Densitometry Information and Courses from MediaLab, Inc.
These are the MediaLab courses that cover Densitometry and links to relevant pages within the course.
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| Hemoglobin Electrophoresis Theory Hemoglobin electrophoresis is the movement of hemoglobin proteins in an electric field at a fixed pH.Because the various hemoglobins are comprised of different combinations of globin chains (normal or abnormal), they will demonstrate different degrees of mobility. Typically, when a thalassemia or hemoglobinopathy is suspected, an alkaline electrophoresis is performed which may be confirmed with acid electrophoresis.For an alkaline hemoglobin electrophoresis, a hemolysate is applied to cellulose acetate which is electrophoresed in a buffer at pH 8.4-8.6. At this pH hemoglobin proteins move from cathode to anode. The proteins are visualized by the application of a dye which also makes them measurable by densitometry. | View Page |
| Hemoglobin Electrophoresis Theory Hemoglobin electrophoresis is the movement of hemoglobin proteins in an electric field at a fixed pH.Because the various hemoglobins are comprised of different combinations of globin chains (normal or abnormal), they will demonstrate different degrees of mobility. Typically, when a thalassemia or hemoglobinopathy is suspected, an alkaline electrophoresis is performed which may be confirmed with acid electrophoresis.For an alkaline hemoglobin electrophoresis, a hemolysate is applied to cellulose acetate which is electrophoresed in a buffer at pH 8.4-8.6. At this pH hemoglobin proteins move from cathode to anode. The proteins are visualized by the application of a dye which also makes them measurable by densitometry. | View Page |
| Hemoglobin electrophoresis on this patient's sample is pictured above and is labeled "patient 2" in lanes 5 and 6. The densitometer tracing of lane 5 is also pictured.What hemoglobin bands are present? | View Page |
| Visualization and Detection Methods 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. | View Page |
| Densitometry After electrophoresis, a stained gel is passed through the optical system of a densitometer to create an electrophoregram, a visual diagram or graph of the separated bands. A densitometer is a special spectrophotometer that measures light transmitted through a solid sample such as a cleared or transparent but stained gel. Using the optical density measurements, the densitometer represents the bands as peaks. These peaks compose the graph or electrophoregram and are printed on a recorder chart or computer display. Absorbance and/or fluorescence can be measured with densitometry.An integrator or microprocessor evaluates the area under each peak and reports each as a percent of the total sample. If the electrophoresis is for separation of serum proteins, the concentration of each band is derived from this percent and the total protein concentration. If the electrophoresis is for separation of enzymes, the enzyme activity of each band is derived from this percent and the total enzyme activity. The densitometer scan below depicts the separated bands from a serum sample electrophoresis. The SPIFE 3000, Helena Laboratories, electrophoresis splits the beta zone into two fractions for easier detection of small beta-migrating monoclonal gammopathies. The densitometer scan from this electrophoresis shows five bands with two peaks in the beta band. | View Page |
| Which statements below are correct descriptions of visualization and detection methods used in electrophoresis? | View Page |
| Lp(a) Testing 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. | View Page |
| Electrophoresis Testing 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. | View Page |