Assessment of precision and concordance of quantitative mitochondrial DNA assays: a collaborative international quality assurance study.
BACKGROUND
A number of international research groups have developed DNA quantitation assays in order to investigate the role of mitochondrial DNA depletion in anti-retroviral therapy-induced toxicities.


OBJECTIVE
A collaborative study was undertaken to evaluate intra-assay precision and between laboratory concordance of measurements of mitochondrial DNA quantity, as a component of a comprehensive quality assurance project.


METHODS
Four laboratories were asked to measure and report mitochondrial DNA and nuclear DNA genome copy number, as well as mitochondrial DNA copy number/cell, for 17 coded aliquots of DNA derived from serial dilutions of pooled DNA from a lymphoblastoid cell line. Samples included masked replicates and five standards. All samples had similar mitochondrial DNA/nuclear DNA ratios. Precision within laboratories was assessed by determining the coefficient of variation of replicates. Concordance between laboratories was assessed by determining the average coefficient of variation of the mean replicate values for each sample. The effect of standardising the assay for these three measurements was also assessed for laboratories A, B and C.


RESULTS
Measurements of mitochondrial DNA and nuclear DNA content for replicate samples varied by an average of less than 6% (based on log(10) values, 72% non-logged values), and measurements of mitochondrial DNA/cell for replicates varied by less than 12% (based on log(10) values, 32% non-logged values), with no improvement of precision after standardisation. Standardisation did significantly improve the concordance of results for measurements of mitochondrial DNA content and mitochondrial DNA/cell. Non-standardised measurements of mitochondrial DNA content for the same sample set varied by 19% between laboratories (based on log(10) values, 96% non-logged values), and after standardisation results varied by less than 3% (based on log(10) values, 54% non-logged values). There was no significant improvement for concordance of measures of nuclear DNA content after standardisation, with results varying by 4.56% between laboratories (based on log(10) values, 45% non-logged values) before standardisation, and by 2.49% (based on log(10) values, 50% non-logged values) after standardisation. Derived values of mitochondrial DNA/cell varied between laboratories by an average of 91% (non-logged, 56% log(10) values) before and by 56% (non-logged, 13% log(10) values) after standardisation.


CONCLUSIONS
All assays demonstrated good precision. The use of common standards is an important step in improving the comparability of data between laboratories.
Centre for Clinical Immunology and Biomedical Statistics, 2nd floor, North Block, Royal Perth Hospital and Murdoch University, Australia.
Emma L
EL
Hammond
David
D
Sayer
David
D
Nolan
Ulrich A
UA
Walker
Anthony de
Ad
Ronde
Julio S G
JS
Montaner
Helene C F
HC
Cote
Michelle E
ME
Gahan
Catherine L
CL
Cherry
Steven L
SL
Wesselingh
Peter
P
Reiss
Simon
S
Mallal
D002460:Cell Line; D004247:DNA / Q000032:analysis; D004272:DNA, Mitochondrial / Q000032:analysis*; D018628:Gene Dosage; D006801:Humans; D007391:International Cooperation; D007753:Laboratories / Q000592:standards*; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D011786:Quality Control; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004272:DNA, Mitochondrial; D004247:DNA