Electrophoresis Information and Courses from MediaLab, Inc.

The normal RBC count (4.84 x 1012/L) in this case, together with the decreased hemoglobin (8.4 g/dL) and MCV (59 fl) is an indicator of ineffective erythropoeisis that often points to thalassemia.The RBC morphology shows slight hypochromic microcytosis with codocytes, schizocytes, and basophilic stippling. Schizocytes form by several mechanisms, one being the removal of RBC inclusions.This patient's elevated bilirubin correlates with her presentation of sclera icterus; her splenomegaly is consistent with increased RBC destruction.The Hb electrophoresis demonstrated a normal pattern, initially, but the unstable Hemoglobin H was revealed upon repeat electrophoresis with reduced incubation time. Hemoglobin H is the result of beta globin chain tetramer formation due to the insufficient supply of alpha globin chains in alpha thalassemia intermedia.People with Hemoglobin H disease (alpha thalassemia intermedia) usually have a normal life expectancy without treatment. However, hemolysis may lead to moderate anemia that may be treated with splenectomy.

Anemia is moderate.RBC count is increased.Hb is moderately decreased.MCV is decreased. MCHC is decreased.RDW is increased.RBC morphology shows slight hypochromic microcytosis with codocytes, schizocytes, and basophilic stippling.Reticulocytes are moderately increased.Hb electrophoresis demonstrates abnormal patterns in both adults and neonates.Adults:HbA decreasedHbA2 decreasedHbF normal to decreasedHb H -2-40% (beta chain tetramers)Neonates: 10-40% Bart's (gamma chain tetramers)Hb H inclusions are frequently seen.Bone marrow demonstrates erythroid hyperplasia.

Anemia is absent.RBC count is within normal limits.Hb is within normal limits.MCV is normal to slightly decrease.MCHC is normal to slightly decrease.RDW is within normal limits.Red Blood Cell morphology is normal.Reticulocytes are within normal limits.Hb electrophoresis demonstrates a normal pattern in adults:Hb A - 97-98%Hb A2 - 1-2.5% Hb F - < 1%. Neonates have 1-2% Bart's Hemoglobin (gamma chain tetramers).Hb H inclusions are rarely seen.Bone marrow is normal.

Of the hemoglobins normally present in an adult, Hb A migrates the fastest, followed by Hb F. Hb A2 moves only slightly from the point of origin near the cathode.Abnormal hemoglobins show the following migration patterns: Hb C migrates with Hb A2 near the cathode. Hb S lies between Hb A2 and Hb F. Hb H and Bart's hemoglobin are unstable and very fast moving placing them past Hb A and near the anode with Hb H being the fastest of the two.Relative migrations of hemoglobin variants on alkaline electrophoresis can be seen below.

This is an example of an electrophoresis on normal blood.

Beta Thalassemia Silent Carrier Beta Thalassemia Minor Beta Thalassemia Intermedia Beta Thalassemia Major Delta-Beta Thalassemia Anemia Absent Mild to absent Moderate Severe Mild Red blood cell (RBC) count Normal Increased Decreased to normal Decreased Decreased to normal Hemoglobin(Hb) Normal Decreased to normal (10 - 12 g/dL) Decreased (7 - 10 g/dL) Marked decrease (<7 g/dL) Decreased to normal (8 - 13 g/dL) Mean corpuscular volume (MCV) Slight to no decrease Marked decrease Marked decrease Marked decrease May be slightly decreased Mean corpuscular hemoglobin concentration (MCHC) Slight to no decrease Marked decrease Marked decrease Marked decrease May be slightly decreased Red blood cell distribution width (RDW) Normal Normal to slightly increased Increased Increased Normal RBC morphology Normal Marked hypochromia and microcytosis Codocytes (target cells) Possible basophilic stippling Nucleated RBCs are usually not present Marked hypochromia and microcytosis Codocytes (target cells) Possible basophilic stippling Nucleated RBCs are usually not present Marked hypochromia and microcytosis Codocytes (target cells) schistocytes ovalocytes basophilic stippling polychromasia nucleated RBCs Possible hypochromia and microcytosis Codocytes (target cells) Basophilic stippling Reticulocyte count Normal May be slightly increased Slightly increased (<5%) Mildly increased (5 - 10%) Mildly increased Hb electrophoresis Normal pattern Decreased amount of Hb A Variable amounts of Hb A2 and Hb F Decreased amount of Hb A Variable amount of Hb A2 Hb F is usually increased Severly decreased amount of Hb A Variable amount of Hb A2 Usually an increased amount of Hb F Decreased amount of Hb A and Hb A2 Increased amount of Hb F (15 - 20%) If red blood cells are normochromic and normocytic, the RBC, Hb, and Hematocrit (HCT) test values follow in three-fold progression (i.e., RBC x 3 = Hb and Hb x 3 = HCT). This is sometimes referred to as "the rule of threes." This rule will usually not apply in cases of beta thalassemia, particularly beta thalassemia minor where the RBCs are not normochromic and are microcytic, and where there is a disproportionate number of RBCs for the amount of hemoglobin that is present.

Of the hemoglobins normally present in an adult, Hb A migrates the fastest, followed by Hb F. Hb A2 moves only slightly from the point of origin near the cathode.Abnormal hemoglobins show the following migration patterns:Hb C migrates with Hb A2 near the cathode.Hb S lies between Hb A2 and Hb F.Hb H and Bart's hemoglobin are unstable and very fast moving, with Hb H being the faster of the two. They are located nearer the anode past Hb A .Relative migrations of hemoglobin variants on alkaline electrophoresis can be seen below.

This densitometer tracing corresponds to the pattern for normal distribution of hemoglobin.

A false positive result for blood on the reagent strip can occur when oxidizing contaminants, such as hypochlorite (bleach), remain in collection bottles after cleaning. Contamination of the urine with provodine-iodine, a strong oxidizing agent, used in surgical procedures can result in a false positive reaction. Microbial peroxide found in association with urinary tract infections may also cause false-positive results. Capoten® (Captopril) can cause decreased reactivity. The muscle tissue form of hemoglobin, myoglobin is a well-known cause of false-positive reactions on the blood portion of the reagent strip. When tissue hemoglobin is present, the urine specimen has a clear red appearance. Patients suffering from muscle-wasting disorders or muscular destruction due to trauma, prolonged coma, or convulsions or individuals engaging in extensive exertion may have myoglobin in their urine. Specific tests for myoglobin, such as immunodiffusion techniques or protein electrophoresis, are needed to confirm the presence of this substance in a urine specimen. Levels of ascorbic acid normally found in urine do not interfere with this test.

Electrophoresis is the migration or separation of charged particles or solutes of a liquid solution in an electrical field. Conventional electrophoresis is tedious and time consuming. Electrophoresis automation and newer electrophoresis techniques have revitalized the utilization of electrophoresis in today's clinical laboratories. Molecular diagnostic analysis using electrophoresis and research in proteomics have also contributed to this revitalization.

Clinical Chemistry Concepts and Applications. Shauna C. Anderson and Susan Cockayne. Long Grove, Illinois: Waveland Press, Inc, 2003.Clinical Laboratory Instrumentation and Automation Principles, Applications, and Selection. Kory M. Ward, Craig A. Lehmann, Alan M. Leiken. Philadelphia: WB Saunders Company, 1994.Laboratory Instrumentation, 4th Edition. Mary C. Haven, Gregory A. Tetrault, Jerald R. Schenken, eds. New York: Van Nostrand Reinhold, 1995.Molecular Diagnostics Fundamentals, Methods, and Clinical Applications. Lela Buckingham and Maribeth L. Flaws. Philadelphia: FA Davis Company, 2007.Principles of Gel Electrophoresis. Available at http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/principles.html accessed 9/29/08.Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 4th Edition. Carl A. Burtis, Edward R. Ashwood, David E. Burns, eds. Philadelphia: Elsevier Saunders, 2005.

The net charge of a molecule is the most important factor affecting the mobility of that molecule. The greater the net charge, the greater the mobility or the more quickly the molecule migrates. The net charge of a particular compound depends upon the buffer and the resultant pH set by that buffer. The size and shape of a molecule also influence the rate of migration in that the larger the size, the slower the molecule will move in electrophoresis.The viscosity and the pore size in the support media or gels used for electrophoresis influence the rate of migration. Increased viscosity slows the migration and increasing pore size speeds up the migration.Increased heat increases the rate of migration. Increasing the strength of the electrical field by increasing voltage and increasing the temperature used for the electrophoresis both increase the mobility and rate of migration. When increasing these factors that affect mobility, caution is necessary. Each will lead to an increase in temperature that can possibly denature the sample and alter the characteristics of the support medium. The ionic strength of the buffer and its effect on mobility are more complicated. The ionic strength of the buffer affects the thickness of the ionic cloud, the rate of migration, and the sharpness of the separated solutes. In electrophoresis, a cloud of ions forms over the medium and is composed of buffer ions, sample ions and other nonbuffer ions. Increasing the buffer ionic strength increases the buffer ions in the cloud and slows the movement of solutes and creates sharper bands. However, this also increases heat production.

The two important purposes of the buffer are to create the pH and to conduct the current. The buffer ions will carry the current during electrophoresis. The pH set by the buffer determines the net charge on the solutes. The pH ionizes these solutes and the resulting net charge determines which electrode the solutes migrate toward. Besides setting the pH, the buffer also maintains the pH throughout the electrophoresis of the sample.

For electrophoretic separation of solutes, the sample of solutes is placed on a gel or membrane in contact with buffer for separation. Common gels are cellulose acetate, agarose, and polyacrylamide gels. These gels are formed into sheets, slabs, or inserted into columns or tubes. The gel can be positioned horizontally or vertically.Cellulose is chemically reacted with acetic anyhdride to form a cellulose acetate gel. Because cellulose requires soaking before sample application and a clearing step for detection of separated solutes or bands, agarose gel is more often used than cellulose acetate gel for clinical electrophoresis.

Polyacrylamide electrophoresis (PAGE) is performed on a gel formed by polymerizing and cross-linking acrylamides. These gels are stronger than agarose gels and also thermostable and transparent. The matrix created by cross-linking the polymer chains is more regular and the pore sizes are more uniform in an individual gel. The pore size can be changed by changing the concentrations of the acrylamides used.In addition to separating fragments by charge and mass, PAGE also separates solutes by molecular size. When using PAGE, the gel allows more fractions of smaller size to be detected than the traditional agarose gel methods.Care is required in polyacrylamide gel preparation and use because acrylamides are carcinogenic.

Automated systems for protein electrophoresis are available for large volumes of samples for electrophoresis. An automated system is capable of separating 10-100 samples simultaneously. There are several different automated systems and the number of process steps automated varies. Automated steps may include reagent addition, sample application, electrophoresis separation, staining, and detection.Below is a stained electrophoresis land for one sample analyzed on the automated instrument, SPIFE 3000, Helena Laboratories. The separated proteins are stained with Acid Blue, a Helena Laboratories' stain developed from Coomassie dye.

Routine electrophoresis is a generic term for the traditional clinical laboratory electrophoresis performed on a rectangle-shaped slab gel. Routine electrophoresis is mostly used for separation of proteins and has some use in separating nucleic acids. Generally several patient specimens and control(s) can be placed on one gel and solutes separated in one run. This type of electrophoresis is sometimes called zone electrophoresis.A serum sample with normal plasma proteins yields five zones or bands of separated proteins: albumin, alpha-1-globulins, alpha-2-globulins, beta-globulins, and gamma-globulins. Proteins in CSF and urine proteins are also separated with routine electrophoresis. Using whole blood treated with a reagent to lyse red blood cells, variant and glycosylated hemoglobins can be detected. With different visualization methods, isoenzymes and lipoproteins in a serum sample can be identified.A manual agarose gel electrophoresis of eight serum samples is pictured below. After electrophoresis, the gel was stained with Ponceau S.

More separations are also achieved with layers of polyacrylamide gels each with a different pore size. The gels can be horizontal or vertical slabs or incorporated into vertical cylinders or rods. Varying the pore size in each layer is significant especially if very small pore sizes are created. DNA of 100 base pairs (bp) or less can be separated.Common applications of PAGE are separation of proteins and nucleic acids. Polyacrylamide gels are also used as the medium in several other types of electrophoresis described in this section.

Capillary electrophoresis (CE) combines electrophoresis and high performance liquid chromatography. CE takes place in a very thin fused silica capillary tube with polyacrylamide or agarose gel. Polyacrylamide is the most common gel used. The ends of the capillary tube are placed in two buffer reservoirs with the anode in one, and the cathode in the other. A high voltage power supply and cooling system are included.One major difference in CE is the detection of separated solutes as migration and separation occur, instead of detection after separation. An optical detector attached to the capillary detects solutes after separation but while still in the capillary; the detector is linked to data collection and storage.

Because of the two separation processes, more information and separated solutes can be gained from a sample. The use of two-dimensional electrophoresis is specialized and most applications are in research fields. It is used to study families of proteins in the field of proteomics and protein content in different types of cells. It is also used extensively in genetics to study differences in diseases, gene mutations, and bacterial DNA. In an effort to find ways to detect malignancies earlier, two-dimensional electrophoresis is used to study tumor cells.

An agarose gel electrophoresis first separates the proteins in a serum sample. Antiserum against the protein of interest is spread directly on the gel. The protein of interest precipitates in the gel matrix. After a wash step to remove other proteins, the precipitated protein is stained. This method is qualitative and is used to identify proteins found in multiple myeloma.Below is the immunofixation electrophoresis gel from a serum sample analyzed on SPIFE 3000, Helena Laboratories. After electrophoresis, the precipitated proteins are stained with Acid Violet, a stain developed and used by Helena Laboratories. The SP lane represents a routine serum protein electrophoresis of this specimen. On the next three protein separations, antiserum against IgG, IgA, and IgM were applied to the G, A, M lanes respectively. Antiserum to kappa light chain was added to the next protein separation and antiserum to lambda light chain to the last protein separation.

Larger fragments of DNA, > 50 kilobases (kb), cannot be separated with AGE or PAGE in routine electrophoresis systems; the gel pore sizes are too small for their migration. Fragment separation can be achieved with alternately applying the power to different pairs of electrodes. The most common method alternates the positive and negative electrodes in cycles during electrophoresis. The DNA fragments must reorient to a new field direction in each cycle. These changing pulses and reorientations separate the large size DNA fragments.Sample runs require longer time periods, some 24 hours or more, special gel boxes, different electrodes and controls for switching the electric fields during electrophoresis.

Separated bands or zones can be visualized with stains and dyes. Densitometry can be used to detect and usually quantitate stained separated fragments. Some electrophoresis methods use labeled probes to detect presence of unknowns in samples.

Minute-size fractions achieved in two-dimensional electrophoresis, IEF and PAGE with SDS, and bands from electrophoresis of nucleic acids are detected differently than protein electrophoresis fractions. Labeled polypeptide probes are used to detect these proteins; labeled single-stranded nucleic acid fragments are used for the detection of nucleic acids. Each probe is made with a label designed to generate a detectable signal. The label is bound to a probe and a system is created such that the signal is visualized when the probe is bound to the target.The most common labels are radioactive isotopes and fluorescence dyes. Chemiluminescence and color or ultraviolet absorbance are also used.

For successful gel electrophoresis, care must be taken with each of the following: Sample application Buffers Support medium StainsElectroendosmosis and wick flow are technical considerations. When irregular, distorted, and atypical bands result, possible causes are investigated.

To prevent microbial growth and contamination, buffers must be refrigerated when not in use. Using a buffer at refrigerator temperature also improves band resolution and lessens evaporation during electrophoresis. It is recommended that an electrophoresis using a small volume of buffer use a fresh buffer for each run. After a large volume buffer electrophoresis, the buffer can be reused if the buffer from each reservoir is combined, mixed well, and refrigerated.

With a pH 8.0-9.0 used for protein electrophoresis, proteins take on a negative charge, that is a negative ion cloud forms. As the negative ion cloud migrates to the anode, the proteins are pulled to the anode. Several gels used routinely for protein electrophoresis attract positive ions from the buffer and form a positive ion cloud. This ion cloud moves in the opposite direction to the cathode. This phenomenon is called electroendosmosis or endosmosis.The tension created by these oppositely moving ion clouds can affect the movement of sample macromolecules. The migration of some proteins can be slowed, some proteins can become immobile, and other proteins are pushed toward the cathode. Many protein electrophoresis methods take advantage of this tension and use it to achieve better separation of protein bands. The gamma globulin band in serum, urine, and other body fluids will separate more sharply by being pushed to the cathode and will appear behind the point of sample application.

Traditionally most clinical laboratory electrophoresis utilizes methods that separate and identify proteins in serum, urine, CSF, and some other body fluids. Most studies are for detecting serum protein abnormalities and gathering more information about gammopathies.In recent years, there has been a resurgence in electrophoresis use and methods. Development of automated methods has enhanced this. The evolution of numerous molecular diagnostic investigations and research in proteomics have also augmented electrophoresis.Applications of two-dimensional electrophoresis discussed the use of electrophoresis in proteomics. Electrophoresis and molecular diagnostics, blotting techniques, and current uses of CE in molecular diagnostics will be discussed now.

Blotting techniques were developed to discriminate fragments of nucleic acids. These techniques involve several processes; electrophoresis is one of the processes and is used to separate fragments of DNA and RNA. In Southern blotting (named after Edward Southern) restriction enzymes cut fragments of DNA are separated by AGE or PAGE, transferred to a membrane or blot, and visualized by hybridization with labeled probes.Northern blotting (not named after an inventor but by analogy to Southern blotting) separates RNA. RNA molecules are shorter and have defined lengths; cutting by restriction enzymes is not required. Denaturing conditions are required because of RNA secondary structures. After membrane blotting, the separated types of RNA are visualized with staining or labeled probes.Western blotting (again not named after an inventor but by analogy to Southern blotting)does not separate nucleic acids; it separates proteins in a mixture. The proteins are usually separated with PAGE, transferred to the membrane and visualized with a labeled antibody against the proteins of interest.

One of the problems with Lp(a) measurement is that the Apo(a) protein has a variable mass. It can have a molecular weight ranging from 275,000 to 800,000 daltons. This is due to variable amounts of repeating regions of the protein. Immunoassay antibodies which recognize these regions will thus give more signal for larger Apo(a) molecules compared to smaller Apo(a) molecules. This is not ideal since again, we would prefer to quantify the number of particles and Lp(a) containing large Apo(a) molecules will produce more signal, skewing the count. One assay system that tries to correct for this is the Lp(a) Cholesterol Electrophoresis Assay sold by Helena Laboratories. This assay uses electrophoresis followed by cholesterol staining and densitometry to calculate the concentration of cholesterol in Lp(a). Although this method still does not enumerate particles, it does appear to have less heterogeneity.Lp(a) is an acute phase reactant. This means that Lp(a) levels will rise in the context of general inflammation. Thus, Lp(a) should not be measured when there is extensive inflammation, such as immediately following a cardiovascular event. Concentrations of Lp(a) above 30 mg/dL are associated with increased cardiovascular risk. The risk of having a cardiovascular event increases 2 to 3 fold if Lp(a) cholesterol is > 30 mg/dL. Fifteen to 20% of the Caucasian population have Lp(a) levels >30 mg/dL. Africans, or people of Aftican descent, generally have levels higher than Caucasians and Asians, however, results must be evaluated in conjunction with clinical history.

Serum lipoprotein electrophoresis is usually performed using fasting serum or plasma. In a fasting sample, large chylomicrons are not normally present and therefore, will not obscure or confound the gel. Because electrophoresis relies on dye-binding and densitometry, samples should have cholesterol > 100 mg/mL. The results of this testing can be used in a variety of ways but typically a report of "type B" or "type A" is sufficient to inform physicians whether there is increased cardiovascular risk.

Detection techniques can vary in both direct and amplified methodologies and can include labeling either the probe or the target molecule of interest:Chemiluminescence: Release of light energy at the end of a chemical reaction that is detected by a luminometer. Uses a label such as acridinium ester. Electrophoresis: movement in a matrix such as a gel that is caused by an electrical field.Enzyme: Uses enzyme and substrate principles to label the appropriate target or probe. Can be combined with fluorescence or dyes for detection.Fluorescence: Molecules that emit light at a longer wavelength when excited at a shorter wavelength. Detection techniques include fluorescent staining of nucleic acids as well as fluorescent labeled probes that are measured in a fluorometer or with fluorescent polarization.Radioactivity: Uses a labeling technique where the radioactive label is then measured in a scintillation counter. The earliest assays utilized radioactive decay.

The family (cited in the previous case history) was from a region of Thailand where the physician knew HbE carriers are prevalent. Homozygous hemoglobin E is common in Southeast Asia and presents with very mild anemia and seldom requires transfusion. Over 30 million people in the world are HbE carriers, making this abnormal hemoglobin almost as common as HbS. Hemoglobin E is uncommon in North America and in Europe, but with changing immigration patterns, hemoglobinopathy E cannot be ignored. Peripheral blood smear findings of target cells, microspherocytes, red cell hypochromia, a few red blood cell fragments, and nucleated red blood cells require evidence from hemoglobin electrophoresis to establish a diagnosis. Clinically, a very important and severe syndrome is hemoglobin E/beta thalassemia in which there is hemolysis requiring repeated transfusions. The patient has a severe anemia, low MCV (50's), and high RBC. This is characteristic of Hgb E/beta thalassemia.

Plasma cells are uncommonly observed in the peripheral blood smear.They are normal constituents of lymph nodes, spleen, connective tissue and bone marrow. The presence of plasma cells in the peripheral blood is indicative of a large number of conditions mostly related to infections , immune disorders, malignancies, toxic exposures, hypersensitivity reactions and their responses.Although mature plasma cells have a distinct appearance, they still may be confused morphologically with immature plasma cells and other cells with inclusions, reactive changes or nucleated red bloods cell with altered identities.In the upper and lower photographs are plasma cells with features mindful of myeloma cellsThe large myeloma cell in the upper photograph has an eccentric immature nucleus with a muddy chromatin pattern.Note also clumping and stacking of the erythrocytes, bordering on rouleaux formation ,implicating an increase in plasma gamma globulin.The plasma cell with the double nucleus in the lower photograph is particularly suggestive of myeloma.Further studies are in order including a bone marrow examination where at least 30% of bone marrow cells should be variations of mature and immature plasma cells.Serum electrophoresis will reveal a monoclonal globulin spike, and light chains in excess of 1.0 gm/24 hours may be seen in the urine.The presence of lytic bone lesions is a convincing clinical clue.With these findings in combination, a diagnosis of myeloma can be made with assurance.