Kirsten Hoad

Vitamin B1 and B6 method harmonization: comparison of performance between laboratories enrolled in the RCPA Quality Assurance Program.

OBJECTIVES The RCPA Quality Assurance Program (RCPA QAP) offers monthly proficiency testing for vitamins A, B1, B6, β-carotene, C and E to laboratories worldwide. A review of the results submitted for the whole blood vitamin B1/B6 sub-program revealed a wide dispersion. Here we describe the results of a methodology survey for vitamins B1 and B6. DESIGN AND METHODS A questionnaire was sent to thirteen laboratories. Eleven laboratories were returning QAP results for vitamin B1 (thiamine diphosphate) and five were returning results for vitamin B6 (pyridoxal-5-phosphate). RESULTS All nine respondents provided a clinical service for vitamins B1 and B6. HPLC with fluorescence detection was the most common method principle. For vitamin B1, six respondents used a commercial assay whilst three used in-house methods; whole blood was the matrix for all. For vitamin B6, five respondents used commercial assays and four used in-house assays. The choice of matrix for vitamin B6 varied with three respondents using whole blood and five using plasma for analysis. Sample preparation incorporated protein precipitation and derivatization steps. An internal standard was employed in sample preparation by only one survey respondent. CONCLUSIONS The immediate result of this survey was the incorporation of plasma vitamin B6 into the RCPA QAP vitamin program. The absence of an internal standard in current vitamin B1 and B6 assays is a likely contributor to the wide dispersion of results seen in this program. We recommend kit manufacturers and laboratories investigate the inclusion of internal standards to correct the variability that may occur during processing.

Serum parathyroid hormone concentrations in patients with HIV infection

Endocrine abnormalities are common in patients infected with the human immunodeficiency virus (HIV) but these rarely cause alterations in calcium homeostasis. 1 However, hypocalcaemia has been described in other acutely ill patients, including patients with infectious diseases.? Following the diagnosis of frank hypoparathyroidism in a patient with symptomatic HIV infection, we have carried out a systematic study of serum parathyroid hormone (PTH) concentration in patients with symptomatic and asymptomatic HIV infection.

Serum vitamin A and E analysis: comparison of methods between laboratories enrolled in an external quality assurance programme

Aim To survey laboratories enrolled in the Royal College of Pathologists of Australasia (RCPA) Chemical Pathology Quality Assurance Programme (QAP) for vitamin A and E testing to determine differences between methods of analysis. Methods A detailed questionnaire covering the major aspects of serum vitamin A and E analysis was sent to all participating laboratories in 2007. Results Thirteen out of the 22 laboratories completed the questionnaire. Methods between laboratories showed a great deal of commonality. All respondents performed a liquid extraction step, which included the addition of an internal standard, followed by high-performance liquid chromatography (C18 columns with predominantly methanol-based mobile phases) with spectrophotometric detection. Major inter-laboratory differences were whether the sample was protected from light, the extraction solvents and ratios used, the drying down temperature used post-liquid extraction and choice of calibrator. Conclusions The questionnaire highlighted discrete methodological differences between laboratories. These findings provide direction to enable the Vitamins Working Party of the Australasian Association of Clinical Biochemists to further investigate the dispersion in results between participants of the RCPA QAP vitamin programme.

Improved plasma free metadrenaline analysis requires mixed mode cation exchange solid-phase extraction prior to liquid chromatography tandem mass spectrometry detection

Background The investigation and effective management of phaeochromocytoma involves biochemical measurement of either conjugated total urine or plasma free metadrenalines. Current analytical methods include enzyme-linked immunosorbent assays, high-performance liquid chromatography (HPLC) with electrochemical detection (ECD) or liquid chromatography tandem mass spectrometry (LCMS/MS). Since the first two methods are either extremely laborious, necessitate low sample run numbers, result in slow turnaround times or are subject to analytical interference, a robust, routine clinical method is not achievable. We established a novel sample preparation method to measure plasma free metadrenalines using LCMS/MS. Methods Three different solid-phase extraction (SPE) methods were compared: hydrophilic–lipophilic balance sorbent (HLB), weak cation exchange (WCX) and mixed mode cation exchange (MCX) and their ability to remove interfering compounds prior to LCMS/MS analysis. Maximum recovery of plasma free metadrenaline and plasma free normetadrenaline were achieved by positively charging compounds prior to SPE application. Results Compared with HLB and WCX cartridges, MCX extraction resulted in chromatography without co-eluting interference with superior assay precision and accuracy. Additionally, samples that could not be quantified because of interference using HPLC/ECD could be readily assayed using this new method. Conclusions The use of the MCX SPE method with LCMS/MS detection provides an improved assay to measure plasma free metadrenalines in comparison to many available alternative methods.

Thyroxine autoantibody interference is an uncommon cause of inappropriate TSH secretion using the Immulite 2000 assay.

BACKGROUND Inappropriate TSH secretion, as defined by an elevated free thyroxine (fT4) and non-suppressed thyrotropin (TSH) result, can be caused by acute illness, medications, TSH secreting tumours, thyroid hormone resistance or immunoassay interference including thyroid hormone autoantibody interference. T4 autoantibody (T4AAb) prevalence has not been determined. We determined the prevalence of inappropriate TSH secretion using the Immulite 2000 assay and the prevalence of T4AAb within this subgroup. METHODS Samples were stored over 13 months and thawed once for batch analysis. T4AAb was detected using radioimmunoprecipitation with >5% of the radiolabel within the immunoprecipitate indicating true positivity. All case notes and medication charts were reviewed. RESULTS Of 13,286 thyroid profiles reviewed, 85 (0.64%) samples demonstrated inappropriate TSH secretion. One of these 85 samples (1.2%) was positive for T4AAb with a radioimmunoprecipitate of 21%. 46/85 (54%) samples were collected on hospitalised patients, 7 patients were prescribed amiodarone, 12 patients were taking beta blockers, 30 patients were on thyroxine replacement and 7/85 (8%) were collected on outpatients not taking medication known to affect thyroid function. CONCLUSION T4AAb interference is an extremely rare explanation for inappropriate TSH secretion with the Immulite 2000 assay but should be excluded before investigating for rarer causes of this biochemical picture.

External quality assurance target setting with NIST SRM 968d material: performance in the 2010 Royal College of Pathologists of Australasia Quality Assurance Program with retinol, α-tocopherol and β-carotene

practices against these guidelines, notably those standards relating to specimen requirements, transport and specimen handling/analysis. A retrospective audit of 281 CSF xanthochromia requests received between 1 September 2008 and 31 October 2009 was undertaken and we also compared the results with clinical outcomes. A number of points and audit failures have emerged. In particular, our standard operating procedures must be improved in order to comply fully with current guidelines to ensure that:

A high pressure liquid chromatography method for separation of prolactin forms

Background Prolactin has multiple forms and macroprolactin, which is thought not to be bioavailable, can cause a raised serum prolactin concentration. Gel filtration chromatography (GFC) is currently the gold standard method for separating macroprolactin, but is labour-intensive. Polyethylene glycol (PEG) precipitation is suitable for routine use but may not always be accurate. We developed a high pressure liquid chromatography (HPLC) assay for macroprolactin measurement. Methods Chromatography was carried out using an Agilent Zorbax GF-250 (9.4 × 250 mm, 4 μm) size exclusion column and 50 mmol/L Tris buffer with 0.15 mmol/L NaCl at pH 7.2 as mobile phase, with a flow rate of 1 mL/min. Serum or plasma was diluted 1:1 with mobile phase and filtered and 100 μL injected. Fractions of 155 μL were collected for prolactin measurement and elution profile plotted. The area under the curve of each prolactin peak was calculated to quantify each prolactin form, and compared with GFC. Results Clear separation of monomeric-, big- and macroprolactin forms was achieved. Quantification was comparable to GFC and precision was acceptable. Total time from injection to collection of the final fraction was 16 min. Conclusions We have developed an HPLC method for quantification of macroprolactin, which is rapid and easy to perform and therefore can be used for routine measurement.