Identification, Measurement, and Assessment of the Clinical Utility of Human Pancreatic Polypeptide by Liquid Chromatography-Tandem Mass Spectrometry.

Human pancreatic polypeptide (HPP) is a 36 amino acid peptide hormone that plays a role in the bidirectional communication between the digestive system and the brain. HPP measurements are used to assess vagal nerve function following sham feeding and to detect gastroenteropancreatic-neuroendocrine tumors. These tests have historically been conducted by radioimmunoassays, but liquid chromatography-tandem mass spectrometry (LC-MS/MS) has several advantages such as improved specificity and elimination of radioactive molecules. Here, we present our LC-MS/MS method. Initially, samples were immunopurified and subjected to LC-high resolution accurate mass tandem mass spectrometry (HRAM-MS/MS) to identify circulating forms of the peptide in human plasma. We identified 23 forms of HPP, including several glycosylated forms. The most abundant peptides then were used for targeted LC-MS/MS measurements. LC-MS/MS performance for precision, accuracy, linearity, recovery, limit of detection, and carryover met our acceptance criteria based on CLIA regulations. Additionally, we observed the expected physiological rise in HPP in response to sham feeding. Our results indicate that HPP measurement by LC-MS/MS produces clinically equivalent results to our established immunoassay when several peptides are monitored, making it a suitable replacement. The measurement of peptide fragments, including modified species, might have additional clinical value.

Interpretation of Abnormal Dexamethasone Suppression Test is Enhanced With Use of Synchronous Free Cortisol Assessment.

CONTEXT: Interpretation of dexamethasone suppression test (DST) may be influenced by dexamethasone absorption and metabolism and by the altered cortisol binding. OBJECTIVE: We aimed to determine the normal ranges of free cortisol during DST in participants without adrenal disorders and to identify the population of patients where post-DST free cortisol measurements add value to the diagnostic workup. DESIGN AND SETTING: Cross-sectional study conducted in a tertiary medical center. PARTICIPANTS: Adult volunteers without adrenal disorders (n = 168; 47 women on oral contraceptive therapy [OCP], 66 women not on OCP, 55 men) and patients undergoing evaluation for hypercortisolism (n = 196; 16 women on OCP). MEASUREMENTS: Post-DST dexamethasone and free cortisol (mass spectrometry) and total cortisol (immunoassay). MAIN OUTCOME MEASURES: Reference range for post-DST free cortisol, diagnostic accuracy of post-DST total cortisol. RESULTS: Adequate dexamethasone concentrations (≥0.1 mcg/dL) were seen in 97.6% volunteers and 96.3% patients. Only 25.5% of women volunteers on OCP had abnormal post-DST total cortisol (>1.8 mcg/dL). In volunteers, the upper post-DST free cortisol range was 48 ng/dL in men and women not on OCP, and 79 ng/dL in women on OCP. When compared with post-DST free cortisol, diagnostic accuracy of post-DST total cortisol was 87.3% (95% CI, 81.7-91.7); all false-positive results occurred in patients with post-DST cortisol between 1.8 and 5 mcg/dL. OCP use was the only factor associated with false-positive results (21.1% vs 4.9%, P = 0.02). CONCLUSIONS: Post-DST free cortisol measurements are valuable in patients with optimal dexamethasone concentrations and post-DST total cortisol between 1.8 and 5 mcg/dL.

Evaluation of the PAX8/PPARG translocation in follicular thyroid cancer with a 4-color reverse-transcription PCR assay and automated high-resolution fragment analysis.

BACKGROUND: Molecular testing of thyroid malignancies, in combination with cytologic and histologic examination, is becoming increasingly attractive as a tool for refining traditional morphologic diagnosis. The molecular changes associated with follicular thyroid carcinoma (FTC) are point mutations in RAS oncogenes or the presence of PAX8/PPARG (paired box 8/peroxisome proliferator-activated receptor gamma) rearrangement. METHODS: We developed and validated a clinical assay for the detection of PAX8/PPARG rearrangements that uses a 4-color reverse-transcription PCR (RT-PCR) assay and high-resolution fragment analysis. RESULTS: The RT-PCR assay is applicable for detecting the various described fusion transcripts of PAX8/PPARG in formalin-fixed, paraffin-embedded thyroid tissue and in fine-needle aspirate biopsy washes from thyroid nodules. The analytical sensitivity of the assay is 1 abnormal cell in a background of 100-10 000 translocation-negative cells. A comparison of the RT-PCR assay with dual-fusion fluorescence in situ hybridization showed an overall concordance of 95%. With this assay, we obtained a prevalence for the PAX8/PPARG rearrangement in FTC of 62% (13 of 21 cases), compared with a 5% prevalence (3 of 55) for other follicular cell-derived neoplasms. CONCLUSIONS: The introduction of this assay into clinical practice could provide useful information for the diagnosis and possibly for the prognosis and treatment of thyroid cancer in the future.