DOI
https://doi.org/10.25772/AW5D-4M90
Defense Date
2019
Document Type
Thesis
Degree Name
Master of Science
Department
Chemistry
First Advisor
Sarah Rutan, Ph.D.
Second Advisor
Maryanne Collinson, Ph.D.
Third Advisor
Ashton Cropp, Ph.D.
Fourth Advisor
Adam Hawkridge, Ph.D.
Abstract
Congenital Adrenal Hyperplasia (CAH) encompasses several disorders related to disruptions in the adrenal steroid production pathway. These disruptions may cause virilization of the external female sex organs, incorrect gender assignment, precocious puberty, and in the most severe form, may cause life-threatening salt wasting and adrenal crisis if not detected and treated early in the newborn period.
17α-Hydroxyprogesterone (17-OHP) is the primary target for immunofluorescence detection of CAH from dried blood spots in newborn screening (NBS). Unfortunately, current immunoassay techniques for the detection of CAH suffer from high false positive rates. The primary factors contributing to false positive determinations can include the natural increase of 17-OHP due to stress stimuli as well as cross-reactivity of the immunoassay antibody with other hormones and endogenous compounds in blood.
Analysis of the adrenal steroid profile and corresponding analyte ratios using high performance liquid chromatography (HPLC)or ultra-high pressure liquid chromatography (UHPLC)combined with tandem mass spectrometry (MS/MS) has been shown to be a sensitive and selective technique for the significant reduction of the false positive reporting rate for CAH in newborn screening.
In working toward optimization, validation, and implementation of an HPLC-MS/MS steroid profile for use by Virginia’s Newborn Screening laboratory as a 2nd tier analysis for CAH screening, a commercially-available core-shell HPLC column with a biphenyl stationary phase was determined to offer adequate retention and selectivity to achieve baseline resolution of isobaric target analytes under rapid reversed phase gradient conditions. Method linearity, precision, and accuracy were assessed using enriched dried blood spot materials. Double-blinded analyses of over 300 newborn dried blood spot specimens were used to determine clinical sensitivity and specificity of the assay, which is projected to substantially reduce the false positive reporting rate for CAH screening while meeting target sample turnaround times.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
12-10-2019
Included in
Analytical Chemistry Commons, Congenital, Hereditary, and Neonatal Diseases and Abnormalities Commons, Endocrine System Diseases Commons, Maternal and Child Health Commons