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The purpose of this study was to determine whether primary breast cancer patients showed evidence of circulating tumour cells (CTCs) during follow-up as an alternative to monitoring disseminated bone marrow tumour cells (DTCs) by immunocytochemistry and reverse transcriptase (RT)–PCR for the detection of micrometastases. We planned to compare CTC and DTC frequency in low-risk and high-risk patients. We identified two cohorts of primary breast cancer patients who were at low (group II, T1N0, n=18) or high (group III, >3 nodes positive (with one exception, a patient with two positive nodes) n=33) risk of relapse who were being followed up after primary treatment. We tested each cohort for CTCs using the CellSearch system on 1–7 occasions and for DTCs by immunocytochemistry and RT–PCR on 1–2 occasions over a period of 2 years. We also examined patients with confirmed metastatic disease (group IV, n=12) and 21 control healthy volunteers for CTCs (group I). All group I samples were negative for CTCs. In contrast, 7 out of 18 (39%) group II primary patients and 23 out of 33 (70%) group III patients were positive for CTCs (P=0.042). If we count only samples with >1 cell as positive: 2 out of 18 (11%) group II patients were positive compared with 10 out of 33 (30%) in group III (P=0.174). In the case of DTCs, 1 out of 13 (8%) group II patients were positive compared with 19 out of 27 (70%) in group III (P<0.001). Only 10 out of 33 (30%) patients in group III showed no evidence of CTCs in all tests over the period of testing, compared with 11 out of 18 (61%) in group II (P=0.033). A significant proportion of poor prognosis primary breast cancer patients (group III) have evidence of CTCs on follow-up. Many also have evidence of DTCs, which are more often found in patients who were lymph node positive. As repeat sampling of peripheral blood is more acceptable to patients, the measurement of CTCs warrants further investigation because it enables blood samples to be taken more frequently, thus possibly enabling clinicians to have prior warning of impending overt metastatic disease.British Journal of Cancer (2009) 100, 160–166. doi:10.1038/sj.bjc.6604773 www.bjcancer.com Published online 25 November 2008 [ABSTRACT FROM AUTHOR]

The flow cytometers that are currently supported by industry provide accurate CD4(+)-T-cell counts for monitoring human immunodeficiency virus disease but remain unaffordable for routine service work under resource-poor conditions. We therefore combined volumetric flow cytometry (measuring absolute lymphocyte counts in unit volumes of blood) and simpler protocols with generic monoclonal antibodies (MAbs) to increase cost efficiency. Volumetric absolute counts were generated using CD45/CD4 and CD45/CD8 MAb combinations in two parallel tubes. The percentage values for the various subsets were also determined within the leukocyte and lymphocyte populations utilizing a fully automated protocol. The levels of agreement between the newly developed method and the present industry standards, including both volumetric and bead-based systems using a full MAb panel for subset analysis, were tested by Bland-Altman analyses. The limits of agreement for CD4 counts generated by the volumetric methods using either CD45/CD4 (in a single tube) or the full Trio MAb panel (in three tubes) on the CytoronAbsolute flow cytometer were between -29 and +46 cells/mm(3) with very little bias for CD4 counts (in favor of the Trio method: +8 CD4(+) lymphocytes/mm(3); 0.38% of lymphocytes). The limits of agreement for absolute CD4 counts yielded by the volumetric CD45/CD4 method and the bead-based method were between -118 and +98 cells/mm(3), again with a negligible bias (-10 CD4(+) lymphocytes/mm(3)). In the volumetric method using CD45/CD8, the strongly CD8(+) cells were gated and the levels of agreement with the full Trio showed a minor bias (in favor of the Trio; +40 CD8(+) cells/mm(3); 5.2% of lymphocytes) without a significant influence on CD4/CD8 ratios. One trained flow cytometrist was able to process 300 to 400 stained tubes per day. This workload extrapolates to a throughput of >30,000 samples per year if both CD45/CD4 and CD45/CD8 stainings are performed for each patient or a throughput of >60,000 samples if only CD45/CD4 counts are tested in a single tube. Thus, on the basis of the high efficiency and excellent agreement with the present industry standards, volumetric flow cytometers with automated gating protocols and autobiosamplers, complemented by generic CD45, CD4, and CD8 MAbs used in two-color immunofluorescence, represent the most suitable arrangements for large regional laboratories in resource-poor settings.