Educational Material: Thrombophilia Tests

1. Educational Material: Thrombophilia Tests

2. Contents • Introduction in thrombophilia • Specific parameters • AT III • Protein C and protein S • Factor V Leiden and APC resistance • Other thrombophilia markers, incl. D-Dimer and antiphospholipid syndrome • Heparin assays and their applications DiaMed Education: Thrombophilia

3. Thrombophilia • Thrombophilia is a disorder in which the blood clots easily or excessively. • Most disorders that cause thrombophilia increase the risk of blood clot formation in veins; some increase the risk of clot formation in both arteries and veins. • Underlying problem: Enhanced generation of thrombin which induces excessive fibrin formation • Thrombin activates other coagulation factors and platelets, and finally produces fibrin clots which are stabilized by F XIIIa (activated also by thrombin) Consequences: Enhanced clot formation  thromboembolic disease DiaMed Education: Thrombophilia

4. Causes for thrombophilia • Some of the disorders that cause thrombophilia are inherited, otheres are acquired. • Examples for congenital causes: • Activated protein C resistance (Factor V Leiden mutation) • Prothrombin 20210 mutation a specific mutation in the prothrombin gene • Deficiency of protein C, protein S, or antithrombin (AT III) • Rare disorders: Deficiency of heparin cofactor II, plasminogen, PAI-1 polymorphisms, disorders In methionine metabolism, PNH etc. • Acquired disorders that cause thrombophilia • Paralysis, prolonged sitting (e.g. in a plane or car), prolonged bed rest, plaster casts etc., which lead to immobilization • The presence of the lupus "anticoagulant," and/ or antiphospholipid antibody syndrome • Recent surgery, and heart attack. heart failure (a condition in which the blood is not pumped sufficiently through the bloodstream) • Increased pressure on veins, including obesity and pregnancy • Inflammatory conditions (leading to elevated concentrations of FVIII and fibrinogen, elevation of PAI-1 etc.) • Hyperhomocysteinemia • Disseminated intravascular coagulation (often associated with cancer or with sepsis) DiaMed Education: Thrombophilia

5. Symptoms and Complications • Most of the inherited disorders do not cause an increased risk of clotting until young adulthood, although thrombi can form at any age (including in newborns). • This is primarily the case in heterozygous cases in which only one version of the protein is abnormal, but the other version is functional. Homozygous forms, however, can lead to severe spontaneous events. • Combination with other risk factors (e.g. pregnancy, high blood pressure, immobilization) during adulthood may precipitate thromboembolic disease, for example a deep vein clot (deep vein thrombosis) in the legs, which can result in leg swelling. • Formation of a deep leg thrombus may be followed by the very dangerous pulmonary embolism. • Less commonly, a thrombus may form in veins of an arm or in the abdomen, and in veins inside the skull. • Hyperhomocysteinemia and the antiphospholipid syndrome may result in venous or arterial clots. When clots obstruct blood flow in arteries, tissues lose their blood supply and may be damaged or destroyed. • Potential consequences: Myocardial infarction, stroke DiaMed Education: Thrombophilia

6. Incidence and progression • Incidence: 1- 5 per 1000 individuals, increases strongly with age • Higher incidence when combined with specific diseases (especially with cancer), with immobilization, or certain treatments • Often thrombosis starts in the distal leg veins, but may progress further. • About 20% remain only in the distal vein, and about 30% progress to proximal leg veins • Up to 15% may embolize to lung  Pulmonary embolism (PE) • High morbidity • High mortality • Thrombosis may be “silent” until embolization DiaMed Education: Thrombophilia

7. Diagnosis and Treatment • A person who has had at least two separate instances of a thrombotic event without an apparent predisposing factor may have an inherited thrombophilia disorder. • An inherited disorder may also be suspected there is a family history of thromboembolic disease. A young – otherwise healthy- person who develops an initial thrombus for no apparent reason may have an inherited disorder. • Laboratory tests are used to identify specific inherited disorders which cause thrombophilia. This includes tests for APC-resistance, protein C or S, and AT III deficiency, and sometimes others. These tests are usually more accurate when performed after a thrombus has been treated. • Inherited disorders that cause thrombophilia are incurable*. Most patients with thrombosis are initially treated with normal or low molecular heparin (LMWH). They are often treated with oral anticoagulants for some time, in recurrent cases for the rest of their lives. • However, the treatment should be started only after a carefully made diagnosis, There can be high risk situations in which therapy may either induce new thromboembolic disease (e.g. necrosis induced by oral anticoagulants, heparin induced thrombocytopenia- HIT) or does not work at all (inefficient heparin therapy in patients with low AT III activity) *except by liver transplantation DiaMed Education: Thrombophilia

8. Antithrombin (AT III)

9. Data on AT III AT III molecule model DiaMed Education: Thrombophilia

10. Antithrombin III: Overview • The serpin antithrombin (AT III) is a major inhibitor of the coagulation factors FXa, FIXa, and thrombin. • AT III forms a stable complex with its target molecules, in which both AT III and the active coagulation factor lose their activity ( consumption!). • This process is accelerated heparin like molecules on the vasculature surface, and of course by heparin therapy. • AT III deficiency can be associated with thrombosis • A low activity of AT III may lead to inefficient therapy with anticoagulants such as heparin, low molecular weight heparin (LMWH), or pentasaccharide, and may have also other adverse effects. • FIXa • ↓|—AT III /heparin • FXa • ↓ |—AT III/ heparin • FIIa • ↓|—AT III/ heparin • Fibrin formation, • Platelet activation • Activation of F V, FVIII, FXI • Activation of TAFI Heparane sulphate FXa AT III Endothelial cell surface Serpin= serine protease inhibitor A class of plasma proteins Examples: AT III, Antiplasmin, C-1-Inhibitor etc. DiaMed Education: Thrombophilia

11. Functional assays for Antithrombin: Principle 1.AT III(sample) + F Xaexcess + Heparin  AT-FXa inactive + F Xaresidual 2.F Xaresidual + chromogenic Substrate  pNA (yellow) + peptide Note: Similar tests via FIIa instead of FXa are used as well. However, FXa based tests do not show interference from other plasmatic inhibitors such as HC II, or during heparin or DTI therapy DTI = direct thrombin inhibitors, e.g. bivalirudin, agatroban, hirudin Example of a calibration curve DiaMed Education: Thrombophilia

12. Determination of Antithrombin: Indications • Venous thrombosis or pulmonary embolism • Congenital AT deficiency (incidence ~ 1/ 5000) • Acquired AT deficiency (may require substitution with concentrates) • DIC / sepsis / SIRS (systemic inflammatory response syndrome) • Liver disease (AT is synthesized in the liver) • Renal disease (loss through increased pore size) • Hormonal therapy (incl. anticontraceptive drugs) • No or inadequate effect of heparin therapy (when aPTT does not show the expected response, AT III may be too low) • Decision if AT III- concentrate therapy (alternatively FFP) is needed • Monitoring of substitution therapy with AT III concentrates or FNP DiaMed Education: Thrombophilia

13. AT III assays: Other methods • Immunoassays (ELISA or immunoturbidimetry) and clotting assays are not very common. • Limitation of immunoassays for AT III: No detection of abnormal forms of AT III, e.g. type II deficiency (normal antigen, decreased function). However useful for characterization of the nature of a specific defect • Clotting tests for AT III are difficult to standardize DiaMed Education: Thrombophilia

14. Protein C and Protein S

15. Data on Protein C DiaMed Education: Thrombophilia

16. Protein C: Overview • After activation, activated protein C (APC) regulates thrombin generation, primarily by proteolytic inactivation of FVa and FVIIIa, a reaction which accelerated by free protein S. • PC has also important anti-inflammatory and cytoprotective functions. • A deficiency of PC is often associated with thromboembolic disease, especially in the homozygous form, or when other risk factors are present. Biological funbction of Protein C (simplified) PC = protein C zymogen, APC = Activated protein C, EPCR = Endothelial Protein C receptor, TM = Thrombomodulin, F Vi = inactivated F V, F VIIIi = inactivated F VIII DiaMed Education: Thrombophilia

17. DiaChrom Protein C: Principle • Protein C(Sample) + Protac®  APC 2. APC + chromogenic substrate  pNA (yellow) + peptide Protac® = a proteolytic enzyme from snake venom (Agkistrodon contortrix) Example of a calibration curve DiaMed Education: Thrombophilia

18. Overview Protein C assays • Methods: • Functional methods • Chromogenic tests (like DiaChrom) • Clot based assays • Immunochemical assays • ELISA • Turbidimetric • Functional methods, especially chromogenic substrate based ones, should be preferably used as a first line assay • Specific disadvantage of clotting assays for protein C function • Somehow less specific • Interference from Lupus anticoagulants or by heparin • Influenced by F V-L mutation or other causes for APC resistance • Interference from alterations of other coagulation factors • May underestimate the PC activity in patients with oral anticoagulants • Immunoassays will not pick up defective protein C (e.g. deficiency type II). However they are useful for characterization of defective forms of protein C DiaMed Education: Thrombophilia

19. Protein C: Indications • Venous thrombosis or pulmonary embolism • Purpura fulminans (especially in newborns with severe deficiency of PC) • Congenital PC deficiency • Acquired PC deficiency (may require substitution with concentrates) • DIC / sepsis / SIRS (systemic inflammatory response syndrome) • Vitamin K antagonists • Liver disease (PC is synthesized in the liver) • Dilution coagulopathy (blood losses, trauma, burns) • Asparaginase therapy • Decision if concentrate therapy (alternatively FFP) is needed • Monitoring of substitution therapy with concentrates or FNP DiaMed Education: Thrombophilia

20. A determination of protein C is useful before oral anticoaguation • Due to its short half live of only 6-8 h, protein C falls rapidly In the induction phase with vitamin K antagonists • At the end of day one or even faster, PC may be already quite low – while other factors, especially prothrombin are relatively high • In this situation, and especially in cases of protein C deficiency, the patient has a risk for the development of cumarin induced skin necrosis • During oral anticoagulation a determination of protein C is difficult because as a vitamin K dependent protein it is decreased by therapy as well FII % activity FX PC Coumadin induced skin necrosis DiaMed Education: Thrombophilia

21. Data on protein S DiaMed Education: Thrombophilia

22. Overview Protein S • A vitamin K dependent plasma protein • A cofactor for APC, involved in the inactivation of FVa and FVIIIa- the motors of thrombin generation • Only the free form of protein S is active. • About 65 % of protein S in plasma is bound the the C4b binding protein (C4bBP), an acute phase protein. • Inflammatory disease, pregnancy, or diabetes mellitus increase C4bBP. This can lead also to a kind of acquired protein S deficiency, in which not the absolute amount of protein S is deficient, but the active free part, • Protein S seems to have also other inhibitory functions, e.g. independent from APC, or as an inhibitor of the activation of TAFI* by APC *TAFI = thrombin activatable fibrinolysis inhibitor, a protein which stabilizes fibrin against premature fibrinolysis DiaMed Education: Thrombophilia

23. Protein S deficiency • Patients with protein S deficiency have an elevated risk for thrombosis, especially deep venous thrombosis • Situations with protein S deficiency are DIC or oral anticoagulation, liver disease, infectious disease (e.g. AIDS), and other diseases • Type I deficiency: Low concentration, low activity • Type 2 deficiency: Normal total antigen, low activity • Type 3 deficiency: Low activity, low free antigen level, normal total antigen DiaMed Education: Thrombophilia

24. Principle of protein S assays • Functional tests: • Various assay principles are used in commercial tests • In general the sample is diluted into protein S deficient plasma (as a source of coagulation factors) and activated protein C is added • After a short incubation time coagulation is triggered by adding a coagulation activator or an activated coagulation factor, together with phospholipids and Ca-ions • The prolongation of clotting time is proportional to the activity of protein S • The protein S induced effect on APC is quite weak, and thus the clotting tests often show only minor clotting time differences in their calibrated ranges 2. Immunological tests (either for total protein S or only the free form) • ELISA • Immunoturbidity DiaMed Education: Thrombophilia

25. FV- Leiden and APC resistance

26. APC Resistance • A clinical state characterized by resistance of plasma against added or induced activated protein C (APC) • Under normal conditions, APC adds as a coagulation inhibitor by inactivation of FVa and FVIIIa, thus prolonging the clotting time • In patients with APCR, this inhibitory effect is either absent or weakened • The majority of cases show APC resistance due to a polymorphism in factor V (“Factor V- Leiden” mutation, 1691G/A) • “APC Resistance” in some clotting assays can have also other causes than the factor V Leiden polymorphism, for example elevated F VIII or FII (for example in carriers of the prothrombin 20210 polymorphism), lupus anticoagulants, or pregnancy. DiaMed Education: Thrombophilia

27. APC Resistance / Factor V Leiden (FV-L) • Factor V-Leiden = A polymorphism in the FV molecule. It makes FVa much more resistant against Activated Protein C (APC), a natural anticoagulant. • On top of that also the cofactor function of FV in the inactivation (!) of FVIIIa and FVa is diminished. • Consequences: • More thrombin is generated  more platelet activation and fibrin formation • Carriers of this polymorphism have an increased risk of thrombosis, especially when other risk factors are present • Homozygotes may have spontaneous thrombosis, e.g. during pregnancy, with anti-baby pills, during hormone therapy or without additional stimulus • On the contrary, the mutation protects against blood losses (e.g. during child birth, or during surgery) • For Europe, the Mediterranean countries, and in many areas of the Middle East, F V-L seems to be the most frequent hereditary cause for thrombosis. In Northern EU up to more than 10 % of the population have FV-L • The polymorphism seems to be absent or extremely rare in Japan, DiaMed Education: Thrombophilia

28. Determination of APCR in the laboratory (1) Clotting Tests („APC resistance“) • First Generation (After Dahlbaeck et al) • 2 x aPTT : With or without added APC • calculation of a ratio of these two tests. Example 75 sec/ 35 sec= 2.14 • Low ratio in patients with APCR, high ratio in F V “wildtype” • Absolute values of ratio are strongly instrument and reagent dependent • Not very specific, multiple interference possible • Strong influence of blood sampling and handling technique • Screening tests (Activation of protein C in plasma with Protac® • Activation of coagulation and PC in the presence of phospholipids and calcium, e.g. via aPTT (ProC Global/ Dade Behring) or via the diluted Russel Viper venom time (dRVVT) test system • Test is sensitive for FVL and several other causes, e.g. protein C or protein S deficiency, high levels of FVIII, prothrombin 20210 mutation, pregnancy, lupus anticoagulants • Is more a kind of global assay for the protein C pathway • Can be made quite specific for FV-L by diluting the sample into an excess of FV deficient plasma DiaMed Education: Thrombophilia

29. Determination of APCR in the laboratory (2) Clotting Tests („APC resistance“) 2nd Generation: • With added FV deficient plasma • Dilute sample into FV deficient plasma (excess of all other coag factors enhanced specificity for FV Leiden) • aPTT or other clotting assay (e.g. with snake venoms/ Pentapharm) with or without added APC  calculation of a ratio (Low ratio in patients APCR, high ratio in FV “wildtype” • Quite specific for FV L, sometimes overlap between homo- and heterozygous form • PCR • Specific test for the mutation • Different techniques possible DiaMed Education: Thrombophilia

30. DiaClot FV L: a simplified method in F V-L testing Principle: • Clotting assay.Sample is highly diluted into a mixture of purified clotting factors, APC and fibrinogen • Under these conditions, only the FV-L (sample) activity determines the clotting time, no ratio required • The clotting time is converted into „%-FV-L“ from a calibration curve obtained with the DiaClot FV-L Calibrator Set • No interference from lupus anticoagulants, high FVIII, oral anticoagulation, heparin etc. • Normal range (in F VL activity-%) < 10 % : Wildtype (Normal F V) 25 – 75 % Heterozygous for F V-L > 75 % Homozygous for F V-L. 2 Reagent System: Reagent 1: Coagulation factor reagent (Human Fibrinogen, Prothrombin, Protein S, APC, heparin neutralizer) Reagent 2 : Factor Xa, phospholipids (both lyophilised) DiaMed Education: Thrombophilia

31. Other rare causes for thrombosis • Dysfibrinogenenmia • a disorder leading for example to defective fibrinolysis • Often not detected via PT, aPTT but via thrombin time or reptilase time assays • Paroxysmal nocturnal haemoglobinuria (PNH) • A disorder induced by dysfunctional regulation of the complement system • Reduced expression of CD55 and CD59 on erythrocytes • Thrombosis in cerebral or intra-abdominal veins • Can be detected by the ID-PNH assays or by flow cytometry • Plasminogen deficiency • A disorder resulting in lowered fibrinolysis • Can be detected with specific chromogenic substrate assays • Deficiency of (or autoantibody) against ADAMTS 13 • ADAMTS 13 (a protease) regulates von Willebrand factor (vWF) by proteolytic cleavage. VWF is essential for primary haemostasis • In the absence of ADAMTS 13, ultra-large and very adhesive forms of VWF lead to massively enhanced platelet adherence which may indirectly induce also thrombin formation • In thrombotic thrombocytopenic purpura a low ADAMTS 13 activity leads to thrombosis, in spite of thrombocytopenia • A diagnosis requires platelet function tests (e.g. DiaMed Impact R) and specific tests for ADAMTS 13 DiaMed Education: Thrombophilia

32. Heparin Induced thrombocytopenia • Ordinarily, heparin prevents clotting and does not affect the platelets, However, heparin is immunogenic. • Triggered by the immune system, heparin may induce a low platelet count, heparin induced thrombocytopenia (HIT) • Two distinct types of HIT can occur: • Non-immune form: Occurs most frequently; is characterized by a mild decrease in the platelet count and is not harmful. • The second type, immune-mediated HIT, occurs much less frequently but is very dangerous. Immune-mediated HIT causes much lower platelet counts, most frequently 5 to 14 days after first beginning heparin therapy • Paradoxically, despite a very low platelet count, patients who suffer from HIT are at risk for major thromboembolic problems. • HIT may affect up to a few % of patents exposed to heparin DiaMed Education: Thrombophilia

33. Pathomechanism • Antibodies can form against a complex of heparin and a platelet factor 4, or "PF4" that is released by platelets. • Antibodies may activate platelets via the fc-γ- receptor, inducing thrombin generation on monocytes and endothelial cells • In rare cases also antibodies against IL-8 or against NAP-2 (neutrophiles) may induce HIT DiaMed Education: Thrombophilia

34. Patients with HIT have a significantly higher thrombotic risk than patients with other well characterized risk factor for thrombosis Therefore an immediate anticoagulation is required, of course with a different drug than heparin Drugs used in this situation are Agathroban Bivalirudin Hirudin (lepirudin) Orgaran® (Fondparinux) Problems with these drugs High expenses (may exceed 1000 USD/day) Difficult to monitor Bleeding not uncommon Therapy of HIT Odds ratio for risk of thrombosis (HIT : range 20 -40) (Modified after Erentin TE Can J Cardiol 1995 and Warkentin TE Thromb Res. 2003) DiaMed Education: Thrombophilia

35. Diagnosis of HIT • The diagnosis of HIT requires a combination of clinical data and laboratory tests • The clinical data can be successfully standardized with the „4-T-Score“ • Lab methods include • Immunochemical tests • Functional tests DiaMed Education: Thrombophilia

36. Lab tests for heparin induced thrombocytopenia Functional tests(the most accurate tests for confirmation of HIT) • Serotonin release assay (SRA) • Limitations: Requires access to fresh platelets and radioactivity, is very time consuming • Heparin induced platelet aggregation • Limitations: Requires access to fresh platelets, is very time consuming Immunochemical methods • ELISA • Limitations: Time consuming, high blank values, many falsely positive values, not a good method for individual test requests, limited value for diagnosis • Advantages: Good negative predictive value • ID-PaGIA assay (gel centrifugation) • Limitations: May give falsely positive values, limited value for diagnosis • Advantages: Very simple and fast, excellent negative predictive value (>99%, Pouplard et al, JTH 2007), suitable for individual requests, • PIFA tests (immune filtration, Akers Biosciences): Gave unreliable results in several clinical trials DiaMed Education: Thrombophilia

37. Other thrombophilia markers

38. Prothrombin mutation 20210 • Risk factor for thrombophilia • Induced by a mutation in the promoter of the prothrombin gene • Induces an enhanced synthesis of prothrombin with higher plasma levels, typically > 125 % of normal activity • Consequences • Venous or other thrombosis • Increased risk for obstetric omplications such as preeclampsia, abruptio placentae, fetal retardation or still birth • Probably also increased risk for coronary heart disease • Risk may be increased by the use of oral contraceptives DiaMed Education: Thrombophilia

39. Lupus Anticoagulants / Antiphospholipid Syndrome • Autoantibodies (IgG, IgA, IgM) directed against phospholipids (PL) or the PL- binding domain of plasma proteins • Some of them prolong PL dependent clotting tests such as the aPTT or the diluted Russel Viper venom time, influence on PT is rather mild • Target antigens are • Phospholipids • Cardioplin • Phosphatidyl serine • Proteins: • 2-glycoprotein 1 • Prothrombin • Protein C • others DiaMed Education: Thrombophilia

40. Clinical consequences of the antiphospholipid syndrome • Venous or arterial thromboembolic disease • DVT • Stroke • Coronary artery disease • Arterial graft occlusion • Obstetric omplications (maternal and fetal) • Intrauterine fetal death • Recurrent abortions • Fetal Growth retardation • Neonatal thrombosis • Others DiaMed Education: Thrombophilia

41. D- Dimer • A marker for enhanced activity of both coagulation and fibrinolysis. • D-Dimer is not a defined analyte. It represents a group of “cross linked fibrin split products” derived from fibrin. • Covalent cross-linking of polymerized fibrin monomers by activated Factor XIII stabilizes the fibrin clot. D-dimers are generated by plasmin lysis of this cross-linked fibrin. • Tests detect split products of various size and may partly react also with fibrinogen degradation products (hyperfibrinolysis) or split products derived from elastase during inflammation (depends on the antibody specificity). • Increased D-Dimer values* indicate a situation with increased fibrin formation and lysis, e.g. like in acute thromboembolism, pulmonary embolism, acute myocardial infarction, during surgery or in trauma patients, in cancer, DIC etc. * Normal values are age dependent DiaMed Education: Thrombophilia

42. Clinical application of D-Dimer tests • Elevated levels are found in many clinical situations. • Therefore the finding of an elevated D-Dimer does not provide very specific information. It rather demonstrates or excludes a clinical state with elevated thrombin formation. • The value of D-Dimer is more the negative result which can be used for exclusion of a thromboembolic state (negative predictive value). • Major applications: • Exclusion of thrombosis, e.g. deep venous thrombosis (DVT) • Exclusion of pulmonary embolism (PE) • Exclusion of disseminated intravascular coagulatiion (DIC) DiaMed Education: Thrombophilia

43. Potential savings when using D-Dimer tests • More than 6 Mio of cases with suspected DVT or PE present in the US to physicians per year, and ~ 200,000 cases of PE are diagnosed. Each case carries a 20% risk of sudden death. • Rapid diagnosis and early treatment of DVT are critical to prevent subsequent morbidity or disabling conditions to the patients • A negative D- Dimer test may prevent more expensive and sometimes invasive methods such as • CT Pulmonary Angiography/ Spiral-Computed Tomography • Ventilation-Perfusion Scan (VQ Scan) • Compression ultrasound of lower extremities • Venography (a radio-imaging method where contrast dye is injected and blood flow can be visualized) • It is imperative to exclude the diagnosis of DVT for various reasons. There is an inherent risk of hemorrhage associated with unnecessary anticoagulant treatment, or the severe risk of heparin induced thrombocytopenia DiaMed Education: Thrombophilia

44. Some precautions when using D- Dimer assays • The assays are only partly standardized. Depending on the calibration and the antibodies selected, values may be quite different between various kits. • D-Dimer values show age dependency (higher values in elderly patients) • A high does hook effect (with falsely low values) may affect certain kits. Very high concentrations of D-Dimer are found in some tumor patients and during thrombolytic therapy. • In hospitalized patients: Numerous diseases and invasive procedures can elevate D-dimer levels without the presence of VTE. Signal D-Dimer High dose hook or prozone effect VTE = venous thromboembolism DiaMed Education: Thrombophilia

45. Determination of heparin

46. Background: Heparin • A negatively charged, sulphated glycosaminoglycan, composed of uronic and glucuronic acid residues. • Commercial heparins are isolated from porcine intestinal mucosa or bovine lung • Heterogeneous mixtures of polysaccharide chains (molecular weight from 3000 to 30 000, with a mean MW of 15 000) • Low molecular weight heparin (LMWH) is prepared from unfractionated heparin. It has a molecular weight which is different between brands • Heparin and UFH exert their anticoagulant effect by activating antithrombin (AT); the heparin-antithrombin (H-AT) complex then inactivates thrombin, activated factor X (FXa) and other activated clotting factors. • LMWH is more efficient against FXa. DiaMed Education: Thrombophilia

47. Important adverse effects of heparin • Heparin induced thrombocytopenia (HIT) • Development of antibodies against the complex of heparin and platelet factor 4 (PF4, a protein released from platelets) • Patients may develop severe thromboembolic disease with high mortality • May affect up to a few percent of patients on heparin or LMWH • Requires immediately a change in the type anticoagulant (e.g. anticoagulation with a direct thrombin inhibitor) • Bleeding (when overdosed). • Occurrence is approximately 10% overall but may increase up to 20% in patients treated with high dose therapy. • Risk of bleeding increases with APTT ratios above the therapeutic range range • Cave: aPTT is a poorly standardized test. Clotting times at the same concentration of heparin may be quite different. DiaMed Education: Thrombophilia

48. Overdosage of heparin increases the bleeding risk 1 These frequencies are statistically significantly different ( 2 test, P <0.037). Source: Use of Anti-Xa Activity as a Marker for Heparin-Induced Bleeding in Patients with APTT >180 s Alexander Haliassosa, Helen Melita-Manolis, Despina Aggelaki, Despina Tassi and George Terzoglou, Clinical Chemistry 43: 1781-1782, 1997; DiaMed Education: Thrombophilia

49. Problems in the application of heparin • The clinical effectiveness of heparin is dependent on achieving an in vitro defined anticoagulant effect (e.g. a certain prolongation of the aPTT, or a certain plasma level of heparin as measured with specific tests). • The dose-response relationship of heparin is not linear • Considerable in vivo variation in response to a fixed dose of heparin between individuals. This is partly reflective of the pharmacokinetic limitations of heparin, caused by the binding of heparin to plasma proteins including platelet factor 4 , fibrinogen, factor VIII and histidine-rich glycoprotein, and probably other factors • The phenomenon known as ‘heparin resistance’ should be taken into consideration to prevent the over-administration of heparin, with potential haemorrhagic consequences, particularly postoperatively and in the setting of cardiac bypass surgery. • In the context of venous thromboembolism, heparin resistance is defined as the need for more than 35 000 U /24 h to prolong the aPTT into the therapeutic range. • A poor response to heparin can of course be related to deficiency of AT III. DiaMed Education: Thrombophilia

50. Heparin assay versus aPTT (1) • Most often heparin therapy is monitored via aPTT, with a target range of a 1.5- 2-fold prolongation of the initial aPTT during heparin therapy. However, the aPTT has limitations: • Results are strongly reagent (and coagulometer) dependent, e.g. the aPTT at the same heparin concentration can be • 70 sec with reagent A • 90 sec with reagent B • Unclottable with reagent C • The measuring range is limited (often unclottable when still in the therapeutic range of heparin therapy [0.3- 0.7 IU/ml]): • Overdose not reliably detectable, bleeding risk • Reducing the dose as a consequence of an unclottable test may lead to subtherapeutic levels of heparin with elevated thrombotic risk • Sometimes an initial value is not available: • 1.5 fold prolongation of initially 25 sec gives only 37.5 sec (almost within the normal range) while 1.5-fold prolongation of initially 35 sec results in an aPTT of 52.5 sec. DiaMed Education: Thrombophilia