Heparin-induced thrombocytopenia (HIT), an adverse drug effect, has gained much attention. Affected patients have a high risk of new thrombotic complications. In addition, HIT is also a model to study mechanisms of immune-mediated disorders. Platelet factor 4 (PF4) is the key protein involved. It is the basis for many diagnostic tests for HIT and is used for in vitro studies and in mouse models on the pathogenesis of HIT. Purified PF4 is known to easily form aggregates, which can cause artifacts in experiments. The impact of storage buffer, storage period, lyophilization, and temperature on the size of PF4 and PF4/heparin (H) complexes was assessed by dynamic light scattering (DLS), while enzyme immunoassay (EIA) was used to test the binding of anti-PF4/H antibodies (aPF4/H Abs) to PF4/H complexes. PF4 size was more stable in Hank's balanced salt solution (HBSS) compared to phosphate-buffered saline (PBS), especially during storage. Lyophilization further facilitated the formation of PF4 aggregates, while incubation of reconstituted lyophilized PF4 in PBS at 37 °C reduced PF4 aggregates. Complexes formed between lyophilized PF4 and heparin were larger, and they enhanced the binding of aPF4/H Abs in EIA compared to complexes between nonlyophilized PF4 and heparin, both in HBSS and PBS, possibly influencing in vitro test results. Our results may be helpful for mechanistic studies on the biological function of PF4 and for the improvement of assays for detecting aPF4/H Abs.