Rnase-free water (4 ml) was routinely added instead of test sample to 16 ml of the master mix and used as a no-template control for every run. At the end of the assay, the specificity of each amplified product was ascertained by means of melting curve analysis. This eliminated false positive detections due to primer dimers or nonspecific amplicons. To confirm that there was no DNA contamination in the input RNA and to assess the specificity of the reverse transcribed rtLC DBS products, initial assays included PCR reactions without the addition of reverse-transcriptase enzyme. Gel electrophoresis of the amplicons was initially performed to further confirm the specificity of the products. Each sample was tested in replicates and a second DBS was independently extracted and run to verify a positive result. The test was repeated if the sample had indeterminate results, and if necessary a new spot was extracted and tested if the results of the repeat test were also indeterminate. Quality control for each experiment was assessed by the performances of the standard curve and the negative control. All DBS prepared by trained personnel were tested for this study as long as multiple filter spots were available for each patient, and the blood spots were good quality with no hemolysis or clots.