Featuring guest speakers from hospital trusts around the country, ‘Current Trends in Electrophoresis Testing’ was chaired by Consultant Clinical Scientist, Dr Robert Beetham, North Bristol NHS Trust. The scientific programme featured presentations on testing strategies for monoclonal proteins and a clinical perspective on multiple myeloma. Topical subjects such as alpha-1 antitrypsin and carbohydrate-deficient transferrin were also covered along with an examination of the benefits of centralised electrophoresis as part of a networked pathology service.
Strategies for detecting monoclonal proteins
As keynote speaker, Dr Robert Beetham provided an outline of how to detect monoclonal proteins and explained the significance of these results for patients.
A monoclonal protein is an immunoglobulin molecule that is the product of a single clone of antibody-producing plasma cells. Monoclonal proteins act as markers for a number of significant diseases including multiple myeloma, a cancer that begins in plasma cells. Monoclonal protein-related disorders are caused by protein aggregation or antibody activity. Electrophoresis testing of serum samples allows intact monoclonal proteins to be detected, while testing of urine samples detects cases where the only abnormality is a free monoclonal light chain. The International Myeloma Working Group1 advises that immunofixation electrophoresis of serum and urine samples is the gold standard for detecting monoclonal protein related disorders.
For serum testing, electrophoresis should be carried out on all immunoglobulin requests, with high resolution electrophoresis indicated for all patients suspected of having a plasma cell dyscrasia. Immunofixation should be carried out if a sample is positive following electrophoresis testing.2 If the electrophoresis is negative but there is a high index of suspicion, then immunofixation is recommended. This could arise from the presence of lytic lesions, spinal cord compression, pathological fractures or free monoclonal lights chains in the urine. Discretionary use of immunofixation applies in the presence of monoclonal protein bands, unusual morphology of the protein profile, or an abnormal pattern of immunoglobulins.
The detection of monoclonal free light chains is dependent on the urine sample, as well as the types and sensitivity of the test. Dr Beetham recommends that for urine electrophoresis to be used as an initial screen, it is important to ensure that the method has appropriate sensitivity and that each urine sample is sufficiently concentrated to contain detectable levels of protein. This can be achieved either by using a sensitive stain on un-concentrated urine samples, or by using a standard stain and routinely concentrating. Immunofixation should be performed if there is a band, or zone, in addition to albumin. Alternatively, immunofixation can be performed as a front-line test.
The strategy of initial detection by serum electrophoresis followed by discretionary immunofixation should be satisfactory if it is accepted that some monoclonal gammopathies may be missed. If quantitative immunoglobulins are included with all electrophoresis then some of these could possibly be detected. The use of urine electrophoresis testing should be maximised, as it is not difficult to obtain samples or carry out the analysis as long as considerations regarding sensitivity are taken into account. Importantly, a suitable strategy for follow-up with haematology departments and indeed follow-up in the community needs to be established.
Myeloma: a clinical perspective
Moving away from the laboratory and into the clinic, Consultant Haematologist, Dr Fenella Willis, from St. George’s Hospital, provided a comprehensive overview of multiple myeloma – from symptoms through to the available treatment strategies.
Myeloma primary affects older patients and represents one per cent of all cancer cases – four per 100,000 in the UK. Caused by unregulated proliferation of abnormal plasma cells, myeloma cells collect in the bone marrow and can crowd out normal blood cells. They can also collect in the solid part of the bone. The disease is called multiple myeloma because it usually affects many bones. Common clinical features include bone pain, broken bones, anaemia, infections and hypercalcaemia.
A number of differentiating investigations aid clinical diagnosis from detection of high paraprotein levels (IgA >20 g/L, IgG >30 g/L, Bence Jones >1 g/24 h), presence of other immunoglobulins and raised beta-2 microglobulin concentrations. A low level of haemoglobin leading to anaemia is another indicator. Myeloma can also be detected through imaging techniques, however bone scans can have poor sensitivity due to low osteoblastic activity.
Myeloma can also damage the kidneys. Light chain immunoglobulins are normally reabsorbed in the renal proximal tubule. As the disease advances, more light chains are produced coupled with a decreased ability to reabsorb. For some patients, up to 50g of Bence Jones protein (BJP) may enter the urine but less than 15g is excreted. The excess BJP precipitates as a viscous mass, which can lead to progressive inflammation, fibrosis and cause epithelial cell atrophy. The Durie Salmon staging system (Table 1) and international prognostic system (Table 2) are used in myeloma.
Multiple myeloma can be managed using a number of treatment strategies, ranging from radiotherapy and chemotherapy to steroids and immunomodulatory drugs. Bisphosphonate treatment is recommended for all myeloma patientsi. Biphosphonates have a high affinity to bone and are rapidly absorbed. Non-nitrogen containing biphosphonates are metabolised by osteoclasts and disrupt cell metabolism, whereas nitrogen containing biphosponates induce apoptosis by disrupting signalling of key regulatory proteins. As the disease progress, it may also be necessary to have orthopaedic input to stabilise pathological fractures. Optimal pain control plays an important role of all treatment strategies for patients suffering from myeloma.
Alpha-1-antitrypsin typing: methodology unravelled
The clinical theme continued with an exploration of alpha-1 antitrypsin typing by Dr Graeme Wild of the Protein Reference Unit at Sheffield Teaching Hospitals NHS Trust.
Different phenotypes of alpha-1-antitrypsin (AAT) were originally discovered by CB Laurell. During an electrophoretic study of lung disease patients in 1962, four phenotypes were identified in the alpha-1 region – F, M, S, Z. Four out of six cases with AAT deficiency had emphysema and a relationship was also noticed with liver disease. Ten per cent of newborns with AAT deficiency have diminished bile flow leading to prolonged conjugated hyperbilirubinemia, known as neonatal cholestasis. One to two per cent goes on to develop juvenile cirrhosis with twenty per cent of adult cirrhosis due to slowly progressive fibrosis of the portal tract.
AAT deficiency causes early onset of lower lobe emphysema, liver cirrhosis, an inflammation of subcutaneous fat know as panniculitis and the autoimmune disease Wegeners granulomatosis. Diagnosis is typically made by lung or liver specialists but it can take a long time to determine AAT deficiency. Although there is no real treatment, lifestyle changes such as reducing alcohol consumption, not smoking and avoiding polluted environments can certainly help. More radical treatment strategies include liver transplants and AAT replacement via intravenous infusions of AAT purified from human plasma. Gene therapy is another suggested option.
Measurement of AAT deficiency is possible using agarose electrophoresis, acid starch gel electrophoresis and isoelectric focussing with protein staining, immunofixation and immunoblotting. It is recommended that all samples for AAT phenotyping should also have AAT quantification. The normal range of AAT in adults is 1.1 – 2.1 g/L. Some potential problems with analysis of AAT are high salt concentrations in samples and changes due to blood transfusions and liver transplants.
Carbohydrate-deficient transferrin: a reliable marker for alcohol abuse
Carbohydrate-deficient transferrin (CDT) is increasingly being used as a marker for detecting long-term alcohol abuse, as Natalie Walsham from King’s College Hospital went on to explain.
In the UK, 90 per cent of the population drinks alcohol with an estimated 750,000 alcohol-dependant individuals. Drink-related deaths and alcoholic liver disease has more than doubled over the past 10 years. Consumption has increased from 4.5 litres per capita in 1961 to 11.3 in 2004. The clinical aspects of increased alcohol consumption include liver disease, pancreatic disease and impaired cognitive function.
Ethanol, acetaldehyde and acetate are typical biochemical markers of alcohol misuse analysed from blood, urine and breath tests respectively. Liver enzymes such as gamma-glutamyl transpeptidase (GGT), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) can also be monitored as well as changes in erythrocytes. A potential drawback of these markers is the relatively short timescale in which alcohol can be measured (blood 24–36 h, breath 1–12 h, urine 24–48 h and saliva 24–26 h).
Transferrin is a plasma protein that transports iron through the blood to the liver, spleen and bone marrow. Transferrin is present in various isoforms which perform the same function but are encoded by different genes. The distribution of these isoforms is modified with repeated and sustained alcohol consumption. Regular alcohol intake of more than 80–100 g/day leads to an increase of the disialo- and asialocarbohydrate-deficient transferrin (CDT) isoforms. These can be used as indicators of long-term alcohol abuse as they reflect alcohol intake during the two weeks before analysis.
There are a number of assays that can be used to measure CDT. Manual and semi-automated methods include isoelectric focusing with immnunoblotting and micro-anion exchange chromatography with immunoassay. Capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) provide automated methods, which measure CDT as a percentage of total transferrin.
Centralised electrophoresis: the Path Links experience
Path Links is a networked pathology service based in greater Lincolnshire. Consultant biochemist Ian Barlow from Scunthorpe General Hospital discussed the benefits of centralising electrophoresis testing.
In 2007, four sites within Path Links were carrying out serum electrophoresis testing, three with serum immunofixation and one with Bence Jones and urine immunofixation. The individual workloads were relatively small, with a total throughput of approximately 20,000 samples per year, split similarly across the four sites. Slow turnaround times, the need for batch analysis and the requirements of clinical pathology accreditation (CPA) were the triggers for service improvements.
The two options investigated were to standardise the electrophoresis instrumentation across all four sites or centralise serum electrophoresis testing to one site. Following comparative analysis between instruments along with alternative equipment evaluation, electrophoresis testing was centralised in May 2008. Scunthorpe was chosen as the primary site as it was already the centre for immunology testing along with specific protein analysis. Importantly, there was existing expertise in serum immunofixation and urine monoclonal protein analysis at this site. Centralised electrophoresis testing has improved turnaround times from an average of 4.8 days down to 3.5. Immunofixation turnaround times have significantly reduced from an average 14.7 days to 4.5. Other benefits reaped through adoption of a single-managed network include harmonisation across sites, consolidated service level agreements, a clear focus and strong corporate image.
Reflex testing
Specific proteins specialist, Mr Jean Deenmamode, from Homerton University Hospital, London gave a thought-provoking presentation on capillary serum electrophoresis and reflex testing based on his experiences over the last seven years.
Until 2001, a relatively small number of serum and urine electrophoresis samples were tested manually at Homerton. Samples with abnormal bands were sent to a specialised laboratory which meant turnaround times were slow. In 2002, a new agarose gel electrophoresis system was installed and application of discretionary or reflex testing was introduced with a globulin level of ?40g/L. After detecting previously unknown positive monoclonal proteins, the globulin level was reduced to ?38g/L. As the number of unknown monoclonal proteins detected has since increased, the question has been raised if it should be reduced further. Possible reasons for these newly detected monoclonal proteins could be the lack of sensitivity of earlier methods or the diverse local patient population. Consequently, the number of electrophoresis samples tested significantly increased and this led to the installation of an automated capillary electrophoresis system in 2004. Although there was some reservations about the application of this new interpretive approach, results with capillary electrophoresis correlated well with those from gel electrophoresis.
A snapshot of patient results for 2007 showed a total of 249 new monoclonal proteins were identified for a population of around 260,000 and a globulin threshold of ?38 g/L. These results raised further questions and so in September 2008, the globulin level was reduced to ?35g/L. In the seven months following this change, the number of new cases totalled 349 and represented almost two per cent of all electrophoresis samples tested.
Discretionary electrophoresis testing using a reduced globulin threshold is not uncommon, however, reducing the globulin level below 35 g/L could be classed as unethical. Small paraproteins may be identified earlier when the globulin level is reduced, but as they could remain small and never present issues – at what point is it correct and ethical to detect them? Do patients need, or indeed want to know earlier, if there is little they can do about it? Moving forward, a clearly defined reporting system between biomedical scientists, clinicians and general practitioners, needs to be established using an agreed globulin threshold.
Ongoing initiative
An informative and diverse seminar programme combined with guest speakers from pre-eminent trusts across the country ensured that the event was both well attended and well received. The Royal Holloway seminar forms part of an ongoing initiative by Sebia UK to help clinical laboratory personnel make best use of their professional skills and understand the context of their work. In addition to external seminars, advice and support is also provided through onsite training and electrophoresis workshops held in the fully equipped laboratory at the Sebia offices in Camberley.