Thrombophilia Testing Robert Gosselin MT (ASCP), CLS

D-dimer –Indicates clot formation –Indicates clot degradation D-dimer test commonly used for exclusion: –Pulmonary embolism –Deep vein thrombosis –Consumptive coagulopathy –Aortic dissection

D E D D E D Plasminogen Plasmin tPA uPA FIBRIN D E D D D E E D D D D E D-dimer Fragment X Fragment D Fragment Y Fragments D & E FIBRINOGEN

Microwell containing target Anti- human-XDP XDP ( + ) Incubate Conjugated Anti-human XDP antibody ¤ ¤ ¤ ¤ Wash +++ ¤¤¤ Incubate Wash Chromogenic tag   Color Amount of color proportional to amount of XDP present. Quantitative result extrapolated from calibration curve Patient XDP  Testing well Reagent beads coated with anti-XDP              Instrument reading— changes in optical density    Incubate Amount of light scattering proportional to XDP present. Quantitative result extrapolated from calibration curve

True Positive True Positives + False Negatives Sensitivity Specificity True Negative True Negatives + False Positives True Negatives All Negatives Negative Predictive Value

Clinical signs and symptoms of DVT+3 Heart rate >100/min+1.5 Hemoptysis +1 Active cancer+1 Bedridden (>3 days) or major (>12 weeks)+1.5 Previously history of DVT or PE+1.5 PE most likely diagnosis+3 Clinical Probability for PE Score:Low 6 Wells PS, et al Thromb Haemost 2000; 83:

Active cancer+1 Paralysis, paresis, recent casting of leg+1 Bedridden (>3 days) or major (>12 weeks)+1 Entire leg swollen+1 Calf swelling (>3cm) compared to other leg+1 Pitting edema greater in symptomatic leg+1 Collateral nonvaricose superficial veins+1 Localized tenderness along deep venous system+1 Previously documented DVT+1 Alternative Dx as or more likely than DVT-2 Score:DVT unlikely 2 Clinical Probability for DVT Wells PS, et al Lancet 1997; 350: ; N Engl J Med 2003;349:

Compression US PositiveNegative Low prob Serial CUS (5-8 days) Mod or High Prob DVT Positive Positive VTE DVT Negative Negative VTE 3 month f/u Positive Negative DVT Algorithm

Spiral CT or Angiogram PositiveNegative PE Positive Positive VTE PE Negative Negative VTE 3 month f/u PE Algorithm

No VTE VTE Innovance D-dimer mg/L

Innovance D-dimer, mg/L LowModHighUnlikelyLikely PE Probability DVT Probability

D-dimer testing pearls Not the silver bullet Method must be highly sensitive Must use clinical probability tools Not useful in high probs Cannot r/o VTE in patients on OAC Heparin Rx can result in false negative Most studies exclude prior Hx Minimal studies on aortic dissection

Common testing: Thrombophilia Protein C: functional preferred Protein S: functional preferred Antithrombin: functional preferred V Leiden (or APC resistance testing) 20210G mutation (prothrombin) MTHFR APS---later! Factor VIII and Fibrinogen (others??) Others… Plasminogen, PAI-I, tPA release, HCFII, TAFI, platelets, etc Molecular testing

Protein C-Amidolytic Protein CActivated Protein C Peptide-pNAPeptide + pNA Copperhead rattlesnake venom

Amidolytic PC-Interferences Protein CActivated Protein C Peptide-pNAPeptide + pNA Copperhead rattlesnake venom Thrombolytics – False  Aprotinin inhibits aPC False 

Protein C-Clotting Protein CActivated Protein C aPTTPronlongation of clotting time Copperhead rattlesnake venom Protein C def plasma Inhibition of Va and VIIIa

Clotting PC-Interferences Protein CActivated Protein C aPTTProlongation of clotting time Copperhead rattlesnake venom Protein C def plasma Inhibition of Va and VIIIa Heparin DTI Falsely  V Leiden mutation Falsely  Aprotinin inhibits aPC False  Increased Fbg or Factor VIII Falsely  LA Falsely  Pre-analytical

Protein S-Clotting Protein S Incubate Prolongation of clotting time Factor Va + aPC Protein S def plasma Inhibition of Va + CaCl 2

Functional PS-Interferences Protein S Incubate Prolongation of clotting time Factor Va + aPC Protein S def plasma Inhibition of Va + CaCl 2 Aprotinin inhibits aPC False  Increased Fbg or Factor VIII Falsely  Heparin DTI Falsely  LA Falsely  V Leiden mutation Falsely  Pre-analytical

Microwell containing capture Ab Anti-PC Anti-PS ** Protein S** or Protein C ( + ) Incubate Conjugated Anti-human PC or PS antibody ¤ ¤ ¤ ¤ Wash +++ ¤¤¤ Incubate Wash Chromogenic tag   Color Amount of color proportional to amount of PC or PS present. Quantitative result extrapolated from calibration curve Patient PS  Testing well Reagent beads coated with C4b              Instrument reading— changes in optical density    Incubate Amount of light scattering proportional to free PS present. Quantitative result extrapolated from calibration curve Anti-human PS  **For PS: Total and Free (PEG pre-treatment of samples to precipitate out bound PS)

Antithrombin testing AT + HeparinAT:Hep complex Excess Activated factor (either Xa or thrombin) AT:Hep:Xa complex + residual Xa S2765 Peptide + pNA Amount of color inversely proportional to amount of AT present. Quantitative result extrapolated from calibration curve

AT testing: interferences AT + HeparinAT:Hep complex Excess Activated factor (either Xa or thrombin) AT:Hep:Xa complex + residual Xa S2765 Peptide + pNA Amount of color inversely proportional to amount of AT present. Quantitative result extrapolated from calibration curve DTI: False  Heparin Rx False 

Causes of   PS or PC Acute phase thrombosis Liver disease Oral vitamin K antagonists (functional assays) Nephrotic syndrome Inflammatory states (PS) Pregnancy Hormonal Rx L-asparaginase Rx Drugs APA  Factor activity Pre-analytical stuff

APC resistance testing Modified aPTT –Factor V deficient plasma increases specificity and sensitivity Plasma + FxV deficient plasma Clotting time #1 aPTT Plasma + FxV deficient plasmaClotting time #2 aPTT CaCl 2 + APC CaCl2 Ratio: CT2 CT1 Normal ratio usually >2.0

APC resistance: Interferences Plasma + FxV deficient plasma Clotting time aPTT Plasma + FxV deficient plasmaClotting time #2 aPTT CaCl 2 + APC CaCl2 Ratio: CT2 CT1 Normal ratio usually >2.0 Pre-analytical Biases usually systematic -- tendency for lower ratios with APA. Patient on Xigris may effect results