Textual Amendments
The common technical specifications set out in this Annex shall apply for the purposes of Annex II List A to Directive 98/79/EC.
The probability that the device gives a positive result in the presence of the target marker.
A specimen known to be positive for the target marker and correctly classified by the device.
A specimen known to be positive for the target marker and misclassified by the device.
The probability that the device gives a negative result in the absence of the target marker.
A specimen known to be negative for the target marker and misclassified by the device.
A specimen known to be negative for the target marker and correctly classified by the device.
Analytical sensitivity may be expressed as the limit of detection, i.e. the smallest amount of the target marker that can be precisely detected.
Analytical specificity means the ability of the method to determine solely the target marker.
The term ‘ NAT ’ is used for tests for the detection and/or quantification of nucleic acids by either amplification of a target sequence, by amplification of a signal or by hybridisation.
‘Rapid test’ means qualitative or semi-quantitative in vitro diagnostic medical devices, used singly or in a small series, which involve non-automated procedures and have been designed to give a fast result.
The robustness of an analytical procedure 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.
The whole system failure rate means the frequency of failures when the entire process is performed as prescribed by the manufacturer.
Confirmation assay means an assay used for the confirmation of a reactive result from a screening assay.
Virus typing assay means an assay used for typing with already known positive samples, not used for primary diagnosis of infection or for screening.
Sero-conversion HIV samples mean:
p24 antigen and/or HIV RNA positive, and
recognised by all of the antibody screening tests, and
positive or indeterminate confirmatory assays.
Early seroconversion HIV samples mean:
p24 antigen and/or HIV RNA positive, and
not recognised by all of the antibody screening tests, and
indeterminate or negative confirmatory assays.
by evaluation of the discrepant sample in further test systems,
by use of an alternative method or marker,
by a review of the clinical status and diagnosis of the patient, and
by the testing of follow-up-samples.
Diagnostic test sensitivity during sero-conversion has to represent the state of the art. Whether further testing of the same or additional sero-conversion panels is conducted by the notified body or by the manufacturer the results shall confirm the initial performance evaluation data (see Table 1). Sero-conversion panels should start with a negative bleed(s) and should have narrow bleeding intervals.
For blood screening devices (with the exception of HBsAg and anti-HBc tests), all true positive samples shall be identified as positive by the device to be CE marked (Table 1). For HBsAg and anti-HBc tests the new device shall have an overall performance at least equivalent to that of the established device (see 3.1.4).
Regarding HIV tests:
all sero-conversion HIV samples shall be identified as positive, and
at least 40 early sero-conversion HIV samples shall be tested. Results should conform to the state of the art.
specimens representing ‘ related ’ infections,
specimens from multipara, i.e. women who have had more than one pregnancy, or rheumatoid factor positive patients,
for recombinant antigens, human antibodies to components of the expression system, for example anti-E. coli, or anti-yeast.
The performance evaluation criteria for NAT assays can be found in Table 2.
Criteria for performance evaluation of reagents and reagent products for determining the blood groups antigens: ABO blood group system ABO1 (A), ABO2 (B), ABO3 (A,B); Rh blood group system RH1 (D), RH2 (C), RH3 (E), RH4 (c), RH5 (e); Kell blood group system KEL1 (K) can be found in Table 9.
by evaluation of the discrepant sample in further test systems,
by use of an alternative method,
‘ Screening ’ assays: anti-HIV 1 and 2, anti-HTLV I and II, anti-HCV, HBsAg, anti-HBc
Anti-HIV-1/2 | Anti-HTLV-I/II | Anti-HCV | HBsAg | Anti-HBc | ||
---|---|---|---|---|---|---|
Diagnostic sensitivity | Positive specimens | 400 HIV-1 100 HIV-2 including 40 non-B subtypes, all available HIV/1 subtypes should be represented by at least 3 samples per subtype | 300 HTLV-I 100 HTLV-II | 400 (positive samples) Including samples from different stages of infection and reflecting different antibody patterns. Genotype 1-4: > 20 samples per genotype (including non-a subtypes of genotype 4); 5: > 5 samples; 6: if available | 400 Including subtypeconsideration | 400 Including evaluation of other HBV-markers |
Sero-conversion panels | 20 panels 10 further panels (at Notified Body or manufacturer) | To be defined when available | 20 panels 10 further panels (at Notified Body or manufacturer) | 20 panels 10 further panels (at Notified Body or manufacturer) | To be defined when available | |
Analytical sensitivity | Standards | 0,130 IU/ml (Second International Standard for HBsAg, subtype adw2, genotype A, NIBSC code: 00/588) | ||||
Specificity | Unselected donors (including first-time donors) | 5 000 | 5 000 | 5 000 | 5 000 | 5 000 |
Hospitalised patients | 200 | 200 | 200 | 200 | 200 | |
Potentially cross-reacting blood-specimens (RF+, related viruses, pregnant women, etc.) | 100 | 100 | 100 | 100 | 100 |
NAT assays for HIV1, HCV, HBV, HTLV I/II (qualitative and quantitative; not molecular typing)
a European Pharmacopoeia guideline. | |||||||||
Notes: Acceptance criteria for ‘whole system failure rate leading to false-neg results’ is 99/100 assays positive. For quantitative NATs a study shall be performed on at least 100 positive specimens reflecting the routine conditions of users (e.g. no pre-selection of specimens). Comparative results with another NAT test system shall be generated in parallel. For qualitative NATs a study on diagnostic sensitivity shall be performed using at least 10 sero-conversion panels. Comparative results with another NAT test system shall be generated in parallel. | |||||||||
HIV1 | HCV | HBV | HTLV I/II | Acceptance criteria | |||||
---|---|---|---|---|---|---|---|---|---|
NAT | qualitative | quantitative | qualitative | quantitative | qualitative | quantitative | qualitative | quantitative | |
As for HIV quantitative | As for HIV quantitative | As for HIV quantitative | |||||||
Sensitivity Detection limit Detection of analytical sensitivity (IU/ml; defined on WHO standards or calibrated reference materials) | According to EP validation guideline a : 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 | Detection limit: as for qualitative tests; Quantification limit: dilutions (half-log10 or less) of calibrated reference preparations, definition of lower, upper quantification limit, precision, accuracy, ‘ linear ’ measuring range, ‘ dynamic range ’ . Reproducibility at different concentration levels to be shown | According to EP validation guideline a : 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 EP validation guideline a : 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 EP validation guideline a : 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 | ||||
Genotype/subtype detection/quantification efficiency | At least 10 samples per subtype (as far as available) | Dilution series of all relevant genotypes/subtypes, preferably of reference materials, as far as available | At least 10 samples per genotype (as far as available) | As far as calibrated genotype reference materials are available | As far as calibrated genotype reference materials are available | ||||
Cell culture supernatants (could substitute for rare HIV-1 subtypes) | Transcripts or plasmids quantified by appropriate methods may be used. | ||||||||
According to EP validation guideline a as far as calibrated subtype reference materials are available; in vitro transcripts could be an option | According to EP validation guideline a as far as calibrated subtype reference materials are available; in vitro transcripts could be an option | According to EP validation guideline a as far as calibrated subtype reference materials are available; in vitro transcripts could be an option | According to EP validation guideline a as far as calibrated subtype reference materials are available; in vitro transcripts could be an option | ||||||
Diagnostic specificity negative samples | 500 blood donors | 100 blood donors | 500 blood donors | 500 blood donors | 500 individual blood donations | ||||
Potential cross-reactive markers | By suitable assay design evidence (e.g. sequence comparison) and/or testing of at least 10 human retrovirus (e.g. HTLV)-positive samples | As for qualitative tests | By assays design and/or testing of at least 10 human flavivirus (e.g. HGV, YFV) positive samples | By assays design and/or testing of at least 10 other DNA-virus positive samples | By assay design and/or testing of at least 10 human retrovirus (e.g. HIV-) positive samples | ||||
Robustness | As for qualitative tests | ||||||||
Cross-contamination | At least 5 runs using alternating high positive (known to occur naturally) and negative samples | At least 5 runs using alternating high positive (known to occur naturally) and negative samples | At least 5 runs using alternating high positive (known to occur naturally) and negative samples | At least 5 runs using alternating high positive (known to occur naturally) and negative samples | |||||
Inhibition | Internal control preferably to go through the whole NAT procedure | Internal control preferably to go through the whole NAT procedure | 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-neg results | At least 100 samples virus-spiked with 3 × the 95 % pos cut-off concentration | At least 100 samples virus-spiked with 3 × the 95 % pos cut-off concentration | At least 100 samples virus-spiked with 3 × the 95 % pos cut-off concentration | At least 100 samples virus-spiked with 3 × the 95 % pos cut-off concentration | 99/100 assays positive |
Rapid tests: anti-HIV 1 and 2, anti-HCV, HBsAg, anti-HBc, anti-HTLV I and II
Anti-HIV 1/2 | Anti-HCV | HBsAg | Anti-HBc | Anti-HTLV I/II | Acceptance criteria | ||
---|---|---|---|---|---|---|---|
Diagnostic sensitivity | Positive specimens | Same criteria as for screening assays | Same criteria as for screening assays | Same criteria as for screening assays | Same criteria as for screening assays | Same criteria as for screening assays | Same criteria as for screening assays |
Sero-conversion panels | Same criteria as for screening assays | Same criteria as for screening assays | Same criteria as for screening assays | Same criteria as for screening assays | Same criteria as for screening assays | Same criteria as for screening assays | |
Diagnostic specificity | Negative specimens | 1 000 blood donations | 1 000 blood donations | 1 000 blood donations | 1 000 blood donations | 1 000 blood donations | ≥ 99 % (anti-HBc: ≥ 96 %) |
200 clinical specimens | 200 clinical specimens | 200 clinical specimens | 200 clinical specimens | 200 clinical specimens | |||
200 samples from pregnant women | 200 samples from pregnant women | 200 samples from pregnant women | 200 samples from pregnant women | ||||
100 potentially interfering samples | 100 potentially interfering samples | 100 potentially interfering samples | 100 potentially interfering samples | 100 potentially interfering samples |
Confirmatory/supplementary assays for anti-HIV 1 and 2, anti-HTLV I and II, anti-HCV, HBsAg
a Acceptance criteria no neutralisation for HBsAg confirmatory assay. | ||||||
Anti-HIV confirmatory assay | Anti-HTLV confirmatory assay | HCV supplementary assay | HBsAg confirmatory assay | Acceptance criteria | ||
---|---|---|---|---|---|---|
Diagnostic sensitivity | Positive specimens | 200 HIV-1 and 100 HIV-2 | 200 HTLV-I and 100 HTLV-II | 300 HCV (positive samples) | 300 HBsAg | Correct identification as positive (or indeterminate), not negative |
Including samples from different stages of infection and reflecting different antibody patterns | Including samples from different stages of infection and reflecting different antibody patterns. Genotypes 1 – 4: > 20 samples (including non-a subtypes of genotype 4); 5: > 5 samples; 6: if available | Including samples from different stages of infection 20 ‘ high pos ’ samples (> 26 IU/ml); 20 samples in the cut-off range | ||||
Sero-conversion panels | 15 sero-conversion panels/low titre panels | 15 sero-conversion panels/low titre panels | 15 sero-conversion panels/low titre panels | |||
Analytical sensitivity | Standards | Second International Standard for HBsAg, subtype adw2, genotype A, NIBSC code: 00/588 | ||||
Diagnostic specificity | Negative specimens | 200 blood donations | 200 blood donation | 200 blood donations | 10 false positives as available from the performance evaluation of the screening assay a . | No false-positive results/ a no neutralisation |
200 clinical samples including pregnant women | 200 clinical samples including pregnant women | 200 clinical samples including pregnant women | ||||
50 potentially interfering samples, including samples with indeterminate results in other confirmatory assays | 50 potentially interfering samples including samples with indeterminate results in other confirmatory assays | 50 potentially interfering samples including samples with indeterminate results in other supplementary assays | 50 potentially interfering samples |
HIV 1 antigen
HIV-1 antigen assay | Acceptance criteria | ||
---|---|---|---|
Diagnostic sensitivity | Positive specimens | 50 HIV-1 Ag-positive 50 cell culture supernatants including different HIV-1 subtypes and HIV-2 | Correct identification (after neutralisation) |
Sero-conversion panels | 20 sero-conversion panels/low titre panels | ||
Analytical sensitivity | Standards | HIV-1 p24 Antigen, First International Reference Reagent, NIBSC code: 90/636 | ≤ 2 IU/ml |
Diagnostic specificity | 200 blood donations 200 clinical samples 50 potentially interfering samples | ≥ 99,5 % after neutralisation |
Serotyping and genotyping assay: HCV
HCV serotyping and genotyping assay | Acceptance criteria | ||
---|---|---|---|
Diagnostic sensitivity | Positive specimens | 200 (positive samples) Including samples from different stages of infection and reflecting different antibody patterns. Genotypes 1 – 4: > 20 samples (including non-a subtypes of genotype 4); 5: > 5 samples; 6: if available | ≥ 95 % agreement between serotyping and genotyping [X1> 95 % agreement between genotyping and sequencing] |
Diagnostic specificity | Negative specimens | 100 |
Editorial Information
HBV markers: anti-HBs, anti HBc IgM, anti-HBe, HBeAg
Anti-HBs | Anti-HBc IgM | Anti-HBe | HBeAg | Acceptance criteria | ||
---|---|---|---|---|---|---|
Diagnostic sensitivity | Positive specimens | 100 vaccinees | 200 | 200 | 200 | ≥ 98 % |
100 naturally infected persons | Including samples from different stages of infection (acute/chronic, etc.) The acceptance criteria should only be applied on samples from acute infection stage. | Including samples from different stages of infection (acute/chronic, etc.) | Including samples from different stages of infection (acute/chronic, etc.) | |||
Sero-conversion panels | 10 follow-ups or anti-HBs sero-conversions | When available | ||||
Analytical sensitivity | Standards | WHO First International Reference Preparation 1977; NIBSC, United Kingdom | HBe — Referenzantigen 82; PEI Germany | Anti-HBs: < 10 mIU/ml | ||
Diagnostic specificity | Negative specimens | 500 | 200 blood donations | 200 blood donation | 200 blood donations | ≥ 98 % |
Including clinical samples | 200 clinical samples | 200 clinical samples | 200 clinical samples | |||
50 potentially interfering samples | 50 potentially interfering samples | 50 potentially interfering samples | 50 potentially interfering samples |
HDV markers: anti-HDV, anti-HDV IgM, delta antigen
Anti-HDV | Anti-HDV IgM | Delta antigen | Acceptance criteria | ||
---|---|---|---|---|---|
Diagnostic sensitivity | Positive specimens | 100 | 50 | 10 | ≥ 98 % |
Specifying HBV markers | Specifying HBV markers | Specifying HBV markers | |||
Diagnostic specificity | Negative specimens | 200 | 200 | 200 | ≥ 98 % |
Including clinical samples | Including clinical samples | Including clinical samples | |||
50 potentially interfering samples | 50 potentially interfering samples | 50 potentially interfering samples |
Blood group antigens in the ABO, Rh and Kell blood group systems
1 | 2 | 3 | |
---|---|---|---|
Specificity | Number of tests per recommended method | Total number of samples to be tested for a launch product | Total number of samples to be tested for a new formulation, or use of well-characterised reagents |
Anti-ABO1 (anti-A), anti-ABO2 (anti-B), anti-ABO3 (anti-A,B) | 500 | 3 000 | 1 000 |
Anti-RH1 (anti-D) | 500 | 3 000 | 1 000 |
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 |
All of the above reagents shall show comparable test results with established reagents with acceptable performance with regard to claimed reactivity of the device. For established reagents, where the application or use has been changed or extended, further testing should be carried out in accordance with the requirements outlined in column 1 (above).
Performance evaluation of anti-D reagents shall include tests against a range of weak RH1 (D) and partial RH1 (D) samples, depending on the intended use of the product.
:
10 % of the test population
:
> 2 % of the test population
:
> 40 % A, B positives
:
> 2 % of RH1 (D) positives
a Only by recommended techniques where reactivity against these antigens is claimed. | ||||||||
Note: Polyclonal reagents must 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 | |||||||
---|---|---|---|---|---|---|---|---|
Positive reactions | Negative reactions | |||||||
A1 | A2B | Ax | B | 0 | ||||
Anti-ABO1 (anti-A) | 2 | 2 | 2 a | 2 | 2 | |||
B | A1B | A1 | 0 | |||||
Anti-ABO2 (anti-B) | 2 | 2 | 2 | 2 | ||||
A1 | A2 | Ax | B | 0 | ||||
Anti-ABO3 (anti-A,B) | 2 | 2 | 2 | 2 | 4 | |||
R1r | R2r | WeakD | r’r | r’r | rr | |||
Anti-RH1 (anti-D) | 2 | 2 | 2 a | 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-RH 3 (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 |
Each batch of reagent must exhibit unequivocal positive or negative results by all recommended techniques in accordance with the results obtained from the performance evaluation data.
The phenotype of red cells used in the control of blood typing reagents listed above should be confirmed using established device.]