Immunocytochemical Phenotyping of Disseminated Tumor Cells in Bone Marrow by uPA Receptor and CK18: Investigation of Sensitivity and Specificity of an Immunogold/Alkaline Phosphatase Double Staining Protocol

Phenotyping of cytokeratin (CK) 18-positive cells in bone marrow is
gaining increasing importance for future prognostic screening of
carcinoma patients. Urokinase-type plasminogen activator receptor
(uPA-R) is one example of a potential aggressive marker for those
cells. However, a valid and reliable double staining method is
needed. Using monoclonal antibodies against uPA-R and CK18, we
modified an immunogold/alkaline phosphatase double staining
protocol. UPA-R/CK18-positive tumor cell controls exhibited black
uPA-R staining in 15–80 of cases and red CK18 staining in almost
100 of tumor cells. Isotype- and cross-matched controls were
completely negative. Bone marrow from healthy donors was always
CK18-negative. Reproducibility of CK18-positive cell detection was
estimated in a series of specimens from 61 gastric cancer patients
comparatively stained with the single alkaline
phosphatase-anti-alkaline phosphatase (APAAP) and our double
staining method (106 bone marrow cells/patient). In four cases,
double staining could not reproduce CK18-positive cells. In 34
cases it revealed fewer or equal numbers, and in 23 cases more
CK18-positive cells than the APAAP method. Overall quantitative
analysis of detected cell numbers (838 in APAAP, range 1–280 in
106; double staining 808, range 0–253) demonstrated relative
reproducibility of APAAP results by double staining of 97.
Correlation of results between both methods was significant
(p