A SIMPLE, FLUORESCENT METHOD TO INTERNALLY LABEL PLATELETS SUITABLE FOR PHYSIOLOGICAL MEASUREMENTS

Current methods for studying platelet survival in vivo are limited by the use of radioisotopes, with their inherent safety and regulatory co ncerns, systemic drug administrations that produce biochemical modific ations of platelet functions, or external labeling techniques, which m ay produce artifacts due to surface modifications. For these reasons, we sought to develop a simple, nonisotopic method for labeling platele ts internally, thereby producing platelets more likely to have in vivo properties equivalent to native cells. Murine platelets in protein-fr ee buffer were fluorescently labeled internally by incubation with 2.5 mu M 5-chloromethyl fluorescein diacetate (CMFDA), and without washin g, were injected into mice for platelet survival studies. CMFDA-labele d platelets were unactivated, as shown by minimal P-selectin expressio n. When tested in vitro for function by aggregometry, the response of CMFDA-labeled platelets to collagen and thrombin was identical to that of unlabeled platelets. Flow cytometric analysis demonstrated that CM FDA platelets were an intensely stained, unimodal population that was completely separated from unlabeled platelets. The mean half-life of l abeled platelets in the murine circulation was 37.5 +/- 4.5 hr (+/- SD ), and the mean survival time was 3.1-3.3 days (n = 24), similar to re sults reported using Cr-51 and In-111. No evidence of in vivo transfer of dye from labeled platelets to unlabeled cells was observed. CMFDA produces a population of platelets that are nonradioactively, internal ly labeled with a highly fluorescent, stable product. The labeled plat elets function equivalently to native platelets, as demonstrated by im munocytometry and aggregometry, and importantly, in vivo, by normal pl atelet survival. (C) 1997 Wiley-Liss, Inc.