Quality assurance and reproducibility of high-resolution two-dimensional electrophoresis and silver staining in polyacrylamide gels.
Qualitative data from high-resolution two-dimensional electrophoresis (2DE) have become clinically useful in immunoglobulin clonality analysis, in resolution of ambiguities in immunofixation typing of paraproteins, and in genetic typing of serum proteins. Since 1986, the authors have been evaluating the College of American Pathologists Reference Preparation for Serum Proteins (RPSP) as a quality control material for 2DE because (1) it is prepared exclusively from pooled human sera, (2) the pool yields a reference pattern of mixed heterozygosity for genetic markers, and (3) RPSP is widely available as a lyophilized preparation that currently serves in the authors' laboratory as a qualitative quality control preparation and that may become a quantitative quality control material or external quality assessment material for 2DE. Using the ISO-DALT 2DE system and silver-staining, the peptide patterns were examined in 11 lots of RPSP and compared with fresh serum and with each other. Consistent differences in the 2DE pattern between RPSP and fresh serum included the presence of freeze-thaw peptides, the presence of degradation spots of apolipoprotein A-I, and the diminution of apolipoprotein spot intensities in RPSP. All lots of RPSP yielded clear identification of the eight serum proteins used for quality control calculations. Run-to-run coefficients of variation for a single lot of RPSP for four parameters of 2DE spot location and gradient reproducibility were comparable with band location reproducibility for the one-dimensional procedures of serum protein electrophoresis and lactate dehydrogenase isoenzyme electrophoresis. It is concluded that the reproducibility, that is, imprecision, of 2DE is the same as one-dimensional clinical electrophoresis techniques and that either RPSP or pooled fresh serum can serve as a satisfactory internal quality control material.
Harrison
H H
HH
Department of Pathology, University of Chicago Pritzker School of Medicine, Illinois 60637.
Miller
K L
KL
Dickinson
C
C
Daufeldt
J A
JA
D001797:Blood Protein Electrophoresis / Q000592:standards*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000592:standards*; D006801:Humans; D011786:Quality Control; D012015:Reference Standards; D015203:Reproducibility of Results; D016622:Silver Staining / Q000592:standards*