Immediate bromodeoxyuridine labelling of unseparated bone marrow cells is superior to labelling after separation

The proliferation of bone marrow cells is important to evaluate in several disease conditions and during treatment of patients with cytokines. Bromodeoxyuridine (BrdUrd) labelling and analysis by flow cytometry is a reliable and reproduceable technique for estimation of the fraction of cells that has incorporated BrdUrd into the DNA during S phase. We have compared immediate bromodeoxyuridine labelling of unseparated bone marrow cells with the previously used labelling in the laboratory after separation of the mononulear cells. Bone marrow aspirates from 7 lymphoma patients (without bone marrow involvement) were studied with these two methods. The unseparated bone marrow aspirates or the mononuclear cell fraction were incubated (37 C) in RPMI 1640 added 20//M BrdUrd and 100 U heparin for 30, 60 and 120 min. After separation of mononuclear cells from the immediately labelled samples, the fraction of BrdUrd labelled cells was estimated by flow cytometry. We found relatively higher BrdUrd labelling indeces (LI), when cells were labelled immediately, and in addition a significant increase of BrdUrd LI when the incubation was prolonged. A large variation in LI was found between patients. Mean LI (%): 30 min 60 min 120 min Before cell separation 8. 9 10. 4 12. 0 After cell separation 6. 1 6. 2 7. 0 Our results strongly suggest that BrdUrd labelling of bone marrow cells should be performed immediately after aspiration, and before separation, because these data are more similar to reported values from in vivo labelling with BrdUrd (approx. 15% at 6 h after injection into the patient; Riccardi et al. , Br J Cancer 59:898, 1989). Thus, immediate labelling of bone marrow aspirates is competitive to in vivo labelling and without any ethical complications.