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Deletion of two out of four alpha chain loci results in alpha thalassemia minor. The deletions may be homozygous (two on the same chromosome) or heterozygous (one from each of two chromosomes). Alpha thalassemia minor does not produce a clinical disease but may be discovered upon routine testing. Both the homozygous and heterozygous form are common in Southeast Asians. The homozygous form is also seen in American Blacks.

Burtis, CA. & Ashwood, ER. Tietz Textbook of Clinical Chemistry 2nd ed. W. B. Saunders. 1994.Harmening, DM. Clinical Hematology and Fundamentals of Hemostatis 5th ed., F.A. Davis, 2008Lotspeich-Steininger, Stiene-Martin and Koepke, Clinical Hematology Principles, Procedures, Correlations, Lippincott 1992McKenzie, SB., Textbook of Hematology 2nd ed., Williams and Wilkins 1996.Miale, JB, Laboratory Medicine Hematology 6th ed., Mosby 1982.Nouwens, J and Spahn, M. Hemoglobin H Disease: A self-instructional unit 3rd ed., Educational Materials for Health Professionals, Inc. 1991.Doig, K. Rodak's Diagnostic Hematology 3rd ed. W.B.Sunders Co., 2007.

Reaction PhaseLook at the phase in which reactions are occuring and see if the results match the reaction patterns on your panel. Reactions occurring only at immediate spin phase could indicate a possible IgM antibody, cold agglutinin, or rouleaux. Reactions occurring in the AHG phase could indicate a possible IgG antibody. Reactions occurring in both reaction phases could indicate a combination of both IgM and IgG antibodies or a strong IgM antibody that carries through to AHG phase. If a tube method is used, reactions are usually read at immediate spin and AHG phase. If a gel method is used, reaction readings are done only at AHG phase. PatternComparing the pattern of reactivity of the sample reactions with that of the panel cells will help to determine which antibody or antibodies are present. If the reactions match, or closely match, a specific panel cell, that could be the antibody.The strength of the reaction does not correlate with the clinical significance of the antibody present. It does correlate to the amount of antibody that is available to react. If reactions are strong at immediate spin (IS) phase and get weaker at antihuman globulin (AHG) phase, it is possible that a strong IgM antibody is present that is carrying through to the AHG phase (e.g., 4+ at IS and 1+ at AHG).The presence of multiple antibodies should be considered if reactions vary in strength or there are two separate reaction patterns in the IS and AHG phases.

Children with beta thalassemia major, also called Cooley's anemia, usually develop clinical signs during their first year of life. They appear to be malnourished and may exhibit abdominal girth expansion. They show skeletal deformations, which are a result of increased erythropoiesis. A common finding is facial bone changes. Other clinical signs include frequent infections, hepatomegaly, splenomegaly, cardiomegaly, gall stones, leg ulcers, and poor growth and sexual development. Death usually occurs by the time these patients are in their early twenties unless treated with blood transfusions along with iron-chelating agents. If no chelating agent is used during treatment life will only be prolonged by about a decade.Beta thalassemia is found most often in populations of people from the Mediterranean, southern China, and India.

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.

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.

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.

Ketone bodies are usually absent in urine. The presence of ketones in the urine probably indicates that the body is using fats rather than carbohydrates for energy. High levels of ketones may be present in the urine of individuals with uncontrolled diabetes 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. Electrolyte imbalance and dehydration occur when ketones accumulate in the blood. If these conditions are not corrected by adjusting the dose of insulin, the patient may develop ketoacidosis and ultimately diabetic coma. Low levels of ketones 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 of carbohydrates or increased loss of carbohydrates.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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).

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).

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.

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.

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.

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.

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.)

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

Electrophoresis is the migration or separation of charged particles or solutes of a liquid solution in an electrical field. Conventional electrophoresis is tedious and time consuming. Electrophoresis automation and newer electrophoresis techniques have revitalized the utilization of electrophoresis in today's clinical laboratories. Molecular diagnostic analysis using electrophoresis and research in proteomics have also contributed to this revitalization.

There are numerous applications of electrophoresis. Routine protein electrophoresis performed in clinical laboratories is the oldest method and therefore the most frequently used method. With the advent of molecular diagnostics, several other electrophoresis methods have become very important, highly automated, and have several important applications.Types of electrophoresis that will be discussed are Routine electrophoresis High resolution electrophoresis Polyacrylamide gel electrophoresis Capillary electrophoresis Isoelectric focusing Immunochemical electrophoresis Two-dimensional electrophoresis Pulsed field electrophoresis

For electrophoretic separation of solutes, the sample of solutes is placed on a gel or membrane in contact with buffer for separation. Common gels are cellulose acetate, agarose, and polyacrylamide gels. These gels are formed into sheets, slabs, or inserted into columns or tubes. The gel can be positioned horizontally or vertically.Cellulose is chemically reacted with acetic anyhdride to form a cellulose acetate gel. Because cellulose requires soaking before sample application and a clearing step for detection of separated solutes or bands, agarose gel is more often used than cellulose acetate gel for clinical electrophoresis.

Risk markers are first hypothesized and then tested. Once a potential marker is identified, concentrations of the serum marker are correlated with patient outcomes. Cardiovascular risk marker studies are typically either retrospective or prospective epidemiology studies. A retrospective study looks backwards at a patient population. For example, we identify (through a hospital database perhaps) patients who have had myocardial infarcts or some other adverse outcome as well as similar subjects without that outcome to use as controls. We then go back and find archived patient serum samples and relate the concentrations of our new risk marker with patient outcomes. Retrospective studies can only be performed if you have archived samples from the patient. Prospective studies look forward in time. For example, we first select a group of subjects and measure our new risk marker in these patients over time. After a few years, we see how the serum concentrations relate to the patient outcomes. Obviously, prospective studies take much longer to perform than retrospective studies. Whatever study model is used, when assessing the value of a cardiovascular risk marker, we must correlate serum concentrations with a specific outcome. The outcome is determined by the study authors. Outcomes could be things like myocardial infarction, stroke, a diagnosis of coronary artery disease, death, or any cardiovascular 'event.'Concentrations of risk markers are divided into tertiles, quatriles or quintiles. This simply means that the top 33%, top 25% or top 20% of the serum concentration values are compared to the bottom 33%, 25% or 20%. For example, risk marker studies will often compare the outcomes of patients with serum concentrations in the upper tertile (those in the top third) with those in the bottom tertile (those in the bottom third) to see if the top 33% had significantly worse outcomes; if so, the risk marker has clinical value.

Measuring ApoB and ApoA1 can be performed using standard immunoassay techniques. Nephelometry is popular, as are ELISA-based methods that are performed on automated chemistry analyzer platforms. The power of the ApoB/ApoA1 ratio as a cardiovascular risk marker is getting widespread attention. An individual with seemingly normal LDL-C may in fact have high ApoB concentrations. When this individual has his or her ApoB/ApoA1 ratio calculated, the risk is evident. Studies have also shown that patients with metabolic syndrome and type-2 diabetes can also easily be identified with the ApoB/ApoA1 ratio, whereas these patients cannot always be identified by measuring LDL-C and HDL-C.In 2004, the global INTERHEART study of risk factors for acute myocardial infarction concluded that the ApoB/ApoA1 ratio was the most important risk factor in all geographic regions. The ApoB/ApoA1 ratio is easy to use because the risk is integrated into a single number that indicates the balance between atherogenic and antiatherogenic particles.There have been many studies concerning the predictive power of the ApoB/ApoA1 ratio. One study, which involved thousands of patients who were followed for an average of 10 years, showed that the ApoB/ApoA1 ratio was a strong predictor of stroke in addition to other cardiovascular events. Due to the evidence presented in studies like these, the National Academy of Clinical Biochemistry (NACB) has recommended that the ApoB/ApoA1 ratio be used as an alternative to the usual total cholesterol (TC)/HDL cholesterol ratio when determining lipoprotein-related risk for cardiovascular disease. Some believe that ApoB/ApoA1 testing will eventually replace traditional LDL-C and HDL-C measurements.

In this course we have described some emerging cardiovascular risk markers. It is important to note that there are many more markers, some of which appear robust and which may have clinical value. Examples of other risk markers that are emerging but were not discussed are listed in the table to the right.An important question that should always be asked is "how many risk markers do we need?" With so many risk markers available, the laboratory needs to research which markers are truly the strongest and most valuable for the patient demographic the facility deals with most and which markers physicians will order and utilize.

Atherosclerosis. U.S. Department of Health & Human Services National Institutes of Health. Available at http://www.nhlbi.nih.gov/health/dci/Diseases/Atherosclerosis/Atherosclerosis_WhatIs.htmlAccessed June 23, 2009.Daniels LB, Barrett-Connor E, Sarno M, Laughlin GA,Bettencourt R, Wolfert RL. Lipoprotein-associated phospholipase A2 (Lp-PLA2) independently predicts incident coronary heart disease (CHD) in an apparently healthy older population: The Rancho Bernardo study. J Am Coll Cardiol. 2008;51:913-919.Executive Summary of the third report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA. 2001; 285:2486-2497. Frostegard, J, Wu R, Lemne C, Thulin T, Witztum JL and de Faire U. Circulating oxidized low-density lipoprotein is increased in hypertension, Clin Sci 2003; 105, 615.Garza CA, Montoir VM, McConnell JP, et al. Association between lipoprotein-associated phospholipase A2 and cardiovascular disease: a systematic review. Mayo Clin Proc. 2007;82(2):159-165.Interpretive Handbook, (MC0440rev0407) Mayo Clinic, Rochester MN;2007. Maksimowicz-McKinnon K, Bhatt DL, Calabrese LH: Recent advances in vascular inflammation: C-reactive protein and other inflammatory biomarkers. Curr Opin Rheumatol. 2004;16:18-24.Mora S, Szklo M, Otvos JD, et al. LDL particle subclasses, LDL particle size, and carotid atherosclerosis in the multi-ethnic study of atherosclerosis. Atherosclerosis. 2007;192:211-217.NACB Laboratory Medicine Practice Guidelines. Emerging biomarkers of cardiovascular disease and stroke. National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines. 2006.PLACtest animation, diaDexus. http://www.plactest.com/laboratorians/action.php Accessed June 23, 2009.Rifai N, Warnick GR. Lipids, lipoproteins, apolipoproteins, and other cardiovascular risk factors. In: Burtis CA, Ashwood ER. Bruns DE. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 4th ed. St. Louis, MO: Elsevier Saunders: 2006; chap. 26.Ridker PM, Rifai N, Rose L, et al. Comparison of C-reactive protein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular events. N Engl J Med. 2002;347:1557-1565.Sniderman AD. Differential response of cholesterol and particle measures of atherogenic lipoproteins to LDL-lowering therapy: Implications for clinical practice. J Clin Lipidol 2008;2:36-42.Tsimikas, S, Brilakis ES, Miller ER, et al. Oxidized phospholipids, Lp(a) lipoprotein, and coronary artery disease, N Engl J Med: 2005;353:46.Tsimikas S, Bergmark C, Beyer RW, et al. Temporal increases in plasma markers of oxidized low-density lipoprotein strongly reflect the presence of acute coronary syndromes. J Am Coll Cardiol. 2003; 41: 360.Tsimikas, S, Lau HK, Han KR, et al. Percutaneous coronary intervention results in acute increases in oxidized phospholipids and lipoprotein(a): Short-term and long-term immunologic responses to oxidized low-density lipoprotein. Circulation. 2004;109, 3164.Tsimikas S, Witztum JL, Miller ER, Sasiela WJ, et al. High-dose atorvastatin reduces total plasma levels of oxidized phospholipids and immune complexes present on apolipoprotein B-100 in patients with acute coronary syndromes in the MIRACL trial, Circulation: 2004;110, 1406. Walldius G, Jungner I, Holme I, et al. High apolipoprotein B, low apolipoprotein A-I, and improvement in the prediction of fatal myocardial infarction (AMORIS study): a prospective study. Lancet. 2001;358:2026-2033.Yusuf S, Hawken S, Ounpuu S, et al. Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study. Lancet. 2004;364:937-952.

The nuclear magnetic resonance (NMR) spectroscopy technique that was developed by LipoScience (LipoScience, Inc., Raleigh, NC), exploits specific magnetic properties of lipoproteins. This technology does not require separation of lipoproteins; serum or plasma can be run through the NMR sensor probe and all lipoproteins can be measured directly and homogeneously. The NMR platform works by subjecting the patient sample to a pulse of radio energy within a strong magnetic field. The energy that is given off by the lipids in the sample results is a signal that can be analyzed by the instrument to determine the number and size of lipoproteins present. Lipids associated with larger lipoproteins produce a signal that is distinct from those of smaller lipoproteins. A computer algorithm developed by LipoScience deconvolutes the signals into lipoprotein subclasses and then quantifies the number of particles in each class.NMR provides a useful and novel way to quantitate lipoprotein particles. However it is currently a proprietary technology and NMR analyzers are not yet readily-available for purchase and use in smaller clinical laboratories.

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 .

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.

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.

Molecular based clinical diagnostic test methodologies differ according to the target of interest. For example, patients suspected of having different diseases will require the identification of different targets. These targets might be found in different cells of the body and may therefore require different specimens to provide the answers. Patient A suspected of having Disease 1-requires the identification of a target of missequenced DNA- might require specimen of whole blood Patient B suspected of having Disease 2-requires identification of a target of antibody production-methodology might require specimen of serum Using this specific approach of disease diagnosis based on unique target identification, tests can provide answers that are more rapid sensitive specific

To aid in the diagnosis of disease or identification of infectious agents, clinical laboratorians use a variety of methodologies to assist them. Knowing what to look for, or the right question to ask, is vital to obtaining the correct answer. Many diseases and agents have unique causes. The cause of the condition then becomes the "target" to be identified and perhaps even quantified. For example: If Patient A is suspected of having disease X, and disease X requires treatment, it is necessary to prove that disease X exists within patient A. We must know something about what causes disease X; is disease X an antigen, a bacteria, a viral particle, a missequenced piece of DNA?Once the target of interest (in this case Disease X) has been identified, the clinical laboratorian can choose the methodology most appropriate to answering the question, "Does disease X exist within Patient A?"

It is imperative to follow the individual package insert procedures when collecting and handling specimens. Reference labs provide specimen requirements as well as collection, handling and transport guidelines.The Clinical and Laboratory Standards Institute (CLSI) formerly known as NCCLS: National Committee for Clinical Laboratory Standards has published procedures for collection including those specific to molecular diagnostics.

As was earlier stated, molecular methodologies are as varied as the targets they are created to seek and there are many ways to arrive at the same answer. There are many combinations of target/amplification/detection.This module will briefly explain the specific uniqueness of each approach. Advantages and disadvantages of each will not be discussed as these will vary by lab, patient population, and other factors discussed in the clinical application section.

Molecular methodologies while highly advantageous do contain limitations and certain disadvantages. These can include:Cost: Molecular methodologies are usually more expensive than standard traditional methodologies. Equipment and reagent costs could be prohibitive to some laboratories. As molecular methods become more standard, the costs could potentially decrease. Currently, laboratories that consider the cost prohibitive prefer to transport molecular specimens to a reference laboratory.Personnel requirements: Depending on laboratory accreditation requirements and testing methodologies some personnel may not be qualified to competently perform molecular testing. Laboratory space requirements: Molecular amplification methods require dedicated space that may not be available in some clinical laboratories.

Burtis CA, Ashwood ER, Bruns DE, eds. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 4th ed. St. Louis, MO: Elsevier Inc; 2006.Clinical and Laboratory Standards Institute (CLSI). Collection, Transport, Preparation, and Storage of Specimens for Molecular Methods; Approved Guideline. CLSI document MM13-A. NCCLS. Wayne, PA: 2005.Clinical and Laboratory Standards Institute (CLSI). Molecular Diagnostic Methods for Infectious Diseases; Approved Guideline. Second ed. CLSI document MM3-A2. NCCLS. Wayne, PA: 2006.

Hereditary hemochromatosis (HH) is a disorder of iron regulation that results in excessive dietary iron absorption through the gastrointestinal tract. Over time, the resultant iron overload and its deposition in tissue may lead to widespread organ damage, a variety of chronic disorders, and even death. Although it is a genetic disorder, clinical symptoms most typically become apparent in middle aged adults. Iron overload occurs in a variety of hereditary and acquired forms, known as iron storage diseases. HH is the most common cause of inherited iron overload. (1) Due to lack of awareness, HH often goes undetected or unrecognized by health care providers. Early detection to prevent the serious complications associated with iron overload has important consequences for reducing morbidity and mortality. Laboratory tests that assess iron levels and molecular assays for genetic mutatations are essential for both its detection and diagnosis.

Hereditary hemochromatosis (HH) is a genetic disorder characterized by iron overload as a result of increased iron absorption. As iron absorption increases, the amount of iron bound to transferrin and transported in the plasma subsequently increases.With no available mechanism for excreting excess absorbed iron, normal iron storage sites become overloaded, resulting in ferritin levels that far exceed normal. As a result, iron is deposited in the parenchymal cells of the liver, pancreas, pituitary, heart, synovium, and other tissues with high concentrations of transferrin receptors. Iron in excess of normal cellular ferritin stores contributes to the generation of free radicals and reactive oxygen intermediates that cause cell damage to organs and tissues. This process results in the clinical condition known as iron overload, a hallmark feature of HH.

Several mutations of the HFE gene have been described. In the C282Y mutation, a base substitution leads to a change in the amino acid in position 282 from cysteine (C) to tyrosine (Y). The loss of the sulfhydryl-containing amino acid disrupts the tertiary structure of HFE so that it no longer binds to beta-2 microglobulin. Beta-2 microglobulin appears to act along with other proteins to chaperone the newly synthesized HFE out of the Golgi apparatus and to the cell surface where it can then bind to TfR. In the C282Y mutation, HFE remains in the Golgi, never making it to the cell surface. The result is that transferrin binding to TfR is enhanced and excessive amounts of iron enter the cells of the small intestine, liver, and other tissues. A second mutation, H63D, causes a histidine (H) residue in position 63 to be replaced by aspartic acid (D). The mechanism by which this mutation leads to increased iron uptake is less well understood when compared to the C282Y mutation. Unlike the C282Y mutation, the H63D mutation does not seem to affect the binding of beta-2 microglobulin and intracellular movement, since detectable concentrations of the mutated protein are found on cell membranes. Some researchers speculate that the H63D mutation affects the binding of proteins involved in iron regulation and uptake at the cell surface.A third mutation, S65C, leads to a serine-to-cysteine substitution in its associated protein. This mutation has been been found in some compound heterozygotes for C282Y or H63D, but is rarely associated with iron overload in HH.Additional mutations of HFE have been identified, but their clinical significance is unclear. Most laboratories performing molecular assays test for only the C282Y, H63D, and S65C mutations.

For reasons as yet unknown, not all individuals who are homozygous for the C282Y mutation display phenotypic features of HH, and persons with H63D polymorphisms rarely develop iron overload. The penetrance (percentage of individuals with a specific genotype who express the associated phenotype) of HFE mutations is generally considered to be low. Results of a recent meta analysis by the US Preventive Services Task Force conclude that 38% to 50% of all C282Y homozygotes develop some evidence of iron overload, but that only 10% to 33% develop clinical disease due to HH. (8) In other words, some individuals may have elevated iron test results such as transferrin saturation, but do not demonstrate significant organ damage. Estimates of penetrance in some studies have found it to be even lower. Penetrance of HFE mutations is currently a controversial subject among experts, and the significance of finding HFE mutations in a given individual is often unclear. The probability that a given individual with HFE mutations will develop clinical disease from iron overload cannot be determined at this time.

Hereditary hemochromatosis (HH) is frequently discovered only during management of associated illness or routine health evaluations. It has been estimated that only a small percentage of all affected persons are actually diagnosed. Individuals with HH may be symptomatic for several years prior to diagnosis and may have consulted multiple health care providers.Under-diagnosis of HH is thought to occur due to:• Lack of specificity of early signs and symptoms• Asymptomatic status of some patients until damage to organs and tissues has occurred• Confusion with liver disease due to other causes• Insufficient awareness and knowledge of HHEarly identification of persons with HH is essential to prevent serious and irreversible complications associated with severe iron overload. A classic triad of skin hyperpigmentation (bronzing), type 2 diabetes, and hepatic cirrhosis has long been recognized as evidence of advanced iron overload. However, persons with HH may present with a much wider variety of signs and symptoms, particularly if they are seen before significant iron accumulation has occurred. Age of presentation and disease severity are highly variable. A diagnosis of HH is based on laboratory evidence of iron overload, genetic mutations associated with HH, and presence of clinical signs and symptoms consistent with HH.(10)

The subject of screening for hereditary hemochromatosis (HH) is controversial and is currently being debated in the medical literature. Using laboratory tests to screen the asymptomatic general population is currently not recommended due to issues of testing costs, low genetic penetrance, and the possible risk of discrimination. Targeted case finding in select high risk populations such as men of Northern European ancestry may be a better approach to screening. (12)Molecular-based (DNA) assays required for confirmation of HH are costly when used for general population screening. Because recent studies have shown that a high percentage of persons with C282Y mutations do not develop iron overload or HH-related clinical conditions, screening for these mutations may falsely label an individual with a disease diagnosis. At the present time, it is impossible to determine which homozygotes or heterozygotes for HFE mutations will eventually develop iron overload. Furthermore, there is potential risk of discrimination in obtaining health insurance for persons identified as having genetic disorders.In contrast, some experts do advocate for screening the general population. Mutations associated with HH are very common in Caucasians in the US. Individuals who know they carry mutations associated with HH may benefit from periodic testing for iron overload. Finally, laboratory tests that assess iron status are relatively inexpensive, widely available, and offer one approach to screening for phenotypic expression of HH. Screening first-degree family members of a person with documented HH is generally considered to be worthwhile. Early detection of HH in relatives with common mutations may permit treatment before the development of substantial iron overload and related disease due to organ damage.

DNA tests for HFE mutations associated with hereditary hemochromatosis (HH) are available in some clinical laboratories and reference laboratories. Testing for the presence of the C282Y is essential, although most labs also test for H63D and S65C mutations. Molecular testing is most appropriate for confirmatory testing of symptomatic individuals with altered iron studies (increased TS and SF), in pre-symptomatic individuals (increased TS, normal SF and liver function tests), and in family members of individuals diagnosed with HH. The use of genetic tests alone for routine screening of asymptomatic persons is not recommended for several reasons. A positive test indicating the presence of HFE mutations does not guarantee that an individual will develop clinically significant iron overload or predict severity of symptoms. A negative result (no HFE mutations present) does not rule out a diagnosis of iron overload because of genetic heterogeneity. Compared to biochemical analyses for iron, molecular assays are expensive. Finally, molecular testing may result in the diagnosis of a genetic disease, thus opening up the possibility for discrimination in health insurance coverage. Using molecular methods, DNA is extracted from leukocytes in whole blood samples or from buccal cells and analyzed for specific HFE mutations using polymerase chain reaction (PCR) with melt curve analysis. Currently there are no FDA-cleared products for HFE testing, and testing laboratories are using "home brew" reagents. This situation is expected to change as manufacturers submit products for FDA approval.

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.    

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.

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.

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.

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.

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.

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. Clinical laboratory personnel who are licensed in the specialties of immunohematology, clinical chemistry, hematology, and serology / immunology may perform all tests in the blood banking specialty.

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.

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.

The laboratory can only perform tests that are ordered by individuals authorized to order tests. If an employee knows that a test has been ordered by someone other then an authorized individual, the employee should report it to their supervisor or the compliance officer (CO). The laboratory must have a system in place to detect tests that are not performed due to a laboratory error and stop or credit the billing for these tests. The laboratory cannot bill for tests that are not performed. An employee who is aware that a test has been canceled or has not been performed for some reason must follow the policies and procedures associated with correcting the billing. Release of test results by phone, fax and other non-routine methods Employees should release test results only to the clinical person who is authorized to receive the test results (i.e., the ordering physician). Never release test results to another physician or entity unless authorized by the ordering physician in writing. Never release the results of a test to a patient unless authorized in writing by the ordering physician.

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).

After the exposure, there is an incubation period that lasts between 45 and 180 days, with an average of 90 days.Many individuals with acute HBV will have no symptoms at all. Some will have a mild illness with loss of appetite, nausea and vomiting, and fatigue. About 30% of infected individuals will develop clinical hepatitis with jaundice (yellow discoloration of the skin and eyes due to liver dysfunction).

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.

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

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Clinical and Laboratory Standards Institute (CLSI). Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays; Approved Guideline. Fourth ed. CLSI document H21-A4. NCCLS. Wayne, PA: 2003.Clinical and Laboratory Standards Institute (CLSI). Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture; Approved Standard. Sixth ed. CLSI document H3-A6. NCCLS. Wayne, PA: 2007.Clinical and Laboratory Standards Institute (CLSI). Procedures for the Handling and Processing of Blood Specimens; Approved Guideline. Third Edition. CLSI document H18-A3. NCCLS. Wayne, PA: 2004.Ernst DJ. Applied Phlebotomy. Baltimore, MD: Lippincott Williams & Wilkins: 2005.Lowe B. Reinforcing safety sticklers. Advance for Medical Laboratory Professionals. May 2004; 16:2A-3A.The Joint Commission. Patient Safety-2009 National Patient Safety Goals. Available at: http://www.jointcommission.org/PatientSafety/NationalPatientSafetyGoals/. Accessed July 18, 2009.

The transfusion service laboratory (TS) instructed clinical staff to draw blood specimens for compatibility testing before transfusing any blood components or products.Once the blood samples were collected, the clinical staff immediately began transfusing the patient with the O Rh-negative blood.

This CaseWith the panel done 2 weeks post-transfusion, 5 panel cells that were Jk(a+) reacted and 5 that were Jk(a-) did not. This yields a p value of 0.004, which is less than the standard of 0.05, and therefore is more than acceptable statistically. In other words, an antibody other than anti-Jka would be expected to produce these panel results only 4 times in 1000 (which is pretty unlikely).Th true p value is much lower because many more cells were tested than in the panel alone.Concluding that the antibody is anti-Jka is further strengthened because the patient's red cells type as Jk(a-).Learning points: The most important things to know about statistical tools such as p values are that they Relate to the probability of getting the observed results if the null hypothesis were true (the panel results were due to another antibody) Do not substitute for technical and clinical judgment.

Some blood safety standards require that a list of common signs and symptoms of suspected adverse reactions be included in both nursing and transfusion service manuals. Several organizations have developed job aids to help clinical staff recognize the signs and symptoms of various suspected transfusion reactions and to suggest appropriate actions (e.g., see REACT in Online Resources).

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

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

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

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.

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.

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.

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.

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.