Search

Background: β-Thalassemia is endemic to many regions in Southeast Asia and India, and Methods: Gap-PCR was used to simultaneously amplify the β-globin gene from genomic DNA and to detect the Δ619bp deletion mutation. Multiplex minisequencing was then performed on the amplified β-globin fragment to detect an additional 15 common Southeast Asian and Indian β-thalassemia mutations. Site-specific primers of different lengths were subjected to multiple rounds of annealing and single-nucleotide extension in the presence of thermostable DNA polymerase and the four dideoxynucleotides, each labeled with a different fluorophore. Minisequencing products were separated and detected by capillary electrophoresis, followed by automated genotyping. The optimized assay was subjected to a double-blind validation analysis of 89 β-thalassemia and wild-type DNA samples of known genotype. Results: Homozygous wild-type or mutant DNA samples produced electropherograms containing only a single colored peak for each mutation site, whereas samples heterozygous for a specific mutation displayed two different-colored peaks for that mutation site. Samples were automatically genotyped based on color and position of primer peaks in the electropherogram. In the double-blind validation analysis, all 89 DNA samples were genotyped correctly (100% assay specificity). Conclusions: The described semiautomated multiplex minisequencing assay can detect the most common Southeast Asian and Indian β-thalassemia mutations, is amenable to high-throughput scale up, and may bring population-based screening of β-thalassemia in endemic regions a step closer to implementation.

Background: Electrophoretic patterns of proteins in serum/plasma are useful in the diagnosis and evaluation of many diseases. Capillary zone electrophoresis (CZE) allows rapid and automated protein separation and produces digital absorbance data, appropriate for mathematical analysis. We previously demonstrated success in detection of monoclonal immunoglobulins in such a system. This study tests new algorithms to produce rapid standardized computer-supported interpretation of the entire electropherogram. Methods: Data from Beckman Paragon CZE 2000 electropherograms were compared with quantitative protein data from >800 routine clinical samples. Algorithms were designed to produce semiquantitative analyses of major proteins and to define different patterns of inflammation based on the electropherogram. Results: The algorithms produced reliable semiquantitative evaluations of prealbumin, albumin, α1-antitrypsin, haptoglobin, and transferrin, but were less accurate for α1-acid glycoprotein. Some genetic variants of albumin and deficiency variants of α1-antitrypsin were easily recognized. Complex clinical traits such as degree and type of inflammation could be evaluated. When used together with previously developed algorithms addressing immunoglobulins, the new algorithms provide relevant clinical interpretation. Selected outputs indicate the need for reflex testing or evaluation by specialists. Conclusions: Automation of both electrophoresis and interpretation can provide a rapid, inexpensive, standardized analysis that can hopefully improve the diagnostic information and clinical outcome for large groups of patients. It also provides objective criteria for clinical interpretations, to be validated or adjusted in future clinical studies.

Background: Development of quality-control criteria to ensure reproducibility of microarray results for potential clinical application is still in its infancy. Methods: In the present studies we developed quality-control criteria and evaluated their effect in microarray data analysis using total RNA from cell lines, frozen tumors, and a commercially available reference RNA. Quality-control criteria such as A260/A280 ratios, percentage of rRNA, and median size of cDNA and cRNA synthesis products were evaluated for robustness in microarray analysis. Furthermore, precision studies using a reference material were performed on the Affymetrix® HG-U133A high-density oligonucleotide microarrays. The same reference RNA sample was examined in 16 different chips run on 2 different days in the four different modules of the Affymetrix fluidics workstation. Fresh and frozen fragmented cRNAs were also compared. An ANOVA model was fit to identify the main sources of variation. Results: Good-quality samples showed >30% rRNA in the electropherograms and cDNA and cRNA synthesis products with median sizes of 2.0 and 3.0 kb, respectively. Precision studies showed that the main source of variation was the day-to-day variability, minimally affecting hybridization exogenous control genes. Altogether, the results showed that the Affymetrix Genechip® system is highly reproducible when RNA that meet the quality-control criteria are used (overall P >0.01). Conclusions: These results confirm the need to establish defined quality-control criteria for sample quality to distinguish between analytical and biological variability.

Background: Hemoglobin-based oxygen carriers (HBOCs) have recently been included in the International Olympic Committee and World Anti-Doping Agency lists of substances and methods prohibited in sports. To enforce this rule and deter abuse of HBOCs in elite sports, it is necessary to develop HBOC-specific screening and confirmation tests that are the usual steps in antidoping control analysis. Methods: We developed a screening method based on electrophoresis of serum samples cleared of haptoglobin (Hp). Four successive steps (immunoprecipitation of Hp, electrophoresis of the cleared serum, Western blotting of the separated proteins, and detection of hemoglobin-related molecules based on the peroxidase properties of the heme moiety), provided electropherograms that could be easily interpreted in terms of the presence of HBOCs. This method was tested with serum samples enriched with various types of HBOCs: polymerized, conjugated, and cross-linked hemoglobins. It was also applied to blood samples collected from 12 healthy volunteers who had been infused with either 30 or 45 g of Hemopure, a glutaraldehyde-polymerized bovine hemoglobin. Results: The method clearly detected the presence in serum of the various types of HBOCs tested and demonstrated no possible confusion with endogenous hemoglobin that may be present in cases of hemolysis. The test was able to detect Hemopure for 4–5 days after administration of 45 g to healthy individuals. Conclusions: The electrophoretic method is a simple, fast, and sensitive procedure that appears to fulfill the criteria of a screening test for the presence of HBOCs in antidoping control samples.

Automated capillary zone electrophoresis (e.g., on the Paragon 2000 CZE, Beckman Instruments) is an alternative to conventional electrophoretic methods for the separation of serum proteins (1)(2)(3). The technique uses ultraviolet (UV) detection at 214 nm for direct quantification of peptide bonds, whereas conventional methods detect the protein fractions by staining with various reagents. CZE has the advantage of improved precision and a faster turnaround (4). Recent reports indicate, however, that the Paragon CZE 2000 masks deficiencies of α1-antitrypsin (5) and that some radio-opaque agents simulate a monoclonal component in the α2-globulin fraction in the CZE electropherogram (6)(7). We encountered an interference from the contrast medium Omnitrast®, which produced an unexpected peak in the α2-zone of the CZE electropherogram. We have now investigated the effect of 12 iodinated radio-opaque media (commercially available in Spain) on instrumentation (Paragon CZE 2000) operated as described by Bossuyt et al. (6). When we used the desalting method described …

Capillary zone electrophoresis (CZE) is an alternative method for separation of serum proteins (1). Two dedicated and automated multichannel instruments are available, the Paragon 2000 (Beckman-Coulter) and the Capillarys (Sebia). The sensitivity and specificity of Paragon 2000 for detection of monoclonal proteins have been reported to be 95%(2)(3)(4) and 78%(4), respectively. The low reported specificity reflects the frequent occurrence of slight abnormalities in the electropherogram at the anodal part of the γ-globulin fraction (fibrinogen region)(4). Only 2 studies (5)(6) evaluated the Capillarys for detection of monoclonal proteins and reported a high sensitivity, and prospective studies in a routine clinical setting are …

Capillary electrophoresis (CE) and immunosubtraction capillary electrophoresis (IS-CE) were compared with the conventional methods agarose gel electrophoresis (AGE) and immunofixation electrophoresis (IFE) for detection and identification of paraproteins. In total, 74 paraproteins out of 468 serum samples were detected by both methods. Seventy-three monoclonal bands with concentrations ranging from 0.6 to 50.9 g/L were detected by the routine method. With CE, 70 paraproteins were detected and quantified on the electropherogram. Four paraproteins were not detected by CE; three of these were IgG (0.6, 1.1, and 2.2 g/L, respectively), and one was a IgM paraprotein (20.3 g/L) that could be visualized by minor changes in the running conditions. In comparison with IFE, 69 paraproteins were typed identically using IS-CE; only one paraprotein (IgMκ, 14.9 g/L) could not be identified. On the other hand, a monoclonal IgA band that had not been detected by AGE was identified by CE and IS-CE. We conclude that, in general, CE could be a useful method for detection of paraproteins and that IS-CE is a good alternative to IFE. Additional studies are required to investigate the ionic strength and pH of the running buffer, because these prove to be the most crucial factors for routine CE separation of paraproteins.

The enhanced resolution of capillary zone electrophoresis (CZE) provides superior sensitivity for the detection of monoclonal (M) proteins in serum samples compared with agarose gel electrophoresis (AGE). This comes at the cost, however, of a lower specificity, calculated as 78% in our hands for the Paragon CZE 2000® with software version 1.602 (Beckman) (1). Abnormalities in the electropherogram that cause confusion are most frequently located in the anodal part of the γ-globulin fraction. Notably, fibrinogen, which remains in incompletely clotted serum samples, migrates at the junction between the β- and γ-fractions. Even when following the strict procedure of keeping serum samples for a minimum of 4 h at room temperature before electrophoresis, we found a suspected fibrinogen or possible M-protein peak in 39 (8.6%) of 454 consecutive routine samples in our laboratory. Recently, a simple method consisting of cold-ethanol precipitation was described for the removal of fibrinogen from serum specimens before agarose protein electrophoresis (2). We evaluated the use of this method in the routine analysis of serum samples by CZE in which a peak at the fibrinogen location could cause confusion with the detection of small M-proteins. Absolute ethanol was added to the specimens at a final concentration of 100 mL/L. …

We describe a computer program system for finding, quantitating, and matching the protein spots resolved on a two-dimensional electropherogram. The programs that locate and quantitate the incorporation of radioactivity into individual spots are totally automatic, as are the programs for matching protein spots between two exposures of the same gel. A semi-automatic method is used to match protein spots between different gels. This procedure is quite fast with the use of a computer-graphic display, which is also helpful in the editing process. A data base is set up and programs have been written to correlate matched protein spots from multi-gel experiments and to efficiently plot out quantitative data from sequences of equivalent spots from many gels or even many multi-gel experiments. The practical use of this system is discussed.

Serum from a 71-year-old man with multiple myeloma complicated with renal failure showed a monoclonal IgG lambda. A second spike appearing in the serum protein electropherogram, suggesting a biclonal gammopathy, was found to be due to lambda Bence Jones protein (29.9 g/liter). Lambda Bence Jones protein was also found in smaller concentration (3.8 g/liter) in the urine. Tetramers of Bence Jones protein were not demonstrable by gel filtration and ultracentrifugation, and renal failure was probably the main reason for this very high concentration of Bence Jones protein in the serum.

Agarose-gel electrophoresis of serum of a 72-year-old woman with liver cirrhosis showed virtually no beta-globulins two weeks before the patient's death. There was marked decrease in the concentrations of transferrin, beta-lipoproteins, hemopexin, complement component C3, beta-glycoprotein I, and cholesterol in serum. Absence of a beta-globulin band appears to signify an ominous prognosis.

We describe the design principles of a photometric flatbed scanner with a scan area of 250 X 250 mm2, a dynamic range of 4000 gray levels, a signal/noise ratio of 2000/1, a step size of 0.1 mm, and a step frequency of 2000 steps per second. It is controlled by a microcomputer and used to acquire data for quantitative evaluation of gels or chromatograms. The performance of the instrument is demonstrated by the data obtained by scanning gratings, gray step filters, and high-resolution electropherograms.