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Laboratories Individuals

Chemical Screening of Urine by Reagent Strip
Precautions

The reagent strips must be handled and stored properly in order to ensure that results are accurate. The following precautions should be observed: Store strips according to the manufacturer's recommendation. DO NOT expose strips to moisture, direct sunlight or volatile fumes. Remove only enough strips for immediate use and immediately recap the bottle. Avoid contamination of test strips. Do not touch the test areas with fingers or do not lay the test strips directly on the workbench. DO NOT use discolored strips. Compare the color of the unused strip to the negative area on the color chart provided by the company. The color should be similar. Check the expiration date. Re-label the container with a revised expiration date if the manufacturer states a shortened usage period once the container has been opened. Reagent strips must be tested periodically (frequency defined by the laboratory) for clinical reactivity with normal and abnormal urine controls. Urine controls are available commercially or may be prepared and preserved in-house.

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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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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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Clinical Significance cont'd

Conditions in which glucose levels in the urine are above 100 mg/dL and detectable include:diabetes mellitus and other endocrine disordersimpaired tubular reabsorption due to advanced kidney diseasepregnancy - glycosuria developing in the 3rd trimester may be due to latent diabetes mellituscentral nervous system damagepancreatic diseasedisturbances of metabolism such as, burns, infection or fractures

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Clinical Significance of Positive Urine Ketone Result

Ketone bodies are usually absent in urine. High levels of ketones are present in the urine of individuals with uncontrolled diabetes. In diabetes the ketones are present because the body's ability to metabolize carbohydrates is defective. Detecting the presence of ketones in the urine is a valuable aid to managing and monitoring individuals with diabetes mellitus. Ketonuria is an indication that the insulin dose needs to be increased. It is also an early indicator of insulin dosage problems in juvenile diabetes or in diabetics experiencing other medical problems. Electrolyte imbalance and dehydration occur when ketones accumulate in the blood. If these conditions are not corrected, the patient may develop acidosis and ultimately diabetic coma. Low levels may be detected during conditions of physiological stress such as fasting, rapid weight loss, frequent strenuous exercise or prolonged vomiting. The presence of ketones in these situations is due to either inadequate intake or increased loss of carbohydrates.

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Clinical Significance

Liver damage or an obstructed bile duct allows conjugated bilirubin to enter the circulation and ultimately to appear in the urine. Patients with clinical jaundice due to hepatitis or cirrhosis will have bilirubinuria. If the jaundice is due to red cell destruction, there is an increase in unconjugated bilirubin which the kidneys cannot excrete.

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Clinical Significance

No blood is found in the urine of healthy individuals although samples from menstruating females, frequently, but not always, test positive for blood. Hematuria is associated with renal or genital urinary disorders in which the bleeding is the result of irritation to the involved organs or trauma. Examples include renal calculi, pyelonephritis, glomerulonephritis, tumors, trauma or exposure to toxic chemicals or drugs and/or strenuous exercise. Hemoglobinuria may be due to the lysis of red cells within the urinary tract. If it is caused by intravascular hemolysis, the hemoglobin is then filtered through the glomeruli. In the normal individual, the hemoglobin molecule attaches to haptoglobin and in this way bypasses the kidney filtration system. When the hemoglobin/haptoglobin system is overwhelmed, as in cases of hemolytic anemia, severe burns, transfusion reaction, infection or strenuous exercise, hemoglobin passes into the urine.

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Clinical Significance

Early detection of bacteria is important in order to prevent cystitis from developing into inflammation or infection involving the kidney and renal pelvis. The nitrite portion of the test strip can be used to screen individuals who are at risk for developing urinary tract infections, such as diabetics, persons with recurrent infections, or pregnant women. The test is also useful in evaluating the success of antibiotic therapy that is used to treat a bladder infection.

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Clinical Significance

Urinary urobilinogen may be increased in the presence of a hemolytic process such as hemolytic anemia. It may also be increased with infectious hepatitis, or with cirrhosis. Comparing the urinary bilirubin result with the urobilinogen result may assist in distinguishing between red cell hemolysis, hepatic disease, and biliary obstruction. Urobilinogen is increased in hemolytic disease and urine bilirubin is negative. Urobilinogen is increased in hepatic disease, and urine bilirubin may be positive or negative. Urobilinogen is low with biliary obstruction, and urine bilirubin is positive. Reagent strips methods however, cannot distinguish normal urobilinogen from absent urobilinogen, as might be seen in complete biliary obstruction.

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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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Clinical Significance

Measurement of specific gravity provides information regarding a patient's state of hydration or dehydration. It also can be used to determine loss of renal tubular concentrating ability.

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CLIA Blood Banking Review
Which of the following is the proper temperature to use when crossmatching in the presence of a cold antibody:View Page

CLIA Chemistry / Urinalysis Review
The elements indicated by the arrows are more likely to be seen in patients with which condition:View Page
Identify the urine sediment element shown by the arrow:View Page
Match collection tube colors and additive type on the right with clinical usage on the left.View Page

CLIA General Laboratory Review
Which one of the following does not directly regulate clinical laboratories:View Page

CLIA Hematology / Hemostasis Review
Pelger-Huet anomaly is characterized by:View Page
Disseminated intravascular coagulation (DIC) is associated with all of the following clinical conditions except:View Page
An India Ink preparation in used to identify:View Page
Match the clinical findings with the associated type of leukemia:View Page

CLIA Microbiology / Serology Review
Which of the following statements about Rickettsia is false:View Page
Which one of the following viruses requires a complex lymphoblastoid cell culture, and is rarely if ever diagnosed by culture:View Page
Which of the following tests would be used to directly document the presence of a specific organism in a clinical specimen:View Page
Which of the following specimens is the most sensitive for detecting active CMV infection:View Page

Confirmatory and Secondary Urinalysis Screening Tests
Microalbumin Test

The presence of low levels of albumin (microalbumin) in the urine is an important finding in an individual with either type 1 or type 2 diabetes. The development of clinical nephropathy leads to reduced glomerular filtration and eventually may lead to renal failure. For this reason, early detection of microalbumin is important in order to avert renal complications in a diabetic patient. The presence of microalbuminuria has also been associated with an increased risk for cardiovascular disease. Reagent strips that are used for routine urinalysis cannot detect low levels of albumin excretion (1 to 2 mg/dL). Special reagent strips that are sensitive for these low levels of albumin are useful for periodic monitoring of patients with diabetes, hypertension, or peripheral vascular disease.

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Causes for Bilirubinuria

A screening test for bilirubin in the urine is included in most urine dipsticks and may be present when liver disease or damage is suspected. Bilirubinuria can be detected before other clinical symptoms such as jaundice are present or recognizable. The detection of small quantities is very important in early diagnosis of obstructive and hepatic jaundice. This test is also useful in the differential diagnosis of obstructive jaundice (positive for bilirubinuria) vs. hemolytic jaundice (negative for bilirubinuria).

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Testing for Reducing Substances Other Than Glucose

Testing pediatric urine specimens for reducing substances other than glucose is a policy that should be implemented in the urinalysis laboratory. The maximum age for this testing is defined by each laboratory and is usually based on consultation with the pediatric clinical staff. The policy that is implemented in most laboratories is to test urine specimens for other reducing substances if the glucose test on the reagent strip is negative and the urine specimen is from a child below the age of one. Verify the policy for your own laboratory because the cutoff age for testing may be different.

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Current Topics in Clinical Microbiology
A 25 year-old female presented in the emergency room with an acute urethral discharge of 2 days duration. A smear for gram stain was obtained (see accompanying image). Many polymorphonuclear leukocytes and intracellular and extracellular gram negative diplococci were observed. Based on the clinical history and the gram stain observation, a diagnosis of gonorrhea can be made.View Page
The carbohydrate utilization reaction seen in the QuadFerm system shown in the picture provides a definitive identification of N. gonorrhoeae:View Page
Review 2

Smith KR, Fisher HC III, Hook, EW III: Prevalence of fluorescent monoclonal antibody-nonreactive Neisseria gonorrhoeae in five North American sexually transmitted disease clinics.J Clin Microbiol 34:1551-1552, 1996We compared a direct fluorescent monoclonal antibody (DFA) test with alternative enzymatic and fermention tests for identifying presumptive gonococcal isolates in a systematic sample from patients attending five sexually transmitted disease clinics in five cities.Fourteen (2.5%) of 556 isolates from three clinics were nonreactive with the DFA confirmatory reagent and reactive by both the Quad-Ferm and Rapid NH tests. The prevalence of DFA-nonreactive Neisseria gonorrhoeae isolates varies geographically and is independent of local methods for the identification of possible gonococci.On the basis of our findings, we recommend that for use in medicolegal and other instances in which a diagnosis of gonorrhea has the potential to have far-reaching effects, it is appropriate to test DFA reagent-nonreactive, oxidase-positive, gram-negative diplococci by alternative methods of gonococcal confirmation.Although the prevalence of such isolates could change, the fluorescent monoclonal antibody confirmation reagents remain useful for many clinical situations. Their ease of use and ready applicability for screening large numbers of isolates make them useful for many laboratories.

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The Gram stain report to be issued based on the microscopic characteristics seen in the accompanying picture would most correctly be, "many wbc with"..View Page
Clinical History

A 67 year-old man entered the hospital with cough, right lower chest pain accentuated by deep breathing, and fever. He had a history of chronic obstructive pulmonary disease secondary to a long history of smoking. The temperature on admission was 39.2C, and auscultation of the chest revealed rales in the right lower lung field. The admission white blood count was 13,500/ml with 80% segmented neutrophils and a shift to the left. A blood culture was obtained.

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Clinical isolates of Escherichia coli and Klebsiella pneumoniae may possess ESBL activity. Therefore, clinical laboratories should be screening all clinically significant isolates of these two species.View Page
Extended Spectrum Beta Lactamases

In follow-up to the observations of the ESBL screening test, the following antibiotic susceptibility profile was later reported: Ampicillin = R; Cefazolin = R; Cefoxitin 1 = S; Ciprofloxacin 0.25 = S; Gentamicin 1 = S; Ceftazidime 32 = R; Imipenem The susceptibility of the 2nd generation drug cefoxitin, with resistance of the 1st generation cefazolin and the 3rd generation ceftazidime, is another way in addition to the screening test in which ESBL activity may be detected. It is recommended that clinical microbiologists check the antibiotic susceptibility profiles for possible ESBL activity of clinically significant isolates of K. pneumoniae and E. coli.Most automated systems have built in methods for automatically detecting an ESBL isolate, or provide an "alert" that such a strain may be present.

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Review 1

Podschun R. Ullmann U.: Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors Clinical Microbiology Reviews. 11(4):589-603, 1998Bacteria belonging to the genus Klebsiella frequently cause human nosocomial infections. In particular, the medically most important Klebsiella species, Klebsiella pneumoniae, accounts for a significant proportion of hospital-acquired urinary tract infections, pneumonia, septicemias, and soft tissue infections.The principal pathogenic reservoirs for transmission of Klebsiella are the gastrointestinal tract and the hands of hospital personnel. Because of their ability to spread rapidly in the hospital environment, these bacteria tend to cause nosocomial outbreaks. Hospital outbreaks of multidrug-resistant Klebsiella spp., especially those in neonatal wards, are often caused by new types of strains, the so-called extended-spectrum-beta-lactamase (ESBL) producersThe incidence of ESBL-producing strains among clinical Klebsiella isolates has been steadily increasing over the past years. The resulting limitations on the therapeutic options demand new measures for the management of Klebsiella hospital infections.While the different typing methods are useful epidemiological tools for infection control, recent findings about Klebsiella virulence factors have provided new insights into the pathogenic strategies of these bacteria. Klebsiella pathogenicity factors such as capsules or lipopolysaccharides are presently considered to be promising candidates for vaccination efforts that may serve as immunological infection control measures.

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Clinical History

A 72- year old woman had a history of recurrent urinary tract infections over the past several months, for which she had received different regimens of antibiotics including ampicillin, trimethoprim-sulfasoxazole, and ciprofloxacin.Relapses often occurred 10 days to two weeks after cessation of therapy.The current flare up, manifest by dysuria, lower abdominal pain and cloudy urine was accompanied by shaking chills and spiking fever.A sterile mid-stream urine specimen was sent to the laboratory for culture.

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PYR Differential

As mentioned before, the spot PYR test is commonly performed to separate Enterococcus species (positive reaction) from the Group D streptococci (S. bovis, S. equinus), which are negative.It should be remembered that Streptococcus pyogenes (group A) also produces PYR; therefore, additional characteristics such as beta hemolysis are important.Some species of Aerococcus and Gemella are also PYR-positive; however, they can be suspected if large cocci in tetrads or clusters are observed on gram stain.These species are rare isolates in most clinical practices.

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Review 2

Suppola JP. Kuikka A. Vaara M. Valtonen VV. Comparison of risk factors and outcome in patients with Enterococcus faecalis vs Enterococcus faecium bacteremia. Scandinavian Journal of Infectious Diseases. 30(2):153-7, 1998.The purpose of our study was to determine retrospectively the risk factors for the acquisition of Enterococcus faecalis vs E. faecium bacteremia, as well as the clinical outcomes of these patients.62 patients with Enterococcus faecalis bacteremia were compared to 31 patients with E. faecium bacteremia. Haematologic malignancies, neutropenia, high-risk source and previous use of aminoglycosides, carbapenems, cephalosporins and clindamycin were significantly associated with E. faecium bacteremia. Instead, urinary catheterization was found to be related to Enterococcus faecalis bacteremia. The mortality rates within 7 d and 30 d were 13% and 27%, respectively, in patients with E. faecalis bacteremia and 6% and 29%, respectively, in patients with E. faecium bacteremia.There was no difference in mortality between E. faecalis and E. faecium bacteremia, nor was there a difference in seriousness of disease at the time of bacteremia. In the subgroups of patients with monomicrobial or clinically significant E. faecalis vs E. faecium bacteremia, the mortality rates were similar to the results of all subjects.Our results do not support the theory that E. faecium would be a more virulent organism than E. faecalis

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The gram stain shown in the photograph was prepared from a positive anaerobic blood culture bottle after 36 hours incubation. Based on the morphology of the bacterial cells (some with spores--blue arrows), the most likely identification is:View Page
Review 2

Citron DM. Appelbaum PC.: How far should a clinical laboratory go in identifying anaerobic isolates, and who should pay? Clinical Infectious Diseases. 16 Suppl 4:S435-8, 1993Identification of anaerobic bacteria in specimens from sites of infection due to mixed organisms can be time-consuming and expensive. Laboratories should limit anaerobic workups by testing only those specimens that have been properly collected and transported to the laboratory.Use of selective and differential media for initial processing can provide rapid and relevant information to the clinician. Anaerobes isolated from normally sterile sites and sites of serious infection should always be completely identified. Group-or genus-level identifications may suffice in other instances.The Bacteroides fragilis group of organisms should always be identified because of their virulence and resistance to many antimicrobial agents.Some of the other organisms that warrant identification include Clostridium septicum (associated with gastrointestinal malignancy); Clostridium ramosum, Clostridium innocuum, and Clostridium clostridioforme (which are resistant to antibiotics); Clostridium perfringens (a cause of myonecrosis and gas gangrene,potentially serious infection); anaerobic cocci (which may be resistant to metronidazole and clindamycin); and fusobacteria (which may be virulent and resistant to clindamycin and penicillin).

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Review 3

Kornbluth AA. Danzig JB. Bernstein LH.: Clostridium septicum infection and associated malignancy. Report of 2 cases and review of the literature. Medicine. 68(1):30-7, 1989We report 2 patients with myonecrosis due to Clostridium septicum and associated colon carcinoma and have reviewed the English language literature for all reported cases of atraumatic C. septicum infection. A total of 162 cases of C. septicum infection have been reported.Eighty-one percent of these patients had an associated malignancy. Thirty-four percent of all patients had associated colon carcinoma, while 40% had a hematologic malignancy. Thirty-seven percent of reported patients had an occult malignancy at the time of their infection with C. septicum. In many patients, the portal of entry was found in the large intestine.In a particularly lethal form (79% mortality) of C. septicum infection, known as "distant myonecrosis," infection metastatic from the initial site of infection causes severe myonecrosis, gangrene, and often death within hours of clinical detection. Overall, survival of patients with C. septicum infection is only 35%.Review of all cases of C. septicum infection suggests several conclusions. 1) Patients with malignancy, particularly colonic or hematologic, and patients with cyclic neutropenia who develop signs and symptoms of sepsis, especially with associated findings of abdominal pain or pain in an extremity, should be treated for possible clostridial infection. 2) C. septicum infection does not appear to be a result of a single specific defect in either humoral or cell-mediated immunity. Rather, it may occur in patients who are granulocytopenic and therefore prone to an enterocolitis. 3) Patients in whom an infection with C. septicum is found must undergo a vigorous search for malignancy following ac

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Clinical History

An 18-year old female incurred a deep penetrating injury of the dorsum of her right foot when a kitchen knife fell from a platter she was carrying while going barefoot. The initial injury partially resolved; however, three days later the foot began to swell, become red, and painful. A deep subcutaneous abscess developed, with a central sinus tract from which a cloudy, serous fluid exuded (see photograph).

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MRSA Screen

Perhaps the most efficient means for detecting methicillin-resistant staphylococci in clinical laboratories is the use of the agar dilution screening test.Illustrated in the photograph is a Mueller-Hinton agar plate containing 6ug/ml of oxicillin, previously inoculated with a strain of Staphylococcus aureus. Oxacillin is used as a marker for methicillin resistance because it is more stable in the agar medium. Growth on this screening medium is presumptive for methicillin resistance.Thus, in the presence of growth, as shown here, a follow-up MIC test must be performed to determine the exact level of resistance.

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Review 2

Hershow RC. Khayr WF. Smith NL.: A comparison of clinical virulence of nosocomially acquired methicillin-resistant and methicillin-sensitive Staphylococcus aureus infections in a university hospital (University of Illinois at Chicago). Infection Control & Hospital Epidemiology. 13(10):587-93, 1992OBJECTIVES: To compare the clinical virulence of nosocomially acquired methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) infections in 1989.DESIGN: A retrospective comparison of host factors, in-hospital exposures, sites of infections, and outcomes of patients with nosocomial MRSA and MSSA infections. PARTICIPANTS: Forty-four adult patients with nosocomial S.aureus infections.RESULTS: The 22 MRSA-infected and 22 MSSA-infected persons were similar regarding mean age, gender, underlying diseases, and exposure to surgery. Before developing infection, MRSA-infected persons were more likely to have received antibiotics and to have stayed in the hospital > 2 weeks. Bacteremia was the most common presentation in the MRSA and MSSA groups (55% and 59%, respectively). Infectious complications and death were infrequent in both groups.CONCLUSIONS: MRSA and MSSA strains infect patients with similar demographic features and underlying diseases, but MRSA infections are significantly more common among patients with previous antibiotic therapy and a prolonged preinfection hospital stay. Clinical presentations and outcomes did not differ significantly between the 2 groups. Thus, similar to studies in the early 1980s, our findings do not suggest greater intrinsic virulence of MRSA.

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Review 3

Ladhani S. Joannou CL. Lochrie DP. Evans RW. Poston SM.: Clinical, microbial, and biochemical aspects of the exfoliative toxins causing staphylococcal scalded-skin syndrome. Clinical Microbiology Reviews. 12:224-242, 1999The exfoliative (epidermolytic) toxins of Staphylococcus aureus are the causative agents of the staphylococcal scalded-skin syndrome (SSSS), a blistering skin disorder that predominantly affects children. Clinical features of SSSS vary along a spectrum, ranging from a few localized blisters to generalized exfoliation covering almost the entire body.The toxins act specifically at the zona granulosa of the epidermis to produce the characteristic exfoliation, although the mechanism by which this is achieved is still poorly understood.Despite the availability of antibiotics, SSSS carries a significant mortality rate, particularly among neonates with secondary complications of epidermal loss and among adults with underlying diseases.

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A clinical condition often associated with Streptococcus milleri (anginosus) is:View Page
Review 1

Piscitelli SC., Shwed J., Schreckenberger P., Danziger LH. Streptococcus milleri group: renewed interest in an elusive pathogen. European Journal of Clinical Microbiology & Infectious Diseases.11:491-8, 1992The following review examines the bacteriological characteristics, epidemiology, pathogenicity and antimicrobial susceptibility of the "Streptococcus milleri group". "Streptococcus milleri group" is a term for a large group of streptococci which includes Streptococcus intermedius, Streptococcus constellatus and Streptococcus anginosus.Usually considered commensals, these organisms are often associated with various pyogenic infections including cardiac, intra-abdominal, subcutaneous and central nervous system infections, particularly with the formation of abscesses.Organisms of the "Streptococcus milleri group" are often unrecognized pathogens due to the lack of uniformity in classifications and difficulties in microbiological identification. Penicillin G, cephalosporins, clindamycin and vancomycin all possess activity against these streptococci.Use of agents with poor activity may promote infections with "Streptococcus milleri group" and allow it to exhibit its pathogenicity. An understanding of these organisms may aid in their recognition and proper treatment.

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The epidural and subdural abscesses in the two patients reported by Gelfand, et al, are clinical manifestations uncommon for S. milleri.View Page
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
Review 1

Spencer RC.: Invasive streptococcEuropean Journal of Clinical Microbiology & Infectious Diseases. 14 Suppl. 1:S26-32, 1995.Before the introduction of antibiotics, serious infections caused by Streptococcus pyogenes (Lancefield Group A streptococci) were common. Before World War II, this bacterium was responsible for as many as 50% of postpartum deaths and was the major cause of death in patients with burns. Also common were the sequelae of streptococcal infections-rheumatic fever and post-streptococcal glomerulonephritis.With the use of penicillin, however, Streptococcus pyogenes was believed to be virtually eliminated as a pathogen. The organism was consigned to the history books, but not for long.In the mid-1980s, focal resurgences of rheumatic fever began to be reported from different areas in the USA, such as Salt Lake City, Utah. In such communities, where increases in cases of rheumatic fever had been reported, the serotypes M-1, 3, 5, 6 and 18 were isolated which, on culture, produced characteristic mucoid colonies. At the same time, reports of increases in invasive streptococcal disease began to surface in both the USA and Europe.Two syndromes were described; invasive streptococcal infection, occurring in previously healthy children and adults, commonly associated with septicaemia resulting from a deep focus of infection such as bone or lung; and streptococcal toxic shock syndrome, involving a cutaneous focus, accompanied by necrotizing or bullous soft tissue changes. Septicaemia is rare in streptococcal toxic shock syndrome, but the most characteristic feature is one of rapidly progressing multi-organ failure. A high proportion of the strains of Streptococcus pyogenes associated with this condition are serotype M-1, and fatality rates approaching 50% have been reported.

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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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Clinical History

The prototype history for this organism is either a still birth or a neonate with death ensuing within 2 or 3 days post-partem due to high fever, sepsis, and respiratory distress. The mother usually experienced a flu-like illness late in the third trimester of pregnancy, characterized by low-grade fever, myalgias, malaise and backache. In this case, biopsy material of brain tissue obtained at autopsy was submitted to the pathology laboratory for tissue diagnosis and fluid from the pia-arachnoid was sent to the microbiology laboratory for culture.

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A Brown and Brenn gram stain was performed on one of the tissue biopsy specimens. Organisms were seen as shown in the photograph. Based on the history and the appearance of the bacteria, the most likely identification is:View Page
In view of the feedback to the previous question, the clinical correlation does not seem to fit in this case. Most likely:View Page
Review 3

Robinson LG. Kourtis AP.: Tale of a toothpick: Eikenella corrodens osteomyelitis. Infection. 28(5):332-3, 2000Tale of a Toothpick is a case of Eikenella corrodens osteomyelitis in a young woman, that resulted from puncture of her foot with a toothpick.The epidemiology, microbiology, common clinical presentations and therapy of E. corrodens are reviewed.A brief summary of the extent of toothpick injuries and their infectious complications are also presented.

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Review 2

Griego RD. Rosen T. Orengo IF. Wolf JE.: Dog, cat, and human bites: a review. Journal of the American Academy of Dermatology. 33:1019-29, 1995It is estimated that half of all Americans will be bitten by an animal or another human being during their lifetimes. The vast majority of the estimated 2 million annual mammalian bite wounds are minor, and the victims never seek medical attention. Nonetheless, bite wounds account for approximately 1% of all emergency department visits and more than $30 million in annual health care costs.Infection is the most common bite-associated complication; the relative risk is determined by the species of the inflicting animal, bite location, host factors, and local wound care. Most infections caused by mammalian bites are polymicrobial, with mixed aerobic and anaerobic species.The clinical presentation and appropriate treatment of infected bite wounds vary according to the causative organisms. Human bite wounds have long had a bad reputation for severe infection and frequent complication. However, recent data demonstrate that human bites occurring anywhere other than the hand present no more of a risk for infection than any other type of mammalian bite.The increased incidence of serious infections and complications associated with human bites to the hand warrants their consideration and management in three different categories: occlusional/simple, clenched fist injuries, and occlusional bites to the hand. This article reviews dogs, cat, and human bite wounds, risk factors for complications, evaluation components, bacteriology, antimicrobial susceptibility patterns, and recommended treatments. Epidemiology, clinical presentation, and treatment of infections caused by Pasteurella multocida, Capnocytophaga canimorsus, Eikenella corrodens, and rhabdovirus (rabies only) receive particular emphasis.

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Department of Transportation (DOT) Regulated Urine Specimen Collection Training
Collection site security requirements

All collection sites must meet the following security requirements: Must be able to prevent unauthorized access to the site during collection. Ensure that the donor does not have access to items that could be used to adulterate or dilute the specimen (e.g. soap, water, cleaning agents, etc.) Secure faucets, toilet tank tops, and other appropriate areas with tamper-evident tape if necessary. Ensure that the donor is at all times under the supervision of the collector or other collection site personnel. Provide for the secure handling and storage of specimens. (Specimens should be stored at 4-6º C. The refrigerator used should not be readily accessible to the general public and should be used only for the storage of urine drug screens and other clinical specimens. The refrigerator should be marked with a biohazard sign. No food or drink should ever be placed in the refrigerator.)

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Descriptive Statistics
Why Statistics?

Many people involved in the clinical laboratory sciences need to be familiar with basic statistics for a variety of reasons.  These reasons include: performing quality control, and interpreting of results of instrument testing determining suitability of different methods or instruments for the same task understanding how acceptable laboratory procedures and methods are established determining ranges for clinical tests of normal, healthy individuals understanding clinical trials and new methods presented in journals and articles performing those trials and research projects yourself

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Fundamentals of Hemostasis
Laboratory Tests of Hemostatic Function – Prothrombin Time

The prothrombin time is a screening test that helps to assess the functionality of both the extrinsic and common pathways. The effectiveness and presence of factors I, II, V, VII, and X are assayed in this diagnostic test, as they are all found in the aforementioned pathways. The results of the prothrombin time are used in conjunction with other diagnostic tests, as well as the clinical picture of the patient, to determine any hemostatic abnormalities which may be present. In addition to being an integral part of the coagulation disorder assessment process, the PT is also used to determine therapeutic effectiveness of oral anticoagulants, by monitoring drugs such as Warfarin, Coumarin, and Dicoumarol. Prothrombin time test results are reported as the number of seconds needed for a clot to form in the patient specimen using the laboratory's instrument/reagent system, and as the International Normalized Ratio (INR).

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Laboratory Tests of Hemostatic Function – Prothrombin Time

The INR component of the laboratory result is a calculated value that is used by the clinician to monitor anticoagulant therapy and adjust dosage as dictated by clinical status. An INR of 2.0 - 3.0 is often desired as the therapeutic range. The following formula is used by the clinical laboratory to derive an INR value. The INR must be adjusted for every new lot of PT reagent. INR= (PT of patient/PT of geometric mean of the normal population)ISI The International Sensitivity Index, or ISI value, is provided by the reagent manufacturer as the relative sensitivity of the reagent itself. The INR is used to standardize PT results, and in turn, anticoagulant therapy, across laboratory instrumentation, methodologies, and locale. Be sure to frequently check that ISI values match those of the lot currently in use as erroneous results may otherwise occur .

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Laboratory Tests of Hemostatic Function - APTT

The activated partial thromboplastin time (APTT) is a screening test that helps to assess the functionality of both the intrinsic and common pathways. The effectiveness and presence of all the coagulation factors are assayed by this diagnostic test with the exception of factors VII and XIII. The results of the activated partial thromboplastin time are used in conjunction with other diagnostic tests, as well as the clinical picture of the patient, to determine hemostatic abnormalities which may be present. In addition to being an integral part of the coagulation disorder assessment process, the APTT is used to determine therapeutic effectiveness of heparin administration. Activated partial thromboplastin time results are presented to the clinician in seconds- the actual time elapsed until a clot was detected using the laboratory's instrument/reagent system.

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Fibrin/Fibrinogen Degradation Products and D-dimers

The presence of D-dimers in plasma or whole blood indicates that fibrin has been formed and degraded (fibrinolysis). Plasmin can also degrade intact fibrinogen, generating fibrinogen degradation products that are detected in fibrin/fibrinogen degradation products (FDP) assays. D-dimers and FDP can become elevated whenever the coagulation and fibrinolytic systems are activated. The presence of D-dimer confirms that both thrombin and plasmin have been generated since it can only be produced as the result of the plasmin degradation of fibrin. This makes the test for D-dimers more specific for fibrinolysis than the FDP test that also detects the products of the direct proteolysis of fibrinogen (fibrinogenolysis).The D-dimer test can be useful in the diagnosis of deep venous thrombosis (DVT) or pulmonary embolism (PE), two forms of venous thromboembolism (VTE). When the test is being used for this purpose, it is important that D-dimer levels are accurately measured and accurately reported because of the serious nature of this clinical decision. If the test is positive in a patient suspected to have DVT or PE, clinicians proceed with further diagnostic tests. If the test is negative, depending on the clinical situation and the sensitivity of the D-dimer assay, DVT or PE is considered unlikely and further diagnostic tests for DVT or PE might not be pursued. D-dimer is a sensitive, but not specific, diagnostic test for disseminated intravascular coagulation, and an indicator of increased risk of future myocardial infarction in patients evaluated for chest pain.

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Tests of Hemostatic Function - Platelet Function Assay

A platelet function assay (PFA) is a screening test for the evaluation of platelets/primary hemostasis. Common clinical applications include the following: Preoperative evaluation of platelet function Determining the presence of drug-induced platelet dysfunction Determining platelet functionality in high-risk pregnancy Evaluation of patients with suspected inherited or acquired platelet disorders such as von Willebrand disease Evaluation of a bleeding patientA PFA instrument is able to differentiate between drug-induced platelet defects and other platelet defects. PFA tests are superior to the bleeding time test. The bleeding time is often not reproducible and, in spite of attempts at standardization, remains prone to variations in test results between persons performing the test. It is also relatively insensitive to platelet function. The bleeding time cannot be used to identify patients who may have recently ingested aspirin or non-steroidal anti-inflammatory drugs or patients who may have a platelet defect attributable to these drugs. The bleeding time is used to assess platelet function, but may be affected by platelet quantity. NOTE: Aspirin, and some other drugs, may falsely prolong bleeding times. Patients must be asked about aspirin use, and be aspirin free for 7-10 days prior to testing, for valid results.

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Anticoagulation Therapy

Anticoagulant therapy is employed in a number of clinical situations Some of these clinical situations include: After an episode of thrombosis, such as deep venous thrombosis (DVT) in the veins of the legs, to prevent reoccurrence. Prophylactically after some surgeries, especially those involving vascular repair such as coronary bypass surgery to prevent clots from blocking newly formed vasculature. In heart valve and chamber disorders where there is an increased risk of thrombosis occurring.

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HIPAA Privacy and Security Regulations
Case Study: Administrative Safeguards You are the technologist in charge of the hematology section in a hospital laboratory, and you are reviewing blood count results for 100 patients as part of an internal quality assurance project. You review the clinical findings in the electronic medical record to correlate with the laboratory results. The following week get a call from your hospital security officer. She says that a routine computer system audit has revealed that you accessed the records of 100 patients and she would like to know why.You tell her:View Page

HIV Safety for Florida

Introduction to Bioterrorism
The Laboratory Response Network (LRN) is a multilevel system designed to link frontline clinical laboratories to advanced capacity testing laboratories. The frontline microbiology laboratories are classified by the LRN as:View Page
Agent: Botulism (bacterium)

Most likely means of dissemination: Aerosol (eating contaminated food)Primary route of entry: Inhalation (oral)General signs and symptoms: Difficulty with speaking, swallowing, or blurred or double vision, drooping eyelids (ptosis), dilated pupils, dry mouth, decreased gag reflex, weakening of the reflexes (hyporeflexia), abnormal sensations such as numbness, prickling, tingling, and arm or leg weakness.Botulism is caused by a neurotoxin and technically could be classified as a chemical WMD. For our discussion it is placed under biological agents because the toxin is derived from a bacterium. Botulism is potentially life-threatening, producing a characteristic clinical picture of muscular paralysis leading to respiratory failure.                Photo courtesy of the CDC archives.    

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Laboratory Response

The broad base of clinical laboratories in this country is an essential component of our nation’s public health and healthcare system and is an essential link in addressing biological and chemical terrorism. In 1999 the Centers for Disease Control and Prevention (CDC) initiated the concept of a Laboratory Response Network (LRN).  The LRN is a network of local, state, federal, and military laboratories across the United States and internationally which work together in an integrated and coordinated way for a rapid response to public health emergencies. The LRN concept of operations is based on a system of safety and proficiency.

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The LRN Pyramid

The LRN is a multilevel system designed to link frontline clinical microbiology laboratories and hospitals and other institutions to state and local public health laboratories in supporting advanced capacity public health, military, veterinary, agricultural, water and food testing laboratories at the federal level. Laboratories within the LRN are divided into 3 levels: Sentinel Labs, Reference Labs, and National Labs.

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Laboratory Response - Chemical, Level 3

Level 3 laboratories are responsible for: Working with hospitals and private laboratories in their jurisdiction Knowing how to properly collect and ship clinical specimens Ensuring that specimens, which can be used as evidence in a criminal investigation, are properly handled and that chain-of-custody procedures are followed Being familiar with chemical  agents and how they can affect health and well-being Training on anticipated clinical sample flow and shipping regulations Working to develop a coordinated response plan for their respective state and jurisdiction

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Sentinel Labs

The frontline clinical microbiology laboratories are known as “sentinel laboratories”. The sentinel laboratories play a key role in the nation’s preparedness efforts. These laboratories perform the initial screening of clinical specimens for potential pathogens (rule-out) and refer specimens or isolates to a state or local public health laboratory at the reference level of the LRN. There are two kinds of sentinel laboratories: advanced and basic. Classification depends on their biological safety level and analytical capability.

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Introduction to Quality Control
Federal Regulations

The importance of quality control is recognized by the federal government. Federal regulations, such as the Clinical Laboratory Improvement Amendments of 1988 (CLIA 88), require successful participation in external quality control programs for clinical laboratories, regardless of size. These regulations are aimed at providing all citizens with the highest quality of health care and at the same time controlling costs.

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Introduction to the ABO Blood Group System
Why Knowledge of A Subgroups Is Important For Laboratorians

For the most part, subgroups are merely of academic interest, but occasionally they present clinical problems. The antigen may be so weak that it is not detected and the red cells are mistyped as group O. This is especially dangerous if the cells are those of a donor. Problems may arise because the serum of an A2 or A2B, A3 or Ax individual might contain anti-A1. This antibody may be detected in serum typing and cause confusion. You would not expect to find a person with A antigen on his red cells and anti-A in his serum. Anti-A1 is produced by about 1-2% of group A2 persons and about 25% of group A2B persons. Subgroups may be determined by reactions with antisera as seen in the table on the next page.

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Laws and Rules of the Florida Board of Clinical Laboratory Personnel
Public health laboratory scientists

Public health laboratory scientists are also regulated by the Board. The table below outlines the various requirements for applicants to receive licensure for a public health laboratory. Public Health Laboratory RequirementsDirectorFulfill the same requirements as a clinical laboratory directorSupervisorBe certified by National Registry in Clinical Chemistry or American Society for MicrobiologyBe licensed as a technologistHave five year's relevant experiencePass the state examTechnician (microbiology)Have a Bachelor's degree in one of the biological sciencesObtain American Society for Microbiology or the National Registry in Microbiology Certification in Public Health Microbiology Technician (chemistry)Have a Bachelor's degree in one of the chemical, biological, or physical sciencesObtain National Registry of Clinical Chemistry Certification in Public Health ChemistryTechnician (conditional)Have a Bachelor's degree in one of the chemical or biological sciencesPerform tests only under the direct supervision of a licensed pathologist, director, supervisor, or technologist.Receives a conditional two-year license, which may be renewed only once A license from the Board of Clinical Laboratory Personnel allows you to work in a public health laboratory at the same level and specialty.

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Director Qualifications

A physician may direct a clinical laboratory without a director's license if he / she is certified in clinical pathology by a national board and has at least four years of relevant experience. Non-physicians may obtain a director's license if he / she:Holds a doctor's degree in chemical, biological, or clinical laboratory scienceIs certified in one of the laboratory specialties by a national boardPasses an exam in supervision and administrationCompletes one hour of HIV / AIDS continuing educationCompletes two hours of medical errors continuing education A director can oversee up to five laboratories.

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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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Competency and Licensing Violations

Clinical laboratory personnel must be licensed and competent to perform their duties. This means holding the appropriate type of license for the task being performed (director, supervisor, technologist, or technician) and being certified in the appropriate specialty for any testing being performed. For example, an individual licensed as a technician in hematology may not perform the duties of a technologist in hematology, nor may that individual perform testing in the microbiology specialty. Showing a lack of competence to perform even licensed duties is a violation of Board rules. Consistent errors can tarnish a laboratory's reputation, and even a single error can harm patient care. Licensed personnel must be certain that they can perform their duties accurately and competently. All of the following are violations of Board rules:Performing clinical duties for which one does not hold a license.Performing services one knows one is not competent to perform.Showing lack of competence or making consistent errors in testing or reporting.Having a license revoked or suspended in another state.

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Kickback and Inducement Violations

Offering or taking a bribe, kickback, bonus, commission, or inducement is against the rules of the Board and against the law. Many companies give away small promotional items, such as pens or note pads, to promote their products. This is legal, but be cautious about accepting more valuable items. This could be seen as a bribe. All of the following are serious violations of Board, state, and federal rules:Participating in any commissions, bonuses, kickbacks, inducements, or split-fee arrangements from physicians, health care providers, suppliers, hospitals, nursing homes, other clinical laboratories, pharmacies, and other facilities.Exploiting or influencing a patient for financial gain, including promoting, selling, or withholding services, drugs, or referrals.

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Summary of Qualifications

The table below summarizes the qualifications for the four types of clinical laboratory personnel licenses. DirectorPhysician certified in clinical pathology OR Non-physician with: Doctoral degreeCertification in a lab specialtyCompleted course on administrationContinuing education in HIV/AIDS and medical errorsSupervisorOne of the following:Doctoral degree + 1 year experienceMaster's degree + 3 years experienceBachelor's degree + 5 years experienceLicensed as a technologist or meets the requirementsOne of the following:Completed course on administration25 hours of CE in administrationCE in HIV / AIDS and medical errors.TechnologistOne of the following:Bachelor's degree + medical technologist training program OR 3 years experienceAssociate's degree + Florida technician's license and completion of a medical laboratory training program OR 5 years experienceCompleted exam in 1+ specialtiesCE in HIV / AIDS and medical errorsTechnicianMeets one of the following:Completed medical lab technology training programHigh school or equivalency diploma + 5 years experienceAssociate's degree + 4 years experienceBachelor's degree + 3 years experienceBachelor's degree in medical technologyCompleted exam (certain specialties only)CE in HIV / AIDS and medical errors

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Specialists in immunohematology, clinical chemistry, hematology, and serology / immunology may perform testing associated radioassay, blood banking, and histology.View Page
Specialists in radioassay, blood banking, and histocompatability may perform all tests associated with immunohematology, clinical chemistry, hematology, and serology / immunology.View Page
Which of the following are violations of Board rules?View Page
A director may only oversee one laboratory.View Page
You cannot work in a clinical laboratory unless you have a four-year college degree.View Page
Specialists in clinical chemistry may perform blood banking testing.View Page

Medical Error Prevention
American Society for Clinical Pathology The American Society for Clinical Pathology, ASCP, promotes medical error prevention through its projects, programs, and activities. It includes this important topic in its many 400 workshops, symposia, teleconferences, and self-study programs. ASCP also promotes error prevention in the medical textbooks, reference manuals, slide atlases, audiovisual materials, and computer software it publishes. Its membership newsletters and The American Journal of Clinical Pathology and LabMedicine journal frequently address error prevention and patient safety.View Page
Human Nature and Error Prevention

The 2000 IOM report states, "It may be part of human nature to err, but it is also part of human nature to create solutions, find better alternatives, and meet the challenges ahead." Everyone should use these positive aspects of human nature to prevent medical errors. Laboratory professionals can do their part by understanding the nature and causes of medical errors and ways to prevent them. They can also benefit by guidance from many clinical, regulatory, and Internet resources that aim to prevent medical errors.

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Medicare Compliance for Clinical Laboratories
Coding

CPT (Current Procedural Terminology) codes are used to describe specific tests or services. The amount of payment for a test is dependent on the CPT code. It is against the law to use the wrong CPT code for a test for the purpose of causing or increasing payment for a test. ICD-9CM (International Classification of Disease, 9th Edition, Clinical Modification) codes are used to classify diseases and conditions, and describe signs, symptoms and medical circumstances. ICD-9CM codes are used to indicate the medical necessity of a particular test. It is against the law to use the wrong ICD-9CM code for the purpose of causing or increasing payment for a test.

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ICD-9CM coding

ICD-9CM (International Classification of Disease, 9th Edition, Clinical Modification) codes are used for the classification of disease and conditions and for describing signs, symptoms and medical circumstances.These codes are used to indicate the medical necessity of a particular test.ICD-9 codes can only be supplied by the ordering physician or a representative of that physician. "Code steering" means to steer or direct a physician to supply an ICD-9 code that is payable. ICD-9 codes cannot be used from a previous laboratory order. If a physician supplies a narrative description instead of an ICD-9 code the laboratory must accurately translate that code using only certified coders.It is against the law to use the wrong ICD-9 code for the purpose of causing or increasing payment for a test.

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Mycology: Hyaline and Dematiaceous Fungi
Based on the structures observed in this photomicrograph, the most probable species of the fungus recovered from an induced sputum specimen is:View Page
The fungus illustrated in this photomicrograph was recovered from an induced sputum specimen from a 74 year old man with chronic obstructive pulmonary disease. This isolate is most likely:View Page
The most helpful feature in differentiating the Zygomycetes from the other hyaline molds in the clinical mycology laboratory is:View Page
Of the following dematiaceous fungi, the black, suede-like colony illustrated here, reaching no larger than the size of a dime after 7 days incubation, most likely can be identified as:View Page
The dematiaceous colony illustrated here grew to a diameter of 3 - 4 cm in 5 days. The dematiaceous fungus that can be ruled out is:View Page

Mycology: Yeasts and Dimorphic Pathogens
Match each of the diseases listed in the drop-down box with the name of its most likely associated dimorphic fungal species.View Page
One of the characteristics common to the dimorphic molds is the ability to convert the mold forms to the yeast forms by incubating subcultures in enriched media at 35°-37°C. The upper image illustrates a subculture of a mold colony suspected of being a dimorphic fungus inoculated to the surface of blood agar and incubated for 3 days at 37°C. Note that the colonies have a prickly appearance, suggesting an intermediate stage of conversion. The lower image is a lactophenol blue mount of a portion of one of the prickly colonies. This fungus can be identified as:View Page
Procedures for the rapid culture confirmation of suspected colonies of B. dermatitidis, C. immitis and H. capsulatum recovered from clinical specimens include:View Page
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
Match each of the fungal species listed below with the appropriate category, indicating whether or not it has the capability of producing pseudohyphae on cornmeal agar.View Page
This photomicrograph is an acid-fast stained smear prepared from a yeast colony growing on ascospore agar. A helmet-shaped, red-staining, acid fast yeast cell is seen in the center of view at the tip of the arrow, against the background, blue-staining blastoconidia. The presumptive identification of Hansenula anomala was made. Predisposing conditions that may indicate that this isolate is more than a contaminant include:View Page

Normal Peripheral Blood Cells
Definition of a Segmented Cell continued.

Since these recommendations have been adopted by many groups, including the College of American Pathologists and the Centers for Disease Control, we will be using them as our criteria for differentiating between bands and segs.This definition was first reported by the Committee for Clarification of the Nomenclature of Cells and Diseases of the Blood and Blood Forming Organs, in the American Journal of Clinical Pathology (18:443-450, 1948).

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OSHA Bloodborne Pathogens
Labeling not Required

The following do not require biohazard labeling: Blood products in clinical use Individual specimen containers However, they are subject to Standard Precautions.

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OSHA Electrical Safety (updated 2007)

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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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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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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Therapeutic Drug Monitoring Definition

Therapeutic Drug Monitoring (TDM) is a branch of clinical chemistry that specializes in the measurement of medication levels in serum. TDM requires quantitative measurements of drugs and/or their metabolites.

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Enzyme Abnormalities and Drugs

The following is a list of enzymes for which known mutations have been associated with clinical effects. Enzymes Substrates (Drugs) Acetylaldehyde dehydrogenase Alcohol Acetylcholinesterase Succinylcholine Alcohol dehydrogenase Alcohol Dihydropyrimidine dehydrogenase Fluorouracil CYP2C9 Warfarin, phenytoin, losartan CYP2C19 Diazepam, omeprazole (Prilosec) CYP2D6 Many antidepressants, opioids, antiarrhythmics Glucose-6-phosphate dehydrogenase Aspirin, quinidine N-acetyltransferase Procainamide, isoniazid Thioprine methyltransferase 6-mercaptopurine UDP-glucuronosyl transferase Acetaminophen, tolbutamide, irinotecan

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Clinical Utility

The ultimate goal in measuring CYP450 function or identifying polymorphisms is to predict effective therapeutic doses and responses in patients.Polymorphisms are identified using molecular techniques (allele-specific PCR, restriction digests, sequencing, hybridization assays, bead-based systems, microarrays, pyrosequencing, et al).Although most clinical labs do not offer PGx testing, reference labs are beginning to market these tests. For example, one reference laboratory in the Midwest that offers CYP2D6 profiling measures about one dozen of the most common and significant mutation sites on this enzyme. This allows for detection of approximately 98% of the known CYP2D6 polymorphisms. The laboratory then generates a report which will advise the physician on the patient's drug-metabolizing status.Estimates show that 6-10% of the general population have a complete deficiency of CYP2D6, with the prevalence of mutations varying from <1% to as much as 21% within a given population.

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Metabolizers

When discussing PGx, we classify a person according to his/her phenotype (metabolic capacity for a given enzyme).A poor metabolizer (PM) is a person who lacks the functional enzyme and therefore exhibits decreased metabolism of drugs. This person would require lower doses of a drug that is metabolized by that enzyme. A PM who receives a standard dose is more likely to experience unwanted side effects or toxicity. A PM can also experience diminished effects with drugs that need to be metabolized to active compounds by the enzyme in question.An ultrarapid metabolizer (UM) will require a higher dose than usual since he/she will eliminate the drug more quickly. A UM may be resistant to standard treatments, and it may take some time to adjust the dosage before therapy is achieved.An intermediate metabolizer (IM) has one wild-type (normal) copy of the gene and one absent or dysfunctional copy. The IM group is very heterogeneous.A person with normal enzyme activity is referred to as an extensive metabolizer (EM). This person should respond to standard dosages of a drug. Most people are EM's. This is the population in which most dosing regimens have been worked out in clinical trials.

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The Bottom Line

By knowing a patient's disposition to specific drugs, the physician should be able to start the patient on an appropriate regimen rather than perfecting treatment based on trial and error. Drugs whose metabolism may prove to be problematic can be avoided, and second-line therapies that are metabolized by different, unaffected enzymes can be chosen. Clinical chemists, pharmacologists, and physicians need to translate knowledge of CYP450 polymorphisms into clinically-validated treatment algorithms. Dosing recommendations for PM, EM, IM and UM patients are beginning to appear in the literature for various classes of drugs, and the FDA is encouraging the incorporation of pharmacogenomic testing in the development process for new drugs.

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CYP450 Induction and Inhibition

CYP450 Inhibitor Inducer CYP1A2 Amiodarone Cimetidine Ciprofloxacin Tobacco CYP2C9 Amiodarone Fluvastatin Isoniazid Fluconazole Rifampin Secobarbital CYP2C19 Cimetidine Indomethacin Ketokonazole Prednisone CYP2D6 Celecoxib Cimetidine Cocaine Methadone Pentazocine Imipramine Desipramine Amitriptyline CYP2E1 Disulfiram Fluoxetine Ethanol Isoniazid CYP3A Midazolam Erythromycin Methadone Phenobarbital Dexamethasone Note: This is not an exhaustive listing of inducers and inhibitors.Reference: Tanaka E, Terada M, Misawa S. Cytochrome P450 2E1: it's clinical and toxicological role. J Clin Pharm Ther. 2000 Jun;25(3):165-75.

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Genotype versus Phenotype

Phenotyping involves measuring the metabolism of a probe drug. For example, with CYP2D6, dextromethorphan or debrisoquine can be given to a patient to see how well the drug is metabolized. Both these drugs are safe and extensively metabolized by CYP2D6. By measuring the parent drug and the metabolite in urine, the metabolic capacity of a CYP450 enzyme can be estimated. Such testing is complex and tedious, however, and has not become routine in clinical laboratories. Therefore, genotyping is likely to be the main tool that is used for assessing the PGx of a patient.

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References

Clinical Chemistry: Theory, Analysis, Correlation, 4th Edition. Lawrence A. Kaplan, Amadeo Pesce, Steven Kazmierczak. New York: Mosby, 2002.FDA Clears Genetic Lab Test for Warfarin Sensitivity. FDA News. U.S. Food and Drug Administration. Available at http://www.fda.gov/bbs/topics/NEWS/2007/NEW01701.html. Accessed June 3, 2008.Goodman & Gilman's The Pharmacological Basis of Therapeutics, 11th Edition. Laurence Brunton, John Lazo, Keith Parker. McGraw-Hill, 2005.Tanaka E, Terada M, Misawa S. Cytochrome P450 2E1: it's clinical and toxicological role. J Clin Pharm Ther. 2000 Jun;25(3):165-75.The Chemistry of Mind-Altering Drugs: History, Pharmacology, and Cultural Context. Daniel Perrine, American Chemical Society Publication, 1996.Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 4th Edition. Carl A. Burtis and Edward R. Ashwood, eds. Philadelphia: WB Saunders, 2005.

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Quality Control
Calibration Curve

Standards are also used to determine the linearity of the testing instrument. This is done by plotting a calibration or standard curve. Most testing instruments must be operated within a linear range. The Clinical and Laboratory Standards Institute or NCCLS defines linearity as “the measure of the degree to which a curve approximates a straight line.The examples to the right show linearity because a change along the x-axis shows a corresponding change along the y-axis, whether the x-value is low or high.

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Reading Gram Stained Direct Smears
A smear with under-decolorized stain should be restained, even if additional clinical material is available to make and stain a new smear.View Page
What is the value of a Direct Smear?

A direct smear is made from a clinical specimen, not a culture. It can be used to:Guide the physician on initial choice of antibiotic, pending results of culture and sensitivity.Judge specimen quality.Contribute to selection of culture media, especially with mixed flora.Provide internal quality control when direct smear results are compared to culture results.

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Cellular elements

The gram stain reaction and appearance can be used to identify most cellular material seen in a direct smear. Identification of cellular elements present in a direct clinical smear is important because most of these elements play an important role in the disease process. For example, the quality of a sputum sample can be assessed by determining the relative numbers of squamous epithelial cells and polymorphonuclear leukocytes (segmented neutrophils) present.

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Significance of Specific Findings:

Epithelial cells in large numbers within sputum smears means that the specimen is predominantly oral saliva, rather than true sputum from the lung. Epithelial cells in urine smears indicate that the sample has been contaminated by organisms found on the vulva or distal urethra. Bacteria found near or on epithelial cells are usually normal contaminating bacterial flora.White blood cells indicate inflammation and possible infection. The direct smear examination should focus within and around these cells.Red blood cells in a direct smear are not usually significant.Yeast may be present as normal flora in upper respiratory tract or genital tract. They may be significant if they predominate, or if budding yeast forms are seen.Hyphae are more likely to indicate the presence of fungal infection, but this determination requires correlation with clinical findings.Bacteria found in spinal fluid, blood, tissue and specimens from other sterile sites are always significant.Body fluids which are normally sterile must be examined carefully. If only one organism per oil immersion field is identified, then there are about 105 organisms per mL present in the sample! Bacteria observed in specimens from the throat, genital tract and other areas containing normal flora suggest infection only if their composition and type varies significantly from the norm.

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Contaminated Gram Stain Solution

Contamination of the staining solutions rarely occurs, but should be suspected when smears repeatedly contain the same organisms, and these organisms do not grow or are inconsistent with the clinical picture. Yeast and gram negative rods can occur as stain contaminants.

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Red Cell Disorders: Peripheral Blood Clues to Nonneoplastic Conditions
Match the form of red cell inclusions in each of the frames of photographs with a corresponding clinical condition.View Page
Match the red cell shapes in each frame of the photograph with its most likely corresponding clinical condition.View Page
An 8 year old girl is protected from severe hemolytic anemia by an elevated fetal hemoglobin level ( hemoglobin F).View Page
An isolated acanthocyte most likely is of little importance on an otherwise normochromic, normocytic peripheral blood smear.View Page
Reticulocyte identification

Reticulocytes are red blood cells prematurely released from the bone marrow. On a Wright-Giemsa stained blood smear, they appear as polychromatic macrocytes. Their presence in the peripheral blood may suggest hemolysis or bleeding. Their presence is expressed as a percentage of the red cell count: newly born= 3-7%; up to one week of age=1-3%; >one week =0.3-1.8%. Automated or manual methods may be used to enumerate reticulocytes. In clinical context, retics must be separated from debris, precipated stain, Pappenheimer bodies, Howell-Jolly bodies, and Heinz bodies.

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Considering the predominance of microspherocytes on the blood smear, and the patient's jaundiced condition, what is the most likely diagnosis?View Page
Sickle cells

This photograph of a peripheral blood smear from an 18-year-old North African woman with anemia reveals sickle cells. Target cells are not conspicuous. This shifts the diagnostic evidence away from HbSC disease. Cells tagged by arrows are variants of sickle cells. These may appear when multiple abnormal hemoglobin combinations are responsible for the clinical problem. The cell marked by the single arrow is an envelope formed not only in HbS disease but in HbC disease as well. Two arrows tag a blister cell, which, when seen in several fields, should prompt a hemoglobin electrophoresis to determine the presence of an undiagnosed hemoglobinopathy. Blister cells with fuzzy edged pseudo-vacuoles (see photo) are to be distinguished from the pseudo-vacuoles (blister)with razor sharp edges suggesting a microangiopathic state.

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The photograph here is of a peripheral smear sent for hematologic review. No clinical information for the patient was sent with the slide. What is the first course of action that the reviewer should take to assist him/her in interpreting the findings on this blood smear?View Page
The photograph is representative of the peripheral blood smear of a five-month-old immigrant from Asia. Her mother was concerned that the child was not eating well. Her spleen was palpable.The hemogram revealed the following:Hb 9.6g/dL (normal 12.0 - 16.0 g/dL)RBC 5.48 X 1012/L (normal 4.2 - 5.9 X 1012/LHCT 30.4% (normal 37 - 48%)MCV 55.4 fl (normal 86 - 98 fl)MCH 17.5 pg (normal 27 - 32 pg)MCHC 31.6 g/dL (normal 31 - 37 g/dL)RDW 34.9% (normal 11 - 15%)Reticulocyte count 10.9% (normal 0.5 - 1.5%)Select the most likely diagnosis based on the clinical information and peripheral blood findings.View Page
Dimorphic RBC population

Illustrated in the photomicrograph of a peripheral smear are two populations of erythrocytes. Approximately 50% of the erythrocytes are normal size and contain a full complement of hemoglobin. The patient had received blood transfusions. The transfused red blood cells are the normocytic, normochromic red cells. Admixed are microcytic erythrocytes and larger erythrocytes, some faintly mottled or smudged, suggestive of reticulocytes. This picture represents a hemolytic process with a reticulocyte response. A similar dimorphic red cell population appears following erythropoietin therapy. It is important to recognize when a population of cells in the peripheral smear is not in context with anticipated laboratory findings and the clinical situation.

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Stomatocytes

Stomatocytes are erythrocytes with a slit-like central pallor. Otherwise, they resemble typical RBC's in size and shape. Unless 10% or more of the RBC's are stomatocytes, their presence is probably artifactual. Stomatocytes form at a low blood acidic pH as seen in exposure to cationic detergents, and in patients receiving phenolthiazine. Hereditary stomatocytosis has some resemblance to hereditary spherocytosis, as stomatocytes may develop into spherocytes with further metamorphosis. In hereditary stomatocytosis, mild anemia and findings of on-going hemolysis should be evident if the condition presents as a clinical problem at all.

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The arrow on this photomicrograh points to a macrocyte. The oval shape should be noted on the patient report.View Page
References

Glassy, Eric F.,(Ed). Color Atlas of Hematology: An Illustrated Field Guide Based on Proficiency Testing. 1998. College of American Pathologists Hematology and Cliical Microbiology Research Committee. College of American Pathologists, Northfield, IL 60093-2750.Hookey,L., Dexter, D., Lee,D. H. The Use and Interpretation Of Quantative Terminology In Reporting Red Blood Cell Morphology. Laboratory Hematology 7:85-88, 2001.Peterson P, Blomberg DJ, Rabinovitch A, Cornbleet PJ. Physician Review of the Peripheral Blood Smear: When and Why. For the Hematology and Clinical Microscopy Resource Committee of the College of American Pathologists. Laboratory Hematology 7:175-179, 2001

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Ways out of the dilemma

What clear courses of action might the clinician take if the technologist reports out from this smear 3+ acanthocytes, 1+ target cells and occasional helmet cells? Gleaning information from the review of peripheral blood smears is important for the technologist, physician, and surely for the patient. Extreme pressures of time constraints and shifting dynamics in communication, from face-to-face encounters to dependency on technology, make innovative solutions to physician-patient information dilemmas imperative. Reporting systems often are geared more toward retrievability, suiting the needs of administrators and record keepers rather than being clearly directed toward improving patient care outcomes. A prime solution to this communication dilemma is to provide technologists with written descriptions and images of specific abnormal findings from peripheral blood smears. With a high degree of probability, these may link directly with underlying information connected to diseases. Mutually understood terms must be established to convert subjective qualitative peripheral blood smear findings into mutually understandable information. For example, regarding the smear shown, it was learned that the patient had recently undergone splenectomy. Creating an integrated communication system for information sharing (providing essential patient information by telephone follow-up or use of a system for e-mail feedback) can help ensure a favorable clinical outcome.

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Reporting of laboratory data in regard to blood cell abnormalities

Laboratory data must be presented to clinicians in a user friendly way to promote effective decision making. Databases must be designed to provide clear information that leads quickly to the best patient care outcome. We continue learning how to collect and retrieve laboratory data from our machines, but we are not always in tune to how entry and retrieval of data is geared to and, more directly, influences patient care outcomes. Examples of blood cell abnormalities on a peripheral blood smear that may immediately direct the physician to a specific diagnosis are: (1) presence of target cells as found in thalassemia or hemoglobinopathies and target cells in liver disease, particularly with obstructive jaundice; (2) burr cells as a signal of chronic renal disease and uremia; and (3)atypical neutrophil inclusions relating to genetic disorders. Critical appraisal of such observations could add valuable clues for a diagnosis. Laboratory professionals must establish a set of principles for orderly observation of blood cell morphology, have a clear vision of the applications of their work, and understand the potential clinical implications of their reports and interpretations. Emphasis on values and relevance focuses on patient care outcomes and their dependency on prompt availability of results and contextual interpretations.

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Criteria for peripheral blood smear review

Initial analysis of the peripheral blood picture is made in most clinical laboratories with an automated instrument. Samples are selected for further analysis when quantitative or qualitative abnormalities beyond a defined standard are found. The following are examples of quantitative RBC abnormalities that may prompt a blood smear review. Each laboratory, however, should develop its own guidelines: Hgb: < 8 or >18 g/dL (<10 or > 21g/dL in a newborn)Hct: <20% or > 60% in adults (<40% or >65% in a newborn)MCHC: <29 g/dLMCV: <69 femtoliters (fl) or >110flFlags generated by the hematology analyzer that indicate possible red cell abnormalities or spurious resultsAny of these findings should be followed up with a peripheral blood smear review.

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Qualitative reports: Follow-up

Any review of a peripheral smear is highly subjective; therefore, each laboratory must establish its own guidelines for quantitating observations and issuing reports in a consistent format. The key question for the laboratory is "How will the clinician use the terms of qualitative results in the reports issued to decide on the next course of action with this patient?" Formats for reporting have been geared more toward the needs of instrumentation facilitation and computer management than toward needs of access and understanding by clinicians working to improve patient care outcomes. Evidence based medicine (EBM) is the formal term used for the process by which research evidence, collective clinical experience, and the user friendly rendering of testing results are integrated to evaluate patient care outcomes.

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Red Cell Morphology
Summary

It is important to differentiate in vitro changes which are secondary to preparing the slide, from in vivo morphology, which is the result of the pathophysiological condition of the patient. Examining erythrocytes in the critical viewing area is extremely important in making this distinction. The determination of the clinical significance of the morphology reported is the responsibility of the physician, who must correlate the blood smear findings with the clinical diagnosis, and other laboratory parameters.

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The identification of which of the following abnormal forms may contribute significantly to specific clinical diagnosis:View Page
Another Target Cell

Another example of a target cell (or codocyte) is seen in the center of this slide. Notice that the hemoglobin in the center of this cell is somewhat lighter in appearance than in the previous slide. A second codocyte can be seen in the upper left portion of the slide. Codocytes appear in conditions which cause the surface of the red cell to increase disproportionately to its volume. This may result from a decrease in hemoglobin, as in iron deficiency anemia, or an increase in cell membrane. Target cells have excess membrane cholesterol and phospholipid and decreased cellular hemoglobin. Examples of other conditions in which target cells may be present include thalassemias, hgb C disease, post splenectomy and obstructive jaundice. Since their presence can be the result of an in vitro artifact, their value in clinical diagnosis is limited.

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Schistocytes

Two slightly larger fragments can be seen in this slide. One is lower center, and the other is lower right. Two dacryocytes or teardrop cells are seen in the upper center. Several ovalocytes are also present in this field. Schistocytes are seen in the same conditions as keratocytes and have a short survival time in circulation. Schistocytes have somewhat more clinical significance than keratocytes.

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The Urine Microscopic: Microscopic Analysis of Urine Sediment
Recognition and Identification

Recognizing and identifying casts is an essential part of the urine microscopic examination. The table below summarizes the different type of casts and their clinical significance. Type Condition Hyaline 0-2/LPF is normal; increase in fever, diuretic therapy, exercise or stress. Glomerulonephritis Pyelonephritis Chronic renal disease White Blood Cell Renal Infection Inflammation of the nephron Red Blood Cell Bleeding in the nephron Glomerulonephritis Strenuous exercise Epithelial Cell Renal tubular damage Coarsely Granular Urinary stasis Gomerular and tubular disease Finely Granular Further degeneration of coarsely granular Amyloid disease Chronic renal disease Fatty Degenerative tubular disease Broad Extreme stasis of urine flow Renal failure

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Squamous Epithelial Cells

The most common type of cell seen in the urine sediment is the epithelial cell. This slide shows squamous epithelial cells under low power brightfield microscopy. They appear as large flattened cells with abundant cytoplasm and small round central nucleus. Although squamous epithelial cells have little clinical significance they must be differentiated from other cellular elements.

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Crystals of Clinical Significance

Crystals of clinical significance include leucine, tyrosine, cystine, cholesterol and bilirubin.

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Specimen #2 - Adult Male

This report indicates that the specimen is normal. The physical characteristics and microscopic tests do not correlate, as 3+ amorphous urates would probably produce a more turbid or cloudy specimen. Amorphous crystals have little clinical significance and may precipitate out of solution with storage temperature changes. Therefore, the results may be reported.

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Review of Common Crystals

The following table lists common crystals found in the urine sediment. Crystals that have no clinical significance must be identified and differentiated from those that can be an indication of a metabolic disorder or other clinically significant conditions. Crystal pH Color Uric Acid Acidic Yellow - Brown Calcium Oxalate Acidic/Neutral Colorless Amorphous Urates Acidic Yellow - Brown Triple Phosphate Alkaline Colorless Ammonium Biruate Alkaline Yellow - Brown Amorphous Phosphate Alkaline/Neutral White - Colorless Calcium Carbonate Alkaline Colorless

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Tuberculosis Awareness for Healthcare Workers
Tuberculosis infection

The natural history of TB infection is usually followed by an immune response and latency after exposure. In about 5-10% of cases, the latent period progresses to an active infection.The organism that causes TB infection is Mycobacterium tuberculosis. This organism is pictured in the photograph to the right as observed when stained with acridine orange stain. Infection occurs when a susceptible person inhales droplet nuclei containing Mycobacterium tuberculosis and the organism reaches the alveoli of the lungs.About 2-12 weeks after infection, the immune system limits multiplication of additional bacteria and the immunological test becomes positive.Latent tuberculosis infection (LTBI) is the stage when the viable organism remains in the body, and the patient has no symptoms and is non-infectious.Most infected persons do not experience clinical illness and are noninfectious. About 5-10% of persons infected with Mycobacterium tuberculosis who are not treated will develop TB during their lifetime. The risk for progression is highest during the first several years after infection.TB infects the lungs most often; however, it can infect almost any organ in the body, including bones and joints.

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High Risk Progression Groups

The following persons are at high risk for progression from LTBI to TB disease: Persons infected with HIV Persons infected with Mycobacterium tuberculosis within the past two years Persons with untreated or inadequately treated TB disease Infants and children <4 years of age Persons with chronic medical conditions or immunocompromising conditions

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TST Interpretation and Classification

The TST interpretation depends on the measured diameter of the induration and the clinical status of the patient.An induration of 15 or more millimeters is considered positive in all persons.An induration of 10 or more millimeters is considered positive in patients in the high risk progression groups and in mycobacteriology laboratory workers.An induration of 5 or more millimeters is considered positive in the high risk infection groups.

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CDC Risk Categories

CDC has identified three risk categories in health-care settings: A low risk healthcare setting is one in which HCWs will most likely not be exposed to persons with TB disease or to clinical specimens that might contain M. tuberculosis. A medium risk healthcare setting is one in which the HCW will or might possibly be exposed to persons with TB disease or to clinical specimens that might contain M. tuberculosis. A potential ongoing transmission healthcare setting is temporarily applied to any setting if there is evidence of person-to-person transmission of M. tuberculosis in the past year.

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Variations in White Cell Morphology - Granulocytes
Barr Body

A Barr body appears as a small drumstick-like projection on one of the lobes of a some of the neutrophil in females. Barr bodies are attached to the nuclear lobe by a single narrow stalk which distinguishes them from other thicker projections, sometimes referred to as "clubs." Clubs have a thicker, and sometimes, a double stalk. This projection can be seen in both males and females and has no clinical significance. Barr bodies must also be distinguished from hair-like projections sometimes seen in the band form, following irradiation or in patients with a malignant tumor that has metastasized. Since Barr bodies are the morphological expression of the inactivated X chromosome, one Barr body can be seen in up to 3% of the neutrophils on a female's peripheral blood slide. In rare chromosome disorders in which three or more X chromosomes are present, two to three Barr bodies per neutrophil can be seen. Recognition of a Barr body in a neutrophil is important in order to avoid reporting it as abnormal unless two or more per neutrophil are seen.

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White Cell and Platelet Disorders: Peripheral Blood Clues to Nonneoplastic Conditions
Match the letters representing the peripheral white blood cells with the most likely associated clinical conditions.View Page
Assume that several other lymphocytes similar to the one in the center of the photograph are found on review of the peripheral smear. A work up for leukemia should be recommended.View Page
Atypical Cells: Quantitative Estimate

A smudge cell is centered in the photograph. In some laboratories, a semi-quantitative estimate of the number of smudge cells may be made; in others, a report of "smudge cells present" may suffice. The point is that a language for reporting semi-quantitative estimates must be established for any atypical cells appearing in the peripheral blood smear. This reporting scheme must be understood by the physician in order to maximize patient care outcomes through his/her decision making process. For example, in the context of this exercise, does it make any difference to the physician if you report few or many smudge cells; or, is a report of smudge cells present sufficient? The answer to this question applies not only to smudge cells, but to the reporting of any other atypical white cells as well. An agreement must be reached between the hematology laboratory and clinical services as to how semi-quantitative estimates will impact the need for further testing in view of patient care outcomes.

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Criteria for evaluation of white blood cells and platelets

In most clinical hematology laboratories, an initial blood count is performed by an electronic instrument. Some of these instruments also produce a differential blood count, and a platelet count. Instruments that provide a 3-part differential indicate the percentage of neutrophils, lymphocytes, and a mixed field group that includes monocytes, eosinophils, basophils, immature and atypical cells. Thus, the atypical cells shown in the photograph would be counted as mixed cells and a smear review would be needed to make an identification. Instruments providing a 5-part differential count include monocytes and eosinophils. In cases where the mixed cell count is high, or there are other indications that atypical cells may be present, a hematologist's review of the smear is indicated.

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The peripheral blood smear noted in the photograph was held for morophological and clinical review as the total platelet count was 10,000/cumm. Conditions fitting this picture include:View Page
Typical cells on a peripheral blood smear as photographed here were repeatedly encountered as the smear was reviewed. The peripheral white blood cell count was 51,000/ml with an orderly maturation sequence. The comment "leukemoid reaction" may properly be appended to the report.View Page
Familial disorders: summary

Several additional familial and congenital disorders associated with atypical inclusions in WBCs are now recorded. These individual syndromes carry the following names: Fechtner, Alport, Epstein, Sebastian, and Paris-Trousseau.Fechtner syndrome( Peterson etal,Blood 65:397-406,1985)was described with 8 family members spanning 4 generations presenting with varying degrees of nephritis, deafness,and congenital cataracts. The syndrome is likely a variant of Alport syndrome with the addition of leukocyte inclusions and macrocytothemia. Several more cases involving other families have been reported. The inclusions resemble toxic Doehle bodies or those of the May-Hegglin anomaly by light microscopy, but are ultrastructurally unique.Alport syndrome in itself is autosomal dominant, X-linked , hereditary and characterized by sensorineural deafness and hereditary nephritis. It is believed to result from abnormal glycopeptide synthesis in renal basement membranes. Recurrent hematuria and slowly progressive renal insufficiency are clinical findings. Cataracts and platelet abnormalities may be added features.Epstein syndrome is essentially Alport syndrome with the addition of macrothrombocytopenia (Seri, et al. Hum Genet 110:182-186, 2002). Neutrophil inclusions are absent in this disorder; neutrophilic inclusions are considered part of the Fechtner syndrome. The Sebastian platelet syndrome is a variant of hereditary macrothrombocytopenia combined with neutrophil inclusions that differ from Doehle bodies, but are similar to those inclusions in Fechtner syndrome. (Greinacher, et al, Blut 61:282-288, 1990).Paris-Trousseau syndrome includes large platelets containing giant alpha granules identifiable in the peripheral blood.(Breton-Gorius, Blood 85:1805,1995)

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Atypical neutrophilic intra-cytoplasmic inclusions ,as noted in the photograph, are present in a peripheral blood smear when one or more of the following underlying conditions are present:View Page
WBC inclusions: summary

The presence of atypical inclusions within the cytoplasm of neutrophils and other leukocytes should lead to a clinical investigation of the setting for these findings.Atypical neutrophil inclusions may be seen in the following disorders: Chediak-Higashi syndrome, May-Hegglin anomaly, Alder-Reilly anomaly, Fechtner , Sebastian, Epstein and Alport-like syndromes and in infectious and toxic conditions (in the form of Doehle bodies).Although a specific entity may not be evident from examination of the peripheral blood alone, it is important that hematology technologists include a comment reporting on the presence of these inclusions or granules. A clinical investigation with further hematologic and genetic studies may then appropriately be considered.Many of the disorders with atypical neutrophil cytoplasmic granules are also associated with platelet abnormalities, particularly giant platelets (lower photograph).Therefore, when atypical granules are recognized, scanning of the peripheral blood smear for atypical platelets may be revealing. These observations serve as readily identifiable markers for acquired and genetic human maladies, and as a guide for unraveling the reasons for a patient's suffering and impaired health.

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Doehle Bodies: Review

Doehle bodies are discrete, round or oval aggregates at the cytoplasmic periphery of neutrophils (blue arrows in figures). They stain sky blue with Romanowsky's stain and often may be deceivingly inconspicuous. In electron-micrographs, Doehle bodies are recognized as lamellar aggregates of rough endoplasmic reticulum. Although not considered a marker for leukemia, Goudsmit, et al (Brit J Hematol 20:447-562, 1971)reported their presence in family members, 2 sisters and 3 brothers. Two of the brothers died of acute myeloblastic leukemia. These testimonials indicate that Doehle bodies, when identified in peripheral blood smears, should be taken seriously so as to stimulate a clinical investigation of the patient.

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The peripheral blood smear presented here was submitted for morphological/clinical review. Conditions in which this picture may be seen include:View Page
The neutrophils seen in two fields in the upper and lower photographs are representative of a majority of the left shift neutrophils found in this peripheral blood smear. The diagnosis of Pelger-Huet anomaly can be made.View Page
Case Follow-up

Illustrated in the upper and lower photographs are two-lobed, eye glass ("pince nez") nuclei of neutrophils typical for patients with Pelger-Huet anomaly. In addition to the characteristic two lobes connected by a delicate bridge, the dense, homogeneous nuclear chromatin helps to define Pelger-Huet anomaly. Since the peripheral blood smear did not support the diagnosis of appendicitis in this patient, and since abdominal pain localized to the right lower quadrant never developed, the boy was hydrated with intravenous fluid and observed. After hydration, his constitutional symptoms improved and the abdominal pain subsided. In fact, the lad was back on the ski slopes the next afternoon. People entering high altitude where the humidity may be very low are susceptible to dehydration and may experience symptoms related to mountain sickness. Therefore, close observation and hydration may be the best practice in monitoring patients with stories and findings similar to this one. A further lesson here is that technologists must be alert to the possibility of Pelger-Huet anomaly if a high white blood cell count with a high percentage of band neutrophils with strikingly uniform morphology and without toxic granulation are found. Inappropriate therapy or an invasive procedure as was contemplated here may be avoided by a proper smear assessment and clinical corroboration.

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The cell photographed here is known as a MOTT cell. The condition in which these cells are associated is:View Page
A peripheral smear was submitted for morphology/clinical because of the number of monocytes as captured in the upper and lower photographs. This picture is consistent with each of the following conditions except: