title: Detection of tumor-associated cells in cryopreserved peripheral blood mononuclear cell samples for retrospective analysis.
abstract: Cryopreserved peripheral blood mononuclear cells (PBMCs) are commonly collected in biobanks. However, little data exist regarding the preservation of tumor-associated cells in cryopreserved collections. The objective of this study was to determine the feasibility of using the CellSieve™ microfiltration assay for the isolation of circulating tumor cells (CTCs) and circulating cancer-associated macrophage-like cells (CAMLs) from cryopreserved PBMC samples.
  
  
  
  Blood samples spiked with breast (MCF-7), prostate (PC-3), and renal (786-O) cancer cell lines were used to establish analytical accuracy, efficiency, and reproducibility after cryopreservation. The spiked samples were processed through Ficoll separation, and cryopreservation was followed by thawing and microfiltration.
  
  
  
  MCF-7 cells were successfully retrieved with recovery efficiencies of 90.5 % without cryopreservation and 87.8 and 89.0 %, respectively, on day 7 and day 66 following cryopreservation. The corresponding recovery efficiencies of PC-3 cells were 83.3 % without cryopreservation and 85.3 and 84.7 %, respectively, after cryopreservation. Recovery efficiencies of 786-O cells were 92.7 % without cryopreservation, and 82.7 and 81.3 %, respectively, after cryopreservation. The recovered cells retained the morphologic characteristics and immunohistochemical markers that had been observed before freezing. The protocols were further validated by quantitation of CAMLs in blood samples from two patients with renal cell carcinoma (RCC). The recovery rates of CTCs and CAMLs from cryopreserved samples were not statistically significant different (P > 0.05) from matched fresh samples.
  
  
  
  To our knowledge, this is the first report that CAMLs could be cryopreserved and analyzed after thawing with microfiltration technology. The application of microfiltration technology to cryopreserved samples will enable much greater retrospective study of cancer patients in relation to long-term outcomes.
authors.affiliation: Creatv MicroTech, Inc., 11609 Lake Potomac Drive, Potomac, MD, 20854, USA.
authors.forename: Peixuan
authors.initials: P
authors.lastname: Zhu
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic, 13400 East Shea Blvd, Scottsdale, AZ, 85259, USA.
authors.forename: Melissa L
authors.initials: ML
authors.lastname: Stanton
authors.affiliation: Department of Urology, Mayo Clinic Hospital, 5777 E Mayo Blvd, Phoenix, AZ, 85054, USA.
authors.forename: Erik P
authors.initials: EP
authors.lastname: Castle
authors.affiliation: Division of Hematology and Medical Oncology, Mayo Clinic, 4500 San Pablo Rd, Jacksonville, FL, 32224, USA.
authors.forename: Richard W
authors.initials: RW
authors.lastname: Joseph
authors.affiliation: Creatv MicroTech, Inc., 11609 Lake Potomac Drive, Potomac, MD, 20854, USA.
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Adams
authors.affiliation: Creatv MicroTech, Inc., 11609 Lake Potomac Drive, Potomac, MD, 20854, USA.
authors.forename: Shuhong
authors.initials: S
authors.lastname: Li
authors.affiliation: Creatv MicroTech, Inc., 11609 Lake Potomac Drive, Potomac, MD, 20854, USA.
authors.forename: Platte
authors.initials: P
authors.lastname: Amstutz
authors.affiliation: Creatv MicroTech, Inc., 11609 Lake Potomac Drive, Potomac, MD, 20854, USA.
authors.forename: Cha-Mei
authors.initials: CM
authors.lastname: Tang
authors.affiliation: Division of Hematology and Medical Oncology, Mayo Clinic, 13400 East Shea Blvd, Scottsdale, AZ, 85259, USA. Ho.Thai@mayo.edu.
authors.forename: Thai H
authors.initials: TH
authors.lastname: Ho
mesh_terms: D002292:Carcinoma, Renal Cell / Q000473:pathology; D045744:Cell Line, Tumor; D015925:Cryopreservation; D005629:Frozen Sections; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007680:Kidney Neoplasms / Q000473:pathology; D007963:Leukocytes, Mononuclear / Q000473:pathology*; D008264:Macrophages / Q000473:pathology; D009369:Neoplasms / Q000097:blood* / Q000473:pathology*; D009360:Neoplastic Cells, Circulating / Q000473:pathology; D012189:Retrospective Studies
keywords: CellSieve™ microfiltration; Circulating cancer-associated macrophage-like cells (CAMLs); Circulating tumor cells (CTCs); Cryopreservation; Ficoll; Fluorescence antibody staining