Plasma Protein Tests: How to Interpret Abnormal Results

Dr Jez Thompson Describes the Range of Plasma Protein Tests Available in Primary Care and Explains what Abnormal Results Might Indicate

Plasma protein is the collective term for the proteins present in the blood. Plasma proteins fall into several different groups and have numerous functions, including maintaining osmotic pressure, and transporting lipids, hormones, vitamins, and minerals. Some plasma proteins are enzymes, while others have functions in blood clotting and the immune system. Excluding immunoglobulins, all major blood proteins are synthesised in the liver.

The total protein test gives an approximate measure of all plasma protein (excluding fibrinogen when testing is on clotted samples). With a typical reference range of 60–80 g/l,¹ plasma proteins constitute around 7% of plasma by weight² and 0.5% of total body mass.

Serum albumin accounts for around 55% of plasma protein (typical reference range: 35–55 g/l).^3,4 It maintains the osmotic pressure of plasma and functions in the transport of calcium, lipids, and steroid hormones.

Globulins make up approximately 35% of plasma protein (typical reference range: 20–35 g/l).^5,6 Globulins are involved in a range of processes including transport of ions, hormones, and lipids; acute-phase responses; and, as immunoglobulins, immune response. Globulins and are divided into four subgroups:²

• alpha₁ (comprising mainly alpha₁ antitrypsin)
• alpha₂ (including haptoglobin and ceruloplasmin)
• beta (comprising transferrin and some complement components)
• gamma (predominantly immunoglobulins and C-reactive protein [CRP]).

Fibrinogen is a soluble protein which constitutes around 6.5% of plasma protein.⁷ Conversion of fibrinogen to the insoluble protein fibrin, is a process central to blood clotting.

The remaining plasma proteins comprise hundreds of distinct protein molecules. Individually, they are present in small amounts, but together they make up approximately 1% of plasma protein, and have crucial roles as regulatory proteins such as enzymes, proenzymes, and hormones.⁸

Reasons for Protein Measurement in Practice

Plasma proteins are heterogenous in nature and involved in many complex functions within the body. Abnormalities in plasma proteins may be primary (the cause of specific pathologies) or secondary (the result of a wide range of disease processes). Abnormal proteinuria can also result from various medical conditions.

The reasons why a GP may request measurement of plasma protein include investigations of symptoms, allergies, and immunity (see Box 1).

Liver panel tests, for example, those requested as part of drug monitoring, may reveal protein abnormalities that require further investigation.¹⁰

Pregnancy-associated plasma protein A (PAPP-A) assessment, together with plasma human chorionic gonadotropin (hCG) measurement and nuchal translucency ultrasound (together known as first-trimester screening), is offered in early pregnancy (10–14 weeks) to assess the risk of a foetal chromosomal disorder such as Down’s syndrome (trisomy 21) or Edwards’ syndrome (trisomy 18).¹¹

Testing urine for protein may assist in the diagnosis of urinary infection, primary renal disease including nephrotic syndrome, secondary renal disease for example in diabetes, multiple myeloma, and pre-eclampsia in pregnancy.¹²

Liver Panel Blood Tests (Liver Function Tests)

‘Routine’ liver panel testing typically includes measurement of total protein and albumin.¹⁰ Total protein is a measure of both serum albumin and globulin, so test results outside the reference range may represent abnormal levels of either one or both of these components. The conditions associated with abnormal globulin and albumin levels are detailed below and summarised in Table 1.

Decreased globulin levels as a fraction of total protein are seen in individuals with malnutrition and patients with nephrotic syndrome when there is renal protein loss. High total protein levels associated with increased globulin may be seen in dehydration, in response to acute infections such as pneumonia and hepatitis, and in chronic inflammatory conditions such as rheumatoid arthritis and systemic lupus erythematosus (SLE). Other causes include Waldenström macroglobulinaemia, a type of non-Hodgkin lymphoma in which abnormal cells synthesise large amounts of macroglobulin, and multiple myeloma, a malignant neoplasm of plasma cells characterised by excessive synthesis of monoclonal globulin that can usually be detected in blood and urine.⁹

Albumin is only synthesised in the liver and serum albumin level may be reduced when the liver’s synthetic function is significantly impaired, for example in long-standing liver disease or advanced cirrhosis. Other causes of a low serum albumin level include severe malnutrition (which may accompany alcohol-related liver disease). Low serum albumin may also be evident when there is protein malabsorption, for example in Crohn’s disease, or when excessive protein is being lost, for example from the gut in protein-losing enteropathy, through the skin in exfoliating dermatitis or after severe burns, or from the kidney in nephrotic syndrome. Severe inflammatory conditions or shock may also be associated with low serum albumin levels, when a catabolic state develops and the synthetic function of the liver switches to the production of other proteins. An important symptom of low serum albumin is the development of peripheral oedema. High serum albumin levels generally reflect dehydration.⁴

Table 1: Conditions Associated with Abnormal Globulin and Albumin Levels^4,9

Urine Protein Testing

Some loss of protein by the kidneys is normal, at levels of up to 150 mg/day. Causes of transient elevated proteinuria include:¹³

• strenuous exercise
• febrile illness
• urinary tract infection
• orthostatic proteinuria (rare after the age of 30 years)
• pregnancy.

Urine may test falsely positive for protein when the sample has been contaminated by vaginal mucous. Causes of persistent proteinuria include:¹³

• primary kidney disease, including nephrotic syndrome and glomerulonephritis
• secondary renal disease, for example, that associated with diabetes and hypertension.

If serum albumin is low and nephrotic syndrome is suspected, testing the urine for protein will help inform the diagnosis.¹⁴ If total protein and globulins are high and multiple myeloma or Waldenström macroglobulinaemia are possible differential diagnoses, dipstick testing for protein followed by measurement of Bence Jones protein (a monoclonal protein found in the urine) may be useful.⁹

C-reactive Protein

C-reactive protein is an acute-phase reactant; a protein synthesised by the liver and released into the blood in response to tissue injury, infection, or other inflammatory processes. Its physiological role is thought to involve binding to the surface of dead or dying cells (and some types of bacteria) to activate the complement system.

C-reactive protein may be acutely raised:¹⁵

• following myocardial infarction
• in sepsis
• in tissue trauma
• after surgery.

In infection or acute inflammation, a patient’s CRP level may rise before the onset of clinical symptoms.

Chronic inflammatory conditions, including rheumatoid arthritis, seronegative arthritides such as Reiter’s syndrome, vasculitic syndromes, and inflammatory bowel disease (although levels tend to be higher in Crohn’s disease than in ulcerative colitis), are also associated with a raised CRP level. However, SLE produces almost no increase in CRP unless there is coincident infection. In chronic inflammatory conditions, CRP level can be valuable in monitoring disease activity, with high levels suggestive of an acute exacerbation or ineffective treatment and falling or low levels indicative of remission.¹⁶

Raised CRP is a feature of infection or inflammation, but it is a non-specific marker of an acute response.¹⁶ Clinical history, examination, and specific diagnostic tests are needed to establish the cause of raised CRP. C-reactive protein typically returns to normal when the acute infective or inflammatory process is resolved.

Protein Electrophoresis

Although standard liver panel tests and on-site urine protein testing give a general indication of protein levels, protein electrophoresis can be used to separate the mixture of proteins present in either plasma or urine into subdivisions to provide additional diagnostic information.⁹ In this process, an electric current is used to move the protein mixture across a thin layer of gel. The distance travelled by each protein depends on a range of variables, including its molecular size and electrical charge. The separated proteins are then visualised using a stain, which reveals a characteristic pattern of bands. Serum proteins are separated into six major groups by protein electrophoresis: albumin and alpha₁, alpha₂, beta₁, beta₂, and gamma globulins. The size of each band gives a qualitative indication of the amount of that protein fraction. This pattern of bands is often converted into a graph, with vertical spikes or peaks where there are large amounts of protein and smaller peaks or valleys where there are small amounts of protein.

Abnormal electrophoresis patterns are associated with a variety of different pathological conditions.

Increased Gamma Fraction

When the gamma fraction is increased, spike-like monoclonal increase is suggestive of malignant or pre-malignant clonal conditions such as multiple myeloma and Waldenström macroglobulinaemia, whereas a broader-based, polyclonal increase typically reflects a more general inflammatory response.¹⁷ Although a monoclonal increase may be of concern, the most common cause is monoclonal gammopathy of uncertain significance (MGUS), which is generally benign but may progress to a malignant condition over time. The most common elevation in gamma globulin levels is polyclonal and typically due to increased immune system activity caused by acute or chronic infection, tissue damage or autoimmune connective tissue diseases such as rheumatoid arthritis, SLE, scleroderma, chronic active autoimmune hepatitis, and primary biliary cholangitis.⁹

Causes of low immunoglobulin levels include:⁹

• congenital and acquired immunodeficiency syndromes
• other conditions associated with reduced immunoglobulin production, such as protein malnutrition
• conditions causing excessive loss of immunoglobulins, such as sepsis, nephrotic syndrome, burns, and protein-losing enteropathy.

Increased Alpha Fraction

Isolated alpha₁ abnormalities are usually due to changes in alpha₁ antitrypsin, with decreased levels occurring in congenital alpha₁ antitrypsin deficiency. Increased levels may be found in acute inflammatory disorders.⁹

Alpha₂ macroglobulin levels may be raised in nephrotic syndrome and haptoglobin levels are increased in stress, infection, inflammation, and tissue necrosis. Haptoglobin levels may be decreased in haemolytic conditions.⁹

Increased Beta Fraction

Beta globulin level may be raised in severe iron deficiency when there are high levels of transferrin. It may be decreased in malnutrition and cirrhosis.⁹

Immunofixation Electrophoresis and Enzyme Immunoassays

When required, specific proteins can be identified using one of a family of tests termed immunofixation electrophoresis.⁹ In these tests, proteins are first separated by electrophoresis and then reacted with a specific antibody. If the antibody reacts with the protein being investigated, the antibody–protein complex remains in the gel while other proteins are washed away, allowing the protein to be identified and quantified. This technique is useful in the assessment of multiple myeloma.

Immunoglobulin E (IgE) antibodies to specific antigens can be measured using the radioallergosorbent testing (RAST) or enzyme-linked immunosorbent assay (ELISA) techniques. These techniques are valuable in the assessment of allergies, for example when there is a risk of anaphylaxis.⁹

A range of other techniques, including enzyme immunoassays, can be used to quantify the serum levels of specific antibodies, such as rubella and hepatitis B antibodies. These techniques are used both to diagnose current infection and to confirm the presence of immunity. A preparation of a specific antigen (for example, hepatitis B surface antigen) is incubated with a specimen of a patient’s serum. If the antibody under investigation is present, it will become coupled to the antigen. Analytic processes are then used to quantify the amount of antibody present in the serum.¹⁸

Conclusion

Plasma proteins have myriad functions and metabolic roles. Abnormalities in their levels can be associated with a range of morbidities, either as a primary cause or as a secondary effect. Understanding of the tests available provides the physician with useful diagnostic tools.

Dr Jez Thompson

RCGP/British Liver Trust Clinical Champion for Liver Disease Clinical Director, Bevan Healthcare