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Serum Protein Electrophoresis/Immunofixation


Definition


  • Serum protein electrophoresis (SPE) is a method of physical separation of protein molecules based on their charge. Changes in both quality and nature of proteins determined by SPE allow clinicians to detect and monitor various pathophysiologic states. SPE, enhanced by follow-up procedures like protein quantification and immunofixation (IF), provides the best tools for general screening of human health state. The monoclonal gammopathies are a group of disorders characterized by the proliferation of a single clone of plasma cells that produces an immunologically homogeneous protein commonly referred to as a paraprotein or monoclonal protein (M-protein). SPE is usually done by the agarose gel electrophoresis or by the capillary zone electrophoretic method. It is the recommended method for the detection of an M-protein. The resulting M-protein, if found, can then be quantitated by means of a densitometer tracing of the gel. In the electrophoretic methodologies (agarose or capillary zone), proteins are classified by their final position after electrophoresis is complete into five general regions: albumin, alpha-1, alpha-2, beta, and gamma. The various immunoglobulin classes (IgG, IgA, IgM, IgD, and IgE) are usually of gamma mobility and make up most of the gamma region, but they may also be found in the beta-gamma and beta regions and may occasionally extend into the alpha-2 globulin area.
  • Other names: serum protein electrophoresis (SPEP).
  • Normal range:
    • SPE:
      • Albumin: 3.5 " “5.0 g/dL
      • Alpha-1 globulin: 0.1 " “0.3 g/dL
      • Alpha-2 globulin: 0.5 " “1.0 g/dL
      • Beta globulin: 0.5 " “0.9 g/dL
      • Gamma globulin: 0.6 " “1.4 g/dL
    • IF: no monoclonal protein detected

Use


  • Monitoring patients with monoclonal gammopathies
  • Diagnosis of monoclonal gammopathies, when used in conjunction with immunofixation
  • Assist in the diagnosis of hepatic disease, hypogammaglobulinemias and hypergammaglobulinemias, inflammatory states, neoplasms, renal disease, and GI disorders
  • SPE should also be considered in any patient with an elevated total serum protein or otherwise unexplained signs and symptoms suggestive of the presence of a plasma cell disorder. These include any one or more of the following:
    • Elevated ESR or serum viscosity
    • Unexplained anemia, back pain, weakness, or fatigue
    • Osteopenia, osteolytic lesions, or spontaneous fractures
    • Renal insufficiency with a bland urine sediment
    • Heavy proteinuria in a patient older than 40 years of age
    • Hypercalcemia
    • Hypergammaglobulinemia
    • Immunoglobulin deficiency
    • BJ proteinuria
    • Unexplained peripheral neuropathy
    • Recurrent infections

Interpretation


Increased In


  • Albumin
    • Usually in hospitalized patients, hemoconcentration, albumin perfusion
    • Normal individuals: no clinical significance
    • Bisalbuminemia (double band), permanent
    • Bisalbuminemia, acquired, transient
      • High doses of beta-lactam antibiotics (complex formation)
      • Hyperbilirubinemia (jaundice, complexed with bilirubin)
      • Azotemia (urea and other N-compounds in blood)
      • Pancreatitis, pancreatic fistulas, or ascites (lysis of albumin by pancreatic enzymes)
  • Alpha-1 globulin
    • Acute inflammatory disorders
    • Severe alcoholism
    • Some hepatic disorders
    • Double bands (AAT phenotypes)
  • Alpha-2 globulin
    • Inflammatory syndromes
    • Nephrotic syndrome
    • Increased estrogen stimulation
    • Double bands in
      • Haptoglobin (Hp) phenotypes (no clinical significance)
      • Hemolysis (Hb-Hp complex)
      • Abnormal migrating beta-lipoproteins (aged samples)
  • Betaglobulin
    • Primary and secondary hyperlipoproteinemias
    • Iron deficiency anemia
    • Estrogen, pregnancy, or anabolic steroids
    • Increase comigrates with transferrin, Hb (in excess to that bound in Hb-Hp complexes)
    • Acute inflammation (later phase)
    • Monoclonal immunoglobulins (IgA is frequent)
    • Double bands
      • Transferrin phenotypes (different degrees of sialation)
      • Alcoholics
  • Gammaglobulin
    • Polyclonal gammopathy: chronic, subacute infections (AIDS, hepatic infections, chronic liver associated with beta " “gamma bridging, autoimmune disorders)
    • Narrow band: monoclonal component, monoclonal gammopathy of undetermined significance, fibrinogen, CRP
    • Two narrow bands:
      • Biclonal or double gammopathies
      • Greater than two bands: oligoclonal hypergammaglobulinemia (present in low concentrations, transient, results in polyclonal processes); autoimmune, viral, bacterial, parasitic infections; restoration of immunoglobulin synthesis of immunosuppressants, 15% normal (no clinical significance)

Decreased In


  • Albumin
    • Congenital analbuminemia
    • Nutritional deficiency
    • Decreased synthesis
    • Hepatocellular insufficiency, damaged liver (cirrhosis, hepatitis)
    • Organ, tissue loss
    • Urinary (nephrotic syndrome), cutaneous (excessive burns) urinary excretion in pregnancy
    • Hypercatabolism
    • Endocrine disorders (thyrotoxicosis, Cushing syndrome)
  • Alpha-1 globulin
    • Hepatocellular insufficiency
    • Malnutrition, protein loss
    • Congenital deficiency of AAT
    • Tangier disease
  • Alpha-2 globulin
    • Hereditary deficiency of Hp phenotypes
    • Nutritional deficiency, hepatocellular insufficiency, protein loss, intravascular hemolysis (decreased Hp)
    • Pancreatitis
  • Betaglobulin
    • Chronic liver or renal disease
    • Hypobetalipoproteinemias
    • Thermal injuries
    • Acute inflammation
    • IgA deficiency
    • C3 degrades and eventually disappears in aged samples
  • Gammaglobulin
    • Physiologic (newborn)
    • Immunodeficiencies, induced (steroids, immunosuppressants, chemotherapy, radiotherapy)
    • Suppressed synthesis caused by monoclonal gammopathies (multiple myeloma, light chain disease, amyloidosis)
    • Lymphomas, leukemias

Limitations


  • The presence of a circulating monoclonal protein may interfere with one of more laboratory tests performed on liquid-based automated analyzers, either by precipitating during the analysis or by virtue of its specific binding properties.
  • A small M-protein may be present even when quantitative immunoglobulin values, beta and gamma mobility components on SPEP, and total serum protein concentrations are all within normal limits.
  • Fibrinogen (in plasma) is seen as a discrete band between the beta and gamma mobility regions. This is indistinguishable from an M-protein; the addition of thrombin to the specimen produces a clot if fibrinogen is present. The presence of fibrinogen is established if the discrete band is no longer detected when electrophoresis is repeated after the addition of thrombin.
  • Hb-Hp complexes secondary to hemolysis may appear as a large band in the alpha-2 globulin region.
  • High concentrations of transferrin in patients with iron deficiency anemia may produce a localized band in the beta region.
  • Nephrotic syndrome is often associated with increased alpha-2 and beta bands, which can be mistaken for an M-protein. Serum albumin and gammaglobulin concentrations are usually reduced in this setting.
  • Nonspecific increases in acute-phase reactants or certain hyperlipoproteinemias may result in increases in alpha-1 bands.
  • Serum IF is more sensitive than SPE and also determines the heavy and light chain type of the monoclonal protein. However, unlike SPE, IF does not give an estimate of the size of the M-protein (i.e., its serum concentration) and thus should be done in conjunction with electrophoresis.
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