Vitamin D A game changer for Clinical Laboratories
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1 Vitamin D A game changer for Clinical Laboratories Michael Wright Clinical Chemistry and Endocrinology Department, SEALS Prince of Wales Hospital Sydney, Australia
2 What is 25OH Vitamin D? 25OH Vitamin D (25OH D) is the most abundant vitamin D metabolite in circulation (nmol/l range) Due to 25OH Vitamin D s longer half-life and the fact that it s hydroxylation step in the liver is not regulated, it is seen as the best measure of Vitamin D status
3 Why would you measure Vitamin D status? Calcium Regulation Immune system Pancreatic Cancer Auto-immune disease Cardiovascular Disease Deficiency is known/ suspected to be involved with Rickets Osteomalacia Osteoporosis Schizophrenia Depression Bone Metastases Diabetes Influenza Breast Cancer Colon Cancer Ovarian Cancer Hypertension Allergy Asthma SLE Rheumatoid Arthritis
4 25OH Vit D requests
5 So how do we measure it in a Clinical Chemistry Laboratory? Weekly workload 10 samples Solvent Extraction Chromatography RIA 100 samples Solvent Extraction Chromatography RIA 1000 samples SolventAutomated Chromatography Extraction Chemiluminescence immunoassay RIA
6 So what is the problem? Patient X 25OH Vitamin D Serum sample taken Monday 25OH Vitamin D = 21nmol/L Repeated the Monday Sample 25OH Vitamin D = 38nmol/L Serum sample taken Thursday Repeated the Thursday Sample 25OH Vitamin D = 53nmol/L 25OH Vitamin D = 35nmol/L 25OH Vitamin D = 48nmol/L 25OH Vitamin D = 27nmol/L 25OH Vitamin D = 44nmol/L So what is the result? Somewhere between 21 and 53!
7 Imprecision Quality Assurance Schemes College of American Pathologists Survey (2008) Chemiluminescence Immunoassays: nmol/L Vitamin D External Quality Assessment Scheme (DEQAS 2014) Number of participating laboratories: 997 Number of different methods: 25 (plus 6 unknown) ALTM = 114.9nmol/L %CV = 16.4% Range = 39 to 176nmol/L!
8 LC-MS/MS and 25OH Vitamin D The introduction of LC-MS/MS represents a hugely important development in the evolution of 25-OH- D testing, as it has for many other clinical analytes. It is a welcome antidote to the onward march of ever-simpler methods that have sacrificed analytical rigor on the altar of expediency. Graham Carter Clinical Chemistry 55: (2009)
9 Liquid Chromatography Triple quadrupole tandem mass spectrometry
10 How we measure 25OH Vitamin D at Prince of Wales Protein PPT 100µL Serum d6-internal Standard 25µL 0.2M ZnSO 4 200µL Methanol Manually process a plate of Calibrators, QC and 84 patient samples in 40min
11 Automated LC-MS/MS Workflow 30min to prepare 96 samples. plus automatic batch upload to the LC-MS/MS Integrating LC-MS into a Clinical Laboratory Hands free data transfer from sample handling to result entry Renee Sahertian etal AACB Conference 2015
12 Online Solid Phase Extraction 2 position divert valve Strata C8 20x2.1mm 20µm On-line SPE cartridge Ascentis Express F5 100x3.0mm 2.7µm Analytical column Pump(s) 1 Pump(s) 2 Auto-sampler Column Oven Mass Spectrometer WASTE
13 Some common issues with LC-MS/MS Suitable for infant samples? Harmonisation/Traceability of calibration
14 Infant Samples A 1.9e5 Intensity, cps 25OHD 3 & 3epi- 25OHD Ascentis Express 50 x 2.1mm 2.7µm C8 B 2.3e4 Intensity, cps Time, min 3epi- 25OHD 3 3-epi 25OHD OHD 3 25OHD Time, min Si O Si Ascentis Express 100 x 2.1mm 2.7µm F5 F F F F F Wright MJ, Halsall DJH and Keevil BG. Clin Chem 2012; v. 58, p
15 Harmonisation of calibrators - NIST Reference Sera
16 Standardisation - VDSP from Centers for Disease Control (CDC)
17 Have the immunoassays improved? VDSP 13x LC-MS/MS laboratories, 11 involved in diagnostic or research testing 2 vendors 6x Immunoassay laboratories All are vendors Siemens, IDS (2), DiaSorin, Roche,Abbott So if I use one of these immunoassays I will be compliant with the VDSP? No.
18 Review of the Medicare Benefits Schedule (a) has signs or symptoms of osteoporosis or osteomalacia; or (b) has increased alkaline phosphatase and otherwise normal liver function tests; or (c) has hyperparathyroidism, hypo- or hypercalcaemia, or hypophosphataemia; or (d) is suffering from malabsorption (for example, because the patient has cystic fibrosis, short bowel syndrome, inflammatory bowel disease or untreated coeliac disease, or has had bariatric surgery); or (e) has deeply pigmented skin, or chronic and severe lack of sun exposure for cultural, medical, occupational or residential reasons; or (f) is taking medication known to decrease 25OH-D levels (for example, anticonvulsants); or (g) has chronic renal failure or is a renal transplant recipient; or (h) is less than 16 years of age and has signs or symptoms of rickets; or (i) is an infant whose mother has established vitamin D deficiency; or (j) is a exclusively breastfed baby and has at least one other risk factor mentioned in a paragraph in this item; or (k) has a sibling who is less than 16 years of age and has vitamin D deficiency
19 Is there any reason to continue using LC-MS? 1) Confirmation testing 2) Research studies 3) Cost
20 You don t need an expensive top-end LC-MS/MS SCIEX API3200 was released in 2005 as a cheaper alternative to the API ,000 samples/year Service cost = $0.54/sample Consumable cost = $1.63/sample Staff time 0.6FTE vs 0.4FTE
21 Future work Measurement of VDBG VDR investigation Free/bioavailable metabolite indexes Dihydroxyl forms 1,25 (OH)2 Vitamin D3/2 24,25 (OH)2 Vitamin D3/2
22 Acknowledgments Prince of Wales Hospital Lawrence Ward Renee Sahertian XuGuang Han Kirsty Whelan Talia Novos Meikei Chan Ah Sim Craig Campbell Vicki Spoulos Paul Stathakis Ben Lu Chris White A.Rita Horvath Sigma-Aldrich/Supelco Craig Aurand Dave Bell SCIEX Chris Hodgkins Brad Patterson
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