INTERNATIONAL WORKING GROUP ON THE STANDARDISATION OF
GENOME AMPLIFICATION TECHNIQUES FOR THE SAFETY TESTING OF
BLOOD, TISSUES AND ORGANS FOR BLOOD BORNE PATHOGENS
SoGAT XVIII: 24-25 May 2005
Co-sponsored by CBER, FDA, USA and NIBSC, UK
Report of the meeting prepared by S Baylis (NIBSC), C Davis (NIBSC),
H Holmes (NIBSC) and J Saldanha (Roche)
The eighteenth meeting of SoGAT was held in the USA for the first time at the Natcher
Facility at NIH campus, Bethesda on the 24th and 25th May 2005. The meeting was
opened by Dr. Karen Midthun, the Deputy Director of CBER, FDA, the organisation
that was hosting the meeting. Dr. Steven Inglis, the Director of NIBSC, informed the
delegates that this was the 10th anniversary of SoGAT and commented on the long history
of standardisation associated with NIBSC.
Standardisation – the way forward (Chaired by Professor Wolfram Gerlich)
In the opening presentation, Dr. Elwyn Griffiths (Health Canada, Canada) gave a brief
overview of the history of standardisation and the role that the World Health
Organisation (WHO) plays in the development of recommendations (previously
requirements) and guidelines on the production of specific standards for the control of
biologicals. The WHO Expert Committee for Biological Standardisation (ECBS) has
been meeting since 1947 and now interacts with numerous organisations. The standards
produced include WHO international Standards which are highly characterised; WHO
reference reagents, which are less characterised interim standards with no unitage
assigned; International Reference Panels for assay and test kit evaluation. Such standards
represent complex biological preparations, by their nature difficult to characterise. With
the implementation of the IVD directive, the status of these standards has come into focus
with the recognition that biological standards and reference materials cannot be
characterised readily by chemical or physico-chemical means. WHO and ISO together
with other standard setting bodies are having on-going discussions of these issues. Many
of these issues are addressed in the new WHO recommendations for the production of
standards. Where appropriate, ISO standards such as 17511 should be used for
metrological traceability. The recommendations cover issues such as, quality assurance;
processing; stability; characterisation and collaborative studies; intended use.
Dr. Robert Wielgosz (Bureau International des Poids et Mesures (BIPM), France)
described the involvement of the Joint Committee for Traceability in Laboratory
Medicine (JCTLM) with BIPM, the International Federation for Clinical Chemistry and
Laboratory Medicine (IFCC), and the International Laboratory Accreditation Cooperation
(ILAC). Dr. Wielgosz highlighted the issues relating to traceability to higher order
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reference materials, particularly in light of the European IVD Directive and the many ISO
standards relating to aspects such as the of nature the measurand, uncertainty of
measurement, and commutability. The BIPM website lists higher order reference
materials, including those designated in SI units, such as electrolytes, non-peptide
hormones and metabolites as well more complex standards expressed in other ways
(www1.bipm.org/en/committees/jc/jctlm/jctlm-db).
Dr. Ana Padilla noted that
following a meeting with WHO and EC representatives, it had been agreed that the
HBsAg WHO International Standard calibrated in IUs would now be recognized as a
higher order standard. Dr. Wielgosz confirmed that the HBsAg IS has been accepted as
an international conventional calibrator in accordance with ISO 17511. He further
commented that a primary calibrator must be defined in SI units.
Dr. Paul Neuwald (AcroMetrix, USA) discussed NAT extraction methods. He started
off by describing the many potential sources of variation in NAT assays. Citing an
example of a CMV reference panel and the results generated from 23 laboratories, Dr.
Neuwald speculated that the variability observed between participating laboratories may
have been due, at least in part, to extraction differences. In a smaller study comparing
two different extraction methods for parvovirus B19 with amplification using the Artus
kit, differences in titre were observed suggesting an extraction issue. He highlighted that
in many previous studies the overall impact of the extraction method had not been fully
investigated using appropriate controls and that AcroMetrix would be prepared to
coordinate such a study. There was general agreement on the affect of the matrix on
extraction efficacy. Professor Gerlich asked if cultured virus had been spiked into
plasma for the CMV study, given that CMV exhibits a high degree of cell association.
Dr. Neuwald confirmed that spiking had been carried out. Dr. Kurt Roth emphasized
the importance of using optimal extraction and amplification methods to avoid widely
varying degrees of sensitivity. Professor Gerlich raised the specific problems associated
with extraction of HBV and on occasion HDV and added that variability in viral lysis
added to the complexity. Dr. Dan Cole stressed the large differences seen between
different batches of plasma.
Dr. Cindy Walker-Peach (Ambion, USA) described the extension of the armored RNA
technology to DNA. In the case of DNA viruses, controls have included plasmids,
infectious virus including attenuated/ inactivated viruses, patient samples and PCR
amplicons. Dr. Walker-Peach proposed that armored DNA was a good alternative
overcoming many of the shortfalls of the alternatives. Ambion have packaged HBV and
internal control amplicons, these are resistant to DNase treatment. QC analysis includes
HPLC of the nucleic acid to confirm the number of nucleotides and NIST-traceable
phosphate determination. Functional assays and stability studies were performed using
real time quantitative PCR demonstrating the utility of the armored DNAs.
Professor Gerlich questioned the need for armored DNA, commenting that for HBV
DNA there were commutability issues that armored DNA could not control for, such as
partial single and partial double stranded regions of the genome and the covalent binding
of protein to the genome.
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Dr. Theo Cuypers (Sanquin, The Netherlands) posed the question as to whether
conversion factors are assay related. He presented data comparing International
Standards and the PeliCheck reagents for HIV-1 RNA, HCV RNA and HBV DNA using
several different extraction and detection methods in multi-centre studies. The data
generated in the studies was summarized and highlighted that there are a wide range of
conversion factors of IU to genome equivalents (geq). Such differences may in part be
due to different extraction techniques used and the various combinations of
amplification/detection platforms. Other factors are things such as the physical and
molecular composition of the reference material which may impact on the commutability.
It was concluded that the conversion from IU to geq may be assay specific.
Professor Gerlich suggested that SoGAT should set up uniform methods to compare
conversion factors, as the use of different methods generates the large ranges observed.
He emphasized the importance of the use of the International Unit to avoid these
conversion factor issues. Dr. Nico Lelie stressed the importance of commutability and
that ideally a standard should reflect what is being measured in a donor sample.
Dr. Susan Bromley (Bayer, USA) and Dr. Bruce Phelps (Chiron, USA) spoke about the
development and characterization of synthetic standards. Dr. Bromley outlined the ideals
of a synthetic standard; of known sequence; readily manufactured; and may be quantified
using traceable methods. In vitro transcribed RNA forms the basis of a synthetic HCV
RNA standard, encompassing the 5’ non-coding region and the core gene.
Characterization and quantification of the RNA transcript is performed using absorbance
(A260), hyperchromatic shift and phosphate determination. This synthetic RNA is used at
Bayer as a reference standard to evaluate other controls and to perform validation of kit
lots. Dr. Bromley concluded that this is a stable method in use at Bayer, and a similar
approach has been used for HIV-1 RNA and HBV DNA. Dr. Bruce Phelps described
feasibility studies to evaluate synthetic RNA standards for HCV NAT. Phase I of the
study was designed by the Industrial Liaison Committee (ILC), using the in vitro
transcribed HCV RNA produced by Bayer. The synthetic RNA was quantified using
several commercially available assays and there was generally good agreement between
these (within 0.6 logs). On the basis of this original study, Phase II is in the process of
being designed to look in more detail at the in vitro transcribed RNA and armored RNA
with commutability to the biological WHO International Standard. Dr. Phelps proposed
that if these studies are successful, then such a synthetic standard could be used in
evaluating replacement standards.
Dr. John Saldanha (Roche Molecular Systems Inc, USA) compared and contrasted the
merits of using biological versus synthetic standards. The main advantages of biological
standards are that the actual agent is quantified in a matrix similar to that of the test
sample and the biological standard contains full length sequence of the agent in question.
With biological standards, different assays yield similar results, but this is not well
defined for synthetic standard materials. The main advantages of synthetic standards
include the possibility to produce large amounts, which should be better characterised
and more homogeneous than a biological standard. Dr. Saldanha stated that there should
be better consistency between replacement lots of a synthetic material which should be
easier to calibrate. Both biological and synthetic standards are stable for long periods.
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Where the 1st WHO International Standard for HCV RNA has been tested by several
laboratories in different collaborative studies, quantification has been inconsistent.
Indeed, regulatory requirements in terms of replacing standards and actual assay
performance call for accurate calibration. Of primary importance is the commutability of
replacement standards to maintain the IU. Dr. Saldanha proposed that in future synthetic
standards could be used as calibrators. Ideally such standards would be expressed in SI
units with traceable reference methods and competent laboratories.
A panel discussion followed this session and topics discussed included the use of
synthetic standards, traceability of reference materials and the use of SI units. Concern
about commutability was expressed by several delegates in that if a synthetic standard
was to be used as a primary standard, then such a preparation would fail to control for the
entire extraction, amplification/detection process. Indeed, given the unique physical
properties of different virus families (e.g. HBV), biological standards would be more
appropriate. If the approach were to be adopted whereby a replacement International
Standard were to be calibrated against a synthetic reference preparation, concern was
expressed that in accordance with ISO 17511, it would require metrological traceability
and at present there are no established reference methods. Dr. Indira Hewlett asked
about the situation in Europe with respect to the use of SI units in the context of the IVD
Directive. Dr. Wielgosz replied that where feasible SI units should be used, however this
was very much dependent upon the availability of appropriate reference methods. An
example was cited for HBsAg where ng values assigned to different standards have
varied. The HBsAg International Standard is now recognized as the higher order
reference material in the IVD context, as no suitable alternative material or reference
method has yet been identified. There was some debate concerning the conversion of IU
to copy number. Dr. Harvey Holmes and Dr. Saldanha reiterated that that such
conversion factors are variable, being assay dependent. Dr. Holmes raised some
additional concerns about synthetic standards. These included the use of sub-genomic
fragments of synthetic material that exclude other regions of a viral genome for which
alternative assays may be available or that may require work up in future.
Standards for NAT (Chaired by Dr. Stephen Inglis)
Dr. Harvey Holmes (NIBSC, UK) reviewed a recent collaborative study to replace the
1st HIV-1 RNA International Standard (IS). One of the candidate standards from the
original collaborative study had been tested in parallel with the 1st IS by eight
laboratories. Statistical analysis showed that the estimated number of RNA copies,
observed in the new study were in line with those seen previously. There was no
evidence of drift or instability suggesting that the candidate standard was a suitable
replacement. Dr Holmes proposed various options to the group regarding assigning a
value to the proposed candidate, these were as follows:
i.
ii.
Use only data from the initial study in 1999.
Use data from the current study, which showed values of 5.44 or 5.46 depending
on whether qualitative data was used.
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iii.
iv.
Pool the data from both studies and take an overall mean, giving values of 5.56 or
5.58 with or without qualitative data respectively.
Produce an overall mean for each assay method.
Dr Holmes informed the group that a report would be submitted to the ECBS this
summer and proposed the replacement IS should be assigned a unitage based on the third
option, using both qualitative and quantitative data. The group was asked for opinions on
this proposal. No comments relating directly to this proposal were raised indicating
overall acceptance from the group. Dr Holmes also asked for feedback regarding the
need for a second HIV Genotype Panel containing more diverse subtypes and groups,
circulating recombinant forms (CRFs) and HIV-2 RNA. Dr. Jean-Pierre Allen
expressed concern that disproportionate attention is given to HIV-1 Group O, which
whilst being a highly diverse group, is in fact rarely encountered. It was agreed that
greater emphasis should be placed on the inclusion of CRFs when designing future
genotype panels. Dr. Hewlett informed the group that the CBER/FDA was evaluating an
HIV-2 NAT panel with some commercial kit manufactures.
Dr. Sally Baylis (NIBSC, UK) talked about the development of a standard for
Plasmodium falciparum the parasite causing for malaria. This particular species is the
most prevalent one infecting humans with the greatest levels of morbidity and mortality.
Although rare, malaria has been transmitted by transfusion with serious consequences in
the recipient. Usually donor selection procedures are sufficient to minimize the risk of
transfusion transmission, however a recent P. falciparum case in the UK, was traced to a
healthy donor previously resident in an endemic area still positive by PCR for parasite
DNA despite not visiting an endemic area for ~9 years. For the standardization studies,
NIBSC has freeze-dried blood from a patient infected with P. falciparum high levels of
parasitaemia. This material has been freeze-dried, showing no loss of titre after freezedrying with good stability. Accelerated degradation studies are in progress. A
collaborative study is underway, looking at the freeze-dried preparation, a high titre
frozen liquid preparation, a low titre liquid preparation and the 3D7 P. falciparum strain
cultured in vitro to a titre comparable to the freeze-dried material. Laboratories involved
in the study include control labs, reference and diagnostic laboratories as well a vaccine
groups. Additional participants were encouraged to take part in the study.
Other NAT issues were discussed including the proposal to replace the first WHO HBV
DNA International Standard (97/746) with one of the materials characterized in the
original collaborative study. The study would be run along the lines for replacement of
the first WHO HCV RNA International Standard mentioned earlier by Dr. John
Saldanha. As the 2nd WHO International Standard for HCV RNA will require
replacement (at current rate of usage this will be exhausted by ~2009) Dr. Sally Baylis
reiterated the new WHO recommendations with like for like replacement of standards
where possible. As the first and second HCV RNA international standards came from a
genotype 1a, anti-HCV positive donation, compliance with the recommendations would
support the use of similar material. However, Dr. Sally Baylis pointed out that a window
period donation may be more appropriate and Dr. Micha Nübling (PEI, Germany) and
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Professor Gerlich agreed with this. Professor Gerlich emphasized the importance of
matching the viral genotype as closely as possible.
Dr. Marie Rios (CBER) described the development of West Nile virus (WNV) standards
for NAT by CBER. Two reagents were produced using the flamingo NY99 strain and a
plasma derived strain FDA-Hu2002. The viruses were cultivated in vitro and examined
by 4 laboratories to determine the RNA copy number. The level of WNV RNA was
found to be 1010 RNA copies/ml by analysis of the titration of intermediate dilutions of
the virus stocks. These preparations contained 107.5 PFU/ml, as determined by two of the
laboratories. Heat inactivation (600C, 2h) was shown to completely remove virus
infectivity, whilst only reducing the NAT reactivity by ~3 log10. Panels prepared from
the heat inactivated stock materials showed a high degree of variability of assay
performance with low copy numbers, with qualitative assays performing better than
quantitative ones. The FDA’s current standard for WNV ID-NAT assays used to screen
blood donations is 100 copies/ml per. Dr. Saldanha commented on the variability of the
calibration of in-house reference reagents and on the value of comparing different
standards with each other.
Dr. Dirk Heckel (Qiagen) described the QIAGEN® BioRobot® MDx diagnostic sample
preparation platform, its application in viral diagnostics and conformity with the
European IVD Directive. The system has undergone extensive performance evaluation
including the platform, operating software, and the reagents and disposable kits. The
validation of downstream applications has been performed specifically looking at the
performance of assays for HIV, HBV and HCV for parameters such as limit of detection,
assay linearity, accuracy, precision etc.
Implementation and quality control I (Chaired by Dr. Nico Lelie)
Dr. Hans-Peter Grunert (Berlin) spoke about the widespread use of external quality
assessment schemes (EQAS) for virus serology and NAT several of which are organized
by the Institute for Standardization and Documentation in Medical Laboratory
(INSTAND, Düsseldorf). Recent studies were described for NAT of HIV-1, HCV and
HBV. The majority of participants perform well in quantitative assays for HIV-1 and
HCV with 93% of results for both viruses within the confidence interval of +/- 0.5 log10
of the median of all quantitative values reported. In the case of HBV however, the
quantitative results are far less reliable, reflected by only 74 to 79% correct results in the
interval of +/- 0.5 log10 around the median. The exact nature of this variability is being
investigated further. EQAS for BSE testing for prion protein in cattle brains has been
performed in Germany since 2002 with a high success rate of correct qualitative results.
Dr. Micha Nübling (PEI, Germany) discussed the most recent study to look at in-house
NAT testing for blood screening in Germany. The study was designed to look at HCV
and HIV-1 NAT with sensitivities of 5,000 and 10,000 IU/ml respectively for individual
donation testing. Laboratories implementing in-house tests must validate and document
assays and are require to seek approval from PEI. In the study, 12 samples were sent to
approximately 20 laboratories. Pool size and extraction volume were tailored to suit each
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participating centre. The samples contained HCV, HIV-1 and mixtures of the two viruses
as well as negative controls. Overall performance was good with higher sensitivity than
the minimal requirements. One laboratory failed to detect a subtype B HIV-1 at
concentration of 10,000 IU/ml (pool size of 20). Two further laboratories failed to detect
the 3160 IU/ml HIV-1 samples (pool size of 96). For HCV, some laboratories missed the
5,000 IU/ml samples and 4 missed 1,500 IU/ml samples. Some false positive results
were reported. On the basis of these results, in-house testing has continued to be found
acceptable. Proficiency testing will continue to be performed. Dr. Lelie, asked about
where the pooling of the samples was performed and Dr. Nübling confirmed that pools
were created prior to dispatch of the panels. Dr. Lelie pointed out that the study didn’t
control for the pooling process itself.
Dr. Mark Manak (BBI, USA) noted that in September 2004 BBI was acquired by
SeraCare Life Sciences, Inc. He went on to describe linearity panels have been produced
for HIV-1, HBV, HCV and CMV. These panels have been designed to mimic clinical
samples as closely as possible using whole virus diluted in defibrinated plasma. Each
panel consists of one negative and nine positive samples. The entire analytical process is
performed by the end-user and the panels are used for quality assurance to evaluate
assays and for training. Professor Gerlich reiterated the importance of the use of IU and
Dr. Saldanha suggested avoiding converting IU to copies for HCV.
The presentation by Dr. Mattias Gessner (Baxter AG, Austria) outlined a study
performed using commercially available preparations (clinical samples, viral
preparations, seroconversion panels) for HCV and HIV-1. These were tested using the
Baxter HI-Q PCR assay, however ~8% of the samples gave unexpectedly negative
results. These samples were sent for independent analysis at the Institute for Virology at
the Medical University of Vienna, using the HIV-1 and HCV Roche COBAS Monitor
assays. These results all gave lower results (many significantly low) for the repeat
samples. These discrepant results will be taken up with the suppliers in the near future.
Dr. Nübling enquired whether the samples were clinical or spiked with virus and how
the materials had been dispatched. Dr. Gessner confirmed that both clinical and spiked
materials had been supplied, dispatched on dry ice, were within the shelf-life and had
been tested immediately upon receipt. Dr. Cuypers wondered whether the panels had
been CE-marked and suggested that sequence analysis may provide some insight into the
discrepancies, although this had not been done at Baxter. Ms. Roberta Madej (Roche
Molecular Systems Inc., USA) commented that Proficiency Testing for Molecular
Methods: Proposed Guideline (MM14-P) would be available from the Clinical
Laboratory Standards Institute (www.clsi.org) covering the principles, manufacturing and
control of such panels.
Implementation and quality control II (Chaired by Dr. Micha Nübling)
Dr. Mike Busch (BSRI, USA) discussed the head to head comparison two NAT testing
platforms available in the USA (COBAS AmpliScreenTM HIV-1 and COBAS
AmpliScreenTM HCV (Roche Molecular Systems) and the Gen-Probe/Chiron ProcleixR
assay). The study was performed on blood donor samples either in a minipool (MP) or
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individual donation (ID) format impacting on the ability to detect RNA early in the HIV
and HCV infection. Differences in detection rates between Roche and Gen-Probe NAT
assays were small and only observed with samples at very low-level viremia. Overall,
the assays give very similar results and reflect the sensitivity levels set by the FDA.
Dr. Thomas Laue (Artus, Germany) described a blood donation, obtained in 2003 in
Germany during the pre-seroconversion diagnostic window period of a hepatitis A virus
(HAV) infection that tested HAV-negative by commercially available HAV reverse
transcription-polymerase chain reaction (RT-PCR) detection assays (Artus and Roche).
The virus was identified using by in-house testing, albeit in a limited number of assays.
Sequence analysis confirmed that the virus strain (HMH) was a genotype IIIA, the first of
this type to be detected in Germany. A new real-time IVD RT-PCR kit was developed at
Artus that allows quantification and detection of all HAV genotypes. Using the new kit,
additional food-borne and travel related cases of genotype 1 HAV cases have been
identified and Dr. Laue stressed that constant monitoring and adaptation of the diagnostic
nucleic acid assays are required to ensure adequate detection.
Dr. Darren Jardine (NRL, Australia) gave an update on quality assurance of NAT in
Australia and the Asian Pacific for blood screening. The function of NRL was outlined
including the evaluation of test kits; on going performance evaluations; quality control
(QC) and external quality assurance schemes (EQAS). Twenty five laboratories take part
in the NAT QC and EQAS programme, using Chiron TMA, Roche AmpliScreen and one
in house HCV RT-PCR assay. QC performance is monitored with the aid of the EDCNet
database permitting the update of proficiency studies, batch analysis and inter-laboratory
comparisons (anonymous). Instruments, operators and reagents are all recorded and
issues rapidly identified impacting on overall blood safety. Dr. Nübling asked if nonregistered users were able to gain access to EDCNet to look at things such as kit
performance and whether lot numbers were recorded for kits. Dr. Jardine replied that
non-registered users can request such information from NRL directly and all lot numbers
were recorded. Dr. Jardine also confirmed that the system is used for viral load
determinations, serological testing or anything that generates a numerical value.
Dr. Phelps enquired if data was entered manually. Dr. Jardine confirmed that this is the
case, however there is a checking procedure for this and NRL are in discussion with
companies to improve data importation. Dr. Roth enquired about the titre of the
controls. Dr. Jardine confirmed that the HCV the controls were 100 IU/ml, were used for
assay validation and in addition to controls supplied in the kits. Dr. Wielgosz commented
that as the QC material is calibrated against the IS, if there are issues when this is
replaced, then a shift may be seen in the values.
Professor Ewa Bröjer (Institute of Hematology and Blood Transfusion, Poland) spoke
about the current status of NAT screening for HCV, HBV and HIV-1 in Poland. HCV
screening has been performed since 2000 using COBAS AmpliScreen (pools of 48) and
Chiron TMA (individual donations). Since 2000, fifty-six HCV “window period”
donations have been identified in Poland, a much higher proportion than in other parts of
Europe and the USA. All seroconverted when followed up and questionnaires introduced
in an attempt to identify the source and date of infection, with look back studies on repeat
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donors. As of 2005, testing has been extended to include HBV and HIV-1 as well as
HCV, utilizing the COBAS AmpliScreen (pools of 24) and Chiron Ultrio (individual
donations, positive results followed up by discriminatory testing). Initial results have
shown that the frequency of occult HBV infection is higher than expected, mostly
detected by single donation testing. Discrepant results have been seen with the Chiron
discriminatory test, probably due to low levels of viraemia. Serological markers are
present in most occult HBV infections that had been followed up. Professor Bröjer
outlined testing algorithm for HBV DNA detection in these HBsAg negative donors
including the options for confirmatory testing. Dr. Lelie stressed the importance of
confirmatory test sensitivity, especially at low levels of viraemia where, due to the
Poisson distribution, positive samples could be missed on re-testing. Dr. Nübling
surmised that anti-HBc testing wasn’t performed in Poland as it was likely that 2-3% of
donors would be positive, hence the use of HBV NAT. He enquired as to why the Ultrio
assay was run on single samples and the AmpliScreen assay was performed using pools
of 24. Professor Bröjer confirmed that the two assays were put into use for contingency
planning and were used as prescribed. Further to the high rate of HCV infection, Dr.
Nübling enquired as to the source of these infections. Professor Bröjer confirmed that
these were mostly due to nosocomial infections in the case of HCV. Replying to
Professor Gerlich, Professor Bröjer stated that the Polish donors were non-remunerated.
Parvovirus B19 (Chaired by Dr. John Saldanha)
Dr. Theo Cuypers (Sanquin, The Netherlands) reviewed the EP monographs for anti-D
immunoglobulin and the limits prescribed for the levels of parvovirus B19 DNA. In the
OMCL guideline for the validation of NAT for parvovirus B19, it was recommended that
assays should also detect recently identified erythrovirus variants, termed genotypes 2
and 3, identified in patients presenting with suspected B19 infection. In a proficiency
study organized by EDQM in April 2004, a genotype 2 erythrovirus was included in the
panel and was not detected by half of the laboratories, who were recommended to review
their assay procedures. Dr. Cuypers reviewed previous studies where the incidence of
genotype 2 and genotype 3 was examined in blood donors and pooled plasma for
fractionation, with no evidence for the presence of these genotypes. In the Sanquin
study, Dr. Cuypers had found no genotype 2 or genotype 3 erythrovirus sequences
present in 321 manufacturing pools tested although a recent study had identified low
levels of genotype 2 in coagulation factor concentrates (~1%). Dr Cuypers concluded
that based on current data, detection of molecular variants (not classified as yet) of
parvovirus B19 was questionable and that additional research on prevalence in plasma
pools and individual donations should be performed. Reference materials would assist
such studies. Dr. Sally Baylis noted that Dr Johannes Blümel from the PEI had
presented data on the chracterisation of a genotype 2 variant received originally from
Immuno in Austria and had presented this work at the previous EPFA meeting in Paris.
Dr. Baylis had also identified a genotype 2 virus in an old batch of cryosupernatant
diluent, not contaminated with B19, that could possibly be used as a reference.
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Dr. Harry Bos (Sanquin, The Netherlands) spoke about the Dutch approach to screening
blood for anti-parvovirus B19 for use in selected at risk patient groups at the request of
the Health Council of the Netherlands. Estimates have suggested that approximately
125,000 such donors are required in the Netherlands providing erythrocyte and
thrombocyte concentrates as well as plasma. Parvovirus B19 IgG antibodies are detected
in donors using the Biotrin ELISA assay looking at donors at six monthly intervals. The
assay is a sandwich ELISA, detecting IgG to the VP2 protein of B19 and has been
automated for large scale screening purposes. The target number of positive donors has
been achieved with an increasing demand from clinician now that such products are
coming on line. The false positive rate will be followed up as more data becomes
available and the implications for the detection of persistent low levels of B19 DNA will
be considered. Professor Allain speculated that the single false positive thus far
identified in the study may have been the result of a waning antibody response. Dr. Roth
commented that there was very little demand for such products in Germany. Dr. Bos
replied that the program for testing was on the advice of the Health Council of the
Netherlands. Dr. Saldanha asked whether the issue of persistence would be determined
by follow up after one year.
Dr Gordon Elliott (Biotrin, Ireland) also spoke about donor screening for parvovirus
B19 antibodies to reduce or eliminate the risk of transmission of the virus. Available
assays include parvovirus B19 IgM and IgG ELISAs which are CE-marked and approved
by the FDA (pregnancy). A new assay is in development for the detection of parvovirus
B19 antigen (assay sensitivity in the order of 5 x 107 IU/ml as determined by qPCR).
Parvovirus B19 antibody testing of all 70 viraemic samples detected at Sanquin over an
18 month period suggested that there were no true IgG positives. The data indicates that
IgG is very rare where viraemia exceeds 106 IU/ml and IgG screening would be expected
to improve identification of parvovirus B19 “safe” donors. In collaboration with
Professor Allain at Cambridge University, sera were examined from UK donors
(genotype 1) and Ghanaian donors (genotype 3) IgG. Expression of the heterologous
VP2 antigens (in the presence or absence of the more variable VP1 antigen) from these
two genotypes and analysis by ELISA demonstrated that there was no difference in the
ability of the current assay to detect IgG derived from individuals infected with different
genotypes. An ELISA based on VP2 capsids or VP1/VP2 co-capsids appear equally
effective in the detection of parvovirus B19 IgG. Dr. Mei-Ying Yu (CBER) commented
on the anti-VP2 neutralisation assay with lack of correlation between EIA titre and
neutralisation titre, Dr. Elliott stated that studies to look at this are in progress.
Dr. Busch (BSRI) asked about genotype cross-neutralisation, and Dr. Elliott stated that
there was cross-protection. Professor Gerlich asked about the expression system, and
Dr. Elliott confirmed that the VP2 protein was produced in insect cells using recombinant
baculoviruses with assembly of VP2 into capsids. Dr. Kevin Brown (NIH) commented
upon the more variable nature of the VP1 protein which contains the majority of
neutralizing epitopes. Detection of anti-VP2 antibodies will miss neutralizing antibodies,
which may have consequences in certain groups of patients such as those with persistent
B19 viraemia. Dr. Brown also commented that ideally, expressed capsids should contain
not only VP2, but also VP1 representing the different genotypes. Professor Allain,
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commented that the VP1 unique region showed cross-reactivity when looking in infected
individuals.
Dr Yoshiaki Okada (National Institute for Infectious Disease, Japan) described studies
to look at inactivation of parvovirus B19 by either dry heat treatment or pasteurization.
Following treatment, infectivity assays were performed on HTB-41 cells (human salivary
gland carcinoma), looking at the levels of spliced mRNA of parvovirus B19. In the
pasteurization study, virus in plasma (10% total volume) was spiked into 5% and 25%
albumin solutions and incubated for 24 hours at 60ºC. In the dry heating experiment,
virus in plasma (10% total volume) was spiked into 5% albumin. Analysis of virus
infectivity following these different treatments showed that B19 was inactivated by the
pasteurization conditions used but was resistant to dry heat treatment. These results were
not specific for the cell line used in the downstream analysis of B19 infectivity.
Dr. Saldanha enquired about the amount of B19 used at the spike in the studies, and Dr.
Okada confirmed that 7 logs of virus had been used. Dr. Saldanha commented that in the
case of certain products contaminated with HAV, drying of products doesn’t result in
complete inactivation, this is more effective in the presence of some moisture.
HBV (Chaired by Dr. Gerold Zerlauth)
Professor Hiroshi Yoshizawa (Hiroshima University, Japan) described the study of
experimental HBV infection using the chimpanzee as an animal model. A viral dose,
equivalent to 10 copies of HBV DNA (genotype A) has been found to be sufficient to
infect a chimpanzee. The doubling time for HBV is approximately 1.6-1.7 days in this
animal model with a HBV genotype A. There was discussion as to how infection varied
between individuals, with things such a virus genotype likely impacting on the doubling
times of the virus in infected individuals. Dr. Busch asked about detection limits and
Professor Yoshizawa confirmed that it wasn’t possible to detect less than 102 copies/ml
and that in the infected chimpanzee, virus was diluted in plasma to give 10 copies of
HBV DNA /ml and 1 ml of inoculum was delivered to the animal. Dr. Busch described
two transfusion transmitted cases of HBV in the USA. In both cases elderly patients
were transfused with HBsAg negative and anti-HBc negative units, only to contract HBV
6 months later. Usually the incubation period is 6-8 weeks, although with more dilute
inocula (typical of transfusion transmitted infections), the incubation period is longer.
Dr. Michael Chudy (PEI, Germany) discussed whether there is any correlation between
HBsAg and the levels of HBV DNA and whether NAT testing could replace assays for
HBsAg. One HBV virion contains approximately 20 ag surface protein, representing all
three forms of HBsAg. During early infection with genotype A HBV, a thousand fold
excess of HBsAg particles was found. Later during infection, the ratio of virion-HBsAg
to total HBsAg is more variable. However in the case of genotype G, there is a
considerable decrease in incomplete forms of the virus, with an early infection ratio of
1:20 of virion-HBsAg to total HBsAg. No correlation was found between HBsAg and
HBV DNA in samples from chronic HBV carriers. One hundred and six samples
positive for HBsAg, anti-HBc and anti-HBe were identified. These were then analysed
SoGAT XVIII, Washington, USA, 24-25 May 2005
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using the Procleix Ultrio assay, however only 89 out of 106 (84%) of these were HBV
DNA positive by NAT. Dr. Chudy concluded by stating that these results support the
continuing use of HBsAg testing as a screening tool. Dr. Busch commented that in the
case of chronic carriers the relationship between HBsAg and DNA levels is very variable;
this relationship is tighter during acute infection.
Dr. Masahiro Satake (JRC, Japan) spoke about look back studies of transfusion related
HBV infection in Japan. Individual samples have been archived for each donor since
1997. Repeat donors, with positive results for pooled NAT for HBV DNA, HBsAg, antiHBc, were tested by individual donation (ID)-NAT. The projected number of screening
negative but HBV ID-NAT positive donations in Japan is estimated to be approximately
40 per year, based upon the minipool-NAT window period (anti-HBc negative) and the
chronic carriers (anti-HBc positive) with low viral loads. Between 11-15 transfusion
transmitted cases of HBV are reported in Japan each year. It was concluded that the
infectivity of a screening negative, but ID-NAT positive component is 13 times higher for
window-period derived ones, than for a chronic-carrier derived ones. Dr. Lelie
suggested that the anti-HBc negatives may not actually be window periods, but may have
been positive previously and just lost anti-HBc.
Professor J.-P. Allain (Cambridge, UK) discussed the characterization of occult HBV
infection in blood donors. Data was presented on HBV genotype E infection in Ghana.
Molecular studies of occult HBV infections are difficult with HBV DNA loads
consistently lower than 500 IU/ml with detection requiring single donor screening, not
pooled. In most cases the only serological marker is anti-HBc.
Tissue and organ screening and emerging issues (Chaired by Dr. Indira Hewlett)
Dr. Dominique Challine (INSERM, France) spoke about the prevention of HBV
infection in recipients of organ, tissue or cell transplants, principally based on screening
for HBV serological markers. More than 11,000 organ, tissue, cell donations donations
were tested over a four year period for HBsAg, anti-HBc, anti-HBs (vaccinated donors
with only the latter marker were excluded) with 520 donors found to be positive.
Samples were tested for HBV DNA using the highly sensitive Roche COBAS TaqMan
assay. HBV DNA was detected in most HBsAg positive donors with the exception of
cornea donors suggesting false positive results for the detection of HBsAg. HBV DNA
was never detected in patients with a recovery profile with the exception of cornea
donors. HBV DNA was detected in a significant number of donors with anti-HBc only.
Dr. Challine concluded by saying that HBV DNA is detected in most HBsAg-positive
organ, tissue and cell donors, but the level of replication is lower than in patients with
chronic hepatitis B. The finding of HBV DNA in donors with anti-HBc alone, supports
testing for the latter marker. Where both anti-HBs and anti-HBc are present, HBV DNA
is not detected (except in corneas). Cornea donors, often sampled after death, may yield
false-positive and false-negative HBsAg results. Testing of organ, tissue and cell donors
for HBV DNA would appear to further improve viral safety in transplantation, however
SoGAT XVIII, Washington, USA, 24-25 May 2005
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technical improvements are necessary. There was general discussion concerning the
quality of samples, particularly in the case of cornea donors, and effects such a
haemolysis of samples collected after death resulting in false positive and false negative
results, particularly affecting serological tests. Professor Gerlich enquired about
screening for anti-HBsAg, Dr. Challine explained that testing for the three serological
markers gave the most information on which to base decisions on whether to proceed
with transplantation e.g. where donors showed vaccination profiles. Dr. Nübling asked
Dr. Challine to comment about HCV RNA positivity in donors and she replied that in the
tissue/organ donors HCV was more prevalent in these groups that are likely to have
higher risk behaviour.
Dr. Theo Cuypers (Sanquin, The Netherlands) spoke about the validation of HCV RNA
detection in small test pools on cadaveric samples. This work is in the wider context of
screening for blood borne infections for the release of a variety of tissues. HCV RNA
testing was performed on plasma samples from bone donors and as a confirmatory test on
anti-HCV positive plasma samples from selected tissue donors (cornea, skin, cardiac).
Plasma was extracted using the NucliSens extractor and HCV RNA tested using the
Amplicor v2.0 assay. Extraction of 1ml of plasma gives a 95% cut-off of 8.7 IU/ml,
however, 15% of results give an invalid result with a negative internal control (IC) signal.
IC signals are improved by using 0.1ml sample plasma diluted with 0.9ml negative
plasma, but sensitivity is lower (87 IU/ml). Further testing of tissue donors in pools of 4,
with 50 μl of plasma per donor gave an acceptable level of invalid results (0.6%), with
95% of the pools giving saturated IC signals and a 95% cut-off of 165 IU/ml. Spiking
experiments confirmed the absence of inhibitors. Dr. Hewlett enquired if there were any
limits of sensitivity for NAT of cadaveric samples in the Netherlands. Dr. Cuypers
replied that testing was voluntary. Sample quality was discussed in relation to
haemolysis, inhibition and sampling times. Professor Gerlich commented that in
Germany, in the case of HCV RNA testing there were many invalid results in the past,
but this has now improved due to the removal of inhibitors in many assays.
Dr. Marta José (Instituto Grifols, Spain) discussed the effect of the storage conditions
on sample stability of RNA/DNA. Following up on previous work examining the
stability of HCV RNA in plasma, Dr. José said that they had found no decrease in the
titre of 100 IU/ml samples HCV RNA after 5 years at -20 ºC. The nucleic acid stability
of other viruses, such as HIV-1 and HBV, was also presented. At -20 ºC, samples
containing HIV-1 were followed up for approximately 3 years with no loss of HIV-1
RNA. Regardless of the HIV-1 RNA concentration, samples stored at 5 ºC maintained
their titre for at least 14 days. At 25 ºC, the HIV-1 RNA half-life was determined at
nearly 7 days. The HBV DNA, at 5 ºC and 25 ºC, was stable for at least 28 days,
regardless of the initial titre. Dr. Lelie confirmed that the test samples were dilutions of
the NIBSC standards.
Dr Saldanha briefly summarised the key points arising from the meeting. In the first
session on standardisation, Dr. Griffiths provided an overview of the guidelines for
WHO standards and Dr Wielgosz highlighted issues related to traceability to higher order
standards in the light of the IVD Directive and certain ISO standards. It was clear from
SoGAT XVIII, Washington, USA, 24-25 May 2005
Page 13 of 14
the subsequent presentations that there was much interest in the development of synthetic
materials as primary standards although issues such as commutability with biological
standards had not been investigated. A study to address this question was proposed.
There was also an update on the replacement HIV-1 and HCV WHO standards from
NIBSC and the development of standard reagents for West Nile Virus NAT by CBER. In
the session on quality control, the problem of quantitation of some commercial panels
was highlighted and the high incidence of HBV, HCV and HIV in Eastern Europe
illustrated through a presentation from Poland by Professor Bröjer. Human parvovirus
B19 is still a major topic especially with the recent descriptions of two new B19
genotypes (genotypes 2 and 3). These genotypes are comparatively rare and preliminary
data from Dr. Cuypers indicated that these genotypes were not detected in the plasma
pools tested although genotype 2 has been reported in commercial available coagulation
factor concentrates. Several problems need to be addressed; the new B19 variants have
not been classified officially, there is a lack of guidance from the control authorities on
the detection of these variants and studies are inhibited by the lack of publicly available
reference and clinical materials. The final presentation in this session was by Dr. Okada
on the use of a B19 infectivity assay using a novel cell line, HTB-41, derived from a
human salivary gland carcinoma, to demonstrate the inactivation of B19 in albumin. A
major topic of discussion, and a challenge to the audience, was the question of NAT
screening for HBV DNA. NAT screening for HBV DNA has not been implemented in
most countries performing NAT testing. An exception is Japan, where, with a high
incidence of HCV, about 11-15 transfusion transmitted cases of HBV are reported each
year. However, samples containing very low titres of virus are able to transmit infection
and the main issue with introducing NAT is the sensitivity of the assay and whether
pooled or individual donor testing should be done. The final session on organ and tissue
screening highlighted the need for validation of assays (both serological and NAT) used
for testing such specimens where the quality of the samples is a major issue.
Dr. Saldanha reported that the SoGAT scientific committee had met during the
workshop and decided that three key areas should be addressed before the next meeting.
These were:
 the feasibility of using synthetic materials as reference standards
 further characterisation of chronic and occult HBV, the challenges facing the
introduction of HBV NAT (sensitivity of assays, pooled or individual testing) and
the identification of well characterised clinical specimens
 the importance of B19 genotypes 2 and 3 in the plasma fractionation setting and
the identification of well characterised samples of these two genotypes.
Three small sub-committees, consisting of members from the workshop, would be set up
to address these issues and report back at the next SoGAT meeting.
Dr Saldanha thanked Dr. Goodman, Dr. Hewlett, and Dr. Yu from CBER, FDA for
hosting the meeting, Dr. Inglis, Mrs. Pamela Lane, Dr. Holmes and Miss Clare Davis
from NIBSC for their role in its organisation and the members of the SoGAT scientific
committee for helping to design such an interesting programme.
SoGAT XVIII, Washington, USA, 24-25 May 2005
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