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Concentration Information and Courses from MediaLab, Inc.

These are the MediaLab courses that cover Concentration and links to relevant pages within the course.

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Laboratories Individuals

Alpha Thalassemia
CBC Results

WBC 6.1 X 10 9/L (Reference range 4.0 - 10.5 X 109/LRBC 4.84 X 1012/L (Reference range 3.50 - 5.50 X 1012/LHb 8.4 g/dL (Reference range 12.0 - 16.0 g/dL)Hct 28.8 % (Reference range 36.0 - 48.0%)MCV 59 fL (Reference range 80.0 - 100.0 fL)MCH 17.4 pg (Reference range 26.0 - 34.0 pg)MCHC 29.3 g/dL (Reference range 32.0 - 36.0 g/dL)RDW 19.5 % (Reference range 11.0 - 15.0 %)Plat 591 X 109/L (Reference range 150 - 400 X 109/L)Even though the RBC count is normal, it is increased for the amount of hemoglobin present. The concentration of hemoglobin in the RBCs is slightly decreased (hypochromic) and the cells are small (microcytic). The variation in RBC size is also slightly increased as are the platelets.

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Beta Thalassemia
Hematologic Findings For Various Types of Beta Thalassemia

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

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Laboratory Test Results

Test Patient Result Reference Intervals (Adult female) White blood cell (WBC) count 3.7 x 109/L 4.4 - 11.3 x 109/L Red blood cell (RBC) count 5.6 x 1012/L 4.1 - 5.1 x 1012/L Hemoglobin (Hb) 10.5 g/dL 12.3 - 15.3 g/dL Hematocrit (HCT) 36.6% 35.9 - 44.6% MCV 65.8 fL 80.0 - 96.0 fL MCH 19.9 pg 27.5 - 33.2 pg MCHC 26.7% 33.4 - 35.5% RDW 14.0 <14.5 Platelets 249.0 x 109/L 100.0 - 450.0 x 109/L Total serum iron 165 µg/dL 60 - 150 µg/dL Iron-binding capacity 230 µg/dL 250 - 400 µg/dL The RBC count is increased for the amount of hemoglobin present. The concentration of hemoglobin in the RBCs is slightly decreased (hypochromic) and the cells are small (microcytic). The variation in RBC size (RDW) is within normal limits.

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Cerebrospinal Fluid
Chemical Substances Present in CSF

The following table lists some of the chemicals present in CSF, and their concentrations: ChemicalLevel sodium 136.0 - 150.0 m Eq/L potassium 2.3 - 2.7 m Eq/L magnesium2.4 - 3.0 m Eq/Lprotein2 - 4 mg/dL (normally diffuses across blood-brain barrier) glucose 45.0 - 60.0 mg/dL calcium2.1 - 2.7 m Eq/dLcholesterolpresent in small amounts creatinine 0.5 - 1.2 mg/dL lactic acid dehyrdogenase (LDH) present in small amounts phosphorus (inorganic)1.0 - 2.0 mg/dLurea6.0 - 16.0 mg/dL uric acid 0.5 - 3.0 mg/dL

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Chemical Screening of Urine by Reagent Strip
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

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. A pH can be falsely interpreted as more acidic than it actually is if improper technique is used and excess urine is allowed to pool on the reagent strip. The reagents from the protein pad, that includes an acid buffer, can run over into the pH pad if the strip has these two tests located next to each other.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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CLIA Blood Banking Review
Which is in the correct order from least concentration of H to most concentration of H:View Page
Which of the following might cause a false positive indirect antiglobulin test:View Page

CLIA Chemistry / Urinalysis Review
Which of the following analytes would not be significantly increased in a plasma sample as a result of hemolysis:View Page
Which of the following will give the best overall picture of a patient's iron stores:View Page
Which of the following electrolytes is most likely to be spuriously elevated in a hemolyzed specimen:View Page
The migration rate of proteins on cellulose acetate is primarily the result of:View Page
The measurement of total glycosylated hemoglobin A1c is an effective means of assessing the average blood glucose levels:View Page
In a normal CSF the protein concentration as compared to that in the serum is generally:View Page
The primary mechanism responsible for glomerular filtration is:View Page
The renal threshold is best described as:View Page

CLIA General Laboratory Review
The direct relationship between the concentration of a substance and its absorbance is referred to as:View Page
The concentration of sodium chloride in an isotonic solution is :View Page
The term TITER ( as it applies to the measurement of antibodies) is best defined as:View Page
An analytical method with a low detection limit would:View Page
Analytical sensitivity of a method generally refers to:View Page
The term analytical specificity refers to:View Page
Electrophoretic separation fundamentally relies on:View Page
Which of the following would most likely occur as the result of hemodilution:View Page
Hematocrit is:View Page
Which of the following would be considered most significant as it relates to serological testing:View Page

CLIA Hematology / Hemostasis Review
Which of the following would not be represented in the usual classification of anemia:View Page
Which of the following observations would best explain why a peripheral blood smear is exhibiting polychromasia:View Page
Hypochromia can best be described as:View Page

CLIA Microbiology / Serology Review
MIC is an acronym for:View Page
The McFarland Comparison Card shown in the illustration is used to:View Page

Confirmatory and Secondary Urinalysis Screening Tests
Diseases Associated with Proteinuria

Normal urine contains very little protein, usually less than 10mg/dL, and the major serum protein that is found in normal urine is albumin. The presence of an increased amount of protein in the urine (proteinuria) can be an indicator of renal disease. The two mechanisms which can lead to proteinuria are glomerular damage or a defect in the reabsorption process of the tubules in the nephron. The concentration of protein in the urine is not necessarily indicative of the severity of renal disease.

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Current Topics in Clinical Microbiology
Beta hemolytic colonies grew from the blood culture bottle after 18 hours incubation (see photograph). The following tests would be helpful in making a preliminary identification:View Page

Descriptive Statistics
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.

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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.

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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

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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

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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.

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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

Electrophoresis
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.

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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.

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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, 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.

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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.

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Which statements below are correct descriptions of visualization and detection methods used in electrophoresis?View Page

Emerging Cardiovascular Risk Markers
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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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Fundamentals of Molecular Diagnostics
Advantages of Molecular Testing

Molecular methodologies offer numerous advantages to the clinical laboratory. These include:Sensitivity: Amplification methodologies are particularly useful in increasing the sensitivity of a methodology and useful in the identification of target molecules of interest that are only present in low concentrations. Specificity: Molecular methods minimize false positive test results by targeting the specific molecule of interest.Turn Around Time: In comparison with standard traditional culture methods, molecular methodologies usually offer better turn around times from receipt to result reporting.Application: broader application can be found with molecular methodologies such as infectious diseases, genetic testing, forensics, drug resistance, and tumor marker detection and monitoring.

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Which of the following are considered advantages of molecular testing?View Page
Amplified Nucleic Acid Tests

Amplification Method Amplifies Use of Thermal Cycling (Thermocycling) Polymerase Chain Reaction (PCR) Target amplification using DNA polymerase Yes Ligase Chain Reaction (LCR) Target amplification using DNA ligase Yes Transcription- based or Transcription-mediated amplification(TMA) Target amplification using reverse transcriptase and RNA polymerase No Strand Displacement (SDA) Target amplification using DNA polymerase that continuously displaces strands of DNA containing the target sequence No Branched DNA (bDNA) Signal amplification using alkaline phosphatase No Loop Mediated (LAMP) Target amplification of multiple DNA sequences in a loop pattern using DNA polymerase No Nucleic acid sequence based (NASBA) Target amplification using 3 enzymes No Q-beta Replicase Probe amplification- The concentration of an RNA probe increases if the target is present No

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Hereditary Hemochromatosis
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

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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.

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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.

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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.

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Specific HFE Mutations

Several mutations of the HFE gene have been described. 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.

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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)

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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)

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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.

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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))

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Introduction to Bioterrorism
Chemical Agents

Chemical warfare agents are poisonous vapors, aerosols, liquids, or solids that have toxic effects on people, animals or plants. They can be released in a number of ways such as by bombs or sprayed from aircraft. Some chemical agents are odorless and tasteless. They can have an immediate effect (such as a few seconds to a few minutes), or a delayed effect (from several hours to several days). Even though chemical agents have the potential to be lethal, they are difficult to deliver in lethal concentrations, particularly in outdoor situations where they tend to dissipate rapidly.

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Lung-damaging or Choking Agents

Example: Phosgene Physical Properties: Heavy gas, smells like fresh cut hay. General Signs and Symptoms: Coughing and choking, followed by tightness in chest, nausea, vomiting and headache. Death is due to the accumulation of fluid in the lungs. Relative Rate of Action: Immediate in high concentrations to several hours in low concentrations.

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Linear Regression Analysis
A Regression Analysis Example

For example, to find a relationship between glucose concentration and absorbance, we could first plot all the points on a scatterplot. Glucose (mg/dL) Absorbance 50 .10 100 .20 150 .30 200 .40 250 .50 300 .60

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Calculating the Y-Intercept

To find the y-intercept, calculate and , the average of the x- and y-values respectively. Then substitute these two values for x and y in the = b + a equation. Finally, solve for the unknown quantity a. Therefore, the complete relationship between glucose concentration and absorbance for the data is y = 0.002x, where y is the absorbance and x is the glucose concentration.

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Prediction Using the Resulting Equation

Once the parameters have been calculated, the resulting equation can be used to make predictions about a value of y given a value of x, provided that the x value is in the same range of x-values that were used to derive the equation. For example, if x = 350 mg/dL, what is the expected value of y? To find the answer, substitute the known value of x into the equation. When the concentration is 350 mg/dL, we expect the absorbance to be about 0.7.

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Medical Error Prevention
Factors that Contribute to Medical ErrorsView Page

Mycology: Yeasts and Dimorphic Pathogens
Arrange the fungal species that are listed in the drop-down box according to length of time of recovery in primary culture; from most rapid to the slowest.View Page

Normal Peripheral Blood Cells
Platelet Kinetics

Platelets are derived from the cytoplasm of megakaryocytes, giant cells in the bone marrow. At any given time, two thirds of the total platelets are in the circulation and one third are present in the spleen. In persons with enlarged spleens 80-90% of the platelets are in the spleen resulting in a decreased concentration of circulating platelets. In individuals who have had a splenectomy all of the platelets will be in the circulating blood. The life span of the platelet is 8-10 days.

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The Process of Phagocytosis

Neutrophils have a relatively short life span.They are produced in the bone marrow, and when they reach the band or segmented stages are released into the peripheral blood.They remain there for approximately ten hours before randomly entering body tissues.Neutrophils in the blood stream can be divided into circulating granulocyte pool(CGP) and marginating granulocytic pool (MGP).The white blood cell count reflects the cells in the circulating pool.The cells in the marginating pool move quickly into the circulating pool when needed.During an infection the neutrophil concentration of the peripheral blood can increase almost immediately due to the shift of these cells from the marginating pool and release from the bone marrow storage pool, if needed.Neutrophils then migrate to areas of tissue damage or infection.Neutrophils do not reenter the blood stream from the tissues, thus end their life in the tissues either as a result of phagocytosis or senescence.

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OSHA Electrical Safety (updated 2007)
Factors that Determine the Degree of Electricity-induced Injury

The degree of electricity-induced injury is dependent on: The amount of electrical energy that is delivered The resistance that is encountered The type of current The current pathway The duration of contact

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OSHA Formaldehyde
PPE

When using formaldehyde in any concentration, with the exception of putting specimens in single vials, you must wear: A cover gown or apron A face shield or safety goggles Gloves This personal protective equipment is provided at no cost to you.

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OSHA Formaldehyde (updated 2009)
Personal Protective Equipment

When using formaldehyde in any concentration, with the exception of placing specimens in single vials, you must wear: A cover gown or apron A face shield or safety goggles Gloves

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What is Formaldehyde?

Formaldehyde solution is a colorless, aqueous liquid with a pungent odor. Concentrated formaldehyde solutions contain not less than 37% of formaldehyde or CH2O. These are usually supplied in 55 gallon drums. Ten percent (10%) aqueous formaldehyde solution, known as formalin , is almost universally used in the histology laboratory to fix and store pathology specimens, and, while still an important potential health hazard, is safer to use because of its lower formaldehyde concentration.

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Parasitology Review
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

Pharmacology in the Clinical Lab: Therapeutic Drug Monitoring and Pharmacogenomics
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.

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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.

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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.

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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.

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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.

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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.

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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. �

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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).

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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.

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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.

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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

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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.

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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.

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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

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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.

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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

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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.

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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.

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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

Phlebotomy
Introduction

Physicians need to know the blood concentration of certain drugs in order to select the best dose for their patients.As a phlebotomist, you might be asked to draw peak (highest), and trough (lowest) levels of various therapeutic drugs.

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Quality Control
Assayed and Unassayed Controls

Assayed controls have been analyzed by the manufacturer so that the range of values for the analytes they contain is known. Unassayed controls are unknowns. The laboratory purchasing the controls must determine the concentration of each analyte.

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What are Standards and Calibrators?

Before controls and patient samples can be run, testing instruments must first be calibrated. This requires standards. Standards are materials which contain accurately determined concentrations of an analyte that are used to either confirm a testing method's validity, or to make sure an instrument reads correctly. Calibrating an instrument allows every unknown patient sample or control to be analyzed from a measured starting point. Standards should not be used in place of daily controls because they do not measure or control other variables in the testing process such as operator technique or sample appropriateness.

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CUSUM Example: Plotting Control Data

To illustrate the use of CUSUM in the laboratory, we'll use daily control values for glucose testing. First, we'll list daily control values under "daily results." Then, we'll calculate mean by using formula A. Next, we can find the difference from the mean for each result, and square that result for the two relevant columns. Using all of the squared differences from the mean, we can find the standard deviation using formula B. Using the mean from formula A and the standard deviation calculations from formulas B and C, we can plot our data points on the Levey-Jennings chart. Formula D helps us calculate the coefficient of variation (CV), which expresses SD as a percentage of mean value and is more reliable for comparing precision at different concentration levels. The lower the CV the greater the precision.

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Red Cell Disorders: Peripheral Blood Clues to Nonneoplastic Conditions
Rouleaux

Rouleaux formation correlates with an increased concentration of serum monoclonal proteins. Rouleaux may be seen as an artifact in the thicker portions of blood smears. The addition of a drop of saline to the blood smear will serve to disperse any artifactual rouleaux formation. The presence of rouleaux formation or RBC agglutination may result in a falsely decreased electronic red blood count and falsely increased MCV, as these clusters may be read as one cell.

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Red Cell Morphology
All of the following statements are true of hypochromic red cells EXCEPT:View Page
Hypochromia

Examples of hypochromic cells are seen in this slide. Notice the thin rim of hemoglobin and the large area of central pallor present in most of these cells. Hypochromic cells are cells which are unusually thin, or in which the hemoglobin concentration is decreased. Decreased hemoglobin concentration can be caused by decreased amounts of iron available for hemoglobin production. The MCHC for this patient was significantly decreased (26 gm/dl of RBCs) indicating a severe degree of hypochromia. When hypochromia is less severe, not all cells will be affected; thus some cells may appear almost normal whereas others show hypochromia.

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Erythrocytes

Erythrocytes, when spread on a glass slide, show varying degrees of central pallor as noted in the previous exercise. This central pallor is related to the hemoglobin concentration present in the red cells.When viewing normal mature red cells, the central area (one-third of the cell) is white, while buff-colored hemoglobin is visible in the outer two-thirds of the cell. The mean corpuscular hemoglobin concentration (MCHC, 32-36 gm/dl of red blood cells), is the indice value which is used to verify the presence of adequate hemoglobin concentration in the cells visible on the peripheral smear.

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Semen Analysis
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

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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.

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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.

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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.

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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.

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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).

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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.

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The Urine Microscopic: Microscopic Analysis of Urine Sediment
All of the following factors favor cast formation except:View Page

Tuberculosis Awareness for Healthcare Workers
How tuberculosis is spread

The Mycobacterium tuberculosis organism is spread through infectious droplet nuclei.When a person infected with pulmonary tuberculosis coughs, sneezes, shouts, or sings, the infectious particles are expelled into the air.The risk of infection is related to both concentration of infectious droplet nuclei and duration of exposure.

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Three levels of TB Infection Control

Administrative controls reduce the risk of exposure to persons who might have TB disease.Environmental controls prevent the spread and reduce the concentration of infectious droplet nuclei in ambient air.Respiratory protection controls are for situations that pose a high risk of exposure to further reduce risk of exposure of HCWs to infectious droplet nuclei that have been expelled into the air from a patient with infectious TB disease.

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