Calibration Verification Guidelines and Regulations

Presentation Topics & Objectives Define difference between Calibration andCalibration Verification What are acceptable Calibration Verificationmaterials How and when Calibration Verification is to beperformed Troubleshooting for a failed CalibrationVerification

Let’s Start From the Beginning January 24th, 2003 Centers for Disease Controland Prevention (CDC) and the Centers forMedicare & Medicaid (CMS) publish laboratoryregulations (CLIA) to become effective April24th, 2003Include requirements for Calibration andCalibration Verification for nonwaived tests Nonwaived tests include moderately and highcomplexity tests

Discussion of Terminology Definitions and applications of following: CalibrationCalibration VerificationReportable RangeAnalytical Measurement Range (AMR)

Definitions Calibration: is the process of testing andadjusting the instrument or test systemreadout to establish a correlation between theinstrument’s measurement of the substancebeing tested and the actual concentration ofthe substance Basically “Tells” the instrument how to read acertain level of the analyte being tested

Definitions Continued Calibration Verification: means testingmaterials of known concentration in the samemanner as patient specimens to assure thetest system is accurately measuring samplesthroughout the reportable range

Definitions Continued Reportable Range: the span of testresult values over which the laboratorycan establish or verify the accuracy ofthe instrument or test systemmeasurement response

Definitions Continued Analytical Measurement Range (AMR):Defined by CAP (College of American Pathologists)as the range of numeric results a method canproduce without any special specimen pre-treatment,such as dilution, that is not part of the usual analyticprocess. (Same as reportable range in CLIAterminology.)

Definitions Continued Linearity As defined by CLSI’s (formerlyNCCLS) EP06-A guideline: The ability (withina given range) to provide results that aredirectly proportional to the concentration(amount) of the analyte in the test sample.Note: “Linearity” does not appear in CLIAregulations.

Calibration Vs. Calibration Verification Calibration Verification essentially checks theCalibration of the analyzer by confirming thatthe test system is able to provide accurateresults throughout the AnalyticalMeasurement Range of the instrument for thatparticular analyte.

In a Nutshell Calibration “tells” the instruments how toread certain analyte concentrations Calibration Verification tests to make sure thecalibrators are accurate across theinstrument's reportable range


Calibration Requirements Laboratory is responsible for performingcalibration as directed by the manufacturer’stest system instructions AND whenCalibration Verification does not produceacceptable results Make sure to document each time calibration isperformed More frequent calibration may be required if thetest system’s calibration is less stable thaninstrument manufacturer’s suggested frequency

Exceptions to Calibration Manual procedures in which an instrument isnot used Microbiology culturesTilt-tube prothrombin timesABO group and D (Rho) typing

Exceptions to Calibration Continued Microscopic procedures Urine sediment examinationKOH and pinworm preparationsAll manual cell differential proceduresManual Cytology screening proceduresProcedures which involve instruments inwhich calibration is not practical Prothrombin procedures

How to Perform Calibration The manufacturer’s instructions of the testsystem should explain the process forcalibration FrequencyReplicatesAnalyte concentrationAcceptable material

Calibration Materials Calibration materials, also called calibratorsare solutions of known analyteconcentrations May contain more than a single analyte The term “standards” generally used to refer tocalibration material


Reasons to Perform Calibration VerificationCan Detect Hook Dose EffectLDL Cholesterol120Actual Value (mg/dL) y 0.4024x 17.346R2 0.876810080Actual Value6040Linear (ActualValue)200050100150200250Predicted Value (mg/dL)300

Reasons to Perform Calibration VerificationContinued Test method may have limited number ofcalibrators May have only one or two calibratorsTest method calibrators may not span theinstrument’s reportable range Even if the test method has three calibrators(minimum number of calibrators required to beexempt from Calibration Verification, may notadequately span AMR)

Reasons to Perform Calibration VerificationContinuedDaily Quality Controls generally do notchallenge lower and upper limits Daily QC normally targets Normal and Abnormalpatient ranges onlyIt is possible to have “Passing” Daily QC, but“Failing” Calibration Verification since controls maynot span entire analyzer range Especially important in Therapeutic Drug Monitoring

Calibration Verification Requirements Labs must perform every 6 months (or morefrequently if specified in the test system’sinstructions) or if: There is a change in reagent lot numbers for aparticular analyte—unless the laboratory canshow that the new lot of reagent does not affectthe range of the patient test results and that dailyquality control results are not adversely affectedby changing reagent lots

Calibration Verification RequirementsContinued There is major preventative maintenance orreplacement of critical instrumentcomponents which may affect test patientresults Includes analyzer being sent to manufacturer forrepairs

Calibration Verification RequirementsContinued Quality control materials begin to shift or areout of the laboratory’s acceptable limits andother methods of checking the accuracy ofthe control results are not able to ascertainand resolve the problem The individual laboratory has determined thereportable range for patient test results are tobe monitored more frequently

Calibration Verification RequirementsContinued Document each time Calibration Verification isperformed IMPORTANT NOTE: The laboratory must alsobe able to verify calibration on factorycalibrated instruments that cannot becalibrated by the end user

What Are Acceptable Calibration VerificationMaterials? Materials of Known ConcentrationsCalibration Materials Proficiency Testing samples with knownresults Commercially available material Controls with known values Patient samples with known values

Calibration Verification Exceptions For Automated Cell Counters: Follow manufacturer’s instructions for analyzeroperation and tests two levels of control eachday of patient testing and results are within lab’sacceptable rangeCalibration Verification is met if: Running 3 or more levels of calibrator andincludes a low, mid, and high value, and isperformed at least once every 6 months

Calibration Verification ExceptionsContinued If laboratory follows manufacturer’sinstruction for analyzer operation and runsthree levels of control material:– on a routine basis (low, mid, and high)– more than once each day patient resultsare reported– and the control results meet the lab’scriteria for acceptability– and the controls are NIST traceable, thecalibration verification requirements havebeen met

Calibration Verification Review When: Every 6 Months or whenever thefollowing occurs: New lot of reagent used (unless lab can verifythat there is no change in results)Major preventative maintenance or replacementof critical test method componentsControls indicate shift or are out of thelaboratory’s acceptable rangeLaboratory determines more frequent verificationrequired

How to PerformCalibrationVerification – Toolsand Resources

How To Perform Calibration Verification Document Date, Analyte, and Calibration Verification material usedRun 3 levels of material Run low, mid, and high levels as if running patient samplesRecord dataCLIA does not state minimum number of replicates Compare results to expected values Document results and determine if results meetlaboratory’s acceptable criteria

Sample Calibration Verification ResultsWorksheet

How To Perform Calibration VerificationContinuedPlot data on Linear Graph Computer program - spreadsheetCommercial ProgramsManufacturer material and programsThird Party control manufacturer and programs

Sample Calibration Verification ResultsSpreadsheet and GraphAnalyteTechnicianGlucoseSmithTerryGen. Chem. Lin. Lot Gen. Chem. Lin. Exp.9/22/201106107Date Lmg/dLmg/dLSLOPEINTERCEPTCOEFFICIENT R1.00160.68430.9999Reagent LotReagent Exp.MethodUnitsmg/dLPredicted Value Actual Value98200202401397597591783785Actual V alue (m g/dL)Glucosey 1.0016x 0.6843R2 0.99991000800600Actual Value400Linear (ActualValue)20000200400600800Predicted Value (mg/dL)1000

Sample Calibration Verification ResultsSpreadsheet and Graph

So, Do I Pass?What areAcceptableResults?

So What Are Acceptable Results?The Million Dollar Question (drum rollplease.):How do I know if my resultsare “Good Enough”?

What is Acceptable CalibrationVerification?—The Ultimate Question Each laboratory is responsible forestablishing acceptable parametersSome labs use CLIA ’88 Proficiency Testing LimitsGeneral Rule of Thumb used by some inspectorsand laboratory managers: R Coefficient (Also R2): 0.98 – 1.00 Tells you is data is linearSlope: 0.90 – 1.10 ( /- 10%) Tells you how well you match up to the expecteddata

Goal of Calibration Verification Confirm that the test system is reportingaccurate results throughout the testsystem’s reportable range

What if CalibrationVerification Fails?

How to Address Failed Cal VerAs per CLIA guidelines: Repeat calibration procedureGood Laboratory Practice to run controls afterrecalibrating and before reporting patient resultsIf the instrument is factory-calibrated, contactinstrument manufacturer

Additional Troubleshooting Calibration Verification Troubleshooting Number of variables to be considered QC MaterialReportable Range of InstrumentChanges in ReagentMaintenance LogsEnvironmental ConditionsInstrument ServicingChanges in OperatorsCompare on similar instrument

Troubleshooting Checklist

Moving Forward –Current and FutureCalibrationVerification Topics

Point-of-Care Testing and CalibrationVerificationIf a lab is running nonwaived tests on POCTinstruments, Calibration Verification may berequired Some anlayte panels on the Abbott i-STAT require Calibration Verification

Top Reasons for Lab CitationsExcerpt from Advance for Administrators ofthe Laboratory November 22, 2010:– Fourth most common reason for lab noncompliance citation:#4 - “not performing calibration verification according to themanufacturer's instructions including: the number, type andconcentration of materials to be used; use of materials at low,medium and high values within the reportable range asdetermined by the laboratory; acceptable limits for calibrationverification, once every 6 months or more often, if required bylaboratory procedures;”

References andResources

References CLIA Brochure #3: LabGuide, 5/07“The State Operations Manual,” Appendix CInterpretive Guidelines, Calibration and CalibrationVerification Procedures (§ 493.1255)—CMS website ronol.aspx accreditation/audio conferences/cvl webinar tions.aspx

Resources Article by Kathryn Connolly Quality ManagementSystems Accreditation Manager for orial/Conte CLSI (Formerly NCCLS) Document EP6-A – Evaluation ofthe Linearity of Quantitative Measurment Procedures: AStatistical Approach; Approved Guideline; ISBN 1-56238498-8; ISSN 0273-3099 dgrhoads website: CMS website:

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