The diagnosis of heparin-induced thrombocytopenia type 2 (HIT) requires a combination of clinical and laboratory data. Rapid exclusion of HIT is supported by sensitive immunoassays. Citrated plasma or whole blood are more often available in the laboratory, and may thus, have practical advantages as compared to serum. In this study, various specimens from patients with suspected HIT were compared using a rapid lateral flow immunoassay (LFI-HIT). Serum, citrated whole blood and plasma were obtained from patients in whom HIT was suspected (n = 211) and were tested with LFI-HIT and several other assays. The comparison of plasma vs. serum in 210 samples showed agreement of 93.6% and between whole blood and serum in 183 samples was 96.7%. All serum-positive cases were correctly detected in plasma. Comparison of samples, which were positive in one of the immunoassays with the functional test heparin-induced platelet aggregation assay revealed a negative predictive value of 98.4% for serum, 97.8% for plasma and 96.2% for citrated whole blood. Performance in whole blood was slightly inferior. These data demonstrate that plasma and serum can be used in LFI-HIT with equivalent performance and that negative test results in LFI-HIT exclude HIT with high probability. The use of whole blood is not recommended by the authors.