Professional Documents
Culture Documents
– Standardisation
‘process of ensuring that all methods for
determining the concentration of a particular analyte
give the same results’
– Calibration
‘process of assigning values to an unknown
samples using a standard’
Immunoassays
• Problem
– for most analytes quantified by immunoassay
there are no reference methods with which to
calibrate standards
Importance of standardisation
• Usual practice to report result and give ref range for
that method next to result
• Once ref range established difficult to avoid it being
used to subconciosly being used to interpret results
eg serum v plasma
or
serum & charcoal stripped serum
• Physiology
• Pathophysiology
• Synacthen test
• Cortisol immunoassays
• Interpretation of the short synacthen test
Physiology
• Steroid hormone: produced
in z. fasiculata of adrenal • Lipophilic. Transported bound
cortex to CBG (82%), albumin (10%).
• Pre- 1962
Research labs only. Req’d several labour –intense steps
• Mid 1990’s
1st automated immunoassay analyser for cortisol 1992
By mid-1990’s automated immunoassay analyser
expansion and most cortisol analyses done on an
automated platform
Cortisol assay : Immunoassay
• 1998
Defining the normal response to the Syncthen test: implications
for the investigation of hypothalamic-pituitary disorders
Penny Clark et al Clin Endo 49: 287-92.
• 1975
1st GCMS – derivatisation & 3H for procedural
losses
• 2013
Owen et al. Developed rapid assay for analysis &
implementation into routine service lab
Ann Clin Biochem 50: 345-52
Clinical Chemistry
Candidate Reference Method
Procedure for the
Quantification of Total Serum
Cortisol with LC-MS/MS.
31
Gender differences
The nature of the
beast is that since we
want to use exciting
endogenous levels in
the Scheme, the male
donations we get in
through the door get
used for Cortisol (and
now General
chemistry) so the
effect that has always
been there was largely
over-looked/ignored
although JGM and
some participants
were aware of the
potential problem
32
EQA sample from the UK NEQAS
scheme June 2015
Reprodu
cibility
on
repeats
34
Pool Stability
35
No recent change in calibration
36
UKNEQAS
41
Female Male
42
Cortisol assays: Binding proteins
• Differences in Ab specificity
• Lack of a single reference material
– At least 6 certified reference materials for cortisol
(current platforms traceable to some of these)
• Lack of single reference method
– Several Mass Spec methods : ID-GCMS initially but
more recently using LC-MS/MS as this technology
has improved
– Current platforms confirming the validity of their
platform against some of these LC-MS/MS
methods
Gordon.avery@abbott.com; HHarper@beckman.com; Robert.Banfield@Roche.Com;
Allan.at.Thompson@Siemens.com; Lisa.Wright@siemens.com
I am writing to the manufacturers of all 5 major Cortisol methods whose products cover around 98% of
participants in the UK NEQAS Scheme.
The five Methods are Abbott Architect, Roche Cobas/Modular, Siemens ADVIA Centaur, Siemens Immulaite
2000/2500 and Beckman Access/Dxi
Letter to the
I am asking if it would it be possible for you to share with me a ‘dossier’ of your most up-to-date validation
data (including recoveries, cross reactants, reference method values, between-method comparisons etc
etc.)?
Diagnostics
I know that when speaking informally to you all before, each of you believes your own assay to be valid and
accurate. Nevertheless, there is a wide spread of biases seen between-methods on our minimally
Industry
manipulated serum pools. From my perspective this is not something I can ignore.
For my part, I can confirm that our material is human serum and is minimally manipulated. Pools are made
from male-only or female-only donations. We mix only like-for-like concentrations. The Pools are filtered to
November
0.5 um and do not contain preservative. I have not tested my material for possible cross-reactants etc etc
but although my working hypothesis is that they are representative and that they are commutable, I would be
happy to investigate both aspects further. The majority of our Pools are endogenous, but we do perform
2013
recovery experiments with exogenous cortisol. Different cortisol powders have been tested in the past and
the results were completely super-imposable between compounds.
I can also confirm that the recovery of added cortisol is not always in step with the method bias. We observe
that there are methods which over-recover and have a high positive bias. We have methods which under-
I took it upon
recover and have a large negative bias. However, there are methods which over-recover, but are essentially
unbiased in the Scheme. The ALTM, our current target value, recovers ~ 104% of added cortisol.
myself to talk to
For operational reasons, the majority of our specimens for Cortisol are from male donors; the female the major
donations being preferentially reserved for our E2 and Progesterone programmes. We do send out a good
number of female specimens and the between-method differences characteristics are different to that of
male serum.
manufacturers.
Please see the attached pdfs which show where we are in terms of B score versus C score Penalty Box I wanted to avoid
Plots and some representative histograms.
Please feel free to give me a call if you require any further background.
the marketing and
I look forward to your replies. get to the brass
Kind regards
tacks
Finlay
Abbott
Architect
Cortisol method bias to CR MS method
Roche E170
Modular
Cortisol method bias to CR MS method
Beckman
Access
Cortisol method bias to CR MS method
Siemens
Advia
Cortisol method bias to CR MS method
Cortisol method bias to CR MS method
UKNEQAS – Dec 2015
• 2015
UKNEQAS announced in annual report
that from April 2016 will abolish ALTM
and use candidate reference LC-MS/MS
target for BIAS calculation
What-if Mass Spec Target
53
February 2016
54
Roche Gen II cortisol assay
A D
B E
C F
Pregnancy v cLCMS/MS
D
A
B E
C F
Cortisol v Cortisol Binding globulin
Prednisolone v cLCMS/MS
A D
1600
(nmol/L)
1000
800
600
400
200
0
-200
0 20 40 60 80 100
B E Certified Target (nmol/L)
C F
Metyrapone v cLCMS/MS
A D
B E
C F
The Way Forward...
• NEQAS gave participants a ‘What-if’ – Mass Spec as Target
report on their own Cortisol performance at Distribution 422
using data from Distributions 401 through to 422.
Intend to use this reference method on all Cortisol pools used in
the Scheme
• Thrust to move towards fully validating the target values used
by all schemes.
• Periodic, retrospective use, of reference values may well be the
practical way forward as batching pools for reference analysis is
a much more cost effective approach than ‘real-time’ reference
values. ‘Reference methodS’ can still have batch-to-batch
‘wobble’, though this is less of a consideration.
• If we validate the ‘field method’ MSMS mean, we could use
this as a real-time target. This is becoming more of a possibility
as we now have 10 MSMS users
The way forward …..
All analytes
• For all analytes we are continuing investigating replacing
the ALTM with a better estimate of the truth as target.
• This follows on from use of ‘Restricted ALTMs’ or MSMS
means for some analytes and (because of the number of
results we were dealing with for some analytes) for
17OHP we moved straight to a MSMS target.
• A validated ‘field method’ MSMS target, like we have for
Female Testosterone, is a good and reliable target and
after April 2016 we may be using this ‘field method’
MSMS mean this as the target for all methods for Female
Testosterone.
• Immunoassay users may find this a bit of a challenge.
LC-MS/MS wider application in larger clinical
labs