	US 7,615,358 B2
	Method for determining the concentration of vital epithelial tumor cells in a body fluid
Ulrich Pachmann, Brandenburger Str. 30, 95448 Bayreuth (Germany); and Katharina Pachmann, Brandenburger Str. 30, 95448 Bayreuth (Germany)
Filed on Apr. 10, 2007, as Appl. No. 11/733,259.
Claims priority of provisional application 60/793462, filed on Apr. 20, 2006.
Prior Publication US 2007/0248999 A1, Oct. 25, 2007
Int. Cl. G01N 33/48 (2006.01); G01N 33/53 (2006.01); G01N 33/547 (2006.01)

U.S. Cl. 435—7.2 [436/63; 436/64; 436/813]

15 Claims

1. A method for determining the concentration of vital epithelial tumor cells in a body fluid, comprising the following steps:

a) obtaining from the body fluid of a test subject a test specimen comprising a mixed cell population suspected of containing epithelial tumor cells,

b) labeling the epithelial tumor cells by addition, to the test specimen or to a preparation obtained from the test specimen, of first antibodies or fragments of first antibodies that specifically bind to epithelial cells, which first antibodies or fragments of first antibodies are bound to a fluorochrome, wherein the first antibodies or fragments of first antibodies are directed against human epithelial antigen, wherein said human epithelial antigen is an antigen that is recognized by the monoclonal antibody HEA 125,

c) applying the test specimen to a first support and incubating the test specimen on said first support to let the epithelial tumor cells in said test specimen adhere to said first support, and

d) identifying and quantitating vital cells of the adhering epithelial tumor cells by means of their morphology and calculating the concentration of vital epithelial tumor cells in the body fluid, wherein the morphology of the adhering epithelial tumor cells is analyzed by means of laser scanning cytometry, wherein living tumor cells are detected due to exclusive surface staining and dead cells are omitted due to intracellular staining,

wherein no enrichment of the epithelial tumor cells is performed before the adhesion of the epithelial tumor cells to said first support, wherein no washing step is performed.