Volume 41, Issue 6

Capillary electrophoresis (CE) is a technique well suited for several separation problems in the life sciences. The main advantages are the higher separation efficacy in comparison to chromatographic methods and the smaller sample volume required. However, due to the limited sensitivity of CE, HPLC remains the method most commonly used for the analysis of drugs in biological fluids. For endogenous compounds like DNA, proteins, or small molecules like purines, CE offers clear advantages over conventional methods and, especially for DNA, CE has already been introduced into clinical routine. Some selected applications will be discussed.

Capillary electrophoresis (CE) is often one of the preferred techniques in pharmaceutical quality control and in clinical chemistry, particularly considering the high selectivity and lower costs compared to HPLC. The precision of CE is as good as in liquid chromatography (LC). The sample-throughput is high due to short analysis times. Efforts for sample pre-treatment are usually minor in CE. Urine and even blood plasma can be directly injected without further pre-treatment. After summarising the basic principles of CE, general strategies for method development are described to achieve selective, efficient, precise, fast, sensitive, and validated methods. Sample pre-treatment requirements are discussed. Standard buffer recipes, surfactants used in micellar electrokinetic chromatography (MEKC), chiral selectors, useful buffer additives, actions to deal with complex matrices, and aspects of validation have been collected. Other techniques that can be performed with CE instruments, such as capillary isoelectric focusing (CIEF), capillary isotachophoresis (CITP), and capillary electrochromatography (CEC), are briefly discussed.

Carbohydrate-deficient transferrin (CDT) is currently considered to be the best available marker for the diagnosis of chronic alcoholism. A large variety of methods have been developed, demonstrating the need for standardisation. Commercially available anion-exchange chromatographic-based assays are easy to use and require no specialised, expensive instruments. However, these methods cannot identify genetic transferrin variants or the carbohydrate-deficient glycoprotein syndrome. In 1989, a capillary isoelectric focusing method was developed for quantitative measurement of CDT. Despite the optimal resolution, this method is not easily applied in a clinical routine environment due to the complexity of analysis. Capillary electrophoresis in a polymer network using coated capillaries allowed full resolution of the sialoforms of human transferrin. The drawbacks due to an expensive and time-consuming sample preparation were eliminated when a method in neat serum was developed. Capillary zone electrophoresis allowed full resolution of the transferrin isoforms with a high analytical performance in a short analysis time thanks to a strong electroosmotic flow. Genetic transferrin variants were easily detected, avoiding false-positive results. Also, using capillary zone electrophoresis, it was shown that CDT is a suitable marker of chronic alcohol abuse detection in transferrin CD (common/cathodal) variants.

Capillary electrophoresis (CE) has been used in a variety of in-house capillary isoelectric focusing (CIEF) and capillary zone electrophoresis (CZE) assays for the detection of hemoglobin (Hb) variants and the quantitation of HbA 2 and HbF. A commercial kit has also been produced for the analysis of hemoglobin variants and thalassemia screening. Though CE methods have been shown to be able to detect many variants, final identification of the variant needs specialized testing such as DNA technology. Over the past 2 years, many instruments that had been used for these hemoglobin variant screening and thalassemia assays have been withdrawn from sale. Although CE HbA 1c analysis is available, it cannot compete in turnaround time or cost with automated HPLC commercial instruments that give accurate HbA 1c results in 3 or 4 minutes. Hence we do not anticipate a bright future for the analysis of hemoglobin by CE.

A systematic review of the literature on capillary electrophoresis applied to short chain organic acid analysis in body fluids has been conducted with special interest on those acids related to inborn errors of metabolism. The technique is briefly described, as well as the choice of the main analytical parameters: sample pre-treatment, polarity, capillary type, background electrolyte, and detection. The applications described in the literature are listed and the main features of the technique are discussed.

Capillary zone electrophoresis (CZE) of serum proteins is increasingly gaining impact in clinical laboratories. Two automated multichannel instruments dedicated to the separation of serum proteins have become available over the last 6 years, the Paragon CZE 2000 ® (Beckman Coulter, CA, USA) and, more recently, the Capillarys ® (Sebia, France). This review focuses on the performance of these commercial instruments to separate serum proteins in a clinical laboratory setting. The utility of CZE to recognize various dysproteinemias and to detect and identify monoclonal proteins will be described and systematically reviewed. The reader will be provided with a summation and an understanding of CZE-specific interference.

This review presents an overview of current research on the use of capillary electrophoretic techniques for the analysis of drugs in biological matrices. The discussion focuses on the applicability of the methods for the identification of unknown toxic compounds, which is defined as systematic toxicological analysis (STA). The aim is to establish whether or not capillary electrophoresis (CE), in one or more of its separation modes, is a method of choice in systematic toxicological analysis. To answer this question, various aspects are discussed, including sample work-up, separation modes, detection techniques, electrophoretic concentration, and identification by database retrieval. Several ways to improve the poor reproducibility and sensitivity are discussed. This leads to the conclusion that CE can be comparable to HPLC in those respects, while it is more favorable in speed, efficiency, and cost. Thus, we conclude that CE is a method of choice for STA, keeping in mind that every method has its limitations and that a combination of several non-correlated methods is always required for the identification of unknown compounds.

Analytical conditions in a system for capillary zone electrophoresis (Beckman Paragon CZE 2000) were originally selected to allow serum protein separation into five discrete protein zones, corresponding to those of conventional clinical electrophoresis. To improve the system's performance, new analytical conditions have been made available. We compared the two sets of conditions ("new" = y; "old" = x) for possible variations of results caused by the change. One hundred thirteen serum samples, covering wide intervals of values, were assayed on two twin instruments working under the old and the new conditions; results were assessed statistically and graphically. Possible clinical significance of differences was checked by comparison with the biological variation-based quality specifications for bias. Statistically significant (y-x) differences were observed for the α 1 -, α 2 - and β-globulin zones; clinically significant differences were observed for all the zones, with the exception of the γ-globulin zone. Therefore, old/new regression equations were calculated, whose reliability was assured by the wide interval of values, by the large sample size, and by the low dispersion of single values around the mean concordance estimates. Such equations may be used to convert "old" into "new" reference values, and for the intercomparison of patient results obtained under different analytical conditions.

Human papillomaviruses (HPV) are known to cause cervical dysplasia and cervical carcinoma. We used a 3-step PCR protocol that allows rapid type-specific HPV testing in a routine laboratory setting: HPV-16-positive samples were determined using a specific Light-Cycler PCR; HPV-16-negative samples were amplified by nested PCR and typed by sequence analysis. During a period of 7 months, 1275 PCR-based HPV tests were performed. Of the 1275 samples, 829 samples tested negative for HPV and 446 tested positive, including 124 positives found in the initial HPV-16-specific Light-Cycler assay. Sequence analysis of 132 samples detected 18 HPV types that are not included in the widely used Hybrid Capture II assay. For comparison, the first 100 cervical specimens were tested in parallel using PCR and direct hybridisation (Hybrid Capture II assay). PCR detected HPV DNA in 23 samples that tested negative in the Hybrid Capture assay. Four out of 37 samples that tested positive for HPV in the Hybrid Capture test may be false positives, because sequence analysis detected HPV types not included in the probe mixtures. As rare and novel HPV types may also confer an oncogenic risk, highly sensitive and specific PCR assays will help in understanding cervical HPV infection and cervical cancer of unknown causes.

Apolipoproteins (apo) B48 and B100 are exclusive markers of lipoproteins derived from the intestine and liver, respectively. Lipoproteins of hepatic origin are causally related to atherosclerosis and are found in plaque. However, lipoproteins of intestinal origin have not previously been reported in human atherosclerotic tissue, although studies in animal models suggest that chylomicrons may contribute to arterial cholesterol entrapment. In this study, we report on the relative distribution of both apoB48 and apoB100 in human atherosclerotic tissue. Lipoproteins were isolated from human femoral and carotid endarterectomy samples, from varicose vein and aortic aneurysms. ApoB was determined by Western blot analysis and quantified based on the signal to apoB48 and apoB100 protein standards of known mass. ApoB48 and apoB100 were found in human carotid and femoral endarterectomy samples, but not in varicose vein or aortic aneurysm tissue. The level of apoB48 relative to hepatic lipoproteins (B100) was found to be much greater than would be predicted based on the relative plasma concentration and arterial exposure of the two lipoprotein groups. Intimal association was substantially greater in carotid endarterectomy samples compared to femoral, however, the ratio of chylomicrons to hepatic lipoproteins was greater in the latter. On the basis that chylomicron apoB48 was found in human atherosclerotic tissue and that each chylomicron particle contains substantial quantities of cholesterol, it is possible that the contribution of intestinal lipoproteins to atherosclerosis may be significant.

We directly sequenced PCR fragments amplified from a porcine thyroid cDNA library with various synthetic primers based on the nucleotide sequence of bovinethyrotropin receptor (bTSHR) cDNA. A nucleotide sequence of 2410 bp was determined and its deduced amino acid sequence contained an open reading frame coding 764 amino acids, which showed 91.8% and 89.5% similarity to bTSHR and human TSH receptor, respectively. A hydrophobicity plot of the porcine protein suggested that it had a putative signal peptide and seven transmembrane domains. Chinese hamster ovary cells stably transfected with the cDNA could be specifically bound to bovine TSH (bTSH) and increased the intracellular cAMP level in response to bTSH and Graves' disease patients' sera stimulation.

Fibrinogen concentration is routinely measured by several methods and the results may influence diagnostic and treatment strategies. It is therefore necessary that results are compatible and transferable between laboratories. In the present study, it is shown that commonly used immuno-nephelometric methods, a commercial variant of the Clauss clotting rate method, and the classical syneresis method, do not differ significantly using patient material, in the interval 2–12 g/l. A research ELISA method that measures intact fibrinogen is not linearly correlated to the syneresis method. The commutability of available calibrators and reference materials (including the WHO 2nd IS) was only 50–80% except for one of the calibrators for which the virtual concentration coincided with that obtained by the

The applicability of point-of-care testing (POCT) glucometers for monitoring blood glucose concentrations has been demonstrated. However, their use in diagnosing type 2 diabetes is still debated. Therefore, a new statistical procedure for estimating discordance rates (DRs) was applied in comparing a well-established laboratory method (Ebio) with another laboratory method Cobas Integra 700) and with several POCT glucometers (Accu-Chek, Accutrend, Elite, HemoCue, Omni) in detecting glucose intolerance states. All procedures led to parallel glucose concentration patterns in capillary blood, venous plasma, and venous blood during oral glucose tolerance tests. However, the mean concentrations differed more or less. The Ebio and Integra results agreed within a maximal deviation of 3%. In blood samples, the HemoCue and Accutrend results were closest to the laboratory procedures (Ebio and Integra) and the highest differences were obtained with the Elite. Comparing whole blood values with those obtained in the aqueous blood compartment (Omni), even greater differences were observed. When all procedures were referred to the same glucose standard, the Ebio, Integra, Accutrend, and Omni results remained almost unchanged, whereas the Elite "moved" toward the Ebio results, and the Accu-Chek results toward the Omni results. Thus, traceability to an aqueous standard was observed with the Ebio, Integra, Accutrend, and Elite in all three sample systems. The Accu-Chek was only traceable in the presence of albumin, and HemoCue was not traceable at all. The clinical relevance of the differences observed between Ebio and POCT glucometers was tested by comparing the relative number of discordant classifications. The highest DRs were observed in the fasting state. They were higher in capillary blood than in the other sample systems. The DRs were found higher with POCT glucometers than with the other established laboratory procedure (Integra). Thus, at least in the fasting state, all POCT glucometers were less reliable than the established laboratory procedures and above the chosen criteria of clinical acceptability (DR≤5%). After transforming all glucometer results with a regression function (bias correction), the DRs were less than 5% if compared with the Ebio procedure in all sample systems. In conclusion, the WHO recommendation not to use POCT glucometers for diagnosing type 2 diabetes must be supported. However, after proper recalibration, the tested systems were acceptable. Therefore, manufacturers should reconsider their calibration procedure. Those POCT procedures should be preferred that can be referred to aqueous glucose solutions.

Hypercholesterolemia is associated with an increased risk of atherosclerosis and coronary heart disease. Therefore, therapeutic lowering of cholesterol is an important preventive measure of cardiac morbidity and death. As one side effect, cholesterol-lowering drugs appear to increase the mortality due to suicides or violence, and low lipid concentrations were found to be associated with trait measures of depression. We compared serum cholesterol concentrations and the Beck Depression Rating Scale (Beck's score) in 604 otherwise healthy outpatients who visited the physician's office for a medical health check-up; 65.4% of individuals presented with serum cholesterol concentrations ≥5.2 mmol/l (>200 mg/dl) and 5.3% had elevated Beck's score (>19), indicative for depression. Beck's score was higher in patients with cholesterol concentrations above the 75th percentile (=6.2 mmol/l; U = 31221, p<0.02, Mann-Whitney U-test), and Beck's score correlated with cholesterol concentrations and with age. Thus, in contrast to the widely accepted view, in our study, higher cholesterol concentrations were associated with signs of depressive mood. Hypercholesterolemia may not necessarily increase the risk of depressive mood, conversely, increased intake of fat and carbohydrates by individuals with depressive mood may increase cholesterol levels.