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laboratoriumdiagnostiek bij heparine geinduceerde trombopenie
Nascholingsbijeenkomst NVKC Regio-Oost
De Lutte
12 januari 2007
Henk Adriaansen
KCHL, Gelre Ziekenhuizen Apeldoorn-Zutphen
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
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
Drugs
• List grows longer every day• Most common:
– Heparin– Antibiotics– Zantac– Quinine– Antiplatelet drugs– Antiepileptics– Antiarrythmics
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
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
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
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
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
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
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
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
Relationship Between Release of 14C-Serotoninand Final Concentration of Heparin in HIT Patients
Adapted with permission from Sheridan D, Carter C, Kelton J G. Blood. 1986;67:27–30.
% 1
4C
-Ser
oto
nin
Rel
ease
Heparin Concentration (µ/mL)
123
4
100
80
60
40
20
00.0010 0.01 0.1 1 10 100 1000
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
Tests for HIT
Serotonin Release Assay
ELISA for heparin-PF4 complex
Sensitivity 88% 97%
Specificity 100% 86%
Positive Predictive Value
100% 93%
Negative Predictive Value
81% 95%
Diagnosis: Heparin/PF4 ELISA
Adapted with permission fromVisentin GP, Aster RH. Curr Opin Hematol. 1995;2:351–357.
Relative Sensitivity in 12 Patients with HIT
Positive Reactions, n
Assay Undiluted 1:10 1:100 1:200 1:500
SerotoninRelease
12 12 2 NotTested
NotTested
ELISA 12 12 12 12 9
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
Ter discussie het Diamed Gelsysteem
voor het aantonen van HIT
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%)
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%
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
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
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., 2004T. Warkentin, Hematology, 2003Newman and Chong, Blood, 2000
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 dense bodies=> Amplificatie
– Vorming van trombocytenaggregaten
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.)