Calibration Verification & Linearity:

Regulatory Requirements and Application to

Coagulation Assays

Lauren Pearson, DO MPH

Assistant Professor
University of Utah Department of Pathology
Assistant Medical Director, University Hospital
Clinical Laboratory

DOI: 10.15428/CCTC.2018.292771

© Clinical Chemistry
Calibration

The process of establishing a correlation between the

measurement signal generated by an instrument and
the true concentration of analyte in the sample.

Calibration verification.
• The process of “testing materials of a known concentration in
the same manner as patient specimens to assure the test
system is accurately measuring samples throughout the
reportable range.”

42 CFR 493.2

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Linearity

Refers to the relationship between the final analytical

result for a measurement and the concentration of the
analyte being measured.
• Analyte concentration versus measurement signal is not
always linear
• Not separately designated by CLIA

Killeen AA, Long T, Souers R et al. Verifying Performance Characteristics of Quantitative Analytical Systems. Arch
Pathol Lab Med 2014;138:1173-1181.

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Analytical measurement range (AMR)

The “range of concentrations of an analyte that a

method can directly measure without any dilution,
concentration, or other pretreatment.”
• Chemistry and Toxicology Checklist, CAP

AMR validation.
• A process used to verify the linear relationship between the
analytical results of a method and the concentration of
analyte over the entire measurement range

42 CFR 493.2

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Regulatory requirements

Calibration verification is required by CLIA.

Laboratories which perform quantitative coagulation

assays must verify:
• Calibration
• AMR validation (linearity)
• Whenever required by the method manufacturer

At least every 6 months.

42 CFR 493.1255
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How to meet minimum requirements

Linearity experiment.
• Analyze 3 samples in duplicate
• Samples must span the AMR
• Include a minimal value, a mid-point value, and a maximum value near
the upper limit
• Sec. 493.1255(b)(2)

Source of materials and acceptability criteria determined by

laboratory director.
• Patient specimens
• Commercial kits
• Standard reference materials
• Calibrators

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Please note:

Re-calibration of a test more frequently than every 6

months meets calibration verification requirements if the
calibration includes samples with low, mid, and high
values near the AMR.

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Why is it important?

Required by CLIA.

If the calibration changes, patient test result values will

change.

Can detect problems earlier than QC or PT.

• If linear range does not cover AMR, may be a problem with
reagents, specimen handling, or analyzer
• Adjustments to reportable range to reflect the linear range

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Why is it relevant to coagulation assays?

Coagulation testing has evolved.

• In the past, primarily clot-based testing
• Some tests and methods now measure a concentration of an
analyte

Requirements apply to methods that are calibrated and

directly measure concentration or activity of an analyte.
• EIA methods
• Immunoturbidity
• Chromogenic methods

http://www.captodayonline.com/Archives/1112/1112g_lap.html

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Examples of applicable assays

EIA or immunoturbidity methods for:

• Coagulation factors
• Protein C and S antigens
• von Willebrand factor antigen
• Quantitative D-dimer
Chromogenic methods for:
• Antithrombin activity
• Protein C activity
• Heparins

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Examples of exempt assays

Clot-based assays.

Platelet function tests.

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Example analyte

Quantitative D-dimer.
• AMR 0.27-4.0 µg/mL FEU
• 5 samples spanning the AMR measured in triplicate
• Slope and intercept calculated

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Sample Expected Value Mean Observed
DDI-01 0.1771 0.177
DDI-02 0.973 0.973
DDI-03 1.807 1.807
DDI-04 2.641 2.590
DDI-05 3.475 3.483

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D-dimer Scatter Plot

Slope 0.992
Intercept -0.001

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 Troubleshooting

Some content adapted from College of American Pathologists Calibration Verification/Linearity Participant Summary

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Non-linearity

Consider sources of error:

• Specimen handling
• Analytical phase of testing
• Clerical errors

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Problems with high or low specimens

Possible manifestations.
• Observed value different than expected
• Samples don’t adequately challenge the upper or lower AMR
How to investigate.
• Assess for recovery issues near the limits of the AMR
• Review dilution protocols
• Assess specimen handling and possible degradation
• Were samples within the AMR for the instrument?
• May need to add samples to adequately challenge the limits

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Bias

Evidence of bias.
• Slope not equal to 1
• Non-zero intercept
• Non-zero percent difference on a bias plot (not shown)
How to investigate.
• Instrument maintenance needed?
• Review QC results for acceptability
• Review recent calibration for error or need for recalibration
• Review reagent handling
• Reagent lot-to-lot comparisons
• Confirm written procedures were followed
• Consider sample mixing or reconstitution problems or improper storage

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Imprecision

Possible manifestations.
• Large difference between replicates for a single specimen
• Standard deviation exceeds allowable random error
How to investigate.
• Exclude clerical error in recording of results
• Review specimen handling (reconstitution, storage, mixing,
etc.)
• Review quality control data
• Perform simple precision study

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References
1. Centers for Medicare & Medicaid Services, Department of Health and Human
Services. Medicare, Medicaid, and CLIA programs; laboratory requirements relating
to quality systems and certain personnel qualifications; final rule [published
correction appears in Fed Regist 2003;68(163):50722–50725]. Fed Regist. 2003;
68(16):3707–3714. Codified at 42 CFR §493.2.
2. Killeen AA, Long T, Souers R et al. Verifying Performance Characteristics of
Quantitative Analytical Systems. Arch Pathol Lab Med 2014;138:1173-1181.
3. Centers for Medicare & Medicaid Services, Department of Health and Human
Services. Medicare, Medicaid, and CLIA programs; laboratory requirements relating
to quality systems and certain personnel qualifications; final rule [published
correction appears in Fed Regist 2003;68(163):50722–50725]. Fed Regist. 2003;
68(16):3707–3714. Codified at 42 CFR §493.1255.
4. College of American Pathologists, Commission on Laboratory Accreditation.
Chemistry and Toxicology Checklist. Northfield, IL: College of American
Pathologists; 2012.
5. Ford, A. As coag tests evolve, so do checklist requirements.
http://www.captodayonline.com/Archives/1112/1112g_lap.htmlAccessed May 18,
2018.
6. College of American Pathologists Calibration Verification/Linearity Participant
Summary.

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Disclosures/Potential Conflicts of Interest
Upon Pearl submission, the presenter completed the Clinical Chemistry
disclosure form. Disclosures and/or potential conflicts of interest:

▪ Employment or Leadership:
Laboratory Director, University of Utah & ARUP Laboratories
▪ Consultant or Advisory Role: No disclosures
▪ Stock Ownership: No disclosures
▪ Honoraria: No disclosures
▪ Research Funding: Sysmex
▪ Expert Testimony: No disclosures
▪ Patents: No disclosures

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