Thrombophilia Testing Robert Gosselin MT (ASCP), CLS.

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

DE

D D

ED

Plasminogen PlasmintPA

uPA

FIBRIN

D

E

D

D

D

E

E

D

DD

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

SpecificityTrue Negative

True Negatives + False Positives

True Negatives

All Negatives

Negative Predictive Value

Clinical signs and symptoms of DVT +3Heart rate >100/min +1.5Hemoptysis +1Active cancer +1Bedridden (>3 days) or major (>12 weeks)

+1.5Previously history of DVT or PE +1.5PE most likely diagnosis +3

Clinical Probability for PE

Score: Low <2 Moderate 2-6 High >6

Wells PS, et al Thromb Haemost 2000; 83:416-20.

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 DVT likely >2

Clinical Probability for DVT

Wells PS, et al Lancet 1997; 350:1795-98; N Engl J Med 2003;349: 1227-35

Compression US

Positive Negative Low prob

Serial CUS (5-8 days) Mod or High Prob

DVT Positive

Positive VTE

DVT Negative

Negative VTE

3 month f/uPositive Negative

DVT

Algorithm

Spiral CT or Angiogram

Positive Negative

PE Positive

Positive VTE

PE Negative

Negative VTE

3 month f/u

PE

Algorithm

0

0.5

1

1.5

2

2.5

3

3.5

4

No VTE VTE

0.8

3.7In

no

van

ce D

-dim

er m

g/L

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

Inn

ova

nce

D-d

imer

, m

g/L

Low Mod High Unlikely Likely

PE ProbabilityPE Probability DVT ProbabilityDVT Probability

D-dimer testing pearls

Not the silver bulletMethod must be highly sensitiveMust use clinical probability tools

Not useful in high probsCannot r/o VTE in patients on OACHeparin Rx can result in false negativeMost studies exclude prior HxMinimal studies on aortic dissection

Common testing: Thrombophilia

Protein C: functional preferredProtein S: functional preferredAntithrombin: functional preferredV Leiden (or APC resistance testing)

20210G mutation (prothrombin)

MTHFRAPS---later!Factor VIII and Fibrinogen (others??)

Others…

Plasminogen, PAI-I, tPA release, HCFII, TAFI, platelets, etc

Molecular testing

Protein C-Amidolytic

Protein C Activated Protein C

Peptide-pNA Peptide + pNA

Copperhead rattlesnake venom

Amidolytic PC-Interferences

Protein C Activated Protein C

Peptide-pNA Peptide + pNA

Copperhead rattlesnake venom

Thrombolytics – Thrombolytics – False False

Aprotinin inhibits aPC

False

Protein C-Clotting

Protein C Activated Protein C

aPTT Pronlongation of clotting time

Copperhead rattlesnake venom

Protein C def plasma

Inhibition of Va and VIIIa

Clotting PC-Interferences

Protein C Activated Protein C

aPTT Prolongation of clotting time

Copperhead rattlesnake venom

Protein C def plasma

Inhibition of Va and VIIIaHeparin

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 plasmaInhibition of Va

+ CaCl2

Functional PS-Interferences

Protein S

Incubate

Prolongation of clotting time

Factor Va + aPC

Protein S def plasmaInhibition of Va

+ CaCl2

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 + Heparin AT: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 + Heparin AT: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 thrombosisLiver diseaseOral vitamin K antagonists (functional assays)

Nephrotic syndrome Inflammatory states (PS)

PregnancyHormonal RxL-asparaginase RxDrugsAPA Factor activity

Pre-analytical stuff

APC resistance testing

Modified aPTT– Factor V deficient plasma increases

specificity and sensitivity

Plasma + FxV deficient plasma Clotting time #1aPTT

Plasma + FxV deficient plasma Clotting time #2aPTT

CaCl2 + APC

CaCl2

Ratio: CT2

CT1Normal ratio usually >2.0

APC resistance: Interferences

Plasma + FxV deficient plasma Clotting timeaPTT

Plasma + FxV deficient plasma Clotting time #2aPTT

CaCl2 + APC

CaCl2

Ratio: CT2

CT1Normal ratio usually >2.0

Pre-analytical

Biases usually systematic -- tendency for lower ratios with APA. Patient on Xigris may effect results