Capillary electrophoresis in the clinical laboratory

Ageing societies in developed countries face increased   economic demands on the practice of modern medicine. In many areas of medicine, the clinical laboratory has become an indispensible instrument for substantiating clinical diagnosis, monitoring therapies and determining the application of modern biological therapeutics individually. Perhaps most important, due to the ease of sample collection, laboratory results have become the cornerstones of preventive medicine. 

 

The prospects of molecular diagnostics on ‘liquid biopsies’ are promising and will hopefully draw more attention to the contribution of laboratory medicine to public health.

 

Economical pressures in the laboratory itself have spurred developments towards increased automation and consolidation of smaller laboratories in larger units, leaving small hospitals frequently with point-of-care tests or small on-site facilities focused on absolute emergency parameters. Furthermore, the laboratory personnel has become a precious asset whose productivity is largely dependent on state-of-the-art laboratory instrumentation, an optimised workflow with a minimisation of preparatory and maintenance chores providing more time to be devoted to the supervision of the analytical process and the evaluation of test results.

 

As a direct consequence of this development, the majority of clinical analysis is performed on modern, high-throughput clinical chemistry or immunological analysers. There are, however, a number of well-established laboratory tests providing generalised information important for clinical practice that do not lend themselves to this approach. 

 

Such a screening test is the serum protein electrophoresis that gives details about acute or chronic inflammation, protein deficiency syndrome or serum dysproteinaemia in general. Traditionally, serum protein electrophoresis was performed on cellulose acetate membranes or agarose gels. Both methods entailed a great amount of manual labour to be performed by trained technical personnel. 

 

The results were often hampered by a variable quality to protein separation and staining resulting in poor precision and reproducibility. The coefficient of variation (CV) for serum protein electrophoresis on acetate membranes reported earlier ranged from 2.6% for albumin to 15.7% for the alpha-1 fraction.¹

 

The advent of capillary electrophoresis as a high-resolution separation method was a great step toward improved analytical quality for serum protein electrophoresis. The CV values for serum protein separation by capillary electrophoresis ranged in our laboratory from 0.8% for albumin to 2.3% for alpha-1 proteins. 

 

Capillary electrophoresis also increased sample throughput while reducing the hands-on time of the laboratory technician. In the last decade, the development of immunosubtraction² has made capillary electrophoresis the preferred method for the screening and monitoring of monoclonal gammopathies due to its increased sensitivity compared to the membrane bound electrophoresis.³ It has to be noted, however, that some free light chains are undetectable by capillary electrophoresis so that the classical membrane bound techniques still have their place in the differential diagnosis of gammopathies.⁴

 

Proteins and bodily fluids

The differentiation of proteins in other body fluids such as urine provides further diagnostic and prognostic data in a number of diseases. The detection and quantification of para-proteins in urine is still an important marker in the follow up of multiple myeloma. The determination of the protein pattern in urine by SDS-polyacrylamide electrophoresis is an important analysis for the staging in renal disease. It has been demonstrated by a number of authors that capillary electrophoresis is a sensitive and powerful tool for urine analysis.⁵ However, in the field urinary protein analysis membrane bound techniques are still very prevalent in routine laboratories. 

 

Haemoglobin variants

Another important domain of separation techniques in the clinical laboratory is the analysis of haemoglobin variants. Haemoglobin variants are monogenic traits that are carried by up to 7% of the world population. There are more than a thousand haemoglobin variants that are listed in the ‘Database of Human Haemoglobin Variants and Thalassemias’ (www.globin.bx.psu.edu/hbvar/menu.html). 

 

The most prevalent haemoglobin variants are HbC and HbD. HbE is predominant in South East Asia, and HbS is found in Africa, on the Arabic peninsula, in India and in the USA where 8.3% of the black population carry a HbS trait. Patients carrying disorders such as homozygous HbS can present with life-threatening conditions; other variants like HbC are clinically predominantly silent. 

 

A further clinically and genetically very heterogeneous group of haemoglobin-related diseases are the alpha and beta thalassaemia syndromes. These are characterised by a reduced synthesis of one of the globin chains resulting in an imbalanced haemoglobin synthesis, that underlies extend and clinical phenotype of these traits.

 

For the screening of these disorders, haemoglobins are usually separated by ion exchange chromatography, by gel-based or capillary electrophoresis. Due to the superior resolution of haemoglobins by capillary electrophoresis it has become the leading technique worldwide for the diagnosis of adult haemoglobin abnormalities.

 

Diabetes mellitus

A medical challenge of a different magnitude is diabetes mellitus. According to the World Health Organization (WHO) the number of people with diabetes will increase from 171,000,000 in the year 2000 to 360,000,000 in the year 2030. There are different types of diabetes: Type I diabetes that renders the patient insulin dependent due to the destruction of pancreatic islet cells; Type II diabetes that is caused by defects in insulin secretion and the resistance of peripheral tissues to insulin. 

 

The maturity-onset diabetes of the young (MODY) is a monogenic form of diabetes typically occurring before the age of 25 and caused by primary insulin secretion defects. Despite its low prevalence, MODY is not a single entity but represents genetic, metabolic, and clinical heterogeneity.⁶ And there is the gestational diabetes mellitus occurring during pregnancy. 

 

For the diagnosis of diabetes mellitus there are various laboratory tests: either repeated fasting plasma glucose >7.0mmol/l is measured, or the patient has a pathological oral glucose tolerance test with a plasma glucose >11.1mmol/l two hours after ingestion of 75g glucose, or a glycosylated haemoglobin A fraction 1c (HbA1c) >6.5% (48mmol/mol). 

 

The importance of a timely diagnosis of diabetes mellitus lies in the long-term complications of untreated, high plasma glucose concentrations. Changes in the microvasculature lead to diabetic cardiomyopathy, nephropathy, retinopathy and neuropathy, while the affection of the large blood vessels will cause coronary artery disease, peripheral vascular disease and stroke. However, the correct determination of a fasting plasma glucose value is a challenging undertaking. 

 

Measuring the fasting plasma glucose concentration requires reliable cooperation of the patient in terms of correct fasting. Furthermore, it is necessary that certain pre-analytical steps are observed. It is mandatory to use a blood sampling system containing efficient inhibitors of cellular glycolysis in order correctly measure the patient’s glucose concentration at the time sample was taken. 

 

In the case of the oral glucose tolerance test, the patient is required to ingest a defined amount of glucose, a rather unpleasant necessity, followed by plasma glucose determination subject to the same pre-analytical requirements as described above for the fasting plasma glucose measurement.

 

HbA1c determination

Recently, HbA1c determination has been recommended for the diagnosis of diabetes mellitus by the WHO.⁷ It is an excellent parameter for the diagnosis of diabetes mellitus as it is not subject to the analytical difficulties mentioned above. However, haemoglobin variants, haemolytic anaemia, chronic kidney disease and iron deficiency alter the life span of the erythrocyte and thereby influence the individual HbA1c concentration. 

 

There are several well established methods for the determination of HbA1c that can be categorised in two groups: separation methods and enzymatic or immunological assays. Immunological assays use antibodies that bind to and quantify HbA1c by turbidimetric or nephelometric methods. Enzymatic assays quantify the generation of hydrogen peroxide after the proteolytic cleavage of the b-chain followed by the reaction of the peptides with fructosyl peptide oxidase. Both are easily automated on clinical analysers. 

 

Alternatively, HbA1c can be measured by physicochemical separation of the different haemoglobins. HPLC ion exchange chromatography separates haemoglobins based on differences in isoelectric points, while affinity chromatography exploits the retention of glycated haemoglobin on a boronate column. Capillary electrophoresis is based on the migration of proteins in a high voltage electrical field and electro-osmotic flow to separate the different haemoglobin molecules (HbA0, HbA1a,b,c, HbA2, HbF) and other haemoglobin variants. 

 

In order to arrive at a correct diagnosis of diabetes, the presence of haemoglobin variants or thalassaemia must be taken into consideration for the correct interpretation of the patients’ HbA1c as these traits potentially alter the life span of the erythrocyte and thereby the patients HbA1c level. It should be emphasised that only ion exchange chromatography and capillary electrophoresis yield information about haemoglobin variants during HbA1c measurement, thereby enabling the laboratory to alert the physician to the possible implications for of the anti-diabetic therapy.

 

CDT determination

Diagnostic questions concerning the patient’s status on alcohol consumption are becoming more frequent recently. Apart from psychometric measures reflecting on the patient’s drinking behaviour and their attitudes, laboratory tests report the organism’s biochemical reaction to prolonged heavy alcohol consumption. The most established biomarkers are gamma-glutamyl-transferase activity, erythrocyte cell volume, ethyl-glucuronide and carbohydrate deficient transferrin (CDT). 

 

The sensitivity and specificity of these tests vary greatly with a clear advantage for CDT over the gamma-glutamyl-transferase. Transferrin is an iron transport-protein that contains two N-linked polysaccharide chains. The composition of these carbohydrate chains is altered when the daily ethanol consumption exceeds 60 grams per day. 

 

Testing for CDT is either carried out by immunological testing, ion exchange chromatography or capillary electrophoresis. In the case of CDT quantification, there appears to however an advantage for the use of immunological tests. Several genetic variants alter the CDT-levels quantified by separation methods because the physicochemical properties of the variants interfere with the correct separation.⁸

 

Conclusions

Capillary electrophoresis is a powerful analytical tool that is eminent for its high analytical resolution of complex protein mixtures. It has found its place not only in specialised areas of laboratory diagnostics such as the differentiation of gammopathies, urinary protein patterns, measurement of CDT or the detection and classification of haemoglobin variants. 

 

But it has also been shown in the past decade to be an important analytical technique for the analysis of high-throughput parameters such as serum protein electrophoresis and HbA1c measurement. This development has been made possible by automated capillary electrophoresis systems that are capable of cap piercing and automated sample mixing. These features combined with the high analytical resolution and an intelligent data processing had severely streamlined our workflow.

 

The next generation of capillary electrophoresis machines has tackled the task of adapting to the variability and speed of clinical chemistry analysers by increasing the number of capillaries and automating the switch been different analytical tests. There are concepts that allow the connection of several machines into single platform that can in combination with a bulk sample loader further decrease the hands on time of the technician. 

 

The connection of electrophoresis systems to automated laboratory tracks is also possible today. The next generation of capillary electrophoresis instruments will thereby further integrate the advantages and the high quality of separation techniques into the modern medical laboratory.

 

1. Kaplan A, Savory J. Evaluation of a cellulose-acetate electrophoresis system for serum protein fractionation. Clin Chem 1965;11(10):937–42. 
2. Bossuyt X et al. Detection and classification of paraproteins by capillary immunofixation/subtraction. Clin Chem 1998;44(4):760–4.
3. Katzmann JA et al. Identification of monoclonal proteins in serum: a quantitative comparison of acetate, agarose gel, and capillary electrophoresis. Electrophoresis 1997;18(10):1775–80. 
4. Clark R et al. Differential diagnosis of gammopathies by capillary electrophoresis and immunosubtraction: analysis of serum samples problematic by agarose gel electrophoresis. Electrophoresis 1998;19(14):2479–84.
5. Jenkins MA. Clinical application of capillary electrophoresis to unconcentrated human urine proteins. Electrophoresis 1997;18(10):1842–6. 
6. Vaxillaire M, Froguel P.  Monogenic diabetes in the young, pharmacogenetics and relevance to multifactorial forms of type 2 diabetes. Endocr Rev 2008;29(3):254–64. 
7. World Health Organizationa. Use of glycated hemoglobin (HbA1c) in the diagnosis of diabetes mellitus. www.who.int/diabetes/publications/report-hba1c_2011.pdf. Last accessed April 2016.
8. Thomas L. Labor und Diagnose. Th Books 2012;8:1212ff.