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Protein Information and Courses from MediaLab, Inc.

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

Learn more about laboratory continuing education for medical technologists to earn CE credit for AMT, ASCP, NCA, and state license renewal and recertification. Or get information about laboratory safety and compliance courses that deliver cost-effective OSHA safety training and continuing education to your laboratory's employees.

Laboratories Individuals

Cerebrospinal Fluid
Normal CSF Protein Level

The composition of CSF has some similarities to plasma. However, the protein level of normal CSF is dramatically lower than that of plasma.

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Clot/Pellicle

Clot formation is always abnormal and is often due to increased levels of protein, especially fibrinogen. When the protein level is 1000 mg/dL, clot formation will most likely occur but clots may also form at lower levels of protein. Some clots may be very fine and appear as a thin membrane or "scum" on the surface of the CSF specimen. This type of clot is referred to as a pellicle. Pellicles are composed of fibrinogen and white blood cells. The type of clot formed may give some specific information about the disease state. Some examples are provided in the following table: Example of ConditionType of Clotbacterial meningitispellicle forms in a short time; large clot formation followsTB meningitisweb-like clot (pellicle) after 12-24 hours (enhanced by refrigeration)paresis (type of neurosyphilis)incomplete clotblockage of CSF circulationcompletely clotted due to protein

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Other Causes of Xanthochromia

Examples of sources of pigment other than oxyhemoglobin and bilirubin that can cause xanthochromia include: methemoglobinincreased CSF protein (> 150 mg/dL)contamination by skin antiseptic (iodine or merthiolate)

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Causes of Xanthochromia in Premature Infants

Xanthochromia may also be present in the cerebrospinal fluid of premature infants. Reasons for this include: elevated bilirubin in the bloodimmaturity of the blood-brain barrierelevated protein in CSF

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Chemical Screening of Urine by Reagent Strip
False-positive tests for protein may be caused by: (Choose ALL of the correct answers)View Page
The reagent strip method for protein primarily tests for which type of protein?View Page
Which of the following reagent strip tests is based on the Ehrlich- aldehyde reaction.View Page
Sulfosalicylic acid can be used to confirm the result of which of the following tests included on a urine reagent strip?View Page
Urine Specimen

The urine specimen should be freshly voided. Urine is an ideal medium for the proliferation of bacteria due to the large amount of urea present. These bacteria metabolize urea, producing ammonia that increases the urine pH. If there is a delay before performance of the test, the sample should be refrigerated. This will: Prevent urease-producing organisms, such as Proteus and Pseudomonas, from converting urine urea to ammonia, which results in an increased pH. Prevent loss of CO2 which increases pH to the alkaline range.The "run-over" phenomenon may occur if excess urine remains on the strip. The protein area, adjacent to the pH area, contains an acid buffer which may "run-over" the pH portion resulting in an acid reading on a neutral or alkaline urine.

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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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False Positive Protein Results

A urine specimen that has remained at room temperature for an extended period of time may produce a false-positive protein result on a reagent strip. A false positive may also occur in the presence of bacterial contamination, alkaline medication, or quaternary ammonium compounds such as disinfectants or drugs, and with skin cleansers containing chlorhexidine.

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Follow-up Testing of Urine Dipstick Protein Results

A 24-hour urine protein may be ordered if a large amount of protein is detected with the dipstick method or if protein persists in the urine. A 24-hour urine protein may also be ordered if the physician suspects the release into the urine of protein other than albumin.

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Confirmatory Testing for Protein

Semiquantitative tests are used in some laboratories to confirm the presence of protein in the specimen when the result is positive on the urine dipstick. Tests that are used for confirmation include: sulfosalicylic acid (SSA); heat and acetic acid; nitric acid ring test; and Roberts' Ring Test. Any one of these procedures may be used for confirmation of the presence of protein. A protein dipstick result that is greater than a trace may be an indication of proteinuria.

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Clinical Significance

The presence of protein in a urine specimen can have serious implications. It may signal severe kidney damage, be a warning of impending kidney involvement, or be transient and unrelated to the renal system. Further quantitative testing of urine for protein may be needed to determine the significance of the proteinuria.

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Clinical Significance cont'd

Proteinuria related to kidney impairment may be due to glomerular membrane damage caused by toxic agents, immune complexes found in lupus erythematosus, or streptococcal glomerulonephritis. The amount of protein present in urine samples from patients with glomerular damage usually ranges from 10-40 mg/dl. If the urinary protein is due to a disorder that affects tubular reabsorption, the urine protein quantities will be much greater. In patients with multiple myeloma, proteinuria is due to the excretion of the Bence Jones protein. This low molecular weight protein produced by a malignant clone of plasma cells circulates in the blood and is filtered in the kidneys in quantities exceeding the tubular capacity. This excess protein is excreted in the urine.

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Clinical Significance cont'd

Individuals with diabetes mellitus may excrete small amounts of protein in the urine which may signal the beginning of reduced glomerular filtration. Stabilizing the blood glucose level at this time may delay progression of diabetic nephropathy. Women in the last month of pregnancy may develop proteinuria as the first sign of impending eclampsia. Eclampsia is the gravest form of toxemia of pregnancy. The presence of protein in this situation must be evaluated by the physician in conjunction with other clinical symptoms.Benign transient proteinuria may be the result of: exposure to cold, strenuous exercise, dehydration, and/or high fever. Benign transient proteinuria may also occur during the acute phase of a severe illness.

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Clinical Significance cont'd

Patients over the age of 60 have a greater chance of having protein in their urine. Occult malignancies and glomerulonephritis, that occur more frequently in the elderly, may be signaled by the presence of proteinuria. Orthostatic proteinuria is a condition seen most often in young adults. The condition may be caused by pressure on the renal nerve. When this condition is suspected, two urine specimens are tested. One specimen is collected upon arising in the morning, and the second is collected several hours later. When this condition is present, the first morning specimen, after the patient has been in a supine position, will be negative for protein. The second specimen, taken after the patient has been upright for several hours, would be positive for protein.

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The ability of proteins to alter the color of tetrabromphenol blue without altering the pH is an application of:View Page
A copper reduction method (e.g. Clinitest® or Benedict's) is performed on pediatric specimens in order to check for the presence of:View Page
False Positive Results

A false positive result for blood on the reagent strip can occur when oxidizing contaminants, such as hypochlorite (bleach), remain in collection bottles after cleaning. Contamination of the urine with provodine-iodine, a strong oxidizing agent, used in surgical procedures can result in a false positive reaction. Microbial peroxide found in association with urinary tract infections may also cause false-positive results. Capoten® (Captopril) can cause decreased reactivity. The muscle tissue form of hemoglobin, myoglobin is a well-known cause of false-positive reactions on the blood portion of the reagent strip. When tissue hemoglobin is present, the urine specimen has a clear red appearance. Patients suffering from muscle-wasting disorders or muscular destruction due to trauma, prolonged coma, or convulsions or individuals engaging in extensive exertion may have myoglobin in their urine. Specific tests for myoglobin, such as immunodiffusion techniques or protein electrophoresis, are needed to confirm the presence of this substance in a urine specimen. Levels of ascorbic acid normally found in urine do not interfere with this test.

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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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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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Clinical Significance

Using the esterase test in conjunction with pH, protein and nitrite provides a combination of tests which can screen for the presence of bacterial infection.

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False-negative results on reagent strips for leukocytes may occur when the specimen contains: (Choose ALL of the correct answers)View Page
Advantages and Limitations of the Reagent Strip Method for Specific Gravity

Specific gravity measured with the reagent strip method correlates well with gravimetric measurement, and, unlike the gravimetric or refractometer methods, does not need to be corrected for glucose or protein. Cloudy/turbid urines do not need to be clarified before measuring specific gravity with the reagent strip method. It is the recommended method for determining specific gravity if a urine specimen contains x-ray contrast media or plasma expanders. Alkaline urine can affect the indicator system and lower the specific gravity result on the reagent pad. If the result is being read visually, it is recommended that .005 be added to the result when the pH is alkaline. Most dipstick readers, however, will automatically adjust the specific gravity reading for pH. A specific gravity reading higher than the reagent strip range would need to be measured by another method, and may require dilution.

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CLIA Blood Banking Review
Which of the following is most commonly associated with febrile non-hemolytic transfusion reactions:View Page
Before testing all cord cells should be thoroughly washed in order to:View Page
Which of the following blood components will provide the best source of fibrinogen for a patient with hypofibrinogenemia:View Page
Antibodies to which of the following are the most frequent cause of febrile transfusion reactions:View Page

CLIA Chemistry / Urinalysis Review
Which of the following tests confirms the presence of Bence-Jones proteinuria:View Page
Which of the following methods is not a quantitative method for the determination of albumin:View Page
Which of the following thyroid function assays is affected least by pregnancy:View Page
Which of the following conditions will not produce a characteristic protein electrophoresis pattern:View Page
Elevation in CSF total protein may be seen in all of the following conditions except:View Page
Which of the following is found in plasma but absent in serum:View Page
The migration rate of proteins on cellulose acetate is primarily the result of:View Page
In a normal CSF the protein concentration as compared to that in the serum is generally:View Page
Which of the following methods would be used to confirm the presence of Bence-Jones protein in the urine:View Page
Label this lipoprotein electrophoresis scan: Ch = Cholesterol, Tr = Triglycerides, Pr = Protein, Ph = Phospholipid.View Page
Chylomicrons are primarily composed of:View Page
This SPE scan most likely represents which of the following disease states:View Page
This serum protein electrophoresis scan most likely represents which condition?View Page
Lipemia in a serum sample is most likely caused by an increase in serum levels of:View Page
Which band on the following serum protein electrophoresis scan is not made up of a mixture of proteins:View Page
Which of the following tests would be used in the assessment of glomerular filtration:View Page
Bence-Jones proteinuria can be seen in all of the following conditions except:View Page
In a patient with acute glomerulonephritis you would expect to find all but the following in the urine except:View Page
Which of the following methods may be employed to definitively identify Bence-Jones proteins:View Page
Which of the following would be the most appropriate method to confirm a positive protein from a urine dipstick:View Page

CLIA General Laboratory Review
Which of the following tests would be useful in the assessment of glomerular filtration:View Page
The assay which is most helpful in identifying specific allergens is:View Page
A hapten is only antigenic when it is coupled with which of the following:View Page
Which of the following is most responsible for increasing the erythrocyte sedimentation rate (ESR):View Page
C-reactive protein:View Page
Nephelometry involves the measurement:View Page
Serum proteins can be separated by cellulose acetate electrophoresis into how many basic fractions:View Page
Which one of the following terms does not describe a significant elevation of a single immunoglobulin class:View Page

CLIA Hematology / Hemostasis Review
This assay would be used to help rule out heparin contamination in a coagulation sample:View Page
An abnormality of which of the following assays would be least likely to be associated with thrombotic tendency:View Page

CLIA Microbiology / Serology Review
The capsular material used to identify capsular subtypes of Pneumococci consists of:View Page
Which of the following substances produced by Group A Streptococci is responsible for producing type specific immunity:View Page
Xanthochromia in CSF is characteristic of: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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Screening and Secondary Tests for Protein

A routine reagent strip protein method, based on the principle of "protein error of indicators," produces a visible colorimetric reaction that is capable of detecting most instances of proteinuria.Traditionally, laboratories have used sulfosalicylic acid (SSA) to confirm all positive protein reagent strip results, but this practice is not as common in today's laboratories. SSA is a precipitation method that reacts with all forms of protein. However, any substance that is precipitated by acid will produce false-positive SSA results. This includes radiographic dyes, cephalosporins, penicillins, and sulfonamides. SSA may be used as a secondary protein detection method if the urine is highly alkaline (pH of 9.0 or greater) which would overwhelm the buffering capacity of the reagent on the protein reagent stick. SSA may also be used as an alternative protein detection method if the urine is highly colored so that the colorimetric reaction is masked on the reagent strip.

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Sulfosalicylic Acid Test (Exton's Modification)

There are several acids which can be used to precipitate proteins - sulfosalicylic, trichloroacetic, nitric, and acetic acids. Sulfosalicylic acid (SSA) is the most frequently used acid test because it does not require the use of heat. Exton’s reagent is 5% sulfosalicylic acid in a solution of sodium sulfate. Exton (1925) found that adding sodium sulfate to the SSA causes a more uniform precipitate to be formed. To perform the SSA procedure mix equal parts of patient urine and the reagent. Rate the amount of turbidity according to the following scale:

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Persons with type 1 or type 2 diabetes should periodically have their urine monitored for:View Page
The screening test most commonly used to detect Bence-Jones protein in urine is:View Page
For which of the following reasons might SSA be used as a secondary urine protein detection method?View Page
Albumin is the major serum protein found in normal urine.View Page
A routine reagent strip test for protein is based on the principle of:View Page
Heat and Acid Test for Urinary Protein

The heat and acetic acid test is another semiquantitative test used to confirm the presence of protein in urine. It is more sensitive than the SSA test because the pH of the sample is brought close to the isoelectric point of proteins. However, this test is sometimes considered too sensitive because it can detect trace amounts of protein which are considered normal. The heat and acetic acid test gives false positive results with inorganic iodides, benzoin, tolutamide, and proteoses, similar to the SSA test. Bence-Jones protein consists of dimers of either kappa or lambda light chains from immunoglobulins. This abnormal protein is most often associated with multiple myeloma, but can also be found in cases of lymphoma, macroglobulinemia, leukemia, and other malignancies (Balant and Fabre, 1978). Testing for Bence-Jones protein is not part of the routine urinalysis. However, if Bence-Jones protein is suspected, the heat precipitation test or immunoelectrophoresis can be performed on a urine specimen. The heat precipitation test is based on the protein’s unusual solubility properties. Bence-Jones protein precipitates at temperatures between 40ºC and 60ºC (56ºC optimum), but dissolves again at 100ºC. Upon cooling, the precipitate will reappear around 60ºC and will dissolve again below 40ºC

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Urine Bilirubin

Normally, small amounts of conjugated bilirubin, regurgitate back from the bile duct and enter the blood stream, so small amounts of conjugated bilirubin can be found in the plasma. Since conjugated bilirubin is not bound to protein, it is easily filtered through the glomerulus and excreted in the urine whenever the plasma level is increased. Normally, no detectable amount of bilirubin (sometimes referred to as “bile”) is found in the urine.

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Current Topics in Clinical Microbiology
Pneumococcal Resistance

Most S. pneumoniae strains gain penicillin resistance by altering the penicillin-binding proteins in their cell wall.Penicillin molecules that cannot find a penicillin binding site cannot interfere with cell wall synthesis.Several different types of penicillin binding proteins may be involved, explaining the various levels of intermediate resistance that may be encountered with different strains of S. pneumoniae.Because different penicillin binding proteins may be involved, the level of penicillin resistance cannot be predicted by the oxacillin screening test.Infections caused by isolates of S. pneumoniae showing penicillin resistance in the intermediate range may be successfully treated by administering high doses of antibiotic.For this reason, the level of resistance with an accurate MIC test must be determined for all clinically significant isolates of S. pneumoniae.

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Middle ear damage in cases of S. pneumoniae infections are caused primarily by:View Page
Review 1

Francois P. Vaudaux P. Foster TJ. Lew DP.: Host-bacteria interactions in foreign body infections. Infection Control & Hospital Epidemiology. 17:514-20, 1996Persistent staphylococcal infections are a major medical problem, especially when they occur on implanted materials or intravascular catheters.This review describes some of the recently discovered molecular mechanisms of Staphylococcus aureus attachment to host proteins coating biomedical implants.These interactions involve specific surface proteins, called bacterial adhesins, that recognize specific domains of host proteins deposited on indwelling devices, such as fibronectin, fibrinogen, or fibrin.Elucidation of molecular mechanisms of S. aureus adhesion to the different host proteins may lead to the development of specific inhibitors blocking attachment of S. aureus, which may decrease the risk of bacterial colonization of indwelling devices.

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Decreasing the risk of staphylococcal colonization of indwelling catheters in the future may involve:View Page
Review 2

Cunningham MW.: Pathogenesis of group A streptococcal infections. Clinical Microbiology Reviews. 13):470-511, 2000Group A streptococci are model extracellular gram-positive pathogens responsible for pharyngitis, impetigo, rheumatic fever, and acute glomerulonephritis. A resurgence of invasive streptococcal diseases and rheumatic fever has appeared in outbreaks over the past 10 years, with a predominant M1 serotype as well as others identified with the outbreaks.Emm (M protein) gene sequencing has changed serotyping, and new virulence genes and new virulence regulatory networks have been defined. The emm gene superfamily has expanded to include antiphagocytic molecules and immunoglobulin-binding proteins with common structural features.At least nine superantigens have been characterized, all of which may contribute to toxic streptococcal syndrome. An emerging theme is the dichotomy between skin and throat strains in their epidemiology and genetic makeup. Eleven adhesions have been reported, and surface plasmin-binding proteins have been defined.The strong resistance of the group A streptococcus to phagocytosis is related to factor H and fibrinogen binding by M protein and to disarming complement component C5a by the C5a peptidase. Molecular mimicry appears to play a role in autoimmune mechanisms involved in rheumatic fever, while nephritis strain-associated proteins may lead to immune-mediated acute glomerulonephritis. Vaccine strategies have focused on recombinant M protein and C5a peptidase vaccines, and mucosal vaccine delivery systems are under investigation.

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Factors related to the strong resistance of certain strains of group A streptococci to phagocytosis include:View Page
Review 3

Rouquette C. Berche P. The pathogenesis of infection by Listeria monocytogenes Microbiologia. 12:245-58, 1996 Listeria monocytogenes is a Gram-positive bacterium responsible for severe infections in human and a large variety of animal species. It is a facultative intracellular pathogen which invades macrophages and most tissue cells of infected hosts where it can proliferate. The molecular basis of this intracellular parasitism has been to a large extent elucidated. The virulence factors, including internalin, listeriolysin O, phospholipases and a bacterial surface protein, ActA, are encoded by chromosomal genes organized in operons. Following internalisation into host cells, the bacteria escape from the phagosomal compartment and enter the cytoplasm. They then spread from cell to cell by a process involving actin polymerisation. In infected hosts, the bacteria cross the intestinal wall at Peyer's patches to invade the mesenteric lymph nodes and the blood. The main target organ is the liver, where the bacteria multiply inside hepatocytes. Early recruitment of polymorphonuclear cells lead to hepatocyte lysis, and thereby bacterial release This causes prolonged septicaemia, particularly in immunocompromised hosts, thus exposing the placenta and brain to infection. The prognosis of listeriosis depends on the severity of meningoencephalitis, due to the elective location of foci of infection in the brain stem (rhombencephalitis). Despite bactericidal antibiotic therapy, the overall mortality is still high (25 to 30%).

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Fundamentals of Hemostasis
Coagulation Disorders and Liver Disease

The liver is the site of production for the vast majority of our clotting factors. Therefore, impaired liver function could adversely affect these hemostatic proteins. Some early indicators of a potential liver problem include: An increase in factor VIII. It is not produced in the liver and will be present in elevated numbers as the body attempts to compensate. The PT is sensitive to liver function, so an unexpected, prolonged PT should be evaluated. A lack of fibrinogen is often indicative of severe liver disease. It is difficult to treat liver disease, so therapy typically centers around replacing the missing factors by way of administration of fresh frozen plasma.

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HIV Safety for Florida
The protein component that surrounds the genome is called a:View Page
Function of HIV Genes

HIV consists of nine genes. Three of the genes provide genetic information for the capsid proteins, envelope proteins, and viral enzymes. The other six genes are regulatory genes, controlling functions such as uncoating of the HIV genome and the penetration of host cells. Gene Number Abbreviation Gene Function 1 gag capsid proteins 2 pol viral enzymes 3 env envelope proteins 4 vif regulatory gene 5 tat regulatory gene 6 vpu regulatory gene 7 nef regulatory gene 8 vpr regulatory gene 9 rev regulatory gene

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Basic Structural Components

HIV consists of two basic components: a core of nucleic acid, called the genome, and a protein component that surrounds the genome, called a capsid. The genome carries the genetic information of the virus, while the capsid gives the virus its shape and protects the genome. The capsid is made up of subunits called capsomeres.

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Proteins Involved in Adsorption

The joining of the HIV and the host cell involves a spike on the HIV envelope and a CD4 molecule on the T-lymphocyte, macrophage, or brain cell.The molecule on the HIV spike is called glycoprotein 120 or gp120. The "120" refers to the molecular weight of the glycoprotein.While the CD4 site is important in viral binding, there is evidence that there are other molecules called co-receptors also involved.These molecules are embedded in the membranes of T-lymphocytes, macrophages, and brain cells. In the T-lymphocyte the abbreviated name of the protein molecule is CXCR4.

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Which structure of HIV contributes to the mutations?View Page

HIV: Structure and Replication
The protein component that surrounds the genome is called a:View Page
What is the function of the majority of HIV's genes?View Page
Function of HIV Genes

HIV consists of nine genes. Three of the genes provide genetic information for the capsid proteins, envelope proteins, and viral enzymes. The other six genes are regulatory genes, controlling functions such as uncoating of the HIV genome and the penetration of host cells. Gene NumberAbbreviationGene Function1gagcapsid proteins2polviral enzymes3envenvelope proteins4vifregulatory gene5tatregulatory gene6vpuregulatory gene7nefregulatory gene8vprregulatory gene9revregulatory gene

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Basic Structural Components

The HIV type-1 belongs to the Family Retroviridae and consists of two basic components: a core of nucleic acid, called the genome, and a protein component that surrounds the genome, called a capsid. The genome carries the genetic information of the virus, while the capsid gives the virus its shape and protects the genome. The capsid is made up of subunits called capsomeres.

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Spread of Infection (2)

At this time an enzyme called protease, using enzymes and proteins from preliminary protein molecules, forms capsomere segments which unite to form an icosahedral capsid.The capsid then changes into a bullet-shaped capsid and surrounds the viral RNA.Next some of the host cell's membrane joins with the viral glycoproteins gp120 and gp41 to form the spikes.Last, part of the host cell's surface membrane encloses the virus and becomes the envelope.

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Spread of Infection (1)

The proviral DNA provides genetic coding that instructs cellular enzymes to construct new HIV genomes, capsid proteins, and reverse transcriptase molecules.All of these are assembled near the edge of the host cell.

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Proteins Involved in Adsorption

The joining of the HIV and the host cell involves a spike on the HIV envelope and a CD4 molecule on the T-lymphocyte, macrophage, or brain cell.The molecule on the HIV spike is called glycoprotein 120 or gp120. The "120" refers to the molecular weight of the glycoprotein.While the CD4 site is important in viral binding, there is evidence that there are other molecules called co-receptors also involved.These molecules are embedded in the membranes of T-lymphocytes, macrophages, and brain cells. In the T-lymphocyte the abbreviated name of the protein molecule is CXCR4.

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Penetration and Infection

After penetration of the cell membrane by the gp41, the HIV capsid enters the cell's cytoplasm. Next, cellular enzymes strip away the capsid so that the HIV genome is released. Also stripped away are proteins p24 and p17. Protein 24 coats the HIV genome and protein 17 lines the inside of the capsid.

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Laws and Rules of the Florida Board of Clinical Laboratory Personnel
Description of Specialties (2)

Specialists in immunohematology perform all testing prior to blood transfusions and work to prevent transfusion infections. They also investigate any post-transfusion reactions. This specialty includes all lab procedures performed in the specialty of histocompatibility. Specialists in clinical chemistry analyze body fluids such as blood, urine, and spinal fluid to determine the chemical makeup, including the amount of carbohydrates, proteins, enzymes, and trace elements. The special covers urine microscopics and chemical evaluation of the liver, kidneys, lungs, heart, and other vital organ systems. This specialty also covers all testing performed in the specialties of radioassay and blood gas analysis. Specialists in blood banking can perform all immunohematology testing as well as testing from the specialties of clinical chemistry, hematology and serology/immunology that relates to donor blood. Specialists in immunohematology, clinical chemistry, hematology, and serology / immunology may perform all tests in the blood banking specialty.

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Mycology: Yeasts and Dimorphic Pathogens
The ingredient added to culture media to enhance the recovery of the dimorphic fungi by preventing the overgrowth of more rapidly growing, saprophytic molds is:View Page
Of the following responses, the one observation that would rule out cryptococcosis as the cause of meningoencephalitis is:View Page
Oral candidiasis may be directly exasperated by the habitual ingestion of:View Page

Normal Peripheral Blood Cells
Basophils in the Blood

Basophils circulate in the blood for a short time and make up only a small percentage (0.5%) of the cells in circulation.They do not migrate to the tissues under normal conditions but may be seen when inflammation resulting from hypersensitivity to protein, contact allergy or skin allograft rejection is present.Basophils are sometimes increased in patients with chronic myeloproliferative disorders.

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Pharmacology in the Clinical Lab: Therapeutic Drug Monitoring and Pharmacogenomics
Protein Binding

Most drugs are bound to proteins when they circulate in the body. Albumin is a major drug-binding protein in serum. Albumin is an alkaline protein, so acidic and neutral drugs primarily bind to it. If albumin binding sites become saturated, acidic and neutral drugs can bind to lipoproteins. Alkaline drugs tend to bind to globulins, particularly to the globulin, alpha-1 acid glycoprotein. Only free, unbound drugs are able to bind drug receptors and have therapeutic effects. An equilibrium exists in the systemic circulation between a free and protein-bound drug and between a free and receptor-bound drug. This is illustrated in the image to the right.

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Other Factors Affecting Drug Absorption and Distribution

In addition to protein availability, other factors may affect drug absorption and distribution in the body as a whole or at specific sites within the body. The following table highlights some of these other factors. Factor Discussion Regional blood flow Reduced area blood flow can be seen in diabetics and enhanced blood flow can be seen in tumors. Lipid solubility of the drug The more lipophilic a drug is, the more likely it will enter the central nervous system. The integrity of the GI tract In a diseased gut, an orally-administered drug may not be absorbed as expected. Age Drug kinetics and dispositions change throughout life. In general, metabolism of drugs is reduced in the elderly. Genetics Mutations or deletions in drug metabolizing enzymes can greatly affect a drug's disposition.

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Given what you have learned thus far, which of the following statements below do you think is true?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.

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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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Laboratory Methods

Immunoassay is the most common technique used by clinical laboratories for therapeutic drug monitoring. Antibodies that recognize drugs can be developed. Although most drugs are much too small to evoke an immune response, scientists can conjugate drugs to immunogenic proteins to produce antibodies that recognize drug-specific epitopes. There are several methods that utilize the principals of immunoassay for detection and quantification of therapeutic drugs in serum. Some of these methods are: Particle-enhanced turbidimetric inhibition immunoassay (PETINIA) Fluorescence Polarization Immunoassay (FPIA) Chemiluminescent assays

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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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Individualized Medicine

It has been said that we live in a new era of "individualized medicine". One of the primary drivers for this idea is the emerging field of pharmacogenomics (PGx). Pharmacogenomics (PGx) is the study of how individual variations in the human genome affect responses to medications. The term "pharmacogenetics" is also used for this discipline (people in the field use both terms); however, the term 'pharmacogenomics' is becoming more popular since we now know the entire human genome. The primary reason that individuals metabolize and respond to drugs differently is the inter-individual differences in receptor proteins and enzymes that metabolize the drugs. Mutations in these receptor proteins and enzymes can give rise to very different responses to drugs. In PGx, these mutations are referred to as variants.

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Warfarin cont.

The genes involved in warfarin metabolism are CYP2C9 and vitamin K epoxide reductase complex subunit 1 (VKOR). Warfarin owes its anticoagulant action to its inhibition of VKOR. This enzyme recycles vitamin K, a critical element for the clotting factors II, VII, IX, and X, as well as for proteins C, S, and Z. There are six CYP2C9 alleles that are known to cause prolonged metabolism of warfarin: CYP2C9 *2, *3, *4, *5, *6, and *11. (Polymorphisms in CYP450 genes are denoted with asterisks.)One-third of the patients that receive warfarin metabolize it differently than expected and experience a higher risk of bleeding.Genetic testing for the two most common polymorphisms (CYP2C9*2 and *3) as well as for VKOR may be able to reduce the variability associated with warfarin dosing response. Labs performing PGx testing can provide general warfarin dosing recommendations based on the patient's genotype analysis. The lab report will indicate whether a patient has a normal, mild, moderate, high, or very high sensitivity to warfarin. For example, a patient who has one CYP2C9 normal wild-type allele (CYP2C9 *1), one polymorphism (CYP2C9*3), and also a VKOR polymorphism is predicted to have a moderate sensitivity to warfarin. This patient should have frequent INR monitoring and possible warfarin dose reduction. It is important to recognize that knowing a genotype does not necessarily guarantee accurate dose prediction; other drugs and/or environmental or disease factors can also alter CYP2C9 activity. Therefore, monitoring the INR is still very important.

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CYP2D6

CYP2D6 has received the most attention: It is estimated that about 25% of common drugs are metabolized by CYP2D6. CYP2D6 accounts for only about 1% of all CYP450 enzymes, but it is important in the metabolism of about 100 drugs. There are more than 80 genetic variants that have been described in the CYP2D6 gene. The normal, wild-type allele displays normal metabolic activity whereas some of the variant forms have enhanced or diminished activity. The variants can be grouped generally according to the resulting alterations in protein function. The groupings correlate with four major enzyme metabolic capacities (phenotypes): poor, intermediate, extensive (normal), or ultra-rapid metabolizers.

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Phlebotomy
Hepatic function panel

Albumin (Alb) Bilirubin (Bili) Alkaline phosphatase (Alk Phos) Total protein (TP) Alanine aminotransferase (ALT) Aspartate aminotransferase (AST)

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Comprehensive metabolic panel (CMP)

Consists of a basic metabolic panel, plus:Albumin (Alb) and Bilirubin (Bili) Alkaline Phosphatase (Alk Phos) Total protein (TP) Alanine aminotransferase (ALT) Aspartate aminotransferase (AST)

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Clots

Blood clots when the coagulation factor proteins within the plasma are activated.Blood starts to clot almost immediately after it is drawn unless it is exposed to an anticoagulant.Clots within the blood specimen, even if not visible to the naked eye, will yield inaccurate results.

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Blood clots

When a blood sample is left standing without anticoagulant, it forms a coagulum or blood clot. The clot contains coagulation proteins, platelets, and entrapped red and white blood cells.

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Plasma components

Plasma is the liquid portion of the blood. It contains many substances including:Water Electrolytes Sugars Proteins Lipids Drugs & Toxins

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Plasma proteins

Numerous types of proteins are dispersed in the plasma. These include: Coagulation proteins (blood clotting factors), which, if activated, will form a blood clot , and Serum proteins, which are left dispersed in liquid after the clot is formed. Serum proteins include: Albumin, a marker of nutrition, and Globulins, or antibodies.

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Whole blood: components

Circulating whole blood is a mixture of: Plasma (which contains fluid, proteins, and lipids), and Formed elements, consisting of red cells, white cells, and platelets.

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Serum

Serum is the fluid that is left over the coagulum after the specimen is centrifuged (spun down). Serum contains all the same substances as plasma, except for the coagulation proteins, which are left behind in the blood clot.

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Platelets

Platelets are small cell fragments present in large numbers in blood.They work together with the blood coagulation proteins to form a blood clot.

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Red Cell Disorders: Peripheral Blood Clues to Nonneoplastic Conditions
Pappenheimer bodies

Pappenheimer bodies are iron-containing granules that aggregate with mitochondria and are deposited in RBC or normoblast cytoplasm. Small and irregular, they are found only in pathological states as thalassemia and sideroblastic anemias(upper image). Wright-Giemsa stain defines the cytoplasmic content (protein), but Prussian blue staining is necessary to define the iron content, the essence of the Pappenheimer body (lower image). Pappenheimer bodies lie typically in small clusters (upper image) and tend to locate at the periphery of the red cell cytoplasm. A cluster is typically smaller than a single Howell-Jolly body.

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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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The arrangement of erythrocytes on this peripheral blood smear may be seen in each of the following conditions except:View Page
The arrangement of the erythrocytes in this peripheral smear should be reported out as rouleaux formation.View Page
Hereditary ovalocytosis and elliptocytosis

Ovalocytes are rod shaped erythrocytes with nearly parallel lateral walls. If the long axis of an erythrocyte is no more than twice as long as the short axis, the cell is an ovalocyte. If the long axis is more than twice as long as the short axis, the cell is an elliptocyte. Hemoglobin tends to collect at each end of these cells. The ends of the cells are rounded and never pointed, to be differentated from sickle cells. Ovalocytes present in greater than 25% of red cells on the blood smear are characteristic of hereditary ovalocytosis. The oval shape is attributed to a defect in horizontal red cell membrane protein interactions. Lesser numbers of circulating ovalocytes may be present in various anemias including megaloblastic, sideroblastic, iron deficiency, and in thalassemias. A rare ovalocyte (less than 1%) may be found on almost any peripheral blood smear. Resistance to malarial infection may be a beneficial attribute of hereditary ovalocytosis.

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Red Cell Morphology
Elliptocytes

Elliptocytes can vary in appearance from slightly oval to thin pencil-shaped forms. Less than 1% of red cells in normal blood are oval. Many examples of elliptocytes can be seen in this smear from a patient with hereditary elliptocytosis(HE). All cases of HE are associated with weakening of membrane skeleton and defective association of proteins that hold the skeleton together. The function of elliptocytes appear to be unaffected in most cases. Notice that the cells vary in shape from slightly oval to cigar-shaped. The largest percentage of elliptocytes is seen on smears from patients with hereditary elliptocytosis. Since many of these patients have no symptoms, the presence of elliptocytes on the smear may be the only diagnostic feature.

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Semen Analysis
Testing collection containers

In order to test collection containers for sperm collection, the sperm must be held in the container for several hours to ensure that neither the numbers nor motility are adversely affected. Numbers will decline if the sperm adhere to the container. Motility will decline if the container is toxic. One method of testing involves removing sperm from semen. The specimen would be centrifuged and the sperm pellet diluted in a small volume of culture medium containing an energy source and at least 0.5% of a protein, such as serum albumin. The processed sperm specimen would be placed in the container to be tested. Total count and motility of the sperm would be tested at the start of incubation and 24 hours later. The container is non-toxic if the motility at the end of 24 hours is no less than 50% of the original value.

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The Urine Microscopic: Microscopic Analysis of Urine Sediment
All of the following results are consistent with the finding of casts in urinary sediment EXCEPT:View Page
The patient was a female and the examination was completed within two hours of collection. Which of the following findings are inconsistent with the rest of the report?View Page
Formation and Significance of Casts

Casts are cylindrical bodies formed either in the distal convoluted tubules or the collecting ducts of the kidney. Since the walls of the tubule act as a mold for cast formation, the width of the tubule determines the width of the cast. Thus, narrow casts are formed in the distal tubules while broad casts are formed in the collecting ducts. The matrix of all casts is thought to be Tamm-Horsfall protein, a glycoprotein secreted by the distal loop of Henle and the distal tubule. This protein entraps cells and granular material of tubular origin. Very few casts are seen in the urine of a person without renal disease, except for hyaline casts, which may be transiently present after strenuous exercise, and during fever, diuretic therapy, and congestive heart failure. A significant number of urinary casts usually indicates the presence of renal disease.

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Granular Casts

Granular casts are composed of plasma protein aggregates and cellular remnants. Granular casts appear as cylinders of coarse, or fine, highly refractive particles. Coarsely granular casts (yellow arrow) contain large, coarse dark yellow or dark brown granules. They are very irregular in shape as shown in this high power magnification under the brightfield microscope. A hyaline cast can be seen just to the left of the coarse granular cast (blue arrow).

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All of the following factors favor cast formation except:View Page
Factors Promoting Cast Formation

The following factors promote the formation of casts in the kidney:Larger than normal amounts of plasma proteins entering the tubules,Decreased pH.Decreased urinary flow rate.Increased urine concentrationAfter formation, casts are washed loose from the tubules and discharged into the urine, where they can be found its sediment.

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The formed elements which may be present in urine sediment include:View Page
Cholesterol Crystals

Cholesterol crystals may be seen in renal tubular disease. These crystals look like plates of glass, sometimes with a notch out of one corner. Under polarized light, they exhibit a stained glass effect. These crystals are rarely seen unless the specimen has been refrigerated, because the lipids remain in droplet form. Large amounts of protein, lipid droplets, fatty casts or oval fat bodies should be found along with cholesterol crystals. Cholesterol crystals are found in acid or neutral urine.

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Specimen #4 - Adult Male

The results of this specimen are abnormal but the abnormalities correlate with each other. The turbidity can be explained by the presence of bacteria and crystals. The presence of RBCs in the microscopic explains the blood found on the dipstick. The casts, bacteria and WBCs can account for the increased protein. The results may be reported.

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Tuberculosis Awareness for Healthcare Workers
Tuberculin Skin Test (TST)

0.1 ml of purified protein derivative (PPD) is injected intradermally on the left arm.The needle tip is inserted so that the needle is visible under the first layer of skin, bevel up.The PPD is injected slowly to produce a small wheal measuring 5-6 mm in diameter.

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Variations in White Cell Morphology - Granulocytes
Toxic Granulation

Toxic granulation is manifested by the presence of large granules in the cytoplasm of segmented and band neutrophils in the peripheral blood. The color of these granules can range from dark purplish blue to an almost red appearance. Toxic granules are azurophilic granules normally present in early myeloid forms, but which are not normally seen at the band and segmented stages of neutrophil maturation. These granules contain peroxidases and hydrolases. Toxic granulation is seen in cases of severe infection, as a result of denatured proteins in rheumatoid arthritis or, less frequently, as a result of autophagocytosis. Infection is the most frequent cause of toxic granulation. This type of granulation may be seen in cells which also contain Dohle bodies and/or vacuoles. Cells containing toxic granules may have decreased numbers of specific granules. Cells containing only a few specific granules, with or without toxic granules, are said to be degranulated. The nucleus in degranulated cells may often be round-bilobed, smooth and pyknotic. This type of nucleus is the result of aging and will disintegrate soon. Increased basophilia of azurophilic granules simulating toxic granules may occur in normal cells with prolonged staining time or decreased pH of the stain.

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Chediak-Higashi

Chediak-Higashi syndrome is a rare autosomal recessive disorder. It results from a mutation of the gene LYST which encodes a protein with multiple phosphorylation sites. This defect causes a cellular abnormality involving the fusion of cytoplasmic granules. Early in neutrophil maturation normal azurophilic granules form, but they fuse together to form megagranules. Later during the myelocyte stage, normal specific granules form. The mature neutrophils contain both normal specific granules and abnormal azurophilic granules. These large abnormal granules can be seen in the cytoplasm of neutrophils, eosinophils, basophils, monocytes and lymphocytes. These abnormal granules are able to kill bacteria in neutrophils and monocytes; however, the process is much less effective than in normal cells in part, because these neutrophils have impaired locomotion. For these reasons, individuals with Chediak-Higashi have recurrent infections. An accelerated lymphoma-like phase occurs, with lymphadenopathy, hepatosplenomegaly, and pancytopenia. Death often occurs at an early age.

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White Cell and Platelet Disorders: Peripheral Blood Clues to Nonneoplastic Conditions
Eosinophil description

The cytoplasm of eosinophils is evenly filled by numerous orange-red granules of uniform size. They do not overlie the nucleus.The eosinophil granules contain numerous enzymes including peroxidase, phospholipase D, catalase, acid phosphatase, and vitamin B12-binding proteins.Their ability to kill bacteria is less than that of neutrophils. Their main purpose is to counteract parasitic infections and to participate in immune allergic reactions.They may also be increased in a variety of nonimmunologic inflammatory responses from bacteria and fungi causing chronic infections. Malignancies, collagen vascular diseases, and myeloproliferative disorders may also may be settings for prominent eosinophils.

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Approximately 10% of the circulating white cells were similar to the one seen in the photograph. The patient was 42 years old and visited his physician because of recent bruising. Note the absence of platelets on the smear. Possible associated conditions include:View Page


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