Core Training

Clinical use and interpretation of serum

protein electrophoresis and adjunct assays

S
erum electrophoresis and related urine and CSF is also possible by another (Figure 1). Sometimes, distinct areas can
tests have a variety of uses in clinical technique called isoelectric focusing that be visualized in the beta fraction which
practice, but their full potential has specific diagnostic indications. This can be subdivided into beta-1 and beta-2
is not often realized. This article article reviews protein electrophoresis and components. Most immunoglobulins (IgM,
summarizes their basis and clinical its associated tests, including indications to IgG, IgD and IgE) exist in the gamma region.
use, and outlines how to interpret the results. order, basic interpretation and steps to take Immunofixation electrophoresis is
next by the generalist. This is supported by an extension of protein electrophoresis,
Introduction: what is the test? literature and the latest guidelines to facilitate in which the proteins are first separated
Separation of different analytes in body clinical practice. by electrophoresis and then exposed to
fluids is useful to distinguish individual antibodies (antisera) specific for certain
components that may be contributing to The basis of the test antigens to identify the components of each
pathology. Electrophoresis is a useful test In clinical laboratories, agarose gel is used to band. In most cases, this is used to identify
to achieve this and helps characterize and separate proteins based on electric charge, the isotype of heavy (IgG, IgM, IgA, IgD or
identify components of biological fluids that which are then visualized using amido IgE) and light chains (kappa or lambda) to
may be a result of, a cause of, or associated black, a protein-binding stain. Serum protein identify a specific paraprotein detected by
with a disease process. Protein electrophoresis electrophoresis is used to examine the general protein electrophoresis.
is a laboratory test that separates out proteins distribution and quantitation of proteins in
in an electric field based on their charge. serum. In an electrical field, the negatively- When should the test be requested?
While protein electrophoresis is commonly charged proteins migrate towards the positive As a diagnostic test, protein electrophoresis has
used for the detection of paraproteins (anode) end and separate according to charge. a variety of uses that may facilitate the direction
(otherwise known as M-bands) for the In protein electrophoresis, two major of a diagnostic workup. It is particularly
diagnosis and monitoring of myeloma, groups of proteins can be distinguished: useful in distinguishing monoclonal from
there are several other uses which are often albumin (50–70% of total serum by weight) polyclonal immunoglobulin expansion when
underappreciated (Spickett, 2013). and globulins (chiefly immunoglobulin G elevated serum immunoglobulin levels are
Electrophoresis can be performed on (IgG) in healthy individuals). Albumin has noted. The classical use of serum protein
any body fluids although by far the most the greatest negative charge and will travel the electrophoresis is in the diagnosis of plasma
common is serum. Separation of proteins in furthest of all proteins. Five distinct bands cell dyscrasias in which excess monoclonal
can be appreciated on zone electrophoresis: immunoglobulins are produced. Conversely,
albumin, alpha-1, alpha-2, beta and gamma identification of polyclonal increases of
Dr Adrian YS Lee, Junior Medical Officer,
Royal Hobart Hospital, Hobart 7000,
Tasmania, Australia; Clinical Lecturer, School Figure 1. Serum protein electrophoresis on normal serum. The protein fractions are labelled
of Medicine, University of Tasmania, accordingly on this electrophoretic strip and a corresponding densitometry graph is also included.
Tasmania, Australia and University Associate,
Menzies Institute for Medical Research,
University of Tasmania, Tasmania, Australia
Mr Paul M Cassar, Senior Scientific Officer,
SEALS Sutherland Centre of Immunology,
Sutherland Hospital, New South Wales,
Australia
Dr Anna M Johnston, Haematologist and
Pathologist, Royal Hobart Hospital,
Tasmania, Australia
Associate Professor Stephen Adelstein,
Clinical Immunologist and Allergist, Royal
Prince Alfred Hospital, New South Wales, Anode (+)
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Australia and Clinical Associate Professor,

Sydney Medical School, University of
Sydney, New South Wales, Australia Albumin a1 a2 b g
Correspondence to: Dr AYS Lee
([email protected])

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 What You Need To Know About

immunoglobulins suggests further tests to disease, large proteins are lost; hence the this diagnosis as they may be found in other
identify an inflammatory disorder such as dominant protein increased on protein inflammatory and autoimmune neurological
an infection, autoimmune disease or, more electrophoresis will be albumin. In contrast, disorders (Ebers and Paty, 1980).
rarely, a malignancy. tubular insult as a result of drug toxicity
As a result, clinical features that suggest will result in inefficient reabsorbtion of Interpretation
these conditions may warrant this test on the low molecular weight proteins, producing Reports of protein electrophoresis are
patient’s serum. These may be isolated and increases in alpha-1 and beta-2 protein returned with quantitative measurements of
sometimes unexplainable features such as fractions on protein electrophoresis each protein group. Protein electrophoresis
fatigue, recurrent infections, or the presence (D’Amico and Bazzi, 2003; Jenkins, 2009). performed on serum can provide several
of CRAB features (hyperCalcaemia, Renal As an adjunct, serum free light chain diagnostic clues depending on which protein
failure, Anaemia and Bone pain/lesions). In analysis is a sensitive assay that detects light fraction is affected (Table 1) and the clinician
addition, serum protein electrophoresis can be chains that may otherwise be missed on can be guided accordingly in the clinical
useful as a monitoring tool for someone with protein electrophoresis or immunofixation investigation and management.
monoclonal gammopathy of undetermined electrophoresis analysis. This is especially In contrast, urine protein electrophoresis
significance. Supplementary tests, such as useful in cases where only free light chains are is more complicated to interpret (Spickett,
measurement of serum free light chains (see secreted at low concentrations. In addition, 2013). Poor renal function, renal damage
below), may then allow stratification into serum free light chain analysis has a place in or a significant systemic inflammatory
low-risk patients who may be monitored the monitoring, prognostication and diagnosis disorder may result in a monoclonal band
clinically, while intermediate and high-risk of monoclonal gammopathies (Dispenzieri as a result of leakage of an intact monoclonal
patients need a yearly test (Jacobs, 2013). et al, 2009), with studies showing high immunoglobulin (Jenkins, 2009), therefore
In addition, in those with established sensitivity and higher detection (screening) urine immunofixation electrophoresis is
M-protein disease, international guidelines rates for this test over urinary protein essential to determine if this is Bence Jones
also recommend monitoring M-protein by electrophoresis (Holding et al, 2011; Graziani protein (monoclonal light chains) from a
protein electrophoresis on a monthly basis and Merlini, 2014; Dejoie et al, 2016). Free possible plasma cell dyscrasia, or some other
while on treatment, or every 3–4 months light chain also showed better concordance to intact protein.
when off therapy (Vincent Rajkumar, 2014). protein electrophoresis and immunofixation
Serum protein electrophoresis may also electrophoresis over 24-hour urinary protein What to do next?
be used to further characterize persistent electrophoresis and is therefore a better What steps to take when an abnormal
protein abnormalities (e.g. elevated IgG). monitoring tool (Dejoie et al, 2014). protein electrophoresis or immunofixation
Consequently, it also has a role to play in Despite these findings, however, the electrophoresis is received depends on the
ambiguous clinical presentations or where latest international guidelines currently do extent of the derangement and in which
standard clinical and laboratory evaluations not support the replacement of 24-hour fraction(s) it is. If the results hint at a
are unclear. In rheumatology, serum protein urinary protein electrophoresis with free possible plasma cell dyscrasia, then care is
electrophoresis is particularly useful in light chain tests (Dispenzieri et al, 2009). vital to ensure all other associated testing
the investigation of general inflammatory Serum free light chain analysis also needs has been carried out. This includes serum
conditions resulting in polyclonal to be interpreted in the patient’s context, immunoglobulins (IgG, IgM and IgA
gammopathy or as a work-up for joint pain as compromised renal function (raised at a minimum), β2-microglobulin for
(which may, in fact, be bone pain seen in a creatinine level) is one common cause of prognostication, serum free light chain,
plasma cell dyscrasia). elevated kappa:lambda ratios which can yield full blood examinations, erythrocyte
It is important that immunofixation false positives (Abadie et al, 2009). sedimentation rate, serum calcium, urea,
electrophoresis is performed in conjunction Free light chain and protein electrophoresis creatinine and skeletal surveys (Firkin, 2009).
with serum protein electrophoresis as it analysis is also useful for the diagnosis of Following this, a referral to a haematologist
is more sensitive for the detection of a primary amyloidosis, since light chains are may be appropriate.
paraprotein and some plasma cell dyscrasia involved in the pathogenesis (Gertz et al, If there is a significant isolated elevation
patients may have a normal or equivocal 2005). Signs and symptoms which suggest or depression of one of the globulin
protein electrophoresis (Kyle, 1994). Urine this diagnosis, e.g. unexpected heart failure, groups, then investigation according to
protein electrophoresis and immunofixation hepatomegaly, proteinuria, or unexplained the corresponding typical proteins found
electrophoresis can be performed in parallel peripheral neuropathy, should warrant in that fraction may be warranted (Table
either on a spot urine sample (morning ordering these tests for possible amyloidosis. 1). For example, if the alpha-1 fraction is
preferable) or 24-hour collection. The Finally, although less commonly significantly and persistently depressed, then
primary use is to detect and characterize ordered, CSF can be subjected to protein an α1-antitrypsin level and phenotype test
free light chains (Bence Jones protein) in the electrophoresis as well in the diagnostic may be requested if clinically appropriate.
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diagnostic workup of plasma cell dyscrasias. workup for multiple sclerosis. CSF Elevation or depression of immunoglobulins
Urinary protein electrophoresis is also oligoclonal IgG bands (two or more bands) must be confirmed with serum quantitative
useful for investigating proteinuria and found on protein electrophoresis help tests and a referral to an immunologist may
distinguishing between glomerular and support a diagnosis of multiple sclerosis be considered if there is no identifiable
tubular protein loss. In glomerular kidney (Polman et al, 2011), but are not specific for secondary cause for the abnormality.

British Journal of Hospital Medicine, February 2017, Vol 78, No 2 C19

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Core Training

KEY POINTS Table 1. Differential diagnoses for protein fraction deviations on serum protein
electrophoresis
■■ Electrophoresis is a laboratory technique
to separate molecules (commonly Protein Typical proteins in this Conditions decreased Conditions increased
proteins) according to their charge. fraction fraction (absent or faint band) (increased band)
■■ The main use for serum protein
Albumin Albumin Negative acute phase Dehydration
electrophoresis is in the workup of
reactant, e.g. inflammation,
plasma cell dyscrasias by differentiating
sepsis, malnutrition, nephrotic
polyclonal vs monoclonal expansion in
syndrome and kidney disease,
immunoglobulins.
liver disease, pregnancy
■■ Immunofixation assays can identify haemodilution (artefact)
the precise heavy and light chains of
monoclonal (M) proteins. Alpha-1 α1-lipoprotein (high- α1-antitrypsin deficiency*, Increased α-fetoprotein – liver
density lipoprotein), α1- liver disease tumours, germ cell tumours,
■■ Laboratory tests need to be interpreted in
antitrypsin, α-fetoprotein pregnancy, inflammatory states
conjunction with clinical situation to be of
maximal benefit. Alpha-2 α2-macroglobulin, Liver disease, haemolysis, Inflammatory states, steroid
haptoglobin, malnutrition, Wilson’s disease use, adrenal insufficiency,
caeruloplasmin nephrotic syndrome, severe
diabetes mellitus
Conclusions
Protein electrophoresis and immunofixation Beta-1 Transferrin, β1-lipoprotein Malnutrition Iron deficiency, pregnancy,
electrophoresis are useful tests to consider on (low-density lipoprotein) inflammatory states,
serum samples, particularly if there are clinical hyperlipidaemia
indications of a possible plasma cell dyscrasia Beta-2 C3 complement, β2- C3 consumption Inflammatory states
or a perturbation in immunoglobulin levels microglobulin
is found. When there is a strong suspicion
of the former, consider requesting a parallel Beta- Fibrinogen (uncoagulated Liver disease, inflammatory
gamma samples), C-reactive states
serum free light chain assay (preferred over
region protein, IgM, IgA
urine electrophoresis). However, a normal
serum protein electrophoresis does not rule Gamma IgG, IgM, IgA, IgD, IgE Inherited humoral Smear – polyclonal
out a monoclonal gammopathy. (+/- monoclonal proteins immunodeficiency, kidney immunoglobulins (infection
Serum protein electrophoresis is a complex which can also migrate disease, sepsis, malnutrition, or inflammation). Monoclonal
test that can give a wealth of information and to alpha-2 region) viral infections, amyloidosis, band – plasma cell dyscrasia,
diagnostic clues to the clinician; however, its leukaemias e.g. multiple myeloma,
lymphoma, Waldenstrom’s
sensitivity means minor abnormalities can be
picked up. If there is a significant abnormality *It is insufficient to make diagnoses based on protein electrophoresis alone; but diagnoses in bold are particularly
that warrants further testing, and if clinically important to consider.
indicated, the clinician may consider a repeat ML, Levoguer AM, Dore PC (2011) Use of
and intact immunoglobulin multiple myeloma.
serum protein electrophoresis a few months Haematologica 101(3): 356–362. https://doi. serum free light chain analysis and urine protein
later, preferably when the patient is ‘well’. org/10.3324/haematol.2015.126797 electrophoresis for detection of monoclonal
Dispenzieri A, Kyle R, Merlini G et al (2009) gammopathies. Clin Chem Lab Med 49(1):
Persistent abnormalities may warrant further 83–88. https://doi.org/10.1515/cclm.2011.010
International Myeloma Working Group guidelines
investigations and/or specialist referral.  BJHM for serum-free light chain analysis in multiple Jacobs CK (2013) How often should serum protein
myeloma and related disorders. Leukemia 23(2): electrophoresis be done in someone with
Conflict of interest: none.
215–224. https://doi.org/10.1038/leu.2008.307 monoclonal gammopathy of undetermined
Abadie JM, Van Hoeven KH, Wells JM (2009) Are Ebers GC, Paty DW (1980) CSF electrophoresis in one significance (MGUS)? Evidence Based Practice
renal reference intervals required when screening thousand patients. Can J Neurol Sci 7(4): 275–280. 16(9): 13.
for plasma cell disorders with serum free light Firkin F (2009) Screening for multiple myeloma. Aust Jenkins MA (2009) Serum and urine electrophoresis
chains and serum protein electrophoresis? Am Prescr 32(4): 92–94. https://doi.org/10.18773/ for detection and identification of monoclonal
J Clin Pathol 131(2): 166–171. https://doi. austprescr.2009.046 proteins. Clin Biochem Rev 30(3): 119–122.
org/10.1309/ajcpr2m4euynhlgm Gertz MA, Comenzo R, Falk RH et al (2005) Kyle RA (1994) The monoclonal gammopathies. Clin
D’Amico G, Bazzi C (2003) Pathophysiology of Definition of organ involvement and treatment Chem 40(11 Pt 2): 2154–2161.
proteinuria. Kidney Int 63(3): 809–825. https:// response in immunoglobulin light chain Polman CH, Reingold SC, Banwell B et al (2011)
doi.org/10.1046/j.1523-1755.2003.00840.x amyloidosis (AL): a consensus opinion from the Diagnostic criteria for multiple sclerosis: 2010
Dejoie T, Attal M, Moreau P, Harousseau J-L, Avet- 10th International Symposium on Amyloid and revisions to the McDonald criteria. Ann Neurol
Loiseau H (2014) Comparing the performance of Amyloidosis, Tours, France, 18-22 April 2004. 69(2): 292–302. https://doi.org/10.1002/
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electrophoresis and immunofixation for monitoring org/10.1002/ajh.20381 Spickett G (2013) Oxford Handbook of Clinical
and assessing response to therapy in patients with Graziani MS, Merlini G (2014) Serum free light Immunology and Allergy. Oxford University Press,
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Dejoie T, Attal M, Moreau P et al (2016) Comparison multiple myeloma and related conditions. Expert Vincent Rajkumar S (2014) Multiple myeloma:
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of monoclonal immunoglobulins in light chain Holding S, Spradbery D, Hoole R, Wilmot R, Shields https://doi.org/10.1002/ajh.23810

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