Epstein-Barr virus-induced transformation of B cells for the diagnosis of genetic metabolic disorders--enumerative conditions for cryopreservation.
Epstein-Barr virus (EBV) infection in vitro causes transformation of B cells and generates B lymphoblastoid cell lines (LCLs). These LCLs have been widely used for the diagnostic of several genetic metabolic disorders. However, up to now, efficiency of LCL generation has been based on misleading subjective analysis. In this study, quantitative analyses have been performed to indicate efficiency of B-cell transformation to measuring human lysosomal acid hydrolases associated with: GM1-gangliosidosis type I, Gaucher disease and mucopolysaccharidosis type I. Peripheral blood mononuclear cells were isolated from 13 subjects, and LCLs were produced by culturing them with EBV for 12 days. Activities of the enzymes beta-galactosidase, beta-glucosidase and alpha-iduronidase were measured before and after cryopreservation in liquid nitrogen for 30 days. Efficiency of the B-cell transformation was screened every 4 days by the enumeration of cell proliferation, cell counts and changes in granularity estimated by flow cytometry. We observed the generation of 13 LCLs. Cell transformation was confirmed by the gradual increase of cellular clusters, cell size and granularity. In addition, we determined that the activity of the enzymes mentioned above did not change following cryopreservation. These data suggest that our enumerative approach for screening of EBV-LCLs is efficient for the enzymatic determination of human lysosomal acid hydrolases and may thus replace misleading subjective analyses.
Mello
A S
AS
Instituto de Pesquisas Biomédicas, Hospital São Lucas, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, RS, Brazil.
Burin
M G
MG
Michellin
K
K
Viapiana
M
M
Giugliani
R
R
Coelho
J C
JC
Bauer
M E
ME
D000328:Adult; D001402:B-Lymphocytes / Q000201:enzymology / Q000473:pathology / Q000821:virology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002472:Cell Transformation, Viral; D015925:Cryopreservation; D016537:Gangliosidosis, GM1 / Q000175:diagnosis / Q000201:enzymology; D005776:Gaucher Disease / Q000175:diagnosis / Q000201:enzymology; D004854:Herpesvirus 4, Human; D006801:Humans; D007068:Iduronidase / Q000378:metabolism*; D018655:Lymphocyte Count; D008247:Lysosomes / Q000201:enzymology; D008059:Mucopolysaccharidosis I / Q000175:diagnosis / Q000201:enzymology; D001616:beta-Galactosidase / Q000378:metabolism*; D001617:beta-Glucosidase / Q000378:metabolism*
D001617:beta-Glucosidase; D001616:beta-Galactosidase; D007068:Iduronidase