Assessment of precision and concordance of quantitative mitochondrial DNA assays: a collaborative international quality assurance study

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Abstract

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. Objectives: 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. Study design: 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 log10 values, 72% non-logged values), and measurements of mitochondrial DNA/cell for replicates varied by less than 12% (based on log10 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 log10 values, 96% non-logged values), and after standardisation results varied by less than 3% (based on log10 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 log10 values, 45% non-logged values) before standardisation, and by 2.49% (based on log10 values, 50% non-logged values) after standardisation. Derived values of mitochondrial DNA/cell varied between laboratories by an average of 91% (non-logged, 56% log10 values) before and by 56% (non-logged, 13% log10 values) after standardisation. Conclusion: All assays demonstrated good precision. The use of common standards is an important step in improving the comparability of data between laboratories.

Introduction

It has been proposed that long-term toxicities associated with the long-term use of nucleoside reverse transcriptase inhibitors (NRTIs) in the context of anti-retroviral therapy for HIV infection are the result of mitochondrial dysfunction. This is based on the knowledge that these drugs are known to inhibit the unique mitochondrial polymerase involved in mitochondrial DNA synthesis (polymerase gamma) as well as their therapeutic target, HIV reverse transcriptase (Brinkman et al., 1999, Kakuda, 2000, White, 2001). Hence, a critical issue in the investigation of tissue specific (including muscle, liver, nerve and adipose tissue) mitochondrial toxicity that is associated with the use of anti-retroviral therapy in the clinical setting, is the development of sensitive and precise mitochondrial DNA quantitation assays.

The establishment of mitochondrial DNA quantitation assays into routine clinical laboratory practice requires the establishment of clinical cut-off points to identify normal from abnormal ranges, as well as the use of controls to monitor quality assurance (QA). However, before these objectives can be met researches must first have sufficient confidence in the validity of the data generated by these assays. A collaborative validation approach that critiques the performance of the assays is therefore important to facilitate the interpretation and comparison of results between independent international research centres.

A number of assays have recently been developed in-house by laboratories to quantify mitochondrial DNA, based on a range of principles that include real-time polymerase chain reaction (PCR) (Hammond et al., 2001, Gahan et al., 2001, Cote et al., 2002), and nucleic acid sequence base amplification (NASBA) (deBaar et al., 2001).

Real-time PCR assays use primers and polymerase in thermocycling reactions to generate an exponential accumulation of the target DNA, the presence of which is detected by various systems, including fluorescence-labelled probes or beacons that are specific for the amplified product. During the exponential phase the amount of amplified DNA is proportional to the amount of starting DNA and quantitative results can be obtained by measuring the PCR product.

Real-time duplex NASBA is a newly developed modification of the standard NASBA, in which both mitochondrial DNA and nuclear DNA are simultaneously amplified in a one-tube format (deBaar et al., 2001). Real-time detection of the isothermal amplification is achieved by including molecular beacons in the reaction, which are specific for either the mitochondrial DNA or nuclear DNA amplification product. The amplification rates of mitochondrial DNA and nuclear DNA are proportional to the content of the mitochondrial DNA and nuclear DNA in the sample. The result of the real-time duplex NASBA is the ratio of mitochondrial DNA to nuclear DNA in a sample, which can be directly converted to the mitochondrial DNA copy number per cell.

Southern blot analysis is an established DNA quantitation technique that has also previously been used in the investigation of mitochondrial DNA depletion syndromes (Barthelemy et al., 2001, Walker et al., 2000).

We report the first QA exercise to evaluate intra- and inter-assay variation and standardisation of results between laboratories. This represents the first stage of a comprehensive international QA exercise, in which the aims include assessing the comparability of data, determining whether further standardisation should be attempted, and enrolling laboratories in a joint effort to determine the clinical relevance of quantifying mitochondrial DNA per cell in vivo.

Precision and accuracy are both important. However, it is neither always practical nor necessary to standardise all assays between multiple laboratories. Individual laboratories may interpret their results in different ways and levels of precision may vary between assays and research centres. For these reasons, QA programs play an invaluable role in disclosing imprecision and/or inaccuracy in an assay. A key aspect of these programs is the distribution of international standards that allow conversion of observed measurements to international assigned units. The conversion of data to a standardised measure may achieve a level of concordance between laboratories that allows data to be compared and interpreted with confidence.

Section snippets

Methods

Seventeen coded wax-sealed tubes, containing DNA (10–20 μl in volume) dissolved in water, were distributed at room temperature to four participating laboratories, including the host laboratory (Table 1). Total DNA was extracted from one human lymphoblastoid cell line using QIAamp DNA blood mini kit columns (Qiagen) according to the manufacturer's instruction. Samples were prepared by making dilutions from the pooled DNA and included masked replicates, as well as five standards prepared by

Results

Standard curves of reported results plotted against pre-assigned values for standards are shown in Fig. 1a–f. Results from all laboratories showed good linearity in that standards were ranked in the correct order from high to low concentration for both mitochondrial and nuclear DNA measurements.

Tables of results (excluding standards) (Table 2a, Table 2b, Table 3a, Table 3b, Table 4a, Table 4b), illustrate the levels of intra-laboratory precision of replicates. For all laboratories, measurements

Discussion

We investigated the precision and concordance of measurements of mitochondrial DNA and nuclear DNA, and of a derived measurement of cellular mitochondrial DNA content, reported for the same set of samples by different laboratories using different real-time assays. The exchange has shown that all participants achieved similar within laboratory precision, and that all laboratories were able to rank samples correctly in terms of both mitochondrial and nuclear DNA content.

The greatest discordance

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