A flow cytometric assay for the confirmation of

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A flow cytometric assay for the confirmation of heparin-induced thrombocytopenia (HIT) Elvira Maličev Blood

A flow cytometric assay for the confirmation of heparin-induced thrombocytopenia (HIT) Elvira Maličev Blood Transfusion Centre of Slovenia 7 th World Hematologists Congress May 08 -09, 2017 Barcelona, Spain

Heparin-Induced Thrombocytopenia (HIT) • • Type 1 HIT presents within the first 4 days

Heparin-Induced Thrombocytopenia (HIT) • • Type 1 HIT presents within the first 4 days after exposure to heparin the platelet count normalizes non-immune disorder that results from the direct effect of heparin on platelet activation Type 2 HIT occurs 4 -10 days after exposure to heparin life- and limb-threatening thrombotic complications immune-mediated disorder In general medical practice, the term HIT refers to Type 2 HIT. 2/17

Heparin-Induced Thrombocytopenia (Type 2 HIT) pathophysiology anti-PF 4/heparin Ig. G antibody platelet PF 4

Heparin-Induced Thrombocytopenia (Type 2 HIT) pathophysiology anti-PF 4/heparin Ig. G antibody platelet PF 4 heparin Fcγ RIIa receptor platelet aggregation and removal, thrombosis platelet activation, release of procoagulant microparticles 3/17

Diagnosis of HIT – clinical scoring tools • determination of the likelihood that a

Diagnosis of HIT – clinical scoring tools • determination of the likelihood that a patient has HIT based on clinical criteria, before laboratory test • clinical scoring tools to help determine clinical probability of HIT: 4 Ts score, HEP score, other scoring systems A total score of 4 Ts: points probability predictive value ≤ 3 low NPV 99. 8% 4 -5 intermediate PPV 14% 6 -8 high PPV 64% Blood 2012; 120: 4160 J Thromb Haemost. 2006; 4: 759 -65 4/17

Diagnosis of HIT – Laboratory testing for anti-PF 4/heparin antibodies Laboratory assays for HIT:

Diagnosis of HIT – Laboratory testing for anti-PF 4/heparin antibodies Laboratory assays for HIT: PRINCIPLES 5/17 CHARACTERISTICS detect anti-PF 4/heparin IMMUNOASSAYS antibodies (plateletactivating + non-platelet activating antibodies) high sensitivity (>95%), limited specificity (30%-70%), widely available FUNCTIONAL ASSAYS high specificity (80 -100%), restricted to specialized laboratories, usually applied as second-line tests detect platelet activating anti-PF 4/heparin antibodies

Immunoassays for HIT � Particle gel immunoassay Pa. GIA � Lateral flow immunoassay �

Immunoassays for HIT � Particle gel immunoassay Pa. GIA � Lateral flow immunoassay � Latex immunoturbidimetric assay � ELISA, enzyme-linked immunosorbent assay The intensity of the colour, measured as optical density (OD), is proportional to the concentration of bound antibodies. 6/17

Functional assays for HIT - based on in vitro activation of platelets as evidence

Functional assays for HIT - based on in vitro activation of platelets as evidence for the presence of platelet-activating antibodies �technically demanding, fresh donor platelets �SRA, 14 C-serotonin release assay (sensitivity 69 -94%, specificity 90 -97%) �HIPA, heparin-induced platelet activation assay (sensitivity 39 -81%, specificity 82 -95%) �flow cytometric assays with indicators of platelet 7/17 activation: - detected surface markers of activated platelets - elevated number of platelet-derived microparticles

Flow cytometric functional assays – surface markers of activated platelets anti-CD 61 antibody for

Flow cytometric functional assays – surface markers of activated platelets anti-CD 61 antibody for β 3 integrin expressed on all platelets CD 61 CD 62 P anti-CD 62 P antibody for CD 62 P (P -selectin) expressed on the activated platelet after α-granule exocytosis Tomer et al, Am J Hematol 1999; 61(1): 53 -61. 8/17

Flow cytometric functional assay – sample preparation patient’s serum fresh donor platelet-rich plasma 0.

Flow cytometric functional assay – sample preparation patient’s serum fresh donor platelet-rich plasma 0. 2 or 200 IU/m. L heparin 1 h incubation flow cytometer 9/17 fluorescently labeled monoclonal antibodies against platelets

Flow cytometric functional assay -analysis The result of the test is expressed as a

Flow cytometric functional assay -analysis The result of the test is expressed as a percentage of activated platelets from all platelets. 47% platelet activation in the presence of pharmacological concentration of heparin. 3% platelet activation in the presence of high concentration of heparin. Positive serum controls 28. 6% ± 12. 3 / excess heparin 3. 0% ± 2. 8 Negative serum controls 4. 7% ± 2. 3 / excess heparin 2. 9. % ± 1. 8 Cut-off for positive test: >10% platelet activation and the activation is at least 50% lower at a high concentration of heparin. 10/17

Comparison of the flow cytometric assay and heparininduced platelet activation assay (HIPA) HIPA n

Comparison of the flow cytometric assay and heparininduced platelet activation assay (HIPA) HIPA n = 41 ELISA positive patients FLOW CITOMETRY Positive Negative 14 3 Negative 4 PPV 14/(14+4)= 0. 78 20 NPV 20/(3+20)= 0. 87 sensitivity: 14/14+3= 0. 82 specificity: 20/20+4=0. 83 accuracy: 34/41= 0. 83 11/17 Limitations of the study: • a small number of the patients • sensitivity and specificity of HIPA is not 100% • correlation with clinical presentation is missing 11

The level of anti-PF 4/heparin antibodies in correlation with result of the flow cytometric

The level of anti-PF 4/heparin antibodies in correlation with result of the flow cytometric functional assay Patient characteristics and the results of ELISA testing Number of all HIT suspected patients 1004 Age, mean (range) 67[1 -95] Gender (M/F) 562/442 Number of Ig. G ELISA positive patients Age, mean (range) Gender (M/F) 107/1004 68 [20 -87] 55/52 ELISA positive patients: 3 groups with increasing OD values were defined: OD <1. 0 47 (44%) OD >1. 0 < 2. 0 26 (24%) 50 OD >2. 0 34 (32%) 40 30 20 10 0 OD <1. 0 12/17 OD >1. 0 < 2. 0 OD >2. 0

Results of flow cytometric assay for patients who met the criteria for a positive

Results of flow cytometric assay for patients who met the criteria for a positive ELISA result (OD>0. 4) 1. 96 ± 0. 13 0. 94 ± 0. 09 The average OD value of patients with platelet activating antibodies was statistically significantly higher then of patients with non-platelet activating antibodies. 13/17

Results of flow cytometric assay for patients who met the criteria for a positive

Results of flow cytometric assay for patients who met the criteria for a positive ELISA result (OD>0. 4) Number of flow cytometric positive patients 64/107 Age, mean (range) Gender (M/F) OD <1. 0 OD >1. 0 < 2. 0 OD >2. 0 94% 50 40 23% 30 58% 20 10 0 OD <1. 0 OD >1. 0 < 2. 0 ELISA positive 14/17 OD >2. 0 PC positive 73 [34 -86] 27/37 11 (23%) 15 (58%) 34 (94%) OD value >2. 0 has a likelihood of 94% to be associated with the presence of platelet activating antibodies. OD value OD >1. 0 < 2. 0 has a likelihood of 58% to be associated with the presence of platelet activating antibodies. OD value OD <1. 0 has a likelihood of 23% to be associated with the presence of platelet activating antibodies.

CONCLUSIONS • Flow cytometers are available in many diagnostic laboratories; the result of the

CONCLUSIONS • Flow cytometers are available in many diagnostic laboratories; the result of the functional assay for HIT can be reached within 2 -3 hours. • The use of different platelet donors increases the reliability of the test. • A higher ELISA OD value represents a higher probability for the presence of platelet-activating anti-heparin/PF 4 antibodies, which can provide additional information for clinicians. 15/17

CONCLUSIONS - limitations • Improvements of the assay to avoid false-negative and false-positive results:

CONCLUSIONS - limitations • Improvements of the assay to avoid false-negative and false-positive results: - selected high reactive (one) donor platelets - using washed platelets instead of PRP - including a weak-positive control serum • To evaluate the results of the flow cytometric functional assay in relation to the 4 T score system/ clinical presentation. 16/17

Thank you for your attention Ljubljana, Slovenia

Thank you for your attention Ljubljana, Slovenia