title: The detection of isolated tumor cells in bone marrow comparing bright-field immunocytochemistry and multicolor immunofluorescence.
abstract: BACKGROUND
  The detection of isolated tumor cells in bone marrow by immunocytochemistry (ICC) has been reported to predict progression of early-stage breast cancer. The most common staining procedure uses bright-field ICC with cytokeratin (CK) antibodies to label isolated tumor cells. However, this method can result in false-positive staining events. We used multicolor immunofluorescence (IF) to develop a more specific assay for detecting isolated tumor cells in marrow samples from breast cancer patients.
  
  
  METHODS
  We compared ICC and IF side by side for detection of cancer cells and false-positive staining events on bone marrow aspirates from breast cancer patients, bone marrow from healthy donors, and healthy donor blood spiked with cancer cells. The primary target for isolated tumor cell detection was CK for both methods. IF used an additional set of antibodies to label hematopoietic cells (HCs).
  
  
  RESULTS
  The detection rate of CK+ events in breast cancer patient bone marrow aspirates was 18 (58%) of 31 for ICC and 21 (68%) of 31 for IF. However, with IF, 17 of 21 CK+ cases were stained with HC markers and thus were identified as false-positive events. A surprisingly high CK+ event rate was observed in healthy donor blood and marrow. In all healthy donor samples, CK+ events were readily identified as HCs by IF. Detection sensitivity of spiked cancer cells in donor blood was similar for both methods.
  
  
  CONCLUSIONS
  There is a high frequency of CK+ events in blood and marrow, and it is important to note that this is observed both in patients with and those without cancer. IF with multiple HC markers allows straightforward discrimination between CK+ cells of hematopoietic and nonhematopoietic origin.
authors.affiliation: Department of Surgery, University of Vermont College of Medicine, Given Building, 89 Beaumont Avenue, Burlington, Vermont, 05405, USA. david.krag@uvm.edu
authors.forename: David N
authors.initials: DN
authors.lastname: Krag
authors.forename: Roberto
authors.initials: R
authors.lastname: Kusminsky
authors.forename: Edward
authors.initials: E
authors.lastname: Manna
authors.forename: Abiy
authors.initials: A
authors.lastname: Ambaye
authors.forename: Donald L
authors.initials: DL
authors.lastname: Weaver
authors.forename: Seth P
authors.initials: SP
authors.lastname: Harlow
authors.forename: Michael
authors.initials: M
authors.lastname: Covelli
authors.forename: Mary A
authors.initials: MA
authors.lastname: Stanley
authors.forename: Laurence
authors.initials: L
authors.lastname: McCahill
authors.forename: Frank
authors.initials: F
authors.lastname: Ittleman
authors.forename: Bruce
authors.initials: B
authors.lastname: Leavitt
authors.forename: Martin
authors.initials: M
authors.lastname: Krag
mesh_terms: D001856:Bone Marrow Examination / Q000379:methods*; D019046:Bone Marrow Neoplasms / Q000473:pathology* / Q000556:secondary; D001943:Breast Neoplasms / Q000473:pathology*; D005260:Female; D005455:Fluorescent Antibody Technique / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D007633:Keratins / Q000032:analysis; D014407:Tumor Cells, Cultured
chemical_list: D007633:Keratins