diagnóstico trombofilia

Diagnosis of thrombophiliaDiagnosis of Thrombophilia - Presentation Diagnostica Stago - all rights reservedrev. CMarch 2012

Training in Haemostasis, 2007 V2

Diagnosis of thrombophiliaDefinition of thrombophilia

Deficiencies in inhibitorsAntithrombinProtein CProtein S


Definition of thrombophiliaThrombophilia can be defined as a tendency to thrombosisOnly weakens the coagulation response to cope with environmental fluctuationsDoes not necessarily cause clinical impairment = RISK FACTORInteraction required with other components before onset of clinical disordersInherited thrombophilia is a genetically determined tendency to develop thrombosis


Inherited thrombophiliaSymptomsFeaturesClinical: venous thromboembolism Spontaneous or out of proportionUsually deep vein thrombosis of lower limbs, pulmonary embolism, superficial thrombophlebitis, sometimes unusual locations

Includes deficiencies of 3 natural anticoagulant proteinsAntithrombinProtein CProtein SIncludes also specific mutations in the genes for factor V (factor V Leiden) and factor II or prothrombin (prothrombin 20210G>A)Prevalence of these deficiencies is low


Acquired thrombophiliaSymptomsFeaturesNo family historyOccurs in associationwith a trigger event e.g. trauma, pregnancy, puerperium, surgery, immobilisation, postoperative period ...

with other clinical disorders, e.g. malignancy, nephrotic syndrome, HIT

with Lupus Anticoagulant (LA) and Antiphospholipid antibodies (APA)


Acquired risk factors and interactions with inherited thrombophiliaVarga EA. Clinical Genetics, 2012, 81(1): 7-17


Situational risk factors (1/2)Cancersix- to sevenfold increased risk of VTE to those without cancermagnitude of risk varies with cancer type, extent of disease and increases during treatment

Central venous cathetersstrongest risk factor for upper extremity thrombosistwo- to threefold higher in factor V Leiden and prothrombin 20210G>A heterozygotesVarga EA. Clinical Genetics, 2012, 81(1): 7-17


Situational risk factors (2/2)Immobility and hospitalizationrisk of VTE increased with all types of immobility including bed rest, lower extremity plaster casts and paralysis or weakness resulting from neurologic diseaseprolonged bed rest confers five- to sixflod increase in riskshorter periods of bed rest and reduced use of an extremity are associated with a two- to threefold increased thrombotic risk

Surgeryall types of surgery increase the risk of VTE

Traumarisk of VTE is nearly 13-fold after major trauma requiring hospitalizationthe risk increases with the severity of the injuries and is highest in those with multiple limb or plevic fracturesVarga EA. Clinical Genetics, 2012, 81(1): 7-17


Inherited thrombophiliasprevalence and relative risksVarga EA. Clinical Genetics, 2012, 81(1): 7-17


Inherited thrombophilia and pregnancy-associated VTEVarga EA. Clinical Genetics, 2012, 81(1): 7-17


Haemostasis balanceActivation and inhibitory pathwaysInhibitors FactorsRISK FACTORS FOR THROMBOSIS


TFPIATPSAPCAT:Antithrombin APC: Activated Protein CPS: Protein STFPI: Tissue Factor Pathway InhibitorTF: Tissue Factor


Heritable thrombophilia screening:who should be screened? (1/6)Neonates and children with purpura fulminans should be tested urgently for protein C and S deficiency (grade 1B).

A variety of functional methods may be required to identify specific severe type 2 functional defects when levels of protein C or S are not

Heritable thrombophilia screening:who should be screened? (2/6)In pregnant women:pregnancy-associated venous thrombosis primarily in relation to clinical risk

previous event due to a minor provoking factor, e.g. travel, should be tested and considered for prophylaxis if a thrombophilia is found.

asymptomatic pregnant women with a family history of venous thrombosis be tested if an event in a first-degree relative was unprovoked, or provoked by pregnancy, combined oral-contraceptive exposure or a minor risk factor (grade 2C). The result will be more informative if the first-degree relative has a known thrombophilia.Baglin T. British Journal of Haematology, 2010, 149: 209-220


Recommendations for laboratory testing in case of venous thrombosis (3/6)Testing at the time of acute venous thrombosis is not indicated.As treatment of acute venous thrombosis is not influenced by test results, testing can be performed later if indicated.

Initiation and intensity of anticoagulant therapy following a diagnosis of acute thrombosis should be the same in patients with and without heritable thrombophilia (grade 1B).

Decisions regarding duration of anticoagulation in unselected patients should be made with reference to whether or not a first episode of venous thrombosis was provoked or not, other risk factors, and risk of anticoagulant therapy-related bleeding, regardless of whether a heritable thrombophilia is known (grade 1B).Baglin T. British Journal of Haematology, 2010, 149: 209-220


Recommendations for assays (4/6)PT testing should be measured to detect the effect of oral VKAs, which will cause a reduction in protein C and S levels.

Functional assays should be used to determine antithrombin and protein C levels.

Chromogenic assays of protein C activity are less subject to interference than clotting assays and are preferable.

Immunoreactive assays of free protein S antigen are preferable to functional assays. If a protein S activity assay is used in the initial screen, low results should be further investigated with an immunoreactive assay for free protein S.

Baglin T. British Journal of Haematology, 2010, 149: 209-220


Recommendations for assays (5/6)If an APC (Activated Protein C) resistance assay is performed to detect the F5G1691A then the modified APC sensitivity test (predilution of the test sample in factor V-deficient plasma), as opposed to the original APC sensitivity tests should be used. If positive the mutation should be confirmed by a direct genetic test. An APC resistance assay is unnecessary if a direct genetic test for F5G1691A is used initially.

Repeat testing for identification of deficiency of antithrombin, protein C and protein S is indicated and a low level should be confirmed on one or more separate samples. Deficiency should not be diagnosed on a single abnormal result.

Baglin T. British Journal of Haematology, 2010, 149: 209-220


Recommendations for interpretation (6/6)Rigorous internal quality control assurance and satisfactory participation in accredited external quality assessment schemes are mandatory.

Thrombophilia testing must be supervised by experienced laboratory staff and the clinical significance of the results must be interpreted by an experienced clinician who is aware of all relevant factors that may influence individual test results in each case.Baglin T. British Journal of Haematology, 2010, 149: 209-220


Laboratory testing for inhibitorsActivity (functional) assaysClotting test

Chromogenic assay

Antigenic assaysImmunological assay


Preanalytical variablesSample:Sampling on citrate 0.109M (1 vol for 9 blood vol blood)No polystyrene tubeTransportation at room temperature Analysis 4 hours after blood samplingFresh or frozen plasma without plateletsPlatelets < 10.109 /L (10 000 / L)Double-centrifugation Quick defrosting in 37C, homogenization of the plasma and the immediate testing


Antithrombin (AT)


Antithrombin (AT)Glycoprotein synthesised in the liverMajor inhibitor of factors IIa and XaNormal plasma level usually between 80 and 120 %AT congenital deficiency: levels of 40 to 60 %


Antithrombin (AT)Physiological variationAT level is low until the age of 6 monthsMarked decrease after the 13th week of pregnancy and during post partum AT level in women up to the menopause is lower than in men

Acquired AT deficiencyHeparin therapy, severe liver disease, nephrotic syndrome, preeclampsia, DIC,


Hereditary AT deficiencyType I:True deficiency Low antigenic and functional levelsType II:Presence of abnormal moleculesNormal antigenic BUT low functional levels RS, effect on Reactive SiteHBS, effect on Heparin Binding SitePE, Pleiotropic (or multiple) effectsPrevalence in general population 1:5000


STA-Stachrom AT IIIChromogenic testDetection of different types of AT deficiencies (I & II)Fully automtaed on STA line

Liatest AT IIIImmuno-turbidimetric assayQuantification of the antigenic ATManual assay

Stago reagents for AT


Diagnosis strategyAllows diagnosis of any type of deficienciesThe more oftenly used

* RS: Reactive Site**HBS: Heparin Binding Site


No AT deficiencyConfirm abnormalityon a new sample, same test

Normal Abnormal> 80% < 80%Chromogenic assay STA-Stachrom AT III

Normal Abnormal > 80% < 80%

Confirm discrepancy AT deficiencyQualitative defect (rare) Proposal for a diagnosis approach+ CriteriaofthrombophiliaAntigenic determinationActivity assayAntigenic determination (LiatestAT III)


STA-Stachrom AT IIISTA-Stachrom AT III 3 (4x3mL)STA-Stachrom AT III 6 (4x6mL)Functional assayAutomated testLong stability7 days on STA -R and STA -Compact8 days sur le STA Satellite 21 days at 2-8C (original vial)Clinically validatedDetection of all AT deficiencies


STA-Stachrom AT III - PrincipleATheparin+Chromogenic substrate CBSParanitroanilineMeasure at = 405 nm+excessIIaIIaIIaIIa+IIaexcess ofIIaPatientIIaATIntensity of the coloration is inversely proportional to the AT level in the plasmaGive the specificity and the sensitivity of the test


STA-Stachrom AT III - SpecificityMain featuresShort time reaction (60 sec)High concentration of heprainBovin thrombin (FII) + specific bufferAn anti-IIa based assay functional assayAllows the detection of some variants not evidenced by other activity techniques (anti-Xa)Allows detection of HBS type II variantsRare: incidence 1:300Cambridge II, Stockholm ATNot sensitive to heparin cofacteur II (HCII)


Perry et al. Thromb Haemost 1998; 79 : 249-253 Ala384Ser mutation: Cambridge type II variant Using an anti-Xa based assay individual with the Ala384Ser mutation, approximately 50% of cases can be missed


Antovic et al. Lab Hematol. 2002; 8: 37-40Some type II deficiencies are not detected with a factor Xa inhibition-based test


ANTITHROMBIN


Competition AT


Siemens versus StagoBerichrom ATIIIThrombin a bovin (Lyo) 5/15 mLSubstrate 3/6 mLBuffer 30/100 mLStability4-48 h on-board (system dependent)2 weeks at 2-8CTurn around time3 minTests number50 testsNot sensitive to Cambridge & Stockholm variantsSTA-Stachrom AT IIIBovine thrombin (Lyo) 3/6 mLSubstrate CBS 61.50 Buffer with heparinStability7 days on-board21 days at 2-8CValidated on instrument

Turn around time60 secTests number30 (x3 vials)60 tests (x6 vials)Detection of all type II Cambridge & Stockholm variantsNot sensitive to heparin cofacteur II (HC II)


Siemens versus StagoInnovance AThuman FXa (liquid) 5/15 mLSubstrate 3/6 mLBuffer 30/100 mLStability4-48 h on-board (system dpt)4 weeks at 2-8CTAT3 minTests number50 testsNot sensitive to Cambridge & Stockholm & Denver variantsSTA-Stachrom AT IIIBovine thrombin (Lyo) 3/6 mLSubstrate CBS 61.50 Buffer with heparinStability7 days on-board21 days at 2-8CValidated on instrument

TAT60 secTests number30 (x3 vials) 60 tests (x6 vials)Detection of type II Cambridge & Stockholm & Denver variantsNot sensitive to heparin HC II


IL versus StagoLiquid Antithrombinbuffered bovine FXa (liquide) 4 mLwith heparinSubstrate 2 mL

Stability48 h on-board5 weeks at 2-8Cfor optimal stability, keep the vial in the fridge between testsPerformancesnot sensitive to heparin HCIInot sensitive to Stockholm & Denver variantsSTA-Stachrom AT IIIbovine thrombin (Lyo) 3/6 mLSubstrate CBS 61.50 Buffer with heparin

Stability7 days on-board21 days at 2-8Cvalidated on instrument

TAT60 secTests number30 & 60 testsDetection of type II Cambridge & Stockholm & Denver variantsNot sensitive to heparin HC II


Conclusion - Antithrombin (1/3)Cooper PC. Int. J. Lab. Hem; 2012. 9: 1-11

Chromogenic assayPrincipleFeaturesStagoSTA-Stachrom ATIIIbovine thrombin functional assay7 days on-board STA line21 days at 2-8Cbuffer with heparinSiemensBerichrom ATIII

Innovance ATbovine thrombin functional assay

anti-Xa functional assay4-48 hours on-board 15 days at 2-8Cbuffer without heparin

4-48 hours on-board28 days at 2-8Cbuffer without heparinILLiquid Antithrombinanti-Xa functional assay48 hours on-board35 days at 2-8Cbuffer with heparin


Conclusion - Antithrombin (2/3)Assays that use factor Xa may fail to detect the common variant, AT Cambridge II, which can be detected by assays using bovine thrombin.

AT activity assays are essential for the detection of AT deficiency because type II defects are relatively common in patients with heritable deficiency.

An excess of antigen over activity level suggests the presence of functionally defective. Consequently the association of a functional and an antigenic assay can detect all forms of AT deficiency.Cooper PC. Int. J. Lab. Hem; 2011. 33(3): 227-237


Conclusion - Antithrombin (3/3)


Protein C


Protein C systemTotalPS Adapted from Esmon CT et al.,Thromb. Haemostasis, 78 (1), 70, 1997FreePSVi: V inactivatedVIIIi: VIII inactivated-PCa: Activated Protein C TM: ThrombomodulinPS: Protein SC4bBP: C4b Binding-ProteinEPCR: Endothelial Cell PC Receptor


Protein CSynthesised by the liverVitamin-K dependant glycoproteinNormal plasma range: 70 - 130 %Levels for heterozygous PC deficient patients between 25 % and 70 %Prevalence of the congenital deficiency in the general population: 0.1 - 0.3 %


Protein CPhysiological variationsAt birth, low PC levels are observed due to liver immaturity PC increases slightly during pregnancy and during the puerperiumIn adults, the protein C level is independent of age and sexAcquired deficienciesOral Anticoagulant therapy, liver disorders, nephrotic syndrome, DIC, ...


Hereditary protein C deficiencyType I: True congenital deficiencyReduced PC antigen and PC activityType II :Abnormal moleculeNormal PC antigen but reduced PC activity Abnormal binding to other proteins (mostly PS)Abnormal binding to PPL (severe types)


Stago reagents for Protein CSTA-Staclot Protein C3 x 1 mLClotting method Detects any type of PC deficiency (I and II)Detects simultanously:catalytic activity FVa / FVIIIa interactions PS / PL / Ca2+ interactions

STA-Stachrom Protein C6 x 3 mLChromogenic methodDetects any type of PC deficiency (I and II partly)Well standardized and less interferences


PC diagnosis strategy*AC: Anticoagulant**AM: Amidolytic


A : Abnormal ( 70 %

N : Normal > 70 %

Type I

Quantitative Deficient

Type II

Qualitative Deficient

AC*

AM**

1st step

STA - Staclot PC

A

A

A

2nd step

STA - Stachrom PC

A

N

A

Asserachrom PC

A

N

N

No PC deficiencyConfirm abnormalityon a new sample, same test

Normal Abnormal> 70% < 70%Activity assay e.g. STA-Staclot PC or STA-Stachrom PC

Normal Abnormal> 70% < 70%

Confirm the discrepancy PC deficiency Qualitative defect (rare) Proposal for a diagnosis approachActivity assayAntigenic determination+ CriteriaofthrombophiliaAntigenic determination Asserachrom PC


STA-Staclot Protein CFunctional assaySnake venom-based assayDetects any deficienciesGolden standard, reference thrombosis centersAutomated on coagulation analysersSpecific No interference by heparin up to 1 IU/ml, by factor deficiencies and protein S levelsStability:8 hours on board


STA-Staclot Protein CPrinciple37C 180 sec.50ml50ml50ml50mlTwo-step reactionFor the quantitative measurement of the functional PC levelAnticoagulant reactionPurified extract of Agkistrodon contortirx venom


STA-Staclot Protein CRole of reagents Deficient plasma which ***adds all the requested factors for coagulationactivates PC > test not directlydependent of the plasma PC Ca2+ to trigger the coagulation reaction************


PC diagnosis strategy*AC : Anticoagulant**AM : Amidolytique


A : Abnormal ( 70 %

N : Normal > 70 %

Type I


Type II

Qualitative Deficient

AC*

AM**

1st step

STA - Staclot PC

A

A

A

2nd step

STA - Stachrom PC

A

N

A

Asserachrom PC

A

N

N

37C MonitoringAbsorbance180 sec.50ml50ml50ml SubstrSTA-Stachrom Protein CPrincipleOne-step reactionFor the quantitative determination of the functional PC levelAmidolytic reactionPurified extract of Agkistrodon contortirx venom


STA-Stachrom Protein CChromogenic assay principleIntensity of the coloration is directly proportional to the PC level in plasma


STA-Stachrom Protein CChromogenic functional assaySnake venom-based assayDetects any type of PC deficiency (I and II partly) Quantitative determination of the functional PC levelAutomated on most analysersSpecificNo interference by heparin up to 1 IU/ml, by factor deficiencies, PS levels or Lupus AnticoagulantExcellent stability21 days on board STA instruments1 month at 4C


PROTEIN C

STA-Staclot PCSTA-Stachrom PCPrincipleR1 PC deficient PlasmaR2 Agkistrodon Contortrix Contortrix (Protac)R1 Agkistrodon Contortrix Controtrix (Protac)R2 CBS 42.46Kit contentref 00737: 3 x 1 mLref 00671: 6 x 3 mLNumber of testsPE = 50 L3 x 20 theoretic tests3 x 16 real testsPE = 100 L6 x 30 theoretic tests6 x 23 real testsStability8 hours on board21 days on boardCalibratorSTA -UnicalibratorSTA -UnicalibratorCalibration4 points / series3 points per lotControlSTA -System Control N+PSTA -System Control N+PMinimum patient per week16 - 4 cal -2ct = 10 patients23 - 3 cal - 2 ct = 18 patients


Competition PC


Siemens versus StagoProtein C Reagentmanual or ou automated methodPC-Activator 1x3 mLAPTT reag. for PC 1x10 mLPC deficient plasma 4x1 mLStability8 hours 15-25C (on-board)2 days at 2-8C7 days < -18CTests number (manual method)60 tests per vial (PC Activator)60 tests per kit

Interferences with LA et FVL

STA-Staclot Protein Cautomated methodPC-Activator 3x1 mLPC-deficient plasmaonly 2 reagentsStability8 hours on-boardvalidated on instrument

Tests number60 tests per kit20 tests per vial

no interferencewith LA or FV leiden


Siemens versus StagoBerichrom Protein CManual or automated methodPC-Activator 5/10 mLSubstrate reagent 3mLHepes Buffer solution 30 mLStabilityPC activator: 2 weeks (2-8C) or 8 h (37C) ; on-board ???Substrate: 6 weeks (2-8C) ou 1 week (37C)Calibrator: 4 hoursControls: 4 hoursTests nb (manual method)10 tests per vial (10 mL)x3 vials = 30 tests

5 tests per vial (5 mL)x4 vials = 20 tests

STA-Stachrom Protein CAutomated methodPC-Activator (3 mL)Substrate reagent (6 mL)Owren on-board StabilityPC Activator: 21 days OBSubstrat: 21days OBCalibrator: 4 hoursControls: 8 hours

Tests nb30 tests per vial180 tests per kit (6 vials)


IL versus StagoHemosIL ProClotAutomated methodPC-Activator 4x1.5 mLPC-deficient plasma 4x1 mLPC control plasma 2x1 mL(low level of Protein C)

StabilityPC-Activator: 15 days (2-8C) or 60 days (-20C flc origine) ; OB ??PC-deficient plasma: 4 hours (ACL Top: 15-25C) or 7 days (-20C)PC control plasma: 4 hours (15-25C)or 7 days (-20C)

LimitationsBilirubin (>20 mg/dl), triglycerides (> 500 mg/dl), hemoglobin (> 150 mg/dl)interferences with LA, high FVIII level, FVLSTA Staclot Protein CAutomated methodPC-Activator 3x1 mLPC-deficient plasmano control in the kit

Stability8 hours OBOB validationSystem controls8 h stability OB

No limites with turbid plasmano interference with LA or FVL

Tests number60 tests per kit20 tests per vial


IL versus StagoHemosIL PCAutomated methodDiluant 1x8 mLPC-activator 2x2.5 mLChromogenic susbtrate 2x2 mLStabilityDiluant: keep at 2-8CPC-activator: 5 days at 15C (Futura, Advance et ACL Top) ; 3 months (2-8C, original vial)chromogenic substrate: 5 days at 15C (Futura, Advance et ACL Top) ; 3 months (2-8C)

Tests number60 tests (kit)STA-Stachrom Protein CAutomated methodPC-Activator (3 mL)Substrate reagent (6 mL)Owren on-board

StabilityPC Activator: 21 days OBSubstrate: 21 days OBCalibrator: 4 hoursControls: 8 hours

Tests number30 tests per vial180 tests per kit (6 vials)


Conclusion - Protein CStago is the only supplier to offer a complete range of reagents for PC determinationidentify the 3 types of deficienciesFunctional assay should be the first choice to determine protein C levels (Cooper, 2012).Chromogenic assay for protein C is less subject to interference than clotting assays and are preferable (Cooper, 2012).Cooper PC. Int. J. Lab. Hem; 2012. 9: 1-11


Protein S


Protein SProduced by the liverVitamin-K dependent glycoproteinNormal range (free PS Ag):Men: 108% (SD 16.5%)Women: 88% (SD 19.5%)2 forms of protein S:Free PS (# 40 %) and PS-C4b BP


Protein SPlasmatic concentration: 25 mg/mlTwo forms of the protein SFree ~ 40 %only active form, cofactor activity on PCBound to C4b-BP not active formC4b-BP: bound the PS to PPL


Protein SPhysiological variationAt birth: low total PS, normal free PS level Total and free PS levels are lower in woman than in man, and increase with agePS levels decrease during pregnancy

Acquired deficiencyWarfarin, DIC, liver disease, oral contraception, nephrotic syndrome, AIDS, inflammatory syndrome (C4bBP ) ...


PS diagnosis strategyAllows diagnosis of any type of deficienciesThe more oftenly used


A: Abnormal ( 70 %

N: Normal > 70 %


Functional Deficient

Free fraction deficient

Type I

Type II

Type III

1st step

STA - Staclot PS

A

A

A

2nd step

STA - Liatest Free PS

A

N

A

Asserachrom Total PS

A

N

N

No PS deficiencyConfirm abnormalityon a new sample, same test

Normal Abnormal> 65 % * < 65% *Activity assay e.g. STA-Staclot Protein S Normal Abnormal> 65% * < 65% *

Confirm discrepancy Protein S deficiency Qualitative defect Proposal for a diagnosis approach* For menActivity assayAntigenic determination+ CriteriaofthrombophiliaAntigenic determination e.g. Asserachrom Free PS or STA-Liatest Free PS


Protein S testingProtein S activitySTA-Staclot Protein S2 x 1 mLClotting test

Free protein S antigenAsserachrom Free Protein SSTA-Liatest Free Protein S6 x 2 mL et 6 x 5 mLImmunological assay

Total protein S antigenAsserachrom Total Protein SLiatest Protein SManual assay


STA-Staclot Protein SPrincipleTwo-step reactionAnticoagulant reactionClotting assay based on the direct PS action on its physiological targets (PCa, factor Va)


STA- Staclot Protein SRole of reagents deficient plasma which adds all the requested factors for coagulation *** Ca2+ to trigger the coagulation reaction************activated factor V added in excess *** > independent test from potential patient's Fact V deficiency

activated PC added in excess > test not directly dependent of the plasma PC ******


STA-Staclot Protein SAdvantagesFunctional assay Clotting assaydetects any type of PS deficiency (type I, II, III)APTT based methodno risk of interference of FVIIa generated in vivo or in vitro (frozen samples)Protein S deficient plasmano interference by factor deficienciesPCa and FVa included in the reagentsoptimal and standardized levels of PCa and FVano in vitro activation of patient PCno interference by APC ResistanceHeparin inhibitor included in reagentno interference by therapeutic heparin up to 1 IU/ml


Diagnostic strategy


A: Abnormal ( 70 %

N: Normal > 70 %


Functional Deficient

Free fraction deficient

Type I

Type II

Type III

1st step

STA - Staclot PS

A

A

A

2nd step

STA - Liatest Free PS

A

N

A

Asserachrom Total PS

A

N

N

Quantitative and direct measurementLatex Immuno Assays (LIA)Based on the use of latex microparticles2 MAbs specific of free PS

Automated on STA lineQuick turn around time (< 10 min)Precalibrated and barcoded assayLiquid and ready to use reagents

Measuring range: 10% -150%Detection limit: 7%No hook effect up to 200%STA-Liatest Free Protein SFeatures


STA-Liatest Free Protein SGood correlation with the referent assay

Blood Coagulation and Fibrinolysis, 2001


Key features of STA-Liatest Free Protein SFeaturesAutomated testQuick & simple to measure Free PSLiquid and ready to usePrecalibrated

AdvantagesVery easy to use5 days OB stabilityOne-shot test or series of batchHigh level of measurement up to 150% (without redilution)StandardizedSecure


LimitationsDosage of the inactive Free protein S:Qualitative deficiencyVKA treatmentRisk of false diagnosis if used first

complementary test to clotting assay, which remains the gold standard


Competition PS


Siemens versus StagoProtein S AcProtein S Ac deficient plasmaProtein S Ac APC reag. (contains human PC, lyo)Protein S Ac starting reag. (contains the activator RVV linked to PL)Stability8 h (15-25C)2 months (< -18C)Tests number?Interference with FV LeidenSTA-Staclot Protein SPS deficient PlasmaPCaBovine FVStability8 h (15-25C)Tests number40 tests per kit20 tests per vialNo interference with FV Leiden and factor deficiencies


IL versus StagoHemosIL ProSPT based assay (rabbit recombinant TF)sensitive to factor VIIfrozen sample can to FVII activation PS reagentPS deficient plasmaPC control plasmaStabilityPS reagent:16 h-2 days (2-8C)1 h (15C - ACL Top)4 hours (ACL 8000/9000)PS control plasma:4 hours on-boardSTA-Staclot Protein SAPTT based assaynot sensitive to FVIIPS deficient PlasmaPCaBovine FVnot sensitive to factor V Leidennot sensitive to factor deficienciesStability8 hours on-boardControls8 hours on-board


IL versus StagoHemosIL Free protein SImmunological test in 2 stepsLatex coated with C4bBPLatex coated with anti PS (Mab)Kit containsBuffer (Liq)Latex C4bBP (Lyo)Latex PS (Liq) to reconstitute Latex C4bBPstabilize 30 minTake care to bubles !!!CalibrationnecessaryOne package3 x 25 tests

Stability1 week on-board (ACL Top)1 months (2-8C)

STA-Liatest Free PS1 steplatex coated with 2 Mabs

Kit containsBuffer (Liquid)Liatest (Liquid) Ready to use

Calibrationprecalibration2 packages :3x2 mL for 20 tests6x5 mL for 50 testsStability5 days on-board (STA Liatest Free PS 6x5 mL)


Thrombophilia screening,in practice


Thrombophilia laboratory testingFunctional assays firstAT activitySTA-Stachrom AT IIIProtein C activitySTA-Staclot Protein CSTA-Stachrom Protein C Protein S activitySTA-Staclot Protein S


Thrombophilia laboratory testingAntigenic assays as complementary testsAT antigenLiatest AT IIIProtein C antigenAsserachrom Protein CFree PS antigenSTA-Liatest Free Protein SAsserachrom Free Protein STotal PS antigenAsserachrom Total Protein S


Thrombophilia laboratory testingOther parameters for a global testing:FV mutation Prothrombin mutationDNA analysislevel of FIIHigh coagulation factor (FVIII, FXI)Antiphospholipid antibodesLAanticardiolipin or anti-b2GP1 antibodiesFactor inhibitors


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pour les variants de Stockholm et Denver soient dtects, il faut utiliser de la bovine thrombin (donc seul STA stachrom ATIII peut les dtecter)

diagnóstico trombofilia

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