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.)