Investigation of cushing’s syndrome

Investigation of cushing’s syndrome

ABSTRACTS accessible to our community. They do not readily invite our strategic involvement and are wary of those inputs which appear to be based on ...

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ABSTRACTS

accessible to our community. They do not readily invite our strategic involvement and are wary of those inputs which appear to be based on self-interest. The enviable quality of Australian pathology is threatened by the absence of an effective strategic interface between laboratory professionals, funders and the community. LC TANDEM MS – OVERVIEW OF TECHNOLOGY James Pitt VCGS Pathology, Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, Vic, Australia Many different types of mass spectrometers are available for the analysis of biological molecules featuring different ionisation methods and technologies for mass analysis. The combination of electrospray ionisation with triple quadrupole or hybrid quadrupole/ion trap mass spectrometry is one of the most versatile and widely-used for quantitative LC-MS analysis in clinical chemistry. Useful data can be obtained using direct injection methods without chromatography, as exemplified by high-throughput screening tests such as neonatal dried blood spot or urine screening. Ion suppression and interferences limit both the sensitivity and imprecision of direct injection methods and chromatography is required for applications requiring improved performance. Short columns are typically used to maintain throughput and columns with particle sizes <2 mm are increasingly used to achieve high chromatographic resolution. Internal standards, preferably 13C analogues, are critical to obtaining good quantitative data and compensating for ion suppression and significant within-batch variations in absolute ion responses. Multiple reaction monitoring is used to monitor analytes within timed chromatographic windows and dozens of analytes can be multiplexed in a single run. With appropriate method design, there appear to be few molecules of clinical interest that cannot be analysed by LC-MS. VITAMIN D STANDARDISATION B. C. McWhinney Chemical Pathology, Pathology Queensland, Brisbane, Qld, Australia Vitamin D has been implicated in a host of conditions, including certain cancers, type 1 diabetes, multiple sclerosis, tuberculosis, Alzheimer’s disease and psoriasis. Some laboratories report test requests increasing by over 100% per year. Vitamin D measurement is performed using a number of technologies including immunoassay, high performance liquid chromatography (HPLC) and LC-MS/MS. The UK-based Vitamin D External Quality Assessment Scheme (DEQAS), the largest vitamin D proficiency testing program with over 600 registered participants, has observed extensive interlaboratory variation. A 2008 review found that the annual mean inter-laboratory imprecision has generally declined from near 30% in the mid 1990 s to under 20% in 2007, with sporadic increases as new methods were introduced and problems with certain assays were identified. Significantly, DEQAS analysis revealed that between April 2007 and January 2008, the LCMS method produced a mean positive bias of >10%. NIST has been developing serum and solvent-based standard reference materials for 25-OH-D (SRM 972 and SRM 2972, respectively). In theory, the SRMs will provide both immunoassay users (in the case of SRM 972) and LC-MS users (in the case of

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SRM 2972) a means to calibrate their assays, reducing the interlaboratory coefficients of variation (CVs) for each method. One study found that harmonisation to a common calibrator reduced average between laboratory variation to 9% from 30%. HORMONE MEASUREMENT TECHNIQUES AND PROBLEMS WITH LC-MS/MS B. R. Cooke PathWest Core Clinical Pathology and Biochemistry, Royal Perth Hospital, Perth, WA, Australia The use of LC-MS/MS for the analysis of serum hormones provides positive identification of steroid compounds at low concentration, without cross-reactivity, or analytical interference problems inherent in immunoassay methods. However, problems exist with LC-MS/MS and include, but are not limited to, matrix ion suppression, isotopic crosstalk from closely related steroids, interference from sample collection vessels and the instability of equipment parameters. Neonatal samples provide unique issues due to the complex nature of the matrix, specific and different collection tubes with their own inherent problems and the need to quantify increasingly lower levels of circulating steroid hormones. The inclusion of isotopic dilution and quantifier ions help identify these issues in routine mass spectrometric analysis. However, it is during the assay developmental stages that the most important decisions are made regarding sample clean up and optimisation of chromatographic separation prior to mass analysis. Consequently, the pre-analytical phase is increasingly recognised as crucial to analysis if accurate quantification of circulating hormone levels are to be determined. Thorough method development protocols have proven to be essential in the optimal development of mass spectrometric, liquid chromatographic methods. INVESTIGATION OF CUSHING’S SYNDROME Warrick J. Inder Department of Endocrinology and Diabetes, St Vincent’s Hospital, and Department of Medicine, University of Melbourne, Melbourne, Vic, Australia In the biochemical investigation of suspected Cushing’s syndrome, screening tests are used to identify potential cases and should have high sensitivity, be inexpensive and easy to perform. Appropriate screening tests are a midnight salivary cortisol and/or the 1 mg dexamethasone suppression test (DST). Normal screening tests indicate the patient is unlikely to have Cushing’s syndrome. An abnormal screening test should be followed up by diagnostic testing using 24 hour urinary free cortisol levels and either a low dose oral or iv DST. After the diagnosis of Cushing’s syndrome is confirmed, differential diagnostic testing should be undertaken. A suppressed plasma ACTH suggests adrenal disease. Those with ACTH-dependent Cushing’s require further localising investigation to distinguish between pituitary and ectopic sources of ACTH. The current gold standard is bilateral inferior petrosal sinus sampling (IPSS). The relative sensitivity and specificity of other biochemical tests in the differential diagnosis of ACTHdependent Cushing’s will be discussed. The majority of cases will have Cushing’s disease, where the treatment of choice is transsphenoidal pituitary surgery. There is no clear consensus of what constitutes biochemical remission post-operatively; the strictest definition requires a plasma cortisol of <50 nmol/L. Long term, up to 25% of cases initially considered ‘cured’ will relapse.

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