Cortisol Assays. The Good, The Bad and The Indifferent. David Ducroq. Cardiff and Vale ulhb WEQAS. Unit 6, Parc Tŷ Glas. Llanishen.

Transcription

1 Cortisol Assays The Good, The Bad and The Indifferent David Ducroq Cardiff and Vale ulhb WEQAS Unit 6, Parc Tŷ Glas Llanishen Cardiff

2 Summary Brief overview of current methods and challenges involved Traceabilty of Cortisol methods Specific studies that have involved the Reference Lab and method comparisons Perfomance of cortisol methods within the Weqas Endocrine program

3 Current Cortisol Methods Immunoassay Most common method in routine use, relatively fast results, prone to interference, variety of assay conformations Tandem MS (LC-MS-MS) Gold standard method (?), becoming used more in routine labs (specialist use, generally urine), potential for standardisation issues, Reference methodology ID-GCMS Gold standard method historically, laborious, not suitable for routine use, Reference methodology HPLC (uv detection) Non-specific method, not routinely used

4 Cortisol Immunoassay 'The antiserum is the most important ingredient in immunoassay. Its specificity and affinity determine not only the specificity and sensitivity of the assay, but also the practicability of many methods' (Pratt, 1978)

5 Immunoassay Issues Cross Reactivity Cortisol Bridge linkage site for immunogen Steroid numbering 11-Deoxycortisol 21-Hydroxycortisol Cortisone

6 Immunoassay Issues Method Setup Differences Label type (homologous v heterologous) Chemilumincesent Enzyme Fluorescent Radioactive Incubation period Modern immunoassays use short incubation times disequilibrium assay Releasing agent (Steroids: Danzol; ph) Standardisation

7 Traceability

8 Primary Calibrator SI Unit Material Procedure Responsibility Secondary Calibrator Primary reference procedure/definitive method Secondary Reference Procedure Manufacturer selected Procedure BIPM, NML, ARML NML, ARML ML Manufacturers Working Calibrator X Manufacturers Standing Procedure End-user s routine procedure ML ML, End user Weqas Ref Lab involvement Routine sample Result Adapted from ISO 17511

9 Clin Chem 55:6 (2009) Cortisol Traceability

10 Cortisol Reference Methods JCTLM Listed Cortisol Reference Methods JCTLM Listed Cortisol Reference Measurement Services

11 Cortisol by Mass Spec LC-MS-MS ID-GCMS (Reference Methods)

12 Standardisation Issues have been observed with the spread of EQA results for testosterone which may be due to standardisation issues across labs Testosterone targeted standards suitable for use in Tandem-MS methods Gravimetric preparation of serum standards Traceable targets assigned using the Weqas JCTLM listed reference measurement service Cortisol standards will be available shortly

13 Testosterone EQA Data RCPA QAP Data courtesy of RCPA QAP

14 Cortisol Studies

15 ACTH Stimulation Test assay comparison Collaboration with Medical Biochemistry, University Hospital Wales

16 Short-Synacthen Test Volunteers recruited at University Hospital Wales: 60 male, 105 female ACTH stimulation tests were carried out in the morning between 0830 and 1130 h. Blood was collected at baseline and 30 min into plain tubes Weqas Reference Lab analysed all samples using the ID-GCMS Reference Method Five different automated immunoassays used to analyse samples

17 Baseline Cortisol No significant gender difference for ID-GCMS results Mean cortisol for all immunoassays was higher than the ID- GCMS value Gender difference observed for all immunoassays Male higher than female Females on OCP gave higher mean cortisol value for all methods

18 ACTH Stimulation Test Correlation with ID-GCMS (a) Centaur (b) Architect (e) Access (c) E170 (d) Immulite (2000) Need method specific cut point for ACTH stimulation test _ line of best fit, males line of best fit, non-ocp females line of best fit, OCP females

19 ACTH Stimulation Test Bias Plot (a) Centaur (b) Architect (e) Access (c) E170 (d) Immulite (2000) Male subjects Non-oral contraceptive pill (OCP) female subjects x, OCP-female subjects.

20 Method Specific Reference Limits for Post ACTH Stimulation Test Reference Limits determined by back transformation of the 25th percentile value (mean 1.97*SD) of the log-transformed data. Recent discussion on ACB Mailbase regarding reference limit in light of the change of Roche II method change

21 Collaboration with Medical Biochemistry, University Hospital Wales

22 Retained samples (70) from routine analysis assayed using ID-GCMS method: Pregnant (2 nd Trimester Downs Screening) ICU (albumin <20g/L; 10 male, 10 female) Renal (creatinine >300 for at least 3 months) Five spiked samples from both male and female serum pool

23 Assay Bias: Spiked Patient Pools Male Female

24 Serum Matrix Roche E170 Abbott Architect Centaur Beckman Access

25 Roche E170 Abbott Architect Centaur Beckman Access

26 EQA Data

27 Weqas Endocrine EQA Scheme Design Single unadulterated donors where possible Reference Target Assignment for available methods Multiple samples in single distribution covering analytical range Checks for: Linearity Bias Systematic errors

28 Steroid Reference Method Traceability Measurand Standard (certified purity) Control material Cortisol NIST 921 DA 192, 193 Progesterone Testosterone NMIJ CRM 6003a M914 (NARL) >99% DA 347, 348 In house * * Moving shortly to NIST971

29 Weqas Cortisol Reference Method Gravimetric Weighing of samples/calibrators Addition of d3-cortisol. Exact matching isotope dilution Solvent extraction (dichloromethane) LH20 gel chromatography Sample derivatisation (heptafluorobutyric acid anhydride) Reconstitution in cyclohexane GCMS analysis. Monitor m/z 489/491

30 Cortisol EQA data comparison All Returns compared to JCTLM listed ID-GCMS Reference Method Data available from May September 2015 Single donor samples where available (male and female) Additional pools with spiked cortisol

31 Cortisol (nmol/l) Cortisol Returns Correlation with ID-GCMS June September Cortisol Correlation with ID-GCMS Overall 400 Access/DxI Elecsys/E Module Advia Centaur 200 Architect 100 Equivalence Linear (+/- 2SD) Cortisol (nmol/l; Reference Method

32 % Bias Cortisol Returns Bias Plot June September Cortisol Bias Plot Overall Mean Value Access/DxI 800 Elecsys/E Module Advia Centaur Architect Cortisol (nmol/l; Reference Method

33 % Bias Cortisol EQA Returns August 2011 Distribution Access/DxI Elecsys 25 Advia 20 Architect Female donor -20 Cortisol (ID-GCMS Reference Target, nmol/l)

34 % Bias 30 Access/DxI Cortisol (nmol/l; Reference Method) Female Male Spike (M/F)

35 % Bias Elecsys/E Module Cortisol (nmol/l; Reference Method) Female Male Spike (M/F)

36 % Bias 30 Advia Centaur Cortisol (nmol/l; Reference Method) Female Male Spike (M/F)

37 % Bias 30 Architect Cortisol (nmol/l; Reference Method) Female Male Spike (M/F)

38 Cortisol (nmol/l) % Bias Distribution S219 closed 17/9/15 Correlation with ID-GCMS Bias Plot Cortisol Correlation with ID-GCMS S219 Elecsys Elecsys Advia Cortisol Bias Plot S Advia ID-GCMS Traceable ID-GCMS Traceable Cortisol (nmol/l; Reference Method) -30 Cortisol (nmol/l; Reference Method) Female sample on OCP?

39 Cortisol (nmol/l) % Bias S219 Correlation with ID-GCMS Bias Plot 600 Beckman ID-GCMS Traceable S Beckman ID-GCMS Traceable Bias Plot S Access DX Access DX Cortisol (nmol/l; Reference Method) -40 Cortisol (nmol/l; Reference Method)

40 % Bias Roche & Roche II Cortisol Methods 40 Elecsys/E Module - S219 M/F spiked sample Cortisol (nmol/l; Reference Method) Elecsys/E Module Roche II

41 In Summary A potential trend towards Tandem MS exists for specialised labs Correct standardisation required for Tandem MS methods Method specific cut points are required Knowledge of assay performance in serum matrix, gender and overall bias are required for result interpretation The methods quoting ID-GCMS traceability show good agreement with the Reference Method Changes to the Roche II method have possibly aligned to ID-GCMS target