[Show abstract][Hide abstract] ABSTRACT: Introduction
In 2010 the Mass Spectrometry Harmonisation Working Group was established to progress the harmonisation and standardisation of mass spectrometry based clinical laboratory methods. The first project is to pursue the harmonisation of the measurement of serum steroids, with serum testosterone as the key analyte. Here we describe the outcome of the first two project goals: 1. provide detailed information on the serum testosterone liquid chromatography tandem mass spectrometry (LC-MS/MS) methods used in Asian-Pacific clinical biochemistry laboratories; and 2. standardise serum testosterone results through the use of a common calibrator.
Materials and Methods
All laboratories participated in the RCPA Quality Assurance Programs (QAP) for serum testosterone. In 2013 a detailed questionnaire was sent to participating laboratories in conjunction with a seven level common calibrator set (from Biocrates), external quality assurance set (from RCPAQAP) and two de-identified human serum samples. Samples were analysed by each laboratory’s (n=8) routine LC-MS/MS method on two separate occasions. In addition, gas chromatography MS analysis was performed by one of these laboratories. Target values were assigned by the National Measurement Institute of Australia (NMIA). ANOVA was performed to determine if recalculation led to a change in results.
The questionnaire demonstrated that most laboratories prepared samples by liquid-liquid extraction and one used solid phase extraction, with consistent MRM’s for the testosterone quantifier/qualifier ions. The source of the laboratory’s calibrator, number of calibration levels and deuterated sites on the internal standard differed. Recalculation of the human testosterone samples against the common calibrator demonstrated a significant change (p<0.05) in results for the male serum (NMIA assigned value 16.19 nmol/L) whereas the change for the female serum (NMIA assigned value 0.57 nmol/L) was not statistically significant for the group.
This is the first such report to fully characterise the LC-MS/MS methods for serum testosterone in common use by clinical diagnostic laboratories. In addition, to characterising the LC-MSMS methods, this pilot study demonstrates the utility of the Biocrates material as a common calibrator and offers 16 initial recommendations to standardise testosterone LC-MS/MS methods. The assignment of reference values to the common calibrator by the reference measurement procedure provides a mechanism to establish traceability. Reference values for future calibrator batches will be made traceable to SI units. Whilst, the common calibrator has achieved a change in results this change is not consistent for all laboratories. Hence further harmonisation of the analytical method through, at least in part, the implementation of the proposed recommendations is supported. This is required before common reference intervals can be recommended for testosterone. Continued monitoring, will be achieved through collaboration and participation in the RCPAQAP Endocrine program.
[Show abstract][Hide abstract] ABSTRACT: Background. In 2010 the Mass Spectrometry Harmonisation Working Group was established under the auspices of the Asian and Pacific Federation of Clinical Biochemistry (APFCB) to progress the harmonisation of mass spectrometry based clinical laboratory methods. The first project is to pursue the harmonisation of the measurement of serum steroids, with serum testosterone as the key analyte. Here we describe the outcome of the first two project goals: 1. provide detailed information on the serum testosterone LC-MSMS methods used in Asian-Pacific clinical biochemistry laboratories; and 2. harmonise serum testosterone results through a common calibrator.
Methods. All laboratories participated in the RCPA Quality Assurance Programs (QAP) for serum testosterone. In 2013 a detailed questionnaire was sent to participating laboratories in conjunction with a seven level common calibrator set (Biocrates), RCPAQAP material and two de-identified human serum samples. Samples were analysed by each laboratory’s (n=8) routine LC-MSMS method on two separate occasions. Target values were assigned by the National Measurement Institute of Australia (NMIA). ANOVA was performed to determine if recalculation led to a change in results.
Results. The questionnaire demonstrated significant commonality in the LC-MSMS methods. Most laboratories prepared samples by LLE and one used SPE. Consistent MRM’s were seen (289→109 and 289→97) for the testosterone quantifier/qualifier. The source of the laboratory’s calibrator, number of levels and deuterated sites on the internal standard differed. Recalculation of the human testosterone samples against the common calibrator demonstrated a significant change (p<0.05) in results for the male serum (NMIA assigned value 16.19 nmol/L) whereas the change for the female serum (NMIA assigned value 0.57 nmol/L) was not statistically significant for the group.
Conclusions. This pilot demonstrates the utility of the Biocrates material as a common calibrator. Reference values for future calibrator batches will be made traceable to SI units. To ensure alignment with other harmonisation initiatives NIST SRM971 will be used as a QC material. Continued monitoring, and on-going method improvements will be achieved through collaboration and participation in the RCPAQAP.
[Show abstract][Hide abstract] ABSTRACT: 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 Program 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.
The Clinical biochemist. Reviews / Australian Association of Clinical Biochemists 05/2014; 35(2).
[Show abstract][Hide abstract] ABSTRACT: CALIBRATORS FOR HUMAN SERUM β-CAROTENE MADE FROM BOVINE SERUM
GA Woollard1,2, A Hammer-Plecas1, KE Hoad2, TA Walmesley2, S Briscoe2, LA Johnson2, , T Harrower2, S Koetsier3, JP Gill3, RF Greaves2
1Department of Specialist Chemical Pathology, Lab Plus, Auckland City Hospital, New Zealand, 2AACB Vitamins Working Party, 3RCPA Quality Assurance Programs, Adelaide, Australia (RCPAQAP)
Beta-carotene is the major dietary carotenoid and also the most important pro-vitamin A precursor. It is measured in serum to demonstrate dietary repletion especially during potential fat malabsorption. RCPAQAP offers a program to measure β-carotene. NIST 968e calibrators have three β-carotene levels up to 0.7μmol/L which covers the lower half of the levels expected in humans (approximate range 0.2-1.5μmol/L). A single level commercial Chromsystems calibrator is available with metrological alignment to this NIST. Multi-level calibrators covering the expected human β-carotene range are inaccessible.
Multi-level calibration set was derived by mixing of human serum with proportions of bovine serum from pasture fed cattle with much higher β-carotene than that achievable by humans. The bovine serum β-carotene concentration was determined by quantitative extraction into hexane and absorbance measurement using its known extinction coefficient. The contribution from minor carotenoids was determined by HPLC and corrections made to the total.
Beta-carotene cannot be added directly to serum because it does not incorporate into lipoproteins. Pasture fed cattle can have β-carotene up to 50µmol/L. They also have much lower levels of the other carotenoids which contribute <10% of the total. The proportion of bovine serum added to human serum is usually <10% because of its very high β-carotene content. Five-point calibration curves are linear up to at least 5.0μmol/L and invariably show coefficients of determination >0.995 in routine service. NIST traceable Chromsystems commercial controls were found at 0.544 ± 0.044μmol/L (Lot #3310, NIST 968d, n=60) and 1.136 ± 0.0.098μmol/L (Lot #3011, NIST 968e, n=30) which is within their stated target limits. Imprecision at 0.267μmol/L near the lower end of reference range is 11.1% (n=46).
By mixing proportions of bovine serum with human serum a wide range of β-carotene levels can be achieved to construct single or multi-level matrix matched calibrations.
[Show abstract][Hide abstract] ABSTRACT: 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).
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: RCPA QAP TARGET SETTING WITH NIST MATERIAL: PERFORMANCE WITH RETINOL, α-TOCOPHEROL AND β-CAROTENE.
Ronda F Greaves, KE Hoad, GA Woollard, TA Walmsley, S Briscoe, LA Johnson, WD Carter, JP Gill.
The AACB Vitamins Working Party
Since 1999, the RCPA Quality Assurance Program (QAP) has conducted an assessment of analytical performance for retinol, α-tocopherol and β-carotene. Previous target setting exercises have been carried out with National Institute of Standards and Technology (NIST) standard reference material (SRM) 968c, provided at two levels; with targets successfully set for retinol but not α-tocopherol or β-carotene. In 2009 NIST released a new, single level, SRM 968d. Here we describe the outcome of the 2010 target setting exercise with NIST SRM 968d.
Laboratories participating in this QAP utilise reverse phase HPLC with spectrophotometric detection. Target setting is conducted using a standardized procedure where selected laboratories are supplied with material for the forthcoming cycle along with the SRM, in this case NIST SRM 968d. The QAP target values are set using the SRM as calibrator.
Assigned values for NIST SRM 968d are: total retinol 1.09±0.17μmol/L; α-tocopherol 13.77±1.28μmol/L; β-carotene 0.145±0.013μmol/L. The linear regression analysis of the NIST SRM 968d target values against all results returned by laboratories in cycle 22 of the RCPA Vitamin QAP are: retinol y=1.0032x–0.0099, r2=0.9987; α-tocopherol y=0.8020x+1.5772, r2=0.9964; β-carotene y=0.8031x–0.1065, r2=0.9914.
Target setting was successful for retinol, but unsuccessful for α-tocopherol and β-carotene. The concentration of β-carotene in NIST SRM 968d is too low. α-Tocopherol is assessed in the NIST material by HPLC with fluorometric detection, which is different to the detection method of QAP participants. Furthermore, the allowable error reported by NIST (≈10%) approximates the total allowable limits for the QAP.
NIST SRM 968d is not usable as a primary calibrator for α-tocopherol and β-carotene.
Australasian Association of Clinical Biochemists' 48th Annual Scientific Conference, Perth; 10/2010
[Show abstract][Hide abstract] ABSTRACT: 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.
A detailed questionnaire covering the major aspects of serum vitamin A and E analysis was sent to all participating laboratories in 2007.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
Clinica chimica acta; international journal of clinical chemistry 02/2009; 403(1-2):136-8. DOI:10.1016/j.cca.2009.02.002 · 2.82 Impact Factor