EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION … · WHO/BS/2015.2262 Page 4 WHO HCV NAT IS...

WHO/BS/2015.2262

ENGLISH ONLY

EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION

Geneva, 12 to 16 October 2015

Collaborative Study to Evaluate the Proposed 5th WHO International

Standard for Hepatitis C Virus (HCV) RNA for Nucleic Acid Amplification

Technique (NAT)-Based Assays

Clare Morris1,3

, Graham Prescott1, Jason Hockley

2 and the Collaborative Study

Group*

1 Division of Virology and 2 Biostatistics, National Institute for Biological Standards and Control,

South Mimms, Potters Bar, Herts, EN6 3QG, UK

3 Study Coordinator; Tel +44 1707 641236, Fax +44 1707 641060,

E-mail: [email protected]

* See Appendix 8

NOTE:

This document has been prepared for the purpose of inviting comments and suggestions on the

proposals contained therein, which will then be considered by the Expert Committee on

Biological Standardization (ECBS). Comments MUST be received by 14 September 2015 and

should be addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention:

Technologies, Standards and Norms (TSN). Comments may also be submitted electronically to

the Responsible Officer: Dr M Nübling at email: [email protected]

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WHO/BS/2015.2262

Summary The previous 4

th HCV IS for NAT (NIBSC code 06/102) was established in 2011, however it

was recognised that the material must be shipped on dry ice in order to prevent loss of titre

during an ambient shipping process.

A small pilot study was conducted at NIBSC to determine the possible effect that the presence or

absence of HCV antibodies may have on the stability of a lyophilised preparation. Following the

assessment of six lyophilised formulations containing lyoprotectants and stabilizing buffers in

the presence and absence of antibodies to HCV, by six laboratories using different commercial

assays, no differences were observed. It was subsequently agreed at the 3rd

joint meeting of the

Blood Virology and Clinical Diagnostics Standardisation of Genomic Amplification Techniques

meeting held on 30/31st May 2014 in Graz, Austria that a candidate standard for the 5th

HCV IS

NAT would comprise an antibody negative preparation of genotype 1a.

The candidate 5th

HCV IS for NAT (NIBSC code 14/150) was filled and freeze dried at NIBSC

in November 2014, 1980 vials were lyophilised with a reconstitution volume of 1.1mL to allow

for single vial use on analysers requiring a 1mL input volume. The subsequent international

collaborative study was performed from December 2014 to March 2015. 17 laboratories from 11

countries participated in the evaluation of 6 samples including the lyophilised candidate standard

and the corresponding frozen liquid bulk. HCV antibody positive plasma and a low titre material

intended for use as a secondary reference material were also assessed. Assays comprised mainly

of commercial real time PCR methods reporting in both quantitative and qualitative values. The

current 4th

HCV IS and the previous 2nd

IS were both included giving the option of deriving

potency comparative to the 2nd

standard.

The range in individual laboratory mean estimates for the candidate material (S5) in quantitative

assays was 4.60 – 5.72 log10 IU/mL and 4.43 – 4.81 log10 IU/mL in qualitative assays. Standard

deviation values for intra laboratory comparisons were low.

Accelerated thermal degradation studies indicate good stability at 3 months however at 6 months

a loss of 0.14Log is observed at +20°C relative to the -20°C baseline sample. Further studies on

real time stability are on-going.

Overall, the results of this study indicate the suitability of the candidate to be established as the

replacement 5th WHO International Standard for HCV (NIBSC code 14/150) with an assigned

potency of 5.0 log10 IU/vial when reconstituted in 1.1mL of nuclease-free water.

Introduction The availability of an international standard for HCV RNA for the past 20 years (1-7) has been

fundamental in driving the close agreement now seen across HCV nucleic acid tests, giving

assurance of results regardless of assays used. HCV RNA assays are used in both a screening

and diagnostic capacity, with regulations in developed countries mandating the screening of

donated blood and plasma for the presence of HCV RNA, whilst in the field of biotherapeutic

medicines (8) European Pharmacopeia guidelines dictate that constituent plasma pools must be

WHO/BS/2015.2262

screened for the presence of HCV RNA and that all assays used for such a purpose must have a

sensitivity of at least 100 IU/mL (9).

Furthermore, with no therapeutic or prophylactic vaccine available there is a global need to use

HCV NAT assays for the diagnosis of patients and the monitoring of those receiving antiviral

therapy (8). In both cases accurate and comparable quantification is required across all HCV

NAT systems.

The 4th

WHO HCV NAT IS (NIBSC code 06/102) was established in 2011 (5); however the

product was established with the knowledge that the material was not able to maintain titre when

shipped to customers at ambient temperature, a practice commonly employed for lyophilised

standards to reduce the shipping costs for the customer. The material did however demonstrate

good stability when shipped on dry ice. The 4th

HCV IS was established with the understanding

that further stability investigation would be carried out and a 5th

standard developed in advance

of the usual time frame of depleted stocks of the previous standard.

A difference in stability has been previously reported (5) between the 1st (99/790) and 2

nd

(99/798) established preparations of the HCV NAT IS and the 3rd

(06/100) and 4th

(06/102)

preparations. The most notable difference between these is the presence of HCV antibody in

preparations 1 and 2 but its absence in preparations 3 and 4. A pilot study was conducted to

determine whether the presence or absence of HCV antibody affected the stability of the product.

This report describes both the pilot study conducted to investigate the stability issues observed in

the previous standard and following the outcome of this study the development and evaluation of

the replacement batch of standard to be established as the 5th

WHO International Standard for

HCV RNA for NAT.

Data from the pilot study were presented at the XXIV Scientific Working Group on the

Standardisation of Genome Amplification Techniques for the Safety Testing of Blood and

Clinical Diagnostics meeting in Graz, Austria in May 2014. The collaborative study to establish

the 5th

HCV NAT IS was presented to the 20th

Anniversary meeting of the Scientific Working

Group on the Standardisation of Genome Amplification Techniques for the Safety Testing of

Blood and clinical diagnostics meeting held on 25/26th

June in London, UK.

In line with previous standards, an international unit derived as the result of a comprehensive

collaborative study incorporating a range of different assays will be assigned to this material. As

with the majority of biological preparations, the material cannot be fully characterized by a

physico-chemical reference method. Therefore, biological assays will be used, these methods are

heterogeneous and the lack of a reference method does not permit the results to be expressed in

absolute values according to the SI system.

Pilot study to investigate stability of freeze-dried HCV formulations

Background

WHO/BS/2015.2262

WHO HCV NAT IS stability discrepancies have previously been reported (5). Briefly the 1st

(99/790) and 2nd

(99/798) established preparations of the HCV NAT IS displayed good stability

throughout their use, whereas a loss of titre over time was observed in the 3rd

(06/100) and 4th

(06/102) IS preparations. The 1st and 2

nd standards were prepared from identical stock material

and lyophilised on two separate occasions. The stock material used to formulate the 3rd

and 4th

standard was different from that used for the 1st and 2

nd standard, however similarly both 3

rd and

4th

standards were produced from the same stock material and lyophilised on two separate

occasions. The main notable difference between the preparations was the presence of HCV

antibody in preparations 1 and 2 but its absence in preparations 3 and 4. High moisture and

oxygen was observed in all preparations.

A pilot study was conducted to determine whether excessive moisture in conjunction with the

presence or absence of HCV antibody affected the stability of lyophilised HCV RNA. The pilot

study was performed between January 2014 and May 2014. Six different HCV RNA candidate

formulations were evaluated for stability through accelerated thermal degradation studies and

subsequently assessed by laboratories using different commercial assays.

Source candidate material The study compared six different HCV RNA preparations comprising of two main groups.

1) HCV window period positive donation, diluted in negative human plasma. 2) Pooled antibody

positive plasma donations.

Six individual HCV RNA positive plasma packs were purchased from the UK NHS Blood and

Transplant Authority. Each pack had previously been screened using the UK blood donation

screening algorithm and rejected due to a positive HCV NAT signal.

Titre determination was carried out at NIBSC using the COBAS® AmpliPrep/COBAS®

TaqMan® HCV v2.0 assay. Three plasma packs, containing a titre that when pooled, would

result in a final concentration of approximately 6 log10 IU/mL of HCV RNA, were selected. The

final volume available for the study, following pooling, was 320mL. This material was used to

formulate the HCV antibody positive preparations.

800mL of HCV RNA positive, HCV antibody negative (window period) plasma was donated by

Public Health England, UK. This was confirmed to be antibody negative at NIBSC using the

Murex EIA and was confirmed to have a titre of 6 log10 IU/mL using the COBAS®

AmpliPrep/COBAS® TaqMan® HCV v2.0 assay.

Table 1 outlines each candidate formulation.

Formulation of candidate pilot study material

Antibody negative material

WHO/BS/2015.2262

A 1/10 dilution into HCV RNA negative, HCV antibody negative plasma was performed on the

window period donation to give a titre in the region of 5 log10 IU/mL.

3x 80mL of material was aliquoted into 120mL sterile bottles and labelled 2, 4 and 6.

Antibody positive material In order to achieve a titre of 1x10

5 IU/mL as the final titre of the antibody positive material, six

HCV positive plasma packs were tested using the Roche COBAS® AmpliPrep/COBAS®

TaqMan® HCV v2.0 assay Two HCV antibody positive plasma packs were selected or intial

pooling to give a concentration of 1.44x103 IU/mL, the flask was labelled pool 6. Pool 6 was

then combined with a single HCV antibody positive donation, with atitre of 8.93x105 IU/mL.

100mLs of Anti HCV positive plasma at a concentration of 1x106 IU/mL was produced and

comprised the HCV antibody positive preparation for this pilot study.

3x 80mL of the pooled HCV antibody positive plasma was aliquoted into 120mL sterile bottles

and labelled 1, 3 and 5.

To assess any stabilising effect during lyophilisation, preparations were made with the presence

or absence of Hepes buffer and trehalose.

Hepes buffer, at a final concentration of 40mmol/L, was added to bottles 3 and 4. Trehalose, at a

final concentration of 10mg/L, was added to bottles 5 and 6. The remaining bottles, 1 and 2,

served as unstabilsed control material for each AB+ and AB- preparation. All preparations were

mixed for 10 minutes thoroughly to ensure homogeneity.

38 x 0.55mL of each preparation was aliquoted in to 2mL sarstedt vials and frozen at -700C to

provide aliquid frozen baseline sample.

Lyophilisation of pilot study candidates Lyophilisation was conducted by Public Health England, Colindale, UK. All preparations were

lyophilised in the same run using a three day plasma cycle. Preparations were then returned to

NIBSC on dry ice.

Accelerated degradation of samples Prior to placing samples at different temperatures, half of each batch of preparation was exposed

to atmospheric conditions to deliberately allow the introduction of moisture and oxygen. The

vials were placed in a microbiological safety cabinet; stoppers were lifted but not fully removed

and the vials left for 60 minutes. After which time all stoppers were closed fully and the screw

cap lids replaced.

In total 25 samples from each of the six lyophilised preparations were placed at -200C and

+200C. At the one month time point samples were removed, 3 vials of each temperature were

assayed by participants in a small collaborative study. Vials of the current 4th

HCV NAT IS were

WHO/BS/2015.2262

evaluated in parallel. 56 vials of 06/102 were placed at +200C for one month. The labelling of

tubes is summarised in table 1.

Statistical analysis

Pilot collaborative study Six laboratories, each using a different, commercially available HCV RNA assay were invited to

participate in a small collaborative study to assess the lyophilised preparations from each

temperature. Frozen liquid bulk material corresponding to each formulation was also included.

Study participants and assays used can be found in appendices 1 and 2.

Participants were sent up to a total of 28 lyophilised preparations and 6 frozen liquid

preparations (Vial numbers varied depending on the volume requirement of the participants

chosen assay). Each participant was requested to perform two independent assays, using a fresh

vial of material each time. For quantitative assays participants were requested to test all vials

neat. For qualitative assays, it was recommended that laboratories tested each sample neat and at

up to four 10-fold dilutions in the first assay to establish the end point and at half log dilutions

around the end point in subsequent assays. A copy of the study protocol is provided in

appendices 3-6.

Results – Pilot study

The mean of all data received from all participants for the analysis of the pilot study samples A-

G are presented in table 2

Observed differences in titre between liquid bulk samples and lyophilised preparations are also

shown. The mean loss in titre due to the lyophilisation of material of the antibody positive

preparations is 0.53 log10 IU/mL whilst for antibody negative preparations it is 0.22 log10 IU/mL.

The mean differences between the presence or absence of moisture or antibodies ranges from

+0.07 to -0.14 log10 IU/mL, this difference is considered within the limits of precision of the

assays used.

Statistical analysis

Statistical analysis using the Arrhenius equation, frequently used to predict loss of titre following

long term storage at -20 through the short term loss at higher temperatures, was not possible due

to the negligible difference in titre between each sample types.

Discussion – Pilot Study

WHO/BS/2015.2262

All panel members used in this study were formulated to contain approximately 1x 105, however

some loss was experienced during lyophilisation giving a range of titres from 4.89 log10 IU/mL

to 5.13 log10 IU/mL. All panel members were compared relative to their -200C counterpart.

Data from the pilot study indicated no loss of titre of HCV RNA across all temperatures and

formulations used. Given the time period available, a timeframe of one month was chosen to

store pilot formulations at -200C and +20

0C. In previous HCV IS formulations a reduction in titre

was observed at temperatures up to 200C over a few days during shipping. By storing samples

for one month it was felt that this would go over and beyond previous scenarios where by a loss

in titre was observed during the shipping process.

In previous HCV IS stability studies it has been demonstrated that there has been a loss in titre

during routine transport at ambient temperature. In this study a temperature of +200C represents

an average ambient shipping temperature, to hold a sample at this temperature for a period of one

month exceeds a usual shipping timeframe and therefore it was expected that if a loss is titre was

to be observed, such a time frame would be suitable to witness this. However as no loss was seen

in any sample, it can be concluded that the presence or absence of HCV antibodies did not

influence the stability of HCV RNA in lyophilised preparations. Indeed the addition of

stabilising compounds also displayed no effect.

Due to the availability of sufficient window period donation at NIBSC it was proposed that the

5th

HCV RNA IS would be formulated from this donation in order to avoid delays in sourcing

sufficient qualities of antibody positive plasma. Delegates of the SoGAT workshop held in Graz,

Austria in May 2014 agreed an antibody negative HCV RNA plasma preparation would be fit for

purpose and development of such a standard should continue in a timely manner.

WHO/BS/2015.2262

Establishment of a 5th International Standard for HCV RNA for

use in NAT - Main collaborative study

Introduction

Following a pilot study (described above) to investigate the stability of lyophilised HCV RNA, it

was agreed at the 3rd

combined Blood Virology and Clinical Diagnostic SoGAT workshop, held

in Graz, Austria (30/31st May 2014) that the 5th

HCV RNA IS for NAT should be formulated

from a window period donation and comprise HCV antibody negative material spiked into

human plasma. It was also agreed that in a change to previous batches of HCV IS, the 5th batch

would be formulated in 1.1mL aliquots to allow for undiluted use on current automated analysers

that require a 1mL input volume.

Material and Methods

Candidate standard The proposed candidate standard comprises (NIBSC code 14/150) a single window period

donation, confirmed by Sanger sequencing to be HCV genotype 1a (sample and sequence

information provided by Public Health England, Colindale UK) spiked into human plasma tested

and found negative for HCV, and HIV-1/2 antibodies, HBsAg and HAV.

Negative human plasma sourced from the National Blood Service, UK (NBS) in the form of

individual plasma packs comprised the diluent for this standard. 12 packs were pooled and

refrozen until required. Each plasma pack was previously screened by the NBS and found

negative for HIV antibodies, HCV RNA, HBsAg and Syphilis. Once pooled the diluent was

further tested at NIBSC and confirmed negative for HIV-1/2 antibodies, HBsAg and HCV RNA.

Preparation of bulk material 300mL of window period material was stored at -70

0C until required. Previous titre

determination using the COBAS® AmpliPrep/COBAS® TaqMan® HCV v2.0 assay, confirmed

the bulk stock material to be 6 log10 IU /mL.

Bulk stock material was thawed in a water bath at 370C, with occasional mixing to encourage

even heat distribution and increase the thawing process. Following thawing the stock was mixed

using a magnetic stirrer and flea for 30 minutes to disperse any aggregates and ensure

homogeneity. Stirring also aided the breakdown of residual lipid clots.

The bulk stock was diluted in human plasma sourced from the UK Blood Transfusion and

Transplant service, all plasma donations were screened and found negative for anti-HIV-1,

HBsAg, and HCV RNA.

280mL of bulk stock was diluted into 2220mL of negative plasma. 2500mL of final preparation

was formulated to contain approximately 1x105 HCV log10 IU/mL. The final formulation was

stirred continuously for 30mins using a magnetic stirrer and flea.

WHO/BS/2015.2262

200mL of liquid bulk stock was dispensed in 1.1mL aliquots into 2mL Sarstedt vials and frozen

at -700C. The remaining bulk preparation was transferred to the Centre for Biological Reference

Materials, located at NIBSC for filling and lyophilisation. The final product was coded, NIBSC

code 14/150.

Filling and lyophilisation of the candidate standard Filling and lyophilisation was performed onsite at NIBSC by the Centre for Biological Reference

Materials (CBRM), Potters Bar, UK in September 2014. The filling was performed in a Metal

and Plastic GmbH (Radolfzell, Germany) negative pressure isolator that contains the entire

filling line and is interfaced with the freeze dryer (CS150 12m2, Serail, Arguenteil, France)

through a ‘pizza door’ arrangement to maintain integrity of the operation.

The bulk material was dispensed in 1.1mL volumes into 5mL screw cap glass vials using a

Bausch & Strobel (Ilfshofen, Germany) filling machine FVF5060. The bulk material stirred

constantly using a magnetic stirrer. The homogeneity of the fill was determined by on-line

check-weighing of the wet weight, a tolerance of 1.093 to 1.108g with a target of 1.100g was

applied; a sample of 3% was check weighed to determine the homogeneity of the fill. Those

outside of this tolerance were discarded.

Filled vials were partially stoppered with halobutyl 14mm diameter cruciform closures and

lyophilized in a CS150 freeze dryer. Vials were loaded onto the shelves at -50°C and held at this

temperature for 4 hrs. A vacuum was applied to 270 ub over 1 hr, followed by ramping to 30 ub

over 1 hr. The temperature was then raised to -40°C, and the vacuum maintained at this

temperature for 42.5 hrs. The shelves were ramped to 25°C over 15 hrs before releasing the

vacuum and back-filling the vials with nitrogen. The vials were then stoppered in the dryer,

removed and capped in the isolator, and the isolator decontaminated with formaldehyde before

removal of the product.

A total of 1980 vials were filled; these vials were then stored at -200C. Continuous temperature

monitoring will proceed for the lifespan of the product. Material characteristics can be found in

table 2.

Post-fill testing Assessments of residual moisture and oxygen content, as an indicator of vial integrity after

sealing, were determined for twelve vials of the freeze-dried product. Residual moisture was

determined by non-invasive near-infrared (NIR) spectroscopy (MCT 600P, Process Sensors,

Corby, UK). NIR results were then correlated to Karl Fischer (using calibration samples of the

same excipient, measured using both NIR and Karl Fischer methods) to give % w/w moisture

readings. Oxygen content was measured using a Lighthouse Infra-Red Analyzer (FMS-750,

Lighthouse Instruments, Charlottesville, USA). The CV of fill mass and mean residual moisture

and Oxygen were within WHO acceptable limits; details can be found in table 3.

Stability of the freeze-dried candidate Accelerated degradation studies are currently on going at NIBSC, where the stability of this

material can be predicted when stored at -200C. Vials of the freeze-dried product were stored at -

WHO/BS/2015.2262

700C, -20

0C, +4

0C, +20

0C, +37

0C and +45

0C in November 2014 and have been tested at 3 and 6

months. At the three month time point two vials were removed from each temperature,

reconstituted and pooled prior to extraction and amplification. At the 6 month time point, three

vials were removed from each temperature, reconstituted and tested individually. At both time

points the HCV RNA concentration was quantified by NAT using the COBAS®

AmpliPrep/COBAS® TaqMan® HCV v2.0.

Study samples The lyophilised candidate material 14/150 was evaluated in parallel to the 2

nd and 4

th WHO

International Standard for HCV for Nucleic Acid Techniques (NIBSC codes 96/798 and 06/102

respectively). Alongside these materials, 3 liquid preparations were provided in this study; a

sample of the candidate liquid bulk material, a HCV antibody positive commutability sample

comprising a single donation HCV antibody positive sample purchased from the UK National

Blood Authority and a candidate working reagent for HCV (14/152-001), consisting of a clinical

samples spiked into HCV negative human plasma. Before shipping, liquid preparations were

stored at -700C, whereas freeze-dried materials were stored at -20

0C.

Collaborative study (NIBSC code CS534) samples were shipped to all participants frozen (on

dry ice). Samples were blinded and coded 1-6 as shown below:

Sample 1 – Candidate HCV International standard liquid bulk material in a 2mL Sarstedt

tube

Sample 2 – HCV antibody positive commutability liquid material in a 2mL Sarstedt tube

Sample 3 – Lyophilized preparation 96/798 in a 3mL crimp cap vial

Sample 4 – Lyophilized preparation 06/102 in a 3 mL crimp cap vial

Sample 5 – Lyophilized preparation 14/150 in a 5mL screw cap vial

Sample 6 – HCV candidate working reagent 14/152-001 in a 2mL Sarstedt tube

Study Design The aim of this collaborative study was to establish a replacement material for the 4th WHO

International Standard for HCV RNA (NIBSC code 06/102) and assign a unitage based on

comparisons to both 2nd and 4th WHO HCV International Standards. This study also includes a

Working Reagent candidate material, which will be calibrated against these preparations in a

range of NAT-based assays. Up to 10 vials of samples 1-6 were sent to participants on dry ice.

Vial numbers varied depending on the volume requirement of the participants chosen assay.

Participants Study samples were sent to 17 participants across 11 countries (Appendix 7). Participants were

selected for their experience in previous collaborative (HCV NAT) studies and their

representative distribution geographically; providing a range of assays across the testing field

(assays used are shown in appendix 8). These included manufacturers of in vitro diagnostic

devices (IVD’s), control and reference laboratories and blood transfusion centres. Laboratories

were designated a number code at random and where a specific laboratory performed more than

one assay their data was analysed separately. These labs were given a decimal number code, for

WHO/BS/2015.2262

example Laboratory 17.1, 17.2 etc. Appendix 7 does not represent the order of designated

laboratory numbers.

Study protocol Participants were requested to test samples 1-6 in their routine HCV NAT-based assays; in three

independent tests. Each participant was given sufficient material for their assay, which had

previously been established through the participant invitation. Where assays required a higher

volume than that of a specific sample, participants were instructed to combine vials and start

their dilutions (outlined below) from the combined material (further details can be found in the

study protocol, appendices 9-10). Samples 3 and 4 were to be reconstituted in 0.5 mL of

deionized, nuclease-free molecular grade water and sample 5 in 1.1 mL. All lyophilized

preparations were left for at least 20 minutes with occasional agitation before use.

Participants were requested to dilute samples in human plasma and extract each dilution before

amplification.

For quantitative assays: Participants were requested to test samples 1, 2 and 6 undiluted only. Sample concentrations of

each sample were reported in IU/mL. For samples 3 – 5, it was suggested that participants

perform a minimum of two serial ten-fold dilutions within the linear range of their assay (e.g. 10-

1 and 10-2).

For qualitative assays Participants were requested to test the dilution at the predicted assay end-point (limit of

detection), with a minimum of two half-log serial dilutions either side of the end-point (i.e. at

least five dilutions in total). Within subsequent assays participants were asked to work around

this end point more precisely. Results were reported as either negative or positive.

In both case all results were returned to NIBSC for analysis with details of methodology used.

Statistical methods Qualitative and quantitative assay results were evaluated separately.

In the case of qualitative assays, for each laboratory and assay method, data from all assays were

pooled to give a number positive out of number tested at each dilution step. A single ‘end-point’

for each dilution series was calculated, to yield an estimate of ‘NAT detectable units/mL. It

should be noted that these estimates are not necessarily directly equivalent to a genuine genome

equivalent number/mL (9). This model assumes that the

probability of a positive result at a given dilution follows a Poisson distribution (with mean given

by the expected number of “copies” in the sample tested), and that a single “copy” will lead to a

positive result. Calculations were performed using the statistical package GLIM (10). When asingle

value is obtained for each laboratory and sample no assessment of within laboratory, i.e

between assay variability can be made.

In the case of quantitative assays, analysis was based on the results supplied by the participants,

reported as IU/mL. For each assay run, a single estimate of log10 IU/mL was obtained for each

sample, by taking the mean of the log10 estimates of IU/mL across replicates, after correcting for

WHO/BS/2015.2262

any dilution factor. A single estimate for the laboratory and assay method was then calculated as

the mean of the log10 estimates of IU/mL across assay runs.

Overall analysis was based on the log10 estimates of IU/mL or ‘NAT detectable units/mL’.

Overall mean estimates were calculated as the means of all individual laboratories. Variation

between laboratories (inter-laboratory) was expressed as standard deviations (SD) of the log10

estimates and % geometric coefficient of variation (%GCV) of the actual estimates. Variation

within laboratories and between assays (intra-laboratory) was expressed as standard deviations of

the log10 estimates and %GCVs of the individual assay mean estimates.

Potencies of sample 5, the candidate International Standard relative to the two previous

International Standards were calculated as the difference in estimated log10 IU/mL (candidate

standard-previous standard) plus the value in International Units/mL (IU/mL) of the previous

standard.

Results

Of the 18 laboratories that were sent samples, 17 laboratories returned data, with two laboratories

returning data from two different assays giving a total of 19 data sets for analysis. The data

returned comprised 12 (63%) quantitative and 7 (37%) qualitative sets. All qualitative data was

produced by assays from a commercial supplier, with the most frequently used assay being the

Roche MPX, two different platforms were represented; MPX Ampliprep and MPX 8800, such

data constituted 86% of qualitative data. The remaining 14% was performed using the Procleix

Ultrio system. All main commercial assays were represented in the quantitative data sets, 4

laboratories used the Roche CAP/CTM v2.0, 2 laboratories reported data from the Roche 6800

platform, single laboratories reported data using the versant kPCR HCV RNA assay, Abbott

RealTime and QIAGEN QiaSymphony. One laboratory reported quantitative data using two

different in house methods. Tables 5 and 6 show the mean laboratory estimates for quantitative

and qualitative assays respectively. Blank spaces in the tables indicate no data was returned for

that sample.

The loss of titre arising from lyophilisation was assessed by participant, mean values for S1

(liquid bulk) with S5 (candidate 14/150). When combining mean values of both quantitative and

qualitative assays for these samples a loss of 0.11 log10 IU/mL is observed. This is similar to that

seen across all assays in the pilot study with the same material. However, taking quantitative and

qualitative data sets individually a loss of +0.09 log10 IU/mL vs -0.44 NAT detectable units are

observed, as demonstrated in table 7.

Inter-laboratory variation for both quantitative and qualitative data is presented in table 7.

Variation is greater amongst qualitative data than quantitative in 4/6 candidates evaluated

demonstrated by higher GCV values. Overall the combined standard deviation values across all

samples were <0.50 log10 IU/mL. Intra-laboratory standard deviations for each quantitative

laboratory data set are presented in table 8, values are very low for each sample indicating

excellent precision.

WHO/BS/2015.2262

Histogram representation of laboratory mean estimates for each sample are shown in figures 1-

6.shaded squares represent qualitative assays and clear squares quantitative assays. Value

agreement is seen across all samples tested with data from qualitative assays flanking the main

consensus group comprising of quantitative assays.

Figure 7 shows the relative potencies for the candidate standard (S5) relative to the previous 2nd

HCV IS (S4), which has previously been assigned a value of 5.00 Log10 IU/ml . In general for

both quantitative and qualitative assays excellent agreement was observed at approximately 5

log10 IU/mL. One outlying data set, (laboratory 15) representing data from the qualitative MPX

assay, gave a value of 3.41 log10 IU/mL. Figure 8 represents quantitative data only; a variation in

potency of only 0.5 log is seen from the mean.

Figure 8 shows the estimated relative potency for the candidate sample (S5) relative to the

current 4th

HCV IS (S4). Harmonisation is seen in all quantitative assays. However outliers are

seen in three separate qualitative assays: the MPX on the 8800 platform, Procleix Ultrio assay

and the MPX on the S201 platform. A reduction in relative potency of -2.05, -1.65 and -1.60

log10 from the mean was observed respectively.

Table 10 shows relative potency estimates for the candidate sample (S5) relative to both the 2nd

and 4th

HCV IS. Mean potencies for qualitative, quantitative and combined data, plus 95%

confidence intervals based on the mean are displayed. Standard deviations for both the

qualitative data sets are higher than quantitative. Results for the candidate standard are

marginally higher across both data sets relative to the 4th

IS than the 2nd

with a difference of 0.59

and 0.44 respectively. A -0.2 log difference is apparent between the 4th

IS and the 2nd

IS

qualitative data sets and potency is also reduced, although to a lesser degree, in the quantitative

data (0.11 log), although these differences are not statistically significant.

Accelerated degradation studies Table 4 shows titres of the candidate standard at following 3 and 6 months storage at elevated

temperatures. Differences between titres compared with the -200C sample at each time point are

shown in parentheses. Changes in titre of up to 0.07 log10 IU/mL were observed in temperatures

up to +200C at the three month time point and titres at the 6 month time point are indicating a

0.14 log10 IU/mL loss at +200C, but all are within the variability of the assays, as presented in

table 4.

Discussion In this study, a range of NAT-based assays for HCV have been used to evaluate the stability and

suitability of the candidate standard as the replacement 5th WHO International Standard for

HCV for NAT-based assays.

A WHO IS for HCV NAT has been available since 1996. During this time, 4 replacement

batches have been established; two comprising antibody positive material and two comprising

WHO/BS/2015.2262

antibody negative, the rationale for the different formulations has circulated around the primary

end user. Initially, due to the availability of material, the 1st and 2

nd standards were developed

with antibody positive sourced plasma from the UK Blood Transfusion Service. The sourcing of

sufficient material for the formulation of a standard can be problematic, with either a small

volume of high titre material needed for spiking or a large volume, <2000mL, that can be

aliquoted and lyophilised directly with no need for a dilution to be made. Following the

development of the first two standards it was identified that the main use of the standard was to

calibrate assays used by the blood donation field, in this case the detection of a positive HCV

NAT sample would be likely to be a window period donation, thus it was subsequently decide to

produce the 3rd

replacement HCV IS using antibody negative material.

The 3rd

and 4th

HCV IS preparations were formulated from at the same time from the same bulk

stock material, they were evaluated in the same collaborative study, suitable performance was

demonstrated by both candidates and thus the 4th

followed on when the 3rd

became depleted. As

assay sensitivity improved a loss in titre in the region of 0.2log10 IU/mL in the 4th

IS became

detectable. A look back study across all HCV IS’s also identified stability issues with the 3rd

preparation, however this was not mirrored in the ‘older’ 1st and 2

nd standard preparations. The

main difference between these preparations was the presence of HCV antibody in standards 1

and 2 and its absence in 3 and 4, ie standards 3 and 4 were formulated from a window period

donation.

It was therefore hypothesised that the presence of HCV antibody created a stabilising effect on

the virus during the lyophilisation process, perhaps by the formation of complexes that protects

the fragile RNA.

Prior to the formulation and study to establish the 5th

standard, a pilot study was performed,

however as demonstrated in this report, with the formulations and protocols used for the study no

loss in titre was observed. The question over antibody positive or negative formulations was

revisited; the rationale for an antibody negative material suiting blood donation centres detecting

window period donations still applies. However it is also recognised that assays would also be

used for patient diagnosis and treatment management, in which case the clinical sample would

most likely be antibody positive. Having established that the presence or absence of stability did

not affect the lyophilised stability of the end product it was agreed to formulate the replacement

5th HCV NAT IS with window period donation. Sufficient volume was available at NIBSC and

its use would enable a timely production of material to ensure continuity of supply.

The candidate was therefore prepared from a new bulk material that had been evaluated for

stability in a pilot study previously described in this report. In line with previous WHO HCV

NAT standards, the selected candidate material was genotype 1a, a circulating strain that still

predominates in the developed world. The selection of genotype was approved at the 3rd

joint

workshop of Blood Virology and Clinical Diagnostics held in Graz, Austria on 30/31st May

2014. It was accepted that whilst a variety of genotypes are in circulation material, the

availability of a WHO International Reference panel for HCV NAT is available for the

optimisation of assays against different genotypes and continuity between WHO HCV NAT

international standards would be prudent.

WHO/BS/2015.2262

Data generated during the pilot study indicated a 0.2 log drop was to be expected during the

lyophilisation of the HCV positive antibody negative material. Appropriate calculations were

made for the production of the final material to adjust for this loss to give a final candidate at 5

log10 IU/mL, as can be seen in table 1, the combined difference for difference between frozen

liquid bulk (S1) and the lyophilised standard (S5) is 0.17 log, however if the data for quantitative

and qualitative data sets is analysed independently there is a larger decrease in titre observed

with qualitative assays (0.43 log10) than quantitative (0.09 log10). The reason for this is unknown,

qualitative assays used in this study utilise similar extraction and amplification mechanisms

compared with those used in quantitative systems so this phenomenon is unlikely to be

attributable to the mechanisms and perhaps more due to analysis methods associated with end

point dilutions.

In this study both the 2nd

and 4th

(current) HCV IS were included for possible relative potency

assignment. Historically it is common practice to assign potency to a replacement standard based

on the immediate previous standard. Theoretically for true continuity of the IU from one

standard to another a direct comparison should always be made back to the 1st IS. However, in

practice, this is not always possible due to limited availability of material. It has discussed

previously in this report that stability issues had been identified with the current 4th

IS. Whilst

these appear to be limited to degradation only at temperatures above -200C it was decided to

include a previous standard to give an alternative reference for relative potency assignment

should the 4th

IS underperform for any reason in this study. Unfortunately there was insufficient

material available to include samples from the 1st HCV IS, however the 1

st and 2

nd standards

were produced from the same bulk material at the same dilution concentration, they were both

assigned a unit of 5 log10 IU/mL in the initial study; the only difference between them being that

the lyophilisation was performed on two different days. Both 1st and 2

nd standard have remained

stable at -200C since their production 19 years ago. Thus it was appropriate to include the stable

2nd

standard in addition to the current 4th

.

Table 4 demonstrates the accelerated degradation stability testing performed to date. Such data

has subsequently been discussed that the 20th

Anniversary SoGAT workshop, previously

referenced, and a WHO collaborating centres meeting held at NIBSC on 2-3rd

July 2015. At both

meetings it was agreed that the data is inconclusive and additional stability testing should be

performed prior to establishment. The six month time point will be repeated and addition 9

month time point performed in advance of the WHO ECBS meeting. In both assays the thermal

degradation samples will be tested alongside the current IS for direct potency calibration instead

of using the assays calibration. Indications at 3months storage at +200C suggest that the material

would remain stable during normal transportation timeframes, this will be confirmed by

simulated transport analysis .

Figures 7-10 and table 10 focus on the relative potency comparisons of both standards to the

proposed 5th

. It can be seen in figure 7 (combined quantitative and qualitative data) that when

comparing the relative potency of S5 relative to S3 (2nd

IS) there is a single outlying data point

from a laboratory using the Roche MPX assay performed on the S201 platform (lab 15). A

second laboratory also provided data for this study using the same platform (lab 9) and it can

been seen that these results are within the main consensus grouping, suggesting that the reduced

WHO/BS/2015.2262

relative potency is a factor of the way the assay has been performed on this occasion and not the

initial assay calibration.

Similarly, when observing data in figure 9, relative potency of S5 compared to S4 (current 4th

IS), qualitative outliers are observed. In addition to the example above, the Procleix Ultrio assay

(lab 10) and Roche MPX performed on the 8800 platform also reports lower potencies than

would be expected. A comparison can be made for the Procleix Ultrio assays with a second

laboratory also using this assay (lab 7), it can be seen that the data set falls within the main

consensus group, therefore as above this is not considered an artefact of the assay system. It is

however harder to draw a conclusion for the final outlier (lab 8) as no other laboratories have

performed this assay in this study.

Given the above comparisons and the potency data presented in table 10, it is apparent that a unit

could be assigned to S5 based on either the 2nd

or 4th

standard. However with the consideration

of stability issues previously identified with the 4th

standard and the ideal scenario, that relative

potencies should be compared directly with the 1st IS, it would seem appropriate to assign the

relative potency of S5 compared with S3 (2nd

IS). S3 has demonstrated good stability and is

identical in formulation and unit assignment to the 1st IS. Furthermore the HCV RNA IS will be

used by users of both qualitative and quantitative assays; it is therefore recommended that at

potency should be derived from the combined data of both data sets.

In a change to previous HCV NAT IS’s, this 5th

replacement has been formulated to be

reconstituted in a final volume of 1.1mL. Previous standards have been reconstituted in a 0.5mL

volume, from the advent of IS development in the blood field 0.5mL provided sufficient volume

for a 1 vial to be required. Current assays now require up to 1mL sample volume, thus with

previous 0.5mL volumes customers were ordering at least two vials to run one assay and stocks

were becoming depleted too quickly. This change will be clearly stated in the information for use

sheet that accompanies each shipment of material. In addition a notification email will also be

sent to all customers who have received the 4th

standard within the last year, notifying them of

the volume change and of the availability of the new standard.

The batch of candidate 5th

HCV IS comprises 1980 vial. With an anticipated dispatch rate of 250

vials/annum; this stock represents a supply that should have is a little over 7.5 years supply.

Whilst it may seem prudent to make a much larger batch, as highlighted above with a biological

preparation such as this, it is always a challenge sourcing a sufficient volume and titre of stock

material. In these situations one way of ensuring the provision of the same batch of material for

an extended period of time is to provide end users with well calibrated secondary reference

materials. Within this study, sample 6 was evaluated for this purpose.

In conclusion a relative potency assigned compared to the 2nd

HCV IS would equal S5 being

assigned a unit of 5.0 log10 IU/mL when reconstituted in 1.1ml.

If it was decided that a potency should be assigned based on the comparison to the current 4th

IS,

then S5 would be assigned a unit of 4.90 log10 IU/ mL when reconstituted in 1.1ml

WHO/BS/2015.2262

Conclusion

Based on the results of this collaborative study, S5 observed in this report (NIBSC code 14/150)

demonstrates continued harmonisation from the availability of a standard for HCV RNA. The

HCV antibody negative formulation used for this candidate performs equally in all assays used.

Further investigation of stability needs to be carried out and data will be available for discussion

at the 2015 WHO ECBS meeting with a recommendation as to how this material should be sent

to participants, supplementary analysis will be submitted to the committee no later than one

month before the meeting.

This standard will continue to be used by IVD manufacturers, blood transfusion centres, control

authorities, and clinical laboratories using both quantitative and qualitative assays, to calibrate

secondary reference materials and in the validation of HCV NAT assays. It is therefore

appropriate to derive a relative potency value from a combined data set comprising quantitative

and qualitative data.

A potency should be assigned against the 2nd

HCV RNA IS (S3), this would determine that when

properly reconstituted, the preparation contains of 5.0 log10 IU/ml

Proposal

It is proposed that a value of 5.0 log10 IU/mL is assigned to the 5th International Standard for

HCV RNA for use in NAT (NIBSC code 14/150).

Comments from Participants

This report was circulated to all participants for comment. Six participants returned a variety of

comments. One laboratory resubmitted data having discovered a calculation error in their data

previously submitted, this required some reanalysis by our statistical department. Three

laboratories retuned minor typographical corrections and clarification of correct table references

in the text. One laboratory asked for the intended unit assignment per mL to be clarified in the

final proposal as the per/vial and per/mL unit was confusing.

Finally one laboratory suggested the qualitative data should not be included in the final relative

potency assignment due to the variability that was observed in these data sets. Whilst this

standard is intended for use in both quantitative and qualitative assays it is agreed that calibration

of a unit assignment in IU/ml will impact largely on the quantitative assays, therefore, in

conjunction with the statistical group at NIBSC the decision has been made to make a unit

proposal based on quantitative data only. It was also suggested that further statistical methods

should be included for some of the data sets, this comment was address by the statistical group at

NIBSC, in this instance further references have been included.

WHO/BS/2015.2262

Acknowledgements

We gratefully acknowledge the important contributions of the collaborative study participants

and the organisations donating material for use in this study

References

1. Saldanha J, Lelie PN and Heath AB. Report on the evaluation of a candidate standard for

GAT assays for HCV RNA. WHO ECBS Report 1997;WHO/BS/97.1861.

2. Saldanha J, Lelie N and Heath AB. Establishment of the First International Standard for

Nucleic Acid Amplification Technology (NAT) assays for HCV RNA. 1999. Vox Sang.

76:149-158.

3. Saldanha J, Heath A, Aberham C, Albrecht J, Gentili G, Gessner M, and Pisani G. WHO

collaborative study to establish a replacement WHO international standard for HCV RNA

NAT. WHO ECBS Report 2003;WHO/BS/03.1958.

4. Saldanha J, Heath A, Aberham C, Albrecht J, Gentili G, Gessner M, Pisani G. World Health

Organization collaborative study to establish a replacement WHO international standard for

hepatitis C virus RNA nucleic acid amplification technology assays. Vox Sang.

2005;88:202-4.

5. Fryer J, Heath AB, Wilkinson DE, Minor PD, Collaborative Study Group. Collaborative

Study to Evaluate the Proposed 4th WHO International Standard for Hepatitis C Virus

(HCV) for Nucleic Acid Amplification Technology (NAT)-Based Assays. WHO ECBS

Report 2011;WHO/BS/2011.2173.

6. Baylis SA, Heath AB and the Collaborative Study Group. WHO collaborative study to

establish a replacement WHO International Standard for Hepatitis C virus RNA nucleic acid

amplification technology (NAT)-based assays. WHO ECBS Report 2007;WHO/BS/07.2055.

7. Baylis SA, Heath AB; Collaborative Study Group. World Health Organization collaborative

study to calibrate the 3rd International Standard for Hepatitis C virus RNA nucleic acid

amplification technology (NAT)-based assays. Vox Sang. 2011;100:409-17.

8. Guidance for Industry Nucleic Acid Testing (NAT) for Human Immunodeficiency Virus

Type 1 (HIV-1) and Hepatitis C Virus (HCV): Testing, Product Disposition, and Donor

Deferral and Reentry. U.S. Department of Health and Human Services Food and Drug

Administration Center for Biologics Evaluation and Research May 2010.

9. Monograph on ‘human plasma for fractionation.’ European Pharmacopoeia. 07/2008:0853. 10. Collet, D., 1991. Modelling Binary Data, Chapman Hall, London.

11. Francis, B., Green, M., Payne, C. (Eds), 1993. The GLIM System Release 4 Manual.

Oxford Science Publications, Clarendon Press, Oxford.

http://www.ncbi.nlm.nih.gov/pubmed/15787732












WHO/BS/2015.2262

Table 1: Formulation of pilot study samples

Study

Sample Code A B C D E F

G

HCV

antibody

status

+ - + - + -

4th HCV

NAT IS

Stabilizer

included - -

Hepes

40mmol/L

Hepes

40mmol/L

Trehalose

10mg/L

Trehalose

10mg/L

WHO/BS/2015.2262

Table 2: Combined pilot study data

Sample Liquid

Bulk

Freeze Dried

Samples

Loss

in FD

(log10)

-20 -M

and -20

+M

-20 -M

and

+20

+M

-20 -

M and

+20 -

M

Moisture (+M)

No Moisture (-

M)

+20

0C -20

0C +20

0C -20

0C

1 (Anti

Pos)

5.12

(0.05)

4.50

(0.11)

4.56

(0.10)

4.54

(0.09)

4.49

(0.11) -0.63 -0.07 -0.01 -0.05

2 (Anti

Neg)

4.93

(0.04)

4.65

(0.02)

4.66

(0.04)

4.72

(0.04)

4.72

(0.09) -0.21 0.06 0.07 0

3 (Anti

Pos +

HEPES)

5.13

(0.05)

4.65

(0.07)

4.69

(0.11)

4.67

(0.12)

4.70

(0.12) -0.43 0.01 0.05 0.03

4 (Anti

Neg +

HEPES)

4.96

(0.18)

4.79

(0.04)

4.81

(0.04)

4.75

(0.06)

4.77

(0.06) -0.19 -0.04 -0.02 0.02

5 (Anti

Pos +

TRE)

5.03

(0.12)

4.46

(0.10)

4.65

(0.13)

4.54

(0.08)

4.51

(0.10) -0.52 -0.14 0.05 -0.03

6 (Anti

Neg +

TRE)

4.89

(0.15)

4.66

(0.03)

4.60

(0.06)

4.67

(0.04)

4.64

(0.06) -0.25 0.04 -0.02 -0.03

4th HCV

IS N/A

5.36

(0.15)

5.36

(0.09)

4.89

(0.23)

4.98

(0.12)

(Standard deviation values are shown in parenthesis)

WHO/BS/2015.2262

Table 3: Details of lyophilised candidate preparation, Study code S5

(NIBSC Code 14/150)

14/150

Number of Vials filled 1980

Mean fill mass (g) 1.1003

CV of fill mass (%) 0.29

Mean residual oxygen (%) 0.87

Mean residual moisture (%) 0.25

Table 4: Accelerated degradation studies of 14/150

-200C +4

0C +20

0C +37

0C +45

0C

3

months 5.09 ( - ) 5.17 ( +0.08) 5.02 (-0.07) 4.86 (-0.23) 4.92 (-0.17)

6

months 5.41 ( - ) 5.35 (-0.06) 5.27 (-0.14) 5.10 (-0.31) NT

*NT = not tested; Values in brackets indicate difference from -200C result

WHO/BS/2015.2262

Table 5: Laboratory Mean Estimates from Quantitative Assays

(log10 IU/mL)

Lab test Lab

code

S1 S2 S3 S4 S5 S6

Roche CAP/CTM v2 1a 4.94 4.13 5.04 5.65 5.06 2.37

Roche COBAS 6800 1b 5.11 4.03 5.14 5.66 5.28 2.73

Versant kPCR HCV RNA 2 5.27 4.47 5.06 5.80 5.07 2.38

Artus HCV QS-RGO Kit

V1 3 5.04 4.10 5.26 5.65 5.05 1.87

Abbott real time HCV

(0.5mL) 4 5.02 4.38 4.99 5.50 4.87 2.05

Roche COBAS 6800 5 5.15 4.48 5.04 5.48 5.10 2.17

Roche CAP/CTM v2 6 5.20 4.60 5.10 5.70 5.13 2.48

Qiagen QiaSymphony 13 4.96 4.32 4.98 5.48 5.18 2.33

Roche CAP/CTM v2 14 4.48 4.32 4.95 5.42 4.60 2.13

In house 17a 5.56 4.98 5.68 6.09 5.72 3.40

In house 17b 4.83 4.31 5.06 5.77 5.47

Roche CAP/CTM v2 18 5.39 4.55 5.22 5.46 5.35 2.59

Table 6: Laboratory Mean Estimates from Qualitative Assays

(NAT-detectable units/mL)

Assay Lab S1 S2 S3 S4 S5 S6

Procliex Ultrio 7 4.82 5.1 4.62

Roche MPX on 8800

analyser 8 4.97 3.64 5 6.73 4.68 1.84

Roche MPX on S201 9 4.67 3.84 4.69 5.36 4.78 1.78

Procliex Ultrio 10 5.07 4.26 5.25 6.26 4.61 1.89

Roche MPX on

Ampliprep 11 4.61 3.74 4.43 4.5 4.43 1.45

Roche MPX on

Ampliprep 12 5.02 3.82 4.5 5.02 4.81 2.6

Roche MPX on S201 15 6.06 3.62 6.06 6.07 4.47

WHO/BS/2015.2262

Table 7: Overall Mean Estimates and Inter-Laboratory Variation (log10

IU/mL for quantitative or NAT-detectable units/mL for qualitative assays)

Sample Assay n Mean SD GCV Min Max

S1 Qualitative 6 5.07 0.52 234% 4.61 6.06

Quantitative 12 5.08 0.28 89% 4.48 5.56

Combined 18 5.07 0.36 129% 4.48 6.06

S2 Qualitative 6 3.82 0.23 71% 3.62 4.26

Quantitative 12 4.39 0.26 81% 4.03 4.98

Combined 18 4.20 0.37 133% 3.62 4.98

S3 Qualitative 7 4.96 0.56 264% 4.43 6.06

Quantitative 12 5.13 0.20 58% 4.95 5.68

Combined 19 5.07 0.37 133% 4.43 6.06

S4 Qualitative 7 5.58 0.80 525% 4.50 6.73

Quantitative 12 5.64 0.19 55% 5.42 6.09

Combined 19 5.61 0.48 205% 4.50 6.73

S5 Qualitative 7 4.63 0.14 39% 4.43 4.81

Quantitative 12 5.16 0.28 93% 4.60 5.72

Combined 19 4.96 0.35 126% 4.43 5.72

S6 Qualitative 5 1.91 0.42 162% 1.45 2.60

Quantitative 11 2.41 0.41 157% 1.87 3.40

Combined 16 2.25 0.46 191% 1.45 3.40

Table 8: Intra-Laboratory standard deviation of log10 IU/mL in Quantitative

Assays

Lab S1 S2 S3 S4 S5 S6

1a 0.09 0.10 0.01 0.07 0.11 0.30

1b 0.16 0.10 0.04 0.04 0.11 0.05

2 0.11 0.03 0.15 0.07 0.04 0.13

3 0.08 0.17 0.03 0.10 0.12 0.11

4 0.08 0.02 0.05 0.04 0.08 0.03

5 0.06 0.07 0.05 0.07 0.05 0.05

6 0.06 0.02 0.02 0.02 0.07 0.24

13 0.02 0.04 0.14 0.08 0.06 0.54

14 0.48 0.04 0.12 0.20 0.14 0.20

17a 0.55 0.13 0.11 0.34 0.21 0.40

17b 0.03 0.02 0.05 0.04 0.03

18 0.03 0.05 0.06 0.07 0.14 0.08

WHO/BS/2015.2262

Table 9: Difference between samples (log10 IU/mL for quantitative

or NAT-detectable units/mL for qualitative assays)

Sample Rel to Assay n Mean SD GCV Min Max

S5 S1 Qualitative 6 -0.44 0.60 295% -1.59 0.11

Quantitative 12 0.08 0.22 67% -0.20 0.64

Combined 18 -0.09 0.45 179% -1.59 0.64

S5 S3 Qualitative 7 -0.34 0.63 329% -1.59 0.31

Quantitative 12 0.03 0.20 57% -0.35 0.41

Combined 19 -0.10 0.44 173% -1.59 0.41

S5 S4 Qualitative 7 -0.95 0.80 528% -2.05 -0.07

Quantitative 12 -0.48 0.21 61% -0.82 -0.11

Combined 19 -0.65 0.54 248% -2.05 -0.07

S2 S5 Qualitative 6 -0.81 0.26 80% -1.04 -0.35

Quantitative 12 -0.77 0.28 92% -1.26 -0.29

Combined 18 -0.78 0.27 85% -1.26 -0.29

S6 S5 Qualitative 5 -2.75 0.32 528% -2.05 -0.07

Quantitative 11 -2.72 0.23 71% -3.19 -2.32

Combined 16 -2.73 0.25 79% -3.18 -2.21

Table 10: Potency Estimates for candidate S5 in log10 IU/mL

Data Relative to n Mean 95% C.I. Stdev Qualitative 2nd IS

(5.00 log10 IU)

7 4.66 4.08 – 5.25 0.63

Quantitative 12 5.03 4.91 – 5.16 0.20

Combined 19 4.90 4.69 – 5.11 0.44

Qualitative 4

th IS

(5.41 log10 IU)

7 4.46 3.73 – 5.20 0.80

Quantitative 12 4.93 4.80 – 5.06 0.21

Combined 19 4.76 4.50 – 5.02 0.54

WHO/BS/2015.2262

Figure 1: Laboratory Mean Estimates for Sample S1 in log10 IU/mL

Num

ber

of Labora

tories

0

1

2

3

4

5

6

7

8

9

10

11

12

13

Log10 IU/ml

1 2 3 4 5 6 7

9

11

14

1a

1b

3

4

5

6

8

10

12

13

17b

2

17a

18

15

Quantitative Assays Qualitative Assays

WHO/BS/2015.2262


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ber

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1

2

3

4

5

6

7

8

9

10

11

12

13

Log10 IU/ml

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8

11

15

1a

1b

3

9

12

2

4

5

6

10

13

14

17b

18

17a


WHO/BS/2015.2262


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2

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4

5

6

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11

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13

Log10 IU/ml

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1a

1b

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WHO/BS/2015.2262


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2

3

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Log10 IU/ml

1 2 3 4 5 6 7

11 7

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1a

1b

3

4

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6

9

13

14

18

2

15

17a

17b

8

10


WHO/BS/2015.2262

Figure 5 Laboratory Mean Estimates for Sample S5 in log10 IU/mL

Num

ber

of Labora

tories

0

1

2

3

4

5

6

7

8

9

10

11

12

13

Log10 IU/ml

1 2 3 4 5 6 7

7

8

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11

14

15

1a

2

3

4

5

6

9

12

13

1b

17a

17b

18


WHO/BS/2015.2262

Figure 6 Laboratory Mean Estimates for Sample S6 in log10 IU/mL

Num

ber

of Labora

tories

0

1

2

3

4

5

6

7

8

9

10

11

12

13

Log10 IU/ml

1 2 3 4 5 6 7

11 3

4

5

8

9

10

14

1a

1b

2

6

12

13

18

17a


WHO/BS/2015.2262

Figure 7 Relative Potency Estimates for Sample S5 relative to Sample S3

Num

ber

of Labora

tories

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

Log10 IU/ml

2 3 4 5 6 7 8

15 8

10

14

1a

1b

2

3

4

5

6

7

9

11

13

17a

18

12

17b


WHO/BS/2015.2262

Figure 8 Relative Potency Estimates for Sample S5 relative to Sample S4

Num

ber

of Labora

tories

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

Log10 IU/ml

2 3 4 5 6 7 8

8 10

15

2

14

1a

1b

3

4

5

6

7

9

12

13

17a

17b

11

18


WHO/BS/2015.2262

Appendices 1- 10

Appendix 1: Pilot study participants

Participant Laboratory Country

Mr. Graham

Prescott

NIBSC UK

Dr. Hong Wang Roche Molecular Systems USA

Dr. Gregg Gorrin Siemens USA

Dr. John Buckels QIAGEN UK

Dr George

Schneider

Abbott Molecular Inc USA

Appendix 2: Assays used in the pilot study

COBAS ® AmpliPrep/COBAS ® TaqMan ® HCV

Test

COBAS ® AmpliPrep/COBAS ® TaqMan ® HCV

Test

VERSANT ® HCV RNA 1.0 Assay (kPCR)

artus HCV QS-RGQ assay

Abbott RealTime HCV Assay

WHO/BS/2015.2262

Appendix 3: covering letter for pilot study

WHO/BS/2015.2262

WHO/BS/2015.2262

Appendix 4: Pilot study protocol and results return sheets

Collaborative Study to evaluate the stability of different candidate materials for the 5th

International standard for HCV

Objective

The aim of the study is to evaluate the stability of different candidate materials for the 5th WHO

International Standard for HCV.

Background

The 4thWHO International Standard (IS) for HCV (NIBSC code: 06/102) was first established in

2011 with sufficient stocks for 3 to 4 years. The collaborative study to established the IS

highlighted some stability issues with the material when stored at elevated temperatures

(+200C). It is now therefore shipped on dry ice and stored at -20 or lower on arrival.

At the SoGAT Blood Virology meeting (2012) it was decided to replace the IS earlier than

planned by first investigating the potential variables that could induce instability over time under

specific conditions.

Materials

The package consists of 28 different freeze dried and 6 liquid preparations (coded as below). The

quantity of each will vary and is dependent on the assay and the volume required for extraction.

There will be enough vials to repeat the assay on a second occasion.

Freeze Dried Liquid Preparations

A1-4 Bulk 1

B1-4 Bulk 2

C1-4 Bulk 3

D1-4 Bulk 4

E1-4 Bulk 5

F1-4 Bulk 6

G1-4

Caution

THESE PREPARATIONS ARE NOT FOR ADMINISTRATION TO HUMANS

Both liquid and freeze dried materials are potentially infectious. Please take care and wear

suitable PPI in a contained lab when opening/preparing vials.

WHO/BS/2015.2262

Study Protocol

For each assay, one panel of the freeze dried material (A1-4 to G1-4) should be reconstituted

with 0.5mL of nuclease free water and left for a minimum of 20 minutes with occasional

agitation before use (please see attached IFU for further details). Frozen liquid preparations

(Bulk 1 to 6) should be removed from -70 storage and thawed just prior to use. All panel

members should be prepared and tested at the same time in a single assay. We do not recommend

that vials should be used after a freeze thaw cycle.

Participants are requested to perform two independent assays. A fresh vial of each reagent should

be used in each assay.

If your assay requires more than 0.5mL; pool the content of two/three vials of the same sample

code. Your shipment should contain sufficient vials to accommodate your assay.

For Quantitative assays

Assay samples A1-4 to G1-4 and Bulks 1-6 undiluted, where possible two assays should be

carried out on separate occasions.

For Qualitative assays

Assay samples A1-4 to G1-4 and Bulks 1-6 to determine the HCV RNA end-point. We would

suggest using 4 tenfold dilutions initially to determine an end point. Samples should be diluted in

human plasma previously determined to be negative for HCV RNA.

In the subsequent assays participants are requested to assay a minimum of two half log dilutions

either side of the pre-determined end point.

Results

Results of each assay should be recorded on the appropriate result form included with this

information sheet.

Please ensure the method details form is also complete.

Please Note: If your lab is not able to test all samples as outlined above due to logistical

problems, then please contact Clare Morris ([email protected]) to discuss a bespoke

protocol.

Data should be returned no later than 21-May-2014 to allow sufficient time for statistical

analysis and preparation for presenting this data at the SoGAT 2nd Joint Blood Virology and

Clinical Diagnostics meeting 2014 to be held in Graz, Austria 28/29th May 2014.

All completed forms should be returned to:


WHO/BS/2015.2262

Clare Morris

Division of Virology

NIBSC

Blanche Lane

South Mimms

Potters Bar

Hertfordshire.

EN6 3QG

UK

Fax: +44 1707 641 060

[email protected]


WHO/BS/2015.2262

Appendix 5: Study results sheets

HCV Stability Collaborative Study

Method Reporting

Name of Participant/Lab Code:

Extraction Method:

Diluent Used

Extraction Protocol

Extraction Instrument (If Used)

Volume Extracted

Volume Eluted

Amplification Method

HCV NAT Assay

HCV gene target

Thermal cycler used

Volume of RNA amplified

Assay 1

Date: Name of Participant/Lab Code:

Operator :

Sample

HCV Result

IU/mL

Crossing point/Ct Value

(if applicable) Comments

A1

A2

A3

WHO/BS/2015.2262

A4

Bulk 1

B1

B2

B3

B4

Bulk 2

C1

C2

C3

C4

Bulk 3

Sample

HCV Result

IU/mL



D1

D2

D3

D4

Bulk 4

E1

E2

E3

E4

Bulk 5

F1

F2

F3

F4

WHO/BS/2015.2262

Assay 2

Date: Name of Participant/Lab Code: Operator :

Bulk 6

G1

G2

G3

G4

Sample

HCV Result

IU/mL



A1

A2

A3

A4

Bulk 1

B1

B2

B3

B4

Bulk 2

C1

C2

C3

C4

Bulk 3

Sample HCV ResultIU/mL



WHO/BS/2015.2262

D1

D2

D3

D4

Bulk 4

E1

E2

E3

E4

Bulk 5

F1

F2

F3

F4

Bulk 6

G1

G2

G3

G4

WHO/BS/2015.2262

Appendix 6: Pilot study IFU

Hepatitis C virus for Nucleic Acid Amplification Techniques

WHO HCV Samples 1-6

Version 1.0, 04th November 2014 1. CONTENTS

Samples 1, 2 & 6 Upon receipt, samples 1, 2 & 6 should be stored at -70°C or below. Samples 1, 2 & 6 contain approximately 1.1 mL of hepatitis C virus positive human plasma. Samples 3 - 5 Samples 3-5 should be stored at -70 or below. Samples 3 & 4 contain 0.5 mL of lyophilized hepatitis C virus (HCV) in a 2 mL crimp top glass vial. Prior to use, these samples must be reconstituted with 0.5 mL of deionised, nuclease-free, molecular-grade water. Sample 5 contains 1.1 mL of lyophilized hepatitis C virus in 5 mL screw cap glass vial. These vials should be left for a minimum of 20 minutes with occasional agitation before use. Further instructions for the dilution and testing of these study samples are provided in the study protocol accompanying the samples.

2. CAUTION THIS PREPARATION IS NOT FOR ADMINISTRATION TO HUMANS OR

ANIMALS. Samples 1-6 contain human plasma and infectious HCV. The human plasma in

which the HCV has been diluted was tested and found negative for HBsAg, HIV antibody, and HCV RNA by PCR.

As with all materials of biological origin, this preparation should be regarded as

potentially hazardous to health. It should be used and discarded according to your own laboratory's safety procedures. Such safety procedures probably will include the wearing of protective gloves and avoiding the generation of aerosols. Care should be exercised in opening ampoules or vials, to avoid cuts.

WHO/BS/2015.2262

3. DIRECTIONS FOR OPENING THE CRIMP TOP VIALS (SAMPLES 3 & 4)

Vials have a ‘flip-up’ circular cap. Either on the cap or the collar of the vial, there is

an indication of the point at which to lever off the cap. This exposes an area of the stopper through which reconstitution and withdrawal of the preparation can be made using a hypodermic needle and syringe. If use of a pipette is preferred, then fully remove the metal collar using, for example, forceps, taking care to avoid cuts by wearing appropriate gloves. Remove the stopper for access.

4. CITATION In any circumstance where the recipient publishes a reference to NIBSC materials,

it is important that the title of the preparation and any NIBSC code number, and the name and address of NIBSC are cited correctly.

5. LIABILITY AND LOSS Information provided by the Institute is given after the exercise of all reasonable

care and skill in its compilation, preparation and issue, but it is provided without liability to the Recipient in its application and use.

It is the responsibility of the Recipient to determine the appropriateness of the

materials supplied by the Institute to the Recipient (“the Goods”) for the proposed application and ensure that it has the necessary technical skills to determine that they are appropriate. Results obtained from the Goods are likely to be dependant on conditions of use by the Recipient and the variability of materials beyond the control of the Institute.

All warranties are excluded to the fullest extent permitted by law, including without

limitation that the Goods are free from infectious agents or that the supply of Goods will not infringe any rights of any third party.

The Institute shall not be liable to the Recipient for any economic loss whether

direct or indirect, which arise in connection with this agreement. The total liability of the Institute in connection with this agreement, whether for

negligence or breach of agreement or otherwise, shall in no event exceed 120% of any price paid or payable by the Recipient for the supply of the Goods.

WHO/BS/2015.2262

6. MATERIAL SAFETY SHEET

Physical properties (at room temperature)

Physical appearance Lyophilised power (samples 3,4 & 5) Frozen Liquid (samples 1,2 & 6)

Fire hazard None

Chemical properties

Stable Yes Corrosive: No

Hygroscopic No Oxidising: No

Flammable No Irritant: No

Other (specify) Contains infectious hepatitis C virus and material of human origin

Handling: See caution, section 2

Toxicological properties

Effects of inhalation: Avoid – contains infectious hepatitis C virus

Effects of ingestion: Avoid – contains infectious hepatitis C virus

Effects of skin absorption: Avoid – contains infectious hepatitis C virus

Suggested First Aid

Inhalation Seek medical advice – contains infectious hepatitis C virus

Ingestion Seek medical advice – contains infectious hepatitis C virus

Contact with eyes Wash with copious amounts of water. Seek medical advice.

Contact with skin Wash thoroughly with water.

Action on Spillage and Method of Disposal

WHO/BS/2015.2262

Spillage of ampoule contents should be taken up with absorbent material wetted with a virucidal agent. Rinse area with a virucidal agent followed by water. Absorbent materials used to treat spillage should be treated as biologically hazardous waste.

Appendix 7: Main study Assay representation

Assay Code Assay No. of data

sets

CTM COBAS® AmpliPrep/COBAS® TaqMan® HCV v2.0 4

COBAS® HCV Test (6800/8800 System) 2

kPCR VERSANT® HCV RNA 1.0 Assay (kPCR) 1

artus® HCV QS-RGQ Kit V1 1

Abbott RealTime HCV 1

In-House HCV 3

Qualitative Assays

Assay Code Assay

Procleix Ultrio Plus® Assay 1

Procleix Ultrio Assay 1

COBAS® MPX test, (6800/8800 Systems) 1

COBAS® TaqScreen MPX Test v2.0 (COBAS® s 201

system)

4

Appendix 8: Main study participants

Participant Laboratory Country

Mr. Graham

Prescott

NIBSC, South Mimms UK

Dr. Thomas Rotten CSL Behring, Bern Switzerland

Dr. YuYany Tangyi NIFDC, Beijing China

WHO/BS/2015.2262

Dr. Agnieszka

Dryla/

Dr. Christina Wolf

Baxter, Vienna Austria

Dr. Saeko Mizusawa National Institute of Infectious

diseases, Tokyo

Japan

Dr. Simon Carne Public Health England, Colindale UK

Dr. Cristina

Alemany

BIOMAT, Barcelona Spain

Dr. Antonia

Zucchini/

Dr. Alessia Monti

Kedrion, Lucca Italy

Dr. Giulio Pisani/

Dr. Karen Cristiano

National Center for

Immunibioloicals Research and

Evaluation (CRICVIB), Rome

Italy

Dr. Hong Wang Roche Molecular Systems,

Somerville

USA

Dr. Lutz Pichl GRC BTS West, Hagen Germany

Dr. Gregg Gorrin/

Dr. Lovedeep

Grewal

Siemens Healthcare Diagnostics,

Berkeley

USA

Dr. George

Schneider/

Dr. William

Camimiti

Abbott Molecular, Des Plaines USA

WHO/BS/2015.2262

Dr. John Buckels

Qiagen, Manchester UK

Dr. Micha Nubling Paul-Ehrlich-Institut (PEI), Langen Germany

Dr. Yi-Chen Yang Food & Drug Administration

(FDA), Taipei

Taiwan

Dr. Susanna

Wessberg/

Dr. Vesa

Kirjavainen

Finnish Red Cross Blood Service,

Helsinki

Finland

Appendix 9: Main study protocol and results sheets

Collaborative study to evaluate the candidate 5th WHO International Standard for

Hepatitis C Virus (HCV) for NAT-based assays

Study Protocol

Background and outline of the study

The World Health Organisation (WHO) Expert Committee on Biological Standardisation

(ECBS) has endorsed a proposal to replace the 4th WHO International Standard for Hepatitis C

Virus (HCV) for NAT-based assays in order to ensure continued supply of this reference

material.

Due to the instability seen in the 4th International standard when stored at elevated temperatures;

a pilot study was carried out earlier this year. This study included both antibody positive and

negative HCV material; held at different temperatures to access the impact on stability. Suitable

material has now been prepared for a larger study to establish the 5th International standard.

WHO/BS/2015.2262

Three lyophilised preparations are to be evaluated alongside three liquid preparations including a

commutability sample. A sufficient number of vials of each study sample are provided for your

assays system. Participants are asked to test dilutions of each sample, using their routine HCV

NAT assay, on three separate occasions. Where possible, we would encourage laboratories to use

quantitative methods, however, data from qualitative assays will also be acceptable.

Study samples

Study samples comprise three liquid preparations in 2 mL screw cap tubes, two lyophilised

preparations in 2 mL crimp top glass vials, and one in a 5 mL screw cap vial; coded sample 1 - 6.

Three vials of each study sample are provided for evaluation on three separate occasions. Upon

receipt, all freeze dried samples should be stored at -20 °C or below. Liquid preparations should

be stored at -70 °C or below

CAUTION: Study samples 1-6 contain infectious HCV and should be handled only in

appropriate containment facilities by fully trained and competent staff in accordance with

national safety guidelines. These preparations contain human plasma, which has been tested and

found negative for HBsAg, and HIV antibody. Care should be taken when opening vials to avoid

cuts. See instructions for use for further details.

IMPORTANT NOTE ON VIAL ALLOCATION

Some vials in this study are in limited supply; in particular samples 3 and 4.

Please be aware that the number of vials sent reflects information that we have received on

volume requirements in your assay system.

Study protocol

Participants are requested to test dilutions of each study sample, using their routine HCV NAT

assay, on three separate occasions.

Prior to each assay run, samples 3 and 4 must be reconstituted with 0.5 mL and sample 5 with

1.1 mL of deionised, nuclease-free molecular-grade water and left for a minimum of 20 minutes

with occasional agitation before use.

Dilutions should be prepared in the diluent normally used in the assay system (e.g. anti-HCV and

HCV RNA negative human plasma).

Each sample must be extracted prior to amplification.

For each independent assay, study samples 1 - 6 should be tested within the same assay run.

Independent assays should be performed on separate days, using a fresh vial of each sample.

WHO/BS/2015.2262

Below, are specific instructions for the dilution and testing of study samples, using either

quantitative or qualitative assays.

For quantitative assays:

For each of three assays, participants are requested to test samples 1, 2 and 6 Neat.

Samples 3 – 5 should be tested, at a minimum of two serial ten-fold dilutions within the linear

range of the assay (e.g. 10-1 and 10-2). If practicable, please test as many replicates as possible

of each dilution of each sample (3 - 5) within the same assay run.

For qualitative assays:

For each of three assays, participants are requested to test the dilution at the assay end-point

(limit of detection), and a minimum of two half-log serial dilutions either side of the end-point

(i.e., at least five dilutions in total). If practicable, please test as many replicates as possible of

each dilution of each sample within the same assay run.

NB: Samples 1-5 contain approximately 1x105 IU/mL HCV RNA when reconstituted in the

specified volume of nuclease-free water.

For sample 6: We suggest starting your testing with the NEAT sample

Additional Guidance notes

For each sample please use all of the vial’s content or combine vial material to prepare both your

neat dilution and subsequent dilutions (10-1, 10-2 etc.) per assay i.e.

For assays requiring 200-350ul sample volume; only 1 vial will be provided for samples 3 and 4,

for each assay (3 in total) Both reconstitute in 0.5mL; Please use the remaining volume 150-

300ul to prepare your first dilution.

Similarly for assays requiring approximately 700ul; two vials will be required for samples 3 & 4.

For this volume please reconstitute as above and combine to create 1mL of each. Use the

remaining volume of 300ul to prepare your first dilution (10-1)

Reporting of results

The results of each assay (HCV concentration in IU/mL or qualitative result; positive / negative)

and methodology used, should be recorded on the Result Reporting Form accompanying the

samples. Where applicable, please also include the crossing point / threshold cycle for each

result. Results should be returned to NIBSC as soon as possible and before 30 January 2015, to

allow sufficient time for statistical analysis and preparation of the final report for submission to

the WHO Expert Committee for Biological Standardisation by July 2015.

WHO/BS/2015.2262

The data should not be published or cited before the formal establishment of the standard by the

WHO ECBS, without the expressed permission of the NIBSC study organiser.

Please Note: If your lab is not able to test all samples as outlined above due to logistical

problems, then please contact Graham Prescott ([email protected]) to discuss a

bespoke protocol.

All completed Result Reporting forms should be returned electronically to Graham Prescott

[email protected]

Alternatively, results may be mailed or faxed to:

Address: Graham Prescott, National Institute for Biological Standards and Control, Blanche

Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG.

Fax: +44 (0)1707 641060

Data analysis

All data from the study will be analysed at NIBSC. The analysis will assess the concentration of

each sample, relative to each other, and the sensitivities of the different assay methods.

Individual participants’ data will be coded and reported “blind” to other participants during the

preparation of the study report, and also in subsequent publications. Participants will receive a

copy of the report of the study and proposed conclusions and recommendations for comment

before it is further distributed. It is normal practice to acknowledge participants as contributors

of data rather than co-authors in publications describing the establishment of the standard.

Results Sheets: Collaborative study to replace the 4th WHO International Standard for

Hepatitis C Virus RNA for nucleic acid amplification techniques

Method Reporting:

Name of Participant/Lab Code:

Extraction Method:

Diluent Used

Extraction Protocol

Extraction Instrument (If Used)



WHO/BS/2015.2262

Volume Extracted

Volume Eluted

Amplification Method

HCV NAT Assay

HCV gene target

Thermal cycler used

Volume of RNA amplified

Assay 1


Sample

HCV Result

IU/mL



Sample 1

Neat

Sample 2

Neat

Sample 3

Neat

Sample 3

1:10

Sample 3

1:100

Sample 4

Neat

WHO/BS/2015.2262

Assay 2


Sample 4

1:10

Sample 4

1:100

Sample 5

Neat

Sample 5

1:10

Sample 5

1:100

Sample 6

Neat

Sample HCV Result/IU/mL



Sample 1

Neat

Sample 2

Neat

Sample 3

Neat

Sample 3

1:10

Sample 3

1:100

Sample 4

WHO/BS/2015.2262

Assay 3


Neat

Sample 4

1:10

Sample 4

1:100

Sample 5

Neat

Sample 5

1:10

Sample 5

1:100

Sample 6

Neat

Sample

HCV Result

IU/mL



Sample 1

Neat

Sample 2

Neat

Sample 3

Neat

Sample 3

1:10

Sample 3

WHO/BS/2015.2262

1:100

Sample 4

1:10

Sample 4

1:100

Sample 5

Neat

Sample 5

1:10

Sample 5

1:100

Sample 6

Neat

WHO/BS/2015.2262

Appendix 10: Main study covering letter:

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