Ensuring quality in 17OHP mass spectrometry measurement: an international study assessing isomeric steroid interference.

OBJECTIVES: Interference from isomeric steroids is a potential cause of disparity between mass spectrometry-based 17-hydroxyprogesterone (17OHP) results. We aimed to assess the proficiency of mass spectrometry laboratories to report 17OHP in the presence of known isomeric steroids. METHODS: A series of five samples were prepared using a previously demonstrated commutable approach. These samples included a control (spiked to 15.0 nmol/L 17OHP) and four challenge samples further enriched with equimolar concentrations of 17OHP isomers (11α-hydroxyprogesterone, 11ß-hydroxyprogesterone, 16α-hydroxyprogesterone or 21-hydroxyprogesterone). These samples were distributed to 38 participating laboratories that reported serum 17OHP results using mass spectrometry in two external quality assurance programs. The result for each challenge sample was compared to the control sample submitted by each participant. RESULTS: Twenty-six laboratories (68 % of distribution) across three continents returned results. Twenty-five laboratories used liquid chromatography-tandem mass spectrometry (LC-MS/MS), and one used gas chromatography-tandem mass spectrometry to measure 17OHP. The all-method median of the control sample was 14.3 nmol/L, ranging from 12.4 to 17.6 nmol/L. One laboratory had results that approached the lower limit of tolerance (minus 17.7 % of the control sample), suggesting the isomeric steroid caused an irregular result. CONCLUSIONS: Most participating laboratories demonstrated their ability to reliably measure 17OHP in the presence of the four clinically relevant isomeric steroids. The performance of the 12 (32 %) laboratories that did not engage in this activity remains unclear. We recommend that all laboratories offering LC-MS/MS analysis of 17OHP in serum, plasma, or dried bloodspots determine that the isomeric steroids are appropriately separated.

Quality assurance programs for vitamin A and E in serum: are we doing enough to assess laboratory performance?

OBJECTIVES: Monitoring serum vitamin A (retinol) and vitamin E (α-tocopherol) concentrations is common practice for assessing nutritional status. Measurement of these vitamins can be challenging due to several factors. Whilst the RCPAQAP Vitamins: Serum Program assists participating laboratories in harmonisation, the materials provided do not contain the analogues of retinol and α-tocopherol that may be present in real patient samples. We aimed to assess participants' capacity to accurately report retinol and α-tocopherol in the presence of the vitamin E analogues tocopherol acetate and γ-tocopherol. METHODS: A supplementary series of a control sample and three matched spiked samples were distributed to each laboratory participating in the Program. Retinol and α-tocopherol results for each spiked sample were compared to the results of the control sample submitted by each participant. Acceptability of retinol and α-tocopherol results was determined based on the RCPAQAP allowable performance specifications (APS). RESULTS: Thirteen participants returned results for the supplementary sample series. Interference from α-tocopherol acetate was observed with results below the APS in 30 % (n=4) of laboratories for retinol quantification and in 23 % (n=3) for α-tocopherol quantification. One laboratory returned results above the APS for α-tocopherol when γ-tocopherol was present. CONCLUSIONS: This supplementary sample series has shown that the presence of vitamin E analogues can lead to the over or under estimation of nutritional status by some participants. Affected laboratories are encouraged to review their analytical procedures. To further assess laboratory competence, EQA providers should consider using patient samples or spiked challenge samples.

Harmonization of LC-MS/MS Measurements of Plasma Free Normetanephrine, Metanephrine, and 3-Methoxytyramine.

BACKGROUND: Plasma-free normetanephrine and metanephrine (metanephrines) are the recommended biomarkers for testing of pheochromocytoma and paraganglioma (PPGL). This study evaluated the status of harmonization of liquid chromatography-tandem mass spectrometry-based measurements of plasma metanephrines and methoxytyramine and clinical interpretation of test results. METHODS: 125 plasma samples from patients tested for PPGLs were analyzed in 12 laboratories. Analytical performance was also assessed from results of a proficiency-testing program. Agreement of test results from different laboratories was assessed by Passing-Bablok regression and Bland-Altman analysis. Agreement in clinical test interpretation based on laboratory specific reference intervals was also examined. RESULTS: Comparisons of analytical test results by regression analysis revealed strong correlations for normetanephrine and metanephrine (R ≥ 0.95) with mean slopes of 1.013 (range 0.975-1.078), and 1.019 (range 0.963-1.081), and intercepts of -0.584 (-53.736 to 54.790) and -3.194 (-17.152 to 5.933), respectively. The mean bias between methods was 1.2% (-11.6% to 16.0%) for metanephrine and 0.1% (-18.0% to 9.5%) for normetanephrine. Measurements of 3-methoxytyramine revealed suboptimal agreement between laboratories with biases ranging from -32.2% to 64.0%. Interrater agreement in test interpretation was >94% for metanephrine and >84% for normetanephrine; improvements in interrater agreement were observed with use of harmonized reference intervals, including age-specific cut-offs for normetanephrine. CONCLUSIONS: Analytical methods for metanephrines are well harmonized between laboratories. However, the 16% disagreement in test interpretation for normetanephrine suggests use of suboptimal method-dependent reference intervals for clinical decision-making for this metabolite. Improved analytical methods and reference interval harmonization are particularly required for 3-methoxytyramine.

Influence of isotopically labeled internal standards on quantification of serum/plasma 17α-hydroxyprogesterone (17OHP) by liquid chromatography mass spectrometry.

Objectives Our recent survey of 44 mass spectrometry laboratories across 17 countries identified variation in internal standard (IS) choice for the measurement of serum/plasma 17α-hydroxyprogesterone (17OHP) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The choice of IS may contribute to inter-method variations. This study evaluated the effect of two common isotopically labeled IS on the quantification of 17OHP by LC-MS/MS. Methods Three collaborating LC-MS/MS laboratories from Asia, Europe and Australia, who routinely measure serum 17OHP, compared two IS, (1) IsoSciences carbon-13 labeled 17OHP-[2,3,4-13C3], and (2) IsoSciences deuterated 17OHP-[2,2,4,6,6,21,21,21-2H]. This was performed as part of their routine patient runs using their respective laboratory standard operating procedure. Results The three laboratories measured 99, 89, 95 independent samples, respectively (up to 100 nmol/L) using the 13C- and 2H-labeled IS. The slopes of the Passing-Bablok regression ranged 0.98-1.00 (all 95% confidence interval [CI] estimates included the line of identity), and intercept of <0.1 nmol/L. Average percentage differences of -0.04% to -5.4% were observed between the two IS materials, which were less than the optimal bias specification of 7% determined by biological variation, indicating no clinically significant difference. The results of 12 Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) proficiency samples (1-40 nmol/L) measured by the laboratories were all within the RCPAQAP analytical performance specifications for both IS. Conclusions Overall, the comparison between the results of 13C- and 2H-labeled IS for 17OHP showed good agreement, and show no clinically significant bias when incorporated into the LC-MS/MS methods employed in the collaborating laboratories.

Pharmacokinetics of flucloxacillin during prolonged intermittent renal replacement therapy in a 76-year-old man.

Prolonged intermittent renal replacement therapy (PIRRT) use has been increasing in critically ill patients with kidney dysfunction. PIRRT can affect the pharmacokinetics of many drugs, although no data is available to guide flucloxacillin dosing in this clinical scenario. Herein, we describe the pharmacokinetics of flucloxacillin, given at 1 g every 4 h during PIRRT, in a 76-year-old, critically ill patient with a methicillin-susceptible Staphylococcus aureus (MSSA) prosthetic joint infection complicated by bacteraemia. Blood samples were taken over 2 days including during a 9-h PIRRT session. A two-compartment model was developed to describe differences in clearance of flucloxacillin during PIRRT and off-PIRRT (9.45 vs. 6.89 L/h). A flucloxacillin dose of 1 g every 4 h during PIRRT therapy appeared to attain adequate exposures for MSSA sepsis in this patient, however higher doses may be required for infection sites with poor drug penetration.

Pharmacokinetics of Benzylpenicillin (Penicillin G) during Prolonged Intermittent Renal Replacement Therapy.

Prolonged intermittent renal replacement therapy (PIRRT) is an increasingly adopted method of renal replacement in critically ill patients. Like continuous renal replacement therapy, PIRRT can alter the pharmacokinetics (PK) of many drugs. In this setting, dosing data for antibiotics like benzylpenicillin are lacking. In order to enable clinicians to prescribe benzylpenicillin safely and effectively, knowledge of the effects of PIRRT on the plasma PK of benzylpenicillin is required. Herein, we describe the PK of benzylpenicillin in 2 critically ill patients on PIRRT for the treatment of penicillin-susceptible Staphylococcus aureus bacteremia complicated by infective endocarditis. Blood samples were taken for each patient taken over dosing periods during PIRRT and off PIRRT. Two-compartment PK models described significant differences in the mean clearance of benzylpenicillin with and without PIRRT (6.61 vs. 3.04 L/h respectively). We would suggest a benzylpenicillin dose of 1,800 mg (3 million units) every 6-h during PIRRT therapy as sufficient to attain PK/pharmacodynamic target.

The challenge of improving the diagnostic yield from metanephrine testing in suspected phaeochromocytoma and paraganglioma.

Background Plasma-free metanephrines (PFM) or urinary fractionated metanephrines (UFM) are the preferred biochemical tests for the diagnosis of phaeochromocytoma and paraganglioma (PPGL). Borderline increased results should be followed up to either exclude or confirm diagnosis. Methods We extracted all PFM and UFM results reported by our laboratory over a six-month period from the laboratory information system. We categorized patients with borderline increased results according to whether follow-up testing had been performed as suggested in the initial laboratory report. Questionnaires were then sent to all requesting doctors and medical notes reviewed where available. Results Two hundred and four patients with borderline increased PFM or UFM were identified. Sixty-five (38.5%) of 169 patients with borderline increased PFM had a repeat test out of which 36 were normal and 29 did not normalize. Of 35 patients with borderline increased UFM, 17 (48.6%) had subsequent PFM measurement, out of which 15 were normal. Questionnaires were returned to 106 (52%) patients. Of these, the most frequent indication for testing was hypertension ( n = 50); 15 patients had an incidental adrenal mass and two of these patients were diagnosed with a phaeochromocytoma. Conclusion Only 38% of patients with borderline increased PFM had a repeat PFM measurement. This was not significantly higher when compared with the 28% in a previous audit that we reported in 2010 ( P = 0.10). Forty-nine per cent of patients with a borderline increased UFM had a repeat UFM or PFM measurement. There remains a substantial possibility of missed detection of PPGL.

Achievements and Future Directions of the APFCB Mass Spectrometry Harmonisation Project on Serum Testosterone.

As an outcome of the 2010 Asian Pacific Conference for Chromatography and Mass Spectrometry in Hong Kong, a collaborative working group was formed to promote the harmonisation of mass spectrometry methods. The Mass Spectrometry Harmonisation Working Group resides under the combined auspices of the Asia-Pacific Federation for Clinical Biochemistry and Laboratory Medicine (APFCB) and the Australasian Association of Clinical Biochemists (AACB). A decision was made to initially focus attention on serum steroids due to the common interest of members in this area; with the first steroid to assess being testosterone. In principle, full standardisation with traceability should be achievable for all steroids as they are small compounds with defined molecular weight and structure. In order to achieve this we need certified reference materials, reference methods, reference laboratories, reference intervals and external quality assurance programs; each being an important pillar in the process. When all the pillars are present, such as for serum testosterone, it is feasible to fully standardise the liquid chromatography - tandem mass spectrometry (LC-MS/MS) methods. In a collaborative process with interested stakeholders, we commenced on a pathway to provide ongoing assessment and seek opportunities for improvement in the LC-MS/MS methods for serum steroids. Here we discuss the outcomes to date and major challenges related to the accurate measurement of serum steroids with a focus on serum testosterone.

Laboratory medicine best practice guideline: vitamins a, e and the carotenoids in blood.

Despite apparent method similarities between laboratories there appear to be confounding factors inhibiting uniform reporting and standardisation of vitamin assays. The Australasian Association of Clinical Biochemists (AACB) Vitamins Working Party, in conjunction with The Royal College of Pathologists of Australasia Quality Assurance Programs, has formulated a guideline to improve performance, reproducibility and accuracy of fat-soluble vitamin results. The aim of the guideline is to identify critical pre-analytical, analytical and post-analytical components of the analysis of vitamins A, E and carotenoids in blood to promote best practice and harmonisation. This best practice guideline has been developed with reference to the Centers for Disease Control and Prevention (CDC) "Laboratory Medicine Best Practices: Developing an Evidence-Based Review and Evaluation Process". The CDC document cites an evaluation framework for generating best practice recommendations that are specific to laboratory medicine. These 50 recommendations proposed herein, were generated from a comprehensive literature search and the extensive combined experience of the AACB Vitamins Working Party members. They were formulated based on comparison between an impact assessment rating and strength of evidence and were classified as either: (1) strongly recommend, (2) recommend, (3) no recommendation for or against, or (4) recommend against. These best practice recommendations represent the consensus views, in association with peer reviewed evidence of the AACB Vitamins Working Party, towards best practice for the collection, analysis and interpretation of vitamins A, E and carotenoids in blood.

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, ß-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.

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.

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.

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.

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.