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Keywords:

  • circulating tumor cells;
  • immunomagnetic cell separation;
  • immunocytochemistry;
  • RT-PCR;
  • squamous cell carcinoma of the head and neck (HNSCC)

Abstract

The optimization of a purely negative depletion, enrichment process for circulating tumor cells (CTCs) in the peripheral blood of head and neck cancer patients is presented. The enrichment process uses a red cell lysis step followed by immunomagnetic labeling, and subsequent depletion, of CD45 positive cells. A number of relevant variables are quantified, or attempted to be quantified, which control the performance of the enrichment process. Six different immunomagnetic labeling combinations were evaluated as well as the significant difference in performance with respect to the blood source: buffy coats purchased from the Red Cross, fresh, peripheral blood from normal donors, and fresh peripheral blood from human cancer patients. After optimization, the process is able to reduce the number of normal blood cells in a cancer patient's blood from 4.05 × 109 to 8.04 × 103 cells/mL and still recover, on average, 2.32 CTC per mL of blood. For all of the cancer patient blood samples tested in which CTC were detected (20 out of 26 patients) the average recovery of CTCs was 21.7 per mL of blood, with a range of 282 to 0.53 CTC. Since the initial number of CTC in a patient's blood is unknown, and most probably varies from patient to patient, the recovery of the CTC is unknown. However, spiking studies of a cancer cell line into normal blood, and subsequent enrichment using the optimized protocol indicated an average recovery of approximately 83%. Unlike a majority of other published studies, this study focused on quantifying as many factors as possible to facilitate both the optimization of the process as well as provide information for current and future performance comparisons. The authors are not aware any other reported study which has achieved the performance reported here (a 5.66 log10) in a purely negative enrichment mode of operation. Such a mode of operation of an enrichment process provides significant flexibility in that it has no bias with respect to what attributes define a CTC; thereby allowing the researcher or clinician to use any maker they choose to define whether the final, enrich product contains CTCs or other cell type relevant to the specific question (i.e., does the CTC have predominately epithelial or mesenchymal characteristics?). Biotechnol. Bioeng. 2009;102: 521–534. © 2008 Wiley Periodicals, Inc.