What Is Protein Electrophoresis Used To Diagnose


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Why Do I Need It

In The Lab – Protein Electrophoresis

If you have an abnormal serum total protein or have certain symptoms your caregiver may want you to have an SPEP. Some diseases and nutrition disorders change the expected amounts of the protein fractions. Your caregiver may suspect that you have a problem with the proteins in your blood. Myeloma is one example of such a problem. Checking and following the SPEP helps your caregiver monitor your condition.

How Is The Specimen Collected

A caregiver will put a wide rubber strap around your arm and tighten it. Your skin will be cleaned with alcohol. A small needle attached to a special test tube will be put into a vein in your arm or hand. The tube has suction to pull the blood into it. When the tube is full, the rubber strap, needle and tube are removed. The caregiver will press a piece of cotton where the needle was removed. You may be asked to hold the cotton on the site for a few minutes to help stop the bleeding. Tape may then be put over the cotton on your arm.

What Are Serum Proteins

Serum is the liquid portion of blood from which the red blood cells, white blood cells, and factors involved in blood clotting have been removed. Serum contains a large number of proteins that perform diverse functions. These functions include providing cellular nutrition, defending against infections, playing a role in inflammation, and acting as hormones or enzymes.

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What Do My Results Mean

There are ranges for each hemoglobin type. For example, if your hemoglobin Type F level is higher than normal, it could be a sign you have a form of thalassemia. But knowing test result numbers is just one part of any diagnosis. Your healthcare provider is your best resource for understanding your specific results.

Serum Protein Electrophoresis Symptoms


Your doctor advises serum protein electrophoresis if you are experiencing symptoms of a condition affecting the proteins in your blood serum. These symptoms could include:

  • unexplained weight loss
  • bone pain or frequent fractures

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When is a protein electrophoresis test advised?

The protein electrophoresis test is advised when a patient is suspected to have a condition that is affecting the bodys plasma cells. In such case, the patient may experience symptoms such as unexplained weight loss, bone pain, fatigue, weakness, nausea, constipation, unusual thirst, frequent urination, frequent illnesses or fevers, bones that fracture easily, back pain, and high levels of calcium in the blood.

What other tests can be advised along with the protein electrophoresis test?

Other tests which can be advised along with protein electrophoresis test include HB electrophoresis, immunofixation electrophoresis, complete blood count, blood calcium and electrolyte test, x-rays, and kidney and liver function tests.

Reference: NCBI

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How Is Protein Electrophoresis Used To Diagnose Myeloma

Myeloma affects white blood cells called plasma cells in the bone marrow. Protein electrophoresis also tests for other proteins and immunoglobulins. The protein electrophoresis test is also used to diagnose other conditions affecting the plasma cells.

How does serum protein electrophoresis work?

Serum protein electrophoresis, SPEP What is this test? Protein electrophoresis is a test that measures specific proteins in the blood. The test separates proteins in the blood based on their electrical charge.

When to use serum protein electrophoresis for pregnancy?

Pregnancy. Electrophoresis on protein in urine may also be done, especially if the results of the serum protein electrophoresis test are abnormal. Normally very little protein is found in urine, but certain diseases cause large amounts of protein to leak into the urine.

Components Of Serum Protein Electrophoresis

The pattern of serum protein electrophoresis results depends on the fractions of two major types of protein: albumin and globulins. Albumin, the major protein component of serum, is produced by the liver under normal physiologic conditions. Globulins comprise a much smaller fraction of the total serum protein content. The subsets of these proteins and their relative quantity are the primary focus of the interpretation of serum protein electrophoresis.1,3

Albumin, the largest peak, lies closest to the positive electrode. The next five components are labeled alpha1, alpha2, beta1, beta2, and gamma. The peaks for these components lie toward the negative electrode, with the gamma peak being closest to that electrode. Figure 1 shows a typical normal pattern for the distribution of proteins as determined by serum protein electrophoresis.

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Gel Buffer System Selection

The decision for the use of chemicals for electrophoresis depends on the size and quantity of proteins and the application of the experiment.

For example, Bis-Tris and Tris-glycine are used to separate a broad range of proteins. However, Bis-Tris has greater sensitivity than Tris-glycine in protein detection. Its suitable to use for applications like mass spectrometry, post-translational modification analysis, or sequencing.

A widely used buffer system is the tris-glycine or Laemmli system. It has stacking gel at pH 6.8 and resolving in the range of ~8.3-9.0 pH. However, the limitation of the system is the formation of disulfide bonds between proteins and the inability of reducing agents to move with the proteins. The challenges can be overcome by using buffers with lower pH, which also provide more stability to the acrylamide gel.

Is There Anything Else I Need To Know About Hemoglobin Electrophoresis

Electrophoresis, Immunoelectrophoresis and Immunofixation

If you are at risk of having a child with an inherited hemoglobin disorder, you may want to speak to a genetic counselor. A genetic counselor is a specially trained professional in genetics and genetic testing. He or she can help you understand the disorder and your risk of passing it along to your child.

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Other Techniques Used With Electrophoresis

There are several techniques, which can be used in conjunction with serum and urine electrophoresis in determining the size and identity of the plasma cell dyscrasia which the patient may have. These techniques include isoelectric focusing of the patients serum and urine,9 immunofixation, either of serum protein electrophoresis to identify particularly low level IgA and IgM bands, or of isoelectric focusing, to distinguish between a monoclonal protein, a prominent IgG or IgG clone, oligoclonal IgG or free light chains. The resolution of immunofixation of isoelectric focusing is approximately five times better than the resolution of immunofixation of electrophoresis. There is also a paper in this issue by FN Cornell on these complementary techniques. Comparison of the techniques of capillary electrophoresis, high resolution electrophoresis and isoelectric focusing are shown in Figures 14.

How Does Protein Gel Electrophoresis Work

Proteins gel electrophoresis involves the use of detergent SDS in the process. The presence of SDS in the buffer causes the denaturation of proteins into short peptide chains and provides them with a negative charge.

Therefore, when the electric field is applied the negatively charged ions will start moving towards the anode from the cathode . The smaller proteins or proteins with less molecular mass will move faster compared to proteins with larger mass.

Figure:An illustration of the separation of proteins from the negative to the positive electrode based on their molecular weight.

After separation, the proteins will appear in the form of solid bands that can be visualized using gel stains.

The separated proteins are used for several experimental purposes, including western blotting assay and mass spectrometry analysis. Thus gel electrophoresis is a fundamental step in proteomics studies.

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When Is It Ordered

Protein electrophoresis is a common test ordered when you have symptoms suggesting certain conditions. Some examples are listed below.

Serum and urine protein electrophoresis may be ordered when you have signs and symptoms of multiple myeloma, such as:

  • Weakened bones that can result in soft spots, fractures, bone lesions and bone pain
  • Carpal tunnel syndrome
  • Destruction of bone that frequently increases the level of calcium in the blood, leading to symptoms such as loss of appetite, nausea, thirst, fatigue, constipation, and confusion
  • Frequent infections
  • Excessive bleeding and bruising
  • Kidney diseaselarge amounts of protein in the urine due to some forms of multiple myeloma can affect the kidneys and may permanently damage them.
  • In some cases, an increase in the thickness of the blood may lead to headaches, problems with vision, seizures, or even coma.

Urine protein electrophoresis may be ordered when you have abnormally high levels of protein in your urine.

Immunofixation electrophoresis or immunosubtraction electrophoresis may be ordered when an abnormal band suggestive of a monoclonal immunoglobulin is detected on either a serum or a urine electrophoresis.

CSF protein electrophoresis may be ordered:

  • When you have headaches or other neurologic symptoms to look for proteins suggestive of inflammation or infection
  • What Is Hemoglobin Electrophoresis

    34 Best Electrophoresis images

    Hemoglobin electrophoresis is one process that healthcare providers use to analyze hemoglobin in your red blood cells.

    Hemoglobin is a protein in your red blood cells that helps cells carry oxygen throughout your body. Sometimes, the gene controlling your hemoglobin changes or mutates, turning healthy red blood cells into damaged cells. These damaged cells can break down and cause anemia and other blood disorders like sickle cell anemia or thalassemias.

    Hemoglobin electrophoresis helps healthcare providers diagnose those conditions. This test is also one of several tests that screen newborn babies for signs of sickle cell anemia and other rare but serious illnesses.

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    Serum Protein Electrophoresis And Cancer

    Some neoplastic diseases cause changes in the concentration of individual protein fractions. Serum protein electrophoresis plays an auxiliary role during oncological diagnostics. This examination, in combination with others, allows to obtain a complete picture about the health of a patient along with the developed health issues. In case of advanced cancer, a decrease in protein is often observed, and thus also the concentration of albumin is lower. SPEP test is most commonly used in the diagnosis and monitoring of myeloma as well as some lymphomas.

    Precast Gels Vs Handcast Gels

    In the majority of labs, researchers cast their gels by following a standard recipe for gel preparation. These gels are known as hand-cast gels.

    However, now ready-to-use precast-protein gels are available that can be used for several applications ranging from SDS-PAGE, isoelectric focusing , to non-denatured protein analysis. The precast gel is more convenient and consistent compared to the hand-cast gels.

    Moreover, to optimize protein resolution, shelf life, and run time of gel, they come in different buffer formulations, including Tris-acetate, Tricine Tris-glycine, Bis-Tris. The use of these precast gels also prevents researchers from touching acrylamide, which is a neurotoxin and carcinogen.

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    Serum Free Light Chain Assay Or Freelite Testing

    The serum free light chain assay is used for diagnosis and monitoring of myeloma. Immunoglobulin proteins are made up of two kinds of molecules, heavy chains and light chains .

    These heavy and light chains are usually bound together as intact immunoglobulins. For reasons we do not know, the plasma cells produce more light chains than heavy chains. The excess, or unbound, light chains circulate freely in the blood. Hence, they are called free light chains. These free light chains are present in both healthy individuals and in patients with myeloma and related disorders. Related disorders include:

    • light chain deposition disease
    • Waldenströms macroglobulinemia

    Patients with myeloma may learn that their cells secrete:

    • both heavy and light chains
    • only heavy chains
    • only light chains

    Some patients, when assessed by SPEP, appear to secrete no M-protein at all. These patients’ myeloma cells secrete no heavy chain protein, only a very small amount of light chain protein. The Freelite test is used for patients who

    • only secrete light chains, which is often called Bence-Jones myeloma.” .
    • secrete both heavy and light chains
    • secrete very low levels of protein, also known as oligosecretory myeloma

    The Freelite assay is also used to:

    • diagnose and monitor patients with MGUS, a non-cancerous elevation in monoclonal protein. The Freelite assay can help to assess if a patient’s MGUS may develop into active myeloma.
    • monitor patients with smoldering or asymptomatic myeloma .

    What Is Protein Electrophoresis

    Clinical Chemistry: Serum Protein Electrophoresis in Multiple Myeloma

    Protein electrophoresis is a specialized test that analyzes specific groups of proteins in the blood serum and measures how much of each group of protein is present. Individual proteins have distinctive sizes and electrical charges. Electrophoresis separates the serum proteins into broad groups based on their size and electrical charge. The results of the analysis are displayed on a special graph, and the pattern of the different proteins is used to help diagnose specific diseases, including some types of cancer.

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    What Specific Laboratory Diagnostic Tests Are Used For The Diagnosis Of Myeloma

    Serum protein electrophoresis by separation of protein upon an agarose gel can detect the whole Ig in the range of 1 to 5 g/dL, but it only detects increased LC in patients who have very high levels of LC-only myeloma, and it is semi-quantitative. Serum immunofixation electrophoresis is around 10 times more sensitive for Igs and LC, but it is not quantitative. Urine IFE requires concentrated urine samples for the detection of FLCs and can detect low levels of LC. The detection of urine LC by the primitive techniques of boiling and precipitation was one of the earliest descriptions of myeloma disease mollities ossium and its manifestations published in 1847 by Dr. Henry Bence-Jones. Subsequently Korngold and Lapiri raised antisera against the two LC domains. Bence-Jones proteins are urinary FLCs detected by urinary protein electrophoresis and immunofixation.

    Nikhil C. Munshi, Sundar Jagannath, in, 2018

    What Happens During Hemoglobin Electrophoresis

    A health care professional will take a blood sample from a vein in your arm, using a small needle. After the needle is inserted, a small amount of blood will be collected into a test tube or vial. You may feel a little sting when the needle goes in or out. This usually takes less than five minutes.

    To test a newborn, a health care provider will clean your baby’s heel with alcohol and poke the heel with a small needle. The provider will collect a few drops of blood and put a bandage on the site.

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    Electrophoretic Techniques In Greater Detail

    High-resolution agarose gel electrophoresis whether commercial or in-house has been used routinely for over 20 years and with serum delivers a separation of between 6 and 17 bands. The technique has been shown to give reproducible quantification of monoclonal bands provided the buffer pH, voltage and type of stain are meticulously reproduced. Immunofixation of high-resolution gels has been successfully used for demonstrating low-level monoclonal IgA and IgM paraproteins. High-resolution agarose gel electrophoresis together with immunoblotting can also be used to separate the various isoforms of transferrin such as asialotransferrin or beta-2-transferrin. This is important in the detection of such proteins in CSF and in other leakage fluid samples.

    The separation of serum proteins by CE was first demonstrated in the early 1990s. CE is a technique that gives excellent separation of serum proteins while reducing the hands-on time required by means of automation.

    With CE, the pH of the buffer used must be constant for a particular system whether a turnkey commercial or research instrument is used. The applied voltage of CE of between 8 and 17 kV is very much higher than the 250400 V used with agarose gels . Use of wavelengths in the far UV to detect light absorption by peptide bonds avoids variable staining of proteins with conventional detection techniques . In this regard, it has been demonstrated that the sensitivity of detection at 200 nm is three times that at 215 nm.

    How Is The Graph Interpreted


    The main consideration when interpreting the electrophoresis graph is whether the globulin is elevated because of an increase in many different globulins or because of an increase in only one type of globulin. When many different globulins are elevated, it is called a polyclonal increase when only a single type of globulin is responsible for the increase, it is called a monoclonal increase . Inflammation is typically polyclonal, while lymphoid neoplasia is more likely to be monoclonal. Unfortunately, there is some overlap between these two general classifications.

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    Blood Tests For Multiple Myeloma

    Blood tests for multiple myeloma involve taking a blood sample from a vein in your arm. There are several blood tests that may be done, including:

    • Complete blood count: A complete blood count looks at the levels of different types of blood cells in a blood sample. If you have multiple myeloma, some of these blood cell counts may be low.
    • Blood chemistry: Blood chemistry tests look at a variety of substances present in your blood. Some substances that may have altered levels in multiple myeloma include calcium, albumin, and creatinine.
    • Immunoglobulins: An immunoglobulin test looks at the levels of different types of antibodies in the blood. In multiple myeloma, levels of one antibody type may be much higher than others.
    • Serum protein electrophoresis and immunofixation: In a serum protein electrophoresis and immunofixation test, a blood sample is placed on a special paper and exposed to an electrical current. This separates the different antibodies in the sample into bands. A healthcare professional can analyze these bands to see if M protein is present.
    • Serum-free light chains: This test looks for a component of antibodies, called light chains, in the blood. It can be helpful in detecting potential multiple myeloma when a SPEP and immunofixation test doesnt find M protein, meaning you may have something called light chain myeloma rather than multiple myeloma.

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