International standards and reference materials for quantitative molecular infectious disease testing

Abstract

The utility of quantitative molecular diagnostics for patient management depends on the ability to relate patient results to prior results or to absolute values in clinical practice guidelines. To do this, those results need to be comparable across time and methods, either by producing the same value across methods and test versions or by using reliable and stable conversions. Universally available standards and reference materials specific to quantitative molecular technologies are critical to this process but are few in number. This review describes recent history in the establishment of international standards for nucleic acid test development, organizations involved in current efforts, and future issues and initiatives.

Figure 1

Evaluation of synthetic and biological HCV reference materials with three commercial HCV quantitative assays. Serial dilutions of the Paul Erlich Institute (PEI) regional HCV standard (80,000 IU/ml), high titer plasma, Armored RNA Quant HCV and synthetic HCV RNA transcript were tested using the RealTime HCV (A), COBAS AmpliPrep/COBAS TaqMan HCV (B), or VERSANT HCV RNA 3.0 assays (C). All test specimens were shipped and stored below −70°C. The HCV Armored RNA was prediluted, shipped, and stored in base human serum. Other specimens were formulated by each laboratory using the same base matrix immediately after thawing of the stock materials. The synthetic HCV RNA transcript was diluted in the presence of lysis reagent to inhibit potential degradation of the RNA by serum nucleases. At least six replicates of each dilution were tested in three independent runs with each assay. The synthetic materials were formulated in copies/ml of base 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, threshold cycle; RLU, relative light units.