Special Protein Testing in the Clinical Laboratory: Overview of Available Assays

The proteins found within the human machine are, quite literally, the building blocks of life: individual cells contain more than 5000 individual proteins. The variety of proteins is seemingly infinite; so, too, is their function, composition, distribution and makeup.

 Figure 1 – Typical clinical samples awaiting analysis for special protein levels.

Proteins in the body form the structure of cells; make up enzymes and hormones that regulate bodily functions; transport analytes, nutrients, and drugs throughout the body; remove metabolic waste; and provide defenses against infection. Among the classes of molecular analytes found inside the body, proteins provide a unique combination of broad clinical significance and accessibility. The analysis of proteins and clinical diagnostic tests for proteins are widely accepted in medical technology. Varying concentrations of proteins are found in blood and its liquid components, plasma and/or serum, and are also present in urine, spinal fluid, feces, amniotic fluids, saliva and pleural fluid, to name a few (see Figure 1). Applications involving protein detection and analysis thus have tremendous diagnostic potential.

Total protein concentration in the body comprises two main components—albumin and globulin. Albumin is relatively straightforward in composition, while globulin contains alpha-1-, alpha-2-, beta- and gammaglobulin fractions. These fractions can be further broken down into individual diagnostic assays, commonly referred to as special protein tests. Along with other patient data, these unique analytes can provide a wealth of clinical information such as definitive diagnosis of acute events, prediction of disease risk and detection of disease recurrence. The most widely used special protein tests are described below.

Albumin: This single-chain protein occurs primarily in plasma and other bodily fluids, albeit at lower concentrations. Its primary functions are the maintenance of osmotic pressure and the binding/transport of substances throughout the body. Albumin is synthesized in the liver and constitutes approximately 60% of the total serum protein. Both increased and decreased levels can be observed with environmental, nutritional, toxic and traumatic stresses to the body.

Alpha (α)-1-acid glycoprotein: Also known as orosomucoid, α-1-acid glycoprotein (AGP) is one of the major acute-phase proteins and is important in drug binding. Elevated AGP levels are found in acute and chronic inflammatory conditions and infections. Low levels of AGP are seen when there is a reduction in synthesis, as in chronic liver disease, or an increased excretion of AGP, as in nephrotic syndrome.

Alpha (α)-1-antitrypsin: This serine protease inhibitor protects the lungs from degradation by neutrophil elastase. Reduced levels are associated with liver disease, but can also occur in early childhood and old age, and with hereditary deficiency. This can cause an imbalance between the neutrophil elastase in the lung and the anti-elastases that are responsible for protecting the lungs, and can potentially lead to emphysema.

Alpha (α)-2-macroglobulin: α-2-Macroglobulin is almost exclusively found and distributed within the intravascular pool. As such, its measurement aids in the diagnosis of blood clotting, or clot lysis, disorders. Levels of α-2-macroglobulin are reported to increase in nephrotic syndrome, liver cirrhosis and diabetes mellitus, where lower-molecular-weight proteins leak from the kidneys into the urine.

Anti-streptolysin O: Group A β-hemolytic streptococci produce a number of exotoxins that can act as antigens. One of these, streptolysin-O, leads to the production of specific antibodies in infected subjects, increasing the serum concentration of anti-streptolysin O (ASO). This can be used to establish the degree of past and present infection by β-hemolytic streptococci. Measurement of ASO levels in sera, in conjunction with other laboratory and clinical findings, is useful in diagnosis of diseases caused by streptococcal infections, including rheumatic fever, scarlet fever, glomerulonephritis, tonsillitis and other upper respiratory infections.

Beta (β)-2-microglobulin: Measurement of β-2-microglobulin aids in the diagnosis of kidney disease. This low-molecular-weight protein is found on the surface of most nucleated cells and is eliminated via the kidneys. Following filtration through the glomeruli, it is reabsorbed and catabolized by the proximal tubular cells. Normally, only trace amounts are excreted in urine; this is markedly increased in renal-tubular disorders. Elevated serum levels of β-2-microglobulin can also occur with rheumatoid arthritis, systemic lupus erythematosus, malignant lymphoma and myelomas.

Ceruloplasmin: Synthesized in the liver, ceruloplasmin plays a major role in copper metabolism, carrying approximately 95% of the total copper in sera. Decreased levels of ceruloplasmin can be caused by hereditary disorders of copper metabolism. In Menkes disease, for example, the body is unable to transport oxidized copper from the gastrointestinal epithelium into the circulation; Wilson’s disease is characterized by the inability to insert oxidized copper (Cu2+) into the developing ceruloplasmin molecule. Dietary copper insufficiency, including malabsorption, also reduces serum ceruloplasmin concentrations. These concentrations may increase as a result of acute-phase reactions, pregnancy or the use of oral contraceptives.

Complement: The complement system enhances (complements) the ability of antibodies and phagocytic cells to clear pathogens from the body. It is part of the body’s innate immune system, which is not adaptable and does not change over the course of an individual’s lifetime. The complement system consists of a number of small proteins found in the blood, synthesized by the liver and normally circulating as inactive precursors. When stimulated by one of several triggers, enzymes in the system cleave specific proteins to release cytokines and initiate an amplifying cascade of further cleavages. The end result of this complement activation, or complement fixation cascade, is massive amplification of the response and activation of the cell-killing membrane attack complex. More than 30 different proteins and protein fragments make up the complement system, accounting for about 10% of the globulin fraction of blood. C3 is the major protein component of the complement system, and has a fundamental role in the inflammatory response and immune system functionality. Deficiencies in C3 are typically seen in liver disease, in severe recurrent infections such as pneumococcal and meningococcal infections and in glomerulonephritis. Elevated serum C3 concentrations, as well as complement levels, are indicative of acute inflammatory reactions and other chronic inflammatory conditions, like rheumatoid arthritis. Another component of the complement system, C4, plays a fundamental role in inflammatory response and immune system functionality. C4 is reduced with severe liver failure; recurrent staphylococcal and streptococcal infections; and immune complex disorders such as glomerulonephritis, vasculitis and endocarditis.

C-reactive protein: Levels of the acute-phase protein C-reactive protein (CRP) increase in response to inflammation. CRP is present in the sera of normal individuals, and elevated levels can indicate infection, tissue injury, inflammatory disorders and associated diseases. Levels of CRP have been used in a variety of population subgroups to assess cardiovascular risk and myocardial infarction. A complete clinical history is required for accurate interpretation of CRP levels, as levels within the normal range may be affected by a number of factors and should always be compared to previous values.

Cystatin C: Measurements of cystatin C aid in the diagnosis and treatment of renal diseases. Cystatin C is produced by all nucleated cells at a constant rate, is freely filtered by the glomerulus and is almost completely reabsorbed and degraded by the proximal tubular cells. The production rate of cystatin C is not affected by age, gender, muscle mass or inflammatory processes. This makes it an ideal marker of glomerular filtration rate (GFR), since a reduction in GFR results in an increase in cystatin C concentrations. Several studies have shown that cystatin C is a better marker for GFR than serum creatinine levels.

Ferritin: Ferritin, an intracellular protein that stores and releases iron in a controlled manner, is produced by almost all living organisms. In humans, it acts as a buffer against both iron deficiency and overload. Ferritin is found in most tissues as a cytosolic protein, but small amounts are secreted into sera, where it functions as an iron carrier. Ferritin levels are indirect markers of the total amount of iron stored in the body, and are thus used as a diagnostic test for iron-deficiency anemia and for those undergoing iron therapies.

Haptoglobin: Measurement of haptoglobin levels is useful in the diagnosis of hemolytic diseases related to the formation of hemoglobin–haptoglobin complexes. Haptoglobin is an acid α-2 acute-phase plasma glycoprotein that binds specifically to free plasma oxyhemoglobin. As a result, the high-molecular-weight complex formed prevents hemoglobin filtering by the kidneys. Low levels of haptoglobin are typically associated with hemolytic anemias and liver disease, while elevated levels can develop in response to inflammatory conditions.

Immunoglobulins (IgA, IgD, IgE, IgG and IgM): The immunoglobulins, or antibodies, are the most significant gammaglobulins, although some immunoglobulins are not gammaglobulins, and some gammaglobulins are not immunoglobulins. Immunoglobulins are glycoprotein molecules produced by white blood cells. They are a critical part of the immune response because they recognize, bind and help to destroy antigens like bacteria and viruses. The antibody immune response is highly complex and exceedingly specific. Immunoglobulin isotypes differ in their biological features, structure, target specificity and distribution. Hence, assessment of the immunoglobulin isotype can provide useful insight into complex humoral immune response. The antibodies produced by plasma cells are classified by isotype that vary in function and antigen responses, mainly due to structure variability. The five major isotypes identified in humans are IgA, IgD, IgE, IgG and IgM.

IgA is the chief immunoglobulin class of sero-mucous secretions, part of the defense system for external body surfaces. The monomeric form is composed of two alpha heavy chains and two light chains. Two subclasses of IgA—IgA1 and IgA2—have been identified in humans. Normal IgA serum levels vary with age. High IgA serum levels are associated with breastfeeding, chronic infections, liver disease and myeloma. Reduced levels are typically seen in immunodeficiency conditions.

IgD is a functionally significant protein. Its precise role is unknown, although there are indications that it functions principally as a cell–surface antigen receptor (triggering lymphocyte differentiation) and as a ligand for IgD receptors on immune-regulatory helper T-cells. While IgD accounts for less than 1% of total plasma immunoglobulin concentration, its levels are influenced by age and inheritance. Very high serum IgD concentrations are found in IgD myeloma patients and in hyperimmunoglobulinemia D syndrome (HIDS), an autosomal recessive disorder characterized by recurrent febrile attacks with abdominal, articular and skin manifestations.

IgE, found in trace amounts in the blood, can be used to diagnose allergic diseases. While IgE is typically the least abundant immunoglobulin (only 0.05% of total immunoglobulin concentration), it is capable of initiating the most powerful inflammatory reactions.

IgG typically constitutes approximately 75% of total serum immunoglobulin. Within the IgG class, the usual order of concentration of the four subclasses is IgG1, IgG2, IgG3 and IgG4, but the actual concentration of each may vary markedly among individuals. There are considerable differences in the properties of IgG subclasses, including the ability to fix complement, bind to macrophages and pass through the placenta. Abnormal levels of one or more subclasses may be associated with conditions like anaphylaxis, autoimmune and gut diseases and hypo- and hypergammaglobulinemia. In particular, reduced production of IgG2 in children may be associated with recurrent infections.

IgM is the first class of immunoglobulin synthesized in response to antigenic attack. This large, multivalent molecule deals most efficiently with polyvalent antigens such as bacteria and viruses. IgM also activates complement. On active immunization, IgM rapidly appears in sera, but levels normally drop after a week, usually in parallel with an increase in IgG. Normal serum levels are dependent on age. Elevated serum levels are associated with hepatitis, myeloma, Waldenstrom macroglobulinemia and other infections. Reduced levels can occur in antibody-deficiency syndrome.

Lipoprotein(a): Lipoprotein(a), also called Lp(a) or LPA, is a lipoprotein subclass. Lp(a) determinations are intended for use in conjunction with clinical evaluation, patient risk assessment and other lipid tests to evaluate disorders of lipid metabolism, and to assess coronary heart disease in specific populations. Genetic studies and numerous epidemiologic studies have identified Lp(a) as a risk factor for atherosclerotic diseases, such as coronary heart disease and stroke.

Microalbumin: When kidneys are functioning normally, there is very little or no albumin in the urine. Abnormal amounts of albumin, or microalbumin, leak into the urine if kidneys are damaged. As such, microalbumin levels can aid in the diagnosis of renal disease. Early detection and treatment are important in preventing renal failure in insulin-dependent diabetics, as elevated urinary albumin is a good indicator of glomerular damage. Additionally, microalbumin levels can be a potential marker for future cardiovascular problems, including hypertension, in noninsulin-dependent diabetes patients.

Prealbumin: Primarily synthesized in the liver, but also by the choroid plexus, prealbumin helps assess nutritional status. It has a high affinity for, and binds with, thyroxine (T4) and retinol-binding protein. Plasma concentrations are dependent on protein and energy intake, with reduced levels associated with malnutrition.

Rheumatoid factor: Rheumatoid factor (RF) is a heterogeneous group of high-molecular-weight autoantibodies directed against the body’s own immunoglobulins. They are produced by plasma cells present at sites of tissue injury. The initiating antigen is thought to be one or more viruses, or viral antigens, that persist in the joint tissues. Research has shown that environmental and genetic factors can affect the production of RF. Rheumatoid factor has also been observed in the sera of patients with lupus erythematosus, hepatitis, liver cirrhosis, syphilis and various other conditions, but the titer is much lower than that seen in rheumatoid arthritis. This abnormal protein is found in the blood and joint fluid of 60–80% of patients with active rheumatoid arthritis.

Transferrin: Transferrin principally occurs in sera, but is found at lower concentrations in other bodily fluids as well. Its main function is the transport of iron to cells. Measurement of transferrin levels aids in the diagnosis of malnutrition, acute inflammation, chronic infection, hepatic disease and iron-deficiency anemia.

 Figure 2 – Analysis of samples for special protein levels using automated instrumentation.

Much research in medical diagnostics, genomics, life sciences and drug discovery focuses on protein chemistry because it has the widest diagnostic potential. Current studies are targeting the diagnosis, monitoring and treatment of cardiovascular disease; spinal injury and neurological disorders (including Alzheimer’s); organ transplant and rejection; new biomarkers for certain cancers; obesity and nutrition; diabetes; and autoimmune conditions (see Figure 2). Successes in medical technology hold promise for new and improved diagnostic tests for many disease indications, and the discovery of novel special protein biomarkers.

References

  1. Bakerman, S.; Bakerman, P. et al. Bakerman’s ABC’s of Interpretive Laboratory Data, 3rd Ed. Interpretive Laboratory Data, Inc.: Valley Stream, N.Y., 1994.
  2. Tietz, N.W. Tietz Fundamentals of Clinical Chemistry, 2nd Ed. WB Saunders: Philadelphia, Penn., 1982.
  3. Pesce, A.J. and Kaplan, L.A. Methods in Clinical Chemistry. CV Mosby: Maryland Heights, Mo., 1987.
  4. Davidsohn, I. and Henry, J. Todd-Sanford Clinical Diagnosis by Laboratory Methods,15th Ed. WB Saunders: Philadelphia, Penn., 1974.

Robert J. Janetschek, MS, MT (ASCP), is affiliated with The Binding Site, 6730 Mesa Ridge Rd., San Diego, Calif. 92121, U.S.A.; tel.: 516-286-5046; e-mail: [email protected]www.thebindingsite.com

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