using a Bland-Altman\cite{10} plot of all samples with values in both assays (35 samples). The overall bias between the laboratory-developed assay and ASR was calculated from this plot using GraphPad Prism 5 Software (GraphPad Software, San Diego, CA). In addition, using proficiency samples provided by the American Society of Transplantation (AST, Mt Laurel, NJ) and Quality Control for Molecular Diagnostics (QCMD, Glasgow, Scotland), we determined results obtained from both assays.

\section{Results}

\subsection{Limits of Detection and Quantification}
The LOD of the ASR was 2.25 $\log_{10}$ copies/mL (180 copies/mL) (Table~\ref{tab:table_1}), which was equivalent to 1.30 $\log_{10}$ copies/mL (20 copies/mL) using the Namalwa cell line. The LOQ based on an SD of 0.15 $\log_{10}$ copies/mL or less was 3.90 $\log_{10}$ copies/mL (Table~\ref{tab:table_1}). Of note, the SDs at 3.53 $\log_{10}$ copies/mL and 2.89 $\log_{10}$ copies/mL were 0.18 and 0.17 $\log_{10}$ copies/mL, respectively.

\subsection{Linearity and Reproducibility}
The assay was linear from 2.17 to 6.21 $\log_{10}$ copies/mL (Figure~\ref{fig:figure_1}), with an $R^2$ value of 0.9925 ($y = 1.0119x - 0.1184$). Reproducibility was assessed by testing 7 replicates of low and high viral load patient samples and 6 replicates of the QSs. The low patient sample had a mean (SD, CV) value of 3.70 $\log_{10}$ copies/mL (0.07, 2.0\%). The high patient sample had a mean (SD, CV) value of 5.40 $\log_{10}$ copies/mL (0.07, 1.4\%). The QSs had mean Ct (SD, CV) values as follows: QS1, 21.8 (0.4, 1.6\%); QS2, 25.1 (0.4, 1.5\%); QS3, 28.5 (0.2, 0.8\%); and QS4, 31.8 (0.3, 1.0\%).

\begin{figure}
\centering
\includegraphics{figure_1.pdf}
\caption{Linearity plot. The mean values for each concentration from 2.44 to 6.21 $\log_{10}$ copies/mL were plotted, with error bars representing the SD. A linear regression line ($y = 1.0119x - 0.1184$) was plotted with Microsoft Excel 2003 software (Redmond, WA), with an $R^2$ value of 0.9925, indicating that the assay is linear at least across this range.}
\label{fig:figure_1}
\end{figure}

\subsection{Agreement}
Of the 61 patient samples tested by the laboratory-developed method and the ASR, 35 were positive in both assays. Of the 6 samples that were positive in the Artus assay but not in the laboratory-developed assay, the average copy number was 1.62 $\log_{10}$ copies/mL, with a range of 1.41 to 2.13 $\log_{10}$ copies/mL. There was only 1 sample that was positive in the laboratory-developed assay but negative in the Artus assay, with a value of 3.23 $\log_{10}$ copies/mL. The remaining 19 samples were negative by both assays. The 35 samples that were positive in both assays were plotted in a standard Bland-Altman\cite{10} plot (Figure~\ref{fig:figure_2}). The mean difference between

\begin{table}
\centering
\caption{Dilution Series}
\label{tab:table_1}
\begin{tabular}{llllll}
EBV Cell Line ($\log_{10}$ copies/mL) & Mean Artus ($\log_{10}$ copies/mL) & $\log_{10}$ Artus SD & $\log_{10}$ Artus CV & No. Tested/No. Positive (\% Positive) \\
6.30 & 6.21 & 0.07 & 0.01 & 8/8 (100) \\
5.30 & 5.23 & 0.11 & 0.02 & 8/8 (100) \\
4.30 & 4.44 & 0.07 & 0.01 & 8/8 (100) \\
4.00 & 3.90 & 0.15 & 0.04 & 12/12 (100) \\
3.70 & 3.53 & 0.18 & 0.05 & 12/12 (100) \\
3.00 & 2.89 & 0.17 & 0.06 & 12/12 (100) \\
2.70 & 2.76 & 0.24 & 0.09 & 16/16 (100) \\
2.40 & 2.17 & 0.39 & 0.02 & 16/16 (100) \\
2.00 & 2.44 & 0.33 & 0.14 & 8/8 (100) \\
1.30 & 2.25 & 0.48 & 0.21 & 8/8 (100) \\
0.60 & 1.60 & 1.06 & 0.66 & 6/8 (75) \\
-0.10 & 1.17 & 1.02 & 0.87 & 5/8 (63) \\
\end{tabular}

CV, coefficient of variation; EBV, Epstein-Barr virus.

* The expected copy number based on the approximate initial number of Namalwa cells (ATCC CRL-1432), the mean measured copy number using the Artus assay, SD, CV, and the number and percentage of positive replicates for each dilution of the Namalwa cell line in EBV--whole blood were tabulated.
\end{table}