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Assay standardisation leads to improved patient outcomes
1. Clare Morris, NIBSC, UK
Assay standardisation – how this
leads to improved patient results
2. 2
National Institute for Biological
Standards and Control
-Establishment of biological standards and
reference materials
-Batch release of vaccines and blood
products
-Underpinning R & D
-Strategic alliances with major stakeholders
(eg USP, WHO)
A UK government funded laboratory with a statutory function in the control
and standardisation of biological medicines.
3. 3
What are we trying to achieve?
Accurate measurement
More robust and reproducible diagnostic assays
Improved health – prevention and clinical
management
4. 4
How can we achieve this?
• Biological medicines are more complex than chemically-
synthesised drugs and cannot be measured using
physicochemical means alone so their biological activity
must be measured using bioassays.
• Its hard to know if you’re correct without a reference to
compare with…..
6. 6
Biological standardisation
Providing laboratories with a means of comparison using the
same unit of measurement
Improve comparability between different lab data regardless
of the assay
In turn leads to better clinical management of patients.
Lack of standards…
No comparability between assays
• Patient movement between hospitals
• Clinical trial comparison
• Treatment implications
7. 7
Types of standard and control
material
High level calibrators
For calibration of an assay or a
product
International standards
Secondary standards
Primary reference
materials
Quality Control (QC) Materials
For regular assay monitoring
Run controls
External QC reagents
CE marked control
8. 8
Hierarchy of standards
WHO International standard (higher order,
international conventional calibrator)
Tertiary Standard (working reagent, Kit
control, run control)
Secondary Standard (calibrator for assays and
tertiary standards)
Traceability
Uncertainty of
measurement
9. 9
Using External Quality Controls
Laboratories should use a
quality control material in
addition to the kit control
Provides intra-laboratory
consistency.
11. 11
NIBSC Study - a less good lab
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Ctvalue
12. 12
NIBSC Study - a poor lab
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25
30
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40
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55
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49
Ctvalue
Quality Manager started to
check assays here
13. 13
Ensuring comparability
QC’s can help individual laboratories ensure quality and
accuracy but how to improve overall agreement between
laboratories, using different assays.
Same patient result
Assay A Assay B
14. 14
Molecular testing is routinely used across diagnostic laboratories to
guide clinical decisions in the management of transplant recipients
Rapid and reliable
Detection of opportunistic BKV infections is essential in the post-
transplantation period
Persistent ≥ 4.0 log10 copies/mL viral load in plasma is
presumptive of disease progression
Treatment threshold is recommended by clinical practice
guidelines to reduce immunosuppression to circumvent organ
damage/loss
BK virus molecular diagnostics in the
transplant setting
Blood
Plasma
Urine
Persistent high viral load
Reduce immune suppression
15. 15
BK virus viral load estimate in copies/mL
Under quantification - increasing BKV lytic infection
- organ damage
Over quantification - immunosupressant reduced too soon
- potential graft rejection
ALL TESTS ARE NOT EQUAL!
Evaluation of a single BKV positive clinical sample by 28 laboratories
Are all tests equal?
A need for standardisation
16. 16
In house
Commercial
Extraction Methods
ABBOTT (M2000SP)
Altona (AltoStar Automation System)
BioMérieux (Nuclisens easyMAG, Nuclisens miniMAG )
ELITech Group (ELITe GALAXY)
Roche ( MagNa Pure 96, MagNA Pure LC 2.0)
Siemens (VERSANT® kPCR Molecular System )
QIAGEN:(QIAcube, QIAcube HT, QIAGEN EZ1,
QIAsymphony)
Commerical Kits
QIAGEN Artus BK Virus QS RGQ kit
Biomerieux BK Virus R-gene
Altona Diagnostics RealStar BKV PCR 1.0
Epoch ASR (formerly Nanogen)
ELITechGroup S.p.A. BKV ELITe MGB® Kit
Luminex BK Primers MultiCode®-RTx Chemistry
Bosphore® BKV Quantification Kit v1 (Anatolia
Geneworks)
Euro-RTBKV kit Eurospital
Thermocycler
ABI Prism 7500 SDS, 7500 Fast, Step One
Eppendorf Mastercycle, 3M Integrated Cycler
Cephid Smart cycler, QIAGEN Rotorgene Q, Agilent
MxPro3000P, VERSANT kPCR Molecular System
Sacace Sacycler-96, Bio-Rad MyiQ, CFX96, QX100
ddPCR
ROCHE Lightcycler 480, 96
Methodology variations
17. 17
Assay Standardisation
Bioassays use complex biological systems to test activity, they
are variable from test to test.
By using a biological reference material or standard of known
activity or potency, bioassay results can be compared and
calibrated to give a consistent result, no matter when or where
the bioassay is performed.
In the case of diagnostic tests, the use of control samples of
known activity allows the sensitivity and specificity of the test
system to be monitored and quality assured.
18. 18
WHO International Standards
Highest order of reference for biological materials medicines
Allows direct comparison between different assays and methodologies
Quantify “relative potency” in a specific but arbitrarily defined International
Unit (IU)
Control all steps of the assay
Behave in similar way to clinical material
Stable over many years
Are quantified in International Units (IU’s) assigned following a multicentre
collaborative study using multiple assays
Intended for calibration of secondary references
19. 19
Standardisation and Comparability
Standard Protocol
(Reference method)
• Allows same assays to
be compared
• Provides assurance of
data within assay
• Limits the
improvement of
assays
• Not practicable in
competitive market
Standard Reference
(Reference material)
• Allows different assays
to be compared
• Provides assurance of
data across hospitals
• Provides framework
for improving all
assays
20. 20
PSA WHO IS – post 2000
Source: Dina Patel, UK NEQAS Immunology,
Immunochemistry & Allergy, Sheffield, UK
• Majority of assays recalibrated to the WHO IS.
• Both introduction of standard and assay improvements reduced variability
• Continued reports that methods are not interchangeable - issue for long
term screening and transfer between hospitals
• Cut-offs for biopsy not clear. PSA is not a perfect tumour marker. Accepted
4µg/L
29. 29
Treating severe Adenosine deaminase
deficiency (ADA-SCID)
• First licensed medicine to treat rare inherited disease ADA-
SCID and clinical trial pipeline X-SCID, WAS, X-CGD, b-
thalassemia, ALD, MLD, are based on the platform
technology involving integrating vectors (RV or LV)
• Two CAR-T products have been approved into US and EU
markets and over 100 CAR-T therapies are at a late stage
of clinical trials
• Patients treated at small number of centers Europe, USA,
Australia, Japan.
• As diseases are rare, patients followed up worldwide
yuan.zhao@nibsc.org
30. 30
Standardisation across technologies
(21 methods; ρC 0.995)
• KRAS mutations in colorectal cancer can lead to some drugs not being
effective.
• Important to understand sensitivities of mutation detection.
• Concordance (medians): MassARRAY<Sanger<Pyro<dPCR & NGS
Jennifer.boyle@nibsc.org
31. 31
Challenges
Early IS’s
– blood borne viruses limited problem
– Plasma almost universal matrix of blood industry
– IS and other references diluted in plasma or equivalent
Clinical Virology markers
– Greater challenge much discussion at SoGAT
– Broader range of matrices tested in labs plasma/WB/CSF
– Too complex to include in initial collaborative study
– Efficiency of extraction of nucleic acid variable
– Evidence of non-commutability- assay or standard?
33. 33
Conclusions
• Different controls have different applications, but
they complement each other in the overall
assurance of a test result.
• The result provided by one assay is not
comparable with another unless they are
calibrated to a common standard.
• Patient outcomes can be affected by using
different assays
• The use of an international standard facilitates the
global harmonisation of assays >> patient results
35. 35
Forthcoming webinar
Internal and external controls
Is EQA sufficient
How frequently should you monitor controls
What are the Westgard rules
Ever used a Levey Jennings plot?
36. 36
Webinar
Quality control materials: how best to
use them, why they can improve your
service, and what this means for your
laboratory accreditation
15 November 2018 | 11:30 - 13:00
The move from CPA to ISO 15189 has left laboratories with many more challenges to address, particularly
around the use of quality control materials.
The webinar will cover:
• ISO15189 and the use of quality control materials
• Types of control material and their use
• How to select the best control materials for your assay
• The benefits of regular monitoring, including the use of Westgard rules
https://nibscsogats.co.uk/home
Editor's Notes
Ask the time…..
Molecular testing is routinely used across diagnostic laboratories to guide clinical decision making in the management of transplant recipients
They allow for rapid and reliable detection of opportunistic BKV infection post-transplantation to predict and diagnose disease course
A persistent viral load of 4.0 log10 copies/mL in plasma is presumptive of BKV disease progression, and is recommended by clinical practice guidelines to reduce immunosuppression
Since there is no effective anti-viral treatment against BK virus, reduction of immunosuppressive drugs allows the reconstitution of the immune response to clear the virus and prevent lytic infection and kidney damage or potential organ loss
However not all tests are equal as illustrated by this evaluation of a single BKV positive sample by 28 labs
and detection in some labs may result in under quantification of BK virus, this would permit BK induced lytic infection to proceed undetected and increase the likelihood of organ damage.
And over-quantification BK virus could result in the removal of immunosuppressive treatment too soon and risk the likelihood of graft rejection.
There is no single reference method for BKV viral load determination, instead there is variation in extraction methods, amplification methods, laboratory developed or commercial assays, targeting different regions of the BKV genome and using different thermocyclers and of course different primer probe sequences and chemistries.
Clear spread of data. Clearly not able to consider all results as one data set
MassARRAY poor (semi-quantitative only)
NGS, dPCR & pyrosequencing excellent, but several outliers with pyrosequencing
NGS & dPCR with strongest concordance (n= 21 methods; Lin’s concordance correlation coefficient ρC 0.995 ) and NGS as most common, increasingly used; dPCR as gold standard
Mutations in KRAS can mean that some drugs will not be effective…. Colorectal cancer
