| Which of the following statements are TRUE for specific gravity measured by the reagent strip method? (Choose ALL of the correct answers) | View Page |
| Acid and alkaline urine pH Reasons for acidic urine pH include: a high-meat diet, respiratory/metabolic acidosis, and hypochloridemia. A urine with a high concentration of glucose may also have a lower pH. An alkaline pH may be the result of a vegetarian diet, respiratory/metabolic alkalosis, or a bacterial infection caused by urease-producing bacteria. Urine that contains bacteria can become more alkaline if the specimen remains at room temperature for an extended period of time. | 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 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 (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.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. | 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, and 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 Women 6 - 8 1 8 - 10 2 10 - 12 10 12 - 14 25 14 - 16 9 16 - 18 1 Figure 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 5 Employees Concentration (mg/dL) 9 7 11 13 10 | View Page |
| Standard Deviation Example (continued) The first step in calculating the standard deviation is to calculate the mean, x. In this case, x = 10.Now, subtract that mean from each of the data values, and then square those results:Table VII Urea Nitrogen Concentration in 5 Employees (mg/dL) Concentration (mg/dL) x- (x-)2 9 -1 1 7 -3 9 11 1 1 13 3 9 10 0 0 Total 20 Use this total to calculate the standard deviation:The standard deviation is about 2.23. | View Page |
| Use the data for the following question:Table VII Urea Nitrogen Concentration in 9 Employees (mg/dL) Concentration (mg/dL)x-(x-)2 10 11 11 13 9 5 15 7 9 Total What is the standard deviation of the above data? You may find it helpful to make a chart similar to the one above. | 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:
Using a microscopePerforming manual cell counts and doing calculations to determine the concentration of those cells per milliliter of fluidMeasuring volumeMeasuring pHMeasuring viabilityKnowledge of OSHA regulations for handling potentially infectious human fluids | View Page |
| Staining and fixation for sperm morphology To examine sperm morphology a semen smear is prepared on a clean glass slide, much like making a blood smear. 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 examination of morphology is made using one of several commonly used stains. These include: Papanicolaou stainDiff QuikShorr stainDetails of these staining methods are available in the WHO IV reference manual.Two slides are prepared and 100 sperm are counted per slide using a bright field 40X or 100X objective. | View Page |
| Summary: Reference values The following are reference values for a normal semen analysis. It should be noted that these are recommended reference ranges only and that they may require adjustment for your particular laboratory or region of the country:Liquefaction: ≤30 minutesVolume: ≥2.0 mlColor: white, yellowish, grayViscosity: non-viscouspH: ≥7.0Sperm count: ≥20 million / mlMotility: ≥50%Leukocytes: ≤1 million / mlWHO III Morphology: ≥30%Strict Morphology: ≥14%
In addition some people find it useful to have a total motile count (TMC). This is calculated by multiplying the concentration x the percent motility x the volume. Normal TMC is 10 million or greater. | 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 prior to collection should be between 2 and 5 days.The entire specimen must be collected because the different portions have varying concentrations of spermatozoa.An appropriate collection container 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.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. Some condoms are toxic to sperm. Collection in condoms often results in inaccurate results for semen volume and other parameters. | 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 hemacytometer Following liquefaction (20-30 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. 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 total sperm count. For high concentration specimens a greater dilution will be necessary. For low concentrations an undiluted or minimally diluted specimen may be required. The appropriate dilution is determined by estimating the concentration needed to do a count of at least 100 cells per side of the loaded hemacytometer.
The diluent that may be used for sperm counts on a hemacytometer can be as follows: 5 gm of sodium bicarbonate in 100 ml of distilled water, plus 1ml of formalin (neutral). | View Page |
| Other counting chambers Some professionals believe that sperm counts done by hemacytometer 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. For each of these methods accurate counts are best obtained when at least 100 sperm per replicate are counted. 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 |