Major improvement of the reference method of the French drug resistance network for P-glycoprotein detection in human haematological malignancies.
The aim of this study was to improve significantly the sensitivity and specificity of the flow cytometric assay of P-glycoprotein (Pgp) implemented and validated by the laboratories of the French Drug Resistance Network [Huet S, Marie JP, Gualde N, Robert J. Reference method for detection of Pgp mediated multidrug resistance in human hematological malignancies: a method validated by the laboratories of the French Drug Resistance Network. Cytometry 1998;34:248-56] in cells displaying low level of resistance. Fluoresceine-conjugated monoclonal antibodies (Mabs) and propidium iodide were respectively replaced by phycoerythrin-conjugated Mabs and Sytox green. The removal of erythrocytes and granulocytes by density gradient was replaced by the lysis of erythrocytes after Mab incubation. Using these conditions, Pgp could be detected in the K-H30 line, which was negative in former studies, with Mab/Control ratios increasing by 3.7- to 5.9-fold, and Mab/Control ratios in the parental sensitive K562 line still ranging between 0.8 and 1.2. When tested on 16 blood samples from patients presenting haematological malignancies, six samples presented low positivity, which was not detected with the former method, while 10 samples remained negative with the two methods. Pgp was specifically detected in pathological blood cells in the six positive samples.
Huet
Sylvie
S
Dpt de Biologie Médicale, Laboratoire de Pharmacologie des Agents Anticancéreux, CNRS FRE 2618, Université Victor Segalen Bordeaux 2 and Institut Bergonié, 229 cours de l'rgonne, F-33076 Bordeaux Cedex, France. huet@bergonie.org
Marie
Jean-Pierre
JP
Laurand
Armelle
A
Robert
Jacques
J
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm; D005434:Flow Cytometry; D019337:Hematologic Neoplasms / Q000378:metabolism*; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D012333:RNA, Messenger