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Deletion of two out of four alpha chain gene 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 of alpha thalassemia minor has been shown in African Americans as well.

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.

The antibody screen can provide sufficient data to make initial hypotheses regarding the likely antibody specificities and may be useful to presumptively rule-out some antibody specificities. When analyzing the antibody screen data, the strength/characteristics of reactions (for example, mixed-field or weak versus strong), the phase of testing (for example, room temperature versus AHG), and the pattern of reactivity (which cells react and which do not) are all important factors that will provide clues about the possible identification of the antibody(ies). Many antibodies exhibit dosage, that is, they react more strongly with homozygous cells than with heterozygous cells. 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. 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. 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.

Why our immune system malfunctions is not completely understood. One current hypothesis is that the following series of events occurs resulting in the initiation of an autoimmune reaction. Gender and Genetic PredispositionA predisposition is usually the first step toward the development of an autoimmune reaction. Women are more likely to develop a systemic autoimmune disease than men. For example in SLE the female to male ratio is 9:1. The genotype of some individuals predetermines that their immune system will be more prone to a break in tolerance. This genetic susceptibility appears to be linked to multiple genes rather than a single gene. This is supported by evidence that some autoimmune diseases are more frequently encountered in certain ethnic groups compared to others. For example in American women between the ages of 15 and 64, the prevalence of SLE is 1 in 700 for Caucasians while it is 1 in 245 for African-American women.(Ref1) Evidence in one recent study suggests that the genes that impart an increased resistance to malaria unfortunately produce an increased susceptibility to the systemic autoimmune rheumatic diseases.(Ref2)Triggering eventThe second step is the occurrence of a triggering event that leads to a break in tolerance. For some very susceptible individuals this event might be exposure to an environmental trigger. These environmental triggers could be ubiquitous such as exposure to the Epstein Barr virus (EBV), or very limited, such as the exposure to leaking silicon from a breast implant. In others, the triggering event might be a change in hormonal balance. Whatever the case, the triggering event initiates the break in tolerance and the cascade of immunological events that eventually lead to the formation of an autoimmune disease begins.Development of autoantibodiesThe third step is the development of autoantibodies and subsequent development of clinical symptoms. Studies have shown that this process can take 3 years or longer and unfortunately, by the time the diagnosis is made, substantial damage to the body may have already occurred.

Slide-based ANA testing using a cell substrate started in the 1950s and continues to be the gold standard method. In the early days of ANA testing, rodent tissue (stomach, liver and/or kidney) was commonly used as the substrate. Rodent tissue however had several drawbacks such as small cell size, a lack of dividing cells (mitotics) and poor antigen expression that made interpretation of ANA patterns difficult. In the 1980s, cultured cell lines were examined for utility as an ANA substrate and the human epithelial- like cell line HEp-2 gained popularity. HEp-2's advantages over rodent tissue are: A large nucleus Better antigen expression Abundant mitotic cells that assist in interpretation of the ANA pattern (if grown properly).More recently a cell line called HEp-2000® has become popular for ANA detection. HEp-2000® is a HEp-2 cell line that has been transfected with the cDNA for overexpression of the SSA/Ro antigen. This results in a substrate with all of the original advantages of HEp-2 plus an added advantage of increased sensitivity for detection of antibodies directed to the SSA/Ro antigen and the ability to identify these clinically significant antibodies during the screening process.(Ref4)It has also been demonstrated that antibodies to SSA/Ro develop early in the disease process.(Ref5) Perhaps most importantly, if a woman has anti-SSA/Ro antibodies and becomes pregnant there is a risk of the antibodies crossing the placenta, resulting in the fetus developing neonatal lupus and congenital heart block in utero.The advantage of using these transfected cells is documented in the current Clinical and Laboratory Standards Institute (CLSI) guidelines for ANA testing. Here they note the "dramatically increased" sensitivity of transfected cells for the detection of SS-A/Ro and the unaltered effect of transfection on other ANA patterns.(Ref6)

American College of Rheumatology, Committee on Rheumatologic Care, Position Statement, Methodology of Testing for Antinuclear Antibodies; Feb, 2009. Available at http://www.rheumatology.org/search/search.asp accessed on June 16, 2010Anuradah V, Chopra A, Sturgess A, Edmonds J. Cost-effective screening method for antinuclear antibody detection. Asian Pacific League of Associations for Rheumatology. 2004(7):13-18.Arbuckle MR, McClain MT, Rubertone MV, et al. Development of autoantibodies before the clinical onset of systemic lupus erythematosus. N Engl J Med. Oct 16 2003;349(16):1526-1533.Bossuyt X, Frans J, Hendrickx A, Godefridis G, Westhovens R, Marien G. Detection of Anti-SSA Antibodies by Indirect Immunofluorescence. Clin Chem. 10 7 2004;50(12):2361-2369.Clinical and Laboratory Standards Institute (formerly NCCLS); Quality Assurance of Laboratory Tests for Autoantibodies to Nuclear Antigens: (1) Indirect Fluorescence Assay for Microscopy and (2) Microtiter Enzyme Immunoassay Methods; Approved Guidelines - Second Edition. CLSI I/LA2-A2. 2006;26(13).Fritzler MJ, Hanson C, Miller J, Eystathioy T. Specificity of autoantibodies to SS-A/Ro on a transfected and overexpressed human 60 kDa Ro autoantigen substrate. J.Clin.Lab.Anal. 2002;16:103-108.Fritzler MJ, Miller BJ. Detection of autoantibodies to SS-A/Ro by indirect immunofluorescence using a transfected and overexpressed human 60 kD Ro autoantigen in HEp-2 cells. J.Clin.Lab.Anal. 1995;9:218-224.Fritzler MJ, Wall W, Gohill J, Kinsella TD, Humbel RL. The Detection of Autoantibodies on HEp-2 Cells Using an Indirect Immunoperoxidase Kit (Colorzyme®). Diag Immunol. 1986;4:217-221. Keech CL, Howarth S, Coates T, Rischmueller M, McCluskey J, Gordon TP. Rapid and sensitive detection of anti-Ro (SS-A) antibodies by indirect immunofluorescence of 60kDa Ro HEp-2 transfectants. Pathology. 1996;28:54-57.Keech CL, McCluskey J, Gordon TP. Transfection and overexpression of the human 60-kDa Ro/SS-A autoantigen in HEp-2 cells. Clin.Immunol.Immunopathol. 1994;73:146-151.Kroshinsky D, Stone JH, Bloch DB, Sepehr A. Case records of the Massachusetts General Hospital. Case 5-2009. A 47-year-old woman with a rash and numbness and pain in the legs. N Engl J Med. Feb 12 2009;360(7):711-720. McCarty, G.A., Valencia, D.W., and Fritzler, M.J., Antinuclear Antibodies-Contempory Techniques and Clinical Application to Connective Tissue Disease. New York: Oxford University Press, Inc. 1984. Murray DL, Homburger HA, Horvat RT, Snyder MR, College of American Pathologists; S-C 2009: Antinuclear Antibody Screening Methods; CAP Surveys S-C Diagnostic Immunology;2009 Pollock W, Toh BH. Routine immunofluorescence detection of Ro/SS-A autoantibody using HEp-2 cells transfected with human 60 kDa Ro/SS-A. J.Clin.Pathol. 1999;52:684-687.Singer, M. and Berg, P., Genes & Genomes-A Changing Perspective. Mill Valley, CA: University Science Books. 1991.Sullivan KE. The complex Genetic Basis of Systemic Lupus Erythematosus, Reprint from 1999 and 2000; Lupus Foundation, Available at http://www.lupus.org/education/articles/geneticbasis.html Accessed June 16, 2010.Wallace DJ. New methods for antinuclear antibody testing: does it cut costs and corners without jeopardizing clinical reliability? Nat Clin Pract Rheumatol. Aug 2006;2(8):410-411.Willcocks LC, Carr EJ, Niederer HA, et al. A defunctioning polymorphism in FCGR2B is associated with protection against malaria but susceptibility to systemic lupus erythematosus. Proc Natl Acad Sci U S A. Apr 27 2010;107(17):7881-7885.

Autoantibodies to the nucleus of the cell are not the only antibodies with clinical significance. Autoantibodies to components of the cytoplasm can be very important pieces in solving the diagnostic puzzle for some patients and should be reported. When reading the ANA results always start by determining if there is a discernible pattern in the nucleus of the cells, if not then the result is ANA negative.Then examine the cytoplasm to see if there is a discernible pattern present there. If so, then the report should contain a comment on the cytoplasmic pattern present.The following photographs demonstrate the more common cytoplasmic patterns.

Clinical symptoms associated with DIC are: Extensive ecchymoses Petechiae Hemorrhage Ultimately, organ failure

The Instrumentation and tools used for paraffin embedding can vary by laboratory. Methods to heat and hold paraffin in the molten state, as well as to harden it by cooling are required. Shown is a typical paraffin embedding center that is used in many clinical histology laboratories.

In order to determine where there is waste, the Lean system recommends taking a waste walk. The table below lists the types of waste that were discussed on the previous page and questions that you might ask as your team proceeds through a waste walk. You will probably not be able to get rid of all waste, but you can continually find ways to reduce it.Waste CategoryQuestionsDefect correctionAre you fixing errors in paperwork?Are you unable to process items because of illegible handwriting, errors, or other causes?OverproductionIs the laboratory producing more reports than needed?Is the laboratory printing, faxing, copying, and/or e-mailing more than is necessary?Are more tests or services being ordered or performed than required by the customer (eg, clinical staff) or for the patient?Excess motionAre you frequently searching for paper documents in cabinets and drawers?Are you regularly hand-carrying paperwork from one laboratory area to another, or one department to another?Excess movement of materialsDo you constantly need to locate and move a piece of equipment?Do you have temporary locations for supplies and equipment?WaitingIs there too much dependency on others to complete a task before you can begin a task?Are there delays in receiving information?Do patients wait for long periods of time before receiving care?Inventory Are files piling up? Is the laboratory purchasing excessive supplies of any kind?Are there obsolete items in the laboratory, eg, files,equipment, supplies, reagents?Excess processingIs the laboratory duplicating reports or information?Are you doing more work than is required for a particular process?Does the laboratory have unnecessary levels of authorization and approval?

After the team has collected enough data, the next step would be to analyze the data, using tools to identify and verify the root cause(s) of the problem. Tools that will be discussed on the following pages include: Pareto chart, control chart, and cause & effect diagram. Numerous advanced statistical tools, such as ANOVA, chi square, t-test, and Z-test, could be used during the "Analyze" phase. Commercial software (Minitab and SPSS) is also available. However, Six Sigma projects in clinical laboratories do not normally require complex statistical analysis techniques.

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 bone marrow expansion and skeletal deformations, which are a result of increased erythropoiesis due to low hemoglobin levels. A common finding is facial bone changes caused by this bone marrow expansion (sometimes referred to as Mongoloid facial features). Other clinical signs include frequent infections, hepatomegaly, splenomegaly, gall stones, leg ulcers, iron toxicity, and poor growth and sexual development. In addition, cardiac failure due to increased burden of the heart attempting to oxygenate the tissues, can lead to serious complications and death if the condition is not treated.In general, 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.The different genotypes associated with beta thalassemia major are: B0/B0, B0/B+, or B+/B+.

Most technologists working in the clinical hematology setting are familiar with the morphology of blood cells found in peripheral blood smears. Many of the same blood cells found in the peripheral blood are also found on cytospin preparations of body fluids. While the morphologies are similar between the two sources, there are changes to the "comfortable/familiar" peripheral blood morphology that are introduced by the cytospin technique. It is paramount to fully conceptualize the appearance of both normal and abnormal blood cell morphologies in body fluids in order to accurately perform fluid differentials.The cytopsin process works by wicking fluid into a filter while fluids samples are spun into a central column and deposited in a mono-layer onto a defined area of a slide. This allows the cells to be concentrated for appropriate identification. This technique can cause pronounced changes to morphology and staining characteristics, as well as cellular destruction if the cytospin malfunctions.The cytospin technique is known to stretch and distort cellular and nuclear morphology and allow nucleoli to appear more prominent than what one would normally see in peripheral smears. Cytospinning, however, does not change nuclear:cytoplasmic ratios nor does it alter relative chromatin textures or clumping patterns. Though this technique can make cells appear larger that on the peripheral blood smear, it can not change cytoplasmic textures and granulation. Focusing on these steadfast features can make cytospin morphology less intimidating.

While the role of the hematopathologist in the interpretation of bone marrow samples is well defined, the role of the technologist can vary greatly depending on the laboratory and the clinical setting.In some cases, physicians deliver prepared bone marrow smears to the laboratory that they have been prepared at the patient's bedside . In other settings, the technologist assists in the bone marrow collection procedure by making bone marrow smears at the bedside. There are also situations where a physician will bring anticoagulated bone marrow in specimen tubes to the laboratory for the technologist to smear, stain, and distribute as the hematopathologist requires.Once the marrow smears are prepared and stained, the next steps will vary depending on the laboratory. In some institutions it may be the hematology or oncology fellows/attending physicians who are responsible for counting and evaluating the aspirate smears, while the biopsy samples go to pathology. In other settings it may be the technologists who perform the differentials while hematopathology completes the evaluation and interpretation.

College of American Pathologists. 2010 Hematology and Clinical Microscopy Glossary. Northfield, IL: College of American Pathologists; 2010.Foucar K, Reichard K, Czuchlewski D. Bone Marrow Pathology 3rd ed. Chicago, IL: ASCP Press; 2010.Glassy EF, ed. Color Atlas of Hematology. Northfield, IL: College of American Pathologists; 1998.Hoffbrand AV, Pettit JE. Color Atlas of Clinical Hematology. St Louis, MO: Mosby; 2001.

When the technologist accompanies the clinician to assist with the bone marrow aspiration procedure to make smears at the bedside, it is necessary to understand the role of the clinician and the technologist.The clinician is responsible for patient positioning and sterile preparation, pain control, and performing the aspirate and biopsy. The clinician often hands off sample syringes to the technologist, once collected. The clinicians are responsible for providing the procedure kit and fixative for the biopsy, all labels, and obtaining the requisitions and a copy of the clinical history for the hematopathologist. The technologist will set up a mini workspace near the bedside where the samples are split into the required tubes. Smears are then prepared from the aspirate as well as biopsy touchpreps before the biopsy is placed in fixative. In this setting the technologist will usually deliver the samples and requisitions to pathology and continue the processing procedure.The kit the technologist brings to the bedside usually contains mini petri dishes, coverslips, slides, microcapilary tubes or Pasteur pipettes, micro-pipette bulb and the various evacuated blood collection tubes and media flasks required for the standard bone marrow draw.Most institutions will have a standard draw and testing protocol designed to ensure that enough sample is obtained to cover all of the usual testing requirements. An example would be a three-syringe-draw with the first two syringes containing no anticoagulant and the third syringe rinsed with preservative-free heparin. The first dry pull would be split between a green and a purple top evacuated blood collection tube and would be used for morphology (EDTA) and flow cytometry and cytogenetics (green) if needed. The second dry pull is split into two additional purple top tubes plus a green top tube and would be used for molecular assays such as SNP array, Flt-3, JAK2, MPL mutation, etc. The final heparinized syringe could be used for other treatment protocol requirements or to provide sample for additional assays.

Many organizations and agencies provide service in healthcare issues relating to cardiac and vascular disease. They provide guidelines, recommendations, and updates on research. Organizations and agencies whose guidelines and recommendations are referenced in this unit are: American Association of Clinical Chemistry (AACC) American College of Cardiology (ACC) American Heart Association (AHA) Centers for Disease Control and Prevention (CDC) European Society of Cardiology (ESC) International Federation for Clinical Chemistry (IFCC) World Health Organization (WHO)

Arneson W, Brickell J, eds.Clinical Chemistry: A Laboratory Perspective. Philadelphia: FA Davis; 2007.Burtis CA, Ashwoood ER, Burns DE, eds. Tietz Fundamentals of Clinical Chemistry. 6th ed. St. Louis, MO: Elsevier Saunders; 2008.Carreiro-Lewandowski E. Update on cardiac markers. Lab Med. 2006;37:597-605.D'Amore PJ. Evolution of C-reactive protein as a cardiac risk factor. Lab Med. 2005;36:234-238.Dotsenko O, Chackathayil J, Lip GY. Cardiac biomarkers:myths, facts and future horizons. Expert Review of Molecular Diagnostics. 2007;7:693-697.Foley, K. BNP: a novel biomarker. Advance for Medical Laboratory Professionals. August 25, 2008:9.Kaplan LA, Pesce AJ, Kazmierczak SC, eds. Clinical Chemistry Theory, Analysis, Correlation. St. Louis, MO: Mosby Elsevier Science; 2007. McDowell J. Reviewing the evidence for BNP, NT-proBNP testing. Clin Lab News. 2006;32:1, 3, 5.Rollins G. cTn and MI - what's the diagnosis? Clin Lab News. 2009;35:1, 3-4.Rollins, G. The BNP debate. Clin Lab News. 2009;35:1,3-4.Schreiber D, Miller SM. Use of cardiac markers in the emergency department. eMedicine. Updated July 2009. Available at: http://emedicine.medscape.com/article/811905-print.Accessed March 25, 2013. Thygesen K, Alpert JS, White HD. Universal definition of myocardial infarction. Circulation 2007;116:2634-2653.

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, 1996 We 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.

Clinical and Laboratory Standards Institute (CLSI). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard-Eighth Edition. CLSI document M07-A8. Wayne, PA: 2009. Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing; Twenty-First Informational Supplement. CLSI document M100-S21. Wayne, PA: 2011. It is important to detect ESBL-producing stains of Klebsiella pneumoniae, K. oxytoca, Escherichia. coli, and Proteus mirabilis as treatment failure may occur if the wrong cephalosporin is selected. These enzymes have the ability to hydrolyze third-generation cephalosporins and aztreonam, but are inhibited by clavulanic acid. ESBL-producing organisms can also exhibit co-resistance to many other classes of antibiotics. In January 2010, the Clinical and Laboratory Standards Institute (CLSI) published revised cephalosporin (cefazolin, cefotaxime, ceftazidime, ceftizoxime, and ceftriaxone) and aztreonam interpretive criteria for Enterobacteriaceae. The criteria for cefepime and cefuroxime (parenteral) did not change. Laboratories using these new criteria detailed in the M100-S21 document, table 2A, published in January 2011, no longer need to routinely test for extended-spectrum beta-lactamases (ESBLs) prior to reporting results. IF using the new criteria, there is no longer a need to change the interpretive criteria for cephalosporin's, aztreonam or penicillin's from susceptible to resistant before reporting. IF reporting moxalactam, cefonicid, cefamandole, or cefoperazone for E. coli, Klebsiella, or Proteus species, ESBL testing should still be performed as outlined in CLSI document M100-S21, supplemental table 2A-S1. If these isolates test ESBL positive, they should be reported as resistant. These drugs have limited availability in many countries, so the interpretive criteria were not evaluated by CLSI. CLSI notes that ESBL testing could still be useful for epidemiology and infection control purposes. ESBL testing should also continue to be performed until the new CLSI interpretive criteria is implemented.

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, 1993 Identification 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 image).

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, 1992 OBJECTIVES: 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 streptococc European 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 US 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 two or three days post-partum 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, 1995 It 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.

Urine pH results must be evaluated in conjunction with a patient's medical condition and clinical history. Factors to be considered include:Respiratory and metabolic statusRenal functionCrystal or calculi formationDietThe table below summarizes dietary and medical conditions as well as preanalytic and analytic errors that may affect urine pH:ConditionAcid pHAlkaline pHHigh meat dietXVegetarian dietXRespiratory/metabolic acidosisXRespiratory/metabolic alkalosisXHypochloridemiaXHigh concentration of urine glucoseXBacterial infection caused by urease-producing bacteriaXProlonged storage of specimen at room temperature, allowing multiplication of urease-producing bacteriaX (above 8.0)Improper procedural technique; excess urine left on reagent strip, allowing acid buffer in protein pad to run over into adjacent pH pad (refers to some reagent strip configurations)XKidney failureXUrinary tract infectionsXVomitingXDiabetic ketoacidosis XDiarrheaXStarvationX

Individuals with diabetes mellitus may excrete small amounts of albumin in the urine (microalbumin) which may signal the beginning of reduced glomerular filtration. Stabilizing the blood glucose level at this time may delay progression of diabetic nephropathy. Both type I and type II diabetes mellitus are leading causes of renal failure. Microvascular damage caused by excessive renal exposure to glucose can lead to diabetic nephropathy. By the time the urine protein level reaches the 30 mg/dL level that is necessary for detection by routine reagent strips, damage to the kidneys may have already occurred. Reagent strips are available that use a dye-binding technique rather than the traditional protein-error of indicators principle. These strips are more sensitive and specific for albumin, detecting levels as low as 8 mg/dL.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. 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.

Ketone bodies are usually absent in urine. The presence of ketones in the urine most likely indicates that the body is using fats rather than carbohydrates for energy. For example, 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 may 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 also be detected in the urine 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.

Blood is normally not present in the urine of healthy individuals, apart from blood during menses that may be detected in urine samples from females, Hematuria is associated with renal or genital disorders in which the bleeding is the result of irritation to the involved organs or some type of trauma. Examples include:Renal calculiPyelonephritisGlomerulonephritisTumorsTraumaExposure to toxic chemicals or drugsStrenuous exerciseHemoglobinuria may be due to the lysis of red blood cells within the urinary tract. This can be caused by intravascular hemolysis, as the hemoglobin is filtered through the glomeruli. In a healthy, normal individual, the hemoglobin molecule attaches to haptoglobin and bypasses the kidney filtration system. When the hemoglobin/haptoglobin system is overwhelmed, hemoglobin passes into the urine. Hemoglobinuria may be associated with:Hemolytic anemiaSevere burnsTransfusion reactionInfection Strenuous exercise

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, as shown in the table below:ConditionUrine Bilirubin ResultUrine Urobilinogen ResultHemolytic diseaseNegativeIncreasedHepatitic diseasePositive or negativeIncreasedBiliary obstructionPositiveNormal* *Urine chemical reagent strip methods 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. There are no "abnormal" specific gravity values. This test simply indicates how concentrated the urine is.

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.

Some laboratories perform testing for other reducing substances on urine specimens from children below a certain age (eg, age 2) whenever a urinalysis is ordered. The purpose is to detect serious inborn errors of metabolism. The reducing substance assay uses the classic Benedict’s copper reduction reaction to detect glucose and other carbohydrate metabolites. If the reducing substance test (ie, Clinitest®) is positive and the reagent dipstick assay for urinary glucose is negative, it is possible that other reducing substances are present. Further testing would be required to diagnose the exact inborn error of metabolism. With the current practice in most U.S. states of screening all newborns for metabolic disorders, the urine test for reducing substances may be performed only when the clinician orders it, rather than the test being part of a routine urinalysis procedure. Clinitest® is not the recommended monitoring test for diabetics; glucose reagent strip testing is a better choice.

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 4-6° 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.)

Bersch C. ed. The ABCs of pre-, neo-, and post-natal testing. MLO. September 2009;41.Clinical and Laboratory Standards Institute (CLSI). Procedures and Devices for the Collection of Diagnostic Capillary Blood Specimens; Approved Standard. Fifth ed. CLSI document H4-A5. NCCLS. Wayne, PA: 2004.Clinical and Laboratory Standards Institute (CLSI). Procedures for the Handling and Processing of Blood Specimens; Approved Guideline. Third ed. CLSI document H18-A3. NCCLS.Wayne, PA: 2004.Ernst DJ. Applied Phlebotomy. Baltimore, MD: Lippincott Williams & Wilkins: 2005.Garza D. Becan-McBride K. Phlebotomy Handbook. 7th ed. Upper Saddle River, NJ: Pearson Prentice Hall: 2005.Stevens B, Yamada J, Ohlsson A. Sucrose for analgesia in newborn infants undergoing painful procedures. Available at http://www.nichd.nih.gov/cochrane/Stevens/Stevens.HTM accessed January 12, 2010.

Many people involved in the clinical laboratory sciences need to be familiar with basic statistics for a variety of reasons. These reasons include:Evaluating quantitative quality control results Evaluating new test methodsUnderstanding how acceptable laboratory procedures and methods are established Determining reference values for laboratory tests Understanding clinical trials and new methods presented in journals and articles Performing research projects

Most clinical laboratories will use a 1:1 mix when performing mixing studies; however, some will use various dilutions of patient plasma and pooled normal plasma for their protocols.In addition, the analysis of the mixing study involves interpreting the pre- and post-mix results. This can be performed using various methods including: the Rosner Index, the <70% correction formula, or a laboratories own calculation and cut-off value. Finally, it is suggested that each laboratory test the sensitivity of their PT and aPTT reagents before running mixing studies. The sensitivity of the reagent system can be tested by running dilutions of the pooled normal plasma controls with specific factor deficient plasma. This ensures that the system will detect a normal result, even if the factor level is as low as 40%.

This course will primarily focus on recommendations made by the American Diabetes Association (ADA) that are related to the diagnosis and monitoring of diabetes. The ADA states on its website, "Our mission is to prevent and cure diabetes and to improve the lives of all people affected by diabetes."*Other important agencies and studies referred to in this course are: International Diabetes Federation (IDF): An alliance of 200 diabetes associations; acts as a global advocate for individuals with diabetes.World Health Organization (WHO): An arm of the United Nations; provides programs for prevention, treatment, and care of those with diabetes worldwide.Diabetes Control and Complications Trial (DCCT): A major clinical study 1983-1993; proved the correlation between control of glucose blood level and lowered onset and severity of the complications of diabetes. *Reference: American Diabetes Association. Available at: http://www.diabetes.org/about-us/. Accessed April 14, 2010.

Categories of increased risk for diabetes is the new designation for individuals whose glucose or HbA1C levels are higher than reference ranges but lower than the diagnostic criteria for diabetes (ADA 2010 Clinical Practice Recommendations). These individuals are at increased risk for development of diabetes and should have intervention initiated. Formerly individuals at increased risk for development of diabetes were called pre-diabetic; the 2010 recommendations recommend use of this new category designation but also state that the term pre-diabetic may still be used. Besides risk of diabetes, these individuals have higher risk for cardiovascular disease.

These are the ranges that are recommended by the 2010 ADA Clinical Practice Guidelines for determining increased risk for diabetes: Glucose test Range indicating increased risk for diabetes Fasting plasma glucose 100 - 125 mg/dL 2-hour plasma glucose following 75g glucose load 140 - 199 mg/dL HbA1C 5.7 - 6.5%

The ADA guidelines include recommendations for screening for diabetes. It is recommended to screen asymptomatic persons for diabetes or their risk of diabetes. Screening is recommended for all individuals age 45 years and older; a negative screen should be repeated every three years. Screening is essential for individuals who are overweight, defined as a body mass index (BMI) > 25 kg/m2. The ADA also recommends earlier screening for many individuals. Among these are individuals who are overweight and have additional risk factors. Additional risk factors include: Physical inactivity Family history of diabetes A member of a high-risk ethnic group Women who have had a large birth weight baby or gestational diabetes diagnosis should have earlier screening. Also included for earlier screening are individuals who are hypertensive or have lipidemia, vascular disease, or other clinical conditions associated with insulin resistance. Individuals who in previous testing had impaired glucose tolerance (IGT), impaired fasting glucose (IFG), or HbA1C in the range of 5.7-6.5% should be screened for diabetes regularly.

In the past twenty years there have been significant improvements in the accuracy of handheld glucose meters. Patient use has resulted in substantial improvements in diabetic control and insulin therapy. Capillary whole blood is easily obtained and glucose concentration is derived on simple to use, portable meters. Since whole blood glucose is lower than plasma glucose, the meters are programmed to correct the value before presenting the result; therefore, the whole blood glucose meter result correlates to serum or plasma results.Clinical and Laboratory Standards Institute (CLSI) has set standards for correlation between glucose meter and laboratory measured glucose levels. If the laboratory measured glucose is > 75 mg/dL, the glucose meter result should be within 20%. For laboratory measured values < 75 mg/dL, the glucose meter result should be within 15 mg/dL.

American Diabetes Association. Standards of medical care in diabetes - 2010. Diabetes Care; January 2010;33:S11-S61.American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care. January 2010;33:S62-S69.Anderson SA, Cockayne S. Clinical Chemistry Concepts and Applications. Long Grove, Illinois: Waveland Press, Inc, 2003.Bell JR. The new glycohemoglobin standard. Clin Lab News, American Association of Clinical Chemistry; October 2008; 34:1, 3-4.Burtis CA, Ashwood ER, Burns DE, eds. Tietz Fundamentals of Clinical Chemistry, 6th ed. St. Louis: Saunders, an imprint of Elsevier, Inc, 2008.Charles MA. Diabetes and the laboratorian: Opportunities for a new role. MLO. May 2001, 16-24.Definition and diagnosis of diabetes mellitus and intermediate hyperglycaemia WHO 2006. World Health Publications. Available at http://www.who.int/topics/diabetes_mellitus/en/ Accessed 1/11/10.Estimated average glucose, eAG. Available at:http://professional.diabetes.org/glucosecalculator.aspxAccessed 1/11/10.Kaplan LA, Pesce AJ, eds. Clinical Chemistry Theory, Analysis, Correlation. St. Louis: Mosby Inc, an affiliate of Elsevier Inc, 2010.Rollin G. A new role for hemoglobin A1C. Clin Lab News, American Association for Clinical Chemistry. December 2008; 34:1, 3.

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 electrophoresisHigh resolution electrophoresisPolyacrylamide gel electrophoresisCapillary electrophoresisIsoelectric focusingImmunochemical electrophoresisTwo-dimensional electrophoresisPulsed 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.html Accessed March 25, 2013.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, RochesterMN;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. NationalAcademy of Clinical Biochemistry Laboratory Medicine Practice Guidelines. 2006.PLACtest animation, diaDexus. http://www.plactest.com/laboratorians/action.php Accessed March 25, 2013.Rifai N, Warnick GR. Lipids, lipoproteins, apolipoproteins, and other cardiovascular risk factors. In: BurtisCA, Ashwood ER. BrunsDE. 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.

George TI, Etzell JE, Bradley KT, et al. 2010 Hematology and Clinical Microscopy Glossary. Northfield, IL: College of American Pathologists; 2010.Glassy EF, ed. Color Atlas of Hematology. Northfield, IL: College of American Pathologists; 1998. McKenzie SB, Williams JL, ed. Clinical Laboratory Hematology. Boston: Pearson Education; 2009.

The INR component of the laboratory PT/INR result is a calculated value that is used by the clinician to monitor warfarin 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 (thromboplastin).INR= (PT of patient/PT of geometric mean of the normal population)ISIThe International Sensitivity Index (ISI) value, is provided by the reagent manufacturer as the relative sensitivity of the reagent itself. When opening a new lot of PT reagent (thromboplastin), it is essential to verify that the ISI value provided by the reagent manufacturer is being used with that reagent lot to prevent the reporting of erroneous INR results, which may have serious consequences for the patient.The INR is used to standardize PT results, and in turn, anticoagulant therapy, across laboratory instrumentation, methodologies, and locale.

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. D-dimer is a sensitive, but non-specific marker of fibrin formation and fibrinolysis that occurs with the formation of blood clots.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 the D-dimer method has been validated by medical literature and 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 also a sensitive, but non-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, including: 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 laboratories provide specimen requirements as well as collection, handling, and transport guidelines.The Clinical and Laboratory Standards Institute (CLSI) 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 laboratory, 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: 2006.Clinical and Laboratory Standards Institute (CLSI). Molecular Diagnostic Methods for Infectious Diseases; Approved Guideline. Second ed. CLSI document MM3-A2. NCCLS. Wayne, PA: 2006.

Sickle cell disease (SCD) manifests itself as a chronic hemolytic anemia. There is slowed growth and development in children with sickle cell anemia, who may present with dactylitis. In addition to the general symptoms of anemia (fatigue, weakness, pallor etc.) patients are prone to infection, cardiomegaly, usually due to iron deposits from frequent transfusions, and bone and organ infarcts. Male patients can experience priapism.Patients with SCD can experience vaso-occlusive, hemolytic, sequestration, and aplastic crises. The major symptom in SCD is pain. Pain is a warning sign that is related to vaso-occlusion and life-threatening complications.

Short chain fatty acids that increase levels of butyrate analogues inhibit the switching of hemoglobin chain production from gamma (HbF) to beta (HbA). Use of these compounds in the treatment of sickle cell is still under investigation.In clinical trials, cells containing HbF have been found to increase in number with the use of decitabine, a DNA hypomethilation agent. Also needing further investigation is the use of erythropoietin for treating sickle cell disease. Various colony stimulating factors have been found to increase the production of HbF.

Before beginning the course take some time to review and think about what you already know about HDFN. For example, jot down brief notes to answer the following questions: Which antibody causes the most severe HDFN? Antibodies in which blood group system are the most common cause of positive direct antiglobulin tests (DATs) in newborns but rarely cause clinically significant hemolysis? Should DATs be performed on all newborns regardless of maternal ABO and Rh blood groups? What is Rh immune globulin (RhIg), its source, constituents, purpose, and mechanism of action? Which tests are used to determine postnatal RhIg dosage? Which type of D variant can produce anti-D? What follow-up tests are typically indicated if a pregnant female has a positive antibody screen when initially tested? Which laboratory findings would suggest that an infant may have ABO HDFN? How can the clinical status of fetuses at risk for HDFN be monitored? What are the characteristics of red cells suitable for intravenous transfusion to fetuses suffering from severe HDFN due to anti-D?

If a pregnant woman is found to have an unexpected clinically significant antibody, routine antenatal serologic tests on the mother include Antibody identification to detect clinically significant antibodies. Antigen typing: Once the antibody is identified, the mother is tested for the corresponding antigen, which she should lack. Antibody titration: Laboratories have different protocols. Depending on the antibody titer, titration may be performed at 2 or 4 week intervals after 18 weeks gestation.Notes (titration): Maternal antibody titer is an unreliable indicator of fetal disease and is mainly done to determine if clinical fetal monitoring is warranted, e.g., Doppler ultrasonography of fetal cerebral blood flow or, more rarely, invasive monitoring such as amniocentesis. Careful quality control is needed for titrations. QC includes using red cells from donors with the same phenotype or likely genotype (e.g., R2r or R2R2) and titrating the new sample in parallel with the prior sample. A two-tube rise or more in a doubling dilution is considered a significant rise in titer. In the case of anti-D, a predetermined critical titer (often 16 or 32 for anti-D depending on the method) indicates the need for clinical fetal monitoring.

Clinically relevant information on D phenotypes can be summarized as follows: D phenotype D antigen expression Rh(D) typing Produce anti-D RBC to transfuse RhIg recommended** D+ normal direct agglutination no D+ or D– no Weak D normal but weak IAT no D+ or D– no Partial D altered direct agglutination* & IAT yes D– yes Partial weak D altered & variable direct agglutination* & IAT yes D– yes D– none IAT yes D– yes * Depending on anti-D reagent used ** USA, UK and parts of Canada

The following published literature and online resources, while useful, should not be used as a substitute for technical and clinical judgment. Medical and technical information becomes obsolete quickly and current sources relevant to the user's location should always be consulted.References indicated by * provide a broad overview of HDFN and are highly recommended.LITERATUREAvent ND, Reid ME. The Rh blood group system: a review. Blood 2000 Jan 15;95 (2):375-87.Bowman J. Thirty-five years of Rh prophylaxis. Transfusion 2003 Dec;43(12):1661-6.* Eder AF. Update on HDFN: new information on long-standing controversies. Immunohematology 2006;22(4):188–195. (scroll to article)Eder, AF, Manno, C.S. Alloimmune hemolytic disease of the fetus and newborn. In Wintrobe's Clinical Hematology, 11th ed. (Greer JP, Foerster J, Lukens JN, Rodgers GM, Paraskevas F, Glader BE, (eds). Philadelphia, PA: Lippincott, Williams & Wilkins, 2004.Flegel WA. Molecular genetics of RH and its clinical application. Transfus Clin Biol. 2006 Mar-Apr;13(1-2):4-12. Kennedy MS, McNanie J, Waheed A. Detection of anti-D following antepartum injections of Rh immune globulin. Immunohematology 1998;14(4):138-40.Koelewijn JM, de Haas M, Vrijkotte TG, van der Schoot CE, Bonsel GJ. Risk factors for RhD immunisation despite antenatal and postnatal anti-D prophylaxis. BJOG. 2009 Sep;116 (10): 1307-14. Epub 2009 Jun 17.* Kumar S, Regan F. Management of pregnancies with RhD alloimmunisation. BMJ. 2005 May 28;330(7502):1255-8. (UK perspective but much valuable information relevant to all)* Murray NA, Roberts IAG. Haemolytic disease of the newborn. Arch Dis Child Fetal Neonatal Ed 2007 Mar; 92(2): F83–F88. Oepkes D, Seaward PG, Vandenbussche FP, Windrim R, Kingdom J, Beyene J, Kanhai HH, Ohlsson A, Ryan G; DIAMOND Study Group. Doppler ultrasonography versus amniocentesis to predict fetal anemia. N Engl J Med. 2006 Jul 13;355(2):156-64.Ramsey G. Inaccurate doses of Rh immune globulin after Rh-incompatible fetomaternal hemorrhage: survey of laboratory practice. Arch Pathol Lab Med 2009 Mar; 133(3):465-9. Reid ME. The Rh antigen D: a review for clinicians. Blood Bulletin 2008 Apr; 10(1).Sandler SG. Effectiveness of the RhIg dose calculator. Arch Pathol Lab Med 2010 Jul;134(7): 967-8.Shulman IA, Calderon C, Nelson JM, Nakayama R. The routine use of Rh-negative reagent red cells for the identification of anti-D and the detection of non-D red cell antibodies. Transfusion 1994 Aug;34(8):666-70.Tamul KR. Determining fetal-maternal hemorrhage with flow cytometry. Advance 2000. Posted online June 5, 2000.Westhoff CM, Sloan SR. Molecular genotyping in transfusion medicine. Clin Chem 2008;54(12): 1948-50.ONLINE RESOURCESPaxton A. Bringing new rigor to RhIg calculations. CAP TODAY. May 2008. Accessed January 18, 2011.*Wagle S, Deshpande PG. Hemolytic disease of the newborn. eMedicine / WebMD. Updated Apr. 9, 2010. Accessed January 18, 2011.

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. The most common mutation in patients with hereditary hemochromatosis is the C282Y mutation. 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.

The PAS staining procedure is most commonly used in the clinical histology laboratory to demonstrate glycogen deposits in the liver. Glycogen granules may also be visible in tumors of the bladder, kidney, ovary, pancreas, and lung. Basement membranes, which are present in various tissues in the body, may also be visualized through the PAS staining procedure. Several disorders can be identified through the demonstration of abnormal basement membranes. The PAS staining procedure can also demonstrate fungi in tissue samples due to the high carbohydrate content of the organisms cellular walls. Neutral mucins in the gastrointestinal tract and some epithelial mucins will also give a PAS-positive staining reaction.

Bancroft JD, Gamble M, et al. Theory and Practice of Histological Techniques. 6th Edition. Philadelphia, PA: Churchill Livingstone; 2008 Carson FL; Hladik C. Histotechnology – A Self Instructional Text. 3rd Edition American Society for Clinical Pathology Press; 2009Carbohydrate Staining and Identification. Bowie, MD: National Society for Histotechnology;1993Guide to Special Stains. Carpinteria, CA: DakoCytomation; 2004Stains File http://stainsfile.info/StainsFile/jindex.htmlAccessed August 21, 2010The Histology Guide - University of Leedshttp://www.histology.leeds.ac.uk/index.phpAccessed August 21, 2010Boston University Histology Learning Systemhttp://www.bu.edu/histology/m/append02.htm Accessed August 21, 2010Biological Stain Commissionwww.biologicalstaincommission.org/ Accessed August 23, 2010University of California, San Francisco Department of Pathology and Lab Medicinehttp://labmed.ucsf.edu/education/residency/fung_morph/fungal_site/yeastpage.htmlAccessed August 23, 2010Special Stains Using the Microwave; Joyce Moore HT/HTL (ASCP); EBSciences http://www.ebsciences.com/papers/moore.htm Accessed August 29, 2010

Due to their transparent nature, the cellular and intracellular structure of tissue samples cannot be microscopically examined until they are colored by dyes. Dyes used to stain tissue samples for microscopic analysis in the clinical histology laboratory are called biological dyes or biological stains. Biological dyes can be grouped into the following two categories:Natural: Dyes that are derived from natural resources. The most important natural dye in the histopathology laboratory is hematoxylin.Artificial: Dyes that are derived through chemical reactions. Artificial dyes greatly outnumber natural dyes.

Many public health and governmental agencies promote awareness and prevention as well as monitor HPV transmission and infections. Additional governmental agencies regulate clinical testing for HPV as well as prevention methods such as vaccines. Clinical organizations recommend testing protocols for the detection of carcinomas that can result from HPV infections. Agencies and organizations referred to in this course are: Centers for Disease Control and Prevention (CDC) Federal Food and Drug Administration (FDA) National Cancer Institute (NCI) American Society for Colposcopy and Cervical Pathology (ASCCP)

Hybridization is the base pairing of complementary strands of RNA and DNA. The complementary base pairing may be between two strands of DNA, two strands of RNA, or a strand of DNA and a strand of RNA. In most clinical molecular testing, one of the strands is synthetic and the resulting double-stranded nucleic acid is referred to as a hybrid. Often the synthetic nucleic acid strand is short and is then called an oligonucleotide. A probe is another name for a synthetic strand of nucleic acid that hybridizes; probes often have attached labels.

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.

Carey J. In: McCoy JP, Keren DE, 4th ed. Flow Cytometry in Clinical Diagnosis. Chicago, IL : American Society for Clinical Pathology Press: 2010; 1-3, 16-21, 35-38.Craig FE. Flow cytometry. In: McKenzie SR, Zeibig E, ed. Clinical Laboratory Hematology.Upper Saddle River, NJ : Pearson Prentice Hall: 2004;419-435.Invitrogen by Life Technologies. Fluorescence tutorials. Available at: http://www.invitrogen.com/site/us/en/home/support/Tutorials.html. Accessed February 10, 2011.McKenzie SR, Williams JL, 2nd ed. Clinical Laboratory Hematology.Upper Saddle River, NJ: Pearson Prentice Hall: 2010;836-849.Stevens CD. In: 3rd ed. Clinical Immunology and Serology.Philadelphia, PA: F. A. Davis Company:2010; 186-196, 399-416.

The quality control (QC) program should make use of whatever tools are available to monitor the method system and ensure reliable test results.The Centers for Medicare and Medicaid Services (CMS) dictates quality control (QC) requirements for laboratories operating in the United States through the Clinical Laboratory Improvement Amendments (CLIA). CLIA requires both intralaboratory (or internal) QC and external quality assessment (QA) measures. Intralaboratory QC is the QC that is performed by the laboratory itself. The external QA required by CLIA is a proficiency testing program.Interlaboratory (between laboratories) QC may also be an option for some method systems. Laboratories using the same instrumentation and same QC lot numbers periodically compare QC results (peer comparisons) to determine target values, QC limits, and data consistency.

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/her red cells and anti-A in the serum. Anti-A1 is produced by about 4% of group A2 individuals and about 25% of group A2B individuals. 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.

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 Laboratory employees should release test results only to a person who is authorized to receive the test results. The Clinical Laboratory Improvement Amendments (CLIA) define an authorized person as: "an individual authorized under State law to order tests or receive test results, or both." Patients may be considered "authorized persons," if State law defines them as such. Refer to your own State laws regarding release of laboratory results directly to patients.

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.

Metabolic syndrome may vary in definition and diagnostic criteria depending on the organization that is consulted. Health-related organizations that have developed diagnostic criteria for metabolic syndrome include: National Heart, Lung, and Blood Institute (NHLBI)/American Heart Association (AHA) World Health Organization (WHO) American Association of Clinical Endocrinologists (AACE) International Diabetes Foundation (IDF) European Group for Study of Insulin Resistance (EGIR)Each organization's set of criteria is slightly different in its parameters and its details for diagnosis. All of the above organizations agree that defining glucose intolerance, obesity, hypertension, and dyslipidemia is important. However, there are varied opinions in how important each risk factor is in relation to the others. Varied criteria to determine obesity is utilized: waist to hip ratio, BMI, and waist circumference. One organization does not include a measurement of obesity. Glucose intolerance is determined by measuring plasma insulin and/or glucose levels. Lack of standardization in insulin measurement and assay availability makes criteria using insulin levels impractical.

Armstrong C. Practice guidelines AHA and NHLBI review diagnosis and management of the metabolic syndrome. Am Fam Physician. 2006;74:891-1062.D'Amore PJ. Evolution of c-reactive protein as a cardiac risk factor. Lab Med. 2005;36:234-238.Devaraj, S, Swarbrick MM, Singh U et al. CRP and adiponectin and its oligomers in the metabolic syndrome evaluation of new laboratory-based biomarkers. Am J Clin Pathol. 2008;129:815-822.Eckel RH, Grundy SM, Zimmet PZ. The metabolic syndrome. Lancet. 2005;365:1415-1428.Expert Panel in Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (authors). 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.Gade W, Gade J, Collins M et al. Failures of feedback: rush hour along the highway to obesity. Clin Lab Sci. 2010;23:39-50.Gade W, Gade J, Collins M et al. Beyond obesity: the diagnosis and pathophysiology of metabolic syndrome. Clin Lab Sci. 2010;23:51-61.Grundy SM. Does a diagnosis of metabolic syndrome have value in clinical practice? Am J Clin Nutr. 2006;83:1248-1251.Grundy SM, Brewer HB, Cleeman JI, et al. Definition of metabolic syndrome: report of the national heart, lung, and blood institute/american heart association conference on scientific issues related to definition. Circulation. 2004;109:433-438.Grundy SM, Cleeman JI, Daniels SR, et al. Diagnosis and management of the metabolic syndrome: An American Heart Association/National Heart, Lung, and Blood Institute scientific statement. Circulation. 2005;112:2735-2752.Grundy SM. Obesity, metabolic syndrome, and cardiovascular disease. J Clin Endocrinol Metab. 2004;89:2595-2600.Mathew B, Francis L, Kayalar A, et al. Obesity: effects on cardiovascular disease and its diagnosis. J Am Board Fam Med. 2008;21:562-568.Metabolic Syndrome. National Heart Lung and Blood Institute. Diseases and Conditions Index. Available at http://www.nhlbi.nih.gov/health/dci/Diseases/ms/ms_whatis.html. Accessed December 5, 2011.Mittal S. The Metabolic Syndrome in Clinical Practice. London, England. Springer-Verlag Springer Science; 2008.Molinaro RJ. Metabolic syndrome: an update on prevalence, criteria, and laboratory testing. MLO. 2007;39:24-27.Ronti T, Lupattelli G, Mannarino E. The endocrine function of adipose tissue: an update. Clin Endocrinol. 2006;64:355-365.

Once relegated to the domain of research laboratories, molecular methods for the diagnosis of infectious disease had little, if any place, in a clinical diagnostic laboratory prior to 1985. Procedurally, molecular methods were very complex and required specialized instrumentation and dedicated laboratory space. They were also susceptible, initially, to the influence of variation of technique. Although they represented valuable research tools, and were helpful as esoteric testing for unique clinical situations, their performance characteristics simply did not fit well into most clinical laboratories.Certain pathogens were logical targets for development. Organisms that were of concern for significant patient populations, were difficult to sustain in transport, and/or were difficult to cultivate and detect by traditional methods represented some of the first targets of commercially offered molecular based assays.Sexually transmitted diseases, affecting significant numbers of people, with key pathogens affected by lability in transport or poor sensitivity with traditional cultivation or antigen detection methods, were among the first targets for development.

Human papilloma virus (HPV) is estimated to be the most common sexually transmitted infection in the United States. Digene's hybrid capture assay for HPV received approval by the Food and Drug Administration (FDA) in 2003. Only in recent years have other manufacturers, such as Third Wave Technologies, added this virus to their testing capabilities.Mycobacterium species represented another desirable target for molecular testing. Although some improvements in cultivation and staining techniques had been realized through the incorporation of broth media and fluorochrome staining, identification is still hampered by the growth rate of the organism. Gen-Probe first marketed probes that would allow identification of tuberculosis, M. avium-intracellulare, and M. gordonae in culture positive specimens. These probes greatly streamlined the workup of culture positive specimens.Of great interest to both clinicians and infection control practitioners, is the direct detection of Mycobacterium in clinical specimens. Gen-Probe received FDA approval for its AMPLIFIED MTDâ product for this specific application (in smear positive specimens) in 1995. This method employs isothermal transcription mediated amplification; the amplicons are detected using the same hybridization as the culture confirmation tests.

Implementing molecular methods may involve purchasing an equipment platform that represents a significant capital investment. Reagents for the assays are frequently more expensive, on a cost per test basis, than either culture or antigen detection methods. Reimbursement issues, although improving, can be more complex. Realistically, implementations of molecular methods are likely to represent increased costs that do need to be weighed against the potential benefits that can be realized.When considering the implementation of a molecular method, the following question should be asked:Will the methods significantly impact/improve clinical management and patient outcomes, reduce antimicrobial costs and lengths of stay, and/or facilitate infection control, epidemiology, or antibiotic stewardship programs?The answer may not be "yes" for every single agent of infectious disease for which molecular methods are now available.

BD GeneOhm™ MRSA [package insert]. Quebec, Qc, Canada: BD Diagnostics; 2009. Available at: http://www.bd.com/geneohm/english/products/pdfs/mrsa_pkginsert.pdf. Accessed February 22, 2012.Bonetta L. Prime time for real-time PCR. Nature Methods. 2005;2:305-312. Available at: http://www.nature.com/nmeth/journal/v2/n4/full/nmeth0405-305.html. Accessed February 22, 2012.Boughton B. Universal PCR Screening for MRSA May Cut Costs, Reduce Infection. In Medscape Medical News. Available at: http://www.medscape.com/viewarticle/708813. Accessed February 22, 2012.CDC Response: A Year in Review. Centers for Disease Control and Prevention Website. Available at: http://www.cdc.gov/h1n1flu/yearinreview.htm. Accessed February 22, 2012.Centers for Disease Control and Prevention. Evaluation of Rapid Influenza Diagnostic Tests for Detection of Novel Influenza A (H1N1) Virus ---United States, 2009. Morbidity and Mortality Weekly Report. August 7, 2009;58(30):826-829. Available at: http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5830a2.htm. Accessed February 22, 2012.Department of Biochemistry. University at Buffalo, School of Medicine and Biomedical Sciences Website. Available at: http://www.smbs.buffalo.edu/bch/Labs/SinhaLab/Protocols/RT-PCR.pdf. Accessed February 22, 2012.Desjardins M, Guibord C, Lalonde B, Toye B, Ramotar K. Evaluation of the IDI-MRSA Assay for the Detection of Methicillin-Resistant Staphylococcus aureus from Nasal and Rectal Specimens Pooled in Selective Broth. J Clin Microbiol. 2006 April;44(4):1219-1223. Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1448652/. Accessed February 22, 2012.Eastwood K, Else P, Charlett A, Wilcox M. Comparison C. difficile detection methods. J Clin Microbiol. 2009;doi:10.1128/JCM.01082-09. Available at: http://jcm.asm.org/cgi/content/short/JCM.01082-09v1Farley JE, Stamper PD, Ross T, Cai M, Speser S, Carroll KC. Comparison of the BD GeneOhm Methicillin-Resistant Staphylococcu aureus (MRSA) PCR Assay to Culture by Use of BBL CHROMagar MRSA for Detection of MRSA in Nasal Surveillance Cultures from an At-Risk Community Population. J Clin Microbiol. 2008;46(2):743-746. Available at: http://jcm.asm.org/content/46/2/743.full. Accessed February 22, 2012.Forrest GN, Mehta S, Weeks E, Lincalis DP, Johnson JK, Venezia RA. Impact of Rapid In Situ Hybridization Testing on Coagulase Negative Staphylocci Positive Blood Cultures. J Antimicrob Chemother. 2006;58(1):154-158. Available at: http://jac.oxfordjournals.org/content/58/1/154.full. Accessed February 22, 2012.Garcia LS, Isenberg HD, eds-in-chief. Clinical Microbiology Procedures Handbook. 2nd ed. Washington, DC: ASM Press; 2007.Hindiyeh M, Hillyard DR, Carroll KC. Evaluation of the Prodesse Hexaplex Multiplex PCR Assay for Direct Detection of Seven Respiratory Viruses in Clinical Specimens. Am J Clin Pathol. 2001;116:218-224. Available at: http://ajcp.ascpjournals.org/content/116/2/218.full.pdf. Accessed February 22, 2012.Hunt M. Real Time PCR. University of South Carolina School of Medicine Website. Available at: http://pathmicro.med.sc.edu/pcr/realtime-home.htm. Accessed February 22,2012.Interim Guidance for Influenza Surveillance: Prioritizing RT-PCR Testing in Laboratories. Centers for Disease Control and Prevention Website. Available at: http://www.cdc.gov/h1n1flu/screening.htm. Accessed February 22, 2012.Interim Guidance for the Detection of Novel Influenza A Virus Using Rapid Influenza Diagnostic Tests. Centers for Disease Control and Prevention Website. Available at: http://www.cdc.gov/h1n1flu/guidance/rapid_testing.htm. Accessed February 22, 2012.Levenson D. Molecular Testing for Respiratory Viruses. In Clinical Laboratory News. March 2008: Vol 34, No 3. Washington, DC: AACC Press; 2008. Available at: http://www.aacc.org/publications/cln/2008/mar/Pages/cover1_0308.aspx. Accessed February 22, 2012.Morshed MG, Lee MK, Jorgensen D, Issac-Renton JL. Molecular methods used in clinical laboratory: prospects and pitfalls. FEMS Immunol Med Microbiol. 2007;49:184-191. Available at: http://www.canlyme.com/morshed_pcr.pdf. Accessed February 22, 2012.Paillard F, Hill CS. Direct nucleic acid diagnostics tests for bacterial infectiousdiseases: Streptococcal pharyngitis, pulmonary tuberculosis, vaginitis, chlamydial and gonococcal infections. MLO-online. 2004;10-15. Available at: http://www.mlo-online.com/articles/0104/mlo0104coverstory.pdf. Accessed February 22, 2012.PCR: an outstanding method. Roche Website. Available at: http://www.roche.com/pages/facets/pcr_e.pdf. Accessed February 22, 2012.Persing DH, ed-in-chief.Molecular Microbiology, Diagnostic Principles and Practice. 2nd ed. Washington, DC: ASM Press; 2010.Pfaller MA. Molecular Approaches to Diagnosing and Managing Infectious Diseases: Practicality and Costs. Emerg Infect Dis. 2001;eid0702. Available at: http://wwwnc.cdc.gov/eid/article/7/2/70-0312_article.htm. Accessed February 22, 2012.Rossney AS, Herra CM, Brennan GI, Morgan PM, O'Connell B. Evaluation of the Xpert Methicillin-Resistant Staphylococcus aureus (MRSA) Assay Using the GeneXpert Real-Time PCR Platform for Rapid Detection of MRSA From Screening Specimens. J Clin Microbiol. 2008;46(10):3285-3290. Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2566096/. Accessed February 22, 2012.The 2009 H1N1 Pandemic: Summary Highlights, April 2009-April 2010. Centers for Disease Control and Prevention Website. Available at: http://www.cdc.gov/h1n1flu/cdcresponse.htm. Accessed February 22, 2012.Timeline of PCR and Roche. Roche Website. Available at: http://molecular.roche.com/About/pcr/Pages/PCRTimeline.aspx. Accessed February 22, 2012.

Clostridium difficile is the cause of antibiotic associated diarrhea (AAD) and pseudomembranous colitis (PMC). PMC is an inflammatory disease of the colon caused by toxins of C. difficile.C. difficile produces two potent toxins: Toxin A (TcdA), an enterotoxinToxin B (TcdB), a cytotoxin It is the production of these toxins in the gastrointestinal tract that ultimately leads to disease. There is a relationship between toxin levels, the development of pseudomembranous colitis (PMC), and the duration of diarrhea. For many years, toxin A was regarded as more important than toxin B in the disease process. Later on, disease producing strains producing only toxin B were identified. These strains produced serious disease, and toxin B was found to be responsible for more serious damage to intestinal cells.

In general, the infection that develops is dependent on the virulence of the particular strain, the inoculum size, and immune status of the host. Staphylococcal infections are typically suppurative, producing abscesses filled with pus and damaged leukocytes surrounded by necrotic tissue. Skin infections range from superficial - boils, carbuncles and furuncles, to bullous impetigo; largely opportunistic infections that develop as a result of previous injury e.g., cuts, burns, surgical wounds - and scalded skin syndrome (extensive exfoliative dermatitis; also known as Ritter's Disease). Other major infections include pneumonia, osteomyelitis (localized infection of bone), and septic arthritis. S. aureus also causes food poisoning as a result of ingestion of food contaminated with an enterotoxin producing strain (enterotoxins A&D) and the potentially fatal toxic shock syndrome, a multisystem disease most often associated with the use of highly absorbent tampons. Toxic shock syndrome is attributed to another toxin (enterotoxin F – TSST1) released by certain strains of S. aureus.Human staphylococcal infections usually remain localized by the normal host defenses. Foreign objects (fomites) such as sutures or intravenous (IV) lines - are readily colonized by S. aureus from skin and can allow the organism to spread systemically via the blood stream – bacteremia/septicemia - leading to more serious infections. Staphylococcal pneumonia is becoming a frequent complication of influenza. Whatever the mode of entry, the invasive nature of S. aureus always poses the threat of more serious deeper tissue invasion and/or bacteremia and hematogenous spread.

A clinical isolate is presumptively identified as Staphylococcus aureus by means of several simple procedures :Gram Stain : Gram-positive cocci, occurring singly, in pairs or "bunches of grapes"Catalase test: Staphylocci are catalase-positive, distinguishes them from Streptococci which are catalase-negative.Coagulase test: S. aureus is coagulase-positive.DNAse Test – S. aureus is DNAse-positiveHeat stable endonuclease – S. aureus is positiveThere are also many commercial kits available for identification of S. aureus based on latex agglutination.

Judicious use of antimicrobials, especially in outpatient settings, can help control the emergence of CA-MRSA and limit the acquisition of additional resistance by existing strains. Regardless of origin, minimizing antibiotic selective pressure that favors the development of resistant strains is essential to controlling the emergence of these strains in both hospital and community settings.The development of vaccines to prevent S. aureus infection in both healthcare and community settings holds great promise. Recently (2007) a vaccine based on an immunotherapeutic licensed to Merck has shown promising results in a clinical trial against hospital acquired S. aureus infections, while Nabi Biopharmaceuticals, Ft. Lauderdale, FL, are developing the "next generation" of StaphVax, which will contain antigen against S. aureus detoxified Panton Valentine Leucocidin and the cytolytic alpha-toxin.

Clostridium are gram-positive or gram-variable, spore-forming, catalase-negative anaerobic bacilli. More than 100 species are currently recognized, though relatively few are encountered in properly collected clinical specimens from humans. There are three types of infection associated with Clostridium species: Non-invasive: Toxin-mediated Invasive: Progressive infection with tissue destruction Purulent disease: Closed space (e.g., in the peritoneal cavity) mixed infection with multiple organisms.Clostridium are well known as the agents of these classic toxin-mediated diseases : DISEASE TOXIN INVOLVED CAUSATIVE ORGANISM Tetanus or "lock jaw" Tetanospasmin Clostridium tetani Myonecrosis/Gas gangrene Exotoxins Clostridium perfringens Botulism (severe food poisoning) Botulin Clostridium botulinum

Clostridial toxins are among the largest bacterial toxins reported to date and C. difficile produces two potent toxins: Toxin A ((TcdA), an enterotoxin and Toxin B (TcdB), a cytotoxin. It is the production of these toxins in the gastrointestinal tract that ultimately leads to disease. There is a relationship between toxin levels, the development of pseudomembranous colitis (PMC), and the duration of diarrhea. Levels of Immunoglobulin G against TcdA correlate directly with protection from disease following colonization, suggesting that a robust immune response is sufficient for protection from C. difficle-associated diarrhea (CDAD). The role of TcdB is not as well understood. Naturally occurring Toxin A negative/Toxin B positive (TcdA-TcdB+) strains have been identified from clinical isolates, which are capable of causing disease, even extensive PMC, suggesting a role for TcdB in CDAD. Toxin A had always been regarded as more important than Toxin B in infection. However, recent work utilizing mutant C. difficile, strains which did not, or could not produce Toxin A, and which were capable of producing very serious disease has led researchers to completely rethink the roles of Toxin A and Toxin B in CDAD. Toxin B was found to be responsible for the more serious damage to intestinal cells. In addition to the primary virulence factors (Toxin A and Toxin B ), Clostridium difficile also produces a third toxin, binary toxin (CDT). The prevalence of CDT in clinical isolates varies widely and its clinical relevance and role in pathogenicity are still not well defined.

In the early 2000's researchers in Quebec, Canada noticed an increase in the number of colectomies being performed as a result of an increase in the frequency and severity of CDAD. At around the same time, doctors at the Centers for Disease Control and Prevention (CDC) were receiving reports of increased frequency and severity of disease in the United States. There were also reports of more disease and more severe forms of C. difficile infection in other areas of the world, suggesting that the experience was very widespread and possibly global. In 2004, analysis of this hypervirulent strain showed a very characteristic strain that had previously been rare but was responsible for the majority of the more serious outbreaks. This strain – BI/NAP1 /027 – has several designations depending on which biological property was examined :- BI: Restriction Endonuclease Analysis (USA)- NAP1: North American PFGE Type 1 based on polyacrylamide gel electrophoresis (USA) - 027: Ribotype 027 by polymerase chain reaction (Europe)There are 5 unique features associated with this strain – It produces the classic toxins A & B, but faster and at much higher levels than other strains. It is Toxinotype III in contrast to the more typical clinical isolates, which tend to be Toxinotype 0. tcdC is deleted from the PaLoc, possibily explaining the observed increase in toxin production. It produces the binary toxin CDT, but its role is still unclear. It exhibits high level in vitro resistance to fluoroquinolones.

High level resistance to aminoglycosides is another significant acquired resistance factor. Since the standard approach for treating systemic infections is a combination of a cell wall targeted antibiotic with an aminoglycoside, assessment of resistance to both classes of antibiotics is important. High level resistance to aminoglycosides will negate the synergistic effect of combined therapy with either penicillin or vancomycin.Standard susceptibility methods (either disk diffusion or broth dilution) will not detect HLR patterns, unless the protocol incorporates testing at increased concentrations of gentamicin and/or streptomycin. The CLSI documents outline recommended protocols for screening for HLR aminoglycoside resitance.Gentamicin HLARDisk diffusion: MHA agar; 120 ug gentamicin disc; standard inoculum and incubation temperature; incubation duration: 16 - 18 hours.Interpretation: Resistant = 6 mm Inconclusive = 7-9 mm Susceptible > 10 mmBroth microdilution: BHI broth; 500 ug/ml gentamicin; standard inoculum and incubation temperature, incubation duration: 24 hours.Interpretation: any growth equates to a resistant interpretation.Streptomycin HLARDisk diffusion: MHA agar; 300 ug streptomycin disc; standard inoculum and incubation temperature; incubation duration: 16 - 18 hours.Interpretation: Resistant = 6 mm Inconclusive = 7-9 mm Susceptible > 10 mmBroth microdilution: BHI broth; 1000 ug/ml streptomycin; standard inoculum and incubation temperature, incubation duration: 24 - 48 hours. If susceptible at 24 hours, reincubate and re-read at 48 hours.Interpretation: any growth equates to a resistant interpretation.Clinical correlationA resistant result indicates that synergistic effects will not be achieved between the indicated aminoglyocside and the cell wall active agent (eg, ampicillin, penicillin, or vancomycin).A susceptible result indicates that synergistic effects are possible.

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.

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

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.

Clinical specimens where organism may be encountered: CSF Blood Stool (rare) Vesicle fluid, skin swab, or biopsy Gram stain morphology from clinical specimens: Large, gram-positive rods with square or concave ends in short chains Spores are usually NOT present Capsule may be viewed in smears from infected tissue, but this is NOT reliable Gram stain morphology from culture material: Large, gram-positive rods with square or concave ends, often in long chains (more than 2-4 cells) Cells easily decolorize as the culture ages Does NOT form capsules in culture Central to sub-terminal, oval spores, with NO significant swelling of the cell It must be noted that spore production increases with the age of the culture. Do NOT keep these cultures in the laboratory for longer than 24 hours for this reason!

Clinical specimens where organism may be encountered: Blood Biopsy, skin scraping, or swab Lymph node aspirate Respiratory secretions - oropharyngeal aspirate, sputum, or bronchial washingsGram stain morphology: Very tiny, gram-negative coccobacillus Pale or weak staining Due to the small size, often difficult to see individual cells

Clinical specimens where Burkholderia species may be encountered: Blood Bone marrow Respiratory specimens - sputum, throat, or nasal Wounds UrineGram stain morphology:B. mallei Gram-negative coccobacillus or small rod Arranged in pairs end-to-end, parallel bundles, or Chinese letter formB. pseudomallei Small, straight, or slightly curved gram-negative rod May demonstrate peripheral or bipolar staining as they age (appear like endospores) Smooth forms are arranged in long, parallel bundles Rough forms more irregularly arranged

Toxin Comment Most Likely Means of Dissemination Primary Route of Entry General Signs and Symptoms Laboratory Testing Botulism toxin: Gram stained image of C. botulinum courtesy of CDC Produced by Clostridium botulinum Could be purified and used in a bioterrorist event to contaminate food or aerosolized to cause disease Aerosol Food contamination Inhalation Ingestion Difficulty speaking or swallowing Blurred or double vision Drooping eyelids (ptosis) Dilated pupils Dry mouth, decreased gag reflex Weakening of the reflexes (hyporeflexia) Abnormal sensations such as numbness, tingling, and progressive arm or leg weakness Flaccid paralysis Culture, anaerobic Digoxigen-labeled IgG ELISA to detect A, B, E, and F toxins Mouse Bioassay for all toxin types and to confirm DIG ELISA Ricin toxin: Extracted from Castor beans Inhibits protein synthesis Causes death approximately 72 hours after initial exposure As an aerosol Inhalation Fever Cough Chest tightness Dyspnea Cyanosis Gastroenteritis Necrosis Antibody detection in clinical specimens Clinical testing not performed unless known exposure has occurred

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 (CLSI) 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.

Gram stain is used primarily as a differential stain for bacteria, although it will also stain most fungi (especially yeasts) and some parasites, including Strongyloides and Trichomonas. The Gram stain procedure is commonly performed on direct smears of clinical specimens and on smears from cultures. This course will focus on Gram-stained direct smears. A direct smear from a clinical specimen can be used to: Judge the quality of the specimen. Provide the clinician with same-day information regarding possible pathogenic organisms, pending results of culture and sensitivity. Contribute to selection of culture media, especially with mixed flora. Provide internal quality control when direct smear results are compared to culture results.

Correlation between a direct Gram-stained smear of the specimen and what grows out in culture should always be attempted. However, some bacterial organisms may appear differently when viewed in direct clinical specimens versus isolates growing on solid or in liquid culture media. For example, if gram-negative organisms are surrounded by large amounts of cellular material in the direct smear, the cellular material may totally or partially prevent decolorization of the bacteria so that the gram-negative bacteria growing in culture may appear gram-positive or gram-variable on direct smear.

When evaluating Gram stains of clinical samples, keep in mind the source of material from which the smear was made. Bacteria found in cerebrospinal fluid (CSF), blood, tissue and specimens from other sterile sites are always significant. Gram stains of body fluids that are normally sterile must be examined carefully. For every one organism per oil immersion field, there are about 105 organisms per mL present in the sample! Examining stained smears of CSF sediment may assist the clinician in establishing a presumptive diagnosis. The Gram stain result and the results of other special stains could also guide in the selection of culture media. If bacteria are observed in a CSF specimen, it is important to determine and report whether the bacteria are inside or outside of white blood cells (intracellular or extracellular). The quantity of organisms seen and the amount and type of host cells are also important to report. 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.

Morris AJ, Tanner DC, Reller LB. Rejection criteria for endotracheal aspirates from adults. J Clin Microbiol. 1993;31:1027-1028.Murray PR, Baron EJ, Jorgensen JH, Landry ML, Pfaller MA, eds. Manual of Clinical Microbiology. 9th ed. Washington, DC: ASM Press; 2007. Thomson RB, Peterson L. Microbiolology laboratory diagnosis of pulmonary infections. In Niederman MS, Sarosi GA, Glassroth G, eds. Respiratory Infections. 2nd ed. Philadelphia, PA: Lippincott, Williams & Wilkins; 2001:541-549.Winn, WC Jr. Koneman's Color Atlas and Textbook of Diagnostic Microbiology. 6th ed. Philadelphia, PA: Lippincott Williams & Wilkins: 2006.

Gram stain is used primarily as a differential stain for bacteria, although it will also stain most fungi (especially yeasts) and some parasites, including Strongyloides and Trichomonas. The Gram stain procedure is commonly performed on direct smears of clinical samples and on smears from cultures. This course will focus on Gram stained direct smears. A direct smear is made from a clinical specimen. 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.

In clinical laboratories today, DNA and RNA are isolated and extracted through both manual and automated techniques.Manual extraction requires many different manipulations to the sample, which increases the risk of contamination. It is also considered high complexity testing so not every laboratory professional is capable of performing this process. Another downside to manual extraction is that the process is extremely time consuming and requires the undivided attention of the technician performing the procedure. Automated extraction has many benefits over the traditional manual methods. The most important benefit is the consistency of the isolated nucleic acid. Manipulation of the sample and reagents is reduced, which dramatically decreases the chance of cross-contamination. Also, automated extraction instruments are considered moderate complexity so that testing can be performed by a greater number of laboratory professionals. Though automated extraction instruments have many benefits over manual methods, the cost per test is high and generally requires a high throughput of samples in order to justify the expense.

Dimmock NJ, Easton AJ, Leppard KN. Introduction to Modern Virology. 6th ed. Malden, MA: Blackwell Publishing; 2007.Foxman B. Molecular Tools and Infectious Disease Epidemiology. San Diego, CA: Elsevier; 2011. Kaplan LA, Amadeo JP. Clinical Chemistry:Theory, Analysis, Correlation. 5th ed. St. Louis, MO: Mosby-Elsevier; 2010.Logan J, Edwards K, Saunders N. Real-Time PCR: Current Technology and Applications. Norfolk, UK: Caister Academic Press; 2009. Ream W, Gellar B, Trempy J, Field K. Molecular Microbiology Laboratory: A Writing-Intensive Course. Boston, MA: Academic Press; 2003. Turgeon ML. Immunology & Serology in LaboratoryMedicine. 3rd ed. St. Louis, MO: Mosby Elsevier; 2003.Walker JM, Rapley, R. Molecular Biology and Biotechnology. Cambridge: The Royal Society of Chemistry; 2009.

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

Dimorphic is a term used to describe two circulating red cell populations. One is the patient's basic red cell population while the other is a second population with distinct morphological features. The distinct populations can be observed in the top image on the right. The bottom image on the right illustrates the two distinct peaks that are observed on the RBC histogram from the automated hematology analyzer.Dimorphic red blood cell populations can be found in conditions/situations such as: red blood cell transfusions, myelodysplasia, refractory anemia with ringed sideroblasts, hemolytic processes involving a reticulocyte response, and when patients are given 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.

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 other laboratory parameters and with the clinical picture.

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 lighter in appearance than in the previous slide. Target cells appear in conditions that 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, Hemoglobin C disease, post splenectomy, and obstructive jaundice.

Clinically relevant information on D phenotypes can be summarized as follows: D phenotype D antigen expression Rh(D) typing Produce anti-D RBC to transfuse RhIg recommended** D+ normal direct agglutination no D+ or D– no Weak D normal but weak IAT no D+ or D– no Partial D altered direct agglutination* & IAT yes D– yes Partial weak D altered & variable direct agglutination* & IAT yes D– yes D– none IAT yes D– yes * Depending on anti-D reagent used ** USA, UK and parts of Canada

Protocols for testing newborns vary internationally and within countries.if the mother is D-negative and has no unexpected antibodies, newborns are always tested at delivery.Many labs do not test all newborns if the mother is Rh positive and especially do not test if the mother is a blood group other than group O. If all infants born to Rh positive women were tested, many positive DATs due to ABO incompatibility would be detected that are of no clinical significance. Instead cord blood is retained for a period (e.g., 7 days) should it be needed, for example, if the mother has an unexpected antibody at delivery or if the newborn develops signs of red cell hemolysis.However, some clinical practice guidelines, such as those of the American Academy of Pediatrics specify that testing infants born to group O Rh positive mothers is optional only if there is appropriate surveillance and risk assessment before discharge and provided there is follow-up. (See Further Reading) Not testing becomes an issue if group O women and their infants are discharged within 24 hours as occurs in some locations, since hyperbilirubinemia due to ABO HDFN may develop later. Therefore, some facilities where early discharge occurs require that all infants born to group O Rh positive mothers be tested.Typical protocols: Infants born to Rh negative mothers are tested; Infants born to Rh positive mothers who are group O are often tested, especially if early discharge is common (limiting surveillance); Infants born to Rh positive mothers who are not group O are often not tested and this is acceptable good practice. Cord blood is typically retained for a period should it be needed for testing later.

This case concerns a common scenario in the TS laboratory, the detection of anti-D at delivery in an Rh negative female who received antenatal RhIG. The case was used to review some of the key learning goals relevant to HDFN and its prevention. More specifically, the case study reviewed the following topics: Historical aspects of HDFN due to anti-D and its prevention; Clinical symptoms and associated laboratory test results in HDFN; Best practices related to perinatal testing programs to prevent HDFN; Interpretation of serologic test results on mother, father, and child; Characteristics and uses of RhIg; Tests to screen for and quantify FMH; Issues related to women with anti-D following RhIg injection; Crossmatch and LIS policies related to passive anti-D from RhIg.Before taking the final quiz, for each of the above topics, list as many of the key learning points that you can recall. As required, review topics that need more study. As well, re-read the learning objectives at the start of the case as these determine assessment questions.It's also worthwhile to read the literature and online resources in Further Reading as these reinforce key points, add to the depth of learning, and enrich course materials.

Several federal agencies share responsibilities for oversight of the healthcare industry in the United States. These agencies include: U.S. Department of Health and Human Services Centers for Medicare and Medicaid Services- Responsible for regulating clinical laboratory testing through the Clinical Laboratory Improvement Amendments of 1988 (CLIA). Food and Drug Administration (FDA)- Responsible for protecting public health through regulation of food, drugs, vaccines, blood and blood products, medical devices, and more. U.S. Department of Labor Occupational Safety and Health Administration (OSHA)- Ensures safe working conditions in healthcare as well as other industries. Some of the federal laws/regulations that affect clinical laboratories in the United States and relate either directly or indirectly to risk management include: Clinical Laboratory Improvement Amendments of 1988 (CLIA) Health Insurance Portability and Accountability Act of 1996 (HIPAA) OSHA standards for hazard communication, chemical hygiene, and bloodborne pathogens Safe Medical Devices Act of 1990 (SMDA) The Patient Safety and Quality Improvement Act of 2005

One of the tools that can be used when performing a root cause analysis is the cause and effect diagram, popularly referred to as the "fishbone diagram" because of its appearance. Cause and Effect (Fishbone) Diagram ExampleThis type of diagram graphically helps identify and organize known or possible causes for a specific problem or area of concern. In this theoretical example, the identified problem is a "near miss." Two units of RBCs were taken to the Dialysis unit for tranfusion of two different patients. The first unit was hung by one clinical person and started just as another clinical person noticed that the unit that he/she picked up for transfusing another patient had the wrong identifying information. The blood was stopped immediately on the first patient. Some of the benefits of constructing a "fishbone diagram" are that it: Helps determine root causes using a structured approach. Encourages group participation and utilizes group knowledge. Indicates possible variations in a process. Indicates areas where more data should possibly be collected.

The Joint Commission (formerly known as the Joint Commission on Accreditation of Healthcare Organizations, or JCAHO) is a private sector, non-profit organization based in the United States. It was created by the merging of the Hospital Standardization Program with similar programs run by the American College of Physicians, the American Hospital Association, the American Medical Association, and the Canadian Medical Association. Unannounced surveys are performed on the entire hospital, including clinical laboratories, in order to gain accreditation for the laboratory and the hospital as a whole. Joint Commission Survey ProcessCompliance with applicable standards is based on the following: Tracing the care delivered to patients Verbal and written information provided to The Joint Commission On-site observations and interviews by Joint Commission surveyors Documents provided by the organizationThe table to the right describes the factors evaluated by the Joint Commission to determine technical competence of a laboratory.

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. National Patient Safety Goals. Available at: http://www.jointcommission.org/standards_information/npsgs.aspx. Accessed July 2, 2012.

Clinical and Laboratory Standards Institute (CLSI). Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture; Approved Standard. 6th ed. CLSI document H3-A6. Wayne, PA: CLSI: 2007.Clinical and Laboratory Standards Institute (CLSI). Procedures and Devices for the Collection of Diagnostic Capillary Blood Specimens; Approved Standard. 5th ed. CLSI document H4-A5. Wayne, PA: CLSI: 2004. Clinical and Laboratory Standards Institute (CLSI). Procedures for the Handling and Processing of Blood Specimens; Approved Guideline. Third Edition. CLSI document H18-A3. Wayne, PA: CLSI: 2004.Ernst DJ. Applied Phlebotomy. Baltimore, MD: Lippincott Williams & Wilkins: 2005.

An arterial line or vascular access device (VAD) is used to provide easy access to a patient's circulatory system for administration of fluids and medications. Occasionally, blood specimens are drawn from a VAD, but phlebotomists are not usually authorized to collect these specimens. However, phlebotomists may sometimes assist when a clinical person is collecting blood from a VAD. If you are present, be certain that the person performing the collection flushes the line with at least 5 mL of saline and the first 5 mL of blood is discarded before collecting the sample. If this procedure is not followed, the specimen may be contaminated with heparin that was used to flush the line or be diluted. This could cause inaccurate test results.

Case study:You work in a large hospital that specializes in pediatrics. The policy of the facility is to encourage the parent or guardian to remain in the room during venipuncture to comfort the child. You have taken steps to prepare the child for the venipuncture, but the child starts to cry and becomes combative. The mother says that she does not want the test done.Suggested plan of action: Do not proceed if the mother has refused the blood collection for her child. The patient's physician or clinical person in charge of the patient should be contacted and informed of the situation. This may not be something that you would do directly. It may be your facility's policy for you to contact your supervisor.

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).

According to the CDC guidelines, patients with clinical illness consistent with uncomplicated influenza who reside in an area where influenza viruses are circulating may not require diagnostic influenza testing for clinical management. Most mild cases of H1N1 infection are self-limiting and do not require confirmation. However, if a patient is hospitalized due to the severity of the symptoms, or if the diagnosis of the patient will provide needed information to the physician to direct clinical care, infection control decisions, or management of close contacts, diagnostic influenza testing should be done. In any case, if a decision to use antiviral treatment is made, the treatment should commence as soon as possible, without waiting for the results of confirmatory diagnostic tests.

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. TypeConditionHyaline0-2/LPF is normal; increase in fever, diuretic therapy, exercise or stress. GlomerulonephritisPyelonephritisChronic renal diseaseWhite Blood CellRenal infectionInflammation of the nephronRed Blood CellBleeding in the nephronGlomerulonephritisStrenuous exerciseEpithelial CellRenal tubular damageGranularMay be seen occasionally in normal urineUrinary stasisGlomerular and tubular diseaseAmyloid diseaseChronic renal diseaseFatty Degenerative tubular diseaseBroadExtreme stasis of urine flowRenal 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.CrystalpHColorUric acidAcidicYellow - brownCalcium oxalateAcidic/neutralColorlessAmorphous uratesAcidicYellow - brownTriple phosphateAlkalineColorlessAmmonium biurateAlkalineYellow - brownAmorphous phosphateAlkaline/neutralWhite - colorlessCalcium carbonateAlkalineColorless

Histology involves studying the structure of body tissues. Because structure is closely related to function, different tissue types are distinguishable from one another by examination of their individual components. Knowledge of the normal histology of the multitude of tissue types within the body is necessary for the recognition and understanding of disease.The tissue on which a diagnosis is made can be taken from a patient in the operating room, a doctor's office, or from an autopsy. Autopsy is an important part of pathology which seeks to establish the cause of sudden or unexpected death, to examine disease progression, and to assist police during the investigation of criminal cases. However, most anatomic pathologists deal with tissue from living patients and a large part of this is the detection and diagnosis of cancer. A tissue diagnosis is essential before a clinician can start treatment involving major surgery, radiation, or drugs.

Although there is no consistent clinical picture of an acute hemolytic transfussion reaction (AHTR), common symptoms include chills, hypotension, and fever. Some patients have experienced pain at the infusion site, flank pain, and anxiety with a feeling of doom. Red or dark urine may be the first sign of intravascular hemolysis. If patients are unconscious or in surgery, changes in vital signs, unexplained bleeding, or hemoglobinuria may be the only signs. Additional signs and symptoms include, but are not limited to: rigors, facial flushing, chest and abdominal pain, nausea and vomiting, dyspnea, oliguria/anuria, diffuse bleeding, shock, and renal failure. The severity of symptoms is related to the amount of incompatible blood transfused. Patients with underlying diseases that involve intravascular hemolysis can make diagnosis extremely difficult.

There are no conclusive tests to diagnosis transfusion-related acute lung injury (TRALI). The condition should be suspected if the clinical picture corresponds with TRALI clinical findings, such as hypoxemia within 6 hours of transfusion. The clinical findings should correlate with chest radiograph findings of bilateral infiltrates. It is important to rule out cardiac causes of pulmonary edema. One way of differentiating is evaluating the B-type natriuretic peptide (BNP) level, which is known to be elevated in congestive heart failure and not TRALI. In the majority of cases, the donor plasma will demonstrate anti-HLA antibodies. Urgent treatment consists of respiratory and volume support. Patients usually require supplemental oxygen, some by a mechanical ventilator. Vasopressor medications can be used to treat the hypotension. Extracorporeal membrane oxygenation (ECMO) and cardiopulmonary bypass have been successful in treating TRALI when conventional methods do not work. Diuretics are contraindicated in TRALI.Patients with TRALI usually improve within 48 to 96 hours. TRALI is fatal in about 5% to 10% of cases.

Delayed hemolytic transfusion reactions (DHTR) are reactions that occurs 3 to 10 days after the transfusion. Usually, the blood appears serologically compatible at initial testing. Delayed reactions are common in patients who have been immunized to a foreign antigen from a previous transfusion or pregnancy, but the antibody titers decrease over time and the antibody is not detectable during pre-transfusion testing. The transfusion leads to a secondary (anamnestic) response, causing increased antibody production that sensitizes antigen-positive donor red cells. Hemolysis is extravascular. Sensitized cells are removed from circulation by the reticuloendothelial system, also called the monocyte-macrophage system. Because there is a delay in the presentation of symptoms, DHTR is not usually considered as a cause of the clinical presentation. The transfusion service usually initiates investigation of a DHTR because of serologic findings in a post-transfusion specimen. DHTRs occur more frequently than acute hemolytic reactions. Approximately 1:2500 transfusions result in a DHTR.

Generally, the clinical symptoms of a delayed hemolytic transfusion reaction (DHTR) resolve within 2-3 weeks without medical intervention other than transfusion support. On the other hand, severe DHTRs can occur with a life-threatening anemia. Severe delayed reactions occur most often in patients with sickle cell anemia. Sickle cell anemia patients have a high alloimmunization rate which puts them at greater risk for developing a DHTR. Diagnosis of a DHTR can be difficult in sickle cell patients because symptoms can be misdiagnosed as sickle cell crisis pain. Delays in medical treatment may lead to death. It is important for the transfusion service to obtain an accurate transfusion history. It is unclear what causes such severe reactions in sickle cell patients. Several explanations include bystander hemolysis, sickle cell hemolytic transfusion syndrome, and hyperhemolysis. In any case, it is important to recognize that severe DHTR in sickle cell patients is not uncommon. Treatment requires rapid diagnosis and transfusion support with antigen-negative red cells.

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.Infection occurs when a susceptible person inhales droplet nuclei containing Mycobacterium tuberculosis and the organism reaches the alveoli of the lungs. The minimal infectious inoculum may be as low as one viable organism.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; the individual has no symptoms and is noninfectious.Most persons infected with M. tuberculosis do not experience clinical illness and are noninfectious. About 5-10% of persons who are infected and are not treated will develop active TB during their lifetime. The risk for progression is highest during the first several years after infection.Most often, M. tuberculosis infects the lungs. However, it can infect almost any organ in the body, including bones and joints. Tuberculosis meningitis is a TB infection that occurs outside the lungs with devastating consequences, most often in young children and patients with AIDS.

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 (see image) appears as a small drumstick-like projection on one of the lobes of some neutrophils 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." Since Barr bodies are the morphological expression of the inactivated X chromosome, Barr bodies (one per neutrophil) 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. 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. 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). The Barr body is considered nonpathological unless associated with rare chromosome disorders.

In most clinical hematology laboratories, an initial blood count is performed by an automated cell counting instrument. Additionally, most of these instruments also produce a five-part differential count, indicating the percentage of neutrophils, lymphocytes, monocytes, basophils, and eosinophils. Some instruments can also provide information about cellular immaturity and abnormal cellular morphologies.Occasionally, atypical cells, similar to those shown in the image to the right, would be flagged or counted as mixed cells, at which point a smear review would be required to make an identification. In cases where there are automated instrument differential flags, mixed cell count is high, or there are other indications that atypical cells may be present, a review of the smear is indicated.