InstrumentationLaboratory

Lab Screening for Lab Screening for Hemorragic Congenital Hemorragic Congenital

CoagulopathiesCoagulopathies

Strategy and approachStrategy and approach

InstrumentationLaboratory

VWD Panel: VWF Antigen & Activity VWD Panel: VWF Antigen & Activity AssaysAssays

FII, FV, F VII, FX Clotting AssaysFII, FV, F VII, FX Clotting Assays FVIII, FIX, FXI, FXII Clotting AssaysFVIII, FIX, FXI, FXII Clotting Assays ACL TOP and ACL Elite Factor ACL TOP and ACL Elite Factor

ParallelismParallelism FXIII AntigenFXIII Antigen

InstrumentationLaboratory

VWD Panel: VWF Antigen & Activity VWD Panel: VWF Antigen & Activity AssaysAssays

InstrumentationLaboratory VWD typesVWD types

InstrumentationLaboratory VWD DiagnosisVWD Diagnosis

VWD typing is a complex and multi-technology process.

InstrumentationLaboratory HemosIL VWD PanelHemosIL VWD Panel

HemosIL VWF AntigenHemosIL VWF Antigen HemosIL VWF ActivityHemosIL VWF Activity

– Automated Latex immunoturbidrimeticAutomated Latex immunoturbidrimetic

– Applications on ACL Elite/Elite Pro, ACL Advance, ACL Applications on ACL Elite/Elite Pro, ACL Advance, ACL TOPTOP

– VWF Antigen and Activity values assigned on the VWF Antigen and Activity values assigned on the HemosIL Plasma Calibrator and ControlsHemosIL Plasma Calibrator and Controls

InstrumentationLaboratory HemosIL VWD PanelHemosIL VWD Panel

HemosIL VWF AntigenHemosIL VWF Antigen

Latex particles coated Latex particles coated with rabbit polyclonal with rabbit polyclonal antibody specific for VWF antibody specific for VWF antigenantigen

HemosIL VWF ActivityHemosIL VWF Activity

Latex particles coated with anti-VWF mouse monoclonal antibody directed against the platelet binding site of VWF (Glycoprotein Ib receptor)

Plasma VWF triggers the agglutination of the latex particles Plasma VWF triggers the agglutination of the latex particles measured at 405 nm.measured at 405 nm.

InstrumentationLaboratory HemosIL VWD panelHemosIL VWD panel

InstrumentationLaboratory VWF Activity: definitionVWF Activity: definition

The activity of VWF is:The activity of VWF is: Binding of circulating FVIII with the function of Binding of circulating FVIII with the function of

stabilizing it and protecting from degradationstabilizing it and protecting from degradation

Adhesion of platelets to injured vesselsAdhesion of platelets to injured vessels– By interacting with the collagen present in the sub-By interacting with the collagen present in the sub-

endotheliumendothelium

– This bound induces VWF to interact with platelet This bound induces VWF to interact with platelet receptors: Glycoprotein Ib of inactivated platelets and receptors: Glycoprotein Ib of inactivated platelets and glycoprotein IIb-IIIa of activated plateletsglycoprotein IIb-IIIa of activated platelets

Multimer size is important for normal biological Multimer size is important for normal biological function: the larger is the most activefunction: the larger is the most active

InstrumentationLaboratory HemosIL VWF ActivityHemosIL VWF Activity

VWF: RcoVWF: Rco– Platelet aggregationPlatelet aggregation

– VWF causes agglutination of VWF causes agglutination of stabilized platelets in the stabilized platelets in the presence of ristocetinpresence of ristocetin

– Ristocetin, in vitro, mimics the Ristocetin, in vitro, mimics the action of collagen in modifying action of collagen in modifying the VWF conformation, thus the VWF conformation, thus facilitating the binding with facilitating the binding with the platelet receptor the platelet receptor glycoprotein Ib.glycoprotein Ib.

– The agglutination degree can The agglutination degree can be measured in be measured in Aggregometers (Biodata, Aggregometers (Biodata, Helena, Dade) or Coagulation Helena, Dade) or Coagulation analyzers (Dade)analyzers (Dade)

HemosIL VWF: HemosIL VWF: AcAc– Latex immunoturbidimetricLatex immunoturbidimetric

– Anti-VWF antibody coated Anti-VWF antibody coated on Latex particles and on Latex particles and directed against the platelet directed against the platelet binding site of VWF binding site of VWF (glycoprotein Ib receptor)(glycoprotein Ib receptor)

– Concept usually applied to Concept usually applied to ELISA (by Axis-Shield; ELISA (by Axis-Shield; Corgenix). Corgenix).

InstrumentationLaboratory Correlation with RCoCorrelation with RCo

InstrumentationLaboratory VWD DiagnosisVWD Diagnosis

InstrumentationLaboratory

InstrumentationLaboratory VWF Activity: ReferencesVWF Activity: References

New Automated von Willebrand Factor Activity Assay to Distinguish Type 1 and Type 2 von Willebrand Disease.M. Piñol1, M. Costa, M. Sales, M.T. Canciani, A.B. FedericiXIX ISTH, Birmingham, UK July 12 – 18, 2003

Comparison of a new rapid automated VWF:Activity assay with a visual agglutination VWF:RCo assay in screening for VWD.JM Smith, A Bowyer, J Storr, M Makris, S KitchenHaemophilia 2004 : 10; 19

Comparison of a von Willebrand Factor Collagen Binding Assay with a Latex-Enhanced Turbidimetric Immunoassay for the Determination of von Willebrand Factor activity in Plasma.Telting d. et al.XVIIth International Symposium on Technologycal Innovation in Laboratory Hemtalogy, May 13-15, 2004 Spain.

InstrumentationLaboratory VWF Activity: ReferencesVWF Activity: References

Determination of von Willebrand Factor Activity: Evaluation of the HemosIL Assay in Comparison with established Procedures .Sucker C. et al.Clinical and Applied Thrombosis/Hemostasis, 12:3, 305 – 310, 2006

Comparison of a new Automated von willebrand Factor Activity Assay With an Aggregation von Willebrand Ristocetin Cofactor Activity Assay for the Diagnosis of von Willebrand Disease.Vleeschawer etal.Blood Coagulation & Fibrinolysis 17(5): 353-358, 2006

Evaluation of a new turbidimetric assay for von Willebrand factor activity useful in the general screening of von Willebrand diseaseM. PIÑOL, M. SALES, M. COSTA, J. SERRA, A. TOSETTO, M. T. CANCIANI, A.B. FEDERICIHaematologica (submitted 2006)

InstrumentationLaboratory

Intrinsic and Extrinsic clotting Intrinsic and Extrinsic clotting AssaysAssays

InstrumentationLaboratory

HemosIL Factor Def PlasmaHemosIL Factor Def PlasmaPanelPanel

InstrumentationLaboratory

HemosIL Factor Def PlasmaHemosIL Factor Def PlasmaPanelPanel

Main FeaturesMain Features– Immuno-depleted Factor Deficient PlasmaImmuno-depleted Factor Deficient Plasma– Factor level < 1% (all remaining at optimal level)Factor level < 1% (all remaining at optimal level)– Format: 5 x 1 mL (lyo)Format: 5 x 1 mL (lyo)– Stability at 2-8 °C : 24h after reconstitutionStability at 2-8 °C : 24h after reconstitution– Stability on-board : 24h after reconstitutionStability on-board : 24h after reconstitution

InstrumentationLaboratory

ACL TOPACL TOPFactor ParallelismFactor Parallelism

InstrumentationLaboratory Factor ParallelismFactor Parallelism

What is Factor Parallelism?What is Factor Parallelism?

– Measurement of Factor Activity Levels at different Measurement of Factor Activity Levels at different sample dilutionssample dilutions

– Comparison of Plasma sample results at different Comparison of Plasma sample results at different dilutions with Calibration results at different dilutionsdilutions with Calibration results at different dilutions

Determination of Factor Activity levelsDetermination of Factor Activity levels Uncover presence of inhibitorsUncover presence of inhibitors

InstrumentationLaboratory Factor ParallelismFactor Parallelism

Why is Factor Parallelism used?Why is Factor Parallelism used?

– Increase the quality of the results for factor assaysIncrease the quality of the results for factor assays

– Uncover possible interferences that might affect the Uncover possible interferences that might affect the test results of the “single dilution test”test results of the “single dilution test”

Lupus AnticoagulantsLupus Anticoagulants HeparinHeparin Factor InhibitorsFactor Inhibitors

InstrumentationLaboratory Factor ParallelismFactor Parallelism

How to use Factor Parallelism:How to use Factor Parallelism:

– 2-3 dilutions of the test plasma2-3 dilutions of the test plasma

– The result of a sample dilution should fall within the The result of a sample dilution should fall within the working range of the assayworking range of the assay

– Results of the various test plasma dilutions should Results of the various test plasma dilutions should demonstrate parallelism with respect to those of the demonstrate parallelism with respect to those of the calibration plasmacalibration plasma

– Ideally test plasma and calibration plasma dilutions Ideally test plasma and calibration plasma dilutions should be the sameshould be the same

InstrumentationLaboratory Factor ParallelismFactor Parallelism

Criteria to define factor parallelism:Criteria to define factor parallelism:

– No official guidelinesNo official guidelines

– Based on mathematical or statistical analysisBased on mathematical or statistical analysis

– Examples:Examples: Comparison of slopes (test plasma dilution curve vs Comparison of slopes (test plasma dilution curve vs

calibration plasma dilution curve)calibration plasma dilution curve)

Check of R2 of test plasma dilution curveCheck of R2 of test plasma dilution curve

Check variance of recalculated results between first Check variance of recalculated results between first dilution (100%) and subsequent dilutionsdilution (100%) and subsequent dilutions

InstrumentationLaboratory Factor ParallelismFactor Parallelism

Effects of Heparin & Lupus Effects of Heparin & Lupus Anticoagulant interference:Anticoagulant interference:

– Prolongation of aPTT clotting times may result on Prolongation of aPTT clotting times may result on falsely detected low factor concentration resultsfalsely detected low factor concentration results

– With test plasma at increasing dilutions may With test plasma at increasing dilutions may decrease final heparin/Lupus concentration, thus decrease final heparin/Lupus concentration, thus recalculated results may increase with the sample recalculated results may increase with the sample dilutiondilution

InstrumentationLaboratory Factor ParallelismFactor Parallelism

Factor inhibitors interference:Factor inhibitors interference:

– Factor inhibitors may be weak or strong inhibitors, Factor inhibitors may be weak or strong inhibitors, therefore the effect of sample dilution may or may therefore the effect of sample dilution may or may not uncover their presencenot uncover their presence

InstrumentationLaboratory Factor Parallelism: ACL TOPFactor Parallelism: ACL TOP

User definable report: may define or select up to 4 Reporting UnitsUser definable report: may define or select up to 4 Reporting Units

InstrumentationLaboratory Factor Parallelism: ACL TOPFactor Parallelism: ACL TOP

Factor Parallelism Criteria:Factor Parallelism Criteria:

InstrumentationLaboratory ACL TOP: Normal sampleACL TOP: Normal sample

InstrumentationLaboratoryACL TOP: Sample with inhibitorsACL TOP: Sample with inhibitors

InstrumentationLaboratory

ACL Elite/Elite ProACL Elite/Elite ProFactor ParallelismFactor Parallelism

InstrumentationLaboratory

ACL Elite/Elite Pro:ACL Elite/Elite Pro:Factor ParallelismFactor Parallelism

3 Sample Dilutions (100%; 50%; 25%)3 Sample Dilutions (100%; 50%; 25%) Dilutions Automatically done by the instrumentsDilutions Automatically done by the instruments Calculations and Results Evaluation Calculations and Results Evaluation

automatically performed by the system and automatically performed by the system and reportedreported

1 Replicate/Level1 Replicate/Level Available for FVIII and FIXAvailable for FVIII and FIX

– APTT -SPAPTT -SP Library 1.01 – 1.09Library 1.01 – 1.09– APTT SynthasilAPTT Synthasil Library in developmentLibrary in development

Representation of results numericalRepresentation of results numerical

InstrumentationLaboratory Parallelism - Calculation SetupParallelism - Calculation Setup

Elite Series performs 3 Fixed Dilutions on

Samples(1 replicate/Level)

Dilution 1 = 1:5

Dilution 2 = 1:10

Dilution 3 = 1:20

InstrumentationLaboratory Parallelism - Calculation SetupParallelism - Calculation Setup

User can select to display and print 4 of the User can select to display and print 4 of the following:following:

CR% = Corrected Result % CR% = Corrected Result % AveCR% = Average of the Corrected Results AveCR% = Average of the Corrected Results

in %in % CV-CR% = CV of the Corrected Results in % CV-CR% = CV of the Corrected Results in % Slope = Slope of the linear regressionSlope = Slope of the linear regression

(seconds vs. uncorrected % result)(seconds vs. uncorrected % result) Intercept = Intercept - linear regression lineIntercept = Intercept - linear regression line RR22 = Correlation coefficient - linear = Correlation coefficient - linear

regression lineregression line

InstrumentationLaboratory Parallelism Screen ReportParallelism Screen Report

Results for 3 dilutions are reported in

•Seconds

•%

•Recalculated %

•Mean Recalculated %•CV Recalculated %•Slope•Intercept •R2

InstrumentationLaboratory Parallelism Printed Detail ReportParallelism Printed Detail Report

This report displays the individual dilution results plus the corrected % activity results.

InstrumentationLaboratory Parallelism Printed Sample ReportParallelism Printed Sample Report

This report displays recalculated results

InstrumentationLaboratory

FXIII Antigen AssayFXIII Antigen Assay

InstrumentationLaboratory FXIII BiochemistryFXIII Biochemistry

InstrumentationLaboratory FXIII BiochemistryFXIII Biochemistry

InstrumentationLaboratory FXIII in the Coag CascadeFXIII in the Coag Cascade

InstrumentationLaboratory FXIII DeficiencyFXIII Deficiency

InheritedInherited– Frequency 1: 5,000,000Frequency 1: 5,000,000

– Types: Subunit A or Subunit BTypes: Subunit A or Subunit B

– In type A usually undetectable levels of FXIIIIn type A usually undetectable levels of FXIII

– No cases of normal Factor XIII antigenic levels with impaired FXIII activity levels have been reported so far

– The bleeding tendency typically becomes obvious when FXIII levels are <1-2%.

– However severe bleeding episodes may occur with FXIII levels in the range of 30% - 50%.

InstrumentationLaboratory FXIII DeficiencyFXIII Deficiency

AcquiredAcquired

– malignant hematological diseases– severe liver disease – DIC – acute stages of inflammatory gastrointestinal

disease – Henoch Schoenlein purpura

InstrumentationLaboratory Measurement methodsMeasurement methods

COMPETITIVE ASSAYS

Katona (Thromb Haemost 2000: 83: 268-73) Lim W et al ( J Thromb Haemost 2004; 2: 1017-1018)

– the clot solubility tests only detect severe FXIII deficiencies with FXIII activity levels of 0 – 3%, thus making these tests unsuitable for detecting FXIII activity levels >5%.

– Concerns regarding the Berichrom FXIII assay analytical sensitivity. Katona reports that the Berichrom assay is relatively insensitive and below FXIII levels of 5% the accuracy and the imprecision are not reliable.

As presented by the survey conducted by the FXIII working party of European Thrombosis Research Organization (ETRO), following a study on 72 patients a FXIII activity >5% can also be severe (Seitz R et al. Sem thromb Hemost 1996; 22: 415-8).

InstrumentationLaboratory HemosIL FXIII AntigenHemosIL FXIII Antigen

CONCEPT: Immunoturbidimetric assay which measures specifically the subunit A of the FXIII tetramer

The inherited FXIII deficiencies reported so far have decreased antigenic and activity levels. There have been no known cases in which the deficiency was caused by decreased activity, with normal antigenic levels.

In addition, the very rare deficiencies affecting the subunit B also influence the plasma levels of the subunit A (Katona E. et al. Thromb Haemost 2000: 83: 268-73). In these cases, the tetramer is not detectable, while the subunit A is detectable but with low concentrations.

InstrumentationLaboratory HemosIL FXIII AntigenHemosIL FXIII Antigen

CONCEPT: Immunoturbidimetric assay which measures specifically the subunit A of the FXIII tetramer

In acquired deficiencies, the measured antigenic levels of FXIII-A would be proportional to the activity levels even though they may not be identical (Lim W et al J Thromb Haemost 2004; 2: 1017-1018) particularly after replacement therapy.

InstrumentationLaboratory HemosIL FXIII AntigenHemosIL FXIII Antigen

InstrumentationLaboratory HemosIL FXIII AntigenHemosIL FXIII Antigen

InstrumentationLaboratory HemosIL FXIII AntigenHemosIL FXIII Antigen

CONCLUSIONSCONCLUSIONS

– HemosIL FXIII Antigen is a liquid ready to use HemosIL FXIII Antigen is a liquid ready to use immunoturbidimetric assayimmunoturbidimetric assay

– Automated on ACL Futura/Advance and ACL TOPAutomated on ACL Futura/Advance and ACL TOP

– Detects the Active subunit of FXIIIDetects the Active subunit of FXIII

– Simplify and facilitates the laboratory investigation Simplify and facilitates the laboratory investigation of FXIII deficiencyof FXIII deficiency