Margaret A Jenkins

The accuracy of cystatin C and commonly used creatinine-based methods for detecting moderate and mild chronic kidney disease in diabetes

Background  The accuracy of measuring serum cystatin C levels for detecting various stages of chronic kidney disease (CKD) in diabetes is still unclear.

Evaluation of serum protein separation by capillary electrophoresis: prospective analysis of 1000 specimens

To critically assess the method of capillary electrophoresis (CE) we examined 1000 prospective serum samples submitted for protein electrophoresis by both high-resolution agarose gel electrophoresis (HRAGE) and CE. CE was performed using a 72 cm (50 cm to detector) x 50 microns I.D. fused-silica capillary with detection of absorbance at 200 nm. The 1000 samples examined contained 362 monoclonal paraproteins with concentrations ranging from 1 to 71 g/l. We evaluated the individual paraprotein correlations, the overall correlation between the two methods being 0.96. We found that HRAGE gave slightly higher values for the monoclonal bands than CE and the difference was statistically significant. We conclude that CE is a viable alternative to HRAGE for the determination of protein dyscrasias in a routine clinical laboratory.

Capillary electrophoresis as a clinical tool

Clinical laboratories must produce accurate results for patients with a minimum turn-around time. Automated commercial capillary electrophoresis instrumentation has been available to the clinical laboratory for the past five years. Our laboratory has utilised capillary electrophoresis (CE) to automate serum protein electrophoresis. We have used the technique of CE to produce clinical results for nearly two years. CE methods are also available for the quantitation of haemoglobin variants, by both isoelectric focusing and free solution techniques. Micellar electrokinetic separations by CE have been developed for some specialised drug assays and for B-group vitamin analysis, while gel-filled capillaries have the capability to separate DNA fragments, such as PCR products. Isoenzyme analysis has shown possibilities by CE, but quantitative results are needed to be clinically useful. Analysis of amino acids for newborn screening programs and as an arterial clotting indicator are being developed. The next five years should see a proliferation of clinical laboratory methods using automated CE.

Capillary isoelectric focusing of haemoglobin variants in the clinical laboratory

For capillary isoelectric focusing (CIEF) to be accepted in the clinical laboratory, it must be reproducible and cost effective. The advent of polyAAEE coated capillaries (Bio-Rad Laboratories, Hercules, CA, USA) has provided the means of obtaining over 100 runs per capillary, something which previously had not always been possible with coated capillaries. Using the Clinical Data Management computer program on the BioFocus 2000 Capillary Electrophoresis System (Bio-Rad Laboratories), we have used a one-step salt mobilization to achieve focusing of haemoglobin variants. Washed red cells are diluted, haemolyzed and separated in the capillary at 8 kV using 1.3% Pharmalyte ampholytes (pH 6.6-7.7/pH 6-8 2:1) in 0.40% methylcellulose. The separated haemoglobins were detected by adsorption at 280 nm. Using published values of haemoglobin variants, we investigated the use of pI markers to confirm the pI of haemoglobin variants detected. CIEF, though more expensive than capillary electrophoretic separations of haemoglobin variants, has greater resolution due to the fact that the separation of variants from pI 6.95 to 7.42 occurs over 4 min, whereas the electrophoretic separation is over 60 s. CIEF is quicker than gel IEF, and shows real-time results as the sample is being processed. The potential for CIEF in the clinical laboratory is not limited to haemoglobin variants, and the technique should become increasingly popular in the near future.

Cystatin C for estimation of glomerular filtration rate in patients with spinal cord injury

Background: Serum creatinine is not a satisfactory marker of glomerular filtration rate (GFR) in patients with spinal cord injury (SCI) who have varying degrees of muscle atrophy. In contrast to serum creatinine, serum cystatin C, a 13-kDa protein, is not affected by muscle mass and is therefore potentially a useful marker of GFR in patients with SCI. In addition, cystatin C is not dependent on sex or age and is not secreted by the renal tubule. Aim: We assessed serum cystatin C as a surrogate marker of GFR in SCI patients. Methods: Cystatin C was analysed using a particle-enhanced immunonephelometric assay (Dade Behring) in serum samples sent for routine measurement of creatinine (64 patients) and creatinine clearance (27 patients) from patients in the Spinal Unit of the Austin Health. We compared these results with serum cystatin C of 57 non-SCI patients who had had a creatinine clearance measurement during the study period. Results: In patients with SCI, the reciprocal of cystatin C had a stronger correlation (r = 0·48, P<0·01) with creatinine clearance than the reciprocal of serum creatinine (r = 0·25, P<0·19). Further, the value of serum creatinine was much lower for a given creatinine clearance in SCI patients than in non-SCI patients; the serum cystatin C concentrations were equivalent. Conclusion: The serum cystatin C is a convenient and more reliable surrogate marker of GFR than serum creatinine and will enable early detection of renal impairment. We need to confirm this finding with a larger study, including comparison with an accepted gold standard for GFR.

Capillary Electrophoresis of Hemoglobin

Abstract 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 HbA2 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 HbA1c analysis is available, it cannot compete in turnaround time or cost with automated HPLC commercial instruments that give accurate HbA1c results in 3 or 4 minutes. Hence we do not anticipate a bright future for the analysis of hemoglobin by CE.

Capillary electrophoresis procedures for serum protein analysis: comparison with established techniques

Methods using automated capillary electrophoresis (CE) instrumentation are available for serum protein electrophoresis with monoclonal band quantitation, isoelectric focusing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis separations. The advantages of CE over previous gel methods relate to the time and labour saved by the automated instrumentation. High pI monoclonal bands and cryoglobulin specimens can be successfully analysed by CE. However, if the CE application uses a standard company supplied kit, then the cost savings are often negated by the high cost of the kit. Improvements such as the inclusion of both a UV-Vis as well as a fluorescence detector as standard within the one commercial instrument, the production of coated IEF capillaries with a useful life of at least 100 samples, and the introduction of a capillary array into all commercial instrumentation would ensure greater use of CE within both the clinical and other protein laboratories.

Recommendations for standardized reporting of protein electrophoresis in Australia and New Zealand

Background Although protein electrophoresis of serum (SPEP) and urine (UPEP) specimens is a well-established laboratory technique, the reporting of results using this important method varies considerably between laboratories. The Australasian Association of Clinical Biochemists recognized a need to adopt a standardized approach to reporting SPEP and UPEP by clinical laboratories. Methods A Working Party considered available data including published literature and clinical studies, together with expert opinion in order to establish optimal reporting practices. A position paper was produced, which was subsequently revised through a consensus process involving scientists and pathologists with expertise in the field throughout Australia and New Zealand. Results Recommendations for standardized reporting of protein electrophoresis have been produced. These cover analytical requirements: detection systems; serum protein and albumin quantification; fractionation into alpha-1, alpha-2, beta and gamma fractions; paraprotein quantification; urine Bence Jones protein quantification; paraprotein characterization; and laboratory performance, expertise and staffing. The recommendations also include general interpretive commenting and commenting for specimens with paraproteins and small bands together with illustrative examples of reports. Conclusions Recommendations are provided for standardized reporting of protein electrophoresis in Australia and New Zealand. It is expected that such standardized reporting formats will reduce both variation between laboratories and the risk of misinterpretation of results.

Multiple sclerosis: use of light-chain typing to assist diagnosis

Although the presence of oligoclonal IgG with abnormal ratio in multiple sclerosis (MS) has been known for many years, this finding has not been put to diagnostic use in most routine clinical laboratories. In a retrospective study we report differences in the oligoclonal banding patterns between multiple sclerosis and non-MS patients. We had sufficient cerebrospinal fluid (CSF) on 36 from 71 patients with oligoclonal bands for immunofixation for κ and λ light chains, and for free κ and free λ. Thirteen out of 14 patients with clinically confirmed MS had predominantly IgG (κ) banding. In contrast, in seven out of eight patients with diagnoses other than MS the IgG was linked to both κ and λ light chains in approximately equal proportions. Nine out of 14 patients with probable/possible/ suspected MS showed predominantly IgG (κ) banding; five others in this group had both IgG (κ) and IgG (λ) and free λ light chains. The finding of IgG (κ) bands in CSF samples with oligoclonal bands supports a diagnosis of MS.

Measurement of Iohexol by Capillary Electrophoresis: Minimizing Practical Problems Encountered:

Iohexol is a non-ionic contrast agent, which has been widely described in recent literature as an accurate marker for the measurement of glomerular filtration rate (GFR). Our aim was to establish a capillary electrophoresis assay, based on a previously described method, that had adequate reproducibility to be used as part of a clinical trial. In this paper, we examine the practical aspects, pitfalls and steps we took to achieve a precise and reproducible assay. To minimize laboratory variation, we examined properties such as the use of an internal standard in a capillary electrophoresis separation, alternative deproteinization methods for serum, the most suitable matrix for the dilution of standards and the implementation of suitable quality control material to ensure that run-to-run variability was minimized. The optimized capillary electrophoretic assay of iohexol was found to be robust, with over 860 runs from the one capillary over a 9-month period. Excluding capital costs of the instrument, the consumable cost of the assay is less than AS0·25 per test, with a run time of 5·25 min and a coefficient of variation (CV) of 4·3% at 80 mg/L. The GFR, calculated from the plasma clearance, had a reproducibility of 5·47%.