Isolation of malignant B cells from patients with chronic lymphocytic leukemia (CLL) for analysis of cell proliferation: validation of a simplified method suitable for multi-center clinical studies.
BACKGROUND
Heavy water ((2)H(2)O) labelling of DNA enables the measurement of low-level cell proliferation in vivo, using gas chromatography/pyrolysis isotope ratio mass spectrometry (GC/P/IRMS), but the methodology has been too complex for widespread use. Here, we report a simplified method for measuring proliferation of malignant B cells in patients with chronic lymphocytic leukemia (CLL).


METHODS
Patients were labelled with (2)H(2)O for 6 weeks; blood samples were obtained at 0, 3, and 6 weeks during (2)H(2)O labelling and 9, 12, and 16 weeks thereafter. Bone marrow was sampled at week 6. Phlebotomy was performed at multiple, non-research clinical sites. CLL cells were isolated in a central laboratory, using a novel RosetteSep-based method; DNA labelling was analyzed by GC/P/IRMS.


RESULTS
In 26 of 29 patients, CLL cell isolation resulted in > or =95% purity for malignant CD5+ B cells; in one patient, malignant cells expressed marginal levels of CD5, and in two others, further sorting of CD5hi malignant cells was required. Cell yields correlated with white blood cell counts and exceeded GC/P/IRMS requirements ( approximately 10(7) cells) >98% of the time; high-quality DNA labelling data were obtained. RosetteSep isolation achieved adequate CLL cell purity from bone marrow in only 64% of samples, but greatly reduced subsequent sort time for impure samples.


CONCLUSIONS
This method enables clinical studies of CLL cell proliferation outside of research settings, using a shorter (2)H(2)O intake protocol, a minimal sampling protocol, and centralised sample processing. The CLL cell isolation protocol may also prove useful in other applications. (clinicaltrials.gov identifier: NCT00481858).
KineMed, Inc., Emeryville, CA 94608, USA. ghayes@kinemed.com
Gregory M
GM
Hayes
Robert
R
Busch
Jason
J
Voogt
Iche M
IM
Siah
Tracy A
TA
Gee
Marc K
MK
Hellerstein
Nicholas
N
Chiorazzi
Kanti R
KR
Rai
Elizabeth J
EJ
Murphy
D000328:Adult; D000368:Aged; D001402:B-Lymphocytes / Q000473:pathology*; D001800:Blood Specimen Collection / Q000379:methods / Q000592:standards; D001854:Bone Marrow Cells / Q000473:pathology; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods; D002986:Clinical Trials as Topic / Q000379:methods; D003581:Cytodiagnosis / Q000379:methods; D004247:DNA / Q000032:analysis; D017666:Deuterium Oxide / Q000737:chemistry; D004526:Efficiency; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000097:blood / Q000175:diagnosis / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D015337:Multicenter Studies as Topic / Q000379:methods
D004247:DNA; D017666:Deuterium Oxide