The primary purpose of newborn screening is to quickly identify children that are at risk of having a specific disorder in order to start treatment, prevent early death and reduce the chances of permanent physical or mental damage. The current and widely accepted approach used for identification of metabolism disorders involves a flow injection analysis with mass spectrometry detection of acylcarnitines and amino acids. Although this approach is widely accepted and has shown to be sufficient for identification of multiple metabolism disorders the method is not fully quantitative and results often have to be confirmed by second-tier tests. The primary focus of this research was to improve the accuracy and selectivity of this screening method by employing a high resolution chromatographic separation for the combined analysis of twelve acylcarnitines and seven amino acids. This method is an improvement over the current methodology allowing for separation of key isomers that are diagnostic for different metabolism disorders, reducing the need for multiple second-tier tests to confirm results and shortening the time to diagnosis. In order to further improve the efficiency of newborn screening we developed an in-line desorption device, which allows for direct analysis of DBS eliminating the need for punching disks from the filter paper cards. Our device was the first published paper that demonstrated the ability to directly analyze dried blood spots, without the need for any offline sample processing. Using this device, we validated a method to quantify biomarkers related to Maple Syrup Urine Disease, a disorder that requires a second-tier test for confirmation. To further improve the accuracy of dried blood spot analysis we evaluated a technique to correct the sample volume in low and high hematocrit samples. The level of hematocrit in blood spotted on filter paper cards affects the volume of sample analyzed, leading to errors in accuracy. Diffuse reflectance was used to relate differences in sample hematocrit on dried blood spots. We validated our technique with eighteen donor samples at various levels of hematocrit. Correcting sample volume for hematocrit showed improved precision and accuracy over the standard approach, ultimately reducing the potential to misidentify samples.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-3905 |
| Date | 21 November 2012 |
| Creators | Miller, John H., IV |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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