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Laboratoriumdiagnostiek bij heparine geinduceerde trombopenie Nascholingsbijeenkomst NVKC Regio- Oost De Lutte 12 januari 2007 Henk Adriaansen KCHL, Gelre.

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Presentatie over: "Laboratoriumdiagnostiek bij heparine geinduceerde trombopenie Nascholingsbijeenkomst NVKC Regio- Oost De Lutte 12 januari 2007 Henk Adriaansen KCHL, Gelre."— Transcript van de presentatie:

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) 14 C-serotonine release test ( 14 C-SRA) ATP release test (lumi-aggregometry) heparine geinduceerde bloedplaatjesactivatie test (HIPA) flow cytometrische bepaling van bloedplaatjes micropartikels flowcytometrische bepaling van annexine-V binding

12 14 C-serotonine release test ( 14 C-SRA) = gouden standaard –PRP+ 14 C-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 Serotonin Release Assay ELISA for heparin-PF4 complex Sensitivity88%97% Specificity100%86% Positive Predictive Value 100%93% Negative Predictive Value 81%95%


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 –FcγRII alfa 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, Ca 2+ uit de dense bodies => 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.)

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