Table 2.

Optimal Specifications and Challenges: Molecular Standards

Characteristic	Optimal specification	Acceptable specification	Challenges
Target nucleic acid	Whole genome as found in virus; intact	Whole genome as found in virus; intact	Is there a representative consensus genome?
Source of target nucleic acid	Virus	Recombinant DNA (DNA viruses) or RNA synthesized in vitro (RNA viruses)	Can the virus be propagated? Is the cultured strain representative?
Length of target nucleic acid	Entire genome	All of the sequences recognized by current and projected future assays	Current assays may not be representative of future technologies
			Cultured organisms, purified NA or synthetic NA may not have important secondary structure
One or several genotypes/subtypes	All relevant genotypes available as standards	One genotype for a “foundation” standard. Genotypes and subtypes calibrated from the foundation standard	One representative organism does not necessarily address technicalities of all variants
			Resources to produce and characterize all variants and perform the global studies to value-assign them would detract from producing standards for more organisms
Which genotype/subtype?	The most globally prevalent	The most globally prevalent	The most globally prevalent or the target of most present and near future testing?
Sequence of target nucleic acid	Known	Known	The proposed material should be sequenced
	Sequence of cDNA clones (from RNA viruses) or DNA clones (from DNA viruses)	Sequence of cDNA clones (from RNA viruses) or DNA clones (from DNA viruses)
Concentration of target nucleic acid	High enough to evaluate calibrators that cover levels of targets found in patients	High enough to evaluate calibrators that cover levels of targets found in patients	Some source material (patient plasma) may not have high enough titers to produce enough standard material.
Matrix	Natural material from which the target is predominantly assayed. (ie, blood-borne viruses: plasma)	Material that will ensure stability and intactness of target nucleic acid and that 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 cellular standards be developed?
Temperature of storage	Ambient or refrigerated	Ambient or refrigerated	Can impact stability
Manufacturing process	Validated, reproducible methods within a validated quality system	Validated methods within a validated quality system	Could limit the number and type of organizations able to produce or contribute materials
Vial-to-vial consistency	SD ≤ 0.15 log10 copies/ml (coefficient of variation ≤ 35%)	SD in target concentration known and published	Could limit the number and type of organizations able to produce or contribute materials
Initial quantification of nucleic acid targets	Independent analytical method(s)	Quantitative NAT using statistically designed protocol and equal weighting of results from assays used	Absence of reference methods requires testing with relevant available methods
Are resources available to adequately test materials with an even representation of the available test methods?