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

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-PF4/heparin IgG antibody PF4 heparin platelet 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 patient has HIT based on clinical criteria, before laboratory test clinical scoring tools to help determine clinical probability of HIT: 4Ts score, HEP score, other scoring systems A total score of 4Ts: 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

Laboratory testing for anti-PF4/heparin antibodies Diagnosis of HIT – Laboratory testing for anti-PF4/heparin antibodies Laboratory assays for HIT: PRINCIPLES CHARACTERISTICS IMMUNOASSAYS detect anti-PF4/heparin antibodies (platelet- activating + non-platelet -activating antibodies) high sensitivity (>95%), limited specificity (30%-70%), widely available FUNCTIONAL ASSAYS detect platelet activating anti-PF4/heparin antibodies high specificity (80-100%), restricted to specialized laboratories, usually applied as second-line tests 5/17

Immunoassays for HIT Particle gel immunoassay PaGIA 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 for the presence of platelet-activating antibodies technically demanding, fresh donor platelets SRA, 14C-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 activation: detected surface markers of activated platelets elevated number of platelet-derived microparticles 7/17

Flow cytometric functional assays – surface markers of activated platelets anti-CD61 antibody for β3 integrin expressed on all platelets CD61 anti-CD62P antibody for CD62P (P-selectin) expressed on the activated platelet after α-granule exocytosis CD62P 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 0.2 or 200 IU/mL heparin 1 h incubation fluorescently labeled monoclonal antibodies against platelets flow cytometer 9/17

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 heparin-induced platelet activation assay (HIPA) ELISA positive patients HIPA   Positive Negative FLOW CITOMETRY 14 4 PPV 14/(14+4)= 0.78 3 20 NPV 20/(3+20)= 0.87 sensitivity: 14/14+3= 0.82 specificity: 20/20+4=0.83 accuracy: 34/41= 0.83 Limitations of the study: a small number of the patients sensitivity and specificity of HIPA is not 100% correlation with clinical presentation is missing 11/17

The level of anti-PF4/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 IgG ELISA positive patients 107/1004 Age, mean (range) 68 [20-87] Gender (M/F) 55/52 OD <1.0 47 (44%) OD >1.0 < 2.0 26 (24%) OD >2.0 34 (32%) ELISA positive patients: 3 groups with increasing OD values were defined: 12/17

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 ELISA result (OD>0.4) Number of flow cytometric positive patients 64/107 Age, mean (range) 73 [34-86] Gender (M/F) 27/37 OD <1.0 11 (23%) OD >1.0 < 2.0 15 (58%) OD >2.0 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. 14/17

A higher ELISA OD value represents a higher 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/PF4 antibodies, which can provide additional information for clinicians. 15/17

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 4T score system/ clinical presentation. 16/17

Thank you for your attention Ljubljana, Slovenia