i am the director of the northwest lipid metabolism and diabetes … · 2019-05-29 · eas 2019 i...
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I am the Director of the Northwest Lipid Metabolism and
Diabetes Research Laboratories (NWRL) at the University of
Washington in Seattle WA, USA
NWRL/UW has received grant and research funding from:
• National Institutes of Health
• Amgen Inc
• Ionic Pharmaceuticals
• Kaiser Permanente
I am consultant to:
• Denka Seiken, Japan
• Roche Diagnostics, Germany
• Medtest DX Inc, USA
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Santica M Marcovina PhD ScD FAHAResearch Professor of Medicine, UW
Director, Northwest Lipid Metabolism and Diabetes Research Laboratories
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Aim: To obtain values in patient samples that are accurate and
comparable independently of the analytical method used for the
measurement. Accuracy is obtained if calibrator values are
traceable to a high order primary reference material directly or via
a suitable secondary reference material and the methods are
certified to guarantee that accuracy of the calibrator results in
accurate values in samples.
Aim: To obtain values in patient samples that are comparable
even though not necessarily accurate.
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• Antibody specificity
• Immunoreactivity of the antibody per particle should be the
same for the assay calibrator and for the samples
• An accuracy-based target value should be assigned to the
assay calibrators
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274
399
649
Kringle 4 Type
V PD
Kringle 4
187
kDa
3-1021
Type 2 repeats
10
20
40
3
30 524
SM Marcovina, University of Washington EAS 2019
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• For Lp(a) the “signal” does not reflect the number of particles
• In samples with apo(a) sizes smaller than the apo(a) size of the
assay calibrator, Lp(a) levels will be
• In samples with apo(a) sized larger than the apo(a) size of the
assay calibrator, Lp(a) levels will be
UNDERESTIMATED
OVERESTIMATED
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• To circumvent this problem, we produced a variety of monoclonal
antibodies against Lp(a) and we were able to obtain and characterize a
high-affinity monoclonal antibody directed to an epitome only present in
the KIV Type 9 region of apo(a)
• Using this unique monoclonal antibody (a-40), we developed and fully
validated an ELISA method that can measure Lp(a) protein on an equimolar
basis
• We demonstrated that Lp(a) contains one molecule of apo(a) per Lp(a)
particle and therefore, the Lp(a) protein values expressed in nmol/L reflect
the number of circulating Lp(a) particles.
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Calibrated with the same sample
containing apo(s) with 21 Kingle 4
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14161820222426283032-50
-25
0
25
50
Kringle 4 number
% Bias
y = 4.05x - 84.7
r = 0.967
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• In the mid 1970’s, John Albers developed the first, high
sensitive RIA to measure Lp(a)
• Lp(a) was isolated from an individual with high Lp(a) levels
and measurements of the lipid and the protein components of
Lp(a) were performed
• This Lp(a) preparation was used as the RIA primary calibrator
with the assigned value being the sum of lipid and protein
values
• Results of Lp(a) were therefore expressed in mg/dL of TOTAL
MASS of Lp(a), not knowing that the Lp(a) mass is highly
variable within and between individuals
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1997: IFCC Working Group
Members:
A. Steinmetz (Chair) F. Dati K. Berg
R. Couderc G. Kostner N. Rifai
I. Sakurabayashi J. Tate
Phase 1: Assessment of analytical performance of 40 test systems by testing serum
samples for precision, linearity, and parallelism.
Results:
❖ A significant number of assays were not optimized and failed to meet the criteria for
precision, linearity, and parallelism.
❖ The among method CV of all systems on reference samples ranged from 22% to 60%.
❖ The among method CV of optimized systems ranged from 16% to 35%
Clin Chem 1998; 44:1629-40
International Federation
of Clinical Chemistry
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1998: Phase 2
Aims:
❖ Selection of a secondary reference material for Lp(a)
❖ Four proposed materials were compared in 27 optimized analytical systems
❖ Results of precision and linearity were comparable
❖ Among-method CV on each of the four materials ranged from 11% to 22%
Based on the overall results, the IFCC Working Group decided to select the material 2B as a
proposed reference material (PRM-2B) for further evaluation and for final target value
assignment. Decision was made to express Lp(a) values in nmol/L.
Clin Chem Lab Med 1999; 37:949-958
International Federation
of Clinical Chemistry
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Principal Investigator:
❖ Santica M Marcovina,
University of Washington
Co-Investigators:
❖ John J Albers,
University of Washington
❖ Angelo Scanu,
University of Chicago
❖ Celina Edelstein,
University of Chicago
National Institutes of Health
National Heart, Lung, and Blood Institute
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Specific Aims:
• Evaluation of Lp(a) assays with particular emphasis on
antibody specificity and sensitivity to apo(a) size
polymorphism
• Feasibility of standardization of Lp(a) assays
• Preparation and characterization of primary reference
material
• Assignment of target value to the IFCC Reference Material
National Institutes of Health
National Heart, Lung, and Blood Institute
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• Study performed in collaboration with the IFCC Working
Group on Lp(a) standardization
• NWRL ELISA served as the reference method
• 21 commercially available methods were evaluated by using
30 fresh-frozen samples spanning a large range of Lp(a)
levels and apo(a) isoforms
• The reference material, PRM-2B, with an assigned value of
107 nmol/L, was used as a common assay calibrator
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• Using PRM-2B to calibrate all the systems, the among-method CVs for each
of the 30 samples ranged from 6% to 31%
• Even though the CVs were lower than those obtained using individual
calibration (CV = 16% to 35%), no harmonization in Lp(a) values measured
by different methods was achieved
• The impact of apo(a) isoform size on Lp(a) concentrations varied among the
different methods as a function of the apo(a) size of the assay calibrators
• Other factors, like difference in instrumentation, also contributed to the
lack of comparability of results
• In 2003, the WHO Expert Committee on Biological Standardization accepted
PRM-2B, with an assigned value of 107 nmol/L, as the “First WHO/IFCC
International Reference Reagent for Lipoprotein(a) Immunoassay”
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10 15 20 25 30 35-100
-50
0
50
100
Major Kringle 4 Repeat
Bias (%)
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10 15 20 25 30 35-100
-50
0
50
100
Major Kringle 4 Repeat
Bias (%)
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10 15 20 25 30 35-50
-25
0
25
50
Major Kringle 4 Repeat
Bias (%)
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• Polyclonal antibody-based. Antibodies strongly reacting with
apo(a) KIV Type 2 repeats.
• Antibodies are coated on the surface of latex particles
(diameter 120 nm)
• Lp(a) particles in plasma are then bound to the antibodies on
the latex particles to form immunocomplexes
• Five independent calibrators with Lp(a) levels from low to high
and apo(a) size from large to small
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Very
Small
Medium
Small
Medium
Large
Very
Large
Lp(a) nmol/L
Signal
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Values expressed in mg/dL of total Lp(a) mass
➔ Single calibrator: no traceability to a common
reference material
• Effect of apo(a) size variation is inevitable
Values traceable to the WHO/IFCC Reference Material
and expressed in nmol/L of Lp(a) protein
➔ 5-point calibrators
• Effect of apo(a) size variation is minimized
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apo(a) Kringle number accounts for 61.1% of the bias variation
ITAITA-DENKA
apo(a) Kringle number accounts for 2.5% of the bias variation
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Denka Method – Instrument 1
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Denka Method – Instrument 2
apo(a) Kringle number accounts for 1.0% of the bias variation apo(a) Kringle number accounts for 0.7% of the bias variation
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Step 1 – Assignment of target values to assay calibrators
• Target values traceable to the WHO-IFCC Reference Material
Step 2 – Validation of the accuracy of value transfer
• Six fresh-frozen samples from individual donors were
prepared at the NWRL and were selected to have a suitable
range of Lp(a) levels and apo(a) isoforms.
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Step 3
• 80 fresh-frozen samples are analyzed, collected from
apparently healthy donors and selected to represent a large
range of Lp(a) levels and apo(a) isoforms
• Detailed certification criteria have been established including
analytical CV, correlation and absolute bias limits between
obtained and assigned values, and the correlation between
the % bias and apo(a) isoform size
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System 1 System 2
System 3 System 4
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• To guarantee that a new lot of reagent and calibrator results in
minimization of the effect of apo(a) size variability on Lp(a) values obtained
by different analytical platforms, requires rigorous work for the distributors
of the Denka assay. Clinical chemistry laboratories using the assay should
ask for demonstration that assay optimization has been performed on their
specific instrument.
• The limitations of the assay should be understood by clinicians and by
clinical chemistry laboratories.
• Claims that the assay is accurate because Denka antibodies do not
recognize the KIV Type 2 repeats of apo(a) or that the values produced by
the assay are not affected by apo(a) size polymorphism are simply false
claims.
• Reviewers of scientific publications should reject false claims about this
assay and request specific demonstration that the impact of apo(a)
variability on their data has been minimized on the instrument used for
performing the analyses.
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• The first small step toward standardization at this point in time is the expression of
Lp(a) values in nmol/L of Lp(a) protein considering that the assays measure apo(a)
and not the total Lp(a) mass.
• Even though the nmol/L protein determined using antibodies, either polyclonal or
monoclonal, directed against the variably expressed KIV Type 2 repeats do not
accurately reflect the number of circulating particles, a common unit for expressing
Lp(a) levels is not only scientifically correct but it’s advisable to avoid the use of
arbitrary, incorrect, and confusing conversion factors to transform values obtained
by different methods.
• The WHO/IFCC Reference Materials is available free of charge from our laboratory
upon request from any manufacturer who wants to change their calibrator units in
nmol/L.
• Manufacturers and clinicians should be aware that traceability of the calibrator to
the reference material does not indicate accuracy of values in patient samples.
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• Little has changed in the arena of Lp(a) measurements since the first attempt by the
IFCC WG to standardize Lp(a) methods by producing the WHO/IFCC First Reference
Material over 20 years ago.
• The problems of Lp(a) measurement have been clearly defined and some methods
able to minimize the effect of apo(a) size variation have been optimized.
• However, we are far away from standardization, or even harmonization, of Lp(a)
values obtained by different methods because the main problem for Lp(a) is not in
the assay calibrator but in the variable immunoreactivity of the assay antibodies
which cannot be eliminated.
• No new reference material is needed at present because no assay will benefit from
its introduction.
• Effort needs to be directed to the development of new approaches to measure Lp(a)
that are antibody-independent, precise, high throughput, and are available to
clinical chemistry laboratories.
• In conjunction to this type of automated instruments, a validated reference material
and an accuracy-based reference method will be necessary.
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