Metanephrine Testing Why, How and When?

Transcription

1 Metanephrine Testing Why, How and When? Gerald Woollard & Malcolm Whiting On behalf of the Working Party on Biogenic Amines SRAC Symposium 16 rd September 2015 Olympic Park Sydney Disclaimers GW & MW members of WPBA GW part funded by AACB and Auckland City Hospital

2 Members of RCPAQAP Working Party on Biogenic Amines (WPBA) Past Chairs John Earl (Westmead Children s hospital, Sydney) Dilo Pillai (Prince of Wales hospital, Sydney) Malcolm Whiting (Flinders Medical Centre, Adelaide) Current membership Gerald Woollard (Incoming Chair, Auckland City Hospital, NZ) Malcolm Whiting (Immediate Past Chair, Adelaide) Brett McWhinney (Brisbane) Kirsten Hoad (Perth) Andrew Ellis (Melbourne) Talia Novos (Sydney) Trisha Anderson (ASE) Sabrina Koestier (RCPAQAP) Members credentials All have longstanding career involvements with biogenic amines Are attached to laboratories performing these tests Have proven expertise in neuroendocrine tumours (NETs) testing and service provision Are involved with method design and development Individually and collectively present and publish their work in various formats

3 Primary role of WPBA Provision of quality management material for the diagnosis of NETs in clinical laboratories Have maintained urinary program since inception early 1990 s (now on cycle 47) Successfully established the plasma metanephrine program for 8 years (now on cycle 15) Linear sets of spiked urine and plasma specimens are supplied by Australian Scientific Enterprises (ASE) Additionally supply of actual patient urine specimens. Deliberately chosen to represent real clinical situations and to test the participant s ability to recognise or cope with analytical situations such as drug or nutritional effects. ASE manager Trisha Anderson is a member of the working party and manufacturers EQA material Direct interaction with RCPAQAP via Sabrina Koestier

4 Secondary roles of WPBA Expert support group for the RCPAQAP EQA program Advisory role to RCPAQAP participants Response to enquiries and concerns from participants on analytical and technical issues Clinical comments for the patient samples. Increasing awareness of the importance of these comments as a learning tool for participants. Comments are being made more informative with extra dialogue especially with regard to unusual aspects of the cases. In urine program only at present Emerging roles outside of the provision of EQA material Promotion of best practice guidelines Harmonized reference ranges via AACB Multi-centre group representing most Australasian biogenic amine labs Anticipated visibility within PRESSOR

5 RCPAQAP biogenic amine program Urine program Catecholamines Noradrenaline Adrenaline Dopamine Metanephrines Normetanephrine Metanephrine 3-methoxytyramine Acidic metabolites HMMA HVA Carcinoid markers Serotonin 5-HIAA Plasma program Plasma metanephrines Normetanephrine Metanephrine 3-methoxytyramine

6 Phaeochromocytoma and Paraganglioma PPGLs Phaeochromocytoma rise from the adrenal medullary chromaffin cells Paraganglioma arise from extra-adrenal chromaffin cells Clinical features secondary to tumoural secretion of catecholamines Phaeochromocytoma in adrenal gland (80-85%) Half produce mixture of noradrenaline and adrenaline Other half produce noradrenaline exclusively Paraganglioma in thorax, abdomen and pelvis (10-15%) Solely produce noradrenaline Rarely produce adrenaline Some also produce dopamine (with or without noradrenaline) Very rarely negligible amounts of any catecholamine Parasympathetic paraganglioma in head and neck (rare) Usually endocrinologically inactive

7 Important laboratory considerations for PPGLs Preferred analyte (decision already clearly made as metanephrines) Catecholamines themselves (active) Metabolites such as metanephrines (inactive) Analytes are mostly conjugated (total, conjugated, free) Preferred specimen (decision not so clear-cut) Urine metanephrines (UMets) Plasma metanephrines (PMets) Preanalytical considerations (critical aspect affecting diagnostic performance) Patient preparation (posture, stress, medications) Sample stability Methodology (still a developing aspect) Older colorimetric assays are obsolete Immunoassays under-estimate the catecholamines LC-ECD is sensitive enough for UMets or PMets LC-MSMS is the preferred method Reference ranges Confounded by SNS activity Age dependency

8 Major contemporary events in PPGL testing in clinical labs What is changing? Advent of Best Practice by Endocrine Society (Lenders JCEM 2014) Previously there were recommendations only Increasing acceptance of mass spectrometry within clinical laboratories LCMSMS is far more expensive than LC-ECD and harder to operate LCMSMS analytical performance for metanephrines is superior LC-MSMS is much more versatile and adaptable for other tests Better understanding of the pathophysiology of catecholamines (Eisenhofer 2014) Catecholamines are responsible for the clinical symptoms and were preferred analyte in the past Metanephrines have better diagnostic performance The reason for this is the regional distribution of COMT and the confounding effect of SNS noradrenaline

9 Best Practice Guidelines (JCEM 2014) SUMMARY OF RECOMMENDATIONS 1.1 We recommend that initial biochemical testing for PPGLs should include measurements of plasma free metanephrines or urinary fractionated metanephrines 1.2 We suggest using liquid chromatography with mass spectrometry or electrochemical detection methods rather than other laboratory methods to establish biochemical diagnosis of PPGLs 1.3 For measurements of plasma metanephrines, we suggest drawing blood with the patient in the supine position and use of reference intervals established in the same position 1.4 We recommend that all patients with positive test results should receive appropriate follow-up according to the extent of increased values and clinical presentation Lenders JWM, Duh Q-Y, Eisenhofer G et al Pheochromocytoma and Paraganglioma: An Endocrine Society Clinical Practice Guideline The Journal of Clinical Endocrinology & Metabolism Volume 99(6), June 2014, p

10 Plasma metanephrines (PMets) or plasma catecholamines (PCats) Marked the demise of the catecholamine test for PPGLs NIH group (JAMA 2002) compared 214 PPGLs for PMets PCats UMets & UCats Marked the turning point for the selection PPGL biomarker selection Since then 14 other studies have agreed 1 st International Symposium on Phaeochromocytoma (Nature 2005) recommended against the use of PCats or UCats

11 RCPAQAP program still has participants who only offer UCats 39 labs report UCats 22 labs report UMets 21 labs report UCats and UMets 2 labs report UMets only (no UCats) 18 labs report UCats only Unlikely they use another EQA for UMets only Possible some labs only screen neuroblastoma patients PCats not offered

12 Best Practice recommends UMets or PMets by LC-ECD and LC-MSMS All methods/matrix require extensive SPE LC-ECD detects by sensitive oxidation but demands chromatographic separation LC-MSMS can separate by m/z with or without chromatographic resolution LC-MSMS can achieve better sensitivity with less risk of interference UMets by LC-ECD PMets by LC-MSMS

13 Best Practice still recommends deconjugated fractionated UMets Deconjugated is the sum total of each metanephrine (conjugated and free) obtained by hydrolysis Fractionated refers to normetanephrine, metanephrine (and 3-MT) being chromatographically separated & individually quantitated Five studies show the diagnostic performances of UMets and PMets are comparable (Lenders 2014) RCPAQAP intends to continue with UMets EQA Table 5. Comparison of Diagnostic Performance of Plasma Free Versus Urinary Fractionated Metanephrines from 5 Available Studies First Author, Year (Ref.) Sensitivity Specificity Plasma Urine Plasma Urine Lenders, 2002 (39) 98.6% (211/214) 97.1% (102/105) 89.3% (575/644) 68.6% (310/452) Unger, 2006 (42) 95.8% (23/24) 93.3% (14/15) 79.4% (54/68) 75.0% (39/52) Hickman, 2009 (46) a 100.0% (14/14) 85.7% (12/14) 97.6% (40/41) 95.1% (39/41) Grouzmann, 2010 (48) 95.7% (44/46) 95.0% (38/40) 89.5% (102/114) 86.4% (121/140) Unger, 2012 (53) 89.5% (17/19) 92.9% (13/14) 90.0% (54/60) 77.6% (38/49) Hickman 2009

14 PMets by LC-MSMS (findings from WPBA survey) Presented yesterday by Malcolm Whiting SUMMARY OF FINDINGS Across Australasia, a variety of LC-MSMS instruments and methods are being used to measure plasma free metanephrines, with two distinct modes of chromatography (HILIC & RPLC) RPLC provides superior chromatographic separation of the 3 metanephrines to HILIC Any gain in sensitivity provided by HILIC is dependent on the MS instrument Implications for method development and instrument selection EQA data suggest that all the different methods produce results that are usually within allowable limits of performance Fit for purpose

15 Consideration of the familiar catecholamine metabolic pathway does not reveal the preferred analyte

16 Eisenhofer Clin Chem 2014 Shows relative production flux of catecholamines and metanephrines into circulation Basal metabolism of cats to mets occurs continuously within chromaffin cells as a result of seepage from the granules This occurs largely independent of moment by moment catecholamine release by SNS Contribution of SNS noradrenaline clouds that from adrenal chromaffin production COMT which produces mets is mostly found in adrenal chromaffin cells and some extra-neuronal cells PNMT is found in adrenal chromaffin cells which produce adrenaline is less affected by SNS activity MAO found in nerve cells and extra-neuronal cells but not in adrenal chromaffin cells. Hence DHPG, MHPG and HMMA unusable for PPGL diagnosis

17 Best practice states fasted-supine patient sampling Many labs accept seated collections Any added SNS activity due to posture, physiological or psychological stress confounds PMets interpretation False positivity due to seated patients (Darr Clin Endo 2013) Prospective Mono-amine Tumour Study 129 PPGLs (67 seated, 62 supine) 633 non-ppgl Multicentre 2 fasting-supine 4 nonfasting-seated labs Seated had higher 97.5 percentiles (63% NMN, 20% MN, 133% 3MT) Seated decreased sensitivity (85% vs 98%) with RR from supine Conversely decreased specificity (71% vs 95%) with supine patients against seated RR Seated vs supine RR 5.7-fold increase false positive PMets UMets

18 Best practice requires age adjustment Most labs have fixed PMets ranges Plasma Mets Upper diagram supine non-ppgl patients Levels increase with age for normetanephrine (dotted line shows age independent median) Metanephrine show shallow age dependence (not shown) Age related ranges decreases false positives Lower diagram PPGL PMets age independent Eisenhofer ACB 2013 Urine Mets 24h urine outputs higher in males Gender and age-dependency requires age partitioning Especially important in children who require multiple age brackets 24h collections age dependent increases Spot urines easier to collect have age dependent decreases against creatinine (reflecting muscle mass)

19 PMets reference ranges are problematic Metanephrine upper limits should be established to ensure optimum diagnostic sensitivity with specificity as secondary consideration Excessive false positivity is expensive for follow-up and is undesirable for patients Development of clinical complacency Normetanephrine will be affected more often than metanephrine Reference ranges should be adjusted for patient age Samples should be taken supine rather than seated Only endocrinology clinics could reasonably adhere to these collection requirements Patient samples accepted from other centres will probably be seated Normal result usually excludes tumour PMets or UMets 3-4 times raised confirms PPGL likelihood Borderline-positive results need re-sampling with strict recumbence and overnight fasting with possible medication withdrawal 3-methoxytyramine is sometimes overlooked by labs Some PPGLs produce dopamine Dietary effect from ubiquitous L-dopa in foods requires fasting specimens Metanephrines are affected by common medications Coffee, tricyclics antidepressants, antihypertensives etc Medication withdrawal not usually sought unless PMets positive

20 How is WPBA responding to these developments and the changing situation WPBA sees itself as an advocate of Best Practice because it is in a unique position to influence the testing of biogenic amines nationally WPBA surveyed RCPAQAP EQA participants regarding their current practices (WPBA presentation yesterday) WPBA is aware of the effects and limitations of the various methodologies in SPE, chromatography and EC and MS detection WPBA should have communications with AACB Harmonization group with regard to Common reference intervals Inclusion of sampling posture and age partition WPBA should promote uniformity in interpretative comments WPBA could get involved with establishing a PMets CRM in whatever capacity WPBA regards prospective involvement with ISP2017 to be held in Sydney will be beneficial WPBA represents most central labs and is already an established multi-centre group WPBA should promote itself as an Australasian point of reference for PPGL analysis