International Standards and Reference Materials for Quantitative Molecular Infectious Disease Testing

ReviewInternational Standards and Reference Materials forQuantitative Molecular Infectious Disease Testing

Roberta M. Madej,* Jack Davis,†

Marcia J. Holden,‡ Stan Kwang,§

Emmanuel Labourier,¶ and George J. Schneider�

From Roche Molecular Diagnostics,* Pleasanton, California; the

Quality Systems Division,† Bio-Rad Laboratories, Diagnostics

Group, San Ramon, California; the Biochemical Science Division,‡

National Institute of Standards and Technology, Gaithersburg,

Maryland; the Quality Systems Division,§ Bio-Rad Laboratories, Irvine,

California; Development,¶ Asuragen, Inc., Austin Texas; and Research

and Development,� Abbott Molecular Inc., Des Plaines, Illinois

The utility of quantitative molecular diagnostics forpatient management depends on the ability to relatepatient results to prior results or to absolute values inclinical practice guidelines. To do this, those resultsneed to be comparable across time and methods,either by producing the same value across methodsand test versions or by using reliable and stable con-versions. Universally available standards and refer-ence materials specific to quantitative molecular tech-nologies are critical to this process but are few innumber. This review describes recent history in theestablishment of international standards for nucleicacid test development, organizations involved in cur-rent efforts, and future issues and initiatives. (J MolDiagn 2010, 12:133–143; DOI: 10.2353/jmoldx.2010.090067)

In the 1990s, it was demonstrated that HIV viral loadtesting could predict and monitor the course of HIV dis-ease.1,2 Similar studies followed for other viruses, andnow quantitative molecular tests are integral to the prog-nosis and monitoring of several infectious diseases.3–5

Early assays, though, lacked adequate reproducibilityand comparability, as illustrated in proficiency testingreports. This inadequacy was attributed to the abundantuse of laboratory-developed protocols, inconsistent train-ing, limited usage of standardized industry-produced as-says, and the absence of adequate universal standardsfor test development.6–8

To mitigate persisting differences, current clinical prac-tice recommendations have been developed whereby se-rial specimens from patients are optimally tested with the

same assay each time they are submitted.9 However, theneed for all tests to report consistently similar results isstill critical and is driven by the increasing length oftime that patients are monitored, the decreasing abilityof practitioners to demand the use of specific labora-tories and tests, diluted knowledge of these practicestandards among the increasing number of nonspe-cialist treatment providers, and the fact that some med-ical decisions are based on absolute values instead ofrelative changes. The requirement for translatablequantification among different molecular tests relies onuniversally available, robust standard reference mate-rials for test developers, manufacturers, and profi-ciency test providers.

Although molecular diagnostics have contributed topatient management for more than a decade, the estab-lishment of standards and reference materials to supportthe field have lagged. The current World Health Organi-zation International Standards address some of theneeds, but they cover only six viruses and one bacterialtarget (National Institute for Biological Standards andControl, http://www.nibsc.ac.uk, last accessed August20, 2009). These materials and the collective efforts toestablish them have played a critical role, particularly inevaluating the sensitivity of molecular screening assaysand in facilitating the relative standardization of quantita-tive assays. However, because of their original intendeduse as sensitivity standards for plasma product manufac-turers’ qualitative assays, they lack some of the charac-teristics desired for the standardization of quantitativediagnostic assays. Another possible resource, the JointCommittee on Traceability in Laboratory Medicine (JCTLM),which was established to “promote and give guidance oninternationally recognized and accepted equivalence ofmeasurements in laboratory medicine and traceabilityto appropriate measurement standards” maintains a

Accepted for publication September 4, 2009.

Certain commercial equipment and materials are identified to specifythe experimental procedure. This does not imply endorsement by theNational Institute of Standards and Technology nor does it imply that thematerial or equipment is the best available for the purpose.

Address reprint requests to Roberta M. Madej, M.S., M.B.A., TethysBioscience, 5858 Horton St., Suite 550 Emeryville, CA 94608 Email:[email protected].

Journal of Molecular Diagnostics, Vol. 12, No. 2, March 2010

Copyright © American Society for Investigative Pathology

and the Association for Molecular Pathology

DOI: 10.2353/jmoldx.2010.090067

133

database of certified reference materials and refer-ence methods (Joint Committee on Traceability in Lab-oratory Medicine, http://www.bipm.org/en/committees/jc/jctlm, last accessed August 20, 2009). However, thereare currently no standards for infectious disease quanti-tative molecular diagnostics either at the level 1 categoryof high-order reference materials (the level of purified chem-icals) or in their level 2 category, which is designed tocatalog biological products and consensus calibrated ma-terials. Laboratory test developers and in vitro diagnostic(IVD) test manufacturers are left with very few options.

In this review we discuss the establishment of thecurrent World Health Organization International Stan-dards, the requirements needed specifically for quantita-tive molecular diagnostics, and current global efforts toaddress these needs. Through scientific, political, andpragmatic perspective guide opinions regarding thesedevelopments, we attempt to present them factually andto provide the clinical community with a foundation forfuture informed discussions.

Characterization, Establishment, andReplenishment of the World HealthOrganization International Standards

The World Health Organization International Standardsare prepared, characterized, and established accordingto “The World Health Organization Guidelines for thePreparation and Establishment of Reference Standardsfor Biological Substances.”10 The first version of this doc-ument was written in 1978. It was revised in 1986, in1990, and most recently in 2004 in a series of meetingswith representatives from country and regional healthdepartments, vaccine manufacturers, standards organi-zations, and diagnostic test manufacturers. It describesthe general principles for the establishment of all WorldHealth Organization biological reference materials, with abroad scope of intended uses: primarily vaccine prepa-rations and immunological and biological assays. Thequantification of viral targets in molecular testing is rec-ognized in the most recent revision.

The most commonly used global standards for thecalibration and characterization of quantitative molecularviral assays are the World Health Organization Interna-tional Standards for hepatitis C virus (HCV), HIV, andhepatitis B virus. The World Health Organization Interna-tional Standard (IS) for HCV was the first of the series andwas established in 1997. It was a preparation of an HCVgenotype 1a high-titered plasma unit diluted into cryosu-pernatant and lyophilized. The preparation was testedwith two other candidate materials in a global collabora-tive study in 22 laboratories during 1996. The study wasdirected by and the data were analyzed by the WorldHealth Organization collaborating center laboratory: theNational Institute for Biological Standards and Controls(NIBSC) in South Mimms, UK. The methods examinedincluded a variety of commercial and laboratory devel-oped assays using single, nested, or heminested prim-ers. Most of the data were generated by testing endpoint

dilutions of the candidate standards using qualitativetraditional (non-real-time) amplification assays, althoughquantitative data submitted by participants were in-cluded in the analysis. The first World Health Organiza-tion HCV IS (96/790) was assigned the value of 105 IU/ml(5 log10 IU/ml) lyophilized in 0.5-ml ampoules.11 The in-ternational unit is considered absolute, and, consistentwith World Health Organization policies, there are nounits of uncertainty associated with this or subsequentreplacements. The IU for each biological preparation“has no existence other than in relation to the preparationthat defines it.”10

Since 1997, the first HCV IS has been replaced by twosubsequent standards: the second World Health Organi-zation HCV IS (96/798) in 2003, which was a secondlyophilization preparation of the original material12 andmost recently a new material prepared and tested in2007.13 The second HCV IS (96/798) was originallytested in the global collaborative study of 1996. Becauseit was the same material as the first IS and was deter-mined in 1996 not to be statistically different from the first,there was no global collaborative study when it assumedits place as a the second IS 6 years later. Stability andcomparative studies were performed in three selectedlaboratories before its use. The second World HealthOrganization HCV IS (96/798) was established with thesame value as the first (96/790): 105 IU/ml (5 log10 IU/ml).

For the recent replacement of the second by the thirdHCV IS, new materials were commissioned, and a fullglobal replacement study directed by NIBSC was per-formed. The candidates were two lyophilized and onefrozen preparation of a genotype 1a, anti-HCV-nega-tive, HCV RNA-positive plasma unit diluted into humanplasma. The candidate materials were tested with thesecond HCV IS (96/798) by 33 participating laboratoriesin 14 countries. This time most assays were quantitative,although data testing the limiting dilutions of the materialswith qualitative molecular assays were also included. Inaddition, real-time amplification assays not present in thefirst study more than 10 years earlier had become com-mon and were reflected in the submission of data. Themean quantification of the second HCV IS (96/798) was5.10 log10 IU/ml with a 95% confidence interval of 5.02 to5.17 log10 IU/ml, which is statistically different from thepreviously assigned 5.0 log10 IU/ml. The mean values ofthe two lyophilized candidates were 5.19 log10 IU/ml(sample 2) and 5.47 log10 IU/ml (sample 3). Sample 2(NIBSC code 06/100) was recommended as the thirdHCV IS to the Expert Committee on Biological Standard-ization (the World Health Organization committee com-missioned “to establish detailed recommendations andguidelines for the manufacturing, licensing, and control ofblood products, cell regulators, vaccines and related invitro diagnostic tests”).13

The World Health Organization HCV IS has been usedas the reference to compare the quantification of HCVgenotype panels provided by the NIBSC14 and regionalreference preparations throughout the world.15 Althoughthe original intended purpose for this IS was for theestablishment of sensitivity criteria for molecular assaysused to screen blood products in the plasma manufac-

134 Madej et alJMD March 2010, Vol. 12, No. 2

turing industry, it has contributed to the standardization ofblood donation screening assays as well as moleculardiagnostic and monitoring assays.16,17

The same process has been applied to other WorldHealth Organization IS preparations for nucleic acid test-ing, and since 1996 standards for HIV,18 hepatitis Bvirus,19 hepatitis A virus,20 parvovirus B19,21 human pap-illomavirus,22 and Plasmodium falciparum23 have beenestablished. The primary targets have been those mostneeded to ensure safety of transfused blood and humanblood-derived biological products (Table 1).

Efforts to Increase the Breadth and Depth ofStandards for Quantitative MolecularInfectious Disease Testing

The traditional intended purpose for international biolog-ical standards was to characterize vaccine preparationsand establish the analytic sensitivity of assays used toscreen blood product materials. This provides insight intothe reasons that these materials today are single-pointreferences with no specification for recoverability when

Table 1. World Health Organization International Standards for Molecular Infectious Disease Testing

TargetYear

established Assigned value* Mean assayed value† Characteristics‡

HCV RNA First InternationalStandard (96/790)

1997 4.70 log10 IU/vial(reconstituted to 0.5 ml)5.00 log10 IU/ml

5.0 log10 NAT detectableunits/ml

HCV-positive donation; genotype1; diluted in cryosupernatant;freeze-dried10

HBV DNA First InternationalStandard (97/746)


6.42 log10 equivalents/ml Single donor material, EurohepR1; genotype A, HBsAgsubtype adw2; diluted inpooled plasma; freeze-dried19

HIV-1 RNA First InternationalStandard (97/656)


4.75 log10 copies/ml inquantitative assays

HIV PCR-positive/antibody-negative plasmapheresisdonation; genotype B; dilutedin defibrinated plasma;freeze-dried18

HCV RNA Second InternationalStandard (96/798)


5.0 log10 NAT detectableunits/ml

HCV positive donation; genotype1; diluted in cryosupernatant;freeze dried; separatelyophilization of same materialas the first HCV IS12

HAV RNA First InternationalStandard (00/560)


5.29 log10 equivalents/ml HAV-positive donation diluted inHCV-, HAV-, HIV-, HBV andHBsAG-, anti-HCV-, anti-HIV-negative human plasma20

Parvovirus B19 DNA FirstInternational Standard (99/800)


5.89 log10 copies/ml Parvovirus B19-positive donation;diluted in pooled plasma;freeze-dried21

HBV DNA Second InternationalStandard (97/750)


6.30 log10 equivalents/ml Single donor material, EurohepR1; genotype A, HBsAgsubtype adw2; diluted inpooled plasma; freeze-dried;second aliquot of originalpreparation19

HIV-1 RNA Second InternationalStandard (97/650)


5.35 log10 copies/ml inquantitative assays; originaltesting

Field isolate: HIV-1, genotype Benv V3, gag; propagated onPBMCs, diluted in pooledhuman cryosupernatant18

Plasmodium falciparum DNA FirstInternational Standard (04/176)


8.51 log10 equivalents/ml Freeze-dried whole blood patientblood collected by exchangetransfusion23

HCV RNA Third InternationalStandard(06/100)


Not applicable Window period anti-HCV-negativeplasma, genotype 1a HCV13

HPV type 16 DNA FirstInternational Standard (06/202)


1 IU/ml is traceable throughcalculations to 0.947 GEq/ml, determined byindependent methods

Freeze-dried recombinantplasmid containing full-lengthHPV-16 DNA in a backgroundof purified human genomicDNA22

HPV type 18 DNA FirstInternational Standard (06/208)


1 IU/ml is traceable throughcalculations to 1.031 GEq/ml, determined byindependent methods

Freeze-dried recombinantplasmid containing full-lengthHPV-18 DNA in a backgroundof purified human genomicDNA22

HAV, hepatitis A virus; HBV, hepatitis B virus; HBsAg, hepatitis B surface antigen; PMBC, peripheral blood mononuclear cell; HPV, humanpapilloma virus; GEq, genome equivalents.

*Concentration is in IU/vial. Some vials are reconstituted only to 0.5 ml.†Assayed value given when determined through global collaborative studies or when assayed by independent methods before assignment of IU.‡Characteristics and instructions for use are at http://www.NIBSC.ac.uk, last accessed August 20, 2009.

Molecular Virology Test Standards 135JMD March 2010, Vol. 12, No. 2

diluted to prepare quantitative standardization curves.Although the establishment of the first World Health Or-ganization HCV IS was consistent with those intendeduses,16,17 the World Health Organization and NIBSChave recognized the role it and subsequent viral IS ma-terials have had in quantitative diagnostics. A 2007 reportof World Health Organization collaborating centers forbiological standards and standardization describes a5-year strategic plan “to prioritize the development ofWorld Health Organization biological reference productsfor the control of blood safety-related IVD [in vitro diag-nostic] tests,” reiterating the safety of the blood supply asthe primary goal.24 Leaders at NIBSC acknowledge thatinternational standards are needed to standardize quan-titative and qualitative molecular infectious disease as-says for patient testing and are proceeding to addressthis area.

Recommendations for the desired specifications ofstandards for quantitative molecular diagnostics havebeen communicated to World Health Organization throughconsultation meetings with the organization and throughworking groups such as Standardization of Genome Ampli-fication Techniques (SoGAT). The group most persistentin continued discussion of performance specifications forquantitative molecular diagnostic international standardshas been the Industrial Liaison Committee (ILC), an or-ganization of molecular assay manufacturers working to-ward the availability of universally accepted referencestandards.

The specific interest of this group has been standardsto serve the development of quantitative molecular diag-nostic assays used in patient testing. ILC and WorldHealth Organization-sponsored meetings with the WorldHealth Organization, US Food and Drug Administration,and other standards organizations were convened in1998, 2000, and 2002 to discuss this topic. In addition,ILC has presented regularly at SoGAT meetings. Speci-fications discussed in these meetings included the es-tablishment of reference materials similar to clinical sam-ples, quantification by methods independent of the currentdiagnostic testing methods, preparations with concentra-tions adequate to evaluate the entire expected clinicalrange, dilution protocols and published dilution recoveryexpectations, and stability testing (2006).25

Studies have shown that for commercial molecularquantitative assays for which international standardshave been available (HCV and HIV), variability has de-creased significantly since the first generation.26,27 How-ever, a recent survey of 28 laboratories examining thevariability of Epstein-Barr virus (EBV) assays across var-ious viral load concentrations, sample types, and assayplatforms demonstrated that significant variation still ex-ists.28 Results of the survey showed that roughly 50% ofall results fell within the “acceptable” parameters of �0.5log10 for quantitative nucleic acid test (NAT) assays. Sim-ilar results from multicenter comparisons have also beenobserved.29 Furthermore, greater variation in results wasobserved among samples containing virally infectedcellular material, suggesting that sample preparationmethodology, such as DNA extraction, needs furtherimprovements. Last, variability was significantly higher in

interlaboratory comparisons versus intralaboratory com-parisons, strongly suggesting a need for assay calibra-tion to a universally accepted reference standard. Thisamount of variability poses significant challenges in clin-ical diagnosis as seen in EBV viral load testing in EBV-related tumors.29

A similar study for human cytomegalovirus (HCMV)assays in the United States, Europe, and Canada dem-onstrated comparable problems. Among 33 laboratoriestesting a panel of seven constructed and three patient-derived samples, 57.6% of quantitative results werewithin the range of �0.5 log10 of expected results. Al-though the authors attributed the variation to several fac-tors, they concluded that a primary measure toward res-olution would be the establishment of an internationalreference standard.30

These observations underscore the need for continuedwork in providing a more diverse toolbox of internationalstandards and the importance these materials have inpatient care. Clinical molecular laboratory directors arenow engaged in the discussion of needed diagnosticstandards for quantitative molecular assays for infectiousdiseases. One challenge for IVD manufacturers will be tomake available affordable reference materials so thatfrequent quality control protocols can be implemented inlaboratories. The following presents singular and com-bined efforts by several groups to fill these gaps, includ-ing work on synthetic materials by the ILC and by theNational Institute of Standards and Technology (NIST)(Gaithersburg, MD) and plans for a broader menu ofWorld Health Organization international standards bySoGAT.

Evaluating the Role of Synthetic Materials

In April 2002, a World Health Organization consultationgroup was convened to consider the role of internationalreference materials in viral load monitoring by NAT and todefine the characteristics of reference materials appropriatefor that purpose. The specifications for an ideal internationalstandard and what might be acceptable in light of thediverse challenges of producing and sustaining them formolecular infectious disease testing were discussed. In thismeeting, HCV and the viral assays were the predominantfocus as the World Health Organization HCV IS had recentlybeen established, although many of the issues were trans-ferrable to other infectious disease targets. Table 2, outlin-ing desired characteristics of molecular standards and thetechnical and strategic challenges in producing them, isadapted from presentations and deliberations at the meet-ing and continued discussions into the present (Report:World Health Organization Consultation on InternationalStandards for in Vitro Clinical Diagnostic Procedures basedon Nucleic Acid Amplification, http://www.who.int/bloodproducts/publications/en/BIVD02apr22.pdf, last accessedAugust 25, 2009). These issues continue to confront orga-nizations attempting to establish globally recognized stan-dard materials.

Because HCV cannot be propagated and measuredby independent methods such as limiting dilution viral


Table 2. Optimal Specifications and Challenges: Molecular Standards

Characteristic Optimal specification Acceptable specification Challenges

Target nucleic acid Whole genome as found invirus; intact

Whole genome as found invirus; intact

Is there a representativeconsensus genome?

Source of target nucleicacid

Virus Recombinant DNA (DNAviruses) or RNAsynthesized in vitro (RNAviruses)

Can the virus be propagated?Is the cultured strainrepresentative?

Length of target nucleicacid

Entire genome All of the sequencesrecognized by current andprojected future assays

Current assays may not berepresentative of futuretechnologies

Cultured organisms, purifiedNA or synthetic NA may nothave important secondarystructure

One or several genotypes/subtypes

All relevant genotypesavailable as standards

One genotype for a“foundation” standard.Genotypes and subtypescalibrated from thefoundation standard

One representative organismdoes not necessarily addresstechnicalities of all variants

Resources to produce andcharacterize all variants andperform the global studies tovalue-assign them woulddetract from producingstandards for moreorganisms

Which genotype/subtype? The most globally prevalent The most globally prevalent The most globally prevalent orthe target of most presentand near future testing?

Sequence of targetnucleic acid

Known Known The proposed material shouldbe sequenced

Sequence of cDNA clones(from RNA viruses) orDNA clones (from DNAviruses)

Sequence of cDNA clones(from RNA viruses) orDNA clones (from DNAviruses)

Concentration of targetnucleic acid

High enough to evaluatecalibrators that coverlevels of targets found inpatients

High enough to evaluatecalibrators that coverlevels of targets found inpatients

Some source material (patientplasma) may not have highenough titers to produceenough standard material.

Matrix Natural material from whichthe target is predominantlyassayed. (ie, blood-borneviruses: plasma)

Material that will ensurestability and intactness oftarget nucleic acid andthat will not affect assay

Is pooled material appropriate?Can the bodily fluid from one

individual be representative?Are decalcified, defibrinated

materials commutable?If the infectious target is tested

from cells, should cellularstandards be developed?

Temperature of storage Ambient or refrigerated Ambient or refrigerated Can impact stabilityManufacturing process Validated, reproducible

methods within a validatedquality system

Validated methods within avalidated quality system

Could limit the number andtype of organizations able toproduce or contributematerials

Vial-to-vial consistency SD � 0.15 log10 copies/ml(coefficient of variation �35%)

SD in target concentrationknown and published

Could limit the number andtype of organizations able toproduce or contributematerials

Initial quantification ofnucleic acid targets

Independent analyticalmethod(s)

Quantitative NAT usingstatistically designedprotocol and equalweighting of results fromassays used

Absence of reference methodsrequires testing with relevantavailable methods

Are resources available toadequately test materialswith an even representationof the available testmethods?


culture, participants at the 2002 meeting designated it asa model for work to explore alternative strategies. Further,considering the desired characteristics discussed, thegroup concluded that synthetic (in vitro synthesized) nu-cleic acids could be considered. The methods for pro-ducing these synthetic materials were not specified at thetime; however, a well-characterized, in vitro-generatedHCV RNA transcript, quantified by physicochemical assaysto a primary phosphate standard was readily available forevaluation through a donation from Bayer Diagnostics (nowSiemens Healthcare Diagnostics, Inc., (Deerfield, IL).31 Thetranscript material included target sequences for all of thetechnologies available.

At the meeting, the ILC was challenged with planningand performing a feasibility study using a synthetic RNAcontaining HCV sequences, and a series of studiesamong manufacturers were initiated. The first feasibilitystudy was performed in 2002. Four NAT methods wereincluded in the study: VERSANT HCV RNA 3.0 Assay(bDNA) (Siemens Healthcare Diagnostics, Inc., Deer-field, IL); COBAS AMPLICOR HCV MONITOR, version 2.0(Roche Molecular Diagnostics, Pleasanton, CA); an inter-nal-use quantitative HCV TMA assay (Gen-Probe, Inc.,San Diego, CA); and LCx HCV RNA Quantitative Assay(Abbott Laboratories, Abbott Park, IL). The original con-centration of the synthetic HCV RNA was determined tobe traceable to a chemical standard (potassium phos-phate) by non-NAT methods.31 Scientists at each siteused the synthetic RNA transcript to quantify HCV RNAconcentrations in members of a patient-derived panel.Feasibility was defined as a demonstration of panel quan-tification value agreement within 1.0 log10 regardless of theNAT method. Values reported by each site as syntheticRNA transcript copies per ml did not differ by more than1.0 log10 regardless of the technology used for analysisand demonstrated the feasibility of a synthetic HCV RNAstandard. Further studies in 2005 and 2006 by this groupevaluated the synthetic material using automated samplepreparation instruments to determine that the materialcould be recovered in full extraction processes. Accept-able recovery was demonstrated; however, handling theRNA transcripts to maintain their integrity required exper-tise that is not universally available. The results of thesestudies were presented at the SoGAT meeting in 2004.

The ILC continued this work in 2007 with two syntheticand two biological materials. Scientists from Roche Mo-lecular Diagnostics, Siemens Healthcare Diagnostics,Inc. Berkeley, CA and Abbott Molecular Inc. (Des Plaines,IL) quantitatively tested dilution panels of these materials intheir respective HCV assays: Roche COBAS AmpliPrep/COBAS TaqMan HCV Assay, Siemens VERSANT HCVRNA 3.0 Assay (bDNA), and Abbott RealTime HCV as-say. The synthetic materials were the HCV RNA transcriptsupplied by Siemens Healthcare Diagnostics, Inc. (de-scribed previously) and an HCV Armored RNA Quantsupplied by Asuragen, Inc. (Austin, TX). Both materialshad been quantified relative to a phosphate standard byindependent, non-NAT methods. The Armored RNA, anRNA transcript synthesized by in vitro transcription, isencapsulated in a bacteriophage protein coat and isstable to nucleases present in normal sample matrices. It

provided a material that could be subjected to the entireassay procedure without special handling. The biologicalmaterials used were the Paul Erlich Institute regional HCVstandard at 80,000 IU/ml, contributed by M. Nubling atthe Paul Erlich Institute (Langen, Germany) and an un-quantified high titer plasma material prediluted into neg-ative plasma prepared by scientists at Abbott Molecular,Inc. Six to eight replicates of four to six linear points ofeach material were tested in each assay in the manufac-turers’ laboratories. The synthetic materials demon-strated full utility based on recovery, range of linearity,and variation. In all three assays, the synthetic targetscould be recovered and quantified with equal or lessvariability than the biological materials (Figure 1). Thestudy, findings, and suggestions of possible roles of bothsynthetic and biological standards were presented at theSoGAT meeting in June 2007.

Because the purpose of international standards is toprovide the initial calibration point for developing assaysand subsequent versions of those assays, it is critical thatthe standard remain reliable and unchanged. This is mostchallenging for biological standards in a field of evolvingtechnologies. Each time a replacement World Health Or-ganization International Standard is assigned a value,several years have passed, and the technologies thatwere used to evaluate the previous standard may notexist. Even when the former and latter are tested in uni-son, there may be small shifts in the valuation of theestablished standard, as described earlier in the WorldHealth Organization HCV IS replacement study and doc-umented in the HIV IS replacement studies. The impactsof these shifts are challenging to project and impossibleto measure over time because of the limited amount ofWorld Health Organization material available. For theWorld Health Organization global collaborative studiesdescribed in this review, four vials of candidates permethod tested are available to the participants, and whenthe standard is established, all those requesting the ma-terial for development purposes are limited to a specificnumber of vials per year. This limitation is necessary sothat the maximum distribution can be provided but hasthe unintended consequence of prohibiting statisticallyrelevant longitudinal studies of the impact of smallchanges in the standards. To mitigate the resource limi-tation, there are several national and regional standardsand secondary reference materials calibrated from theWorld Health Organization International Standards. In ad-dition, diagnostic test manufacturers have procedures fordeveloping secondary, internal use references calibratedagainst and traceable to the World Health OrganizationInternational Standards. Clinical scientists constructinglaboratory developed tests are expected to act in a sim-ilar manner; either by generating or purchasing second-ary reference materials for their internal development andcalibration activities. However, with each dilution of the ISand calibration of secondary materials, there is inherenterror as demonstrated in ISO 17511:2003. Because themolecular community may not be able to adequatelyproject or quantify this impact, it is even more critical thatwe contribute to support, critique, and maintain the integ-rity of continued replacement of biological standards.


To contend with these issues and the limitations of thematerials, a scenario was proposed whereby well char-acterized and independently tested synthetic nucleicacid material could serve as the constant in an environ-ment in which reference methods are not available. Ifstable synthetic materials could be established alongwith the international biological standard and the relation-ship between the two could be quantified for relevanttechnologies, the synthetic material could be tested with

each replacement lot of the biological standard. Testdevelopers would continue to use the biological stan-dards, but each replacement lot of those materials wouldbe tested against the “unchanging” synthetic material.Because of the potential greater supply of synthetic ma-terials, they could also be used in the characterization ofsecondary reference materials. This concept has notbeen tested for molecular diagnostics; however, currentdevelopment of an HCMV nucleic acid material at theNIST and a biological standard for NIBSC provides atimely opportunity.

Development of a HCMV Standard ReferenceMaterial at NIST

Because HCMV resides intracellularly and is also presentin plasma, this virus and those like it present an addedlevel of complexity for producing standards: determiningthe biological matrix that most universally representswhat is tested in clinical practice. Considering this, astandard that is free of biological material may be anappropriate reference for the development of severaltypes of biological standards. The NIST has undertaken aproject to develop a standard reference material for hu-man cytomegalovirus. The material will consist of pureHCMV DNA. The goal is to develop a reference thatwould be traceable to the SI (International System ofUnits, the metric system) and suitable as a calibrant forquantitative PCR assays of HCMV viral load but perhapsmore importantly as a tool to standardize various NATassay reagents and calibrants produced by manufactur-ers of diagnostics or laboratory developed protocols.

Quantitative real-time PCR is rapidly becoming themethod of choice for HCMV quantification over previouslydeveloped assays such as antigenemia because of itssensitivity and speed32 and the possibility of highthroughput implementation. But there are issues such asDNA sequence diversity among targets used for quanti-fication that can result in false-negative results.33,34 Thereis a proliferation of assays with different target sequencesin the HCMV genome. In a recent survey, 10 differentopen reading frame targets were used, glycoprotein Bbeing the most frequent.35

The HCMV standard reference material will consist ofpure DNA from a Towne strain HCMV bacterial artificialchromosome (BAC).36 The BAC DNA insert is 10 kbp andreplaces the US1 to US12 open reading frames in theTowne cytomegalovirus DNA by homologous recombina-tion. The final construct, �UL147 Towne BAC, will be usedto produce viral DNA.37 The Towne BAC is stable andcan provide large quantities of consistent viral DNA,whereas cultured virus, especially from laboratory strainsof HCMV, can produce truncated genomes.38 Genomesize consistency is important for calculation of genomecopy number.

The HCMV Towne BAC DNA reference material will becertified for sequence. The intent is to sequence all of therelevant regions of the genome that are used as targetsfor PCR assays. Two approaches will be used for quan-tification of the BAC DNA. Digital PCR is a primary

RealTime HCV

15

20

25

30

35

40

2 3 4 5 6 7

Expected Log(IU/mL)

Ct

PEI

Plasma

Armored RNA

RNA transcript

COBAS TaqMan HCV

15

20

25

30

35

40

2 3 4 5 6 7

Expected Log(IU/mL)

Ct

PEI

Plasma

Armored RNA

RNA transcript

VERSANT HCV RNA 3.0

0.0

1.0

2.0

3.0

4.0

5.0

2 3 4 5 6 7

Expected Log(IU/mL)

Lo

g(R

LU

) PEI

Plasma

Armored RNA

RNA transcript

A

B

C

Figure 1. Evaluation of synthetic and biological HCV reference materialswith three commercial HCV quantitative assays. Serial dilutions of the PaulErlich Institute (PEI) regional HCV standard (80,000 IU/ml), high titer plasma,Armored RNA Quant HCV and synthetic HCV RNA transcript were testedusing the RealTime HCV (A), COBAS AmpliPrep/COBAS TaqMan HCV (B),or VERSANT HCV RNA 3.0 assays (C). All test specimens were shipped andstored below �70°C. The HCV Armored RNA was prediluted, shipped, andstored in base human serum. Other specimens were formulated by eachlaboratory using the same base matrix immediately after thawing of the stockmaterials. The synthetic HCV RNA transcript was diluted in the presence oflysis reagent to inhibit potential degradation of the RNA by serum nucleases.At least six replicates of each dilution were tested in three independent runswith each assay. The synthetic materials were formulated in copies/ml ofbase matrix (2.5 � 103, 5 � 103, 1 � 104, 1 � 105, 1 � 106, and 1 � 107

copies/ml) and converted to IU/ml (IU/ml � copies/ml � 5). Ct, thresholdcycle; RLU, relative light units.


method for counting copies of genomes or plasmids. Thisapproach could provide traceability to the mole. PureDNA can be accurately and precisely quantified basedon phosphorus measurements using high-performanceinductively coupled plasma-optical emission spectros-copy.39 Once the HCMV standard reference material hasbeen certified, interlaboratory studies will be conducted.

SoGAT Committee for Viral Diagnostics

Originally sponsored by the World Health Organization toaddress the safety of transfused blood, tissue, and or-gans, with emphasis on viral, bacterial, and parasiticcontamination, SoGAT has convened at least annually formore than 12 years. NIBSC has maintained leadership ofthis group with representatives from public health andother government agencies, IVD and independent con-trol manufacturers, and diagnostic and university labora-tories. The meeting provides a forum for discussion onassays, reference materials, and regulatory issues aswell as for organizing collaborative studies. At theSoGAT meeting in June 2007, it was announced that asecond series of meetings would be started with thefocus on molecular diagnostics for clinical virology.The goals for the new group will be patterned on thosefor the original group, and it is expected there will becrossover interests between the two groups.SoGAT-CD (SoGAT Clinical Diagnostics) convened forthe first time in June 2008.

At the first meeting of SoGAT-CD, issues specific tomolecular testing for infectious diseases were discussed.The lack of quantitative reference materials for the grow-ing number of viral molecular tests was a persistent con-cern. Within months of the meeting, the NIBSC issueddraft protocols for comment regarding the establishmentof two reference materials: one for HCMV and one forEBV quantitative assays. At the next meeting, in Septem-ber 2009, participants discussed the progress and pathforward of the CMV and EBV standards.

The Joint Committee for Traceability inLaboratory Medicine

The JCTLM was formed in 2002 with a Declaration ofCooperation between the International Committee ofWeights and Measures, the International Federationof Clinical Chemistry and Laboratory Medicine, and theInternational Laboratory Accreditation Cooperation and issponsored by the International Bureau of Weighs andMeasures. The stimulus for this committee came from theIVDD, Directive 98/79/EC of the European Parliament andof the Council of October 27, 1998, on in vitro diagnosticmedical devices. The work of the committee is to facilitatethe implementation of traceability to higher order refer-ence materials and measurements that are required bythe IVDD. Committee participants include nationalmeasurement institutes of several countries, other gov-ernment agencies (such as US Centers for DiseaseControl and Prevention), and industry and laboratoryassociations.

Global standardization can lead to the ultimate goal ofachieving correct and equivalent measurements on pa-tients’ samples anywhere, at any time, and independentof specific assay. Different assays should be able toproduce comparable results with consistent uncertaintiesand intervals. As an important step in that direction, thereneeds to be higher-order reference materials and refer-ence measurements. The term “higher-order” was notdefined in the IVDD; but the requirements are describedin ISO 15193 and ISO 15194.40,41 The JCTLM WorkingGroup 1 evaluates submitted nominations for referencematerials and methods by determining compliance withthese ISO standards. Based on this, two lists are pub-lished in the JCTLM database. List 1 describes certifiedreference materials and measurement methods that aretraceable to the SI. The kinds of materials include smallmolecules, such as electrolytes, metabolites, and someproteins. List 2 includes reference materials, not trace-able to the SI, but traceable to an internationally agreedon protocol. Examples here are coagulation factors orblood typing. Working Group 1 is divided into reviewteams for nine categories of analytes, including nucleicacids. Announcements for new nominations to be consid-ered for inclusion on the lists are made periodically, andlisted materials are removed when they are no longer avail-able. The JCTLM database is hosted on the InternationalBureau of Weighs and Measures website, where documen-tation to provide transparency to the decision-making pro-cess is available (Joint Committee for Traceability inLaboratory Medicine, http://www.bipm.org/en/committees/jc/jctlm, last accessed August 26, 2009). Armbruster andMiller42 have written a comprehensive review article onJCTLM and its role in global standardization.

The JCTLM Working Group 1 Nucleic Acid ReviewTeam has experts from Japan, Europe, and the UnitedStates representing bioindustry, molecular diagnosticsmanufacturers, reference material producers, and publichealth organizations. The group is preparing the crite-ria that will be applied to assess the “quality” and“higher-order traceability” of JCTLM database-nomi-nated nucleic acids reference materials, which onlyhave stated “nominal qualities,” most usually se-quence. There is limited ISO standard guidance on thecriteria that need to be fulfilled to consider a referencematerial’s nominal properties as those of higher-orderor high-quality (although issues of identity, stability, ho-mogeneity, and commutability need to be considered). De-veloping clear guidance on internationally accepted criteriaagainst which the traceability of nucleic acid reference ma-terials can be reviewed will hopefully encourage the nomi-nation of more nucleic acids reference materials to theJCTLM database.

Association for Molecular Pathology

The Association for Molecular Pathology is a not-for-profitscientific society dedicated to the advancement, prac-tice, and science of clinical molecular laboratory medi-cine and translational research based on the applicationsof genomics and proteomics (Association for Molecular


Pathology, http://www.amp.org, last accessed August27, 2009). The Association for Molecular Pathology Clin-ical Practice Committee Working Group on InfectiousDisease has focused on the need for standardization ofquantitative molecular viral load testing. Quantitative PCRprocedures, in particular real-time PCR, are popular waysto monitor viral load because of the reliability and broadlinear range of quantification. Many different assays havebeen developed, often in individual laboratories. Theneed for reference standards for normalization of labora-tory measurements is continually recognized and empha-sized by the Association for Molecular Pathology in meet-ings, workshops, discussion threads, and collaborativestudies.28,29,32 The organization is working with the NISTto produce an HCMV Standard Reference Material. Fu-ture projects include assessing the need for referencestandards for quantitative BK virus and EBV viral loadtesting.

Perspective and Future Issues

The benefit of having internationally recognized refer-ence materials for quantitative molecular tests is nowwell recognized by all of the constituencies involved indevelopment and testing: clinical laboratories, inde-pendent control and reference material manufacturers,IVD manufacturers, and regulatory and policy makers.Challenges now involve communication of the complexissues across these diverse stakeholders and explor-ing different approaches to strategic, technical, andapplications issues. Strategic issues include agree-ment on target priorities and design and managementof studies. For technical issues, linearity of the materi-als, the relationship of synthetics to biological prod-ucts, biological matrices, possible standard referencemethods, and commutability are topics that aresurfacing.

Until recently, the impact of commutability on stan-dardization of laboratory results has not been adequatelystressed. A review in 2006 of the Joint Committee forTraceability in Laboratory Medicine’s list of approvedreference materials revealed very few that were validatedfor commutability against native clinical samples.43 Oneof the most important considerations in the develop-ment of effective standards and reference materials isthat the material be commutable, ie, behave as closelyas possible to the test samples in the full range ofmeasurement procedures expected to make use of thematerial. Commutability is described as equivalence ofthe mathematical relationships, or ratios, between theresults of different measurement procedures for a ref-erence material and for test samples of normal andpathological origin.44

Commutability does not apply solely to synthetic refer-ence materials. Biological standards are often assumedto be commutable because of their native origin but maynot have been specifically validated as such. In 2005 acommittee was formed by the Clinical and LaboratoryStandards Institute for the development of guidelines toestablish consistent assessment of commutability of refer-

ence materials.45 Application of commutability to standardsand secondary reference materials is expected to be aseminal topic for diagnostics in the future and moleculardiagnostics will necessarily be part of that discussion.

Other important technical issues include understand-ing the relationship between the different approaches tostandards that technological advancements may providein the near future. Plans for the establishment of bothsynthetic and biological standard materials for HCMVhave already been discussed in this review. Experimentsto understand the relationship of each of these materialsto each other and the role each will play in standardiza-tion of HCMV assays will include discussions of traceabil-ity, commutability, and strategy.

The recent interest from all levels of stakeholders in theeffort to establish international standards for quantitativemolecular diagnostics will lead to further inclusivity, trans-parency, and critical evaluation. It will be important tomaintain avenues of communication that have opened upthrough professional organizations such as Associationfor Molecular Pathology, European Society for ClinicalVirology, and Pan American Society for Clinical Virology.The ILC has reaffirmed its primary objective to work in thearea of these technical and strategic issues regarding theestablishment and replenishment of international stan-dards and is encouraged by the significant attentionbeing given to these critical issues by others. Discussion,critique, and constructive efforts specific to the issues inquantitative molecular diagnostic standards are reallyjust beginning. Continued and active involvement of allstakeholders will be necessary to achieve the commongoal of reliable and commutable patient results in molec-ular infectious disease testing.

Acknowledgments

We acknowledge Jan Turczyn and the scientists atSiemens Healthcare Diagnostics (Berkeley, CA) for theircontribution of HCV standard materials and their partici-pation in the synthetic materials feasibility experiments.We also acknowledge Micha Nubling and the Paul ErlichInstitute for the contribution of the HCV Paul Erlich Insti-tute biological standard reference used in the syntheticmaterials feasibility experiments. Finally, we acknowl-edge the framework and support of the Industrial LiaisonCommittee and the chair, Jim Reid. The need for thisreview was recognized, formed, and supported throughthe organization’s discussions, workshops, and experi-mentation. Particular thanks are given to GiuseppeColucci for editorial input.

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Documents

International Standards and Reference Materials for Quantitative Molecular Infectious Disease Testing