Instrument Calibration

Dr Faryal Husnain
PGR 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.”
Calibration
• Calibration is the foundation of all clinical laboratory testing that
insures the accurate reporting of patient results. Calibration is the
process that links the analytical signal with the concentration of
analyte present in serum, urine or other body fluid.
Calibration
• Before beginning calibration a medical laboratory scientist programs the
instrument with the concentration of each analyte according to the
information provided on the package insert supplied with the calibrator kit.
• The instrument then measures the calibrator and adjusts the signal to match
the given values. Depending on the method, this signal might be
potentiometric, photometric, fluorometric, chemiluminescent,
nephelometric or turbidimetric. 
Calibration Materials / Calibrators:

•Calibration materials, also called calibrators are solutions of known

analyte concentrations. Previously, the term “standards” was used
when referring to calibration materials.
Calibration
•  Plotting signal on the Y-axis versus analyte concentration on the X-
axis creates a calibration curve. The purpose of a calibration curve is
to establish the relationship between the concentration of an analyte
and the magnitude of the signal given by the measuring device. The
relationship can be linear or nonlinear.
• Calibration verification: Confirms that the current calibration
settings remain valid
 Calibration Curve

signal

concentration
 Calibration Curve – Two Points

signal

concentration
 Calibration Curve – Three Points

signal

concentration
Requirements of Calibration
• Validate or verify
oReportable range:
oAnalytical measurement range
Calibration
• Two components:
oThe primary range of measurement
−Analytical measurement range
oAnything done to the system to expand this
range
−“ Clinical reportable 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
No of samples required for AMR validation
• Three
o CLIA minimal requirement (low, mid-point, high)
Other Considerations for Calibration
• Set criteria of acceptance
• Requirements of CLIA,CAP,other accreditation body
• Established protocol
• Medical relevance(calibration errors lead to medical decisions that
affect pateints,increase lab costs)

o All of this should be established by the laboratory director

o All of this should be documented formally
General Principles of Calibration
• Establish a target value
o May use a patient sample’s result as the “target”
o May use peer group mean of PT material
o May be established by the provider of the material

• Establish an acceptable range around the target

o May be a laboratory-assigned range
o May be provided by the manufacturer

• Document your protocol (approved by director)

When to Calibrate?
• Both CAP and CLIA '88 require that calibration or calibration
verification be performed at least every six months,also after
• reagent lot changes,
• unacceptable quality control,
• major service to the instrument,
• when recommended by the manufacturer.
• Any additional requirements by an accreditation body
 Step by Step Calibration

•Calibration frequency for each analyte is

mentioned in kit insert / SOP for each analyte.
•It is also required when QC showing any shift
or trend & with each reagent lot change.
• Lab technologist / Technician needs to
calibrate the assay when ever above mentioned
status is observed.
Step by Step Calibration
•Calibrators are obtained from storage place and allowed to attain
room temperature. If calibrator stored in freezers, it should be properly
thawed and mixed before using
• Note down the date of opening at box of calibrator.
• Check test count of onboard reagent status, that require calibration.
• Lot number of calibrator should be entered in analyzer, along with
concentration of each calibrator. Also verify units of calibrators.
Step by Step Calibration
•In some cases concentration is available by default; only calibrator Lot number is
required (e.g. in case of Abbott Alinity i system).
•Carefully read instructions available in calibrator insert. Prepare calibrators as
written in kit insert or SOPs of that analyte.
• Give the calibration order for the analyte which require calibration in Analyzer
•Get the required volume of calibrator in Eppendroff or specific cup of analyzer.
Check each calibrator for bubbles & volume very carefully. If any bubble or droplet
present remove, before loading them.. Load the rack in priority or Stat bay.
Calibration Step by Step
•Note down calibration date at the calibrator box with signatures of
operator and store accordingly.
•Note down the all the details in calibration log sheet & attach
calibration detail printout with the sheet.
•As calibration successfully is completed, compare the calibration
details with previously done calibration. If no significant difference
observed proceed forward for Calibration verification procedure.
Calibration Verification
•To perform calibration verification, materials with known concentrations
are tested in the same manner as patient sample, which can be:
•Proficiency testing material
• Patient samples with known values
•Control material with different lot number.
•Calibration verification needs to be done with at least 3 different
concentrations specimen that should cover the reportable range of an
analyte (Low, medium & high).
• Compare the result, if falls within acceptable range, proceed along with
the patient testing.
Calibration Verification
• Calibration verification” means the assaying of
materials of known concentration in the same
manner as patient specimens to substantiate the
instrument or test system’s calibration throughout the
reportable range for patient test results.
CAP Interpretation of CLIA Calibration and
Verification of Calibration
•If calibration satisfies the CLIA requirements for calibration
verification [i.e., calibrated at least every six months with
appropriate calibrators], no further action is necessary
•CAP requirements
• Prove the calibration still is valid (CAP Calibration
Verification)
• Prove response over the entire analytical measurement
range (CAP AMR validation)
Components of CAP Calibration
Verification
• Participants receive a set of vials with varying concentrations of
analyte(s)
• Participants submit results for two assays from each vial, within the same
run if possible
• The CAP provides two individual evaluations and several peer group
summaries
o Calibration verification evaluation
o Peer group summary statistics
o Peer group performance summaries
How to Calibrate
•Linearity Study
• Linearity studies will be performed as part of the procedure
"Evaluation of Automated Test Methods" in order to determine
linear reportable range. For each analyte, a set of linearity
standards will be tested in the same manner as patient
samples.
• Testing should be performed in triplicate, and at a minimum, in
duplicate, when performed within a single run.
• The test results will be graphed and statistically analyzed
Linearity Study
• Review the linearity data for acceptable accuracy and precision.
Ideally, endpoint assays should be within 10% of the
standard’s stated value or peer group comparison value
• Coefficient of Variation, which is a measure of precision, and is
the standard deviation expressed as a percentage of the mean,
ideally should also be less than 10%, or at a minimum, remain
within the threshold of the manufacturer’s stated acceptable
performance.
Criteria for acceptability: 
•  The CLIA criteria for acceptable performance can be applied in
the following way

• For replicate measurements on each level, plot the average

value of the results vs the assigned value. Then the graph is
compared with the Tea(plus minus1 Tea)
• TEa =The sum of random error (imprecision) and systematic error
(bias/inaccuracy) can be calculated from instrument performance
data according to the formula), 2CV + bias(%) or 2SD + bias (analyte
units)