Cortisol Assays
– The Good, The Bad and The Indifferent
David Ducroq
Cardiff and Vale uLHB
WEQAS
Unit 6, Parc Tŷ Glas
Llanishen
Cardiff
www.weqas.com
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
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
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)
Immunoassay Issues – Cross Reactivity
Cortisol
Steroid numbering
Bridge linkage site for immunogen
11-Deoxycortisol
21-Hydroxycortisol
Cortisone
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
Traceability
Material
SI
Unit
Primary
Calibrator
Responsibility
Procedure
Primary reference
procedure/definitive method
Secondary Reference
Procedure
Secondary
Calibrator
Manufacturers
Working Calibrator
NML, ARML
Manufacturer selected
Procedure
X
BIPM, NML,
ARML
Manufacturers Standing
Procedure
ML
ML
Weqas Ref
Lab
involvement
End-user’s routine procedure ML, End
user
Routine sample
Result
Adapted from ISO 17511
Cortisol Traceability
Clin Chem 55:6 (2009)
Cortisol Reference Methods
JCTLM Listed Cortisol
Reference Methods
JCTLM Listed Cortisol
Reference
Measurement
Services
Cortisol by Mass Spec
LC-MS-MS
ID-GCMS (Reference
Methods)
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
Testosterone EQA Data – RCPA QAP
Data courtesy of RCPA QAP
Cortisol Studies
ACTH Stimulation Test – assay comparison
Collaboration with Medical Biochemistry, University
Hospital Wales
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
Baseline Cortisol
• No significant gender difference for ID-GCMS results
• Mean cortisol for all immunoassays was higher than the IDGCMS value
• Gender difference observed for all immunoassays – Male higher
than female
• Females on OCP gave higher mean cortisol value for all methods
ACTH Stimulation Test – Correlation with
ID-GCMS
(a) Centaur
(c) E170
(b) Architect
(e) Access
(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 ▲
ACTH Stimulation Test – Bias Plot
(a) Centaur
(c) E170
(b) Architect
(d) Immulite (2000)
● Male subjects
○ Non-oral contraceptive pill (OCP) female subjects
x, OCP-female subjects.
(e) Access
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
Collaboration with Medical Biochemistry, University
Hospital Wales
•
Retained samples (70) from routine analysis assayed
using ID-GCMS method:
Pregnant (2nd 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
Assay Bias: Spiked Patient Pools
Male
Female
Serum Matrix
Roche E170
Centaur
Abbott Architect
Beckman Access
Roche E170
Centaur
Abbott Architect
Beckman Access
EQA Data
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
Steroid Reference Method Traceability
Measurand
Standard
(certified
purity)
Control
material
Cortisol
NIST 921
DA 192, 193
Progesterone
NMIJ CRM
6003a
DA 347, 348
Testosterone
M914 (NARL)
>99%
* Moving shortly to NIST971
“In house” *
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
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
Cortisol Returns Correlation with ID-GCMS
June – September 2015
Cortisol Correlation with ID-GCMS
900
800
Cortisol (nmol/L)
700
600
500
Overall
400
Access/DxI 800
300
Elecsys/E Module
Advia Centaur
200
Architect
Equivalence
100
Linear (+/- 2SD)
0
100
200
300
400
500
600
Cortisol (nmol/L; Reference Method
700
800
900
Cortisol Returns Bias Plot
June – September 2015
Cortisol Bias Plot
Overall Mean Value
Access/DxI 800
40
Elecsys/E Module
Advia Centaur
30
Architect
20
% Bias
10
0
0
100
200
300
400
500
600
-10
-20
-30
Cortisol (nmol/L; Reference Method
700
800
900
Cortisol EQA Returns August 2011
Distribution 172
35
Access/DxI 800
30
Elecsys
25
Advia
Architect
20
% Bias
15
10
5
0
-5100
150
200
250
300
350
400
-10
-15
Female donor
-20
Cortisol (ID-GCMS Reference Target, nmol/L)
450
Access/DxI 800
30
20
% Bias
10
0
0
100
200
300
400
500
600
-10
-20
-30
Cortisol (nmol/L; Reference Method)
Female
Male
Spike (M/F)
700
800
900
Elecsys/E Module
40
30
% Bias
20
10
0
0
100
200
300
400
500
600
700
-10
-20
-30
Cortisol (nmol/L; Reference Method)
Female
Male
Spike (M/F)
800
900
Advia Centaur
30
20
% Bias
10
0
0
100
200
300
400
500
600
-10
-20
-30
Cortisol (nmol/L; Reference Method)
Female
Male
Spike (M/F)
700
800
900
Architect
30
20
% Bias
10
0
0
100
200
300
400
500
600
-10
-20
-30
Cortisol (nmol/L; Reference Method)
Female
Male
Spike (M/F)
700
800
900
Distribution S219 – closed 17/9/15
Correlation with ID-GCMS
700
Bias Plot
Cortisol Correlation with ID-GCMS
S219
600
50
Elecsys
Cortisol Bias Plot
S219
40
Elecsys
Advia
Advia
30
ID-GCMS
Traceable
400
ID-GCMS
Traceable
20
% Bias
Cortisol (nmol/L)
500
300
10
0
200
0
200
400
-10
100
-20
0
0
200
400
Cortisol (nmol/L; Reference Method)
600
-30
Female sample on OCP?
Cortisol (nmol/L; Reference Method)
600
S219
Bias Plot
Correlation with ID-GCMS
Beckman ID-GCMS Traceable
S219
600
Beckman ID-GCMS Traceable Bias Plot
S219
15
10
500
5
0
400
Cortisol (nmol/L)
0
100
200
300
400
500
-5
300
% Bias
-10
-15
200
-20
DX
Access
100
Access
-25
DX
-30
0
0
100
200
300
400
500
Cortisol (nmol/L; Reference Method)
600
-35
-40
Cortisol (nmol/L; Reference Method)
600
Roche & Roche II Cortisol Methods
Elecsys/E Module - S219
M/F spiked
sample
40
30
% Bias
20
10
0
0
100
200
300
400
500
600
-10
-20
-30
Cortisol (nmol/L; Reference Method)
Elecsys/E Module
Roche II
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