National Institutes of Health Amgen Inc Ionic Pharmaceuticals - - PowerPoint PPT Presentation

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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

Santica M Marcovina PhD ScD FAHA

Research 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

3-10 2 1

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

1 4 1 6 1 8 2 2 2 2 4 2 6 2 8 3 3 2

• 5
• 2

5 2 5 5 K r i n g l e 4 n u m b e r % B i a s

y = 4 . 5 x

• 8

4 . 7 r = . 9 6 7

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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

• f 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

• f 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
• f 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

50 100 Major Kringle 4 Repeat Bias (%)

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10 15 20 25 30 35

• 100
• 50

50 100 Major Kringle 4 Repeat Bias (%)

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10 15 20 25 30 35

• 50
• 25

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

ITA ITA-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

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

• btained 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

• f 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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