TITLE: Calibration Verification & Linearity: Regulatory Requirements and Application to

Coagulation Assays

PRESENTER: Lauren Pearson, DO MPH

Slide 1:

Hello, my name is Lauren Pearson. I am an Assistant Professor of Pathology at the University

Of Utah Department Of Pathology and Medical Director of clinical laboratories at University of
Utah Hospital and Clinics. Welcome to this Pearl of Laboratory Medicine on “Calibration
Verification & Linearity: Regulatory Requirements and Application to Coagulation Assays.”

Slide 2:

We will first begin this Pearl with some relevant definitions. Calibration verification is the process
ss
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. It is
different from calibration, which is the process of establishing a correlation between the
measurement signal generated by the instrument and the true concentration of the analyte in
the sample. Samples can be tested in duplicate for calibration verification which may be slightly
different than the process for testing patient samples.

Slide 3:

Linearity refers to the relationship between the final analytical result for a measurement and the
concentration of the analyte being measured. This distinction is relevant because a plot of
analyte concentration versus measurement signal from the instrument may not be linear. The
concept of “linearity” is not separately designated by CLIA. Related to linearity is the concept of
the analytical measurement range (AMR).

Slide 4:

© 2016 Clinical Chemistry

Pearls of Laboratory Medicine
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The AMR is the range of concentrations of an analyte that a method can directly measure
without any dilution, concentration, or other pretreatment. AMR validation is 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.

Slide 5:

CLIA regulations require that laboratories perform calibration verification at least every six
months. Calibration verification is also indicated in the following situations: whenever there is a
complete change in the set of reagents to a new lot, there is major preventative maintenance or
replacement of critical parts of the instrument, relocation of the instrument, quality control data
show a trend, shift, or are outside of acceptable limits. College of American Pathologists (CAP)
checklist requirements break this down into calibration verification and AMR validation
(linearity).

Slide 6:

How can labs meet the regulatory requirements? By performing a linearity experiment! The
minimum requirement is to analyze three samples in duplicate that span the AMR of the assay.
The samples must include a minimal value near the lower limit, a mid-point value, and a
maximum value near the upper limit of the AMR. The source of materials as well as the
acceptability criteria for accepting or rejecting tests during calibration verification are determined
by the laboratory director. Patient samples may be used, so long as they sufficiently challenge
the upper and lower ends of the AMR and are of acceptable quality and stability. Commercial
kits, control materials, calibrators of a different lot than the current calibration, proficiency testing
materials, and reference materials are an alternative to using patient samples, and are available
for purchase from a number of vendors. It is important to ensure that samples of the appropriate
matrix are used.

Slide 7:
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.

Slide 8:

Calibration verification is required by CLIA, but why else is it important? Calibration verification
is helpful for monitoring assay performance over time and maintaining quality results. If the
calibration changes, patient results will change. It may also detect accuracy and precision
problems earlier than quality control or proficiency testing data. If the assay is shown to be non-
linear within the AMR, the laboratory is alerted to possible problems with reagents, specimen
handling, or the instrument itself. The range of values reported on patient specimens may need
to be changed accordingly.

© 2016 Clinical Chemistry 2

Pearls of Laboratory Medicine
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Slide 9:

These concepts are comfortable and familiar to many laboratorians in clinical chemistry, but are
newly applied to other areas of laboratory medicine, such as thrombosis and hemostasis
testing. This is because in the past, coagulation testing was primarily clot-based testing using
instruments that were not calibrated to measure the concentration of an analyte. Methodology
has evolved since then and many coagulation laboratories use methods which may be
calibrated and measure a concentration of an analyte. Hence, the requirements for calibration
verification now apply in the coagulation laboratory.

Slide 10:

Examples of assays which meet this criteria include EIA methods, immunoturbidity methods,
and chromogenic methods. This slide shows many examples of such applicable assays, some
of which are often available in routine or stat laboratory settings as well as reference laboratory
settings.

Slide 11:

Not all coagulation assays are calibratable, and thus these requirements will not apply.
Examples of exempt assays include clot-based assays and platelet function tests.

Slide 12:

We will now transition to applying these concepts to a specific example, quantitative D-dimer. In
this example, the AMR of the assay is 0.27-4.0 micrograms per milliliter. The linearity
experiment I will show in the following slides consisted of analyzing five samples spanning the
AMR, each measured in triplicate. Linear regression analysis was performed and slope and
intercept were calculated. In this example, the source of the samples was a commercially
produced kit.

Slide 13:

Here is a table listing the mean observed values of the raw data for the measurements for D-
dimer obtained for each sample. Notice that for each sample, the mean observed measurement
is close to, or equal to the expected value.

Slide 14:

Here is a scatter plot of the data. Each of the individual measurements for each sample are
plotted. The x-axis is the expected concentration of D-dimer for each sample, and the y-axis is
the measured concentration. A linear regression line with a slope of 0.992 and intercept of -
0.001 was fit to the points. The data appear to be linear visually, and the plot demonstrates

© 2016 Clinical Chemistry 3

Pearls of Laboratory Medicine
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minimal scatter of the data points, with even coverage of the AMR throughout the range and
adequate coverage to the limits at the high and low ends. All differences between the observed
values and the expected values are within allowable error limits. The slope and intercept
indicate minimal proportional and constant bias.

Slide 15:

We will now discuss what to do if you observe that an assay is not linear over its AMR, or if
unexpected bias or imprecision is present.

Slide 16:

If the results show that the assay is non-linear over the full range or even a partial range, there
are three areas to focus your troubleshooting steps. First, review specimen handling steps.
Were the samples used for testing stored appropriately? If a kit was used, were the kit’s
instructions followed? If patient samples were used, were they processed according to standard
operating procedure prior to testing to ensure adequate mixing, centrifugation, or were other
necessary processing steps were taken? Next, examine the analytic phase of testing. Were
standard operating procedures followed appropriately? Was instrument maintenance performed
as applicable? Were quality control results acceptable? Were reagents used within stability?
Were any flags or errors generated by the instrument during testing? Was testing performed by
an individual deemed competent to perform testing? Lastly, consider the possibility of clerical
errors if results from the instrument were transcribed into another file for data analysis.

Slide 17:

It is fairly common to encounter situations where an assay is linear over the tested range, however, the

samples tested at the low end or the high end of the AMR are problematic. Trouble at the low and high

end is observed when the samples don’t come close enough to the limits of the AMR, or when samples

do adequately challenge the ends but the observed values are different than expected. For the former,

the lab may need to acquire additional samples near the low end and the high end for analysis. If the

lower or upper end of the presumed AMR cannot be verified, then labs have the option of using a

narrower AMR. If the observed values are different than expected, it could be the case that the analyte

concentrations of the samples were not within the AMR of the instrument, so this should be verified as

well. For other problems with high or low specimens, assess pre-analytic variables including sample

handling and degradation. Consider errors due to recovery of the analyte, dilution protocols, etc.

Slide 18:

An assay may be proven to be linear but show unacceptable bias. Bias is evident when the
linear regression analysis produces a slope that is not equal to 1, a non-zero intercept, or
differences on a bias plot. What constitutes acceptable bias is at the discretion of the laboratory
director. Investigate possible sources of bias by examining quality control results, instrument

© 2016 Clinical Chemistry 4

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maintenance records, recent calibration data, standard operating procedures, reagent lot-to-lot
comparisons, and sample quality.

Slide 19:

An assay may be proven to be linear but show unacceptable imprecision. Possible

manifestations include unexpected increased scatter in the data, large differences between
replicates for specimens, or a standard deviation which exceeds allowable error. Begin the
investigation by reviewing specimen handling steps and quality control data. If the source of
imprecision is not evident, you may elect to perform a simple precision study using a set of
samples, preferably patient samples, to further investigate.

References:

There are numerous useful resources available for assistance with meeting regulatory
requirements for calibration verification and linearity, many of which are listed on this slide.
Additionally (not cited here), there is a Clinical Laboratory Standards Institute document, EP-6,
which may be useful.

Slide 19: Disclosures

Dr. Pearson is employed by the University of Utah and ARUP Laboratories.

Slide 20: Thank You from www.TraineeCouncil.org

Thank you for joining me on this Pearl of Laboratory Medicine on “Calibration Verification &
Linearity: Regulatory Requirements and Application to Coagulation Assays.”

© 2016 Clinical Chemistry 5

Pearls of Laboratory Medicine
Title

© 2016 Clinical Chemistry 6

 PEARLS OF LABORATORY MEDICINE

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:

© 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
2
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
4
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:
 Consultant or Advisory Role:
 Stock Ownership:
 Honoraria:
 Research Funding:
 Expert Testimony:
 Patents:

***This slide will be completed by CCJ Staff based on

disclosure form completed electronically in the submission
site.***

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