactivity

Potentially cross-reacting specimens

≥100 in total

(e.g. RF+, from related virus infections, from pregnant women)

Table 2. Rapid tests: anti-HTLV I/II

| Performance characteristic | Specimen | Specimen numbers, features, use | Acceptance criteria |
| --- | --- | --- | --- |
| Diagnostic sensitivity | Positive specimens | ≥300 HTLV-I
  ≥100 HTLV-II | all true positive specimens shall be identified as positive |
| Seroconversion panels | To be defined when available | diagnostic sensitivity during seroconversion shall represent the state of the art, if applicable |
| Diagnostic specificity | Unselected blood donors (including first-time donors) | ≥1 000 | ≥ 99% |
| Hospitalised patients | ≥200 | Potential limitations for specificity, if any, shall be identified |
| Cross-reactivity | Potentially cross-reacting specimens | ≥200 specimens from pregnant women
  ≥100 other potentially cross-reacting specimens in total (e.g. RF+, from related infections) |

Performance characteristic

Specimen

Specimen numbers, features, use

Acceptance criteria

Diagnostic sensitivity

Positive specimens

≥300 HTLV-I

≥100 HTLV-II

all true positive specimens shall be identified as positive

Seroconversion panels

To be defined when available

diagnostic sensitivity during seroconversion shall represent the state of the art, if applicable

Diagnostic specificity

Unselected blood donors (including first-time donors)

≥1 000

≥ 99%

Hospitalised patients

≥200

Potential limitations for specificity, if any, shall be identified

Cross-reactivity

Potentially cross-reacting specimens

≥200 specimens from pregnant women

≥100 other potentially cross-reacting specimens in total (e.g. RF+, from related infections)

Table 3. Confirmatory assays: anti-HTLV I/II

| Performance characteristic | Specimen | Specimen numbers, features, use | Acceptance criteria |
| --- | --- | --- | --- |
| Diagnostic sensitivity | Positive specimens | ≥200 HTLV I
  ≥100 HTLV II | Identification as “confirmed positive” or “indeterminate”, not as “negative” |
| Seroconversion panels | To be defined when available | diagnostic sensitivity during seroconversion shall represent the state of the art, if applicable |
| Diagnostic specificity | Blood donors | ≥200 | No false positive results |
| Hospitalised patients | ≥200 |
| Cross-reactivity | Potentially cross-reacting specimens | ≥50 in total (including specimens from pregnant women, specimens with indeterminate results in other confirmatory assays) |

Performance characteristic

Specimen

Specimen numbers, features, use

Acceptance criteria

Diagnostic sensitivity

Positive specimens

≥200 HTLV I

≥100 HTLV II

Identification as “confirmed positive” or “indeterminate”, not as “negative”

Seroconversion panels

To be defined when available

diagnostic sensitivity during seroconversion shall represent the state of the art, if applicable

Diagnostic specificity

Blood donors

≥200

No false positive results

Hospitalised patients

≥200

Cross-reactivity

Potentially cross-reacting specimens

≥50 in total (including specimens from pregnant women, specimens with indeterminate results in other confirmatory assays)

Table 4. NAT devices for HTLV I/II

| 1. | For target sequence amplification devices, a functionality control for each specimen (internal control) shall reflect the state of the art. This control shall as far as possible be used throughout the whole process, i.e. extraction, amplification/hybridisation, detection. |
| --- | --- |

1.

| 2. | Genotype and/or subtype detection shall be demonstrated by appropriate primer or probe design validation and shall also be validated by testing characterised genotyped specimens. |
| --- | --- |

2.

| 3. | Potential cross-reactivity of non-target nucleic acid sequences shall be analysed by appropriate primer or probe design validation and shall also be validated by testing selected specimens. |
| --- | --- |

3.

| 4. | Results of quantitative NAT devices shall be traceable to international standards or calibrated reference materials, if available, and be expressed in international units utilised in the specific field of application.








  Performance characteristic | Specimen | Specimen numbers, features, use | Acceptance criteria |
| --- | --- | --- | --- | --- |
| Analytical sensitivity | International reference preparations | NAT sensitivity and NAT LOD shall be validated by dilution series of reference materials, testing of replicates (minimum 24) at different analyte concentrations, including those with transition from positive to negative results with the respective NAT device.
  LOD shall be expressed as 95% positive cut-off value (IU/ml) after statistical analysis (e.g. Probit). (2)

  quantitative NAT: definition of lower, upper quantification limit, precision, accuracy,
  ‘linear’ measuring range, ‘dynamic range’.
  Reproducibility at different concentration levels | According to the state of the art |
| HTLV I and HTLV II genotype sensitivity | all relevant genotypes, preferably from international reference materials
  potential substitutes for rare HTLV genotypes (to be quantified by appropriate methods): cell culture supernatants; in vitro transcripts; plasmids | Qualitative NAT: at least 10 specimens/genotype or subtype
  Quantitative NAT: dilution series for demonstration of quantification efficiencies | According to the state of the art |
| Diagnostic specificity | Blood donor specimens | Qualitative NAT: ≥500
  Quantitative NAT: ≥100 | According to the state of the art |
| Cross-reactivity | Potentially cross-reacting specimens | ≥10 human retrovirus positive specimens (e.g. HIV-1, HIV-2) | According to the state of the art |
| Carry-over | High HTLV RNA positive;
  HTLV RNA negative | At least five runs with alternating high-positive and negative specimens shall be performed during robustness studies. The virus titres of the high positive specimens shall be representative of high virus titres occurring naturally. | According to the state of the art |
| Detection in relation to antibody status | HTLV-RNA positives: anti-HTLV negative, anti-HTLV positive | Pre-seroconversion (anti-HTLV negative) and post-seroconversion (anti-HTLV positive) specimens | According to the state of the art |
| Whole system failure rate | HTLV RNA low-positive | ≥100 HTLV RNA low-positive specimens shall be tested. These specimens shall contain a virus concentration equivalent to three times the 95 % positive cut-off virus concentration. | ≥99% positive |

4.

Performance characteristic

Specimen

Specimen numbers, features, use

Acceptance criteria

Analytical sensitivity

International reference preparations

NAT sensitivity and NAT LOD shall be validated by dilution series of reference materials, testing of replicates (minimum 24) at different analyte concentrations, including those with transition from positive to negative results with the respective NAT device.

LOD shall be expressed as 95% positive cut-off value (IU/ml) after statistical analysis (e.g. Probit). (2)

quantitative NAT: definition of lower, upper quantification limit, precision, accuracy,

‘linear’ measuring range, ‘dynamic range’.

Reproducibility at different concentration levels

According to the state of the art

HTLV I and HTLV II genotype sensitivity

all relevant genotypes, preferably from international reference materials

potential substitutes for rare HTLV genotypes (to be quantified by appropriate methods): cell culture supernatants; in vitro transcripts; plasmids

Qualitative NAT: at least 10 specimens/genotype or subtype

Quantitative NAT: dilution series for demonstration of quantification efficiencies

According to the state of the art

Diagnostic specificity

Blood donor specimens

Qualitative NAT: ≥500

Quantitative NAT: ≥100

According to the state of the art

Cross-reactivity

Potentially cross-reacting specimens

≥10 human retrovirus positive specimens (e.g. HIV-1, HIV-2)

According to the state of the art

Carry-over

High HTLV RNA positive;

HTLV RNA negative

At least five runs with alternating high-positive and negative specimens shall be performed during robustness studies. The virus titres of the high positive specimens shall be representative of high virus titres occurring naturally.

According to the state of the art

Detection in relation to antibody status

HTLV-RNA positives: anti-HTLV negative, anti-HTLV positive

Pre-seroconversion (anti-HTLV negative) and post-seroconversion (anti-HTLV positive) specimens

According to the state of the art

Whole system failure rate

HTLV RNA low-positive

≥100 HTLV RNA low-positive specimens shall be tested. These specimens shall contain a virus concentration equivalent to three times the 95 % positive cut-off virus concentration.

≥99% positive

---

(1)  Blood donor populations shall be investigated from at least two blood donation centres and consist of consecutive blood donations, which have not been selected to exclude first-time donors.

(2)  Reference: European Pharmacopeia 9.0, 2.6.21 Nucleic acid amplification techniques, Validation.

---

ANNEX V

COMMON SPECIFICATIONS FOR DEVICES INTENDED FOR DETECTION OR QUANTIFICATION OF MARKERS OF HEPATITIS C VIRUS (HCV) INFECTION

Scope

This Annex applies to devices intended for detection or quantification of markers of hepatitis C virus (HCV) infection.

Table 1 applies to first-line assays for anti-HCV antibodies (anti-HCV) and combined antigen/antibody tests for HCV (HCV Ag/Ab) which are not rapid tests.

Table 2 applies to first-line assays for anti-HCV and HCV Ag/Ab which are rapid tests.

Table 3 applies to confirmatory and supplemental assays for anti-HCV.

Table 4 applies to HCV antigen tests and HCV Ag/Ab.

Table 5 applies to qualitative and quantitative NAT devices for HCV RNA.

Table 6 applies to HCV self-tests.

Table 1. First-line assays: anti-HCV, HCV Ag/Ab (requirements for antibody detection)

| Performance characteristic | Specimen | Specimen numbers, features, use | Acceptance criteria |
| --- | --- | --- | --- |
| Diagnostic sensitivity | Positive specimens | ≥400
  Including specimens from different stages of infection and reflecting different antibody patterns
  HCV genotype 1-4: > 20 specimens per genotype (including non-a subtypes of genotype 4); HCV genotypes 5 and 6: > 5 specimens each;
  including 25 positive ‘same day’ fresh serum specimens (≤ 1 day after specimen taking) | all true positive specimens shall be identified as positive |
| | Seroconversion panels | ≥30 panels
  HCV seroconversion panels for the evaluation of HCV antigen and antibody combined tests (HCV Ag/Ab) shall start with one or more negative bleeds and comprise panel members from early HCV infection (HCV core antigen and/or HCV RNA positive but anti-HCV negative). | diagnostic sensitivity during seroconversion shall represent the state of the art
  HCV Ag/Ab tests shall demonstrate enhanced sensitivity in early HCV infection when compared to HCV antibody only tests. |
| Diagnostic specificity | Unselected blood donors (including first-time donors) (1) | ≥5 000 | ≥99,5% |
| Hospitalised patients | ≥200 | Potential limitations for specificity, if any, shall be identified |
| Cross-reactivity | Potentially cross-reacting specimens | ≥100 in total
  (e.g. RF+, from related virus infections, from pregnant women) |

Performance characteristic

Specimen

Specimen numbers, features, use

Acceptance criteria

Diagnostic sensitivity

Positive specimens

≥400

Including specimens from different stages of infection and reflecting different antibody patterns

HCV genotype 1-4: > 20 specimens per genotype (including non-a subtypes of genotype 4); HCV genotypes 5 and 6: > 5 specimens each;

including 25 positive ‘same day’ fresh serum specimens (≤ 1 day after specimen taking)

all true positive specimens shall be identified as positive

Seroconversion panels

≥30 panels

HCV seroconversion panels for the evaluation of HCV antigen and antibody combined tests (HCV Ag/Ab) shall start with one or more negative bleeds and comprise panel members from early HCV infection (HCV core antigen and/or HCV RNA positive but anti-HCV negative).

diagnostic sensitivity during seroconversion shall represent the state of the art

HCV Ag/Ab tests shall demonstrate enhanced sensitivity in early HCV infection when compared to HCV antibody only tests.

Diagnostic specificity

Unselected blood donors (including first-time donors) (1)

≥5 000

≥99,5%

Hospitalised patients

≥200

Potential limitations for specificity, if any, shall be identified

Cross-reactivity

Potentially cross-reacting specimens

≥100 in total

(e.g. RF+, from related virus infections, from pregnant women)

Table 2. Rapid tests: anti-HCV, HCV Ag/Ab (requirements for antibody detection)

| Performance characteristic | Specimen | Specimen numbers, features, use | Acceptance criteria |
| --- | --- | --- | --- |
| Diagnostic sensitivity | Positive specimens | ≥400
  including specimens from different stages of infection and reflecting different antibody patterns.
  HCV genotype 1-4: > 20 specimens per genotype (including non-a subtypes of genotype 4); HCV genotypes 5 and 6: > 5 specimens each; | all true positive specimens shall be identified as positive |
| Seroconversion panels | ≥30 panels
  HCV seroconversion panels for the evaluation of HCV antigen and antibody combined tests (HCV Ag/Ab) shall start with one or more negative bleeds and comprise panel members from early HCV infection (HCV core antigen and/or HCV RNA positive but anti-HCV negative). | diagnostic sensitivity during seroconversion shall represent the state of the art
  HCV Ag/Ab tests shall demonstrate enhanced sensitivity in early HCV infection when compared to HCV antibody only tests. |
| Diagnostic specificity | Unselected blood donors (including first-time donors)1 | ≥1 000 | ≥ 99 % |
| Hospitalised patients | ≥200 | Potential limitations for specificity, if any, shall be identified |
| Cross-reactivity | Potentially cross-reacting specimens | ≥200 specimens from pregnant women
  ≥100 other potentially cross-reacting specimens in total (e.g. RF+, from related infections) |

Performance characteristic

Specimen

Specimen numbers, features, use

Acceptance criteria

Diagnostic sensitivity

Positive specimens

≥400

including specimens from different stages of infection and reflecting different antibody patterns.

HCV genotype 1-4: > 20 specimens per genotype (including non-a subtypes of genotype 4); HCV genotypes 5 and 6: > 5 specimens each;

all true positive specimens shall be identified as positive

Seroconversion panels

≥30 panels

HCV seroconversion panels for the evaluation of HCV antigen and antibody combined tests (HCV Ag/Ab) shall start with one or more negative bleeds and comprise panel members from early HCV infection (HCV core antigen and/or HCV RNA positive but anti-HCV negative).

diagnostic sensitivity during seroconversion shall represent the state of the art

HCV Ag/Ab tests shall demonstrate enhanced sensitivity in early HCV infection when compared to HCV antibody only tests.

Diagnostic specificity

Unselected blood donors (including first-time donors)1

≥1 000

≥ 99 %

Hospitalised patients

≥200

Potential limitations for specificity, if any, shall be identified

Cross-reactivity

Potentially cross-reacting specimens

≥200 specimens from pregnant women

≥100 other potentially cross-reacting specimens in total (e.g. RF+, from related infections)

Table 3. Confirmatory and supplemental assays: anti-HCV

| Performance characteristic | Specimen | Specimen numbers, features, use | Acceptance criteria |
| --- | --- | --- | --- |
| Diagnostic sensitivity | Positive specimens | ≥300
  Including specimens from different stages of infection and reflecting different antibody patterns.
  HCV genotypes 1 – 4: > 20 specimens (including non-a subtypes of genotype 4; HCV genotype 5: > 5 specimens; HCV genotype 6: as far as available | identification as “confirmed positive” or “indeterminate”, not as “negative” |
| Seroconversion panels | ≥15 seroconversion panels/low titre panels | diagnostic sensitivity during seroconversion shall represent the state of the art |
| Diagnostic specificity | Blood donors | ≥200 | No false positive results/ no neutralisation |
| Hospitalised patients | ≥200 |
| Cross-reactivity | Potentially cross-reacting specimens | ≥50 in total (including specimens from pregnant women, specimens with indeterminate results in other confirmatory assays) |

Performance characteristic

Specimen

Specimen numbers, features, use

Acceptance criteria

Diagnostic sensitivity

Positive specimens

≥300

Including specimens from different stages of infection and reflecting different antibody patterns.

HCV genotypes 1 – 4: > 20 specimens (including non-a subtypes of genotype 4; HCV genotype 5: > 5 specimens; HCV genotype 6: as far as available

identification as “confirmed positive” or “indeterminate”, not as “negative”

Seroconversion panels

≥15 seroconversion panels/low titre panels

diagnostic sensitivity during seroconversion shall represent the state of the art

Diagnostic specificity

Blood donors

≥200

No false positive results/ no neutralisation

Hospitalised patients

≥200

Cross-reactivity

Potentially cross-reacting specimens

≥50 in total (including specimens from pregnant women, specimens with indeterminate results in other confirmatory assays)

Table 4. Antigen tests: HCV antigen, HCV Ag/Ab (requirements for antigen detection)

| Performance characteristic | Specimen | Specimen numbers, features, use | Acceptance criteria |
| --- | --- | --- | --- |
| Diagnostic sensitivity | Positive specimens | ≥25 HCV core antigen and/or HCV RNA positive but anti-HCV negative specimens, comprising HCV genotypes 1-6 (if a genotype is not available, a justification shall be made) | all true positive specimens shall be identified as positive |
| Seroconversion panels | ≥20 seroconversion panels/low titre panels
  HCV seroconversion panels for the evaluation of HCV antigen and antibody combined tests shall start with one or more negative bleeds and comprise panel members from early HCV infection (HCV core antigen and/or HCV RNA positive but anti-HCV negative). | diagnostic sensitivity during seroconversion shall represent the state of the art
  HCV antigen and antibody combined tests shall demonstrate enhanced sensitivity in early HCV infection when compared to HCV antibody only tests. |
| Analytical sensitivity | WHO International Standard HCV core (PEI 129096/12) | Dilution series | |
| Diagnostic specificity | Blood donors | ≥200 | ≥ 99,5 % after neutralisation or, if no neutralisation test available, after resolution of the specimen status |
| Hospitalised patients | ≥200 | Potential limitations for specificity, if any, shall be identified |
| Cross-reactivity | Potentially cross-reacting specimens | ≥50 |

Performance characteristic

Specimen

Specimen numbers, features, use

Acceptance criteria

Diagnostic sensitivity

Positive specimens

≥25 HCV core antigen and/or HCV RNA positive but anti-HCV negative specimens, comprising HCV genotypes 1-6 (if a genotype is not available, a justification shall be made)

all true positive specimens shall be identified as positive

Seroconversion panels

≥20 seroconversion panels/low titre panels

HCV seroconversion panels for the evaluation of HCV antigen and antibody combined tests shall start with one or more negative bleeds and comprise panel members from early HCV infection (HCV core antigen and/or HCV RNA positive but anti-HCV negative).

diagnostic sensitivity during seroconversion shall represent the state of the art

HCV antigen and antibody combined tests shall demonstrate enhanced sensitivity in early HCV infection when compared to HCV antibody only tests.

Analytical sensitivity

WHO International Standard HCV core (PEI 129096/12)

Dilution series

Diagnostic specificity

Blood donors

≥200

≥ 99,5 % after neutralisation or, if no neutralisation test available, after resolution of the specimen status

Hospitalised patients

≥200

Potential limitations for specificity, if any, shall be identified

Cross-reactivity

Potentially cross-reacting specimens

≥50

Table 5. Qualitative and quantitative NAT devices for HCV RNA

| 1. | For target sequence amplification devices, a functionality control for each specimen (internal control) shall reflect the state of the art. This control shall as far as possible be used throughout the whole process, i.e. extraction, amplification/hybridisation, detection. |
| --- | --- |

1.

| 2. | Genotype and/or subtype detection shall be demonstrated by appropriate primer or probe design validation and shall also be validated by testing characterised genotyped specimens. |
| --- | --- |

2.

| 3. | Potential cross-reactivity of non-target nucleic acid sequences shall be analysed by appropriate primer or probe design validation and shall also be validated by testing selected specimens. |
| --- | --- |

3.

| 4. | Results of quantitative NAT devices shall be traceable to international standards or calibrated reference materials, if available, and be expressed in international units utilised in the specific field of application.








  Performance characteristic | Specimen | Specimen numbers, features, use | Acceptance criteria |
| --- | --- | --- | --- | --- |
| Analytical sensitivity | WHO International Standard HCV RNA(or calibrated reference materials) | NAT sensitivity and NAT LOD shall be validated by dilution series of reference materials, testing of replicates (minimum 24) at different analyte concentrations, including those with transition from positive to negative results with the respective NAT device.
  LOD shall be expressed as 95% positive cut-off value (IU/ml) after statistical analysis (e.g. Probit). (2)

  quantitative NAT: definition of lower, upper quantification limit, precision, accuracy,
  ‘linear’ measuring range, ‘dynamic range’.
  Reproducibility at different concentration levels | According to the state of the art |
| HCV genotype sensitivity | all relevant genotypes/subtypes, preferably from international reference materials
  potential substitutes for rare HCV genotypes (to be quantified by appropriate methods): in vitro transcripts; plasmids | Qualitative NAT: ≥10 specimens/genotype or subtype
  Quantitative NAT: dilution series for demonstration of quantification efficiencies | According to the state of the art |
| Diagnostic sensitivity | Positive specimens reflecting the routine conditions of users (e.g. no pre-selection of specimens) | Quantitative NAT: ≥100
  Comparative results with another NAT system shall be generated in parallel | According to the state of the art |
| Seroconversion panels | Qualitative NAT: ≥10 panels
  Comparative results with another NAT system shall be generated in parallel | According to the state of the art |
| Diagnostic specificity | Blood donor specimens | Qualitative NAT: ≥500
  Quantitative NAT: ≥100 | According to the state of the art |
| Cross-reactivity | Potentially cross-reacting specimens | >10 human flavivirus (e.g. HGV, YFV) positive specimens | According to the state of the art |
| Carry-over | High HCV RNA positive;
  HCV RNA negative | At least five runs with alternating high-positive and negative specimens shall be performed during robustness studies. The virus titres of the high positive specimens shall be representative of high virus titres occurring naturally. | According to the state of the art |
| Detection in relation to antibody status | HCV RNA positives: anti-HCV negative, anti-HCV positive | Pre-seroconversion (anti-HCV negative) and post-seroconversion (anti-HCV positive) specimens | According to the state of the art |
| Whole system failure rate | HCV RNA low-positive | ≥100 HCV RNA low-positive specimens shall be tested. These specimens shall contain a virus concentration equivalent to three times the 95 % positive cut-off virus concentration. | ≥99% positive |

4.

Performance characteristic

Specimen

Specimen numbers, features, use

Acceptance criteria

Analytical sensitivity

WHO International Standard HCV RNA(or calibrated reference materials)

NAT sensitivity and NAT LOD shall be validated by dilution series of reference materials, testing of replicates (minimum 24) at different analyte concentrations, including those with transition from positive to negative results with the respective NAT device.

LOD shall be expressed as 95% positive cut-off value (IU/ml) after statistical analysis (e.g. Probit). (2)

quantitative NAT: definition of lower, upper quantification limit, precision, accuracy,

‘linear’ measuring range, ‘dynamic range’.

Reproducibility at different concentration levels

According to the state of the art

HCV genotype sensitivity

all relevant genotypes/subtypes, preferably from international reference materials

potential substitutes for rare HCV genotypes (to be quantified by appropriate methods): in vitro transcripts; plasmids

Qualitative NAT: ≥10 specimens/genotype or subtype

Quantitative NAT: dilution series for demonstration of quantification efficiencies

According to the state of the art

Diagnostic sensitivity

Positive specimens reflecting the routine conditions of users (e.g. no pre-selection of specimens)

Quantitative NAT: ≥100

Comparative results with another NAT system shall be generated in parallel

According to the state of the art

Seroconversion panels

Qualitative NAT: ≥10 panels

Comparative results with another NAT system shall be generated in parallel

According to the state of the art

Diagnostic specificity

Blood donor specimens

Qualitative NAT: ≥500

Quantitative NAT: ≥100

According to the state of the art

Cross-reactivity

Potentially cross-reacting specimens

>10 human flavivirus (e.g. HGV, YFV) positive specimens

According to the state of the art

Carry-over

High HCV RNA positive;

HCV RNA negative

At least five runs with alternating high-positive and negative specimens shall be performed during robustness studies. The virus titres of the high positive specimens shall be representative of high virus titres occurring naturally.

According to the state of the art

Detection in relation to antibody status

HCV RNA positives: anti-HCV negative, anti-HCV positive

Pre-seroconversion (anti-HCV negative) and post-seroconversion (anti-HCV positive) specimens

According to the state of the art

Whole system failure rate

HCV RNA low-positive

≥100 HCV RNA low-positive specimens shall be tested. These specimens shall contain a virus concentration equivalent to three times the 95 % positive cut-off virus concentration.

≥99% positive

Table 6. Additional requirements for HCV self-tests

| Performance characteristic | Specimens (3) | Number of lay persons |
| --- | --- | --- |
| Result interpretation (4) | Interpretation of results (5) by lay persons reflecting the following range of reactivity levels:






  — | non-reactive |

Performance characteristic

Specimens (3)

Number of lay persons

Result interpretation (4)

Interpretation of results (5) by lay persons reflecting the following range of reactivity levels:

—

non-reactive

| — | reactive |
| --- | --- |

—

reactive

| — | weak reactive (6) |
| --- | --- |

—

weak reactive (6)

| — | invalid |
| --- | --- |

—

invalid

≥ 100

Diagnostic sensitivity

lay persons that are known positive

≥ 200

Diagnostic specificity

lay persons that do not know their status

≥ 400

lay persons that are at high risk of acquiring the infection

≥ 200

---

(1)  Blood donor populations shall be investigated from at least two blood donation centres and consist of consecutive blood donations, which have not been selected to exclude first-time donors.

(2)  Reference: European Pharmacopeia 9.0, 2.6.21 Nucleic acid amplification techniques, Validation.

(3)  For each body fluid claimed for use with the device, e.g. whole blood, urine, saliva, etc., the sensitivity and specificity of the device for self-testing in the hands of lay persons shall be defined against the confirmed patient infectious status.

(4)  The result interpretation study shall include reading and interpretation of the test results by at least 100 lay persons with each lay person subjected to reading results covering the specified range of result reactivity levels. The manufacturer shall determine the concordance between lay person reading and professional user reading.

(5)  Tests shall be performed prior to the result interpretation study using whenever possible the specimen type intended by the manufacturer. The tests may be performed on contrived specimens based on the natural matrix of the respective specimen type.

(6)  A higher proportion of the specimens shall be in the weak-positive range close to the cut-off or LOD of the test.

---

ANNEX VI

COMMON SPECIFICATIONS FOR DEVICES INTENDED FOR DETECTION OR QUANTIFICATION OF MARKERS OF HEPATITIS B VIRUS (HBV) INFECTION

Scope

This Annex applies to devices intended for detection or quantification of markers of hepatitis B virus (HBV) infection.

Table 1 applies to first-line assays for hepatitis B surface antigen (HBsAg), and for antibodies against hepatitis B core antigen (anti-HBc) which are not rapid tests.

Table 2 applies to first-line assays for HBsAg and anti-HBc which are rapid tests.

Table 3 applies to confirmatory assays for HBsAg.

Table 4 applies to assays for the hepatitis B virus markers: hepatitis B surface antibodies (anti-HBs), IgM antibody against the hepatitis B core antigen (anti-HBc IgM), antibodies against the hepatitis Be antigen (anti-HBe) and hepatitis Be antigen (HBeAg).

Table 5 applies to qualitative and quantitative NAT devices for HBV deoxyribonucleic acid (DNA).

Table 6 applies to HBV self-tests.

Table 1. First-line assays: HBsAg, anti-HBc

| Performance characteristic | Specimen | Specimen numbers, features, use | Acceptance criteria |
| --- | --- | --- | --- |
| Diagnostic sensitivity | Positive specimens | ≥400
  anti-HBc: including evaluation of different HBV markers
  HBsAg: including different HBV genotypes / subtypes / mutants
  anti-HBc or HBsAg: including 25 positive ‘same day’ fresh serum (≤ 1 day after specimen taking) | Overall performance shall be at least equivalent to the comparator device |
| Seroconversion panels | HBsAg assays:
  ≥30 panels
  anti-HBc assays:
  to be defined when available | diagnostic sensitivity during seroconversion shall represent the state of the art (for anti-HBc this shall be the case if applicable) |
| Analytical sensitivity | WHO Third International Standard HBsAg (subtypes ayw1/adw2, HBV genotype B4, NIBSC code: 12/226) | | For HBsAg assays: <0,130 IU/ml |
| Diagnostic specificity | Unselected blood donors (including first-time donors) (1) | ≥5 000 | ≥99,5% |
| Hospitalised patients | ≥200 | Potential limitations for specificity, if any, shall be identified |
| Cross-reactivity | Potentially cross-reacting specimens | ≥100 in total
  (e.g. RF+, from related virus infections, from pregnant women,) |

Performance characteristic

Specimen

Specimen numbers, features, use

Acceptance criteria

Diagnostic sensitivity

Positive specimens

≥400

anti-HBc: including evaluation of different HBV markers

HBsAg: including different HBV genotypes / subtypes / mutants

anti-HBc or HBsAg: including 25 positive ‘same day’ fresh serum (≤ 1 day after specimen taking)

Overall performance shall be at least equivalent to the comparator device

Seroconversion panels

HBsAg assays:

≥30 panels

anti-HBc assays:

to be defined when available

diagnostic sensitivity during seroconversion shall represent the state of the art (for anti-HBc this shall be the case if applicable)

Analytical sensitivity

WHO Third International Standard HBsAg (subtypes ayw1/adw2, HBV genotype B4, NIBSC code: 12/226)

For HBsAg assays: <0,130 IU/ml

Diagnostic specificity

Unselected blood donors (including first-time donors) (1)

≥5 000

≥99,5%

Hospitalised patients

≥200

Potential limitations for specificity, if any, shall be identified

Cross-reactivity

Potentially cross-reacting specimens

≥100 in total

(e.g. RF+, from related virus infections, from pregnant women,)

Table 2. Rapid tests: HBsAg, anti-HBc

| Performance characteristic | Specimen | Specimen numbers, features, use | Acceptance criteria |
| --- | --- | --- | --- |
| Diagnostic sensitivity | Positive specimens | ≥400
  including evaluation of different HBV markers
  including different HBV genotypes / subtypes / mutants | Overall performance shall be at least equivalent to that of the comparator device |
| Seroconversion panels | HBsAg assays:
  ≥30 panels
  anti-HBc assays:
  to be defined when available | Diagnostic sensitivity during seroconversion shall represent the state of the art (for anti-HBc this shall be the case if applicable) |
| Diagnostic specificity | Unselected blood donors (including first-time donors) | ≥1 000 | HBsAg assays: ≥ 99 %
  anti-HBc assays: ≥ 99 % |
| Hospitalised patients | ≥200 | Potential limitations for specificity, if any, shall be identified |
| Cross-reactivity | Potentially cross-reacting specimens | ≥200 specimens from pregnant women
  ≥100 other potentially cross-reacting specimens in total (e.g. RF+, from related infections) |

Performance characteristic

Specimen

Specimen numbers, features, use

Acceptance criteria

Diagnostic sensitivity

Positive specimens

≥400

including evaluation of different HBV markers

including different HBV genotypes / subtypes / mutants

Overall performance shall be at least equivalent to that of the comparator device

Seroconversion panels

HBsAg assays:

≥30 panels

anti-HBc assays:

to be defined when available

Diagnostic sensitivity during seroconversion shall represent the state of the art (for anti-HBc this shall be the case if applicable)

Diagnostic specificity

Unselected blood donors (including first-time donors)

≥1 000

HBsAg assays: ≥ 99 %

anti-HBc assays: ≥ 99 %

Hospitalised patients

≥200

Potential limitations for specificity, if any, shall be identified

Cross-reactivity

Potentially cross-reacting specimens

≥200 specimens from pregnant women

≥100 other potentially cross-reacting specimens in total (e.g. RF+, from related infections)

Table 3. Confirmatory assays: HBsAg

| Performance characteristic | Specimen | Specimen numbers, features, use | Acceptance criteria |
| --- | --- | --- | --- |
| Diagnostic sensitivity | Positive specimens | ≥300
  Including specimens from different stages of infection
  Including 20 ‘high positive’ specimens (>26 IU/ml); 20 specimens in the cut-off range | Correct identification as positive (or indeterminate), not negative |
| Seroconversion panels | ≥15 seroconversion panels/low titre panels | Diagnostic sensitivity during seroconversion shall represent the state of the art |
| Analytical sensitivity | WHO Third International Standard for HBsAg, subtypes ayw1/adw2, HBV genotype B4, NIBSC code: 12/226 | | |
| Diagnostic specificity | Negative specimens | ≥10 false positives as available from the performance evaluation of the first-line assay | No false positive results/ no neutralisation |
| Cross-reactivity | Potentially cross-reacting specimens | ≥50 |

Performance characteristic

Specimen

Specimen numbers, features, use

Acceptance criteria

Diagnostic sensitivity

Positive specimens

≥300

Including specimens from different stages of infection

Including 20 ‘high positive’ specimens (>26 IU/ml); 20 specimens in the cut-off range

Correct identification as positive (or indeterminate), not negative

Seroconversion panels

≥15 seroconversion panels/low titre panels

Diagnostic sensitivity during seroconversion shall represent the state of the art

Analytical sensitivity

WHO Third International Standard for HBsAg, subtypes ayw1/adw2, HBV genotype B4, NIBSC code: 12/226

Diagnostic specificity

Negative specimens

≥10 false positives as available from the performance evaluation of the first-line assay

No false positive results/ no neutralisation

Cross-reactivity

Potentially cross-reacting specimens

≥50

Table 4. Assays for the HBV markers: anti-HBs, anti-HBc IgM, anti-HBe, HBeAg

| Performance characteristic | | anti-HBs | anti-HBc IgM | anti-HBe | HBeAg | Acceptance criteria |
| --- | --- | --- | --- | --- | --- | --- |
| Diagnostic sensitivity | Positive specimens | ≥100 vaccinees
  ≥100 naturally infected persons | ≥200
  Including specimens from different stages of infection (acute/chronic, etc.) | ≥200
  Including specimens from different stages of infection (acute/chronic, etc.) | ≥200
  Including specimens from different stages of infection (acute/chronic, etc.) | ≥ 98 %
  (for anti-HBc IgM: applicable only on specimens from acute infection stage) |
| Seroconversion panels | 10 anti-HBs seroconversion panels or follow-up series | When available | When available | When available | Diagnostic sensitivity during seroconversion shall represent the state of the art (for anti-HBc IgM, anti-HBe, HBeAg this shall be the case if applicable) |
| Analytical sensitivity | Standards | WHO Second International Standard for anti-hepatitis B surface antigen (anti-HBs) immunoglobulin, human NIBSC code: 07/164 | | WHO First International Standard anti-hepatitis B virus e antigen (anti-HBe), PEI code 129095/12 | WHO First International Standard for Hepatitis B Virus e Antigen (HBeAg) PEI code 129097/12 HBe | anti-HBs: < 10 mIU/ml |
| Diagnostic specificity | Negative specimens | ≥500
  Including clinical specimens
  ≥50 potentially interfering specimens | ≥200 blood donations
  ≥200 clinical specimens
  ≥50 potentially interfering specimens | ≥200 blood donations
  ≥200 clinical specimens
  ≥50 potentially interfering specimens | ≥200 blood donations
  ≥200 clinical specimens
  ≥50 potentially interfering specimens | ≥ 98 % |

Performance characteristic

anti-HBs

anti-HBc IgM

anti-HBe

HBeAg

Acceptance criteria

Diagnostic sensitivity

Positive specimens

≥100 vaccinees

≥100 naturally infected persons

≥200

Including specimens from different stages of infection (acute/chronic, etc.)

≥200

Including specimens from different stages of infection (acute/chronic, etc.)

≥200

Including specimens from different stages of infection (acute/chronic, etc.)

≥ 98 %

(for anti-HBc IgM: applicable only on specimens from acute infection stage)

Seroconversion panels

10 anti-HBs seroconversion panels or follow-up series

When available

When available

When available

Diagnostic sensitivity during seroconversion shall represent the state of the art (for anti-HBc IgM, anti-HBe, HBeAg this shall be the case if applicable)

Analytical sensitivity

Standards

WHO Second International Standard for anti-hepatitis B surface antigen (anti-HBs) immunoglobulin, human NIBSC code: 07/164

WHO First International Standard anti-hepatitis B virus e antigen (anti-HBe), PEI code 129095/12

WHO First International Standard for Hepatitis B Virus e Antigen (HBeAg) PEI code 129097/12 HBe

anti-HBs: < 10 mIU/ml

Diagnostic specificity

Negative specimens

≥500

Including clinical specimens

≥50 potentially interfering specimens

≥200 blood donations

≥200 clinical specimens

≥50 potentially interfering specimens

≥200 blood donations

≥200 clinical specimens

≥50 potentially interfering specimens

≥200 blood donations

≥200 clinical specimens

≥50 potentially interfering specimens

≥ 98 %

Table 5. Qualitative and quantitative NAT devices for HBV DNA

| 1. | For target sequence amplification devices, a functionality control for each specimen (internal control) shall reflect the state of the art. This control shall as far as possible be used throughout the whole process, i.e. extraction, amplification/hybridisation, detection. |
| --- | --- |

1.

| 2. | Genotype and/or subtype detection shall be demonstrated by appropriate primer or probe design validation and shall also be validated by testing characterised genotyped specimens. |
| --- | --- |

2.

| 3. | Potential cross-reactivity of non-target nucleic acid sequences shall be analysed by appropriate primer or probe design validation and shall also be validated by testing selected specimens. |
| --- | --- |

3.

| 4. | Results of quantitative NAT devices shall be traceable to international standards or calibrated reference materials, if available, and be expressed in international units utilised in the specific field of application.








  Performance characteristic | Specimen | Specimen numbers, features, use | Acceptance criteria |
| --- | --- | --- | --- | --- |
| Analytical sensitivity | WHO International Standard HBV DNA (or calibrated reference materials) | NAT sensitivity and NAT LOD shall be validated by dilution series of reference materials, testing of replicates (minimum 24) at different analyte concentrations, including those with transition from positive to negative results with the respective NAT device.
  LOD shall be expressed as 95% positive cut-off value (IU/ml) after statistical analysis (e.g. Probit). (2)

  quantitative NAT: definition of lower, upper quantification limit, precision, accuracy,
  ‘linear’ measuring range, ‘dynamic range’. Reproducibility at different concentration levels | According to the state of the art |
| HBV genotype sensitivity | WHO International Reference Panel HBV DNA (HBV genotypes)
  all relevant genotypes/subtypes, preferably from international reference materials
  potential substitutes for rare HBV genotypes (to be quantified by appropriate methods): plasmids; synthetic DNA | Qualitative NAT: at least 10 specimens/genotype or subtype
  Quantitative NAT: dilution series for demonstration of quantification efficiencies | According to the state of the art |
| Diagnostic sensitivity | Positive specimens reflecting the routine conditions of users (no pre-selection of specimens) | Quantitative NAT: ≥100
  Comparative results with another NAT system shall be generated in parallel | According to the state of the art |
| Seroconversion panels | Qualitative NAT: ≥10 panels
  Comparative results with another NAT system shall be generated in parallel | According to the state of the art |
| Diagnostic specificity | Blood donor specimens | Qualitative NAT: ≥500
  Quantitative NAT: ≥100 | According to the state of the art |
| Cross-reactivity | Potentially cross-reacting specimens | | According to the state of the art |
| Carry-over | High HBV DNA positive;
  HBV DNA negative | At least five runs with alternating high-positive and negative specimens shall be performed during robustness studies. The virus titres of the high positive specimens shall be representative of high virus titres occurring naturally. | According to the state of the art |
| Detection in relation to antibody status | HBV DNA positives: anti-HBV negative, anti-HBV positive | Pre-seroconversion (anti-HBV negative) and post-seroconversion (anti-HBV positive) specimens | According to the state of the art |
| Whole system failure rate | HBV DNA low-positive | ≥100 HBV DNA low-positive specimens shall be tested. These specimens shall contain a virus concentration equivalent to three times the 95 % positive cut-off virus concentration. | ≥99% positive |

4.

Performance characteristic

Specimen

Specimen numbers, features, use

Acceptance criteria

Analytical sensitivity

WHO International Standard HBV DNA (or calibrated reference materials)

NAT sensitivity and NAT LOD shall be validated by dilution series of reference materials, testing of replicates (minimum 24) at different analyte concentrations, including those with transition from positive to negative results with the respective NAT device.

LOD shall be expressed as 95% positive cut-off value (IU/ml) after statistical analysis (e.g. Probit). (2)

quantitative NAT: definition of lower, upper quantification limit, precision, accuracy,

‘linear’ measuring range, ‘dynamic range’. Reproducibility at different concentration levels

According to the state of the art

HBV genotype sensitivity

WHO International Reference Panel HBV DNA (HBV genotypes)

all relevant genotypes/subtypes, preferably from international reference materials

potential substitutes for rare HBV genotypes (to be quantified by appropriate methods): plasmids; synthetic DNA

Qualitative NAT: at least 10 specimens/genotype or subtype

Quantitative NAT: dilution series for demonstration of quantification efficiencies

According to the state of the art

Diagnostic sensitivity

Positive specimens reflecting the routine conditions of users (no pre-selection of specimens)

Quantitative NAT: ≥100

Comparative results with another NAT system shall be generated in parallel

According to the state of the art

Seroconversion panels

Qualitative NAT: ≥10 panels

Comparative results with another NAT system shall be generated in parallel

According to the state of the art

Diagnostic specificity

Blood donor specimens

Qualitative NAT: ≥500

Quantitative NAT: ≥100

According to the state of the art

Cross-reactivity

Potentially cross-reacting specimens

According to the state of the art

Carry-over

High HBV DNA positive;

HBV DNA negative

At least five runs with alternating high-positive and negative specimens shall be performed during robustness studies. The virus titres of the high positive specimens shall be representative of high virus titres occurring naturally.

According to the state of the art

Detection in relation to antibody status

HBV DNA positives: anti-HBV negative, anti-HBV positive

Pre-seroconversion (anti-HBV negative) and post-seroconversion (anti-HBV positive) specimens

According to the state of the art

Whole system failure rate

HBV DNA low-positive

≥100 HBV DNA low-positive specimens shall be tested. These specimens shall contain a virus concentration equivalent to three times the 95 % positive cut-off virus concentration.

≥99% positive

Table 6. Additional requirements for HBV self-tests

| Performance characteristic | Specimens (3) | Number of lay persons |
| --- | --- | --- |
| Result interpretation (4) | Interpretation of results (5) by lay persons reflecting the following range of reactivity levels:






  — | non-reactive |

Performance characteristic

Specimens (3)

Number of lay persons

Result interpretation (4)

Interpretation of results (5) by lay persons reflecting the following range of reactivity levels:

—

non-reactive

| — | reactive |
| --- | --- |

—

reactive

| — | weak reactive (6) |
| --- | --- |

—

weak reactive (6)

| — | invalid |
| --- | --- |

—

invalid

≥100

Diagnostic sensitivity

lay persons that are known positive

≥200

Diagnostic specificity

lay persons that do not know their status

≥400

lay persons that are at high risk of acquiring the infection

≥200

---

(1)  Blood donor populations shall be investigated from at least two blood donation centres and consist of consecutive blood donations, which have not been selected to exclude first-time donor.

(2)  Reference: European Pharmacopeia 9.0, 2.6.21 Nucleic acid amplification techniques, Validation.

(3)  For each body fluid claimed for use with the device, e.g. whole blood, urine, saliva, etc., the sensitivity and specificity of the device for self-testing in the hands of lay persons shall be defined against the confirmed patient infectious status.

(4)  The result interpretation study shall include reading and interpretation of the test results by at least 100 lay persons with each lay person subjected to reading results covering the specified range of result reactivity levels. The manufacturer shall determine the concordance between lay person reading and professional user reading.

(5)  Tests shall be performed prior to the result interpretation study using whenever possible the specimen type intended by the manufacturer. The tests may be performed on contrived specimens based on the natural matrix of the respective specimen type.

(6)  A higher proportion of the specimens shall be in the low-positive range close to the cut-off or LOD of the test.

---

ANNEX VII

COMMON SPECIFICATIONS FOR DEVICES INTENDED FOR DETECTION OR QUANTIFICATION OF MARKERS OF HEPATITIS D VIRUS (HDV) INFECTION

Scope

This Annex applies to devices intended for detection or quantification of markers of hepatitis D virus (HDV) infection.

Table 1 applies to devices intended for detection (including confirmation) or quantification of the following hepatitis D virus markers: antibodies against hepatitis D virus (anti-HDV), IgM antibodies against hepatitis D virus (anti-HDV IgM), the delta antigen.

Table 2 applies to qualitative and quantitative NAT devices for HDV RNA.

Table 1. Assays for HDV markers: anti-HDV, anti-HDV IgM, delta antigen

| Performance characteristic | | anti-HDV | anti-HDV IgM | Delta antigen | Acceptance criteria |
| --- | --- | --- | --- | --- | --- |
| Diagnostic sensitivity | Positive specimens | ≥100
  Specifying markers of HBV coinfection | ≥50
  Specifying markers of HBV coinfection | ≥10
  Specifying markers of HBV coinfection | ≥ 98 % |
| Diagnostic specificity | Negative specimens | ≥200
  Including clinical specimens
  ≥50 potentially interfering specimens | ≥200
  Including clinical specimens
  ≥50 potentially interfering specimens | ≥200
  Including clinical specimens
  ≥50 potentially interfering specimens | ≥ 98 % |

Performance characteristic

anti-HDV

anti-HDV IgM

Delta antigen

Acceptance criteria

Diagnostic sensitivity

Positive specimens

≥100

Specifying markers of HBV coinfection

≥50

Specifying markers of HBV coinfection

≥10

Specifying markers of HBV coinfection

≥ 98 %

Diagnostic specificity

Negative specimens

≥200

Including clinical specimens

≥50 potentially interfering specimens

≥200

Including clinical specimens

≥50 potentially interfering specimens

≥200

Including clinical specimens

≥50 potentially interfering specimens

≥ 98 %

Table 2. Qualitative and quantitative NAT devices for HDV RNA

| 1. | For target sequence amplification devices, a functionality control for each specimen (internal control) shall reflect the state of the art. This control shall as far as possible be used throughout the whole process, i.e. extraction, amplification/hybridisation, detection. |
| --- | --- |

1.

| 2. | Genotype and/or subtype detection shall be demonstrated by appropriate primer or probe design validation and shall also be validated by testing characterised genotyped specimens. |
| --- | --- |

2.

| 3. | Potential cross-reactivity of non-target nucleic acid sequences shall be analysed by appropriate primer or probe design validation and shall also be validated by testing selected specimens. |
| --- | --- |

3.

| 4. | Results of quantitative NAT devices shall be traceable to international standards or calibrated reference materials, if available, and be expressed in international units utilised in the specific field of application.








  Performance characteristic | Specimen | Specimen numbers, features, use | Acceptance criteria |
| --- | --- | --- | --- | --- |
| Analytical sensitivity | WHO First International Standard HDV RNA, PEI code 7657/12 | NAT sensitivity and NAT LOD shall be validated by dilution series of reference materials, testing of replicates (minimum 24) at different analyte concentrations, including those with transition from positive to negative results with the respective NAT device.
  LOD shall be expressed as 95% positive cut-off value (IU/ml) after statistical analysis (e.g. Probit). (1)

  quantitative NAT: definition of lower, upper quantification limit, precision, accuracy,
  ‘linear’ measuring range, ‘dynamic range’. Reproducibility at different concentration levels | According to the state of the art |
| HDV genotype sensitivity | all relevant genotypes/subtypes, preferably from international reference materials
  potential substitutes for rare HDV genotypes (to be quantified by appropriate methods): plasmids; synthetic RNA | Quantitative NAT: dilution series for demonstration of quantification efficiencies | According to the state of the art |
| Diagnostic specificity | Blood donor specimens | Qualitative NAT: ≥100
  Quantitative NAT: ≥100 | According to the state of the art |
| Cross-reactivity | Potentially cross-reacting specimens | | According to the state of the art |
| Carry-over | High HDV RNA positive;
  HDV RNA negative | At least five runs with alternating high-positive and negative specimens shall be performed during robustness studies. The virus titres of the high positive specimens shall be representative of high virus titres occurring naturally. | According to the state of the art |
| Whole system failure rate | HDV RNA low-positive | ≥100 HDV RNA low-positive specimens shall be tested. These specimens shall contain a virus concentration equivalent to three times the 95 % positive cut-off virus concentration. | ≥99% positive |

4.

Performance characteristic

Specimen

Specimen numbers, features, use

Acceptance criteria

Analytical sensitivity

WHO First International Standard HDV RNA, PEI code 7657/12

NAT sensitivity and NAT LOD shall be validated by dilution series of reference materials, testing of replicates (minimum 24) at different analyte concentrations, including those with transition from positive to negative results with the respective NAT device.

LOD shall be expressed as 95% positive cut-off value (IU/ml) after statistical analysis (e.g. Probit). (1)

quantitative NAT: definition of lower, upper quantification limit, precision, accuracy,

‘linear’ measuring range, ‘dynamic range’. Reproducibility at different concentration levels

According to the state of the art

HDV genotype sensitivity

all relevant genotypes/subtypes, preferably from international reference materials

potential substitutes for rare HDV genotypes (to be quantified by appropriate methods): plasmids; synthetic RNA

Quantitative NAT: dilution series for demonstration of quantification efficiencies

According to the state of the art

Diagnostic specificity

Blood donor specimens

Qualitative NAT: ≥100

Quantitative NAT: ≥100

According to the state of the art

Cross-reactivity

Potentially cross-reacting specimens

According to the state of the art

Carry-over

High HDV RNA positive;

HDV RNA negative

At least five runs with alternating high-positive and negative specimens shall be performed during robustness studies. The virus titres of the high positive specimens shall be representative of high virus titres occurring naturally.

According to the state of the art

Whole system failure rate

HDV RNA low-positive

≥100 HDV RNA low-positive specimens shall be tested. These specimens shall contain a virus concentration equivalent to three times the 95 % positive cut-off virus concentration.

≥99% positive

---

(1)  Reference: European Pharmacopeia 9.0, 2.6.21 Nucleic acid amplification techniques, Validation.

---

ANNEX VIII

COMMON SPECIFICATIONS FOR DEVICES INTENDED FOR DETECTION OF MARKERS OF VARIANT CREUTZFELDT-JACOB (vCJD) DISEASE

Scope

This Annex applies to devices intended for detection of markers of variant Creutzfeldt-Jakob disease (vCJD).

Table 1 applies to devices intended for detection of markers of vCJD.

Table 1. Devices for detection of markers of vCJD

| Performance characteristic | Material | Number of specimens | Acceptance criteria |
| --- | --- | --- | --- |
| Analytical sensitivity | vCJD brain spikes in human plasma (WHO reference number NHBY0/0003) | ≥24 replicates of each of three dilutions of the material WHO number NHBY0/0003 (1×104, 1×105, 1×106) | 23 of the 24 replicates detected at 1×104 |
| vCJD spleen spikes in human plasma (10% spleen homogenate — NIBSC reference number NHSY0/0009) | ≥24 replicates of each of three dilutions of the material NIBSC number NHSY0/0009 (1×10, 1×102 , 1×103 ) | 23 of the 24 replicates detected at 1×10 |
| Diagnostic sensitivity | Specimens from appropriate animal models | As many specimens as reasonably possible and available, and ≥10 specimens | 90% |
| Specimens from humans with known clinical vCJD | As many specimens as reasonably possible and available, and ≥10 specimens | 90% |
| Only in cases where 10 specimens are not available:






  — | the number of specimens tested shall be between 6 and 9 |

Performance characteristic

Material

Number of specimens

Acceptance criteria

Analytical sensitivity

vCJD brain spikes in human plasma (WHO reference number NHBY0/0003)

≥24 replicates of each of three dilutions of the material WHO number NHBY0/0003 (1×104, 1×105, 1×106)

23 of the 24 replicates detected at 1×104

vCJD spleen spikes in human plasma (10% spleen homogenate — NIBSC reference number NHSY0/0009)

≥24 replicates of each of three dilutions of the material NIBSC number NHSY0/0009 (1×10, 1×102 , 1×103 )

23 of the 24 replicates detected at 1×10

Diagnostic sensitivity

Specimens from appropriate animal models

As many specimens as reasonably possible and available, and ≥10 specimens

90%

Specimens from humans with known clinical vCJD

As many specimens as reasonably possible and available, and ≥10 specimens

90%

Only in cases where 10 specimens are not available:

—

the number of specimens tested shall be between 6 and 9

| — | all available specimens shall be tested |
| --- | --- |

—

all available specimens shall be tested

max. one false negative result

Analytical specificity

Potentially cross-reacting specimens

≥100

Diagnostic specificity

Normal human plasma specimens from area of low bovine spongiform encephalopathy (BSE) exposure

≥5 000

≥ 99,5 %

---

ANNEX IX

COMMON SPECIFICATIONS FOR DEVICES INTENDED FOR DETECTION OR QUANTIFICATION OF MARKERS OF CYTOMEGALOVIRUS (CMV) INFECTION

Scope

This Annex applies to devices intended for detection or quantification of markers of cytomegalovirus (CMV) infection.

Table 1 applies to first-line assays for total antibodies against CMV (total anti-CMV) and IgG antibodies against CMV (anti-CMV IgG).

Table 2 applies to qualitative and quantitative NAT devices for CMV DNA.

Table 1. First-line assays: total anti-CMV and anti-CMV IgG

| Performance characteristic | Specimens | Specimen number, features, use | Acceptance criteria |
| --- | --- | --- | --- |
| Diagnostic sensitivity | Positive specimens | ≥400
  including specimens from recent and past CMV infection,
  low and high positive titre specimens | ≥ 99% sensitivity for confirmable past infection (1);
  overall sensitivity including recent infection (2) shall be at least equivalent to the comparator device |
| Seroconversion panels | To be tested when available | Diagnostic sensitivity during seroconversion shall represent the state of the art |
| Analytical sensitivity | Standards | WHO International Standard anti-CMV IgG (PEI-code 136616/17)
  In case of titre determinations and quantitative statements | |
| Diagnostic specificity | Negative specimens | ≥400 (3) CMV negative specimens from unselected donors, as compared to another CMV test. | ≥ 99% |
| Hospitalised patients (4) | ≥200 | Potential limitations for specificity, if any, shall be identified |
| Cross-reactivity | Potentially cross-reacting (5) specimens | ≥100 in total
  (e.g. RF+, related viruses or other infectious agents, pregnant women, etc.) |

Performance characteristic

Specimens

Specimen number, features, use

Acceptance criteria

Diagnostic sensitivity

Positive specimens

≥400

including specimens from recent and past CMV infection,

low and high positive titre specimens

≥ 99% sensitivity for confirmable past infection (1);

overall sensitivity including recent infection (2) shall be at least equivalent to the comparator device

Seroconversion panels

To be tested when available

Diagnostic sensitivity during seroconversion shall represent the state of the art

Analytical sensitivity

Standards

WHO International Standard anti-CMV IgG (PEI-code 136616/17)

In case of titre determinations and quantitative statements

Diagnostic specificity

Negative specimens

≥400 (3) CMV negative specimens from unselected donors, as compared to another CMV test.

≥ 99%

Hospitalised patients (4)

≥200

Potential limitations for specificity, if any, shall be identified

Cross-reactivity

Potentially cross-reacting (5) specimens

≥100 in total

(e.g. RF+, related viruses or other infectious agents, pregnant women, etc.)

Table 2. Qualitative and quantitative NAT devices for CMV DNA

| 1. | For target sequence amplification devices, a functionality control for each specimen (internal control) shall reflect the state of the art. This control shall as far as possible be used throughout the whole process, i.e. extraction, amplification/hybridisation, detection. |
| --- | --- |

1.

| 2. | Genotype and/or subtype detection shall be demonstrated by appropriate primer or probe design validation and shall also be validated by testing characterised genotyped specimens. |
| --- | --- |

2.

| 3. | Potential cross-reactivity of non-target nucleic acid sequences shall be analysed by appropriate primer or probe design validation and shall also be validated by testing selected specimens. |
| --- | --- |

3.

| 4. | Results of quantitative NAT devices shall be traceable to international standards or calibrated reference materials, if available, and be expressed in international units utilised in the specific field of application.








  Performance characteristic | Specimens | Specimen numbers, features, use | Acceptance criteria |
| --- | --- | --- | --- | --- |
| Analytical sensitivity | WHO First International Standard Human CMV DNA (09/162; 5 000 000 IU/vial) (or calibrated reference materials) | NAT sensitivity and NAT LOD shall be validated by dilution series of reference materials, testing of replicates (minimum 24) at different analyte concentrations, including those with transition from positive to negative results with the respective NAT device.
  LOD shall be expressed as 95% positive cut-off value (IU/ml) after statistical analysis (e.g. Probit). (6)

  quantitative NAT: definition of lower, upper quantification limit, precision, accuracy,
  ‘linear’ measuring range, ‘dynamic range’.
  Reproducibility at different concentration levels | According to the state of the art |
| Diagnostic sensitivity
  CMV Strain sensitivity | Patient specimens determined as CMV DNA positive by comparator device
  Dilution series of CMV positive cell cultures may serve as potential substitutes | Qualitative NAT: ≥100
  Quantitative NAT: ≥100
  dilution series for demonstration of quantification efficiencies | According to the state of the art |
| Diagnostic specificity | Blood donor specimens | Qualitative NAT: ≥500
  Quantitative NAT: ≥100 | According to the state of the art |
| Cross-reactivity | Potentially cross-reacting specimens | ≥20 specimens in total
  Including human specimens positive for related human herpesviruses, e.g. EBV, HHV6, VZV
  Herpesvirus positive cell cultures may serve as potential substitutes | According to the state of the art |
| Carry-over | High CMV DNA positive;
  CMV DNA negative | At least five runs with alternating high-positive and negative specimens shall be performed during robustness studies. The virus titres of the high positive specimens shall be representative of high virus titres occurring naturally. | According to the state of the art |
| Whole system failure rate | CMV DNA low-positive | ≥100 CMV DNA low-positive specimens shall be tested. These specimens shall contain a virus concentration equivalent to three times the 95 % positive cut-off virus concentration. | ≥99% positive |

4.

Performance characteristic

Specimens

Specimen numbers, features, use

Acceptance criteria

Analytical sensitivity

WHO First International Standard Human CMV DNA (09/162; 5 000 000 IU/vial) (or calibrated reference materials)

NAT sensitivity and NAT LOD shall be validated by dilution series of reference materials, testing of replicates (minimum 24) at different analyte concentrations, including those with transition from positive to negative results with the respective NAT device.

LOD shall be expressed as 95% positive cut-off value (IU/ml) after statistical analysis (e.g. Probit). (6)

quantitative NAT: definition of lower, upper quantification limit, precision, accuracy,

‘linear’ measuring range, ‘dynamic range’.

Reproducibility at different concentration levels

According to the state of the art

Diagnostic sensitivity

CMV Strain sensitivity

Patient specimens determined as CMV DNA positive by comparator device

Dilution series of CMV positive cell cultures may serve as potential substitutes

Qualitative NAT: ≥100

Quantitative NAT: ≥100

dilution series for demonstration of quantification efficiencies

According to the state of the art

Diagnostic specificity

Blood donor specimens

Qualitative NAT: ≥500

Quantitative NAT: ≥100

According to the state of the art

Cross-reactivity

Potentially cross-reacting specimens

≥20 specimens in total

Including human specimens positive for related human herpesviruses, e.g. EBV, HHV6, VZV

Herpesvirus positive cell cultures may serve as potential substitutes

According to the state of the art

Carry-over

High CMV DNA positive;

CMV DNA negative

At least five runs with alternating high-positive and negative specimens shall be performed during robustness studies. The virus titres of the high positive specimens shall be representative of high virus titres occurring naturally.

According to the state of the art

Whole system failure rate

CMV DNA low-positive

≥100 CMV DNA low-positive specimens shall be tested. These specimens shall contain a virus concentration equivalent to three times the 95 % positive cut-off virus concentration.

≥99% positive

---

(1)  Including testing of other CMV parameters (e.g. CMV-IgM, avidity, immunoblot) or previous / follow-up specimens to assess true specimen status.

(2)  Supplementary testing to confirm recent CMV infection (primary or re-infection): e.g. CMV-IgM, IgG-avidity, immunoblot analysis.

(3)  Corresponding to an initial number of 1000 donors at an assumed CMV prevalence of 60 %.

(4)  Including pre-transplant recipients.

(5)  Including related β-herpes viruses (HHV-6, HHV-7).

(6)  Reference: European Pharmacopeia 9.0, 2.6.21 Nucleic acid amplification techniques, Validation.

---

ANNEX X

COMMON SPECIFICATIONS FOR DEVICES INTENDED FOR DETECTION OR QUANTIFICATION OF MARKERS OF EPSTEIN-BARR VIRUS (EBV) INFECTION

Scope

This Annex applies to devices intended for detection or quantification of markers of Epstein-Barr virus (EBV) infection.

Table 1 applies to first-line assays for IgG antibodies against viral capsid antigen of EBV (anti-EBV VCA IgG).

Table 2 applies to qualitative and quantitative NAT devices for EBV DNA.

Table 1: First-line assays: anti-EBV VCA IgG

| Performance characteristic | Specimens | Specimen number, features, use | Acceptance criteria |
| --- | --- | --- | --- |
| Diagnostic sensitivity | Positive specimens | ≥400
  including specimens from recent and past EBV infection,
  low and high positive titre specimens | ≥ 99% for confirmable past infection (1); overall sensitivity including recent infection (2) shall be at least equivalent to the comparator device |
| Seroconversion panels | To be tested when available | diagnostic sensitivity during seroconversion shall represent the state of the art |
| Analytical sensitivity | Standards | International reference reagents, when available | |
| Diagnostic specificity | Negative specimens | ≥ 200 (3) EBV negatives from unselected donors as compared to another EBV device | ≥ 99% |
| Hospitalised patients (4) | ≥200 | Potential limitations for specificity, if any, shall be identified |
| Cross-reactivity | Potentially cross-reacting specimens | ≥100 in total
  (e.g. RF+, related viruses or other infectious agents, pregnant women, etc.) |

Performance characteristic

Specimens

Specimen number, features, use

Acceptance criteria

Diagnostic sensitivity

Positive specimens

≥400

including specimens from recent and past EBV infection,

low and high positive titre specimens

≥ 99% for confirmable past infection (1); overall sensitivity including recent infection (2) shall be at least equivalent to the comparator device

Seroconversion panels

To be tested when available

diagnostic sensitivity during seroconversion shall represent the state of the art

Analytical sensitivity

Standards

International reference reagents, when available

Diagnostic specificity

Negative specimens

≥ 200 (3) EBV negatives from unselected donors as compared to another EBV device

≥ 99%

Hospitalised patients (4)

≥200

Potential limitations for specificity, if any, shall be identified

Cross-reactivity

Potentially cross-reacting specimens

≥100 in total

(e.g. RF+, related viruses or other infectious agents, pregnant women, etc.)

Table 2. Qualitative and quantitative NAT devices for EBV DNA

| 1. | For target sequence amplification devices, a functionality control for each specimen (internal control) shall reflect the state of the art. This control shall as far as possible be used throughout the whole process, i.e. extraction, amplification/hybridisation, detection. |
| --- | --- |

1.

| 2. | Genotype and/or subtype detection shall be demonstrated by appropriate primer or probe design validation and shall also be validated by testing characterised genotyped specimens. |
| --- | --- |

2.

| 3. | Potential cross-reactivity of non-target nucleic acid sequences shall be analysed by appropriate primer or probe design validation and shall also be validated by testing selected specimens. |
| --- | --- |

3.

| 4. | Results of quantitative NAT devices shall be traceable to international standards or calibrated reference materials, if available, and be expressed in international units utilised in the specific field of application.








  Performance characteristic | Specimens | Specimen numbers, features, use | Acceptance criteria |
| --- | --- | --- | --- | --- |
| Analytical sensitivity | WHO First International Standard Human EBV DNA (09/260; 5 000 000 IU/vial) (or calibrated reference materials) | NAT sensitivity and NAT LOD shall be validated by dilution series of reference materials, testing of replicates (minimum 24) at different analyte concentrations, including those with transition from positive to negative results with the respective NAT device.
  LOD shall be expressed as 95% positive cut-off value (IU/ml) after statistical analysis (e.g. Probit). (5)

  quantitative NAT: definition of lower, upper quantification limit, precision, accuracy,
  ‘linear’ measuring range, ‘dynamic range’.
  Reproducibility at different concentration levels | According to the state of the art |
| Diagnostic sensitivity
  EBV strain sensitivity | Patient specimens determined as EBV DNA positive by comparator device
  Dilution series of EBV positive cell cultures may serve as potential substitutes | Qualitative NAT: ≥100
  Quantitative NAT: ≥100
  dilution series for demonstration of quantification efficiencies | |
| Diagnostic specificity | Negative specimens | Qualitative NAT: ≥500
  Quantitative NAT: ≥100 | According to the state of the art |
| Cross-reactivity | Potentially cross-reacting specimens | ≥20 specimens in total
  Including human specimens positive for related human herpesviruses, e.g. CMV, HHV6, VZV
  Herpesvirus positive cell cultures may serve as potential substitutes | According to the state of the art |
| Carry-over | High EBV DNA positive;
  EBV DNA negative | At least five runs with alternating high-positive and negative specimens shall be performed during robustness studies. The virus titres of the high positive specimens shall be representative of high virus titres occurring naturally. | According to the state of the art |
| Whole system failure rate | EBV DNA low-positive | ≥100 EBV DNA low-positive specimens shall be tested. These specimens shall contain a virus concentration equivalent to three times the 95 % positive cut-off virus concentration. | ≥99% positive |

4.

Performance characteristic

Specimens

Specimen numbers, features, use

Acceptance criteria

Analytical sensitivity

WHO First International Standard Human EBV DNA (09/260; 5 000 000 IU/vial) (or calibrated reference materials)

NAT sensitivity and NAT LOD shall be validated by dilution series of reference materials, testing of replicates (minimum 24) at different analyte concentrations, including those with transition from positive to negative results with the respective NAT device.

LOD shall be expressed as 95% positive cut-off value (IU/ml) after statistical analysis (e.g. Probit). (5)

quantitative NAT: definition of lower, upper quantification limit, precision, accuracy,

‘linear’ measuring range, ‘dynamic range’.

Reproducibility at different concentration levels

According to the state of the art

Diagnostic sensitivity

EBV strain sensitivity

Patient specimens determined as EBV DNA positive by comparator device

Dilution series of EBV positive cell cultures may serve as potential substitutes

Qualitative NAT: ≥100

Quantitative NAT: ≥100

dilution series for demonstration of quantification efficiencies

Diagnostic specificity

Negative specimens

Qualitative NAT: ≥500

Quantitative NAT: ≥100

According to the state of the art

Cross-reactivity

Potentially cross-reacting specimens

≥20 specimens in total

Including human specimens positive for related human herpesviruses, e.g. CMV, HHV6, VZV

Herpesvirus positive cell cultures may serve as potential substitutes

According to the state of the art

Carry-over

High EBV DNA positive;

EBV DNA negative

At least five runs with alternating high-positive and negative specimens shall be performed during robustness studies. The virus titres of the high positive specimens shall be representative of high virus titres occurring naturally.

According to the state of the art

Whole system failure rate

EBV DNA low-positive

≥100 EBV DNA low-positive specimens shall be tested. These specimens shall contain a virus concentration equivalent to three times the 95 % positive cut-off virus concentration.

≥99% positive

---

(1)  Including testing of other EBV markers and parameters (e.g. VCA-IgM, EBNA-1 IgG, immunoblot) or previous / follow-up specimens to assess the true specimen status.

(2)  Supplementary testing to confirm recent EBV infection: e.g. VCA-IgM, IgG-avidity, immunoblot analysis.

(3)  At an assumed EBV prevalence of 80 % corresponding to an initial number of 1000 donors.

(4)  Including pre-transplant recipients.

(5)  Reference: European Pharmacopeia 9.0, 2.6.21 Nucleic acid amplification techniques, Validation.

---

ANNEX XI

COMMON SPECIFICATIONS FOR DEVICES INTENDED FOR DETECTION OF MARKERS OF TREPONEMA PALLIDUM INFECTION

Scope

This Annex applies to devices intended for detection of markers of Treponema pallidum (T. pallidum).

Table 1 applies to first-line assays for antibodies against T. pallidum (anti-T.pallidum).

Table 2 applies to confirmatory and supplemental anti-T.pallidum assays.

Table 1. First-line assays: anti-T.pallidum

| Performance characteristic | Specimens | Specimen number, features, use | Acceptance criteria |
| --- | --- | --- | --- |
| Diagnostic
  sensitivity | Positive specimens | ≥200 positive specimens in total,
  at different stages of the infection if available,
  including high positive and low positive specimens,
  identified as positive by at least two different serological tests (one of which is an enzyme immunoassay) for different antibodies to T.pallidum | ≥99.5% overall sensitivity |
| Seroconversion panels | At least 1 seroconversion panel, ≥1 if possible, including individual specimens from the early infection phase | Diagnostic sensitivity during seroconversion shall represent the state of the art. |
| Analytical
  sensitivity | Standards | WHO international standards
  NIBSC code 05/132, when available | |
| Diagnostic specificity | Unselected blood donors (including first-time donors) (1) | ≥5 000 | ≥99,5% |
| Hospitalised patients | ≥200 | Potential limitations for specificity, if any, shall be identified |
| Cross-reactivity | Potentially cross-reacting specimens | ≥100 in total
  including the following specimens: positive for Borrelia burgdorferi sensu lato confirmed by IgG immunoblot; anti-HIV positive; RF+; other related microbial/infectious agents; systemic lupus erythematosus (SLE) patients; antiphospholipid antibody positive; pregnant women etc. |

Performance characteristic

Specimens

Specimen number, features, use

Acceptance criteria

Diagnostic

sensitivity

Positive specimens

≥200 positive specimens in total,

at different stages of the infection if available,

including high positive and low positive specimens,

identified as positive by at least two different serological tests (one of which is an enzyme immunoassay) for different antibodies to T.pallidum

≥99.5% overall sensitivity

Seroconversion panels

At least 1 seroconversion panel, ≥1 if possible, including individual specimens from the early infection phase

Diagnostic sensitivity during seroconversion shall represent the state of the art.

Analytical

sensitivity

Standards

WHO international standards

NIBSC code 05/132, when available

Diagnostic specificity

Unselected blood donors (including first-time donors) (1)

≥5 000

≥99,5%

Hospitalised patients

≥200

Potential limitations for specificity, if any, shall be identified

Cross-reactivity

Potentially cross-reacting specimens

≥100 in total

including the following specimens: positive for Borrelia burgdorferi sensu lato confirmed by IgG immunoblot; anti-HIV positive; RF+; other related microbial/infectious agents; systemic lupus erythematosus (SLE) patients; antiphospholipid antibody positive; pregnant women etc.

Table 2. Confirmatory and supplemental assays: anti-T.pallidum

| Performance characteristic | Specimens | Specimen number, features, use | Acceptance criteria |
| --- | --- | --- | --- |
| Diagnostic sensitivity | Positive specimens | ≥300 positive specimens at different stages of the infection (early primary syphilis, secondary stage, and during late syphilis ) including high positive specimens, 50 low positive specimens,
  by at least two different serological tests (one of which is an enzyme immunoassay) for different antibodies to T.pallidum | 99% identification as “confirmed positive” or “indeterminate” |
| Seroconversion panels | At least 1 seroconversion panel, ≥1 if possible, including individual specimens from the early infection phase | Diagnostic sensitivity during seroconversion shall represent the state of the art |
| Analytical sensitivity | Standards | WHO international standards
  NIBSC code 05/132 | |
| Diagnostic specificity | Blood donors | ≥200 | ≥ 99%; |
| Clinical specimens | ≥200 | Potential limitations for specificity, if any, shall be identified |
| Cross-reactivity | Potentially cross-reacting specimens | ≥50 in total, including specimens from pregnant women and specimens with indeterminate results in other confirmatory assays. |

Performance characteristic

Specimens

Specimen number, features, use

Acceptance criteria

Diagnostic sensitivity

Positive specimens

≥300 positive specimens at different stages of the infection (early primary syphilis, secondary stage, and during late syphilis ) including high positive specimens, 50 low positive specimens,

by at least two different serological tests (one of which is an enzyme immunoassay) for different antibodies to T.pallidum

99% identification as “confirmed positive” or “indeterminate”

Seroconversion panels

At least 1 seroconversion panel, ≥1 if possible, including individual specimens from the early infection phase

Diagnostic sensitivity during seroconversion shall represent the state of the art

Analytical sensitivity

Standards

WHO international standards

NIBSC code 05/132

Diagnostic specificity

Blood donors

≥200

≥ 99%;

Clinical specimens

≥200

Potential limitations for specificity, if any, shall be identified

Cross-reactivity

Potentially cross-reacting specimens

≥50 in total, including specimens from pregnant women and specimens with indeterminate results in other confirmatory assays.

---

(1)  Blood donor populations shall be investigated from at least two blood donation centres and consist of consecutive blood donations, which have not been selected to exclude first-time donors.

---

ANNEX XII

COMMON SPECIFICATIONS FOR DEVICES INTENDED FOR DETECTION OR QUANTIFICATION OF MARKERS OF TRYPANOSOMA CRUZI INFECTION

Scope

This Annex applies for devices intended for detection or quantification of markers of Trypanosoma cruzi (T. cruzi) infection.

Table 1 applies to first-line assays for antibodies against T. cruzi (anti-T. cruzi).

Table 2 applies to confirmatory and supplemental anti-T. cruzi assays.

Table 3 applies to qualitative and quantitative NAT devices for T. cruzi DNA.

Table 1. First-line assays: anti-T. cruzi

| Performance characteristic | Specimens | Specimen number, features, use | Acceptance criteria |
| --- | --- | --- | --- |
| Diagnostic sensitivity | Positive specimens | ≥400 positive specimens, including highly positive confirmed by at least two different serological tests for different antibodies to T.cruzi.
  Of those 400, ≥25 parasite positive specimens which have been confirmed by direct detection. | 99.5% overall sensitivity |
| Seroconversion panels | To be defined when available | Diagnostic sensitivity during seroconversion shall represent the state of the art |
| Analytical sensitivity | Standards | WHO international standards
  NIBSC code: 09/186
  NIBSC code: 09/188 | |
| Diagnostic specificity | Unselected donors (including first-time donors) (1) | ≥5 000 | ≥99,5% |
| Hospitalised patients | ≥200 | Potential limitations for specificity, if any, shall be identified |
| Cross-reactivity | Potentially cross-reacting specimens | ≥100 in total
  including the following specimens: positive for anti-Toxoplasma gondii; at least 5 specimens positive for anti-Leishmania; RF+; related microbial agents or other infectious agents; SLE patients; antiphospholipid antibody positive patients; pregnant women, etc. |

Performance characteristic

Specimens

Specimen number, features, use

Acceptance criteria

Diagnostic sensitivity

Positive specimens

≥400 positive specimens, including highly positive confirmed by at least two different serological tests for different antibodies to T.cruzi.

Of those 400, ≥25 parasite positive specimens which have been confirmed by direct detection.

99.5% overall sensitivity

Seroconversion panels

To be defined when available

Diagnostic sensitivity during seroconversion shall represent the state of the art

Analytical sensitivity

Standards

WHO international standards

NIBSC code: 09/186

NIBSC code: 09/188

Diagnostic specificity

Unselected donors (including first-time donors) (1)

≥5 000

≥99,5%

Hospitalised patients

≥200

Potential limitations for specificity, if any, shall be identified

Cross-reactivity

Potentially cross-reacting specimens

≥100 in total

including the following specimens: positive for anti-Toxoplasma gondii; at least 5 specimens positive for anti-Leishmania; RF+; related microbial agents or other infectious agents; SLE patients; antiphospholipid antibody positive patients; pregnant women, etc.

Table 2. Confirmatory and supplemental assays: anti-T. cruzi

| Performance characteristic | Specimens | Specimen number, features, use | Acceptance criteria |
| --- | --- | --- | --- |
| Diagnostic sensitivity | Positive specimens | ≥300 positive specimens, including highly positive confirmed by at least two different serological tests for different antibodies to T.cruzi.
  Of those 300, ≥25 parasite positive specimens, which have been confirmed by direct detection. | ≥99% identification as “confirmed positive” or “indeterminate” |
| Seroconversion panels | As available | Diagnostic sensitivity during seroconversion shall represent the state of the art, if applicable |
| Analytical sensitivity | Standards | WHO international standards
  NIBSC code: 09/186
  NIBSC code: 09/188 | |
| Diagnostic specificity | Negative specimens | ≥200 | ≥99% |
| Clinical specimens | ≥200 | Potential limitations for specificity, if any, shall be identified |
| Cross-reactivity | Potentially cross-reacting specimens | ≥50 in total, including specimens from pregnant women and specimens with indeterminate results in other confirmatory assays |

Performance characteristic

Specimens

Specimen number, features, use

Acceptance criteria

Diagnostic sensitivity

Positive specimens

≥300 positive specimens, including highly positive confirmed by at least two different serological tests for different antibodies to T.cruzi.

Of those 300, ≥25 parasite positive specimens, which have been confirmed by direct detection.

≥99% identification as “confirmed positive” or “indeterminate”

Seroconversion panels

As available

Diagnostic sensitivity during seroconversion shall represent the state of the art, if applicable

Analytical sensitivity

Standards

WHO international standards

NIBSC code: 09/186

NIBSC code: 09/188

Diagnostic specificity

Negative specimens

≥200

≥99%

Clinical specimens

≥200

Potential limitations for specificity, if any, shall be identified

Cross-reactivity

Potentially cross-reacting specimens

≥50 in total, including specimens from pregnant women and specimens with indeterminate results in other confirmatory assays

Table 3: NAT devices for T. cruzi DNA

| 1. | For target sequence amplification devices, a functionality control for each specimen (internal control) shall reflect the state of the art. This control shall as far as possible be used throughout the whole process, i.e. extraction, amplification/hybridisation, detection. |
| --- | --- |

1.

| 2. | Genotype and/or subtype detection shall be demonstrated by appropriate primer or probe design validation and shall also be validated by testing characterised genotyped specimens. |
| --- | --- |

2.

| 3. | Potential cross-reactivity of non-target nucleic acid sequences shall be analysed by appropriate primer or probe design validation and shall also be validated by testing selected specimens. |
| --- | --- |

3.

| 4. | Results of quantitative NAT devices shall be traceable to international standards or calibrated reference materials, if available, and be expressed in international units utilised in the specific field of application.








  Performance characteristic | Specimens | Specimen number, features, use | Acceptance criteria |
| --- | --- | --- | --- | --- |
| Analytical sensitivity | Characterized in-house reference preparation (as long as international reference materials are not available) | NAT sensitivity and NAT LOD shall be validated by dilution series of reference materials, testing of replicates (minimal 24) at different analyte concentrations, including those with transition from positive to negative results with the respective NAT device.
  LOD shall be expressed as 95% positive cut-off value (IU/ml) after statistical analysis (e.g. Probit). (2) | According to the state of the art |
| Diagnostic sensitivity: different T.cruzi strains / isolates | Patient specimens from different regions determined as T.cruzi DNA positive by comparator device; sequence variants | ≥100
  Dilution series of T.cruzi positive cell cultures (isolates) or T.cruzi positive materials from animal models may serve as potential substitutes | According to the state of the art |
| Diagnostic specificity | Negative specimens | ≥100 | According to the state of the art |
| Cross-reactivity | Potentially cross-reacting specimens | ≥10 human specimens positive for other parasites, e.g. Plasmodium species, Trypanosoma brucei. Positive cell cultures may serve as potential substitutes | According to the state of the art |
| Carry-over | | At least five runs with alternating high-positive and negative specimens shall be performed during robustness studies. The T.cruzi titres of the high positive specimens shall be representative of high T.cruzi titres occurring naturally. | According to the state of the art |
| Whole system failure rate | | ≥100 T.cruzi DNA low-positive specimens shall be tested. These specimens shall contain a T.cruzi concentration equivalent to three times the 95 % positive cut-off T.cruzi concentration. | ≥99% positive |

4.

Performance characteristic

Specimens

Specimen number, features, use

Acceptance criteria

Analytical sensitivity

Characterized in-house reference preparation (as long as international reference materials are not available)

NAT sensitivity and NAT LOD shall be validated by dilution series of reference materials, testing of replicates (minimal 24) at different analyte concentrations, including those with transition from positive to negative results with the respective NAT device.

LOD shall be expressed as 95% positive cut-off value (IU/ml) after statistical analysis (e.g. Probit). (2)

According to the state of the art

Diagnostic sensitivity: different T.cruzi strains / isolates

Patient specimens from different regions determined as T.cruzi DNA positive by comparator device; sequence variants

≥100

Dilution series of T.cruzi positive cell cultures (isolates) or T.cruzi positive materials from animal models may serve as potential substitutes

According to the state of the art

Diagnostic specificity

Negative specimens

≥100

According to the state of the art

Cross-reactivity

Potentially cross-reacting specimens

≥10 human specimens positive for other parasites, e.g. Plasmodium species, Trypanosoma brucei. Positive cell cultures may serve as potential substitutes

According to the state of the art

Carry-over

At least five runs with alternating high-positive and negative specimens shall be performed during robustness studies. The T.cruzi titres of the high positive specimens shall be representative of high T.cruzi titres occurring naturally.

According to the state of the art

Whole system failure rate

≥100 T.cruzi DNA low-positive specimens shall be tested. These specimens shall contain a T.cruzi concentration equivalent to three times the 95 % positive cut-off T.cruzi concentration.

≥99% positive

---

(1)  Blood donor populations shall be investigated from at least two blood donation centres and consist of consecutive blood donations, which have not been selected to exclude first-time donors.

(2)  Reference: European Pharmacopeia 9.0, 2.6.21 Nucleic acid amplification techniques, Validation.

---

ANNEX XIII

COMMON SPECIFICATIONS FOR DEVICES INTENDED FOR DETECTION OR QUANTIFICATION OF MARKERS OF SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 INFECTION

Scope

This Annex applies to devices intended for detection or quantification of markers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

Table 1 applies to the following first-line assays (including rapid tests) for antibodies against SARS-CoV-2 (anti-SARS-CoV-2): total antibody, IgG-only, IgG combined with IgM and/or IgA.

Table 2 applies to first-line assays (including rapid tests) for detection of anti-SARS-CoV-2 IgM and/or IgA.

Table 3 applies to confirmatory or supplemental assays for anti-SARS-CoV-2.

Table 4 applies to antigen SARS-CoV-2 tests, including rapid antigen tests.

Table 5 applies to NAT assays for SARS-CoV-2 RNA.

Table 6 applies to SARS-CoV-2 antigen self-tests which have already undergone a performance evaluation for professional use.

Table 7 applies to SARS-CoV-2 antibody self-tests which have already undergone a performance evaluation for professional use.

Table 1: First-line assays (including rapid tests) for anti-SARS-CoV-2: total antibody, IgG-only, IgG combined
   (1)
  with IgM and/or IgA

| Performance characteristic | Specimen | Specimen number, features, use | Acceptance criteria |
| --- | --- | --- | --- |
| Diagnostic sensitivity | Positive specimens | ≥400
  including specimens from early infection and post seroconversion (2) (within the first 21 days and after 21 days following the onset of symptoms);
  including specimens from asymptomatic or subclinical and mildly symptomatic (outpatient treatment) individuals;
  including specimens with low and high titers;
  including specimens from vaccinated individuals where appropriate (3);
  consideration of genetic variants | ≥90% sensitivity (4) for specimens taken >21 days after onset of symptoms (5);
  overall sensitivity including the early infection phase shall be at least equivalent to the comparator device (6) |
| Seroconversion panels | As far as available | Seroconversion sensitivity comparable to other CE-marked devices |
| Analytical sensitivity | Reference preparations | WHO International Standard (IS) for anti- SARS-CoV-2 (NIBSC code 20/136);
  WHO International Reference Panel (RP) for anti-SARS-CoV-2 antibodies (NIBSC codes 20/140, 20/142, 20/144, 20/148, 20/150) | IS: for titre determinations / quantitative (7) result output;
  RP: all antibody assays |
| Diagnostic specificity | Negative specimens (8) | ≥400
  specimens from non-infected and non-vaccinated individuals (9) | >99% specificity (10) |
| | ≥200
  hospitalised patients (without SARS-CoV-2 infection) | Potential limitations for specificity, if any, shall beidentified |
| Cross-reactivity | Potentially cross-reacting specimens | ≥100 in total
  including RF+, pregnant women, specimens with antibodies against endemic human coronaviruses 229E, OC43, NL63, HKU1 and other pathogens of respiratory diseases such as influenza A, B, RSV etc. |

Performance characteristic

Specimen

Specimen number, features, use

Acceptance criteria

Diagnostic sensitivity

Positive specimens

≥400

including specimens from early infection and post seroconversion (2) (within the first 21 days and after 21 days following the onset of symptoms);

including specimens from asymptomatic or subclinical and mildly symptomatic (outpatient treatment) individuals;

including specimens with low and high titers;

including specimens from vaccinated individuals where appropriate (3);

consideration of genetic variants

≥90% sensitivity (4) for specimens taken >21 days after onset of symptoms (5);

overall sensitivity including the early infection phase shall be at least equivalent to the comparator device (6)

Seroconversion panels

As far as available

Seroconversion sensitivity comparable to other CE-marked devices

Analytical sensitivity

Reference preparations

WHO International Standard (IS) for anti- SARS-CoV-2 (NIBSC code 20/136);

WHO International Reference Panel (RP) for anti-SARS-CoV-2 antibodies (NIBSC codes 20/140, 20/142, 20/144, 20/148, 20/150)

IS: for titre determinations / quantitative (7) result output;

RP: all antibody assays

Diagnostic specificity

Negative specimens (8)

≥400

specimens from non-infected and non-vaccinated individuals (9)

>99% specificity (10)

≥200

hospitalised patients (without SARS-CoV-2 infection)

Potential limitations for specificity, if any, shall beidentified

Cross-reactivity

Potentially cross-reacting specimens

≥100 in total

including RF+, pregnant women, specimens with antibodies against endemic human coronaviruses 229E, OC43, NL63, HKU1 and other pathogens of respiratory diseases such as influenza A, B, RSV etc.

Table 2: First-line assays (including rapid tests) for anti-SARS-CoV-2: IgM and/or IgA detection

| Performance characteristic | Specimen | Specimen number, features, use | Acceptance criteria |
| --- | --- | --- | --- |
| Diagnostic sensitivity | Positive specimens | ≥200 (11)

  Specimens (12) with a significant proportion from the early phase of the infection (within 21 days after onset of symptoms) compared to specimens past seroconversion (>21 days after onset of symptoms);
  including specimens from asymptomatic, subclinical, mildly symptomatic (outpatient treatment) individuals;
  including freshly (13) vaccinated individuals if appropriate;
  consideration of genetic variants | ≥80% sensitivity (14) for specimens taken during the first 21 days after symptom onset (15);
  overall sensitivity shall be at least equivalent to the comparator device (16) of the same type (i.e. IgM and/or IgA) |
| Seroconversion panels | As far as available | Seroconversion sensitivity comparable to other CE-marked devices |
| Analytical sensitivity | Standards | N/A | N/A |
| Diagnostic specificity | Negative specimens (17) | ≥200
  specimens from non-infected and non-vaccinated individuals (18) | ≥98% specificity (19) |
| | ≥100
  from hospitalised patients (without SARS-CoV-2 infection) | Potential limitations for specificity, if any, shall be identified |
| Cross-reactivity | Potentially cross-reacting specimens | ≥100 in total
  including RF+, pregnant women, specimens with antibodies against endemic human coronaviruses 229E, OC43, NL63, HKU1 and other pathogens of respiratory diseases such as influenza A, B, RSV etc. |

Performance characteristic

Specimen

Specimen number, features, use

Acceptance criteria

Diagnostic sensitivity

Positive specimens

≥200 (11)

Specimens (12) with a significant proportion from the early phase of the infection (within 21 days after onset of symptoms) compared to specimens past seroconversion (>21 days after onset of symptoms);

including specimens from asymptomatic, subclinical, mildly symptomatic (outpatient treatment) individuals;

including freshly (13) vaccinated individuals if appropriate;

consideration of genetic variants

≥80% sensitivity (14) for specimens taken during the first 21 days after symptom onset (15);

overall sensitivity shall be at least equivalent to the comparator device (16) of the same type (i.e. IgM and/or IgA)

Seroconversion panels

As far as available

Seroconversion sensitivity comparable to other CE-marked devices

Analytical sensitivity

Standards

N/A

N/A

Diagnostic specificity

Negative specimens (17)

≥200

specimens from non-infected and non-vaccinated individuals (18)

≥98% specificity (19)

≥100

from hospitalised patients (without SARS-CoV-2 infection)

Potential limitations for specificity, if any, shall be identified

Cross-reactivity

Potentially cross-reacting specimens

≥100 in total

including RF+, pregnant women, specimens with antibodies against endemic human coronaviruses 229E, OC43, NL63, HKU1 and other pathogens of respiratory diseases such as influenza A, B, RSV etc.

Table 3: Confirmatory or supplemental
   (20)
  assays for anti-SARS-CoV-2

| Performance characteristic | Specimen | Specimen number, features, use | Acceptance criteria |
| --- | --- | --- | --- |
| Diagnostic sensitivity | Positive specimens | ≥200
  including specimens pre and post seroconversion (within the first 21 days and after 21 days following the onset of symptoms) | Correct determination as “positive” (or “indeterminate”) |
| Seroconversion panels/low titre panels | as far as available |
| Analytical sensitivity | Standards | N/A | N/A |
| Diagnostic specificity | Negative specimens (21) | ≥200 from non-infected / non-vaccinated population | No false positive results;
  correct determination as “negative” (or “indeterminate”) |
| | ≥200 from hospitalised patients (without SARS-CoV-2 infection) |
| Cross-reactivity | Potentially cross-reacting specimens | ≥50 in total
  including specimens with antibodies against endemic human coronaviruses 229E, OC43, NL63, HKU1 and other pathogens of respiratory diseases such as influenza A, B, RSV etc.;
  including specimens with indeterminate or false positive results in other anti-SARS-CoV-2 assays |

Performance characteristic

Specimen

Specimen number, features, use

Acceptance criteria

Diagnostic sensitivity

Positive specimens

≥200

including specimens pre and post seroconversion (within the first 21 days and after 21 days following the onset of symptoms)

Correct determination as “positive” (or “indeterminate”)

Seroconversion panels/low titre panels

as far as available

Analytical sensitivity

Standards

N/A

N/A

Diagnostic specificity

Negative specimens (21)

≥200 from non-infected / non-vaccinated population

No false positive results;

correct determination as “negative” (or “indeterminate”)

≥200 from hospitalised patients (without SARS-CoV-2 infection)

Cross-reactivity

Potentially cross-reacting specimens

≥50 in total

including specimens with antibodies against endemic human coronaviruses 229E, OC43, NL63, HKU1 and other pathogens of respiratory diseases such as influenza A, B, RSV etc.;

including specimens with indeterminate or false positive results in other anti-SARS-CoV-2 assays

Table 4: Antigen assays (including rapid tests): SARS-CoV-2

| Performance characteristic | Specimen | Specimen number, features, use | Acceptance criteria |
| --- | --- | --- | --- |
| Diagnostic sensitivity | Positive specimens | ≥100 (22)

  NAT positive specimens (23) from early infection within the first 7 days after symptom onset (24);
  specimens shall represent naturally occurring viral loads (25);
  consideration of genetic variants (26);
  consideration of variations in specimen collection and/or specimen handling (27) | Detection of >80% (rapid tests);
  detection of >85% (lab-based assays (28));
  relative to SARS-CoV-2-NAT (29), (30) |
| Analytical sensitivity | Standards | As soon as available | Establishment of a LOD (31) |
| Diagnostic specificity | Negative specimens | ≥300
  from non-infected individuals | Specificity >98% (rapid tests)
  Specificity >99% (lab-based assays (28)) |
| ≥100 from hospitalised patients | Potential limitations for specificity, if any, shall be identified |
| Cross-reactivity | Potentially cross-reacting specimens | ≥50 in total
  including virus-positive specimens of endemic human coronaviruses 229E, OC43, NL63, HKU1; influenza A, B, RSV, and other pathogens of respiratory diseases, eligible for differential diagnosis; including bacteria (32) present in the specimen taking area |

Performance characteristic

Specimen

Specimen number, features, use

Acceptance criteria

Diagnostic sensitivity

Positive specimens

≥100 (22)

NAT positive specimens (23) from early infection within the first 7 days after symptom onset (24);

specimens shall represent naturally occurring viral loads (25);

consideration of genetic variants (26);

consideration of variations in specimen collection and/or specimen handling (27)

Detection of >80% (rapid tests);

detection of >85% (lab-based assays (28));

relative to SARS-CoV-2-NAT (29), (30)

Analytical sensitivity

Standards

As soon as available

Establishment of a LOD (31)

Diagnostic specificity

Negative specimens

≥300

from non-infected individuals

Specificity >98% (rapid tests)

Specificity >99% (lab-based assays (28))

≥100 from hospitalised patients

Potential limitations for specificity, if any, shall be identified

Cross-reactivity

Potentially cross-reacting specimens

≥50 in total

including virus-positive specimens of endemic human coronaviruses 229E, OC43, NL63, HKU1; influenza A, B, RSV, and other pathogens of respiratory diseases, eligible for differential diagnosis; including bacteria (32) present in the specimen taking area

Table 5: NAT devices for SARS-CoV-2 RNA

| Performance characteristic | Specimen | SARS-CoV-2 RNA qualitative | SARS-CoV-2 RNA quantitative |
| --- | --- | --- | --- |
| Sensitivity |
| Analytical sensitivity: LOD | WHO First International Standard SARS-CoV-2 RNA (NIBSC code 20/146; 7.70 Log10 IU/mL)
  Secondary standards calibrated against WHO IS | According to Ph. Eur. NAT validation guideline:
  several dilution series into borderline concentration; statistical analysis (e.g. Probit analysis) on the basis of at least 24 replicates; calculation of 95 % cut-off value | According to Ph. Eur. NAT validation guideline:
  several dilution series of calibrated reference preparations into borderline concentration; statistical analysis (e.g. Probit analysis) on the basis of at least 24 replicates; calculation of 95 % cut-off value as LOD |
| Quantification limit; quantification features | WHO First International Standard SARS-CoV-2 RNA (NIBSC code 20/146; 7.70 Log10 IU/mL)
  Secondary standards calibrated against WHO IS | | Dilutions (half-log10 or less) of calibrated reference preparations; determination of lower, upper quantification limit, LOD, precision, accuracy, “linear” measuring range, “dynamic range”. Synthetic target nucleic acid may be used as secondary standard to achieve higher concentration levels. Reproducibility at different concentration levels to be shown |
| Diagnostic sensitivity: different SARS-CoV-2 RNA strains | Patient specimens determined as SARS-CoV-2 RNA positive by comparator device from different regions and outbreak clusters; sequence variants
  Dilution series of SARS-CoV-2 positive cell cultures (isolates) may serve as potential substitutes | ≥100 (33) | |
| Quantification efficiency | SARS-CoV-2 RNA positive patient specimens from different regions and outbreak clusters; sequence variants
  with quantitative values obtained by comparator device
  Dilution series of SARS-CoV-2 RNA positive cell cultures may serve as potential substitutes | | ≥100 |
| Inclusivity | In silico analysis (34);
  at least two independent target gene regions in one test run (dual-target design) | Evidence of suitable device design: primer/probe sequence alignments with published SARS-CoV-2 sequences | Evidence of suitable device design: primer/probe sequence alignments with published SARS-CoV-2 sequences |
| Specificity |
| Diagnostic specificity | SARS-CoV-2 RNA negative human specimens | ≥500 | ≥100 |
| In silico analysis (34) | | Evidence of suitable device design (sequence alignments); regular check of primer/probe sequences against sequence data bank entries | Evidence of suitable device design evidence (sequence alignments); regular check of primer/probe sequences against sequence data bank entries |
| Cross-reactivity | specimens positive (various concentrations) for related human coronaviruses 229E, HKU1, OC43, NL63, MERS coronavirus; SARS CoV-1 if available; Influenza virus A, B; RSV; Legionella pneumophila;
  positive cell cultures may serve as potential substitutes | ≥20 in total | ≥20 in total |
| Robustness |
| Carry-over | | At least 5 runs using alternating high positive and negative specimens. The virus titres of the high positive specimens shall be representative of high virus titres occurring naturally. | At least 5 runs using alternating high positive (known to occur naturally) and negative specimens |
| Inhibition | | Internal control preferably to go through the whole NAT procedure | Internal control preferably to go through the whole NAT procedure |
| Whole system failure rate leading to false negative results: 99/100 assays positive | | ≥100 specimens virus-spiked with 3 × the 95 % positive cut-off concentration (3 x LOD) | ≥100 specimens virus-spiked with 3 × the 95 % positive cut-off concentration (3 x LOD) |

Performance characteristic

Specimen

SARS-CoV-2 RNA qualitative

SARS-CoV-2 RNA quantitative

Sensitivity

Analytical sensitivity: LOD

WHO First International Standard SARS-CoV-2 RNA (NIBSC code 20/146; 7.70 Log10 IU/mL)

Secondary standards calibrated against WHO IS

According to Ph. Eur. NAT validation guideline:

several dilution series into borderline concentration; statistical analysis (e.g. Probit analysis) on the basis of at least 24 replicates; calculation of 95 % cut-off value

According to Ph. Eur. NAT validation guideline:

several dilution series of calibrated reference preparations into borderline concentration; statistical analysis (e.g. Probit analysis) on the basis of at least 24 replicates; calculation of 95 % cut-off value as LOD

Quantification limit; quantification features

WHO First International Standard SARS-CoV-2 RNA (NIBSC code 20/146; 7.70 Log10 IU/mL)

Secondary standards calibrated against WHO IS

Dilutions (half-log10 or less) of calibrated reference preparations; determination of lower, upper quantification limit, LOD, precision, accuracy, “linear” measuring range, “dynamic range”. Synthetic target nucleic acid may be used as secondary standard to achieve higher concentration levels. Reproducibility at different concentration levels to be shown

Diagnostic sensitivity: different SARS-CoV-2 RNA strains

Patient specimens determined as SARS-CoV-2 RNA positive by comparator device from different regions and outbreak clusters; sequence variants

Dilution series of SARS-CoV-2 positive cell cultures (isolates) may serve as potential substitutes

≥100 (33)

Quantification efficiency

SARS-CoV-2 RNA positive patient specimens from different regions and outbreak clusters; sequence variants

with quantitative values obtained by comparator device

Dilution series of SARS-CoV-2 RNA positive cell cultures may serve as potential substitutes

≥100

Inclusivity

In silico analysis (34);

at least two independent target gene regions in one test run (dual-target design)

Evidence of suitable device design: primer/probe sequence alignments with published SARS-CoV-2 sequences

Evidence of suitable device design: primer/probe sequence alignments with published SARS-CoV-2 sequences

Specificity

Diagnostic specificity

SARS-CoV-2 RNA negative human specimens

≥500

≥100

In silico analysis (34)

Evidence of suitable device design (sequence alignments); regular check of primer/probe sequences against sequence data bank entries

Evidence of suitable device design evidence (sequence alignments); regular check of primer/probe sequences against sequence data bank entries

Cross-reactivity

specimens positive (various concentrations) for related human coronaviruses 229E, HKU1, OC43, NL63, MERS coronavirus; SARS CoV-1 if available; Influenza virus A, B; RSV; Legionella pneumophila;

positive cell cultures may serve as potential substitutes

≥20 in total

≥20 in total

Robustness

Carry-over

At least 5 runs using alternating high positive and negative specimens. The virus titres of the high positive specimens shall be representative of high virus titres occurring naturally.

At least 5 runs using alternating high positive (known to occur naturally) and negative specimens

Inhibition

Internal control preferably to go through the whole NAT procedure

Internal control preferably to go through the whole NAT procedure

Whole system failure rate leading to false negative results: 99/100 assays positive

≥100 specimens virus-spiked with 3 × the 95 % positive cut-off concentration (3 x LOD)

≥100 specimens virus-spiked with 3 × the 95 % positive cut-off concentration (3 x LOD)

Table 6: Additional requirements for SARS-CoV-2 antigen self-tests
   (35)

| Performance characteristic | Specimens (36) | Number of lay persons |
| --- | --- | --- |
| Result interpretation (37) | Interpretation of results (38) by lay persons reflecting the following range of reactivity levels:






  — | non-reactive |

Performance characteristic

Specimens (36)

Number of lay persons

Result interpretation (37)

Interpretation of results (38) by lay persons reflecting the following range of reactivity levels:

—

non-reactive

| — | reactive |
| --- | --- |

—

reactive

| — | weak reactive (39) |
| --- | --- |

—

weak reactive (39)

| — | invalid |
| --- | --- |

—

invalid

≥100

Diagnostic sensitivity (40)

Lay persons that are known antigen positive (41)
  ,
   (42)

≥30

Diagnostic specificity (43)

Lay persons that do not know their status (39)

≥60

Table 7: Additional requirements for SARS-CoV-2 antibody self-tests
   (44)

| Performance characteristic | Specimens (45) | Number of lay persons |
| --- | --- | --- |
| Result interpretation (46) | Interpretation of results (47) by lay persons reflecting the following range of reactivity levels:






  — | non-reactive |

Performance characteristic

Specimens (45)

Number of lay persons

Result interpretation (46)

Interpretation of results (47) by lay persons reflecting the following range of reactivity levels:

—

non-reactive

| — | reactive |
| --- | --- |

—

reactive

| — | weak reactive (48) |
| --- | --- |

—

weak reactive (48)

| — | invalid |
| --- | --- |

—

invalid

≥100

Diagnostic sensitivity (49)

Lay persons that are known antibody positive (50)

≥100

Diagnostic specificity (51)

Lay persons that do not know their status (48)

≥100

---

(1)  Performance claim of the combined overall result; for devices with separate claims for IgM and/or IgA, see table 2.

(2)  Details on the time interval between specimen taking and onset of symptoms (or time of infection, if available) shall be provided.

(3)  The manufacturer shall provide a justification of the suitability and timing for sensitivity evaluation of the relevant antibodies in vaccinated individuals.

(4)  Based on confirmed positive SARS-CoV-2 NAT result.

(5)  Claims for sensitivity shall be specified in relation to the time between specimen taking after symptom onset or the initial PCR diagnosis and the test.

(6)  CE marked under Regulation (EU) 2017/746 as class D, if available.

(7)  This applies to quantitative assays if they are also first-line assays.

(8)  Negative specimens shall be from individuals with no history of SARS-CoV-2 infection (if available pre-pandemic).

(9)  Individuals vaccinated against an antigen different from that used in the device may be included, if appropriate.

(10)  False positive results shall be resolved by re-testing in other SARS-CoV-2 serological assays, if necessary with different test design and antigen coating compared to the initial test, and/or confirmatory testing.

(11)  In case of devices detecting both IgM and IgA, 200 per marker IgM and IgA.

(12)  Details on the time interval between specimen taking and onset of symptoms (or time of infection, if available) shall be provided.

(13)  The manufacturer shall provide a justification of the suitability and timing for sensitivity evaluation of IgM and IgA in vaccinated individuals.

(14)  Diagnosis based on confirmed positive SARS-CoV-2 NAT result.

(15)  Claims for sensitivity shall be specified in relation to the time between specimen taking after symptom onset or the initial PCR diagnosis and the test.

(16)  CE marked under Regulation (EU) 2017/746 as class D, if available.

(17)  Negative specimens shall be from individuals with no history of SARS-CoV-2 infection (if available pre-pandemic).

(18)  Individuals vaccinated against an antigen different from that used in the device may be included, if appropriate.

(19)  False positive results shall be resolved by re-testing in other SARS-CoV-2 serological assays, if necessary with different test design and antigen coating compared to the initial test, and/or confirmatory testing. Clarification of false positive results may additionally include testing for presence of other anti-SARS-CoV-2 antibody types (IgA, IgG, total antibody).

(20)  E.g. immunoblot with antigens different from those used in the initial antibody test.

(21)  Negative specimens shall be from individuals with no history of SARS-CoV-2 infection (if available pre-pandemic).

(22)  If the device is intended to be used for more than one specimen type, 100 specimens shall be required for each specimen type. If this is not possible in exceptional circumstances (e.g. if specimen collection is very invasive), the manufacturer shall provide a justification and evidence of matrix equivalence.

(23)  Specimen taking shall be matched for antigen and NAT testing, e.g., two simultaneous specimens from each individual or optimally NAT- and antigen testing from the same specimen (e.g. from the eluate of one swab); the buffer/transport medium shall be compatible with antigen testing; any volume change in the buffer/medium for specimen uptake between antigen and NAT device shall be clearly communicated.

(24)  Or time of infection, if known, taking into account the incubation time.

(25)  I.e., without preselection; the viral loads and their distribution shall be shown, e.g. characterized by Ct-values of RT-PCR; or transformed into viral load per ml of specimen, if applicable.

(26)  Depending on the design of the device and nature of the genetic variant. For the purpose of evaluation, at least 3 specimens shall be represented for each relevant genetic variant.

(27)  Specimen collection and extraction items such as swabs, extraction buffers, etc., shall be part of the evaluation. If proprietary specimen taking/preparation is not included in the device, device performance shall be investigated for an applicable range of specimen taking devices. If the specimen is not tested immediately, e.g. after a certain transport time, stability of the antigen shall be investigated.

(28)  Other than rapid tests, i.e. formal laboratory-based devices e.g. enzyme immunoassay, automated tests, etc.

(29)  The sensitivity of ≥80%, ≥85% respectively, shall be for all specimen types claimed. All claimed specimen types shall be compared with paired NAT results from nasopharyngeal specimens.

(30)  The relationship between the sensitivity of the antigen test and of the NAT shall be demonstrated; sensitivity may be shown relating to different viral load ranges and to the threshold of infectivity. The NAT and extraction method used shall be described.

(31)  Unless there is an available international standard, analytical sensitivity may be tested by dilution series of in-house virus preparations, comparatively with other antigen tests and NAT; if inactivated virus is used, the effect of inactivation and freeze/thawing on the antigen shall be investigated.

(32)  E.g. staphylococci and streptococci expressing protein A or G.

(33)  If the device is intended to be used for more than one specimen type, 100 specimens shall be required for each specimen type. If this is not possible in exceptional circumstances (e.g. if specimen collection is very invasive), the manufacturer shall provide a justification and evidence of matrix equivalence.

(34)  The manufacturer shall document evidence of proactive regular surveillance checks against updated data bank entries in the post-market performance follow-up report.

(35)  It is assumed that the underlying performance of the self-test has already been previously demonstrated with the evaluation/assessment of a professional test of the same design as the respective self-test under evaluation. In case for the self-use specimens in question there is no corresponding professional test variant, comparison shall be made with the standard specimen type (e.g. nasopharyngeal swabs for antigen test, serum or plasma for antibody test) of the corresponding professional test.

(36)  For each self-use specimen type claimed with the device (e.g. nasal specimen, sputum, saliva, whole blood, etc.).

(37)  The result interpretation study shall include reading and interpretation of the test results by at least 100 lay persons, with each lay person subjected to reading results covering the specified range of result reactivity levels. The manufacturer shall determine the concordance between lay person reading and professional user reading.

(38)  Tests shall be performed prior to the result interpretation study using whenever possible the specimen type intended by the manufacturer. Tests may be performed on contrived specimens based on the natural matrix of the respective specimen type.

(39)  A higher proportion of the specimens shall be in the low-positive range close to the cut-off or LOD of the test.

(40)  In comparison to RT-PCR. The manufacturer shall determine the concordance between lay person reading and professional user reading.

(41)  Individuals unaware of the professional diagnostic result prior to self-testing, and performing the entire test procedure from specimen collection and specimen pre-treatment (swab, buffer extraction, etc.) to reading.

(42)  Subjects up to about 7 days after symptom onset.

(43)  The manufacturer shall determine the concordance between lay person reading and professional user reading.

(44)  It is assumed that the underlying performance of the self-test has already been previously demonstrated with the evaluation/assessment of a professional test of the same design as the respective self-test under evaluation. In case for the self-use specimens in question there is no corresponding professional test variant, comparison shall be made with the standard specimen type (e.g. nasopharyngeal swabs for antigen test, serum or plasma for antibody test) of the corresponding professional test.

(45)  For each self-use specimen type claimed with the device (e.g. nasal specimen, sputum, saliva, whole blood, etc.).

(46)  The result interpretation study shall include reading and interpretation of the test results by at least 100 lay persons, with each lay person subjected to reading results covering the specified range of result reactivity levels. The manufacturer shall determine the concordance between lay person reading and professional user reading.

(47)  Tests shall be performed prior to the result interpretation study using whenever possible the specimen type intended by the manufacturer. Tests may be performed on contrived specimens based on the natural matrix of the respective specimen type.

(48)  A higher proportion of the specimens shall be in the low-positive range close to the cut-off or LOD of the test.

(49)  With previous history of initial RT PCR-confirmed infection for SARS-CoV-2; in comparison to a previous confirmed antibody result. The manufacturer shall determine the concordance between lay person reading and professional user reading.

(50)  Individuals unaware of the professional diagnostic result prior to self-testing, and performing the entire test procedure from specimen collection and specimen pre-treatment (swab, buffer extraction, etc.) to reading.

(51)  The manufacturer shall determine the concordance between lay person reading and professional user reading.

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