laboratoriumdiagnostiek bij heparine geinduceerde trombopenie

1. laboratoriumdiagnostiek bij heparine geinduceerde trombopenie Nascholingsbijeenkomst NVKC Regio-Oost De Lutte 12 januari 2007 Henk Adriaansen KCHL, Gelre Ziekenhuizen Apeldoorn-Zutphen

2. Etiology of Thrombocytopenia • Decreased production • Infection, chemo, alcohol, B12/folate, MDS, leukemia • Increased destruction • DIC, TTP, PTP, drugs, infection, HELLP, cardiopulmonary bypass • Dilutional or distributional • Excessive pRBC transfused, splenomegaly, pseudothrombocytopenia

3. Pseudothrombocytopenia • About 0.3% of population • Caused by EDTA • Benign, lab only phenomena, may be intermittant • Platelet clumps on smear • Re-run CBC in citrate or heparin

4. Drugs • List grows longer every day • Most common: • Heparin • Antibiotics • Zantac • Quinine • Antiplatelet drugs • Antiepileptics • Antiarrythmics

5. Thrombocytopenia due to drugs • Heparin is a whole separate issue • Stop the drug & wait for recovery • Improvement usually in few days-week • No effective growth factors to date • IL-11 (Neumega) approved for chemotherapy related thrombocytopenia • Doesn’t work

6. Heparin Induced Thrombocytopenia (HIT) • Seen in up to 10% of patients on heparin • Most are non-immune • More common with UFH than LMWH • Up to 5% with UFH • 1% with LMWH • Can be seen with heparin flushes, heparin coated catheters, heparin during dialysis

7. HIT Type 1 • Non-immune response to heparin • Usually mild drop in platelets (>100,000) • 1-2 days after start of heparin • Often returns to normal on heparin • Usually no clinical consequence • Usually responds to simple cessation of heparin

8. HIT Type 2 • Immune mediated • Antibody against heparin-platelet factor 4 complex • Antibody binds to Fc receptor & activates platelet • Potentially life/limb threatening condition • Leads to thrombocytopenia, arterial and venous thromboses

10. Tests for HIT • Diagnosis is clinical • Do not wait for tests to start therapy- both are send outs and take days • Serotonin release assay – • Functional assay that looks for platelet activation • ELISA for PF 4 • Antigen assay for heparin/PF4 complex • Platelet aggregation • Geltest Diamed

11. Functionele testen voor HIT • bloedplaatjesaggregatie test (PAT) • 14C-serotonine release test (14C-SRA) • ATP release test (lumi-aggregometry) • heparine geinduceerde bloedplaatjesactivatie test (HIPA) • flow cytometrische bepaling van bloedplaatjes micropartikels • flowcytometrische bepaling van annexine-V binding

12. 14C-serotonine release test (14C-SRA) = gouden standaard • PRP+14C-serotonine: 15’ op 37°C • + test serum + heparine(1): 60’ op 22°C • +0.5% EDTA • centrifugatie • scintillatieteller

13. Serotonin Release Assay • Measures the release of serotonin from aggregated platelets in serum of patient with HIT; relies on platelet aggregation in the presence of heparin • Advantages • High specificity and sensitivity • Validated in blinded assessment of a clinical trial • Disadvantages • Technically demanding and time-consuming • Requires the use of radioactive materials • Not widely available

15. ELISA • Immunologische bepaling van HIT antilichamen • ELISA test voor heparine-PF4 antistoffen • microtiterplaat gecoat met hep-PF4 +patientenserum of positieve controle of negatieve controle, 60’ op KT • 5x wassen + antihumaan IgG,A,Mperoxidase conjugaat, 60’ op KT+ 5x wassen • ortho-phenyleen diameer/ureumperoxidase substraat,5’ op KT

16. Tests for HIT

18. Platelet Aggregation Assay • Measures platelet aggregation of IgG in serum or plasma of a HIT patient treated with heparin • Donor platelets can be washed or suspended in citrated plasma • Advantages • Easily performed in most laboratories • Specificity greater than 90% • Disadvantages • Low sensitivity: 35%–81%; sensitivity higher using washed platelets • Reactivity varies among donor platelets

19. Ter discussie het Diamed Gelsysteem voor het aantonen van HIT

20. HIT Type 2 • Thrombotic Sequelae of HIT: • Venous: arterial thrombosis = 4:1 • DVT (50%) • PE (25%) • Acute limb ischemia (10-20%) • Warfarin-associated venous limb gangrene (5-10%) • Acute thrombotic stroke or MI (3-5%)

21. HIT Type 2 • 50% risk of thrombosis over 30 days with cessation of heparin alone • Thrombotic tendency exists for at least 40 days after stopping heparin • Overall risk of thrombotic complication: 38-76%

22. HIT Type 2 Time course • Typically occurs 4-14 days after starting heparin • Has occurred as soon as 10 hours after re-exposure to heparin • Has occurred 3-4 days after cessation of heparin

23. HIT Diagnosis • Consider in anyone with unexplained drop in platelets to < 150,000 or 50% decrease while on heparin • Diagnosis is clinical • Do not wait for lab test results to start treatment

24. Pathofysiologie HIT • Verschillende componenten betrokken bij het ontstaansmechanisme van HIT: • Trombocyten • HIT-antistoffen • FcγRIIalfa isovormen • Endotheliale cellen • Leukocyten • Inflammatoire toestand J. Walenga et al., Sem. Throm. Hem., 2004 T. Warkentin, Hematology, 2003 Newman and Chong, Blood, 2000

25. Pathofysiologie • HIT-antistoffen en bloedplaatjes • Binding van heparine aan PF4 tetrameer  neoepitope (IL8, NAP2 = CXC chemokines) • Vorming van AL tegen heparine-PF4 complex (IgG1 > IgG3 > IgG2 > IgG4 >> IgA en IgM) • Fab-regio van het AL bindt heparine-PF4 complex, terwijl het Fc-gedeelte FcγRII op de Blp-membraan bindt • Activatie van de trombocyten met • secretie van meer PF4, stollingsfactoren,... uit de alfa-granules • secretie van serotonine, ADP, Ca2+ uit de densebodies => Amplificatie • Vorming van trombocytenaggregaten

26. Pathofysiologie • Receptor isovormen • 2 isovormen op aminozuurplaats 131: een arginine (R) of een histidine (H) • FcγRII-H bindt IgG2 met hogere affiniteit dan de FcγRII-R  hogere frekwentie HIT bij patienten met H/H genotype (Brandt et al., Denomme et al.)