| Monitoring Lipids Most often a lipid panel measures concentrations of total cholesterol, HDL-C, LDL-C, and triglycerides. HDL-C is measurement of the cholesterol in the lipoprotein HDL, and LDL-C, the measurement of cholesterol in LDL.Cardiovascular disease (CVD) is associated with elevations in LDL-C; increased LDL-C in individuals puts them at risk for CVD and is sometimes considered a pre-AMI condition. The opposite is true for HDL-C. One of the functions of this lipoprotein is to remove excess cholesterol, transporting it to the liver for reprocessing or excretion. To prevent cardiac disease, HDL-C levels should remain up and if below recommended range, steps are prescribed to raise the HDL-C concentration.Recommended ranges for lipids from the 2001 National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATP III). These were developed in the US as recommended levels that decrease risk for CHD in adults: Cholesterol <200 mg/dL Triglyceride <150 mg/dL LDL-C <100 mg/dL HDL-C >59 mg/dL | View Page |
| Optimal Cardiac Biomarkers An ideal marker for cardiac disease should have these qualitites: Should be specific to myocardial tissue Be in low concentrations in normal peripheral blood Be rapidly released after myocardial injury Should be detected in low quantities with little interference from like compounds Should remain in circulation for a sufficient length of time for detection The plasma concentration of the marker should be directly related to the extent of injury. The test for the biomarker should be easily automated and relatively inexpensive to run, and results should be obtained rapidly. | View Page |
| History In the past, an AMI was primarily diagnosed by evaluating symptoms at patient presentation, ECG measurement, and results of enzyme assays that were considered cardiac enzymes. The enzymes, creatine kinase (CK), lactate dehydrogenase (LD), and aspartate aminotransferase (AST) were assayed several times a day often for several days to observe peak concentration and return to normal level for each enzyme. The first assay result was the baseline level or baseline concentration. Isoenzymes of CK and LD were later added for AMI diagnosis. All three of these enzymes are found in other tissues, making the diagnosis difficult and lengthy. In the 1980s, CK isoenzyme, CK-MB, though not totally cardiac specific, became the benchmark marker for an AMI. None of these enzymes are in any of the current recommendations except for CK-MBCurrent diagnosis, monitoring, and screening relating to heart disease includes measurement of lipids, proteins, enzymes, and other biomolecules. Risk stratification for cardiac and vascular disease is an additional role for measurement of these analytes. The physiological changes in the development of heart disease are better understood and the role of the clinical laboratory is greatly expanded.Today's markers are significant because of their location in the myocyte, the kinetics of their release in myocyte damage, and their rate of clearance from peripheral blood. | View Page |
| cTnT cTnT is also encoded by a separate gene and after translation, an 11-amino acid chain is added giving this protein its cardiac specificity. Its release characteristics and elevation periods are similar to cTnI. In some AMI patients, a second increase in analyte concentration occurs. It is believed that there are two pools of cTnT, cystolic and structurally bound pools. The first elevation is from the cystolic pool and the second elevation is from the structurally bound pool released later in the necrotic process. | View Page |
| Use of Troponins Both cTnI and cTnT are cardiac specific, rapidly released after injury, remain in circulation for several days, normally in low concentration in serum or plasma, and can be rapidly assayed at relatively low cost.Currently cTnI and cTnT are considered the best markers in diagnosing ACS. Either protein is assayed to detect an AMI or other myocardial injury. These markers are especially helpful when the patient with chest pain and symptoms of an AMI does not have a diagnostic ECG. Cardiac troponin levels are used in risk stratification for a patient with chest pain that is not diagnosed with an AMI at presentation. Elevations of cardiac troponins are especially significant when other markers are normal. These elevations predict higher risk of severe cardiac events in the coming month. In other patients with ACS, troponin elevations identify those who are at risk for cardiac events for up to six months. | View Page |
| 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 | View Page |
| CK-MB Before troponins were used in cardiac disease diagnosis, CK-MB, an isoenzyme of creatine kinase (CK), was the marker of choice for AMI diagnosis. CK-MB is released in circulation 4-6 hours after symptoms of an AMI and usually peaks within 24 hours. Levels of CK-MB are back to normal range in 48-72 hours. The latter is different from the cardiac troponin pattern. Use of CK-MB in diagnosis of an AMI varies. Some institutions have discontinued assaying CK-MB in suspected AMIs; others use CK-MB measurements in conjunction with cTnI or cTnT. Because CK-MB returns to normal much faster than cardiac troponins, CK-MB measurements can be used when a reinfarction is suspected. In reinfarction, CK-MB concentration rises again after the return to baseline levels. Currently, CK-MB results do not predict future adverse cardiac events and do not have any prognostic or risk stratification use. | View Page |
| Which one of the following cardiac biomarkers could detect a reinfarction? | View Page |
| High Sensitivity C-Reactive Protein (hs-CRP) C-reactive protein (CRP) is an acute-phase protein produced by the liver in response to injury or tissue damage. It has been assayed for many years as a non-specific marker of acute inflammatory diseases, infections, neoplastic diseases, and other conditions where inflammation occurs. It is still assayed in this manner as a marker of inflammation by immunoassay methods that are sensitive to concentrations of 5-20 mg/L. Atherosclerosis is a subclinical chronic inflammatory condition. Highly sensitive measurements of CRP have been developed to detect this protein in lower levels that are sensitive to 0.5-10.0 mg/L. This assay is referred to as high sensitivity C-reactive protein (hs-CRP). | View Page |
| Myeloperoxidase Myeloperoxidase (MPO) is an enzyme released by leukocytes and some macrophages and elevated levels indicate an ongoing inflammatory process. MPO is also involved in the degradation of the plaque matrix in atherosclerosis. Increased serum concentrations of MPO would indicate both an inflammatory process and plaque instability. An immunoassay for MPO has been approved for use in high risk patients with ACS. More study is required to learn if the MPO levels provide additional information than troponin levels in risk stratification. | View Page |
| Comparisons of Concentrations of CRP and hs-CRP Laboratory Test Test Result Interpretation CRP 10.2 mg/L Acute inflammation hs-CRP 0.5 mg/L Low risk for cardiac disease hs-CRP 1.5 mg/L Average risk for cardiac disease hs-CRP 3.5 mg/L High risk for cardiac disease | View Page |
| Comparisons of Concentrations of CRP and hs-CRP The following page illustrates the difference in concentrations for the traditional acute inflammation marker, CRP, and the CVD inflammation risk marker, hs-CRP. Values of CRP in an acute inflammation and hs-CRP in the three ranges of risk for CVD were selected and inserted into a table and graph to depict these concentration differences. | View Page |
| Urine pH: Acidic and Alkaline Urine pH results must be evaluated in conjunction with a patient's medical condition and clinical history. Factors to be considered include:Respiratory and metabolic statusRenal functionCrystal or calculi formationDietThe table below summarizes dietary and medical conditions as well as preanalytic and analytic errors that may affect urine pH:ConditionAcid pHAlkaline pHHigh meat dietXVegetarian dietXRespiratory/metabolic acidosisXRespiratory/metabolic alkalosisXHypochloridemiaXHigh concentration of urine glucoseXBacterial infection caused by urease-producing bacteriaXProlonged storage of specimen at room temperature, allowing multiplication of urease-producing bacteriaX (above 8.0)Improper procedural technique; excess urine left on reagent strip, allowing acid buffer in protein pad to run over into adjacent pH pad (refers to some reagent strip configurations)XKidney failureXUrinary tract infectionsXVomitingXDiabetic ketoacidosis XDiarrheaXStarvationX | View Page |
| Protein Error of Indicators Testing for protein in the urine is based on the phenomenon called the "Protein Error of Indicators" (ability of protein to alter the color of some acid-base indicators without altering the pH). In a solution void of protein, tetrabromphenol blue, buffered at a pH of 3, is yellow. However, in the presence of protein, particularly albumin, the color changes to green, then blue, depending upon the concentration. This method is more sensitive to albumin than to globulin, detecting as little as 5 mg albumin/dL urine. Bence Jones protein and mucoprotein are examples of globulin components that are sometimes present in urine, but are not distinguishable by the chemical reagent strip method for urine protein. False positive results can occur when testing for urine protein. A urine specimen that has remained at room temperature for an extended period of time may produce a false-positive protein result on a reagent strip. A false positive may also occur in the presence of bacterial contamination, alkaline medication, quaternary ammonium compounds such as disinfectants or drugs, and with skin cleansers containing chlorhexidine. | View Page |
| Clinical Significance of Glucose in the Urine In a healthy individual, almost all of the glucose filtered by the renal glomerulus is reabsorbed in the proximal convoluted tubule. The amount of glucose reabsorbed by the proximal tubule is determined by the body's need to maintain a sufficient level of glucose in the blood. If the concentration of blood glucose becomes too high (160-180 mg/dL), the tubules no longer reabsorb glucose, allowing it to pass through into the urine. It is important to note that glucose may appear in the urine of healthy individuals after consuming a meal that is high in glucose. Fasting prior to providing a sample for screening eliminates this problem. Conditions in which glucose levels in the urine are above 100 mg/dL and detectable include: diabetes mellitus and other endocrine disordersimpaired tubular reabsorption due to advanced kidney diseasepregnancy - glycosuria developing in the 3rd trimester may be due to latent diabetes mellituscentral nervous system damagepancreatic diseasedisturbances of metabolism such as, burns, infection or fractures | View Page |
| The ketone component that is measured by the nitroprusside reaction is: | 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. | View Page |
| Measuring Specific Gravity The chemical reagent strip measures specific gravity (SG) in increments of 0.005 with readings from 1.000 to 1.035. The test principle is based on a change in pKa (the negative log of the acid disassociation) of certain pretreated electrolytes (methylvinyl ether/maleic anhydride) in relation to ionic concentration of the urine. These electrolytes in the reagent area contain acid groups which disassociate according to the ionic concentration of the specimen. The more ions in the specimen, the more acid groups will become disassociated, releasing hydrogen ions and causing a more acid pH. The reagent area contains a pH indicator (bromthymol blue) which demonstrates the change in pH. The higher the specific gravity of the urine specimen, the more acidic the reagent area will become. The colors of the reagent area will range from deep blue-green in urines of low ionic concentration to green-to-yellow green in urines of increasing ionic concentration, and consequently, higher specific gravity. | View Page |
| How does ion concentration in the urine relate to specific gravity? | View Page |
| Semi-Automated Instruments Several manufacturers offer semi-automated instruments (chemical reagent strip, a.k.a. dipstick readers) for reading reagent strips. Use of an instrument removes the subjectivity of visually interpreting color changes on reagent strips, and assures that tests will be read at the correct time. Transcription errors can also be avoided if the instrument is interfaced with the laboratory information system as well. The technology employed is based on the principle of reflectance, with the amount of light reflected being inversely related to the concentration of substances present. An example of reflectance is the light which is scattered after light strikes an unpolished surface. Since each component on the dipstick produces a different color reaction, the light source for each test must be at the appropriate wavelength. This is accomplished either by using filters or monochromatic light sources. The percent reflectance is determined by dividing the test reflectance by the calibration reflectance and multiplying by 100. Algorithms are used to change the results obtained into a linear relationship with concentration of analyte. | View Page |
| Which of the following statements are TRUE for specific gravity measured by the reagent strip method? (Select ALL that apply) | View Page |
| Acid and Alkaline Urine pH Urine pH results must be evaluated in conjunction with a patient's medical condition and clinical history. Factors to be considered include:Respiratory and metabolic statusRenal function Crystal or calculi formationDietThe table below summarizes dietary, medical, and artifactual conditions that may affect urine pH:ConditionAcid pHAlkaline pHHigh meat dietXVegetarian dietXRespiratory/metabolic acidosisXRespiratory/metabolic alkalosisXHypochloridemiaXHigh concentration of urine glucoseXBacterial infection caused by urease-producing bacteriaXProlonged storage of specimen at room temperature, allowing multiplication of urease-producing bacteriaX (above 8.0)Improper procedural technique; excess urine left on reagent strip, allowing acid buffer in protein pad to run over into adjacent pH pad (refers to some reagent strip configurations)X | View Page |
| Protein Error of Indicators Testing for protein is based on the phenomenon called the "Protein Error of Indicators" (ability of protein to alter the color of some acid-base indicators without altering the pH). In a solution void of protein, tetrabromphenol blue, buffered at a pH of 3, is yellow. However, in the presence of protein (albumin), the color changes to green, then blue, depending upon the concentration. This method is more sensitive to albumin than to globulin, detecting as little as 5 mg albumin/dL urine. Bence Jones protein and mucoprotein are examples of globulin components that are sometimes present in urine, but are not distinguishable by the dipstick method for protein. | View Page |
| Clinical Significance In the healthy individual, almost all of the glucose filtered by the renal glomerulus is reabsorbed in the proximal convoluted tubule. The amount of glucose reabsorbed by the proximal tubule is determined by the body's need to maintain a sufficient level of glucose in the blood. If the concentration of blood glucose becomes too high (160-180 mg/dL), the tubules no longer reabsorb glucose, allowing it to pass through into the urine. It is important to note that glucose may appear in the urine of healthy individuals after consuming a meal that is high in glucose. Fasting prior to providing a sample for screening eliminates this problem. | View Page |
| False Negative Results False negative results may occur with some 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. | View Page |
| Test Sensitivity This test is sensitive to 0.06-0.1 mg/dL nitrite ion in urines with a low specific gravity and ascorbic acid concentrations of less than 25 mg/dL. Pink spots or pink edges should not be interpreted as a positive result because some medications can color urine red or turn red in an acid environment. Any degree of uniform pink color should be considered positive, suggesting the presence of 105 organisms/mL. Detection of low levels of nitrite ion may be enhanced by comparing the activated test strip to a white background. It is important to note that color development is NOT proportional to the number of bacteria present. The test is specific for nitrites and does not react with any other substances normally present in urine. Negative results do not necessarily rule out a urinary tract infection because yeasts or gram-positive bacteria unable to reduce nitrites may be the causative agent. | View Page |
| False Positive Urobilinogen Results A false positive urobilinogen reaction may occur with the dipstick method when substances known to react with Ehrlich's reagent such as sulfonamides and p-aminosalicylic acid are present in the urine. Drugs that contain Azo dyes, such as Azo Gantrisin®, have a gold color that masks the reaction, causing a false positive reaction. Atypical color reactions may be obtained in the presence of high concentrations of p-aminobenzoic acid. The dipstick urobilinogen test cannot detect porphobilinogen in a urine specimen. Porphobilinogen is a molecule formed during the synthesis of the heme portion of hemoglobin. | View Page |
| False Negative False negative results may occur in the presence of significant levels of protein or glucose and in urines with high specific gravity which may crenate the white blood cells causing them to be come unable to release esterases. Some drugs such as Cephalexin (Kelfex®), Cephalothin Keflin®) or high concentrations of oxalic acid may also cause decreased test results. Tetracycline may cause decreased activity, and high levels of the drug may cause a false negative reaction. Large amounts of ascorbate may cause false negative results. | View Page |
| Measuring Specific Gravity The reagent strip measures specific gravity (SG) in increments of 0.005 with readings from 1.000 to 1.035. The test principle is based on a change in pKa (the negative log of the acid disassociation) of certain pretreated electrolytes (methylvinyl ether/maleic anhydride) in relation to ionic concentration of the urine. These electrolytes in the reagent area contain acid groups which disassociate according to the ionic concentration of the specimen. The more ions in the specimen, the more acid groups will become disassociated, releasing hydrogen ions and causing a more acid pH. The reagent area contains a pH indicator (bromthymol blue) which demonstrates the change in pH. The higher the SG of the urine specimen, the more acidic the reagent area will become. The colors of the reagent area will range from deep blue-green in urines of low ionic concentration to green-to-yellow green in urines of increasing ionic concentration, and consequently, higher SG. | View Page |
| How does ion concentration in the urine relate to specific gravity (SG)? | View Page |
| Semi-Automated Instruments Several manufacturers offer semi-automated instruments (dipstick readers) for reading reagent strips. Use of an instrument removes the subjectivity of visually interpreting color changes on reagent strips, and assures that tests will be read at the correct time. Transcription errors will also be avoided if the instrument is interfaced with the laboratory information system. The technology employed is based on the principle of reflectance, with the amount of light reflected being inversely related to the concentration of substances present. An example of reflectance is the light which is scattered after light strikes an unpolished surface. Since each component on the dipstick produces a different color reaction, the light source for each test must be at the appropriate wavelength. This is accomplished either by using filters or monochromatic light sources. The percent reflectance is determined by dividing the test reflectance by the calibration reflectance and multiplying by 100. Algorithms are used to change the results obtained into a linear relationship with concentration of analyte. | View Page |
| Discrete and Continuous Data There are two main types of data that you might encounter. The first is discrete data, which is a count of whole events, objects or persons. For example, the number of people with a certain illness is a discrete quantity, ie, countable.The other type of data is continuous data, which is the measure of a quantity such as length, volume, or time, which can occur at any value. For example, the concentration of glucose in the blood is a continuous quantity. Even if the instrument you are using rounds off values to whole numbers, these quantities are still continuous; ie, not countable. | View Page |
| Independent and Dependent Variables In statistics, a variable is any quantity that is a part of a data point. Variables can either be dependent or independent. An independent variable is a quantity that is directly controlled by the observer or experimenter. The dependent variable, as its name suggests, depends on the independent variable. The dependent variable is often the quantity you want to measure; it the result of the experiment or test.For example, you may want to determine the relationship between hemoglobin concentration and age. You select people of various ages, and then test their hemoglobin concentrations. Age is the independent variable, and is controlled by the experimenter (you can select which ages are in the experiment). The dependent variable is the resulting hemoglobin concentration.In some cases, these criteria may not be useful in determining which variable should be the independent variable, such as determining the correlation between the readings given by two different instruments for the same samples. In that case, there might be other criteria for selecting the independent variable. | View Page |
| Using Frequency Distributions A frequency distribution is a chart that groups data into different classes and then graphically shows how many data points fall into each class. A frequency distribution allows the reader to see easily the approximate center and spread of the data. Table II shows the frequencies of different hemoglobin concentrations. Figure 2 is a histogram of the data. Table II Frequency distribution of blood hemoglobin levels from healthy women determined on the Coulter Gen S Hemoglobin (gm/dL)Number of Women6 - 818 - 10210 - 121012 - 142514 - 16916 - 181Figure 2 Frequency Distribution Blood Hemoglobin Levels from 48 Healthy Women Determined on the Coulter Gen S | View Page |
| Standard Deviation Example Now we will do an example calculation of the standard deviation of a set of data. Here are the data we will use:Table VII Urea Nitrogen Concentration in Five EmployeesConcentration (mg/dL)97111310 | View Page |
| Standard Deviation Example (continued) The first step in calculating the standard deviation is to calculate the mean, . In this case, = 10.Now, subtract that mean from each of the data values, and then square those results: Table VIIUrea Nitrogen Concentration in 5 Employees (mg/dL) Concentration (mg/dL)x-(x-)29-117-39111113391000Total20Use this total to calculate the standard deviation:The standard deviation is about 2.23. | View Page |
| Using the standard deviation formula,what is the standard deviation of the following data? You may find it helpful to duplicate this chart to perform your calculations: Table VII Urea Nitrogen Concentration in 9 Employees (mg/dL) Concentration (mg/dL)x-(x-)210111113951579Total | View Page |
| Introduction Diabetes is a metabolic disorder caused by impaired pancreatic function, resulting in decreased insulin concentration and activity. This causes the patient with diabetes to have elevated blood glucose concentrations (hyperglycemia). Hyperglycemia leads to serious risk factors and life-threatening complications for the individual. Because of these risks and the ensuing chronic illness for diabetic patients, ongoing medical care and education for self-management are required. Diabetes is a national and international healthcare issue due to its high incidence and healthcare costs. According to the World Health Organization (WHO) in 2000, there were 171 million individuals worldwide with diabetes. That number is projected to increase to 366 million by 2030. | View Page |
| Blood Glucose and Hormonal Control Several hormones regulate blood glucose concentration. Insulin, the main regulatory hormone, is produced by and secreted from the pancreatic beta-cells. Insulin stimulates the uptake of glucose and the movement of glucose from blood to cells for energy production. Insulin also stimulates glycogenesis, inhibits glycogenolysis, and regulates protein synthesis.Other hormones that are also involved in carbohydrate metabolism include: Pancreatic glucagon- stimulates glycogenolysis and gluconeogenesis Adrenal gland cortisol- promotes gluconeogenesis Epinephrine- a neurotransmitter that increases glycogenolysis | View Page |
| Which of the following hormones increase plasma glucose concentration by converting glycogen to glucose? | View Page |
| Which of the following hormones is mainly responsible for the entry of glucose into the cell for energy production? | View Page |
| Other Causes of Diabetes Diabetes may be caused by conditions other than absolute insulin deficiency, decreased insulin, or impaired insulin activity. Because of the other hormones that regulate glucose concentration, increased glucagon, cortisol and epinephrine will cause hyperglycemia. Genetic defects in beta-cell function and insulin action, and other diseases of the pancreas result in diabetes. Also some medications, drugs, or chemicals can induce hyperglycemia. | View Page |
| Other Complications Ketoacidosis is always a serious complication for type 1 diabetics. Due to lack of uptake of glucose into cells by insulin, proteins and fats are utilized as energy sources. This results in excess acetyl CoA which is converted to ketone bodies. A serious acidosis results and if untreated or not resolved by the body, coma and death can occur.Most often the acetyl CoA in a type 2 patient is converted to cholesterol and results in hyperlipidemia and heart disease in these patients.The elderly type 2 diabetic is at risk for a hyperosmolar nonketotic coma. The patient becomes dehydrated due to increased urine excretion to lower the blood glucose. If reduced renal or cardiac function is also present, glucose excretion is impaired and blood glucose concentrations can become extremely high. Ketones are not produced in excess, thus the patient remains nonketotic. Insufficient hydration, elevated blood glucose, and decreased renal excretion of waste products result in an increased osmolality and total concentration of all plasma components. | View Page |
| Whole Blood Glucose Testing In the past twenty years there have been significant improvements in the accuracy of handheld glucose meters. Patient use has resulted in substantial improvements in diabetic control and insulin therapy. Capillary whole blood is easily obtained and glucose concentration is derived on simple to use, portable meters. Since whole blood glucose is lower than plasma glucose, the meters are programmed to correct the value before presenting the result; therefore, the whole blood glucose meter result correlates to serum or plasma results.Clinical and Laboratory Standards Institute (CLSI) has set standards for correlation between glucose meter and laboratory measured glucose levels. If the laboratory measured glucose is > 75 mg/dL, the glucose meter result should be within 20%. For laboratory measured values < 75 mg/dL, the glucose meter result should be within 15 mg/dL. | View Page |
| Urine Glucose Before glucose meters were available, urine glucose was frequently used to approximate diabetic glucose levels. Blood glucose levels can be related to urine glucose concentration because of urinary excretion of glucose. Physician offices, clinics, and patients at home tested urine with reagent strips for a semi-quantitative measurement of urine glucose and adjustments in insulin therapy were made. Monitoring a diabetic carbohydrate management is seldom performed this way today. Portable meter measurement of blood glucose is a much better management method. Urine glucose measurement is neither sensitive nor specific and does not give information about blood glucose below the renal threshold (usually 180 mg/dL).As a semiquantitative measurement, urine glucose is a routine assay on urinalysis test and an abnormal result would be investigated with blood levels. If quantitative measurements are needed, a timed urine specimen is collected and measured for glucose by blood glucose methods. | View Page |
| HbA1C Hemoglobin A comprises the majority of normal adult hemoglobin (Hb) and includes the minor hemoglobins, Hb A1a, Hb A1b, and Hb A1c. Sometimes these three are referred to as Hb A1 but A1C is the major fraction and composes 80% of Hb A1. Following synthesis of Hb A, a nonenzymatic reaction adds glucose to the N-terminal valine on either beta chain forming glycated Hb. The pre-A1C molecule is a labile Schiff base and this reaction is reversible. As the red blood cells circulate, an irreversible Amadori rearrangement of the pre-A1C base occurs forming a stable ketoamine, A1C. Over the life span of the red blood cells (120 days) this process continues and the concentration of A1C is proportional to the concentration of the blood glucose. The concentration of A1C then relates to an individual's average glucose over time and can be used as an index relating to the extent of carbohydrate control during a 2 - 3 month period. There is also a direct relationship between the concentration of HbA1C and risk of complications in diabetic patients. Therefore, the ADA has recommended using HbA1C measurements to monitor glycemic control. | View Page |
| Fructosamine Fructosamines are formed by the nonenzymatic addition of glucose to proteins other than hemoglobin. These reactions form ketoamines; glycated albumin is the primary fructosamine. Fructosamine is not ordinarily measured clinically. However, when abnormal hemoglobins or rapid hemoglobin turnover is present, the glycated hemoglobin assay may not be accurate and a fructosamine assay is recommended for evaluation of long-term diabetic control. The half-life for albumin is approximately twenty days. The concentration of glycated albumin is an index of glucose control over a period of 2 to 3 weeks. When earlier detection of carbohydrate management changes is needed, fructosamine would detect changes earlier than HbA1C. | View Page |
| Estimated Average Glucose Estimated average glucose (eAG) is a glucose concentration level calculated from a patient's HbA1C result. In 2008, the ADA recommended the use of this new term and that this calculation be performed and reported routinely with the measured A1C result. The formula for conversion of HbA1C to glucose in mg/dL is eAG = 28.7 x A1C – 46.7. A web calculator is located at: http://professional.diabetes.org/glucosecalculator.aspx. Accessed January 11, 2010. | View Page |
| Specimens Serum and plasma are the most common clinical specimens used for electrophoresis applications. Urine and cerebrospinal fluids (CSF) are also suitable. Other body fluids such as pleural fluid and pericardial fluid are analyzed less frequently. Some specimens require pretreatment before electrophoresis. Low concentrations of proteins normally in urine and CSF are concentrated in order to have enough proteins for detectable separations. Some body fluids require removal of pigments, salts, and other compounds that interfere with electrophoresis or the detection of separated solutes. In molecular diagnostic testing of DNA and RNA, the nucleic acids must first be isolated from the specimen and then purified before separation with electrophoresis. | View Page |
| After reviewing the information on specimen samples for electrophoresis, select the one correct statement. | View Page |
| Polyacrylamide Gels 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. | View Page |
| Electroimmunoassay Electrophoresis In electroimmunoassay electrophoresis, the antiserum is mixed in the gel during preparation. In the electrophoresis of the serum sample, the voltage drives the sample antigen into the antiserum creating a precipitin line in the shape of a rocket. This line is proportional to the concentration of the antigen, which is the protein to be detected. Each gel contains several serum samples, one antibody suspended in the gel, and standards of known concentration of antigen. Quantitation of the unknown antigen is derived from the height of the sample rockets compared to the height of the standard rockets. Electroimmunoassay electrophoresis is often referred to as rocket electrophoresis. | 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. Recall the order of protein fractions from left to right is: Albumin, alpha 1, alpha 2, beta, and gamma. | View Page |
| Which statements below are correct descriptions of visualization and detection methods used in electrophoresis? | View Page |
| Introduction We are all aware of the clinical laboratory's role in assessing overall health and we are also aware that measuring a patient's serum lipids will provide some insight into their cardiovascular health. The traditional measurements of low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides are the 'classic' cardiovascular risk markers.Laboratorians, and even the general public are now well-aware that LDL-C ('bad' cholesterol) concentrations should be low while HDL-C ('good' cholesterol) concentrations should be high. Triglycerides should be kept in check as well. Optimal levels are shown in the table below. So what is the risk if these values are not within optimal ranges?Cardiovascular risk can be simply defined as increasing the odds of having a pathology which affects blood flow and/or the heart. The most common cardiovascular pathology is atherosclerosis. Other cardiovascular pathologies whose odds increase as serum lipids and other cardiovascular markers become suboptimal are myocardial infarction (heart attack), stroke, congestive heart disease and coronary artery disease. Other diseases such as diabetes and the metabolic syndrome are also strongly associated with the classic cardiovascular risk markers LDL-C, HDL-C and triglycerides. | View Page |
| Risk Markers We have listed the 'classic' cardiovascular risk markers as LDL-C, HDL-C and triglycerides. But there are many more cardiovascular risk markers as well as cardiovascular risk factors. A cardiovascular risk factor is a condition (not a laboratory analyte) that is associated with an increased risk of developing cardiovascular disease. Examples include: Age Gender (males are at increased risk) Heredity Hypertension Cigarette Smoking Obesity Diabetes StressThere are also negative risk factors, factors which decrease a person's risk of cardiovascular disease. Examples include: Optimal HDL-C concentration Exercise Estrogen Moderate alcohol intakeThis course will not focus on cardiovascular risk factors. Instead we will focus on newer, emerging cardiovascular risk markers. There are well over twenty well-studied cardiovascular risk markers; in this course we will focus on some of the more established markers and the ones which are becoming more commonly measured in the clinical laboratory. These include apolipoprotein A1/apolipoprotein B100, Lp(a), oxidized LDL, LpPLA2, hsCRP and lipoprotein particle size and concentration.It is important to remember that the association between a cardiovascular risk marker and actually having or developing cardiovascular disease is a statistical one. The fact that a patient has a particular risk marker which is abnormal simply increases the probability of developing cardiovascular disease, it does not mean that he or she is certain to develop cardiovascular disease. Conversely, if an individual does not have a particular cardiovascular risk marker present it does not guarantee protection against cardiovascular disease. We must always remember that some percentage of individuals who have heart attacks or strokes will not have abnormal risk markers present. | View Page |
| Patient Studies to Validate Risk Markers Risk markers are first hypothesized and then tested. Once a potential marker is identified, concentrations of the serum marker are correlated with patient outcomes. Cardiovascular risk marker studies are typically either retrospective or prospective epidemiology studies. A retrospective study looks backwards at a patient population. For example, we identify (through a hospital database perhaps) patients who have had myocardial infarcts or some other adverse outcome as well as similar subjects without that outcome to use as controls. We then go back and find archived patient serum samples and relate the concentrations of our new risk marker with patient outcomes. Retrospective studies can only be performed if you have archived samples from the patient. Prospective studies look forward in time. For example, we first select a group of subjects and measure our new risk marker in these patients over time. After a few years, we see how the serum concentrations relate to the patient outcomes. Obviously, prospective studies take much longer to perform than retrospective studies. Whatever study model is used, when assessing the value of a cardiovascular risk marker, we must correlate serum concentrations with a specific outcome. The outcome is determined by the study authors. Outcomes could be things like myocardial infarction, stroke, a diagnosis of coronary artery disease, death, or any cardiovascular 'event.'Concentrations of risk markers are divided into tertiles, quatriles or quintiles. This simply means that the top 33%, top 25% or top 20% of the serum concentration values are compared to the bottom 33%, 25% or 20%. For example, risk marker studies will often compare the outcomes of patients with serum concentrations in the upper tertile (those in the top third) with those in the bottom tertile (those in the bottom third) to see if the top 33% had significantly worse outcomes; if so, the risk marker has clinical value. | View Page |
| Which of the following is NOT a cardiovascular risk factor? | View Page |
| ApoB and ApoA1 By measuring ApoB we can quantify the amount of all atherogenic or potentially atherogenic lipoproteins that carry this apolipoprotein. Although lipoprotein particles other than LDL can carry ApoB, LDL accounts for the vast majority of ApoB; therefore, it is a good index of LDL particle number. Furthermore, the other particles that can have ApoB (such as IDL and Lp(a)) are also atherogenic and so it is not problematic if they are counted along with LDL, since they also contribute to cardiovascular risk. What about ApoA1? HDL-C is known as 'good cholesterol'. The role for HDL in the body is to sequester excess cholesterol and bring it back to the liver. Since HDL can remove cholesterol and transport it back to the liver for excretion or re-utilization it is indeed good. HDL is a negative cardiovascular risk factor; as its concentration goes up, a person's cardiovascular risk decreases. A person with low cardiovascular risk would have low ApoB levels and high ApoA1 levels. If we measure both ApoB and ApoA1 and express them as a ratio of ApoB/ApoA1 we get a powerful cardiovascular risk marker. The ratio should be approximately 0.3-0.9. Patients with a higher ratio have elevated ApoB (LDL) and/or low ApoA1 (HDL) and are thus at increased risk. By combining these two markers in a ratio, we get synergy and enhanced predictive power. | View Page |
| ApoB/ApoA1: The Test Measuring ApoB and ApoA1 can be performed using standard immunoassay techniques. Nephelometry is popular, as are ELISA-based methods that are performed on automated chemistry analyzer platforms. The power of the ApoB/ApoA1 ratio as a cardiovascular risk marker is getting widespread attention. An individual with seemingly normal LDL-C may in fact have high ApoB concentrations. When this individual has his or her ApoB/ApoA1 ratio calculated, the risk is evident. Studies have also shown that patients with metabolic syndrome and type-2 diabetes can also easily be identified with the ApoB/ApoA1 ratio, whereas these patients cannot always be identified by measuring LDL-C and HDL-C.In 2004, the global INTERHEART study of risk factors for acute myocardial infarction concluded that the ApoB/ApoA1 ratio was the most important risk factor in all geographic regions. The ApoB/ApoA1 ratio is easy to use because the risk is integrated into a single number that indicates the balance between atherogenic and antiatherogenic particles.There have been many studies concerning the predictive power of the ApoB/ApoA1 ratio. One study, which involved thousands of patients who were followed for an average of 10 years, showed that the ApoB/ApoA1 ratio was a strong predictor of stroke in addition to other cardiovascular events. Due to the evidence presented in studies like these, the National Academy of Clinical Biochemistry (NACB) has recommended that the ApoB/ApoA1 ratio be used as an alternative to the usual total cholesterol (TC)/HDL cholesterol ratio when determining lipoprotein-related risk for cardiovascular disease. Some believe that ApoB/ApoA1 testing will eventually replace traditional LDL-C and HDL-C measurements. | View Page |
| What can be said of a patient who has high ApoB and low ApoA1 concentrations? | View Page |
| Lp(a) Lipoprotein (a) is a modified version of LDL containing a unique protein, apolipoprotein (a). It was discovered in 1963 and is well-associated with vascular disease. Do not confuse apolipoprotein (a) with apolipoprotein A that is found on high density lipoprotein particles. Lipoprotein (a) is abbreviated as Lp(a). Lp(a) is an LDL particle whose ApoB molecule has formed a disulfide bond with another protein called Apo(a), see figure. Apo(a) is a protein very similar in structure to plasminogen. Numerous retrospective case control studies and prospective studies have shown Lp(a) to be an independent risk factor for vascular disease. This means that Lp(a) levels alone (not in conjunction with LDL, or patient risk factors) can predict cardiovascular risk. Lp(a) has been called the most atherogenic lipoprotein. Serum concentrations of Lp(a) are related to genetic factors; drugs and diet changes do not typically lower Lp(a) as they do LDL. | 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 |
| High Sensitivity-C-Reactive Protein C-reactive protein (CRP) is a very sensitive acute phase reactant. Serum CRP levels increase following a variety of pro-inflammatory events such as infection, tissue necrosis, trauma, surgery and even malignancy. CRP levels can increase quickly and dramatically (often 100 fold) during inflammation. CRP can activate compliment, bind Fc receptors and can function as an opsonin, enhancing phagocytosis with certain infections. Measurement of CRP is not new, it has been on clinical laboratory testing menus for decades. However, a newer version of the CRP test is now in use to assess cardiovascular risk.High sensitivity-CRP (hs-CRP) assays have been developed that are more sensitive to the more subtle changes that can occur during chronic vascular inflammation. (Recall that atherosclerosis is an inflammatory process.) By measuring hsCRP we can get a glimpse at vascular function. CRP has been shown to be an independent risk factor for atherosclerotic disease and cardiac death. A 2002 prospective study of more than 27,000 patients showed that the CRP concentration is a stronger predictor of cardiovascular events than the LDL-cholesterol level. | View Page |
| LpPLA2 and Cardiovascular Risk There have been dozens of clinical studies demonstrating LpPLA2's ability to predict cardiovascular risk. A 2008 study showed that people whose LpPLA2 concentrations were in the upper quartile were 1.64 times more likely to have a cardiac event than those in the lowest quartile. A meta-analysis (a study that sums the results of several other studies) performed by researchers at the Mayo Clinic showed that the unadjusted odds ratio for the association between elevated Lp-PLA2 levels and cardiovascular disease risk was 1.51, indicating that patients with elevated LpPLA2 patients had 1.51 times the risk of cardiovascular disease or events. | View Page |
| Regulation of Iron Equilibrium Regulation of iron equilibrium occurs mainly through the process of absorption. Iron is absorbed through the mucosal cells lining the duodenum. A variety of proteins are involved in this process. Hepcidin, an antimicrobial protein primarily produced in the liver, has been recently found to be a major (negative) regulator of dietary iron absorption by disrupting cellular iron transport in the intestine. Decreased levels of hepcidin are related to increased iron absorption into the bloodstream. Hepcidin is increased in response to iron overload and inflammation. (4)Additional proteins involved in iron metabolism include transferrin (Tf), transferrin receptor (TfR), ferroportin, HFE protein, hemojuvelin, and others. Their roles in iron absorption are complex and in some instances incompletely understood.Factors affecting iron absorption include: Tissue stores, e.g., decreased stored iron is associated with a decrease in hepcidin and increase in iron absorption. Rate of hematopoietic activity, e.g., an increased rate of erythropoiesis is associated with a decrease in hepcidin and an increase in iron absorption. Oxygen concentration in tissues, e.g., hypoxia decreases hepcidin and increases iron absorption, thereby promoting increased erythopoiesis. Dietary intake, including form of iron ingested, e.g., heme iron is more readily absorbed than non-heme forms of iron. Condition of GI tract mucosal cells Intraluminal factors, e.g. intestinal motility | View Page |
| Iron Transport Once absorbed through the mucosal cells of the duodenum, iron is bound to a carrier plasma protein, transferrin (Tf), for movement to sites of utilization. Almost all iron in plasma is bound to Tf, and most Tf-bound iron is carried to the bone marrow to be incorporated into developing erythrocytes. Transferrin is normally about 20% to 40% saturated with iron. (5)Transferrin releases iron to specific transferrin receptors (TfRs) for movement into cells. Transferrin receptors are found on all cells, but are found in relatively high concentration in erythroid precursors, hepatocytes, and placental cells. When the capacity of plasma Tf to bind iron is exceeded, i.e., transferrin saturation (TS) is higher than normal, excess iron is taken up by hepatocytes and other cells. A brief summary of iron metabolism is illustrated. | View Page |
| Altered Iron Absorption Hereditary hemochromatosis (HH) is a genetic disorder characterized by iron overload as a result of increased iron absorption. As iron absorption increases, the amount of iron bound to transferrin and transported in the plasma subsequently increases.With no available mechanism for excreting excess absorbed iron, normal iron storage sites become overloaded, resulting in ferritin levels that far exceed normal. As a result, iron is deposited in the parenchymal cells of the liver, pancreas, pituitary, heart, synovium, and other tissues with high concentrations of transferrin receptors. Iron in excess of normal cellular ferritin stores contributes to the generation of free radicals and reactive oxygen intermediates that cause cell damage to organs and tissues. This process results in the clinical condition known as iron overload, a hallmark feature of HH. | View Page |
| HFE and Other Genes A hemochromatosis gene, HFE, was identified in 1996. Mutations in the HFE gene are found in the majority of patients diagnosed with hereditary hemochromatosis (HH). The locus for the gene is on the long arm of chromosome 6 where it codes for a membrane protein, HFE. The exact mechanism of the role of HFE protein in iron metabolism is incompletely understood. It is thought that HFE, along with a second protein, beta-2 microglobin, interacts with transferrin receptors (TfR) on cell membranes. This interaction supresses the affinity of transferrin for TfR, thus lowering the uptake of transferrin--and its attached iron--into the cell. Transferrin receptors have been found on the surface of a variety of cells, with the greatest concentration on cell membranes of intestinal cells, hepatocytes, and RBC precursors. In addition to HFE, HH is also associated with mutations in other genes involved in iron homeostasis, including hemojuvelin (HJV), TfR, hepcidin, and ferroportin. Hepcidin production is reduced in HH due to all of these genetic causes, with a resulting increase in iron absorption. Mutations in HFE are the most common cause of HH. | View Page |
| Specific HFE Mutations Several mutations of the HFE gene have been described. The most common mutation in patients with hereditary hemochromatosis is the C282Y mutation. In the C282Y mutation, a base substitution leads to a change in the amino acid in position 282 from cysteine (C) to tyrosine (Y). The loss of the sulfhydryl-containing amino acid disrupts the tertiary structure of HFE so that it no longer binds to beta-2 microglobulin. Beta-2 microglobulin appears to act along with other proteins to chaperone the newly synthesized HFE out of the Golgi apparatus and to the cell surface where it can then bind to TfR. In the C282Y mutation, HFE remains in the Golgi, never making it to the cell surface. The result is that transferrin binding to TfR is enhanced and excessive amounts of iron enter the cells of the small intestine, liver, and other tissues. A second mutation, H63D, causes a histidine (H) residue in position 63 to be replaced by aspartic acid (D). The mechanism by which this mutation leads to increased iron uptake is less well understood when compared to the C282Y mutation. Unlike the C282Y mutation, the H63D mutation does not seem to affect the binding of beta-2 microglobulin and intracellular movement, since detectable concentrations of the mutated protein are found on cell membranes. Some researchers speculate that the H63D mutation affects the binding of proteins involved in iron regulation and uptake at the cell surface.A third mutation, S65C, leads to a serine-to-cysteine substitution in its associated protein. This mutation has been been found in some compound heterozygotes for C282Y or H63D, but is rarely associated with iron overload in HH.Additional mutations of HFE have been identified, but their clinical significance is unclear. Most laboratories performing molecular assays test for only the C282Y, H63D, and S65C mutations. | View Page |
| Serum Iron Serum iron (SI) is a measure of circulating iron bound to transferrin and is reflective of total body iron. SI is elevated in hereditary hemochromatosis (HH) and acute hepatitis. SI is decreased in iron deficiency anemia and chronic inflammation. SI concentrations exhibit diurnal variation, with the lowest values occurring around midnight. In addition, specimens collected from the same individual at the same time of the day may exhibit day to day variations as high as 40%. SI determinations are also affected by diet, menstrual cycle, pregnancy, ingestion of iron supplements, and oral contraceptive use. SI levels alone are considered insensitive indicators of HH. SI is typically measured on automated analyzers using spectrophotometric methods. Iron in the sample is released from transferrin with an acid reagent, reduced to the ferrous state, and reacted with a chromogen such as bathophenanthroline or ferrozine. The intensity of the color change is proportional to the iron concentration. Interference can arise from the use of a hemolyzed sample and contamination of reagents and water with iron. A typical reference interval for SI is 60 - 150 micrograms/dL. SI is usually ordered along with its companion test, the total iron binding capacity (TIBC), or with transferrin (Tf).(2) | View Page |
| Transferrin and Total Iron Binding Capacity The test for transferrin (Tf) measures the concentration of the primary carrier protein for iron. Measuring total iron binding capacity (TIBC) is an indirect method of assessing transferrin and provides comparable information. The TIBC (or transferrin) are typically performed along with the SI. Taken together, these determinations are useful in the differential diagnosis of many disorders affecting iron metabolism, including hereditary hemochromatosis (HH) and iron deficiency anemia. Tf and TIBC are typically low-normal or decreased in HH and are increased in iron deficiency. Serum transferrin can be measured directly using immunochemical methods such as nephelometry and turbidimetry. TIBC is performed in a 2-step method by adding ferric iron to the specimen in sufficient quantity to completely fill all of the iron binding sites on transferrin. Excess, unbound iron is removed by adsorption with magnesium carbonate, alumina, or ion resin. The iron content of the saturated binding protein is then measured as described for SI. Serum is the specimen of choice for Tf and TIBC. TIBC is less subject than SI to day-to-day variation and other causes of variability.A typical reference interval for TIBC is 300 - 360 micrograms/dL.(2) | View Page |
| Initial Treatment Phlebotomy is considered the treatment of choice for patients with iron overload due to hereditary hemochromatosis (HH). Each unit of blood contains approximately 200 to 250 mg of iron. As erythrocytes are removed by phlebotomy, iron stores are mobilized and utilized in the production of new, circulating erythrocytes. Through periodic phlebotomies, stored iron is removed until iron-deficient erythropoiesis is induced. The initial, or iron reduction, phase of treatment typically consists of removing one unit (450 mL) of whole blood once or twice weekly. Prior to beginning phlebotomy, the patient's hemoglobin and hematocrit must be checked to ensure that the patient is not anemic. A sample for serum ferritin is also collected at this time.Initial treatment goals include inducing iron deficient hematopoiesis without the development of debilitating symptoms of anemia. A hemoglobin concentration of 10.0 to 12.0 g/dL is often used as a target range. The initial treatment phase continues until excess stored iron is removed and ferritin levels decrease to approximately 50 ng/mL. (13) Ferritin and hemoglobin levels are periodically monitored during this phase. The number of phlebotomies needed to reduce iron levels and induce anemia is related to the degree of initial iron overload. Patients may be referred to a hematologist or gastroenterologist during the initial treatment phase. Many patients receive therapeutic phlebotomy services in a hospital or doctor's office, but patients may also undergo phlebotomy at a blood center. Blood collected from persons with HH may be used for transfusion or as blood products if it has been collected from a facility with an approved variance from the US Food and Drug Administration. Not all blood centers have applied for or been granted this variance.(14)The initial treatment phase continues until excess stored iron is removed and ferritin levels decrease to approximately 50 ng/mL. Removal of excess stored iron may take from one month to three years. | View Page |
| Maintenance Therapy Lifelong treatment of hereditary hemochromatosis (HH) is needed to keep iron at low levels. Without regular treatment, iron stores will re-accumulate. The primary care physician may manage patient care during long-term maintenance. Long-term maintenance typically consists of removal of an average of 2 to 6 units of whole blood yearly, although this number is variable. Monitoring of hemoglobin and serum ferritin levels determine the frequency of phlebotomy. Serum ferritin levels should be maintained at concentrations of no more than 50 ng/mL. (10,13)) | View Page |
| Arrange the following steps of a typical O & P examination in order of typical completion: | View Page |
| Arrange the major steps of the ethyl acetate concentration procedure in order of completion: | View Page |
| Which of the following specimen processing techniques is based on the principle that parasites are heavier than sample debris and will be present in the sediment after being processed? | View Page |
| Match each parasite listed here with the appropriate laboratory technique that may be used for its identification: Each answer may only be used once. | View Page |
| Arrange the four layers of material visible in the conical tube after the ethyl acetate concentration procedure has been performed in order from top to bottom: | View Page |
| Suppose that a stool specimen was received in the laboratory for an O & P examination. The clinical laboratory scientist on duty performed direct wet preparations and found suspicious forms. An ethyl acetate concentration procedure was done, the top layer was examined, and no suspicious forms were seen. A slide of the sample was stained with Trichrome and again suspicious forms were noted. Which of the following is the most likely explanation for these discrepant results? | View Page |
| Which of the following is considered as the technique of choice for identifying the oocysts of Isospora belli and Cryptosporidium parvum? | View Page |
| Which of the following is considered as the best fixative for maintaining specimen integrity during and following permanent staining? | View Page |
| A 50 year old male domestic airline pilot was rushed to the hospital after complaining of tremendous fluid loss due to severe diarrhea. History revealed that the patient was diagnosed with AIDS 6 months ago. The doctor ordered a battery of tests including a stool for parasite examination. Since the sample was properly labeled indicating that the patient was immunocompromised, the lab performed both the standard processing procedures and a modified acid-fast (mod AFB) stain. The mod AFB stain revealed this suspicious form which measured a mere 4 µm. This patient is most likely infected with: | View Page |
| Basic Pharmacokinetics In order to discuss TDM and PGx we need to also introduce the concept of pharmacokinetics. Pharmacokinetics is the study of drug disposition in the body: how and when drugs enter the circulation, how long they remain in the blood, and how they are eliminated. TDM is the clinical assessment of a drug's pharmacokinetic properties. Physicians and pharmacists need to establish that a drug is present at an effective concentration but not at a toxic concentration. The next few pages will describe some of the factors that determine a drug's disposition in the body. These factors ultimately decide the need for therapeutic drug monitoring. | View Page |
| Given what you have learned thus far, which of the following statements below do you think is true? | View Page |
| Steady State Most drugs are not given as a single dose but are part of a regimen. It is the physician's responsibility to prescribe a drug so that the concentration of that drug reaches a safe and effective level. The dosing-goal for the prescribing clinician, if multiple doses of a drug will be given, is for both the peak and the trough drug levels to be consistently within the therapeutic range. If a drug is given at intervals that are the same as its half-life, it will take about 5 half-lives to reach steady state. | View Page |
| Why TDM? Pharmacologists determine a drug's pharmacokinetic characteristics empirically during clinical drug trials. From these studies, they are able to determine the solubility and distribution, the average half-life, the levels of protein binding, and the effective concentrations needed for treatment. | View Page |
| Unexpected Concentrations TDM provides a quantitative measure of the circulating concentration of a drug. The physician determines if the dosage of the drug needs to be adjusted based on this information.If a drug concentration is determined to be outside the therapeutic range, it may be for one of the reasons listed in the table below. Reason Discussion Noncompliance Patients may (intentionally or unintentionally) not take the drug. TDM can thus help monitor compliance. Dosing errors The dose may have been erroneous or inappropriate given the patient's condition. Malabsorption The TDM result will reveal if the drug cannot be absorbed well through the gut and an alternative route of administration will be needed. Drug interactions Many drugs interfere with the absorption or metabolism of other drugs. These interactions will be revealed by TDM. Kidney or liver disease Any pathology that affects elimination will cause an elevation in a drug level that will be unmasked by TDM. Altered protein binding Changes in serum proteins can lead to big changes in the amount of free drug in serum. Variations in the genetics of drug-metabolizing enzymes can also affect drug concentrations in the body. This is the field of pharmacogenomics that will be discussed later in the course. | View Page |
| TDM for all drugs? Can all drugs benefit from TDM? Not really. For TDM to be effective and useful, one or more of the following should apply: The effective concentration and toxic concentrations must be well-defined. The pharmacokinetics of the drug are known to be variable. The drug is given chronically. There is the potential for drug-to-drug interactions. The drug exhibits high protein binding. The toxicity will mimic the indication for the drug; toxicity may not be visible during an exam but will only be revealed with TDM. The patient is pregnant, very young, or elderly. Compliance or history with the drug is poor. | View Page |
| A physician needs to prescribe a drug with a narrow therapeutic window. He is concerned about possible toxic effects. To assess the upper concentration of such a drug, which time for drawing the specimen do you think makes the most sense? | View Page |
| Sampling Ideally, a drug level would be monitored frequently and consistently, providing the clinician with a detailed pharmacokinetic profile over time. In reality, serum samples are often measured only during relatively infrequent clinic visits, meaning that many days or weeks may pass before a drug concentration 'snap-shot' is taken. | View Page |
| Protein Availability and Drug Dosing Drug-binding proteins in serum can fluctuate in disease states. For example, if albumin levels fall, as can occur in liver failure or nephrotic syndrome, less albumin will be available for drug binding; a subsequent dose may produce a toxic concentration of free drug.The image on the right illustrates the loss of equilibrium between a protein-bound drug and a free drug when drug-binding proteins are diminished.Doses of drugs that are highly protein-bound may need to be adjusted in patients with lower drug-binding protein levels. Examples of some common drugs that are highly protein-bound include thyroxine, warfarin, diazepam, heparin, imipramine and phenytoin. | View Page |
| Drug Concentration Over Time When a drug enters the body, it reaches a peak concentration that starts to fall as the drug is eliminated. The figure on the right shows a typical kinetic with a drug given intravenously (IV). | View Page |
| Half-life The amount of time it takes for a drug's concentration in the body to decrease by 50% is called the drug's half-life (t1/2).The longer a drug's half-life, the slower it is removed from the body. Most drugs are eliminated from the body in 1 to 3 days, but some drugs with longer half-lives can still be detected in the body weeks after the initial dose. The figure below illustrates a typical kinetic pattern for an oral drug. | View Page |
| Bioavailability Bioavailability refers to the amount of drug that actually reaches the circulation. It is calculated by comparing (in the same subjects) the area under the serum concentration - time curve (AUC) of an equivalent dose of the intravenous form and oral form. This is illustrated in the diagram on the right.For IV drugs, the bioavailability is 100%For oral medications, the bioavailability will be less than 100%, due in part to any of these reasons:* Oral drugs take longer to enter the circulation.* Oral drugs have slower absorption and distribution than IV drugs.* The amount of drug that is absorbed can depend on the status of the GI tract (stomach pH, presence of food, integrity/health of the intestines, speed of the GI tract, etc.)For oral drugs to be effective, bioavailability typically should be greater than 70%.Not all of a drug taken orally is able to have a pharmacologic effect; the dose would need to be higher than an IV dose.Since the absorption of an oral drug is slower than an IV drug and the drug takes longer to enter the circulation, clearing the drug will also most likely take a longer time. | View Page |
| Peak and Trough Sampling Times To assess drug concentrations during the trough phase, blood should be drawn immediately before the next dose. To assess peak levels, the time for drawing depends on the route of administration: Oral: One hour after drug is taken (assumes a half-life of > two hours) IV: 15-30 minutes after injection/infusion Intramuscular (IM): 30 minutes - one hour after injection | View Page |
| Drug Elimination Most water-soluble drugs are eliminated from the body through hepatic metabolism. renal filtration, or a combination of the two.An alteration in renal function will have a major effect on the clearance of the drug or its active metabolite(s). Decreased renal function results in elevated serum drug concentrations. | View Page |
| Why TDM? Every drug has a sub-clinical concentration (a concentration at which effective therapy won't be achieved) and a toxic concentration (a concentration at which the drug will be harmful to the patient.)For some drugs, the range between the minimum effective concentration and the toxic concentration is large. These drugs are thus relatively safe. Other drugs have a very narrow therapeutic window and need closer monitoring. This is the role of TDM.Medications with narrow therapeutic windows, like the anticonvulsant carbamazepine (Tegretol), should be closely monitored since elevated doses can cause serious conditions such as agranulocytosis. | View Page |
| When is TDM Not Useful? TDM is not useful for these drugs or in these specific situations: Intracelluar drugs that need to be converted to active forms (like AZT) Drugs in which the effects last much longer than the serum concentrations of the drugs; examples include antineoplastics (cancer chemotherapies) and warfarin Narcotic pain medications where continued use can lead to tolerance such that the levels needed for pain relief in one person would be toxic to another person | View Page |
| TDM for Antibiotics Infection is obviously a very serious indication, and effective antibiotic levels must be achieved as soon as possible. However, many antibiotics also have nephrotoxic or ototoxic effects; the concentrations of these antibiotics need to be monitored. Examples of antibiotics that are monitored by TDM include: Amikacin Gentamicin Tobramycin VancomycinAntibiotics such as ampicillin that are readily cleared and have a wide therapeutic window are not usually monitored by TDM. | View Page |
| TDM for Immunosuppressants Drugs used to inhibit the immune system are part of standard treatment after transplant surgeries. Regarding the use of TDM, there are some reports of hepatotoxicity and nephrotoxicity with some agents, but the main reason for TDM is to ensure that concentrations are adequate to suppress the immune response and prevent rejection. Examples of immunosuppressants that are monitored by TDM include: Cyclosporine Methotrexate Tacrolimus FK778 | View Page |
| PETINIA Particle-enhanced turbidimetric inhibition immunoassay (PETINIA) is a homogeneous competitive immunoassay.Antibody fragments and drug-latex particles will bind to form aggregates that increase the turbidity of the solution. Free drug from the sample competes for the antibody fragment, thereby decreasing the rate of particle aggregation. The rate of aggregation is inversely proportional to the concentration of drug in the sample. | View Page |
| FPIA Fluoresence polarization immunoassay (FPIA) is also a homogenous competitive immunoassay. In this system, fluorescein-labeled drug competes with unlabeled drug from the patient's serum sample for binding sites on an antibody reagent. The patient's sample, presumably containing the therapeutic drug that is being monitored, and the fluorescein-labeled drug are added to a chamber containing antibody for that drug. The labeled and unlabeled drug will compete for binding sites on the antibody. The greater the amount of drug in the sample, the fewer the number of binding sites that are available for the labeled analyte, leaving a greater number of small, free fluorescein-labeled molecules in the solution.When the chamber is excited with plane polarized light, fluorescein will absorb the light and emit it at a higher wavelength as fluorescent light. A small, free fluorescein-labeled drug rotates randomly and faster than it would if it were bound to antibody, interrupting the light and leading to less emission of light. The larger antibody-drug-fluorescein complexes rotate slower and emit more light in the measured plane. A lower level of drug in the patient's sample results in greater emission of polarized light because there are more antibody-drug-fluorescein complexes present to produce light in the measured plane. A higher level of drug in the patient's sample results in a lower emission of polarized light. This inverse relationship between the concentration of the drug and the polarization units (signal) is illustrated in the image below. | View Page |
| A patient is taking cimetidine for a stomach ulcer. This drug inhibits CYP2D6. The patient is now prescribed amphetamine for narcolepsy. Amphetamine is metabolized by CYP2D6. What would you predict? | View Page |
| Prerequisites The basic laboratory skills that you will need to do a semen analysis include: Use of a microscopePerformance of manual cell countsMeasuring volumeMeasuring pHMeasuring viabilityKnowledge of OSHA regulations for handling potentially infectious human fluids | View Page |
| Preparing Semen Smears for Evaluation of Sperm Morphology To examine sperm morphology, prepare at least two smears after the semen has liquified (> 30 minutes). The specimen should be thoroughly mixed before pipetting the aliquot onto the slide and then mixed again before preparing the second slide. If the sample is undiluted, the technique that is used is the same technique used to make a peripheral blood smear or bone marrow smear. This technique is demonstrated on this page. It is important that the sperm be spread evenly on this slide and that the concentration be such that individual sperm can be clearly viewed. Too many sperm per slide makes evaluation difficult. Too few, makes it hard to find enough sperm for an adequate count. The smears are then labeled, air-dried, fixed, and stained. Some of the more commonly used stains include: Papanicolaou stainDiff QuikShorr stain Details of these staining methods are available in the WHO 5th edition. | View Page |
| Summary: Reference Values The following are lower reference limits for a normal semen analysis. It should be noted that these are recommendations only, as stated in World Health Organization. WHO Laboratory Manual for the Examination and Processing of Human Semen. 5th ed. The limits may differ from those used in your laboratory, if laboratory studies or different reference materials were used to establish the values.Liquefaction: ≤30 minutes (no greater than 1 hour)Volume: >1.5 mLpH: ≥7.2Sperm concentration: ≥15 x 106 / mLMotility: Progressive motility 32%. The lower reference limit for total motility (progressive + non-progressive) 40%.WHO 5th edition: ≥4% normal forms | View Page |
| Collection Accurate semen analysis results require appropriate sample collection. Patients must receive detailed directions for proper specimen collection and transport. Directions should be in writing. Specific instructions should include: The period of abstinence from ejaculation prior to collection should be at least 2, but no more than 7 days.Avoid lubricants and other contaminating substances.The entire specimen must be collected because different portions of the ejaculate have varying concentrations of spermatozoa. The first portion of the ejaculate is rich in sperm whereas the later portions of the ejaculated specimen are mainly seminal vesicular fluid. If the first portion is lost, the semen analysis results will be greatly affected. An appropriate collection container (supplied by the physician's office or laboratory) must be used.Each laboratory should designate an appropriate, wide mouth, collection container.Each lot of collection containers should be tested to ensure that it is non-toxic to sperm or contain confirmatory information from the manufacturer that this testing was completed.Alternative collection containers should be discouraged because their level of toxicity is unknown.Use of condoms for collection should be discouraged particularly when the purpose of the semen analysis is to test for fertility. Condoms are toxic to sperm unless the brand that is used is specifically for the purpose of specimen collection for semen analysis. Collection in a condom may also cause inaccurate semen volume measurement. | View Page |
| High viscosity If the specimen is more viscous than normal, it may be difficult to dilute it or to load it onto counting chambers in the undiluted condition. In this rare situation the semen may need to be manipulated to reduce the viscosity before a count is done. One method to do this is to repeatedly pipet the specimen up and down with an equal volume of culture medium. Care must be taken to avoid foaming. Other methods include enzyme digestion, for example with bromelain at a concentration of 1 gm / liter, or addition of a small amount of emulsifier, such as Alevare or chymotrypsin. Any manipulation of this type must be recorded on the report sheet. Calculation of the number of sperm per milliliter will also have to be corrected for any dilution. | View Page |
| Diluting a Specimen for Counting on a Hemocytometer Semen must be diluted prior to counting sperm if a hemocytometer is used. In additon to preventing overlapping of sperm cells, the diluting fluid immobilizes the sperm in the chamber to further facilitate counting.Following liquefaction (at about 30 minutes), mix the sample manually by swirling the container several times. Thorough mixing is essential for accurate counting. Calibrated automatic pipettes are used to prepare a dilution. Some laboratories may require two separate dilutions, as recommended by WHO 5th edition. Because of the viscosity of semen, the semen should be added to the diluent using a positive pressure pipettor. The dilution often used for routine sperm counts is 1:20 but the actual dilution factor will vary depending on the sperm concentration that was noted during the initial microscopic evaluation. If a high concentration of sperm are noted, a greater dilution will be necessary. For low concentrations, a minimally diluted (e.g., 1:2) specimen may be required. The appropriate dilution is determined by estimating the concentration needed to achieve a count of at least 200 spermatozoa in the area that is counted on each side of the hemocytometer. | View Page |
| Other Counting Chambers Some professionals believe that sperm counts done by hemocytometer are not accurate because of the need to dilute the viscous semen prior to counting. There are several other counting methods available to assess sperm concentration. The advantages of the following methods are: The specimen does not have to be diluted Motile and non-motile sperm can both be counted avoiding the need for wet mount evaluation of motile cells. Note that counting moving sperm can be difficult and takes significant practice to avoid error. Makler (Zygotek Systems, Inc.). An undiluted sample is placed on the chamber and covered with the coverglass. Ten squares on the grid contain 0.000001ml. CellVu (Millennium Sciences, Inc). Two sides of a special slide are loaded with a drop of undiluted semen. Coverslips with special grids are placed on top of the sperm according to manufacturer's directions. Sperm on both sides are counted.MicroCell (Conception Technologies) has two chambers on a single, disposable slide. A special eyepiece with a grid is needed for counting. | View Page |
| In Vivo Red Cell Destruction Important events that occur in an immune-mediated hemolytic transfusion reaction (HTR) include: Antibody Binding to Red Blood Cells Antibodies may be either IgM or IgG class. IgM antibodies activate complement and lead to intravascular hemolysis where free hemoglobin is released into the plasma. IgG antibodies rarely activate complement but they are often involved in effecting phagocytosis. The concentration of the antibody is directly related to the severity of the HTR. Activation of Complement The end result of complement activation is red cell lysis. Activation of Mononuclear Phagocytes and Cytokines Sensitized red cells are removed from circulation by mononuclear phagocytes. Macrophages in the spleen and Kupffner cells in the liver are active in this process. Activation of Coagulation Antibody-antigen complexes may initiate coagulation and cause disseminated intravascular coagulation (DIC). Shock and Renal Failure Hemolysis can be intravascular or extravascular. In intravascular hemolysis, free hemoglobin, RBC stroma, and intracellular enzymes are released into the blood stream. This results in hemoglobulinemia and hemglobinuria which can lead to kidney damage. In extravascular hemolysis, there is no release of free hemoglobin. Sensitized red cells are removed from the circulation by the monocytes and macrophages in the reticuloendothelial system. | View Page |
| Additional Testing If preliminary testing suggests hemolysis or if the results are misleading, additional testing may be required. If human error has been ruled out during the clerical check, repeat ABO/Rh testing should be performed on the unit of blood or its segment and the pre-transfusion sample to detect any sample mix ups and clerical errors. Antibody detection studies should be performed on the pre- and post-transfusion samples to look for any unidentified antibodies. If an antibody is identified, the donor cells should be tested for the corresponding antigen. The crossmatch should be repeated with pre-and post-tranfusion specimens using the indirect antiglobulin test (IAT). An incompatible crossmatch with the pre-transfusion sample indicates an original error, either clerical or technical. Incompatibility with only the post-transfusion sample indicates a possible anamnestic response, as in a delayed hemolytic transfusion reaction (DHTR), or sample misidentification. The patient's first voided urine specimen should be examined for the presence of free hemoglobin. The patient's bilirubin levels may also be evaluated. A change from normal pale yellow serum to a post-transfusion bright or deep yellow serum should prompt an investigation for hemolysis. The maximum concentration of bilirubin following hemolysis is not usually detectable until 3 to 6 hours after transfusion. The hemoglobin and hematocrit can be tested to detect a drop in hemoglobin or failure of the hemoglobin to rise after transfusion. Important information about physical or chemical hemolysis may be gained from examining the returned unit bag. If hemolysis is present in the bag or tubing, a process which affected the blood, such as inappropriate warming or faulty infusion pump, should be suspected. If bacterial contamination is suspected, the unit can be cultured. A positive culture indicates a reaction due to bacterial contamination. | View Page |
| Presentation and Prevalence Although the risk of acquiring transfusion transmitted viral infections is low due to donor testing, bacterial infections are still reported. Platelets are the most implicated product in bacterial contamination reports because they are stored at room temperature (20-24oC) and provide a favorable environment for bacterial growth. Sespis occurs in about 1 in 25,000 platelet transfusions. It may be fatal in about 1 in 60,000 transfusions. Bacteria can be present in other components as well, such as red blood cells (RBCs), cryoprecipitate, and plasma. Contamination in red cell components is rare with events occurring 1 in 250,000 transfusions. This low incidence is due to the refrigerated storage requirements for red cells at 1-6oC. Because plasma and cryoprecipitate are stored frozen, they are least likey to contain bacteria. Contamination usually occurs when these products are thawed in a water bath that contains bacteria. Reactions range from minimal or no symptoms to fatal septic shock and death. Severity of the reaction depends on the bacterial species involved, the concentration and growth rate of the organisms, and the recipient's immune status. Septic reactions can present with a fever of higher than 38.5oC, rigors, and hypotension that begin during the transfusion. Patients may also have nausea, vomiting, dyspnea, and diarrhea. Septic shock, oliguria, and disseminated intravascular coagulation (DIC) are also complications. | View Page |
| Reducing Transfusion-Associated Septic Reactions Measures taken to reduce bacterial contamination of blood components include donor screening, improved skin disinfection, diversion of the first aliquot of blood, and pretransfusion bacterial detection. Screening of donors is done by questioning them about fever occurrence and dental or medical procedures that occurred days before donation. Donors who develop symptoms of an infection may be asked to notify the blood bank. Complete skin disinfection is not possible because of organisms living in places that are inaccessible, such as sebaceous glands and hair follicles. Factors affecting skin disinfection are the type and concentration of antiseptic, use or single or multiple antiseptics, method and steps of application, and contact time. Studies have shown that a two-stage method using a sponge scrub and ampule with tincture of iodine is the most effective method. The AABB recommends an initial 30 second scrub with a 0.7% iodophor solution followed by the application of a 10% iodophor compound, which must be allowed to dry for 30 seconds. To avoid normal flora contamination, blood may be diverted into a satellite bag at the beginning of donation. These bags are developed so that backflow is prevented. Blood contained in the satellite bag is used for blood grouping and infectious disease testing. Blood diversion is not a mandatory practice in the United States. The AABB requires that the transfusion service have a method to detect bacteria in all platelet components. Culture-based methods are used at blood collecting facilities near the time of collection. Hospital-based transfusion services use other less costly non-culture based methods such as gram staining or pH and glucose analysis prior to releasing the product for transfusion. Recently, a qualitative immunoassay for the detection of bacteria in platelets has been developed. This test detects antigens on the cell walls of the bacteria. It has been documented to be more sensitive than other non-culture based methods. | View Page |
