Muscle Information and Courses from MediaLab, Inc.
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This sample is demonstrating an anti-cytoskeletal pattern in the cytoplasm of the cells.The cytoskeleton of eukaryotic cells is comprised of microfilaments such as actin, intermediate filaments such as vimentin and microtubules such as tubulin. Autoantibodies to these proteins are seen in a variety of autoimmune diseases.Whenever an ANA is read the first step is to see if there is a clearly discernible pattern in the nucleus of the interphase cells. In this case the answer is no (a). However, there is considerable staining in the cytoplasm. This fibrous staining of the cytoskeleton is a discernible cytoplasmic pattern and should be reported (b).This sample is reported as ANA Negative, suspect cytoskeletal antibodies present. (The term "suspect" is used because the ANA substrate is not considered "confirmatory" for the identification of cytoskeletal antibodies. Additional follow-up testing is required).Follow-up testing would include testing for anti-smooth muscle antibodies. Anti-smooth muscle antibodies are seen in autoimmune liver disease.Reporting anti-Cytoskeletal Antibodies:Cytoplasmic pattern:• Fibrous strands in cytoplasmReport as:• Suspect cytoskeletal, suggest follow-up Clinical Significance:• Actin: Autoimmune hepatitis, PBC• Vimentin and others: SLE, RA, others
|The Human Heart|
The human heart is a muscular organ that is formed into four chambers with an interconnecting vascular system. Venous blood from the body enters the right atrium, is pumped into the right ventricle and from there is pumped to the lungs for reoxygenation. Oxygenated blood from the lungs accumulates in the left atrium and is pumped into the left ventricle and out to the body. Myocytes (muscle cells) require large amounts of energy and oxygen to accomplish this. The capacity of the heart to pump adequately is regulated by the volume of blood, systemic blood pressure, and the force of contraction achieved in the left ventricular wall.
|Infarction is an inadequate blood supply that decreases the availability of oxygen.||View Page|
|Terminology Describing Ischemic Heart Disease and Heart Failure|
Descriptions of cardiac biomarkers and their use require knowledge of several terms. These terms and their definitions follow.Acute Coronary Syndrome (ACS) Includes all the ischemic events that can occur in the heart. These events range from angina (where there is no cell death or reversible cell injury) to an AMI with large areas of cell necrosis. A continuum of events that are involved in ACS is illustrated on the page that follows this glossary of terms.Acute Myocardial Infarction (AMI) Commonly referred to as a heart attack. A sudden loss of circulating blood and oxygen that causes necrosis of myocardial tissue. It is most often caused by the narrowing of coronary arteries by atherosclerosis, a thrombus, or dislodged plaque material.Angina Chest pain caused by inadequate supply of oxygen to heart myocardium. It is synonymous with angina pectoris, pectoris meaning chest.Congestive Heart Failure (CHF) Usually, a left ventricular dysfunction resulting from aging, hypertension, atherosclerosis or muscle damage from an AMI or repeated AMIs. In CHF, the heart is not able to effectively pump blood through its chambers and to the body. Fluid accumulates in the lungs and tissues causing edema because less blood leaves through the arteries than what entered the heart from the veins. Electrocardiogram (ECG or EKG) The tracings of the electrical current that passes through the myocardium. The heart contractions are stimulated by this current. In areas of myocyte necrosis, the current does not pass and the tracings display abnormal patterns.Infarction An area of tissue death that occurs due to lack of oxygen. Clogging of an artery will cause dead muscle tissue or infarction.IschemiaAn inadequate blood supply that decreases availability of oxygen. Atherosclerosis is the main cause of myocardial ischemia.
Currently, the troponins are the definitive markers of myocyte injury. The troponins are contractile proteins that regulate muscle contractions; they work with calcium ions and another protein, tropomyosin. There are three types of troponin: Troponin C -- Calcium-Binding Component Troponin I -- Inhibitory Component Troponin T -- Tropomyosin-Binding ComponentIsoforms of the troponins are found in cardiac muscle and in slow-twitch and fast-twitch skeletal muscle. Troponin C is found in both cardiac and skeletal muscle in the same form. Troponin I has a specific form found in cardiac muscle, cTnI; cTnT is the cardiac specific form of troponin T.
|Troponin Measurement and Ranges|
Rapid immunoassays provide concentration levels of cTnI and cTnT that are approximately 96% sensitive and 94% specific for cardiac injury.Each diagnostic company develops their unique antibody against epitopes on the proteins. There is only one assay available for cTnT. However, there are several different antibodies that are used by manufacturers to detect cTnI. Consequently, different assay methods may not correlate well. Standardization is needed for intra-laboratory comparisons. Reference Ranges for an adult: cTnT <0.01 ng/mL No cardiac injurycTnI references may vary with different assay methods, but approximate these values: Equal to or < 0.03 ng/mL -- No detectable cardiac injury 0.04-0.49 ng/mL -- Cardiac muscle injury Equal to or > 0.5 ng/mL-- Myocardial infarction
|CK-MB Measurement and Ranges|
CK-MB assays used in cardiac damage diagnosis are mass immunoassay measurements. CK-MB is also present in skeletal muscle; therefore, skeletal muscle injury can also cause CK-MB elevation. CK-MB normal value for an adult: < 5.0 ng/mL
Myoglobin can also be used as a diagnostic indicator of an AMI. Myoglobin is an oxygen-binding protein in cardiac and skeletal muscle. It is released earlier after muscle injury than cardiac troponins and CK-MB and returns to normal faster than either of these other markers. It rises within 2 - 4 hours after chest pain, peaks in 6 - 12 hours, and is usually normal within 24 - 36 hours.Because of myoglobin's increase after skeletal muscle injury, it lacks the needed specificity for diagnosis of ACS and an AMI. False-positive elevation of myoglobin may also occur in a patient with impaired renal function since myoglobin is cleared through the kidneys.Myoglobin reference ranges for adults when an immunoassay method is used are approximately: Male 17-106 ng/mL Female 1-66 ng/mL Variation in ranges may be seen with different measurement methods.
|Select the correct statements regarding troponins.||View Page|
|Which ones of the following cardiac biomarkers can be used as a stand-alone marker, and is therefore the preferred biomarker to indicate the presence of an AMI?||View Page|
|While driving home after work, a 45-year-old male construction worker is involved in a collision with another car. Upon arrival in the emergency department, the patient complains of chest pain along with severe pain from accident injuries. X-rays and laboratory assays including cardiac biomarkers are ordered. BiomarkerReference ValuePatient Result at PresentationPatient Result at 6 HoursPatient Result at 12 Hours CK-MB< 5.0 ng/mL7.0 ng/mL6.0 ng/mL6.5 ng/mL cTnI<0.4 ng/mL0.03 ng/mL< 0.03 ng/mL< 0.03 ng/mLDo the cardiac marker results indicate that an AMI is causing his chest pain?||View Page|
|Gas gangrene may be seen in infections with all the following clostridia EXCEPT:||View Page|
|False Positive and Negative Results|
False Positives:A false positive result for blood on the urine chemical 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 also 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. False Negatives:False negative results may occur in some analysis methods when the concentration of ascorbic acid is greater than 5 mg/dL. The sensitivity of the blood portion of the test strip is decreased in specimens with a high specific gravity and increased protein. High levels of nitrites may delay the reaction, causing a false negative to be reported. If the pH of a urine sample is below 5, hemolysis of red cells as part of the test reaction is inhibited which results in a false negative reaction. An improperly mixed specimen may test negative if the red blood cells are in the sediment.
|False Positive Results|
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.
|Creatinine excretion correlates best with which of the following parameters:||View Page|
|Following a myocardial infarction which of the following enzymes will be the first to
become elevated:||View Page|
|Increases in the MB fraction of CK is associated with:||View Page|
|Increases in LD fractions 4 and 5 are indicative of:||View Page|
|Match the tissues on the left with the corresponding LDH isoenzyme peak on the right.||View Page|
|The following LDH Isoenzyme pattern would be seen in:||View Page|
|The following LDH Isoenzyme pattern would be seen in:||View Page|
|The following LDH Isoenzyme pattern would be seen in:||View Page|
|Label the scan with CK isoenzyme fractions:||View Page|
|Label the scan:||View Page|
|The following CK isoenzyme pattern would be seen in:||View Page|
|What is Connective Tissue?|
Connective tissue offers structural and metabolic support structure for organs and tissue. It is the most abundant tissue type in the body and can be found throughout. Cells and extracellular material called connective tissue matrix make up connective tissue. Fibroblasts, mast cells, macrophages, adipose cells, blood leukocytes, and plasma cells can all be found to some degree in connective tissue. In addition to cells, the matrix has 3 different fibers present:Reticular fibers - Support soft organs and the network around nerve fibers, fats cells, lymph nodes, and muscle fibers.Collagenous fibers - Found in ligaments, tendons, cartilage, and bone.Elastic fibers - Allow tissue to expand and are typically located in skin and blood vessel walls.
|Where are reticular fibers found?||View Page|
|Masson's Trichrome Staining - Chemistry|
Using acid-base chemistry, three dyes are employed to selectively stain muscle, collagen fibers, fibrin, and erythrocytes. Bouin’s solution is used first as a mordant to link the dye to the targeted tissue components. Nuclei are stained with Weigert’s hematoxylin, an iron hematoxylin, which is resistant to decolorization by the subsequent acidic staining solutions. Biebrich scarlet acid fuchsin stains all acidophilic tissue elements such as cytoplasm, muscle, and collagen. Subsequent application of phosphomolybdic/phosphotungstic acid is used as a decolorizer causing the Biebrich scarlet acid fuchsin to diffuse out of the collagen fibers while leaving the muscle cells red. Application of aniline blue will stain the collagen after which, 1% acetic acid is applied to differentiate the tissue section.
|Masson's Trichrome Staining - Diagnostic Applications|
Masson's is used to differentiate between collagen and smooth muscle in tumors and identify an increase in collagen in diseases such as cirrhosis. This is a routine stain for liver and kidney biopsies. Trichrome stains are used to distinguish collagen from muscle and aid in the diagnosis of fibrotic changes, neuromuscular diseases, and tumors of muscle origin.
|Masson's Trichrome Staining - Staining Protocol|
Sample type required: Deparaffinized and rehydrated tissue section (3-5 microns) on positively (+) charged slidesPreferred fixative: 10% neutral buffered formalin (NBF)Control: Internal; appendix, fallopian tube, uterus, or small intestine are recommended choicesReagentTimeTechnical NotesBouin's solution15 minutes (in heated solution)Used as both a fixative and mordant.Microwave solution for 1 minute before placing slides inside.Running water wash5 minutesEnsure that ALL picric acid (yellow) has been rinsed clear.Weigert's working hematoxylin10 minutesRunning water wash5 minutesRequired for nuclear bluing.Distilled water3 changesBiebrich scarlet acid fuchsin5 minutesDistilled water3 changesPhosphotungstic/phosphomolybdic acid10 minutesDiscard solution after use.Aniline blue5 minutesDistilled water3 changes< 1% acetic acid1 minuteDiscard solution after use.Post staining procedure: Tissue section should be dehydrated with 95% and absolute alcohols followed by two changes of xylene and then coverslip. Expected results:Nuclei - BlackCytoplasm, muscle, erythrocytes - RedCollagen - Blue
|Phosphotungstic Acid-Hematoxylin (PTAH) Staining - Chemistry|
The PTAH staining method relies on acid-base chemistry to stain collagen and muscle fibers. These fibers are demonstrated using a tungsten mordant provided by the phosphotungstic acid. This mordant binds hematein and stains selective tissue components blue, while the phosphotungstic acid is believed to stain other tissue components a red-brown color. Tissue should preferably be fixed in Zenkers, which is believed to intensify staining reactions. However, tissues fixed in formalin are commonly post-fixed in Zenker’s solution for this purpose. A section of skeletal muscle tissue can be used for quality control.The amount of phosphotungstic acid in the staining solution is far greater than the amount of hematein and it is believed that tungsten binds all available components blue, while the phosphotungstic acid is thought to stain the red-brown components. This stain has been referred to as a polychrome stain because one solution gives two major colors. The components colored red-brown will lose this color with water or prolonged alcohol washes. Therefore, dehydration of the tissue section following staining MUST be rapid. (Ref4)
|Phosphotungstic Acid-Hematoxylin (PTAH) Staining - Diagnostic Applications|
Although trichrome techniques will demonstrate muscle fibers, PTAH is preferred for demonstrating cross-striations of skeletal muscle, which may be lost in certain muscle diseases.
|Which of the following staining procedures can be used to demonstrate elastic fibers in connective tissues?||View Page|
|Agent: Tularemia (bacterium)|
Most likely means of dissemination: Solid or aerosolPrimary route of entry: Inhalation, absorption, or ingestionGeneral signs and symptoms: Sudden fever, chills, headaches, muscle aches, joint pain, dry cough, progressive weakness, and pneumonia.The disease is not transmissible through human contact. When used as a WMD, infection would be acquired by handling infected material, eating or drinking contaminated food or water or by breathing in the bacterium.
|Agent: Viral hemorrhagic fevers (Ebola, Marburg, Lassa and Argentine)|
Most likely means of dissemination: Solid, liquid or aerosolPrimary route of entry: Absorption, inhalation, ingestionGeneral signs and symptoms: Vary by type of viral hemorrhagic fever (VHF), but initial signs and symptoms often include marked fever, fatigue, dizziness, muscle aches, loss of strength, and exhaustion. Severe cases of VHF often show signs of bleeding under the skin, in internal organs, or from body orifices like the mouth, eyes, or ears. Photo courtesy of the CDC archives.
|Causes of Work-related Musculoskeletal Disorders|
The primary goal of ergonomics is the prevention of musculoskeletal disorders (MSDs). There are many potential causes of MSDs. Injury can occur from a single event (strain, sprain, slip, or fall) or result from build-up of tissue damage from many small injuries. An MSD can develop over time if a motion is repeated consistently so that the constant trauma causes damage to a muscle, tendon, bone, or bursa of a joint. Force, vibration, or maintenance of an awkward position for a prolonged period of time can cause MSDs.Some specific causes of MSDs that are related to laboratory tasks are:Prolonged use of a keyboard or mouse Prolonged sitting at a microscope Pipetting Screwing and unscrewing vial caps Standing at a laboratory instrument for a prolonged period of time Lack of rest-- intensive hours at the workstation with few breaks Sustained awkward position
|Role of Adipokines|
Over twenty adipokines have been identified. Some adipokines are secreted solely by adipocytes; several are secreted by adipocytes and other body cells. Their role is very widespread as they integrate with various body organs and tissues: skeletal muscle, adrenal cortex, brain and sympathetic nervous system. Adipokines function in: Energy balance Immune reactions Insulin sensitivity Angiogenesis Blood pressure maintenance Lipid metabolism Hemostasis
IL-6 responds to tissue injury. IL-6 is synthesized and secreted by many different cells in addition to adipocytes including immune cells, fibroblasts, endothelial cells and skeletal muscle. IL-6 is increased in obesity and insulin resistance and those with elevated levels are at higher risk for type 2 diabetes and myocardial infarction. Similar to TNF-a, IL-6 increases NEFA release and reduces adiponectin secretion. IL-6 increases insulin resistance by inhibiting insulin receptor signal transduction in liver cells. It also increases other inflammatory cytokines, interleukin-1 (IL-1) and TNF-a, and stimulates the liver to produce C-reactive protein (CRP), an important protein marker of inflammation.
Adiponectin is very different from TNF-a, IL-6, and PAI-1. It is synthesized and secreted almost exclusively by the adipocytes and is an anti-inflammatory cytokine. Levels of adiponectin are decreased in weight gain, obesity and in those who are insulin resistant. Secretions of TNF-a and IL-6 reduce adipocyte secretion of adiponectin. Adiponectin is a protective adipokine. It inhibits several steps in the inflammatory process and increases insulin sensitivity by enhancing glucose transport into muscle cells. Adiponectin also decreases liver glucose production. Adiponectin slows and inhibits steps in plaque formation in blood vessels and is thus antiatherogenic.
Insulin is a pancreatic hormone that plays a vital role in carbohydrate and lipid metabolism. Insulin regulates glucose concentrations by: Promoting glycolysis - the uptake of glucose by cells for energy Stimulating glycogenesis - the conversion of excess blood glucose to glycogen storage in the liver Inhibiting glycogenolysis - the conversion of glycogen back to glucose Inhibiting gluconeogenesis - the formation of glucose from noncarbohydrates Insulin increases lipid synthesis in the liver and fat cells and inhibits lipolysis, the release of non-esterified fatty acids (NEFAs) from triglycerides in fat and muscle cells. Insulin also promotes protein synthesis.If insulin resistance occurs, carbohydrate and lipid metabolism are impaired. Insulin resistance ordinarily results in increased insulin levels as the body senses a need for more insulin action. The impaired insulin action results in elevated plasma glucose levels. The increase in lipolysis increases blood concentrations of NEFAs and causes abnormal blood lipid levels.
|The most common eye complication in patients with candidiasis is:||View Page|
|Which of the following identifies a major mechanism of electricity-induced injury?||View Page|
The major mechanisms of electricity-induced injury include: Electrical energy causing direct tissue damage Conversion of electrical energy into thermal energy causing massive tissue destruction Direct trauma resulting from falls or violent muscle spasms
|Which of the parasites listed here is/are transmitted via ingestion of contaminated pork?||View Page|
|This suspicious form was recovered in muscle tissue.||View Page|
|This parasite, found in striated muscle, is responsible for which of the following conditions?||View Page|
|Stool is the specimen of choice for the recovery of which of the following parasites?||View Page|
|Which of the following parasites lay live larvae?||View Page|
|Match each parasite listed here with its corresponding optimal specimen type from which it may be recovered:||View Page|
|A 29 year old male steak house owner from Arizona presented to his doctor complaining of weight loss, abdominal pain and diarrhea. Patient history revealed that the man eats all of his meals at his restaurant and his favorite meat is rare sirloin steak. The man also noted that he had recently been on anti-parasitic medication. The doctor ordered a stool for parasitic examination. These two suspicious forms were seen. The patient is most likely suffering from an infection with:||View Page|
|Which of the following parasites may be recovered in the peripheral blood?||View Page|
|In reference to semen analysis microscopic examination, "round cells" could refer to which of the following types of cells?||View Page|
|Signs and Symptoms of the H1N1 Virus|
Symptoms of the Influenza A 2009 H1N1 virus are similar to what is currently recognized as typical flu-related symptoms. The most common symptoms for the H1N1 virus include: fever (greater than 100.0° F or 37.8 ° C) sore throat fatigue cough runny / stuffy nose chills diarrhea nausea / vomiting headaches body aches (muscle & joint pain) More severe symptoms may be present.
Differentiation of the eosin staining occurs in the subsequent water wash and continues as the tissue goes through the dehydration alcohols. The desired result should yield the following three shades of pink: Orange-pink: Red blood cells Light pink: Connective tissue Dark pink: Muscle fibersThe cytoplasm of the cells will contain varying shades of pink.
|Other than the cytoplasm of cells, what other tissue constituents are stained with eosin? (Choose all that apply.)||View Page|
Uterine tissue is displayed in the image. The red blood cells (RBCs) are the orange-pink clusters.The connective tissue, which is interspersed throughout the tissue, is staining light pink. The tissue that is staining darker pink is smooth muscle. The purple staining is the nuclei. Note the lack of uniformity of these cells, especially in the right portion of the picture. This indicates a neoplasm of some kind.
|Three Broad Categories of Biological Stains|
There are three broad categories of biological stains: General or Routine Stains: Used to differentiate between the nucleus and the cytoplasm. This allows for the different tissue types to be distinguished from one another. These stains use one, two, or sometimes three dyes. The H&E stain falls into this category. Special Stains: These are used to demonstrate specific elements within the tissue, such as connective tissue, muscle, carbohydrates, lipids, pigments, and nerve tissue. Heavy Metal Impregnation: Involves the deposit of metallic substances onto targeted cells. This is often used to demonstrate nerve cells and certain microorganisms.
|Which category of stain would you employ if asked to demonstrate muscle in a tissue sample ?||View Page|
A unit of Red Blood Cells contained 250 mg of iron as part of the hemoglobin molecule. A long-term complication of red cell transfusion is iron overload, or hemosiderosis. As red cells are destroyed, they release iron. The iron cannot be excreted and is stored as hemosiderin and ferritin. Iron accumulates in the liver, heart, spleen, and endocrine organs. Tissue damage, heart failure, liver failure, diabetes, and hypothyroidism can occur. Patients who are transfused frequently are at the greatest risk for iron overload. Diseases such as sickle cell disease, thalessemia, aplastic anemia, and other chronic anemias usually require frequent transfusions. Signs and symptoms of hemosiderosis include muscle weakness, fatigue, weight loss, mild jaundice, anemia, and cardiac arrhythmia. Ferritin levels and other iron studies should be assessed. Specific stains may be used to detect iron in tissue biopsies. Iron chelation may be used to treat and prevent iron overload. Chelation works by using an agent that binds to iron and helps remove it through the urine or feces.