A METHOD FOR TRACKING THE MIGRATION OF BLOOD-LYMPHOCYTES

A method has been devised for labeling whole blood with the fluorescen t dye fluorescein isothiocyanate (FITC) so the migration of blood lymp hocytes can be studied in the sheep. Although lymphocytes can be purif ied from blood using density gradient media or elutriation it is diffi cult to obtain a large number of cells, because many cells are usually lost during the purification steps. It is desirable to label at least 10(8)-10(9) lymphocytes for lymphocyte tracking studies, because a sm aller number is difficult to subsequently detect and quantitate in blo od and lymph even using flow cytometry. Also, it is desirable to minim ize the in vitro manipulation of lymphocytes, because dead or damaged lymphocytes will not recirculate. By labeling all the cellular compone nts of a sample of whole blood rather than first purifying the lymphoc ytes we have been able to satisfy both of these criteria. Although lab eled blood cells of all types are reinjected into the animal, the lymp hocytes are easily distinguishable from other cells using a flow cytom eter. In these studies between 2.4 - 12.4 x 10(8) lymphocytes were inj ected intravenously, and they were detectable in the blood and lymph f or at least 10 days. The recovery of FITC-labeled (FITC+) lymphocytes in efferent lymph is comparable to that of lymphocytes labeled with ot her fluorescent or radioactive markers. The presence of labeled non-ly mphoid cells in the animal makes this technique impractical for studie s of lymphocyte localization within histologic sections. However, it i s useful for studies in animals in which lymphatic vessels can be cann ulated and the blood-to-lymph recirculation of labeled lymphocytes mon itored, and it also may be applicable for studies in which lymphoid or gan suspensions are analyzed using flow cytometry.