Heparin-induced thrombocytopenia (HIT) remains a challenge, with diagnosis confirmed only by functional assays. The gold standard 14C-serotonin release assay (SRA) is highly sensitive but technically challenging and unsuitable for routine use. We conducted a large study to validate whole blood impedance aggregometry (WBIA) as a suitable diagnostic tool for HIT. WBIA and SRA were used to test 181 samples positive for H-PF4 antibodies by PaGIA or ELISA. Using the same high responder donor, 77 samples were positive by WBIA (aggregation with low-dose but not high-dose heparin). Using the strict definition for SRA positivity, 72 samples were true HIT. In nine samples, serotonin release with high-dose heparin dropped by > 50 but was still >20 ; these were retested after a one-half dilution and 8/9 became positive. Ten other samples were discrepant between the two assays: one strongly positive (89 release) and six weakly positive samples by SRA (average release 56 ) were WBIA negative. When these samples were retested using a random donor, only two remained SRA positive. Three samples were strongly WBIA positive but SRA negative; two were retested by SRA with 0.5IU/ml heparin and one became positive. Under controlled conditions, using the same selected high-responder donor, WBIA and SRA performed similarly with slightly increased sensitivity of the WBIA when using the strict definition of SRA positivity. WBIA is easy to perform with rapid turn-around time and warrants a multi-laboratory trial to complete its validation as a confirmatory assay for platelet-activating HIT antibodies. ? Schattauer 2012.