Table 4.
Selected factors contributing to interassay variability in viral load reporting among quantitative nucleic acid amplification testsa
| Major factor | Specific variable(s) | Comments |
|---|---|---|
| Sample selection and specimen volume | Blood compartments | Higher viral load obtained with whole blood and cell-containing compartments than with plasma or serum |
| Pipetting technique | Automated vs manual pipetting devices | |
| Nucleic acid extraction | Liquid-phase magnetic beads, silica membrane/column | Viral load reporting may depend on the efficiency of viral nucleic acid extraction; in one study, the lowest level of variability in viral load reporting was observed for those methods using liquid-phase extraction compared to magnetic bead and silica membrane extraction (see the text); the volume of sample for nucleic acid extraction may also affect viral load reporting |
| PCR instrument | TaqMan, LightCycler or iCycler, others | Multiple studies have demonstrated differences in viral load reporting depending on the assay platform (see the text) |
| Molecular amplification targets | CMV polymerase gene, glycoprotein B gene, immediate-early gene, others | Differences in amplification efficiency have been demonstrated among various gene targets; the presence of gene polymorphisms in the gene target can reduce sensitivity and quantitative results (see the text) |
| Probe chemistry | Hydrolysis (TaqMan), fluorescence resonance energy transfer (FRET) | |
| Detection reagents | Real-time EIA Southern blot | One study identified detection reagents as the biggest contributor to overall assay variability |
| Calibrators | Abbott, Acrometrix, Advanced Biotechnologies, Qiagen, Roche | One of the most significant variables associated with interassay variability; different quantitative standards can have dramatic effects on viral load results; the introduction of the first WHO international CMV reference standard and the U.S. National Institute of Standards and Technology standard reference material can be expected to address this issue |
| Interaction among variables | The interaction among any of the above variables may compound and increase the variability of viral load reporting |
a
Data were gathered mostly from a large study of laboratory proficiency testing (85). See the text for further discussion of these variables.