TSS-2In vitro diagnostic medical devices to identify Glucose-6-phosphate dehydrogenase (G6PD) activity

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Technical Specifications Series

for submission to WHO Prequalification – Diagnostic Assessment

TSS-2

In vitro diagnostic medical

devices to identify Glucose-6-

phosphate dehydrogenase

(G6PD) activity

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Technical Specifications Series for submission to WHO Prequalification – Diagnostic Assessment:

IVDs to identify G6PD activity TSS-2 Technical Specifications Series for submission to WHO prequalification – diagnostic assessment: in vitro diagnostics medical devices to identify Glucose-6-phosphate dehydrogenase (G6PD) activity Published 21 December 2016

ISBN 978-92-4-151186-5

© World Health Organization 2016

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Table of contents ... 3

Acknowledgements ... 4

List of contributors ... 4

A. Introduction ... 6

B. Other guidance documents ... 6

C. Performance principles for WHO prequalification ... 6

C.1 Intended use ... 6

C.2 Diversity of specimen types, users and testing environments and impact on required studies ... 7

C.3 Applicability of supporting evidence to IVD under review ... 7

D. Table of Requirements ... 9

Part 1 Establishing analytical performance ... 10

Part 2 Establishing clinical performance ... 17

E. References ... 19

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IVDs to identify G6PD activity TSS-2

Acknowledgements

The document “TSS-2 Technical Specifications Series for submission to WHO Prequalification – Diagnostic Assessment: In vitro diagnostics medical devices to identify Glucose-6-phosphate dehydrogenase (G6PD) activity” was developed with support from the Bill & Melinda Gates Foundation and UNITAID. The document was prepared in collaboration with Dr M. Lanigan, Geneva, Switzerland and R. Meurant, WHO and with input and expertise from D. Healy; H. Ardura and Dr J.

Cunningham, WHO. This document was produced under the coordination and supervision of R.

Meurant and I. Prat, Prequalification Team, WHO, Geneva, Switzerland.

List of contributors

A technical consultation on WHO prequalification requirements for in vitro diagnostic medical devices (IVDs) to detect G6PD deficiency was held in Geneva, Switzerland from 26 to 28 September 2016.

Meeting participants

B. Ayalew, Ethiopian Food, Medicine and Healthcare Administration and Control Authority, Addis Ababa, Ethiopia (representing Pan African Harmonization Working Party PAHWP); Dr G. Bancone, Shoklo, Malaria Research Unit, Thailand

¹

; Dr L. Carrington, Center for Devices and Radiological Health, US Food and Drug Administration, MD, USA

²

; Dr M. Gatton, Queensland University of Technology, Australia

³

; Dr M. Guimaraes de Lacerda, Fundaco de Medicana Tropical do Amazonas Manaus, Brazil; N. Kitiwouranon, Food and Drug Administration, Bangkok, Thailand; Dr M. Lanigan, Geneva, Switzerland; J. Luchavez, Research Institute for Tropical Medicine, Muntinlupa City, The Philippines; L. Luzzatto, Dar es Salaam, The United Republic of Tanzania, M. McNiven, Therapeutic Goods Administration, Canberra, Australia; L. Musset, Institut Pasteur de la Guyane, Guyana;

Professor A. Poon, Asian Harmonization Working Party, Hong Kong, China; Dr J. Richards, Burnet Institute, Melbourne, Australia; Dr L. Robertson, Imperial College Healthcare, London, United Kingdom.

Meeting participants with observer status

Dr G. Domingo,

PATH, Seattle, USA; D

r S. Pal, PATH, Seattle, USA.

WHO Secretariat

H. Ardura; D. Healy; R. Meurant; I. Prat; A Sands; Dr W. Urassa, Prequalification team, Essential Medicines Programme; Dr A. Bosman

;

Dr J. Cunningham,

Prevention Diagnostics & Treatment, Global Malaria Programme.

The draft technical specifications document has been posted on the WHO website for public consultation on 07 November 2016. Various stakeholders, including manufacturers submitting to WHO Prequalification of IVDs, IVD manufacturing industry associations, various national and international regulatory bodies, and IVD standards organizations were informed of the consultation in order to solicit feedback. A two month response period was provided.

First round of public comment were received for consideration from Dr .L Carrington, Center for Devices and Radiological Health, US Food and Drug Administration, MD, USA; Dr G. Domingo,

PATH, Seattle, USA; Dr J. Duncan, London, The United Kingdom,

Dr S. Hojvat, MD, USA; N. Kitiwouranon, Food

1 Participated via web conferencing

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and Drug Administration, Bangkok, Thailand; Dr M. Kondratovich, Center for Devices and Radiological Health, US Food and Drug Administration, MD, USA; M. McNiven, Therapeutic Goods Administration, Canberra, Australia; Dr J. Richards, Burnet Institute, Melbourne, Australia; Dr R.

Rong, Center for Devices and Radiological Health, US Food and Drug Administration, MD, USA;

D

r S.

Pal, PATH, Seattle, USA.

Second round of public comments were received for consideration from Dr G. Domingo,

PATH, Seattle, USA;

Dr. A. Liebmann-Vinson, BD, NC, USA.

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A. Introduction

The purpose of this document is to provide technical guidance to in vitro diagnostic medical device (IVD) manufacturers that intend to seek WHO prequalification of IVDs for the detection of glucose-6- phosphate dehydrogenase (G6PD) deficiency.

Where possible, WHO performance conditions are aligned with published guidance, standards and/or regulatory documents. Although references to source documents are provided, in some cases WHO has additional requirements. For prequalification purposes, manufacturers must provide evidence in support of the clinical performance of an IVD which can demonstrate that reasonable steps have been taken to ensure that a properly manufactured IVD, being correctly operated in the hands of the intended user, will detect the target analyte and fulfil its indications for use.

Prequalification requirements summarized in this document do not extend to the demonstration of clinical utility, i.e. the effectiveness and/or benefits of an IVD, relative to and/or in combination with other measures, as a tool to inform clinical intervention in a given population or healthcare setting.

To demonstrate clinical utility, a separate set of studies is required. Clinical utility studies usually inform programmatic strategy and are thus the responsibility of programme managers, ministries of health and other related bodies in individual WHO Member States. Such studies do not fall under the scope of prequalification.

B. Other guidance documents

It is recommended that this document is read in conjunction with other WHO guidance documentation, including:



WHO Prequalification Team: Diagnostics “Technical Guidance Series (TGS)”



Sample Product Dossiers for WHO prequalification



WHO document PQDx_018 “Instructions for Compilation of a Product Dossier”

These documents are available at: http://www.who.int/diagnostics_laboratory/evaluations/en/

C. Performance principles for WHO prequalification C.1 Intended use

An IVD intended for prequalification must be accompanied by a sufficiently detailed intended use statement. This should allow an understanding of at least the following:



the function of the IVD (e.g. to differentiate G6PD deficient and intermediate from G6PD normal specimens, etc.) and whether it is qualitative, semi-quantitative or quantitative;



the intended testing population for which functions are intended (e.g. detection of deficient males; detection of deficient and intermediate females; etc.) and the intended operational setting (e.g. for use in near-patient testing); and



clinical indication (e.g. To assess the potential for susceptibly to a serious side effect to a drug or group of drugs for the purpose of guiding appropriate patient therapy and management).

G6PD exhibits considerable genotypic and phenotypic variation and it is expected that there will be a

broad range of enzyme activities observable in a population where deficiency is prevalent. For the

purposes of PQ, the G6PD status of a specimen is defined with reference to the adjusted male

median of a given population

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and differs between males and females

(2)

. For the purposes of

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prequalification, G6PD activities that correspond to ‘deficient’, ‘intermediate’ or ‘normal’ in males or females are defined as:

Male

G6PD deficient G6PD activity < 30% of the adjusted male median G6PD normal G6PD activity > 30% of the adjusted male median Female

G6PD deficient G6PD activity < 30% of the adjusted male median G6PD intermediate G6PD activity 30-80% of the adjusted male median G6PD normal G6PD activity > 80% of the adjusted male median

C.2 Diversity of specimen types, users and testing environments and impact on required studies

For WHO purposes, performance studies should be conducted using the specimen types most likely to be used in resource-limited WHO Member States (e.g. capillary whole blood). If this is not possible, substantial data should be presented to show the equivalence between specimen types used in performance studies.

Prequalified IVDs in low- and middle-income countries are likely to be used by laboratory professionals

⁴

and at point-of-care by healthcare workers or trained lay providers

⁵

. Depending on the intended use of an IVD, performance studies must be designed to take into account not only the diversity of knowledge and skills across the population of IVD users, but also the likely operational settings in which testing will occur including environmental attributes (temperature, humidity) and access to a consistent source of power and clean water.

C.3 Applicability of supporting evidence to IVD under review

Performance requirements summarized in this document correspond to IVDs that are designed to detect G6PD deficiency:



by way of either qualitative, semi-quantitative or quantitative test result; and



that are reported as qualitative, semi-quantitative or quantitative interpretations.

For example, an IVD may be designed to provide numerical (quantitative) output as a test result, which is then interpreted as ‘G6PD deficient’, ‘G6PD intermediate’ or ‘G6PD normal’.

When establishing performance, the true G6PD status of a specimen must be determined using a suitable quantitative reference test, justification for which must be provided. The corresponding quantitative values of G6PD activity (International units per gram of haemoglobin IU/g Hb), and how these were calculated should be reported. Estimation of G6PD activity using a quantitative reference test must take into account the imprecision and bias inherent in that test. Where appropriate specimen characterization should also include testing that ensures that G6PD activity correlates with the genotype of a specimen. Where the reported G6PD activity has been normalised to haemoglobin concentration, traceability to a suitable biological reference material must be provided.

4 Medical technologists, medical laboratory technicians or similar, who have received a formal professional or paraprofessional certificate or tertiary education degree.

5 Any person who performs functions related to healthcare delivery and has been trained to deliver specific services but has received no formal professional or paraprofessional certificate or tertiary education degree.

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IVDs that rely on detection of G6PD activity are highly sensitive to ambient operating conditions, especially temperature. It is important that performance studies are, to the greatest extent possible, conducted using identical operating conditions and that those conditions are reported as part of the performance results, unless the test uses an instrument that automatically corrects for temperature.

Moreover, estimation (and reporting) of IVD performance must include the rate of invalid test results.

For certain analytical studies, it may be acceptable to use contrived specimens (e.g. where G6PD deficiency has been simulated by mixing, or enzyme activity has been artificially reduced). Although all reasonable attempts should be made to use natural specimens, justification should be provided where contrived specimens are used in the submitted studies. Clinical performance studies should be based on testing in natural specimens only.

Studies should be undertaken using the final, or locked-down version of the IVD that is intended to be prequalified. Where this is not possible (e.g. because of design variation) a justification must be provided; additional supporting evidence may also be required. This can occur as minor variations to design where no negative impact on performance has been demonstrated.

Where applicable, several different lots of the IVD should be used, such that each lot comprises

different batches of critical components. Specific information is provided in this document for the

numbers of lots required for particular studies. However, it is a manufacturer’s responsibility to

ensure, via risk analysis of its IVD that the numbers of lots chosen for estimating performance

characteristics takes into account the variability in performance likely to arise from the diversity of

key components and their formulation.

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D. Table of Requirements

PART 1 Establishing analytical performance

1.1 Specimen type

1.1.1 Demonstration of equivalence between specimen types 1.1.2 Demonstration of equivalence of claimed anticoagulants

1.2 Specimen collection, storage and transport

1.2.1 Specimen stability

1.3 Precision of measurement

1.3.1 Repeatability, reproducibility

1.4 Performance panels

1.4.1 Genotype panels

1.5 Validation of reading time

1.5.1 Validation of reading time

1.6 Analytical sensitivity

1.6.1 Analytical sensitivity

1.6.2 Performance at critical decision points 1.6.3 Measuring range and correlation

1.7 Analytical specificity

1.7.1 Potentially interfering substances

1.8 Metrological traceability of calibrators and control material values

1.8.1 Metrological traceability of calibrators and control material values

1.9 Stability

1.9.1 IVD stability 1.9.2 Shelf life 1.9.3 In-use stability

1.10 Flex studies

1.10.1 Flex studies

PART 2 Establishing Clinical Performance 2.1 Diagnostic sensitivity and specificity

2.1.1 Diagnostic sensitivity and specificity

2.2 Qualification of usability

2.2.1 Label comprehension study

2.2.2 Results interpretation study

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Part 1 Establishing analytical performance Technical Specifications Series for submission to WHO Prequalification – Diagnostic Assessment:

Part 1 Establishing analytical performance

Aspect Testing requirements Comments References

1.1 Specimen type 1.1.1

Demonstration of equivalence between specimen types

For each claimed specimen type, testing to demonstrate equivalent performance in at least:

 25 G6PD-normal;

 25 G6PD-intermediate; and

 25 G6PD-deficient specimens.

1. All specimen types and anticoagulants claimed for use with the IVD must be validated.

2. The relationship should be established between claimed specimen types and reference materials for analytical studies such that the equivalence of performance can be understood.

The design of subsequent studies must then take that relationship into account.

3. If there is no equivalence between claimed specimen types then additional testing must be performed so that the impact this will have on each subsequent performance claim can be fully understood.

Example: an IVD intended for testing capillary whole blood for which analytical sensitivity is estimated using panels of venous whole blood specimens.

 The relationship between analytical sensitivity in venous whole blood to that of the same characteristic in capillary whole blood must be understood.

 This might be achieved by mixing G6PD-normal and G6PD- deficient specimens in different proportions to produce a range of G6PD activities and comparing performance between matched specimen types (capillary vs. venous whole blood) and/or anticoagulants. Mixtures should be chosen that allow the performance at decision points (i.e.

deficient/intermediate/normal) to be understood.

WHO TGS-3 (3)

1.1.2

Demonstration of equivalence of claimed anticoagulants

For each claimed anticoagulant, testing to demonstrate equivalent performance in at least:

 25 G6PD-normal;

 25 G6PD-intermediate; and

 25 G6PD-deficient specimens.

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1.2 Specimen collection, storage and transport 1.2.1

Specimen stability

Real time studies taking into account:

 storage conditions (duration at different temperatures and variation in humidity, temperature limits,

freeze/thaw cycles, where appropriate)

 transport conditions, where applicable (see comment 1)

 specimen collection and/or transfer devices (including filter paper for dried-blood spots), whether these contain anticoagulants and whether they can be sealed.

1. Particular attention should be paid to the length of time likely to elapse between specimen collection and its addition to the IVD in the setting this IVD may be used.

2. Where specimens are transported for testing with a reference method, these conditions should also be monitored and reported.

WHO TGS-1 (4)

1.3 Precision of measurement 1.3.1

Repeatability, reproducibility

Both repeatability (within-condition – see comment 1) and reproducibility (between-condition– see comment 1) estimated using, depending on intended use, panels of at least:

 1 G6PD-normal specimen;

 1 G6PD-intermediate specimen; and

 1 G6PD-deficient specimen.

Specimens chosen for the testing panel should, wherever possible, include panel members that reflect the main specimen types intended for use with the IVD (e.g. capillary whole blood).

Specimens should be chosen that ensure good representation of G6PD activity near critical decision points. The IVD must demonstrate acceptable precision at critical decision points.

Each panel member tested:

 in five replicates;

 using three different lots;

 over 3-5 runs;

 at each of three different testing sites; and

 at two temperatures within the claimed operating range (for enzymatic IVDs), where appropriate.

1. E.g. within- or between-run, -lot, -day, -site, etc.

2. Where possible, the testing panel should be the same for all operators, lots and sites.

3. The numbers of invalid tests must be reported.

4. Lots should be composed of different batches of critical components.

5. Results must be statistically analyzed (e.g. using ANOVA to identify and isolate the sources and extent of any variance.) 6. In addition to ANOVA, the percentage of correctly-identified,

incorrectly-identified and invalid results should be tabulated for each specimen and be separately stratified according to each of site, lot, etc. This type of analysis is especially important for rapid tests which may not have any numerical values for ANOVA analysis.

7. The effect of operator-to-operator variation on IVD

performance is also to be considered as a human factor when designing robustness (flex) studies (see 1.10.1 Flex studies). The results of estimating operator-to-operator variation on IVD performance may be used in conjunction with studies to qualify the usability of the IVD.

8. Users should be selected based on a pre-determined and contextually appropriate level of education, literacy and auxiliary skills that will challenge the usability of the IVD and

CLSI EP05-A3 (5)

ISO 13612:2002 (6)

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IVDs to identify G6PD activity TSS-2 The effect of operator-to-operator variation on IVD

performance is to be included as part of the precision studies (see also Comment 7).

Testing should be performed:

 by personnel representative of expected end users ,

 unassisted; and

 using only those materials provided with the IVD (e.g.

Instructions for use, labels and other instructional materials); and

 using at a minimum, G6PD specimens close to the critical decision points for the assay, as appropriate.

reflect the diversity of intended users and operational settings.

These characteristics should be detailed in the submission.

1.4 Performance panels 1.4.1

Genotype panels

For ‘capture’-type IVDs (e.g. detection by antigen-antibody binding) all reasonable attempts should be made to show the ability of the IVD to detect a diversity of prevalent genotypes.

WHO (9)

1.5 Validation of reading time 1.5.1

Validation of reading time

For IVDs where a reading interval is specified (i.e. time when result can first be read; time beyond which result should not be read) validation of critical time points must be provided.

Performance studies should be conducted at each of three temperatures (at the mid-point and two extremes of the claimed operating range); the effect of humidity on reading times should also be investigated.

1. The ranges of humidity tested for should be risk-based, taking into consideration likely operational settings.

2. The intended operating temperature, upon which reading time has been validated, must be clearly stated in the instructions for use.

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1.6 Analytical sensitivity 1.6.1

Analytical sensitivity

Limit of detection may be determined by testing in at least 8- member dilution panel. At least 15 replicates tested per dilution. Analytical sensitivity estimated by determining the lowest enzyme activity for which the rate of detection is 95%, and expressed as IU/g Hb.

The enzyme activity of specimens in the testing panel should be calibrated against a suitable biological reference material and appropriate reference method.

1. Optimally, testing should be conducted using more than one final design (locked-down) lot.

EU IVD (17)

CLSI EP17-A2 (7)

1.6.2 Performance at critical decision points

The ability of an IVD to differentiate G6PD activity (e.g.

deficient or normal; deficient, intermediate or normal; etc.) must be validated.

This may be achieved by mixing G6PD-normal and G6PD- deficient specimens in different proportions to produce a broad range of G6PD activities that allow the performance at decision points (i.e. deficient/intermediate/normal) to be understood. Mixtures from at least three separate specimen pairs should be tested.

1. Validation of IVD performance at critical decision points (e.g.

deficient or normal; deficient, intermediate or normal; etc.) must include consideration of the range of haemoglobin concentrations over which the IVD is able to perform.

2. Performance of the IVD at critical decision points may be validated as part of a determination of the relationship between specimen types and/or anticoagulants (see Specimen type, Aspect 1.1.2).

1.6.3 Measuring range and correlation

For IVDs that report quantitative test results the measuring range should be determined using specimens with known values of G6PD activity which have been calibrated against a suitable biological reference material and appropriate reference method. Results of parallel testing of specimens with known values of G6PD activity should be subjected to suitable statistical analyses.

The extent of correlation of quantitation with a suitable reference test should also be determined.

3. The influence of haemoglobin on IVD performance should be understood. Several determinations of performance parameters (measuring range and correlation) should be made at different concentrations of haemoglobin representing specimens with normal haemoglobin as well as mild and severe anaemia. The methods of determining haemoglobin concentration should be clearly described.

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IVDs to identify G6PD activity TSS-2 The measuring range and extent of correlation of quantitation

for determination of haemoglobin concentration must also be established for those IVDs that either report a concentration of haemoglobin or include normalisation to haemoglobin concentration as part of the reported result.

1.7 Analytical specificity 1.7.1

Potentially interfering substances

The potential for false results (falsely normal or falsely deficient) must be investigated in specificity studies. Studies can be addressed using either natural or contrived specimens.

1. In some cases (refer those identified in the adjacent “Testing Requirements” column), access to appropriate compounds or specimens may be challenging. Provided justification is given, it may be possible to address these potential sources of

interference as part of clinical studies in representative populations (see Part 2).

2. The risk assessment conducted for an IVD should identify substances for which the potential for interference is reasonably foreseeable.

3. Where either the scientific literature and/or risk analysis identifies the potential for false results in co-infected individuals (e.g. decreased sensitivity or specificity), further investigation should be undertaken using G6PD-normal and -deficient specimens.

4. Any observed interference should be investigated and

performance limitations of the IVD reported in the IFU. Results should be reported with respect to each condition and not be reported as an aggregate of the total number of specimens tested in the study.

5. Testing should be performed in G6PD-normal, -intermediate and -deficient specimens, in the presence or absence of each potentially interfering substance at physiologically relevant dosages. Where natural specimens cannot be obtained, it may be acceptable to artificially generate G6PD-deficiency. Non- clinical, laboratory testing should, where possible, include a minimum of 200 specimens, with each substance/condition represented by at least 3-5 specimens from different individuals.

EU IVD (17)

CLSI EP07-A2 (8)

Endogenous  Total cholesterol

 Elevated lipid, bilirubin and protein

 Lactic acid

 Lactate dehydrogenase

 Copper containing compounds: e.g. Copper chloride and Copper sulfate

 Abnormally high (54-65%) and abnormally low (17-18%) haemocrit

 Elevated reticulocytes, leukocytes and thrombocytes

 Malaria, HAV, HBV, HCV, HIV, pneumonia,

 Typhoid fever*

 Sickle cell anaemia, variant haemoglobin (A, D, E ,S, C) anaemias, haemoglobinopathies, iron deficiency, leukaemia, and/or other red blood cell dyscrasias*

 Diabetes.*

Exogenous  Relevant medicines, including: antiparasitic, antimalarial, antiretroviral and anti-tuberculosis medications

 Common over-the-counter anti-inflammatory medications (aspirin, paracetamol, ibuprofen)

 Ethanol, caffeine

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 Certain steroids (aldosterone-related)

* see comment 1

1.8 Metrological traceability of calibrators and control material values 1.8.1

Metrological traceability of calibrators and control material values

The traceability of an assay-specific quality control specimen to a validated reference material must be demonstrated.

1. Where a G6PD IVD uses an in-line procedural control band, the extent to which the presence or absence of this band

corresponds to a valid test (identification of and traceability to a suitable reference) should be demonstrated.

WHO (9)

1.9 Stability 1.9.1 IVD stability

Replicate testing of a panel consisting of at least:

 1 G6PD-normal specimen

 1 G6PD-intermediate specimen

 1 G6PD-deficient specimen

Specimens chosen for the testing panel should, wherever possible, include panel members that reflect the main specimen types intended for use with the IVD (e.g. capillary whole blood).

Specimens should be chosen that ensure good representation of G6PD activity near critical decision points.

1. Lots must comprise different batches of critical components.

2. Determination of shipping stability must be performed using simulated extreme stress conditions, ensuring that application of those conditions is consistent and controlled.

3. Claims for stability must be based on the second-last successful data point from the least stable lot, with, if lots are different, a statistical analysis showing that the bulk of lots will be expected to meet the claimed life. For example: for testing conducted at 3, 6, 9, 12 and 15 months, if stability was observed at 15 months, then the maximum stability claim can be 12 months.

4. Accelerated studies do not replace the need for real time studies.

5. In-use stability of labile components should be conducted using components in their final configuration.

ISO 23640:2011 (10)

CLSI EP25-A (11)

WHO TGS-2 (12)

ASTM D4169-14 (13)

1.9.2 Shelf life

 Real time, minimum of three lots of final design product

 Transport stressed (simulated) before real time studies are undertaken

 IVD in final packaging also subjected to drop-shock testing

1.9.3

In-use stability

 Minimum of one lot, using panel(s) compiled as above

 Testing of all labile components (e.g. buffers vials, sealed cartridges, etc.; see comment 5)

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1.10 Flex studies

1.10.1 Flex studies

The influence of the following factors on expected results (G6PD-normal, -intermediate and –deficient, as appropriate) should be considered:

 temperature (see Comment 1)

 reading time

 specimen and/or reagent volume

 buffer pH

 buffer concentration (to account for evaporation, whether in single- or multiple-use containers)

 lighting and humidity

1. In addition to investigating deviations of temperature within those claimed in the instructions for use (in the middle and at both lower and upper extremes of a claimed temperature range), temperatures ranges should be investigated that exceed those of claimed operating conditions and which cause test failure (incorrect/invalid results).

2. Refer to WHO document PQDx_018 “Instructions for

compilation of a product dossier”(9) for other flex studies that may be relevant, taking into consideration the broad range of operational and environmental conditions consistent with intended use.

WHO (9)

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Part 2 Establishing clinical performance

2.1 Diagnostic sensitivity and specificity 2.1.1

Diagnostic sensitivity and specificity

Diagnostic sensitivity and specificity should be determined principally in capillary whole blood.

Testing should be conducted:

 at different geographical settings representative of intended use (minimum of two regions, including at least one region where Plasmodium vivax is prevalent);

 by a variety of intended users (i.e. 9 - 12 users); and

 using more than one lot

Clinical performance studies should be designed to recruit specimens from regions of high G6PD-deficiency in sufficient numbers so as to ensure the testing of a total of:

 200 G6PD deficient specimens

 200 G6PD intermediate specimens

 1000 G6PD normal specimens (1)

1. Prequalified G6PD IVDs will generally be used by trained lay providers and trained health care workers. For prequalification purposes, these should be considered as the intended user, rather than a laboratory professional.

2. A separate, venous whole blood specimen should be collected in parallel to establish the reference result. The testing algorithm used to determine the reference results should include:

 Quantitative determination of G6PD activity, expressed as IU/g Hb; and

 Lots should comprise different batches of critical components.

3. Where possible, all discrepant results (between assay under evaluation and the reference results) should be repeated on the same lot, and then on all available lots and the variability noted.

Performance characteristics must be reported using initial results, only. The results of further testing of specimens with discrepant results must be reported separately as additional information about IVD performance.

4. All invalid test results must be recorded.

5. Estimates of diagnostic/clinical sensitivity and specificity should be reported with two-sided 95% confidence intervals.

6. For IVDs that report three or more results (e.g. ‘deficient’,

‘intermediate’ or ‘normal’), sensitivity and specificity refer to probability that the IVD is able to report a true result;

performance characteristics should be reported for each type of result (e.g. ‘deficient’, ‘intermediate’ or ‘normal’), as

appropriate.

EU IVD (17) WHO (14) FDA (15)

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Part 2 Establishing clinical performance Technical Specifications Series for submission to WHO Prequalification – Diagnostic Assessment:

2.2 Qualification of usability 2.2.1

Label comprehension study

Questionnaire-based testing of subjects to assess ability of intended users to correctly comprehend key messages from packaging and labelling:

 Understanding key warnings, limitations and/or restrictions.

 Proper test procedure.

 Test result interpretation.

Questionnaire to be administered to at least 30 intended users, in order to demonstrate comprehension of key messages in each population described above.

1. Instructions for use and labelling should be clear and easy to understand; use of pictorial instructional material is

encouraged. (16)

2. Prequalified G6PD IVDs will generally be used by trained lay providers and trained health care workers. For prequalification purposes, these should be considered as the intended user, rather than a laboratory professional.

EU IVD (17) FDA (18)

2.2.2 Results interpretation study

Subjects to interpret the results of contrived IVDs (e.g.

static/pre-made tests) to assess their ability to correctly interpret pre-determined test results. Contrived tests should be made to demonstrate the following potential test result interpretations:

 Non-reactive.

 Range of invalid results.

 Reactive.

 Weak reactive.

Testing subjects to consist of at least 30 intended users from at least two geographically diverse populations to demonstrate correct interpretation of simulated test results.

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E. References

1 Domingo GJ et al., G6PD testing in support of treatment and elimination of malaria:

recommendations for evaluation of G6PD tests. Malar J. 2013 Nov 4;12:391.

2 Point-of-care G6PD testing to support safe use of primaquine for the treatment of vixax malaria. WHO Evidence Review Group meeting report. Malaria Policy Advisory Committee Meeting, Geneva, Switzerland. 2015. Available at:

http://www.who.int/malaria/mpac/mpac-march2015-erg-g6pd.pdf, accessed 16 September 2016.

3 WHO Prequalification Team – Diagnostic Assessment. Technical Guidance Series (TGS).

Principles for Performance studies TGS–3. Geneva: World Health Organization; 2016.

Available at:

http://www.who.int/diagnostics_laboratory/guidance/technical_guidance_series/en/, accessed 15 July 2015.

Standards applicable to the WHO Prequalification of in vitro diagnostics TGS–1. Geneva:

World Health Organization; 2016. Available at:

5 CLSI. Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline - Third Edition. CLSI document EP05-A3. Wayne, PA: Clinical and Laboratory Standards Institute;

2014.

6 ISO 13612:2002. Performance evaluation of in vitro diagnostic medical devices. Geneva:

International Organization for Standardization/British Standards Organization; 2002.

7 CLSI. Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures;

Approved Guideline- Second Edition. CLSI document EP17-A2; Wayne, PA; 2012.

8 CLSI. Interference Testing in Clinical Chemistry. Approved Guideline - Second Edition. CLSI document EP07-A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2005.

9 WHO Prequalification Team – Diagnostic Assessment. Instructions for Compilation of a Product Dossier. WHO document PQDx_018 v3, 27 August 2014. Available at:

http://www.who.int/entity/diagnostics_laboratory/evaluations/141015_pqdx_018_dossier_i nstructions_v4.pdf?ua=1, accessed 18 July 2016.

10 ISO 23640:2011. In vitro diagnostic medical devices - Evaluation of stability of in vitro diagnostic reagents. Geneva, Switzerland: International Organization for Standardization;

2011.

11 CLSI. Evaluation of Stability of In Vitro Diagnostic Reagents; Approved Guideline. CLSI document EP25-A. Wayne, PA: Clinical and Laboratory Standards Institute; 2009.

Establishing stability of an in vitro diagnostic for WHO Prequalification TGS–2. Geneva: World Health Organization,. 2016. Available at:

13 ASTM International. ASTM D4169-14. Standard Practice for Performance Testing of Shipping Containers and Systems. West Conshohocken, PA: ASTM International; 2014.

14 WHO Technical Report Series No. 366, 1967, Standardization of Procedures for the Study of Glucose-6-Phosphate Dehydrogenase.

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15 U.S. Food and Drug Administration. Guidance for Industry and FDA Staff, Statistical Guidance on Reporting Results from Studies Evaluating Diagnostic Tests. Rockville, MD. Available at:

http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/GuidanceDo cuments/ucm071287.pdf, Accessed 3 November 2016.

16 How to use a rapid diagnostic test (RDT): A guide for training at a village and clinic level (Modified for training in the use of the Generic Pf Test for falciparum malaria). The USAID Quality Assurance Project (QAP), University Research Co., LLC, and the World Health Organization (WHO), Bethesda, MD, and Geneva. Available at:

http://www.wpro.who.int/malaria/NR/rdonlyres/23DD7DCB-48C4-4CFF-BD45- 332F0BE3DCC7/0/generic_PfPan_training_manual_web.pdf, accessed 15 July 2016.

17 Committee of the European Parliament, Agenda 15 June 2016. Proposal for a regulation of the European Parliament and of the Council on in vitro diagnostic medical devices. European Parliament, eMeeting for Committees. Available at:

http://www.emeeting.europarl.europa.eu/committees/agenda/201606/ENVI/ENVI(2016)061 5_1/sitt-2571599, accessed 5 October 2016.

18 U.S. Food and Drug Administration. Write It Right. Rockville, MD: HHS Publication FDA 93- 4258; 1993. Backinger CL and Kingsley PA. [Internet]. Available from:

http://www.fda.gov/downloads/MedicalDevices/.../ucm070771.pdf, accessed 3 November 2016.

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List of related WHO Publications of related interest

WHO Prequalification Team- Diagnostic Assessment. Technical Guidance Series for WHO Prequalification – Diagnostic assessment (available online)

WHO Prequalification Team – Diagnostic Assessment. Instructions for Compilation of a Product

Dossier. WHO document WHO/PQDx_18 (available online)

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The Technical Specifications Series for submission to WHO Prequalification – Diagnostic Assessment set out appropriate performance evaluation criteria to meet prequalification requirements. Each Technical Specification provides information on the minimum performance requirements for WHO prequalification that should be met by a manufacturer to ensure that their