title: Optimal immunocytochemical and flow cytometric detection of P-gp, MRP and LRP in childhood acute lymphoblastic leukemia.
abstract: The clinical relevance of multidrug resistance (MDR)-related proteins in childhood acute lymphoblastic leukemia (ALL) is largely unknown. The diversity of techniques, fixation methods, storage of cells (fresh or cryopreserved) etc, may contribute to discrepancies observed between several studies. We therefore optimized the detection of P-glycoprotein (P-gp), MDR-associated protein (MRP) and lung resistance-related protein (LRP) by immunocytochemistry and flow cytometry in childhood ALL cells. Thirteen fixation methods were compared using six antibodies in both immunocytochemistry and flow cytometry. The optimal fixation for P-gp (C219, MRK16), MRP (MRPr1) and LRP (LRP56) was a mixture of 2% (v/v) formaldehyde solution and acetone incubated for only 10 s at room temperature (FAc). For MRP recognized by MRPm6, the optimal fixation condition was acetone for 5 min at room temperature in immunocytochemistry, and methanol for 15 min at -20 degrees C in flow cytometry. P-gp staining by 4E3 was strongly antibody batch-dependent; on cytospins FAc fixation was optimal, but inconclusive data were obtained by flow cytometry. The optimized fixation conditions on fresh samples revealed a day-to-day variation in staining (both increasing and decreasing) in one third of the immunocytochemical tests. In flow cytometry the day-to-day variation in the fluorescence index was -1 +/- 22%. In both techniques, staining was comparable between fresh and cryopreserved cells. We recommend the use of the above mentioned fixation methods in order to study the clinical relevance of P-gp, MRP and LRP in childhood ALL.
authors.affiliation: Department of Pediatric Hematology/Oncology, Academic Hospital Vrije Universiteit, Amsterdam, The Netherlands.
authors.forename: M L
authors.initials: ML
authors.lastname: Den Boer
authors.forename: C M
authors.initials: CM
authors.lastname: Zwaan
authors.forename: R
authors.initials: R
authors.lastname: Pieters
authors.forename: K M
authors.initials: KM
authors.lastname: Kazemier
authors.forename: M M
authors.initials: MM
authors.lastname: Rottier
authors.forename: M J
authors.initials: MJ
authors.lastname: Flens
authors.forename: R J
authors.initials: RJ
authors.lastname: Scheper
authors.forename: A J
authors.initials: AJ
authors.lastname: Veerman
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis*; D018528:ATP-Binding Cassette Transporters / Q000032:analysis*; D002648:Child; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins / Q000032:analysis*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy* / Q000378:metabolism; D015203:Reproducibility of Results; D014407:Tumor Cells, Cultured; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein