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). DESIGN AND 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. CONCLUSION: 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).

Heavy water labeling of DNA for measurement of cell proliferation and recruitment during primary murine lymph node responses against model antigens.

Lymph node (LN) responses to antigens involve inflammatory lymphocyte recruitment and proliferation of rare antigen-specific precursors; the relative contributions of these processes have not been well quantified. The popliteal LN assay (PLNA), used for immunotoxicity screening, measures LN swelling as a surrogate of antigen-specific immunity, but nonspecific irritants cause false-positive results. Quantification of proliferating cells may improve specificity, but commonly-used biosynthetic labels (e.g., BrdU) have limitations. In vivo labeling with heavy water ((2)H(2)O) is nontoxic and (2)H incorporation into the DNA of dividing cells highly consistent, even in apoptotic microenvironments such as the thymus. Here, we have used continuous (2)H(2)O labeling and GC/MS analysis to quantify the cumulative fraction of recently divided cells (f) in draining LN of mice. Priming of BALB/c mice with model antigens (KLH, DNCB) increased both LN cell counts and f in responding lymphocyte subsets, whereas lymphocyte recruitment to LN by irritants (IFA, DMSO) increased cell counts but had little effect on f. Thus, antigen-driven proliferation (possibly including a bystander component) was reflected in f, whereas LN cellularity was primarily increased by recruitment. Cell counts responded differentially to changes in Ag dose and immunization with IFA, whereas f was unaffected by these variables. GC/MS analysis of (2)H(2)O-labeled lymphocyte DNA affords sensitive, precise measurements of fractional lymphoproliferation. Dissection of proliferation and cell recruitment by this approach may be useful for preclinical in vivo screening of novel adjuvants and immunomodulatory agents, for studying their mechanism of action, and for immunotoxicity screening in the PLNA.

Isolation of peripheral blood CD4(+) T cells using RosetteSep and MACS for studies of DNA turnover by deuterium labeling.

Tracking deuterium ((2)H) incorporation into cellular DNA, after administration of (2)H(2)O or (2)H(2)-glucose, is a recently developed, broadly applicable method for measuring in vivo cell proliferation and turnover that can be used safely in humans. This approach has been used to evaluate the turnover of T-cell subpopulations purified from the peripheral blood of HIV-1-infected patients using fluorescence-activated cell sorting (FACS). A requirement for widespread adoption of this approach for medical decision-making and for use in larger clinical trials is a simple, reproducible, high-throughput method for isolation of highly purified CD4(+) T cells from peripheral blood. Here, we present a simple method, which does not require FACS, for isolating these cells in sufficient purity and yield for analysis of (2)H incorporation into DNA. When blood from HIV-1-infected patients was used, neither the depletion of unwanted cell lineages by erythrocyte crosslinking (RosetteSep) nor the enrichment of CD4(+) cells by immunomagnetic beads (MACS) individually resulted in sufficient purity. The successive application of the two techniques, however, permitted isolation of >95% pure CD4(+) T cells in adequate yield (>10(6) cells/10 ml blood) from healthy donors and HIV-1-infected patients with CD4 counts between 300 and 700 cells/microl. Moreover, (2)H incorporation into cellular DNA after administration of (2)H(2)O to HIV-1-infected patients was indistinguishable between CD4(+) T cells isolated by RosetteSep/MACS and FACS. Thus, both FACS and the new method isolate a similar mixture of long- and short-lived CD4(+) T cells. In practice, the RosetteSep/MACS method is simple, rapid, robust and capable of high throughput.