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Hemostasis Test Validation, Performance and
Reference Intervals
Richard A. Marlar, Ph.D.Pathology and Laboratory MedicineOklahoma City VA Medical Center
University of Oklahoma Health Sciences Center
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DISCLOSURE Richard A. Marlar, PhD
Relevant Financial RelationshipsNone
Off Label UsageNone
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Chemistry and Hematology Lab Values
Results expressed as amounts or participlesAlbumin 3-4 g/dLSodium 135-145 mEq/LRBC 4.3-5.9 x106/ mm3
International StandardsSeamless values between methodsExcellent reproducibility
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Chemistry and Hematology Testing Accuracy
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Coagulation Lab Values
Results expressed as arbitrary valuesPTT 32 secFactor VIII 34%FVIII Inhibitor 12 Bethesda units
Few International StandardsValues vary between methods
Heparin Therapeutic range:Reagent A 65-85 secReagent B 85-120 sec
Moderate reproducibility
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Coagulation Testing Accuracy
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, Performance and Reference
Intervals Outline
Validation of New AssayComponents required for validationProtocol for validation
Assay Method PerformanceInternal QC performanceExternal Proficiency Testing
Reference IntervalsBasic principlesCommon methods
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Hemostasis Test Validation
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Bottom-line for Hemostasis Testing
All aspects of hemostasis testing leads to Post Medical Decision MakingTesting parameters should be based on “Evidence Based Medicine”, however Tradition is more commonMust compromise between statistical number and minimal number for evaluation of a test
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Coagulation Laboratory Assays and Procedures
Evaluation of a pathophysiologic condition
Assist in the diagnosis of the cause of bleeding or thrombosisMonitoring Therapy
To have clinical value as an assay:Total error must be low to reflect the biological condition
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New Test Implementation and Validation
Range of tests:FDA approvedAnalyte Specific Reagent (ASR)Research Use Only (RUO)“Home Brew”
All new tests must be systematically validated and regulated
Level of evaluation variesAll new tests must be validatedAll tests must meet a defined level of reliabilityAll tests must continue to be validated on a regular basis
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Coagulation Laboratory Analysis Assay Error
For an analysis to have value:Total error must be low to differentiate between normal and abnormal
General types of error:Individual biological variabilityPre-Analytical variabilityAnalytical variability in test performanceStatistical errorInterfering substances
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New Test Implementation and Validation
Assay Validation ProtocolPurpose of validating is to insure the generation of high quality data for accurate diagnosis of diseaseTo ensure a proper validation, a pre-approved protocol must be usedProtocol must include:
All administrative aspects of testStatistical validation of assayAll test parameters evaluated
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Coagulation Test Implementation and Validation Protocol
Responsibilities
Ultimate responsibility:Laboratory DirectorDepartmental Director
Specific responsibilities:Department SupervisorLaboratory Information Manager (LIM)Quality Assurance Technologist
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Coagulation Test Implementation and Validation Protocol
Supervisor’s Responsibilities
Existence of patentsWritten procedure
Proper formatNot final draftUsually provided by manufacturer
Slide #1
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Coagulation Test Implementation and Validation Protocol
Supervisor’s ResponsibilitiesStatistical Validation study
Varies depending on type of testIncluded Parameters
AccuracyPrecisionAnalytical Sensitivity and SpecificityInterferencesReference Range and AMRLimits of DetectionCorrelation
Slide #2
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Coagulation Test Implementation and Validation Protocol
Supervisor’s Responsibilities
Quality Control procedureControl materialsFrequencyCriteria for acceptability
MSDS InformationComplete test information for LISNecessary information to cliniciansCost analysis and test charge
Slide #3
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Coagulation Test Implementation and Validation Protocol
Supervisor’s Responsibilities
Training Plan:Training methods and materialsList of staff requiring trainingDocumentation of completionDocumentation of competency assessmentNew employee training checklist
Slide #4
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Coagulation Test Implementation and Validation Protocol
LIM’s Responsibilities
Add test information to LIS filesVerify report format
Lab Information SystemHospital Information System
Establish CPT coding and other coding necessary for cost recovery
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Coagulation Test Implementation and Validation Protocol
QA Technologist’s Responsibilities
Add procedure to Document Inventory LogAssign document number
Add signed procedure to SOP manualAdd to Specimen Collection manualAdd to Activity Menu
For accrediting organizationImplement Proficiency Testing procedure
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Validation Parameters Coagulation's Dilemma
Validation varies on type of testFDA approvedResearch Use Only“Home Brew”
AccuracyPrecisionInterferencesReference RangeAMRLimits of DetectionCorrelation
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Hemostasis Test Performance
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Performance of Hemostasis Tests
Necessary to ensure accurate results over timeMust have on-going evaluation of the methodMust be able to compare results
From validation until current runInternal QC:
On-going periodic analysisRequired by regulatory agenciesSet of rules to determine acceptance or failure
External Quality AssessmentPeriodic comparison with other labsBest measure of accuracySet of rules to determine acceptance or failure
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Reference Interval in Coagulation and Hemostasis
INTERPRETATION OF COAGULATION DATA IS A COMPARATIVE DECISION-MAKING PROCESS!Compare patient result to reference intervalReference interval set up for:
“Normal” or healthyPhysiologic conditionsPathologic conditions
Therapeutic reference interval is determined in similar manner
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Establishing Reference Intervals for Coagulation Assays
Ref Interval Criteria
New Analyte Determination is difficultLack data on physiology, pathology and medical aspects
New Method Determination somewhat easierPhysiology and medical aspects known
Transference Easier to determineMust use caution in establishing
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Use of “Transference” for Establishing Reference Interval
Applying previously established ranges (by lab, others or literature) to new assay methods.Methods to accept transference:
Use of inspection and subjectivity of method procedure to determine similar rangeValidation of range with small number of reference samples
Methods not well established for coagulation assays
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Validation of Coagulation Tests Summary
Validation of a coagulation test can be difficultSet up protocol for validation:
Protocol written Approved before starting
Protocol to Include:Administrative issuesValidation with statistical analysis
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Performance Criteria of Coagulation Tests
Summary
Continued evaluation of assay Internal Quality Control
Periodic testing of same sampleMaintain precision and accuracy over timeEstablished rules for acceptability
External Quality ControlMeasure of accuracy and precision
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Reference Intervals Determinations for Coagulation Tests
SummaryReference interval is critical for clinical decision makingDifferent types of reference intervals:
NormalPhysiologicPathologicTherapeutic
Reference interval determinations:New analyteNew methodTransference
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Reference Literature for Validation, Performance and Ref Intervals
Guidelines for Validation of Assays:CLSI Evaluation Documents for Validation Studies (EP05-EP21)
Guidelines for Reference Intervals:CLSI Document on Reference Intervals in the Clinical Laboratory (C28-A2)
Guidelines for Specific Coagulation Tests:CLSI Document on PT and APTT (H47-A2)Numerous CLSI Documents on Coagulation Testing (H series)
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Questions?
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Testing Validation Parameters Coagulation's Compromise
Optimum Practical MeasureAccuracy 100 10-20 +5%Precision 10 within
10 between5 within runs
5 between runs%CV
Interferences 25 of each ~5 of eachReference Range 100 normals 20-40 normals Mean +2 SD
AMR 50-100 20-40Limits of Detection 25
undetectable5-10
undetectable+3 SD of
zeroCorrelation 200 20-25 R > 0.90
Slope ~1.0