| Autoimmune Disease (continued) 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. | View Page |
| Antinuclear Antibody Test The antinuclear antibody test (ANA) is a test used to screen for the presence of autoantibodies that are directed toward components in the nucleus of the cell. Clinicians use the ANA test to assess the likelihood that a given patient has a SARD. The results of the ANA test alone are not diagnostic for the SARD. The patient must also have clinical evidence of the disease as well. Because the early clinical presentation for many of the SARDs are nonspecific, the results of the ANA test and subsequent follow-up testing are key pieces to making the correct diagnosis.Rheumatoid arthritis (RA) is the most prevalent disease in this group; however, the ANA assay is not the primary laboratory test for RA. Instead, the test for RA looks for the presence of rheumatoid factor (RF) or more recently, cyclic citrullinated peptide antibodies (anti-CCP).For the other diseases in the SARD group, especially SLE and SSc, the results of the ANA test can be useful in determining a correct diagnosis. The utility of the ANA test is to detect the antibodies early in the disease process.The ANA results in conjunction with clinical presentation give the clinician solid evidence to intervene with an appropriate treatment. Studies have shown that once treatment is started, the formation of new antibodies slows or even halts.(Ref3)Currently there are no cures for the SARDs. Treatments primarily focus on keeping the patient comfortable and the immune response in check. Treatments can vary from non-steroidal anti-inflammatory drugs, to immuno-suppressive drugs, to stem cell transplants. Individual treatment is often dependent on the severity of the disease and the response to the selected drug regimen. | View Page |
| History of ANA Testing 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) | View Page |
| Centromere This is an example of a centromere ANA pattern.This pattern is characterized by staining of discrete speckles in the nucleus of the interphase cells (a). This is staining of the centromere. There are usually 46 speckles, one for each set of chromosomes. Notice the discrete speckling is also seen in the chromosomal area of the metaphase mitotics (b). The presence of anti-centromere antibodies can be determined by the staining pattern alone. No specific follow-up testing is necessary. Anti-centromere antibodies are most often seen in patients with the limited form of systemic sclerosis called the CREST syndrome. CREST is an acronym for the clinical features:Calcinosis cutisRaynaud's phenomenonEsophageal dysfunctionSclerodactylyTelangiectasiaAnti-centromere antibodies have been detected years prior to the onset of symptoms. Finding these antibodies in patients with Raynaud's phenomenon or other risk factors is an important indicator for the potential development of systemic sclerosis.This pattern is reported as ANA positive Centromere; titering is necessary.Centromere:Interphase cells Usually 46 discrete speckled Evenly distributed in the nucleusMetaphase mitotic cells Discrete speckles align in the chromosomal region of the mitotics No staining outside of the chromosomal area | View Page |
| References 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. | View Page |
| Scl-70 Antibodies to Scl-70, also known as anti-topoisomerase I antibodies, produce three ANA patterns: homogeneous, speckled and nucleolar. This is because the Scl-70 antigen is present in all three areas. In photographs it is difficult to capture the fine granular speckled staining (a), therefore, in the following photos the patterns are best described as Homogeneous (b) and Nucleolar (c).In cases of mixed homogeneous and speckled ANAs, follow-up testing for anti-dsDNA and anti-ENA antibodies is necessary.When mixed patterns are titered, the endpoint for each pattern is reported.This pattern is reported as ANA positive, Homogeneous, Nucleolar and Speckled; titering is necessary. (It should be noted that anti-Scl-70 cannot be identified simply by the ANA patterns. Follow-up ENA testing is necessary.)Scl-70:Antibodies target topoisomerase IDetection methodsANA: Three patterns present Homogeneous, Specked and Nucleolar Rarely seen with other marker antibodies Confirm by: ID, ELISA, WB, others Clinical significance Poor prognosis May precede onset of symptoms | View Page |
| Overview 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. | View Page |
| Cytoskeletal This sample is demonstrating an anti-cytoskeletal pattern in the cytoplasm of the cells.The cytoskeleton of eukaryotic cells is comprised of microfilaments such as actin, intermediate filaments such as vimentin and microtubules such as tubulin. Autoantibodies to these proteins are seen in a variety of autoimmune diseases.Whenever an ANA is read the first step is to see if there is a clearly discernible pattern in the nucleus of the interphase cells. In this case the answer is no (a). However, there is considerable staining in the cytoplasm. This fibrous staining of the cytoskeleton is a discernible cytoplasmic pattern and should be reported (b).This sample is reported as ANA Negative, suspect cytoskeletal antibodies present. (The term "suspect" is used because the ANA substrate is not considered "confirmatory" for the identification of cytoskeletal antibodies. Additional follow-up testing is required).Follow-up testing would include testing for anti-smooth muscle antibodies. Anti-smooth muscle antibodies are seen in autoimmune liver disease.Reporting anti-Cytoskeletal Antibodies:Cytoplasmic pattern:• Fibrous strands in cytoplasmReport as:• Suspect cytoskeletal, suggest follow-up Clinical Significance:• Actin: Autoimmune hepatitis, PBC• Vimentin and others: SLE, RA, others | View Page |
| Cytoplasmic patterns are interesting to look at but have no clinical significance and should NOT be reported. | View Page |
| Clinical Laboratory's Role: Bone Marrow Aspirates and Biopsies 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. | View Page |
| Responsibility of the Technologist versus Hematopathologist Depending on the laboratory protocols and the hematopathologist's preference, the technologist's responsibilities in bone marrow aspirate processing and reviewing can vary from simply staining slides to complex tasks such as smearing, staining, counting and distributing samples based on laboratory standard protocols. While laboratory professionals may perform bone marrow differentials, it is the hematopathologist's responsibility to review, interpret, and verify those counts after evaluating all of the samples that were provided. This includes both the bone marrow aspirate and biopsy smears, as well as the flow cytometry data, if required. When differentiating and counting samples with increased numbers of blasts, the technologist may make a determination of cell line for the blasts, according to observations on a Wright or Wright-Giemsa stained slide, but the hematopathologist may modify this placement based on flow cytometry data and additional special stains.The laboratory technologist can assess cellularity, presence of megakaryocytes, and possible presence of tumor cells on the bone marrow smears. Ultimately, the hematopatholgist is responsible for interpreting the bone marrow differential results after meticulous review of all smears stained, as well as any biopsy sections available. It is also the hematopatholgist's responsibility to identify tumor cells that may be present.In laboratories that use a standard order set with sample drop off directly from bedside, it is the technologist's responsibility to split and distribute the marrow based on laboratory protocol. It is hematopathologist's responsibility to verify that standard order testing was actually sent and to add on any additional testing deemed necessary, based on clinical history and lab findings. Once you become comfortable with bone marrow morphology and more familiar with your laboratory's protocols, performing marrow differentials will become less intimidating and more of a collaboration between technologist and hematopathologist. | View Page |
| References 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. | View Page |
| Bone marrow Smear Preparation Accurate interpretation of bone marrow aspirate specimens is highly dependent on two factors: The quality of the sample obtained by the clinician The quality of the prepared bone marrow smears While the technologists have no control over the quality of the aspirate obtained by the clinician, in most cases they are able to impact the quality of laboratory prepared smears.The method used for preparing slides may be dependent on sample volume, staining techniques, storage space, clinical setting, patient status, and hematopathologist's preference. Bone marrow sample preparations can be made on slides or coverslips from a direct marrow sample or a sample that is manipulated to enhance cellularity (selected for marrow fragments or spicules). In situations where the technologist goes to the bedside, touch-preparation (touch-prep) smears from the biopsy core can be prepared before it is placed in fixative.Regardless of which method is the preferred primary technique at your institution, it is useful to be familiar with other methods, since there may be a situation when the use of an alternate method is the only way to obtain an interpretable smear.When adequate samples are provided, it is desirable to make several slides or coverslips so that there will be enough preps available for all the possible tests/special stains that may be requested. The extra smears can be stored at room temperature and protected from light in an envelope or sleeve for further testing, if required. | View Page |
| Bone Marrow Collection: Patient Bedside 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. | View Page |
| Hematogone Hematogone is a term applied to a subset of early B-lymphocytes, found in normal bone marrow, whose morphology greatly resembles that of leukemic lymphoblasts. These cells are larger than the average mature lymphocyte, have scant cytoplasm, and a fine, soft chromatin texture; however, they are not quite as immature in appearance as a true leukemic lymphoblast. Hematogones are more common in younger children but can be found in bone marrow samples of patients at any age. They tend to be found in increased numbers within the bone marrows of patients recovering from bone marrow suppression. Common causes of increased concentrations of hematogones include: viral illness, chemotherapy recovery, and immune mediated cytopenias, such as idiopathic thrombocytpenic purpura (ITP). Hematogones are also common in patients with neuroblastoma.With experience, knowledge of the patients underlying clinical condition, and the ability to review a patient's bone marrow, it is possible to distinguish hematogones from blasts. When necessary, a hematogone flow cytometry panel can be obtained to distinguish these benign cells from lymphoblasts.Notice the size of these blast-like hematogones (see red arrows). They are larger than the few background lymphocytes present in these images. Notice the fine chromatin and scant cytoplasm. They are usually found mixed in with the full range of bone marrow cellular lineages, but can cluster with other lymphocytes within the spicules. | View Page |
| Organizations and Agencies 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) | View Page |
| Cardiac Biomarkers and Congestive Heart Failure A patient with congestive heart failure (CHF) may exhibit signs and symptoms that are nonspecific; among these are edema, hypertension, shortness of breath, and weakness. Until recently the diagnosis of CHF was difficult, lengthy, and often concluded by ruling out other conditions. B-type natriuretic peptide (BNP) and/or the N-terminal fragment, NT-proBNP, are now routinely measured to diagnose CHF. ProBNP is the precursor of BNP. It is released from the left ventricle myocardium in response to mechanical stretch. This stretch is described as an increase in ventricular wall tension because of pressure and volume overload that occurs in CHF. ProBNP is then enzymatically cleaved to produce BNP and NT-ProBNP. BNP is the active hormone composed of 32 amino acids. The N-terminal fragment is a larger chain of 76 amino acids; this fragment is inactive. Studies indicate that NT-proBNP has the same clinical utility as BNP.Besides diagnosing CHF, the levels of BNP and NT-ProBNP correlate to the severity of the heart disease, assist in detection of CHF where patients are asymptomatic, and differentiate patients whose pulmonary disease presents with symptoms similar to CHF. | View Page |
| A 79-year-old diabetic and retired homemaker lives alone since the death of her husband 5 years ago. At age 75, she was diagnosed with an AMI and had another AMI last year. In the past 6 weeks, she has often been weak and often lacks energy. For the past 2 days, her symptoms have worsened and she is short of breath upon any exertion. Her daughter brings her to see the cardiologist who orders a BNP to evaluate her cardiac disease.BNP 520 pg/mL (Reference value = <100 pg/mL)What may this BNP result indicate when considered together with the patient's clinical symptoms? | View Page |
| Use of hs-CRP, Measurement, and Ranges In 2002, the AHA and CDC recommended measurement of hs-CRP as an aid in the diagnosis and treatment of CVD. At low levels, it can detect those at risk for cardiac heart disease. At high levels in those with no history of heart disease, it indicates a high risk for AMI, stroke, or peripheral vascular disease. For patients with ACS or stable coronary disease, hs-CRP is used to predict future coronary events.Nephelometry and immunoturbidimetric measurement methods provide lower limits needed for hs-CRP assays. Due to variation in results among clinical laboratories, work is underway for standardization of measurements. Ranges of hs-CRP in prediction of risk for CVD are: <1.0 mg/L Low CVD risk 1.0-3.0 mg/L Average risk for CVD >3.0 mg/L High risk for future CVDIf results are >10.0 mg/L, the patient should be evaluated for an acute inflammatory condition. | View Page |
| References 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. | View Page |
| A 25-year-old female presented in the emergency room with an acute urethral discharge of two days duration. The image to the right shows the Gram stained smear that was obtained. Many polymorphonuclear leukocytes and intracellular and extracellular gram-negative diplococci were observed. Based on the clinical history and the Gram stain observation, a diagnosis of gonorrhea can be made. | View Page |
| The carbohydrate utilization reaction seen in the QuadFerm system shown in the image provides a definitive identification of N. gonorrhoeae. | View Page |
| Review 1 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. | View Page |
| The Gram stain report to be issued based on the microscopic characteristics seen in the image on the right would most correctly be, "many WBC with..." (Choose the BEST response) | View Page |
| Clinical History A 67-year-old man entered the hospital with cough, right lower chest pain accentuated by deep breathing, and fever. He had a history of chronic obstructive pulmonary disease secondary to a long history of smoking. The temperature on admission was 39.2C, and auscultation of the chest revealed rales in the right lower lung field. The admission white blood count was 13,500/ml with 80% segmented neutrophils and a shift to the left. A blood culture was obtained. | View Page |
| Isolates of Escherichia coli, Klebsiella pneumoniae, K. oxytoca and clinically significant isolates of Proteus mirabilis may possess extended-spectrum beta-lactamase (ESBL) activity. | View Page |
| Extended-Spectrum Beta-Lactamases (ESBLs) Review 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. | View Page |
| Review 1 Podschun R. Ullmann U.: Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors Clinical Microbiology Reviews. 11(4):589-603, 1998 Bacteria belonging to the genus Klebsiella frequently cause human nosocomial infections. In particular, the medically most important Klebsiella species, Klebsiella pneumoniae, accounts for a significant proportion of hospital-acquired urinary tract infections, pneumonia, septicemias, and soft tissue infections. The principal pathogenic reservoirs for transmission of Klebsiella are the gastrointestinal tract and the hands of hospital personnel. Because of their ability to spread rapidly in the hospital environment, these bacteria tend to cause nosocomial outbreaks. Hospital outbreaks of multidrug-resistant Klebsiella species, especially those in neonatal wards, are often caused by new types of strains, the so-called extended-spectrum-beta-lactamase (ESBL) producers The incidence of ESBL-producing strains among clinical Klebsiella isolates has been steadily increasing over the past years. The resulting limitations on the therapeutic options demand new measures for the management of Klebsiella hospital infections. While the different typing methods are useful epidemiological tools for infection control, recent findings about Klebsiella virulence factors have provided new insights into the pathogenic strategies of these bacteria. Klebsiella pathogenicity factors such as capsules or lipopolysaccharides are presently considered to be promising candidates for vaccination efforts that may serve as immunological infection control measures. | View Page |
| Clinical History A 72- year old woman had a history of recurrent urinary tract infections over the past several months, for which she had received different regimens of antibiotics including ampicillin, trimethoprim-sulfasoxazole, and ciprofloxacin.Relapses often occurred 10 days to two weeks after cessation of therapy.The current flare up, manifest by dysuria, lower abdominal pain and cloudy urine was accompanied by shaking chills and spiking fever.A sterile mid-stream urine specimen was sent to the laboratory for culture.
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| Pyrrolidonyl Arylamidase (PYR) Differential As mentioned before, the spot pyrrolidonyl arylamidase (PYR) test is commonly performed to separate Enterococcus species (positive reaction) from the Group D streptococci (S. bovis, S. equinus), which are negative. It should be remembered that Streptococcus pyogenes (group A) also produces PYR; therefore, additional characteristics such as beta hemolysis are important. Some species of Aerococcus and Gemella are also PYR-positive; however, they can be suspected if large cocci in tetrads or clusters are observed on gram stain. These species are rare isolates in most clinical practices. | View Page |
| Review 2 Suppola JP. Kuikka A. Vaara M. Valtonen VV. Comparison of risk factors and outcome in patients with Enterococcus faecalis vs Enterococcus faecium bacteremia. Scandinavian Journal of Infectious Diseases. 30(2):153-7, 1998. The purpose of our study was to determine retrospectively the risk factors for the acquisition of Enterococcus faecalis vs E. faecium bacteremia, as well as the clinical outcomes of these patients. 62 patients with Enterococcus faecalis bacteremia were compared to 31 patients with E. faecium bacteremia. Haematologic malignancies, neutropenia, high-risk source and previous use of aminoglycosides, carbapenems, cephalosporins and clindamycin were significantly associated with E. faecium bacteremia. Instead, urinary catheterization was found to be related to Enterococcus faecalis bacteremia. The mortality rates within 7 d and 30 d were 13% and 27%, respectively, in patients with E. faecalis bacteremia and 6% and 29%, respectively, in patients with E. faecium bacteremia. There was no difference in mortality between E. faecalis and E. faecium bacteremia, nor was there a difference in seriousness of disease at the time of bacteremia. In the subgroups of patients with monomicrobial or clinically significant E. faecalis vs E. faecium bacteremia, the mortality rates were similar to the results of all subjects. Our results do not support the theory that E. faecium would be a more virulent organism than E. faecalis. | View Page |
| The Gram stain shown in the image was prepared from a positive anaerobic blood culture bottle after 36 hours incubation. Based on the morphology of the bacterial cells (some with spores, noted by the blue arrows), what the most likely identification? | View Page |
| Review 2 Citron DM. Appelbaum PC.: How far should a clinical laboratory go in identifying anaerobic isolates, and who should pay? Clinical Infectious Diseases. 16 Suppl 4:S435-8, 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). | View Page |
| Review 3 Kornbluth AA. Danzig JB. Bernstein LH.: Clostridium septicum infection and associated malignancy. Report of 2 cases and review of the literature. Medicine. 68(1):30-7, 1989 We report 2 patients with myonecrosis due to Clostridium septicum and associated colon carcinoma and have reviewed the English language literature for all reported cases of atraumatic C. septicum infection. A total of 162 cases of C. septicum infection have been reported. Eighty-one percent of these patients had an associated malignancy. Thirty-four percent of all patients had associated colon carcinoma, while 40% had a hematologic malignancy. Thirty-seven percent of reported patients had an occult malignancy at the time of their infection with C. septicum. In many patients, the portal of entry was found in the large intestine. In a particularly lethal form (79% mortality) of C. septicum infection, known as "distant myonecrosis," infection metastatic from the initial site of infection causes severe myonecrosis, gangrene, and often death within hours of clinical detection. Overall, survival of patients with C. septicum infection is only 35%. Review of all cases of C. septicum infection suggests several conclusions. 1) Patients with malignancy, particularly colonic or hematologic, and patients with cyclic neutropenia who develop signs and symptoms of sepsis, especially with associated findings of abdominal pain or pain in an extremity, should be treated for possible clostridial infection. 2) C. septicum infection does not appear to be a result of a single specific defect in either humoral or cell-mediated immunity. Rather, it may occur in patients who are granulocytopenic and therefore prone to an enterocolitis. 3) Patients in whom an infection with C. septicum is found must undergo a vigorous search for malignancy. | View Page |
| Clinical History An 18-year-old female incurred a deep penetrating injury of the dorsum of her right foot when a kitchen knife fell from a platter she was carrying while going barefoot. The initial injury partially resolved; however, three days later the foot began to swell, become red, and painful. A deep subcutaneous abscess developed, with a central sinus tract from which a cloudy, serous fluid exuded (see image). | View Page |
| Methicillin-Resistant Staphylococcus aureus (MRSA) Screen Perhaps the most efficient means for detecting methicillin-resistant staphylococci in clinical laboratories is the use of the agar dilution screening test. Illustrated in the image is a Mueller-Hinton agar plate containing 6 ug/mL of oxicillin, previously inoculated with a strain of Staphylococcus aureus. Oxacillin is used as a marker for methicillin resistance because it is more stable in the agar medium. Growth on this screening medium is presumptive for methicillin resistance. Thus, in the presence of growth, as shown here, a follow-up minimum inhibitory concentration (MIC) test must be performed to determine the exact level of resistance. | View Page |
| Review 2 Hershow RC. Khayr WF. Smith NL.: A comparison of clinical virulence of nosocomially acquired methicillin-resistant and methicillin-sensitive Staphylococcus aureus infections in a university hospital (University of Illinois at Chicago). Infection Control & Hospital Epidemiology. 13(10):587-93, 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. | View Page |
| Review 3 Ladhani S. Joannou CL. Lochrie DP. Evans RW. Poston SM.: Clinical, microbial, and biochemical aspects of the exfoliative toxins causing staphylococcal scalded-skin syndrome. Clinical Microbiology Reviews. 12:224-242, 1999 The exfoliative (epidermolytic) toxins of Staphylococcus aureus are the causative agents of the staphylococcal scalded-skin syndrome (SSSS), a blistering skin disorder that predominantly affects children. Clinical features of SSSS vary along a spectrum, ranging from a few localized blisters to generalized exfoliation covering almost the entire body. The toxins act specifically at the zona granulosa of the epidermis to produce the characteristic exfoliation, although the mechanism by which this is achieved is still poorly understood. Despite the availability of antibiotics, SSSS carries a significant mortality rate, particularly among neonates with secondary complications of epidermal loss and among adults with underlying diseases. | View Page |
| A clinical condition often associated with Streptococcus anginosus ("milleri") is: | View Page |
| Review 1 Piscitelli SC., Shwed J., Schreckenberger P., Danziger LH. Streptococcus milleri group: renewed interest in an elusive pathogen. European Journal of Clinical Microbiology & Infectious Diseases.11:491-8, 1992 The following review examines the bacteriological characteristics, epidemiology, pathogenicity and antimicrobial susceptibility of the "Streptococcus milleri group". "Streptococcus milleri group" is a term for a large group of streptococci which includes Streptococcus intermedius, Streptococcus constellatus, and Streptococcus anginosus. Usually considered commensals, these organisms are often associated with various pyogenic infections including cardiac, intra-abdominal, subcutaneous and central nervous system infections, particularly with the formation of abscesses. Organisms of the "Streptococcus milleri group" are often unrecognized pathogens due to the lack of uniformity in classifications and difficulties in microbiological identification. Penicillin G, cephalosporins, clindamycin and vancomycin all possess activity against these streptococci. Use of agents with poor activity may promote infections with "Streptococcus milleri group" and allow it to exhibit its pathogenicity. An understanding of these organisms may aid in their recognition and proper treatment. | View Page |
| The epidural and subdural abscesses in the two patients reported by Gelfand, et al, are clinical manifestations uncommon for S. anginosus ("milleri"). | View Page |
| Beta hemolytic colonies grew from the blood culture bottle after 18 hours incubation (see image). Which of following tests would be helpful in making a preliminary identification? (Choose all that apply) | View Page |
| Review 1 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. | View Page |
| Review 2 Cunningham MW.: Pathogenesis of group A streptococcal infections. Clinical Microbiology Reviews. 13):470-511, 2000 Group A streptococci are model extracellular gram-positive pathogens responsible for pharyngitis, impetigo, rheumatic fever, and acute glomerulonephritis. A resurgence of invasive streptococcal diseases and rheumatic fever has appeared in outbreaks over the past 10 years, with a predominant M1 serotype as well as others identified with the outbreaks. Emm (M protein) gene sequencing has changed serotyping, and new virulence genes and new virulence regulatory networks have been defined. The emm gene superfamily has expanded to include antiphagocytic molecules and immunoglobulin-binding proteins with common structural features. At least nine superantigens have been characterized, all of which may contribute to toxic streptococcal syndrome. An emerging theme is the dichotomy between skin and throat strains in their epidemiology and genetic makeup. Eleven adhesions have been reported, and surface plasmin-binding proteins have been defined. The strong resistance of the group A streptococcus to phagocytosis is related to factor H and fibrinogen binding by M protein and to disarming complement component C5a by the C5a peptidase. Molecular mimicry appears to play a role in autoimmune mechanisms involved in rheumatic fever, while nephritis strain-associated proteins may lead to immune-mediated acute glomerulonephritis. Vaccine strategies have focused on recombinant M protein and C5a peptidase vaccines, and mucosal vaccine delivery systems are under investigation. | View Page |
| Clinical History 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. | View Page |
| A Brown and Brenn gram stain was performed on one of the tissue biopsy specimens. Organisms were seen as shown in the image. Based on the history and the appearance of the bacteria, the most likely identification is: | View Page |
| In view of the feedback to the previous question, what is the most likely reason that the clinical correlation does not seem to fit in this case? | View Page |
| Review 3 Robinson LG. Kourtis AP.: Tale of a toothpick: Eikenella corrodens osteomyelitis. Infection. 28(5):332-3, 2000 Tale of a Toothpick is a case of Eikenella corrodens osteomyelitis in a young woman, that resulted from puncture of her foot with a toothpick. The epidemiology, microbiology, common clinical presentations and therapy of E. corrodens are reviewed. A brief summary of the extent of toothpick injuries and their infectious complications are also presented. | View Page |
| Review 2 Griego RD. Rosen T. Orengo IF. Wolf JE.: Dog, cat, and human bites: a review. Journal of the American Academy of Dermatology. 33:1019-29, 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. | View Page |
| Urine pH: Acidic and Alkaline 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 | View Page |
| Clinical Significance of Urine Protein The presence of an increased amount of protein in a urine specimen is often the first indicator of renal disease. Proteinuria 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. Proteinuria related to kidney impairment may be due to glomerular membrane damage caused by toxic agents, immune complexes found in lupus erythematosus, or streptococcal glomerulonephritis. The amount of protein present in urine samples from patients with glomerular damage usually ranges from 10-40 mg/dL. If the urinary protein is due to a disorder that affects tubular reabsorption, the urine protein quantities will be much greater. In patients with multiple myeloma, proteinuria is due to the excretion of the Bence Jones protein. This low molecular weight protein produced by a malignant clone of plasma cells circulates in the blood and is filtered in the kidneys in quantities exceeding the tubular capacity. This excess protein is excreted in the urine. | View Page |
| Clinical Significance of Urine Protein (continued) 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. | View Page |
| Clinical Significance of Glucose in the Urine In a 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. Conditions in which glucose levels in the urine are above 100 mg/dL and detectable include: diabetes mellitus and other endocrine disordersimpaired tubular reabsorption due to advanced kidney diseasepregnancy - glycosuria developing in the 3rd trimester may be due to latent diabetes mellituscentral nervous system damagepancreatic diseasedisturbances of metabolism such as, burns, infection or fractures | View Page |
| Clinical Significance of Positive Urine Ketones 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. | View Page |
| Clinical Significance of Urine Bilirubin Liver damage or an obstructed bile duct allows conjugated bilirubin to enter the circulation and ultimately to appear in the urine. Patients with clinical jaundice due to hepatitis or cirrhosis will have bilirubinuria. If the jaundice is due to red cell destruction, there is an increase in unconjugated bilirubin which the kidneys cannot excrete. | View Page |
| Clinical Significance of Blood in Urine 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 | View Page |
| Clinical Significance of Nitrites in Urine Early detection of bacteria is important in order to prevent cystitis from developing into inflammation or infection involving the kidney and renal pelvis. The nitrite portion of the test strip can be used to screen individuals who are at risk for developing urinary tract infections, such as diabetics, persons with recurrent infections, or pregnant women. The test is also useful in evaluating the success of antibiotic therapy that is used to treat a bladder infection. | View Page |
| Clinical Significance of Urobilinogen in 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, 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. | View Page |
| Clinical Significance of Leukocyte Esterase in Urine Using the esterase test in conjunction with pH, protein and nitrite provides a combination of tests which can screen for the presence of bacterial infections in the urinary system. | View Page |
| Clinical Significance of Specific Gravity 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. | View Page |
| Reagent Strip Precautions The reagent strips must be handled and stored properly in order to ensure that results are accurate. The following precautions should be observed:Store strips according to the manufacturer's recommendation. DO NOT expose strips to moisture, volatile fumes, or direct sunlight (emphasized in the image on the right). Remove only enough strips for immediate use and immediately recap the bottle. Avoid contamination of test strips. Do not touch the test areas with fingers and do not lay the test strips directly on the workbench. DO NOT use discolored strips. Compare the color of the unused strip to the negative area on the color chart provided by the company. The color should be similar. Check the expiration date. Re-label the container with a revised expiration date, if the manufacturer states a shortened usage period once the container has been opened. Reagent strips must be tested periodically (frequency defined by the laboratory) for clinical reactivity with normal and abnormal urine controls. Urine controls are available commercially or may be prepared and preserved in-house. | View Page |
| Clinical Significance The presence of protein in a urine specimen can have serious implications. It may signal severe kidney damage, be a warning of impending kidney involvement, or be transient and unrelated to the renal system. Further quantitative testing of urine for protein may be needed to determine the significance of the proteinuria. | View Page |
| Clinical Significance cont'd Proteinuria related to kidney impairment may be due to glomerular membrane damage caused by toxic agents, immune complexes found in lupus erythematosus, or streptococcal glomerulonephritis. The amount of protein present in urine samples from patients with glomerular damage usually ranges from 10-40 mg/dl. If the urinary protein is due to a disorder that affects tubular reabsorption, the urine protein quantities will be much greater. In patients with multiple myeloma, proteinuria is due to the excretion of the Bence Jones protein. This low molecular weight protein produced by a malignant clone of plasma cells circulates in the blood and is filtered in the kidneys in quantities exceeding the tubular capacity. This excess protein is excreted in the urine. | View Page |
| Clinical Significance cont'd Individuals with diabetes mellitus may excrete small amounts of protein in the urine which may signal the beginning of reduced glomerular filtration. Stabilizing the blood glucose level at this time may delay progression of diabetic nephropathy. Women in the last month of pregnancy may develop proteinuria as the first sign of impending eclampsia. Eclampsia is the gravest form of toxemia of pregnancy. The presence of protein in this situation must be evaluated by the physician in conjunction with other clinical symptoms.Benign transient proteinuria may be the result of: exposure to cold, strenuous exercise, dehydration, and/or high fever. Benign transient proteinuria may also occur during the acute phase of a severe illness. | View Page |
| Clinical Significance cont'd Patients over the age of 60 have a greater chance of having protein in their urine. Occult malignancies and glomerulonephritis, that occur more frequently in the elderly, may be signaled by the presence of proteinuria. Orthostatic proteinuria is a condition seen most often in young adults. The condition may be caused by pressure on the renal nerve. When this condition is suspected, two urine specimens are tested. One specimen is collected upon arising in the morning, and the second is collected several hours later. When this condition is present, the first morning specimen, after the patient has been in a supine position, will be negative for protein. The second specimen, taken after the patient has been upright for several hours, would be positive for protein. | View Page |
| Clinical Significance In the healthy individual, almost all of the glucose filtered by the renal glomerulus is reabsorbed in the proximal convoluted tubule. The amount of glucose reabsorbed by the proximal tubule is determined by the body's need to maintain a sufficient level of glucose in the blood. If the concentration of blood glucose becomes too high (160-180 mg/dL), the tubules no longer reabsorb glucose, allowing it to pass through into the urine. It is important to note that glucose may appear in the urine of healthy individuals after consuming a meal that is high in glucose. Fasting prior to providing a sample for screening eliminates this problem. | View Page |
| Clinical Significance cont'd Conditions in which glucose levels in the urine are above 100 mg/dL and detectable include:diabetes mellitus and other endocrine disordersimpaired tubular reabsorption due to advanced kidney diseasepregnancy - glycosuria developing in the 3rd trimester may be due to latent diabetes mellituscentral nervous system damagepancreatic diseasedisturbances of metabolism such as, burns, infection or fractures | View Page |
| Clinical Significance of Positive Urine Ketone Result 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. | View Page |
| Clinical Significance Liver damage or an obstructed bile duct allows conjugated bilirubin to enter the circulation and ultimately to appear in the urine. Patients with clinical jaundice due to hepatitis or cirrhosis will have bilirubinuria. If the jaundice is due to red cell destruction, there is an increase in unconjugated bilirubin which the kidneys cannot excrete. | View Page |
| Clinical Significance No blood is found in the urine of healthy individuals although samples from menstruating females, frequently, but not always, test positive for blood. Hematuria is associated with renal or genital urinary disorders in which the bleeding is the result of irritation to the involved organs or trauma. Examples include renal calculi, pyelonephritis, glomerulonephritis, tumors, trauma or exposure to toxic chemicals or drugs and/or strenuous exercise. Hemoglobinuria may be due to the lysis of red cells within the urinary tract. If it is caused by intravascular hemolysis, the hemoglobin is then filtered through the glomeruli. In the normal individual, the hemoglobin molecule attaches to haptoglobin and in this way bypasses the kidney filtration system. When the hemoglobin/haptoglobin system is overwhelmed, as in cases of hemolytic anemia, severe burns, transfusion reaction, infection or strenuous exercise, hemoglobin passes into the urine. | View Page |
| Clinical Significance Early detection of bacteria is important in order to prevent cystitis from developing into inflammation or infection involving the kidney and renal pelvis. The nitrite portion of the test strip can be used to screen individuals who are at risk for developing urinary tract infections, such as diabetics, persons with recurrent infections, or pregnant women. The test is also useful in evaluating the success of antibiotic therapy that is used to treat a bladder infection. | View Page |
| Clinical Significance Urinary urobilinogen may be increased in the presence of a hemolytic process such as hemolytic anemia. It may also be increased with infectious hepatitis, or with cirrhosis. Comparing the urinary bilirubin result with the urobilinogen result may assist in distinguishing between red cell hemolysis, hepatic disease, and biliary obstruction. Urobilinogen is increased in hemolytic disease and urine bilirubin is negative. Urobilinogen is increased in hepatic disease, and urine bilirubin may be positive or negative. Urobilinogen is low with biliary obstruction, and urine bilirubin is positive. Reagent strips methods however, cannot distinguish normal urobilinogen from absent urobilinogen, as might be seen in complete biliary obstruction. | View Page |
| Clinical Significance Using the esterase test in conjunction with pH, protein and nitrite provides a combination of tests which can screen for the presence of bacterial infection. | View Page |
| Clinical Significance Measurement of specific gravity provides information regarding a patient's state of hydration or dehydration. It also can be used to determine loss of renal tubular concentrating ability. | View Page |
| Organizations and Agencies 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. | View Page |
| Diagnosis of Diabetes In 1997, the ADA recommended significant changes in the diagnosis of diabetes. The poorly reproducible oral glucose tolerance test (OGTT) was replaced with easier to use and more patient-friendly diagnostic criteria. An elevated fasting plasma glucose (FPG) was the preferred test to document hyperglycemia according to the 1997 ADA Clinical Practice Recommendations. An elevated casual plasma glucose with symptoms of diabetes and 2-hour plasma glucose after an ingestion of 75 grams of dissolved glucose were also used for diagnosis. In 2010, the ADA affirmed the decision of an international expert committee's recommendation to use the HbA1c test to diagnose diabetes with a threshold > 6.5%.Any one of the four criteria can be used. The hyperglycemia should be demonstrated a second time by any of the four criteria unless the glucose level is significantly high and diabetes is unquestionable. The table below lists the diagnostic assays and criteria. Assay Description Criteria for Diabetes HbA1C Performed in laboratory by method NGSP certified and standardized to DCCT assay > 6.5 % Fasting plasma glucose At least 8 hour fast > 126 mg/dL Casual plasma glucose Symptoms of diabetes; Blood glucose measured at any time of day > 200 mg/dL Two-hour plasma glucose Following a glucose load of 75g anhydrous glucose dissolved in water > 200 mg/dL These criteria are reviewed regularly by ADA, WHO, and IDF. | View Page |
| Categories of Increased Risk for Diabetes 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. | View Page |
| Hemoglobin A1C and Diabetes Diagnosis The addition of hemoglobin A1C (HbA1C) measurement to the diagnosis of diabetes is a significant change. HbA1C assay is currently the standard biomarker for glycemic management. Mainly due to lack of standardization, HbA1C measurement had not been a component for diagnosis of diabetes. HbA1C assays are now highly standardized and recommended usage expanded. The 2010 ADA Clinical Practice Recommendations specifically states that the HbA1C measurement be a National Glycohemoglobin Standardization Program (NGSP) method and traceable to the Diabetes Control and Complications Trial (DCCT) reference assay. Note that point-of-care HbA1C methods do not currently meet this standardization criteria for diagnostic use. | View Page |
| Categories of Increased Risk for Diabetes 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% | View Page |
| Classification of Diabetes In 1997, the ADA also revised the classification of diabetes. The new designations are based upon the cause, not treatment, for each class of diabetes. Where numbers are used for type classification, Arabic numerals have replaced Roman numerals for greater clarity and ease. There are four clinical classes of diabetes: Type 1 Type 2 Gestational Diabetes Other | View Page |
| Screening for Diabetes 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. | View Page |
| Clinical Testing A large number of assays related to carbohydrate management and diabetes monitoring are performed in clinical laboratories, hospital nursing units, nursing homes, physician offices, clinics, and by patients at home, school, or work.Assays that will be discussed are: Blood Glucose Urine Glucose Ketones Microalbuminuria Insulin and C-Peptide Insulin Antibodies Glycosylated Proteins | View Page |
| Whole Blood Glucose Testing 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. | View Page |
| The Laboratory's Role in Diagnosis and Monitoring of Diabetes Even though most diabetics, physician offices, clinics, nursing homes, and nursing units use glucose meters for monitoring glucose levels, the laboratory's role in diagnosis is vital. The function of the laboratory is crucial in diagnosis, monitoring, and management of diabetes. Diabetic patients can go into severe metabolic imbalances that are life threatening. These metabolic conditions include: diabetic ketoacidosis, hyperosmolar nonketotic coma, and hypoglycemia. Laboratory testing is essential in diagnosing and monitoring these conditions.Laboratory blood glucose and HbA1C levels are used to demonstrate the level of hyperglycemia required for diagnosis. If an OGTT is needed for classification or characterization of hyperglycemia, a patient is sent to a hospital or clinical laboratory for the test. Detection of elevated microalbumin levels that can signal early stages of renal impairment is accomplished through laboratory testing. There are many other disease states and complications associated with diabetes. Clinical laboratories detect these diseases and monitor the complications that result. Important among these assays are urea, creatinine, and serum lipids. If a diabetic does have a pancreatic transplant, serum C-peptide and insulins levels monitor transplant success and viability of transplanted organ. | View Page |
| References 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. | View Page |
| Introduction 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. | View Page |
| Specimens Serum and plasma are the most common clinical specimens used for electrophoresis applications. Urine and cerebrospinal fluids (CSF) are also suitable. Other body fluids such as pleural fluid and pericardial fluid are analyzed less frequently. Some specimens require pretreatment before electrophoresis. Low concentrations of proteins normally in urine and CSF are concentrated in order to have enough proteins for detectable separations. Some body fluids require removal of pigments, salts, and other compounds that interfere with electrophoresis or the detection of separated solutes. In molecular diagnostic testing of DNA and RNA, the nucleic acids must first be isolated from the specimen and then purified before separation with electrophoresis. | View Page |
| Types of Electrophoresis 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 | View Page |
| References Clinical Chemistry Concepts and Applications. Shauna C. Anderson and Susan Cockayne. Long Grove, Illinois: Waveland Press, Inc, 2003.Clinical Laboratory Instrumentation and Automation Principles, Applications, and Selection. Kory M. Ward, Craig A. Lehmann, Alan M. Leiken. Philadelphia: WB Saunders Company, 1994.Laboratory Instrumentation, 4th Edition. Mary C. Haven, Gregory A. Tetrault, Jerald R. Schenken, eds. New York: Van Nostrand Reinhold, 1995.Molecular Diagnostics Fundamentals, Methods, and Clinical Applications. Lela Buckingham and Maribeth L. Flaws. Philadelphia: FA Davis Company, 2007.Principles of Gel Electrophoresis. Available at http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/principles.html accessed 9/29/08.Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 4th Edition. Carl A. Burtis, Edward R. Ashwood, David E. Burns, eds. Philadelphia: Elsevier Saunders, 2005. | View Page |
| Types of Support Media 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. | View Page |
| Agarose Gel Agarose gels are chemically purified forms of agar, a polysaccharide extracted from seaweed. The gel pores allow for separation of proteins based on their individual charge and mass. Agarose gel will naturally clear after drying the separated proteins.Common clinical uses of agarose gel electrophoresis (AGE) are separations of plasma proteins, hemoglobin variants, lipoproteins, and isoenzymes. The gels come prepackaged with a plastic template to lay over gel for sample application or slots etched in the gel for these samples. | View Page |
| Currently there has been a revitalization in the clinical usage of electrophoresis. Previously, methods were primarily used to separate proteins in blood and other body fluids. From the following statements, select the statements that correctly describe newer applications of electrophoresis. | View Page |
| Patient Studies to Validate Risk Markers Risk markers are first hypothesized and then tested. Once a potential marker is identified, concentrations of the serum marker are correlated with patient outcomes. Cardiovascular risk marker studies are typically either retrospective or prospective epidemiology studies. A retrospective study looks backwards at a patient population. For example, we identify (through a hospital database perhaps) patients who have had myocardial infarcts or some other adverse outcome as well as similar subjects without that outcome to use as controls. We then go back and find archived patient serum samples and relate the concentrations of our new risk marker with patient outcomes. Retrospective studies can only be performed if you have archived samples from the patient. Prospective studies look forward in time. For example, we first select a group of subjects and measure our new risk marker in these patients over time. After a few years, we see how the serum concentrations relate to the patient outcomes. Obviously, prospective studies take much longer to perform than retrospective studies. Whatever study model is used, when assessing the value of a cardiovascular risk marker, we must correlate serum concentrations with a specific outcome. The outcome is determined by the study authors. Outcomes could be things like myocardial infarction, stroke, a diagnosis of coronary artery disease, death, or any cardiovascular 'event.'Concentrations of risk markers are divided into tertiles, quatriles or quintiles. This simply means that the top 33%, top 25% or top 20% of the serum concentration values are compared to the bottom 33%, 25% or 20%. For example, risk marker studies will often compare the outcomes of patients with serum concentrations in the upper tertile (those in the top third) with those in the bottom tertile (those in the bottom third) to see if the top 33% had significantly worse outcomes; if so, the risk marker has clinical value. | View Page |
| Lipoprotein Particles Lipid-carrying particles are collectively known as lipoproteins. Lipoproteins are classified according to their densities. The names of these particles, from least to most dense, are: chylomicrons, very-low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). Most laboratorians are familiar with these particles as many, especially LDL and HDL, are routinely measured in clinical labs. In this course we will not focus on the physiology of all of these particles or the differences between them all. However it is important to understand the basic structure and function of a lipoprotein particle. | View Page |
| ApoB/ApoA1: The Test Measuring ApoB and ApoA1 can be performed using standard immunoassay techniques. Nephelometry is popular, as are ELISA-based methods that are performed on automated chemistry analyzer platforms. The power of the ApoB/ApoA1 ratio as a cardiovascular risk marker is getting widespread attention. An individual with seemingly normal LDL-C may in fact have high ApoB concentrations. When this individual has his or her ApoB/ApoA1 ratio calculated, the risk is evident. Studies have also shown that patients with metabolic syndrome and type-2 diabetes can also easily be identified with the ApoB/ApoA1 ratio, whereas these patients cannot always be identified by measuring LDL-C and HDL-C.In 2004, the global INTERHEART study of risk factors for acute myocardial infarction concluded that the ApoB/ApoA1 ratio was the most important risk factor in all geographic regions. The ApoB/ApoA1 ratio is easy to use because the risk is integrated into a single number that indicates the balance between atherogenic and antiatherogenic particles.There have been many studies concerning the predictive power of the ApoB/ApoA1 ratio. One study, which involved thousands of patients who were followed for an average of 10 years, showed that the ApoB/ApoA1 ratio was a strong predictor of stroke in addition to other cardiovascular events. Due to the evidence presented in studies like these, the National Academy of Clinical Biochemistry (NACB) has recommended that the ApoB/ApoA1 ratio be used as an alternative to the usual total cholesterol (TC)/HDL cholesterol ratio when determining lipoprotein-related risk for cardiovascular disease. Some believe that ApoB/ApoA1 testing will eventually replace traditional LDL-C and HDL-C measurements. | View Page |
| Lp(a) Testing One of the problems with Lp(a) measurement is that the Apo(a) protein has a variable mass. It can have a molecular weight ranging from 275,000 to 800,000 daltons. This is due to variable amounts of repeating regions of the protein. Immunoassay antibodies which recognize these regions will thus give more signal for larger Apo(a) molecules compared to smaller Apo(a) molecules. This is not ideal since again, we would prefer to quantify the number of particles and Lp(a) containing large Apo(a) molecules will produce more signal, skewing the count. One assay system that tries to correct for this is the Lp(a) Cholesterol Electrophoresis Assay sold by Helena Laboratories. This assay uses electrophoresis followed by cholesterol staining and densitometry to calculate the concentration of cholesterol in Lp(a). Although this method still does not enumerate particles, it does appear to have less heterogeneity.Lp(a) is an acute phase reactant. This means that Lp(a) levels will rise in the context of general inflammation. Thus, Lp(a) should not be measured when there is extensive inflammation, such as immediately following a cardiovascular event. Concentrations of Lp(a) above 30 mg/dL are associated with increased cardiovascular risk. The risk of having a cardiovascular event increases 2 to 3 fold if Lp(a) cholesterol is > 30 mg/dL. Fifteen to 20% of the Caucasian population have Lp(a) levels >30 mg/dL. Africans, or people of Aftican descent, generally have levels higher than Caucasians and Asians, however, results must be evaluated in conjunction with clinical history. | View Page |
| Summary 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. | View Page |
| Adult Treatment Panel How do physicians interpret risk marker results? Assuming the laboratory offers, and physicians order, cardiovascular risk marker tests, how are these results used? The National Cholesterol Education Program periodically assembles scientists and physicians to create lipid treatment guidelines for patients. These panels are referred to as the Adult Treatment Panel (ATP). The third assembly of the ATP did not give specific guidelines regarding risk marker use in patients but they did acknowledge their potential utility. The general consensus is that novel cardiovascular risk markers should be used in selected patients, such as those who already have significant risk factors (hypertension, smoking, obesity, etc.) or in patients who have family histories of cardiovascular disease. The value in using risk markers is that they will not only uncover cardiovascular risk but they can also be used to motivate patients to alter lifestyle and diet. It is expected that as these emerging cardiovascular risk markers continue to be validated in clinical studies, they will become very useful and perhaps even be part of a new standard of care for patients.If risk marker levels can be correlated to treatment strategies, physicians will find them especially useful in tracking patient success. | View Page |
| References 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. | View Page |
| LpPLA2 and Cardiovascular Risk There have been dozens of clinical studies demonstrating LpPLA2's ability to predict cardiovascular risk. A 2008 study showed that people whose LpPLA2 concentrations were in the upper quartile were 1.64 times more likely to have a cardiac event than those in the lowest quartile. A meta-analysis (a study that sums the results of several other studies) performed by researchers at the Mayo Clinic showed that the unadjusted odds ratio for the association between elevated Lp-PLA2 levels and cardiovascular disease risk was 1.51, indicating that patients with elevated LpPLA2 patients had 1.51 times the risk of cardiovascular disease or events. | View Page |
| Nuclear Magnetic Resonance 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. | View Page |
| Prothrombin Time (PT)/INR PT is a screening test that helps to assess the functionality of both the extrinsic and common pathways. The effectiveness and presence of factors I, II, V, VII, and X are assayed in this diagnostic test, as they are all found in the aforementioned pathways. The results of the PT test are used in conjunction with other diagnostic tests, as well as the clinical picture of the patient, to determine any hemostatic abnormalities that may be present.In addition to being an integral part of the coagulation disorder assessment process, the PT is also used to determine therapeutic effectiveness of the oral anticoagulant, warfarin. PT test results are reported as the number of seconds needed for a clot to form in the patient specimen using the laboratory's instrument/reagent system, and as the International Normalized Ratio (INR). | View Page |
| PT/INR, continued 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. | View Page |
| Activated Partial Thromboplastin Time (aPTT) APTT is a screening test that helps to assess the functionality of both the intrinsic and common pathways. The presence and effectiveness of all the coagulation factors are assayed by this diagnostic test, with the exception of factors VII and XIII. In other words, factors XII, XI, X, IX, VIII, V, II, and I are all involved in this assay.The results of the aPTT are used in conjunction with other diagnostic tests, as well as the clinical picture of the patient, to determine hemostatic abnormalities that may be present. In addition to being an integral part of the coagulation disorder assessment process, the aPTT is used to determine therapeutic effectiveness of heparin administration. APTT results are reported in seconds, which represent the actual time elapsed until a clot was detected using the laboratory's instrument/reagent system. | View Page |
| Fibrin/Fibrinogen Degradation Products and D-Dimers The presence of D-dimers in plasma or whole blood indicates that fibrin has been formed and degraded (fibrinolysis). Plasmin can also degrade intact fibrinogen, generating fibrinogen degradation products that are detected in fibrin/fibrinogen degradation products (FDP) assays. D-dimers and FDP can become elevated whenever the coagulation and fibrinolytic systems are activated. The presence of D-dimer confirms that both thrombin and plasmin have been generated, since it can only be produced as the result of the plasmin degradation of fibrin. 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. | View Page |
| Platelet Function Assay A platelet function assay (PFA) is a screening test for the evaluation of platelets/primary hemostasis. Common clinical applications include the following:Preoperative evaluation of platelet functionDetermining the presence of drug-induced platelet dysfunctionDetermining platelet functionality in high-risk pregnancyEvaluation of patients with suspected inherited or acquired platelet disorders such as von Willebrand diseaseEvaluation of a bleeding patientA PFA instrument is able to differentiate between drug-induced platelet defects and other platelet defects. PFA tests are superior to the bleeding time test. The bleeding time is often not reproducible and, in spite of attempts at standardization, remains prone to variations in test results between persons performing the test. It is also relatively insensitive to platelet function. The bleeding time cannot be used to identify patients who may have recently ingested aspirin or non-steroidal anti-inflammatory drugs or patients who may have a platelet defect attributable to these drugs. The bleeding time is used to assess platelet function, but may be affected by platelet quantity. NOTE: Aspirin, and some other drugs, may falsely prolong bleeding times. Patients must be asked about aspirin use, and be aspirin free for 7-10 days prior to testing, for valid results. | View Page |
| Anticoagulation Therapy 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. | View Page |
| Targets 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 | View Page |
| Overview Molecular diagnostics have begun to play an integral part in clinical laboratory diagnostic testing. Traditionally, molecular diagnostics have been utilized in three major clinical areas: Infectious diseases Genetics Tumor markers These molecular based diagnostic tests, while historically reserved for specialty/reference labs, have recently seen expansion of their utility within the scope of routine clinical laboratories. Molecular based diagnostics can be utilized by small labs as well as large ones, and can be found in virtually every department of the clinical laboratory. | View Page |
| Overview 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?" | View Page |
| Pre-analytical Variables Pre-analytical variables are those that affect the specimen before the actual testing begins. Some of the pre-analytical variables to consider with molecular testing include those that are applicable to all clinical specimens but should be emphasized when discussing molecular methodologies. Some of these include but are not limited to:Receipt of valid orderProper patient identificationProper venipuncture procedure for blood collection Use of correct anticoagulant Collection of correct specimen type (eg - plasma, serum, whole blood)Order of drawProper storage Proper transportProcedures if there is a delay in testing and/or transport | View Page |
| Resources 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. | View Page |
| Transport Many clinical laboratories utilize reference laboratories for molecular methodology testing. Transport and shipping of biological specimens must follow laws and regulations governing these types of specimens. Consult your laboratory's accrediting agency and reference laboratory for specific polices and procedures. | View Page |
| Overview 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. | View Page |
| Infectious Diseases Molecular methodologies can be useful in the detection of a variety of diseases that are important public health issues such as:Chlamydia trachomatis (CT) Neisseria gonorrhoeae (GC)Human papillomavirus (HPV)Human immunodeficiency virus(HIV)Herpes simplex virus(HSV)Cytomegalovirus(CMV)In many clinical laboratories, traditional methods have been replaced by molecular methodologies because testing can occur for several pathogens in a single specimen. This is termed multiplex testing. | View Page |
| Disadvantages of Molecular Testing 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. | View Page |
| Methods for Detection of Mutations The reason to chose a particular molecular method can be influenced by disease detection, monitoring, or therapy in certain patient populations. Molecular methodologies can be used to identify alterationsor variations or changes in DNA sequencing that can cause disease. Sequence alterations that are known to cause disease are termed mutations. These changes or mutations can be applied to areas of the clinical laboratory such as infectious disease, paternity, genetic testing, and pharmacogenetics. Some of the more common alterations are:Deletion: A missing nucleotide or other portion of DNA sequence Insertion: An extra DNA nucleotide or other portion of DNA sequence Missense: A nucleotide or sequence substitution that codes for a different amino acidNonsense: A nucleotide substitution that ends in early termination of the protein manufacturing process; usually due to a stop codon.The most common alteration is a single base change or single nucleotide polymorphism (SNP). | View Page |
| References 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. | View Page |
| Clinical Manifestations 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. | View Page |
| References Afenyi-Annan, A., Kail, M., Combs, M.R., Orringer, E.P., Ashley-Kock, A., & Telen, M.J. Lack of Duffy antigen expression is associated with organ damage in patients with sickle cell disease. Transfusion. 2008;48:917-924. Ataka, K. I. et. al.Efficacy and safety of the Gardos channel blocker, senicapoc (ICA-17043), in patients with sickle cell anemia. Blood: 2008; 11(8) 3991-3997.Ballas, S.K., Sickle Cell Anaemia: Progress in Pathogenesis and Treatment. Drugs 2002: 62(8); 1143-1172.Bianchi, N., Zuccato, C., Lampronti, I., Borgatti, N., and Gambari, R. Fetal Hemoglobin Inducers from the Natural World: a novel approach for the identification of drugs for the treatment of B-thalassemia and Sickle-cell anemia. eCAM: 2009; 6(2)141-151.Centers for Disease Control and Prevention. Sickle cell disease: Symptoms and treatments. Available at: http://www.cdc.gov/ncbddd/sicklecell/symptoms.html. Accessed January 21, 2010.Harmening, Denise M., Clinical Hematology and Fundementals of Hemostatis 4th., F.A. Davis, 2001.Inati, A., Koussa, S. Taher, A., & Perrine, S. Sickle cell disease: New insights into pathophysiology and treatment. Pediatr Ann. May 2008.Kaushansky, K., Lichtman, M.A., Beulter, E., Kipps, T.J., and Prchal, J.T. Williams Hematology 8th Ed. McGraw Hill 2010.Lotspeich-Steininger, Stiene-Martin and Koepke, Clinical Hematology Principles, Procedures, Correlations, Lippincott 1992. McKenzie, Shirlyn B., Textbook of Hematology 2nd ed., Williams and Wilkins 1996. Miale, John B, Laboratory Medicine Hematology 6th ed., Mosby 1982. Niscola, P., Sorrentino, F., Scaramucci, L., de Faritiis, P., & Cianciulli, P. Pain syndromes in Sickle Cell Disease: An update. American Academy of Pain Medicine. 2009:470-480.Rodak, Bernadette, Diagnostic Hematology, W.B.Saunders Co., 1995.Yoon, S.L. & black, S. Comprehensive, integrative management of pain for patients with Sickle-Cell Disease. Journal of Alternative and Complementary Medicine. 2006: 12; 995-1001. | View Page |
| Investigative Therapies 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. | View Page |
| Advance Organizer 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? | View Page |
| ABO HDFN - Diagnostic Tests Before ABO HDFN is considered as a possible cause of jaundice and anemia in the newborn, other causes should be considered, for example, erythrocyte membrane defects or red cell enzyme deficiencies. The diagnosis of ABO HDFN in the laboratory differs from diagnosing Rh and other types of HDFN in which clinically significant antibodies must be identified. Diagnosis may be difficult, because the DAT on the newborn's red cells is unreliable. Indeed, many labs do not routinely do a DAT on infants born to Rh positive females, since many will be positive in the absence of clinically significant hemolysis. Cord blood is often retained (e.g., for 7 days) should the infant develop signs of HDFN and required testing.If ABO HDFN is possible, based on incompatible ABO blood groups and a positive DAT, and the mother's antibody screen is negative, many laboratories do not investigate the positive DAT as would be done for unexpected antibodies like anti-D or anti-K (the laboratory does not perform an elution on the newborn's red cells). Instead, the infant's plasma is tested against group A1 (or B cells) and group O screen cells using the indirect antiglobulin test (IAT). A positive reaction with A1 or B cells, but not group O cells, would suffice to report a case of possible ABO HDFN. | View Page |
| Follow-up Investigative Tests (Mother) 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. | View Page |
| Maternal antibody titer is a good indicator of severity of HDFN. | View Page |
| Clinical Relevance of D Phenotypes 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 | View Page |
| Main Learning Goals This course reviewed some of the key learning goals relevant to HDFN and its investigation, prevention, and treatment. More specifically, the course reviewed the following topics: Historical aspects of HDFN due to anti-D and its prevention; HDFN due to antibodies in the ABO, Rh, and other blood group systems; Clinical symptoms and associated laboratory test results in HDFN; Best practices related to perinatal testing programs to prevent and diagnose HDFN; Characteristics and uses of RhIg; Interpretation of typical serologic test results when investigating HDFN.Before taking the final quiz, for each of the above topics, list as many of the key learning points that you can recall, then review topics that need more study. As well, re-read the learning objectives at the start of the course 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 the course materials. | View Page |
| Literature and Online Resources 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. | View Page |
| Introduction 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. | View Page |
| References 1. Beutler E. Iron storage disease: Facts, fiction and progress. Blood Cells Mol Dis. 2007;39:140-7.2. Higgins T, Beutler E, Doumas BT. Hemoglobin, iron, and bilirubin. In: Burtis CA, editor. Teitz Fundamentals of Clinical Chemistry. 6th ed. Saunders Elsevier, 2008.3. Ganz T. Hepcidin, a key regulator of iron metabolism and mediator of anemia and inflammation. Blood 2003;102(3):78-8.4. Andrews NC, Schmidt PJ. Iron homeostasis. Annu Rev Physiolo. 2007;69:69-85.5. Murtagh LJ, Whiley M, Wilson S, et al. Unsaturated iron binding capacity and transferrin saturation are equally reliable in detection of HFE hemochromatosis. Am J Gastroenterol. 2002;97(8):2093-9.6. Haddy TB, Castro OL, Rana SR. Hereditary hemochromatosis in children, adolescents, and young adults. Am J Pediatr Hematol Oncol 1988;10:23-4.7. Edwards CQ, Ajoika RS, Kushner JP. Hemochromatosis: A genetic definition. In Barton JC, Edwards CQ, eds. Hemochromatosis: Genetics, Pathophysiology, Diagnosis and Treatment. Cambridge, UK:Cambridge Univ Pr 2000:8-11.8. Whitlock EP, Garlitz BA, Harris EL , et al. Screening for Hereditary Hemochromatosis: A Systematic Review for the U.S. Preventive Services Task Force. Ann Intern Med. 2006; 145: 209-23.9. Wallace DF, Subramaniam VN. Non-HFE haemaochromatosis. World J Gastroenterol. 2007;13(35):4690-8.10. Tavill AS. Diagnosis and management of hemochromatosis. Hepatology. 2001;33:1321-811. Qaseem A, Aronson M, Fitterman N, Snow V, Weiss KB, Owens DK, et al. Screening for hereditary hemochromatosis: a clinical practice guideline from the American College of Physicians. Ann Intern Med. 2005;143:517-21.12. Phatak PD, Bonkovsky HL, and Kowdley KV. Hereditary Hemochromatosis: time for targeted screening. Ann Intern Med. 2008; 149(4): 270 – 2.13. Brissot P, deBels F. Current approaches to the management of hemochromatosis. Hematology Am Soc Hematol Educ Program. 2006:36-41. 14. Guidance for industry: Variances for blood collection from individuals with hereditary hemochromatosis. http://www.fda.gov/cber/gdlns/hemchrom.htm Accessed 12/17/08. | View Page |
| Altered Iron Absorption Hereditary hemochromatosis (HH) is a genetic disorder characterized by iron overload as a result of increased iron absorption. As iron absorption increases, the amount of iron bound to transferrin and transported in the plasma subsequently increases.With no available mechanism for excreting excess absorbed iron, normal iron storage sites become overloaded, resulting in ferritin levels that far exceed normal. As a result, iron is deposited in the parenchymal cells of the liver, pancreas, pituitary, heart, synovium, and other tissues with high concentrations of transferrin receptors. Iron in excess of normal cellular ferritin stores contributes to the generation of free radicals and reactive oxygen intermediates that cause cell damage to organs and tissues. This process results in the clinical condition known as iron overload, a hallmark feature of HH. | View Page |
| Development of Iron Overload The amount of time needed for iron to increase to levels causing organ damage is variable and may be partially dependent on gender, dietary or other environmental factors, and unknown genetic factors. Blood loss through menstruation and pregnancy are thought to delay the onset of iron overload, and therefore symptoms of HH, in women. Similarly, regular blood donation may confer some degree of protection. The loss of hemoglobin within intact erythrocytes reduces the amount of iron available for recycling.As levels of storage iron increase, clinical features of iron overload, including hepatic dysfunction or failure, diabetes, hypogonadism, arthritis, cardiomyopathy, hyperpigmentation, and fatigue, may become evident.Symptomatic patients typically present in middle age between the ages of 30 and 60, although this is quite variable. Persons as young as 20 may show clinical signs and symptoms of HH.(6) In the US, males are more than twice as likely as females to be diagnosed with HH, and the majority of cases are found in Caucasians. | View Page |
| Specific HFE Mutations 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. | View Page |
| Epidemiology of HFE Mutations The prevalence of common HFE mutations among persons with hereditary hemochromatosis (HH) has been reported in numerous studies conducted in the US, France, Australia, and other countries. Homozygous C282Y mutation (C282Y/C282Y) is present in 82% to 90% of Caucasian patients diagnosed with iron overload due to HH.(7) This suggests a strong link between the genotype and the phenotypic presentation of clinical iron overload. Much lower percentages of persons diagnosed with HH do not have two C282Y mutations. A small percentage of persons diagnosed with HH are compound heterozygotes for C282Y and H63D (C282Y/H63D), are homozygous for H63D (H63D/H63D), heterozygous for C282Y (C282Y/wild type) or for H63D (H63D/wild type), or carry S65C or other HFE mutations.It may be that symptomatic heterozygotes are actually HFE-compound heterozygotes with additional unidentified mutations modifying the expression of the more severe known mutation. It is quite possible that more mutations of HFE and elucidation of other gene mutations modifying HFE will be discovered in the future enabling scientists to better explain the phenotypic heterogeneity of this disorder.In the US, the C282Y mutation is most prevalent in the non-Hispanic white population. It is much less common among Hispanics and African Americans. | View Page |
| Incomplete Penetrance 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. | View Page |
| Non-HFE Mutations Genes in addition to HFE have been linked to hereditary hemochromatosis (HH). They include the hepcidin, hemojuvelin, transferrin receptor, and ferroportin genes. Mutations of some are linked with dominantly inherited HH, juvenile HH, and African iron overload. Unlike HH due to HFE mutations, these clinical disorders are rarely observed in the US population.(9) | View Page |
| General Clinical Considerations 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) | View Page |
| Secondary Disorders of Iron Overload In addition to hereditary hemochromatosis (HH), there are other conditions of iron overload that must be considered in a differential diagnosis. Disorders such as sickle cell disease, thalassemia, sideroblastic anemia, congenital dyserythropoietic anemia, and liver disease may also cause iron overload. Transfusion-dependant patients and persons who abuse iron-containing vitamin supplements are also at risk. These conditions are usually described as secondary iron overload, in contrast to the primary iron overload of HH.Patient history, clinical signs and symptoms, biochemical and hematologic laboratory analyses, and possibly results of a liver biopsy may be needed to establish a diagnosis of a condition causing secondary iron overload. DNA tests for common HFE mutations are very likely the most important diagnostic tool for identifying HH as the cause of iron overload. In some patients, both secondary causes and HH may be contributing to iron overload. Differentiating the secondary causes of iron overload from HH is heavily dependent on the results of laboratory assays, but a complete discussion is beyond the scope of this course. | View Page |
| Which of the following is (are) needed for a diagnosis of hereditary hemochromatosis (HH)? | View Page |
| Why is serum ferritin (SF) a less than optimal screening test for hereditary hemochromatosis (HH)? | View Page |
| Screening Controversies 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. | View Page |
| How would you interpret the result of a UIBC test that is less than the lower limit of the reference interval? | View Page |
| Molecular Tests 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. | View Page |
| Which of the following does NOT apply to the use of molecular assays in testing for hereditary hemochromatosis (HH)? | View Page |
| Public health laboratory scientists Public health laboratory scientists are also regulated by the Board.
The table below outlines the various requirements for applicants to receive licensure for a public health laboratory.
Public Health Laboratory RequirementsDirectorFulfill the same requirements as a clinical laboratory directorSupervisorBe certified by National Registry in Clinical Chemistry or American Society for MicrobiologyBe licensed as a technologistHave five year's relevant experiencePass the state examTechnician (microbiology)Have a Bachelor's degree in one of the biological sciencesObtain American Society for Microbiology or the National Registry in Microbiology Certification in Public Health Microbiology
Technician (chemistry)Have a Bachelor's degree in one of the chemical, biological, or physical sciencesObtain National Registry of Clinical Chemistry Certification in Public Health ChemistryTechnician (conditional)Have a Bachelor's degree in one of the chemical or biological sciencesPerform tests only under the direct supervision of a licensed pathologist, director, supervisor, or technologist.Receives a conditional two-year license, which may be renewed only once
A license from the Board of Clinical Laboratory Personnel allows you to work in a public health laboratory at the same level and specialty. | View Page |
| Director Qualifications Effective date March 17, 2008All applicants for a Director license must meet the qualifications for a high complexity laboratory director that are defined in 42 CFR 493.1443 as published on October 1, 2007.A licensed physician may direct a clinical laboratory without a separate laboratory director's license if he / she is certified in clinical pathology by the American Board of Pathology (ABP) or the American Osteopathic Board of Pathology (AOBP); is board-certified in the pertinent laboratory speciality; and/or has four years of pertinent clinical laboratory experience (post-graduate) with two years experience in the speciality that will he/she will direct.A non-physician may obtain a director's license for a specialty area if he / she: Holds an earned doctoral degree in a chemical, biological, or clinical laboratory science Is certified in the pertinent laboratory specialty by an approved national board A director can oversee up to five laboratories. | View Page |
| Description of Specialties (2) Specialists in immunohematology perform all testing prior to blood transfusions and work to prevent transfusion infections. They also investigate any post-transfusion reactions. This specialty includes all lab procedures performed in the specialty of histocompatibility. Specialists in clinical chemistry analyze body fluids such as blood, urine, and spinal fluid to determine the chemical makeup, including the amount of carbohydrates, proteins, enzymes, and trace elements. The special covers urine microscopics and chemical evaluation of the liver, kidneys, lungs, heart, and other vital organ systems. This specialty also covers all testing performed in the specialties of radioassay and blood gas analysis. Specialists in blood banking can perform all immunohematology testing as well as testing from the specialties of clinical chemistry, hematology and serology/immunology that relates to donor blood. 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. | View Page |
| Competency and Licensing Violations Clinical laboratory personnel must be licensed and competent to perform their duties. This means holding the appropriate type of license for the task being performed (director, supervisor, technologist, or technician) and being certified in the appropriate specialty for any testing being performed. For example, an individual licensed as a technician in hematology may not perform the duties of a technologist in hematology, nor may that individual perform testing in the microbiology specialty.
Showing a lack of competence to perform even licensed duties is a violation of Board rules. Consistent errors can tarnish a laboratory's reputation, and even a single error can harm patient care. Licensed personnel must be certain that they can perform their duties accurately and competently.
All of the following are violations of Board rules:Performing clinical duties for which one does not hold a license.Performing services one knows one is not competent to perform.Showing lack of competence or making consistent errors in testing or reporting.Having a license revoked or suspended in another state. | View Page |
| Kickback and Inducement Violations Offering or taking a bribe, kickback, bonus, commission, or inducement is against the rules of the Board and against the law.
Many companies give away small promotional items, such as pens or note pads, to promote their products. This is legal, but be cautious about accepting more valuable items. This could be seen as a bribe.
All of the following are serious violations of Board, state, and federal rules:Participating in any commissions, bonuses, kickbacks, inducements, or split-fee arrangements from physicians, health care providers, suppliers, hospitals, nursing homes, other clinical laboratories, pharmacies, and other facilities.Exploiting or influencing a patient for financial gain, including promoting, selling, or withholding services, drugs, or referrals. | View Page |
| Summary of Qualifications The table below summarizes the qualifications for the four types of clinical laboratory personnel licenses.
DirectorPhysician certified in clinical pathology OR Non-physician with: Doctoral degreeCertification in a lab specialtyCompleted course on administrationContinuing education in HIV/AIDS and medical errorsSupervisorOne of the following:Doctoral degree + 1 year experienceMaster's degree + 3 years experienceBachelor's degree + 5 years experienceLicensed as a technologist or meets the requirementsOne of the following:Completed course on administration25 hours of CE in administrationCE in HIV / AIDS and medical errors.TechnologistOne of the following:Bachelor's degree + medical technologist training program OR 3 years experienceAssociate's degree + Florida technician's license and completion of a medical laboratory training program OR 5 years experienceCompleted exam in 1+ specialtiesCE in HIV / AIDS and medical errorsTechnicianMeets one of the following:Completed medical lab technology training programHigh school or equivalency diploma + 5 years experienceAssociate's degree + 4 years experienceBachelor's degree + 3 years experienceBachelor's degree in medical technologyCompleted exam (certain specialties only)CE in HIV / AIDS and medical errors | View Page |
| Clinical laboratory personnel who are licensed in the specialties of immunohematology, clinical chemistry, hematology, and serology / immunology may perform testing in the specialty of blood banking. | View Page |
| Specialists in radioassay, blood banking, and histocompatability may perform all tests associated with immunohematology, clinical chemistry, hematology, and serology / immunology. | View Page |
| Which of the following are violations of Board rules? | View Page |
| A director may only oversee one laboratory. | View Page |
| You cannot work in a clinical laboratory unless you have a four-year college degree. | View Page |
| Clinical laboratory personnel who are licensed in the specialties of immunohematology, clinical chemistry, hematology, AND serology / immunology may perform testing in the specialty of blood banking. | View Page |
| Prior to 1985 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. | View Page |
| Chlamydia trachomatis and Neisseria gonorrhoeae In 1988, Gen-Probe marketed the PACE® System, using non-amplified probes for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae. This product was later followed by the PACE® 2 product line.Amplified assays for both Chlamydia and Neisseria followed in subsequent years, offered by several manufacturers. Automation of at least some parts of the process made it more feasible for clinical laboratories to incorporate molecular methods into their test menus.Roche developed a polymerase chain reaction (PCR) methodology focusing on specific nucleic acid sequences for both organisms. The Roche COBAS® AMPLICOR assay, on a semi-automated platform, obtained Food and Drug Administration (FDA) clearance in 1999. The Abbott LCx® semi-automated platform, based on ligase chain reaction, was also introduced around the same time. Shortly thereafter, Gen-Probe offered their APTIMA Combo 2® Assay, an amplification assay that utilized target capture. Later on, the TIGRIS® automated system by PROCLEIX® was added to provide automated specimen processing, enhancing the efficiency of the product line. Becton Dickenson then entered the arena, with the ProbeTec™, another semi-automated system based on strand displacement amplification.Digene also marketed its hybrid capture assay for Chlamydia and Neisseria. Unlike the other commercial assays, this method did not amplify the target DNA sequence, but instead employed a chemiluminescent methodology to amplify the signal of RNA:DNA hybrids. | View Page |
| Human Papilloma Virus (HPV) and Mycobacterium 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. | View Page |
| Hepatitis and Viral Load Testing Platforms for qualitative and quantitative viral testing, such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), or hepatitis C virus (HCV), were another key area of development. Roche launched its AMPLICOR® quantitative MONITOR assays for HIV and HCV outside of the United States in 1995. The HIV-1 assay was FDA approved in 1996; the HCV assay was not cleared until 2001. The HCV and HIV assays were cleared for use on the COBAS® AMPLICOR instrumentation in 2001 and 2005, respectively. In 2005, Roche launched its next generation platform, the AmpliPrep/COBAS® TaqMan®, outside the United States. This platform was designed for automated sample preparation, amplification, and quantitation of HIV, HBV, and HCV. FDA approval was obtained for HIV in 2007; HCV and HBV followed in 2008.Bayer Versant utilized branched chain DNA (bDNA) technologies for its assays. Their HIV viral load assay obtained FDA approval in 2002; both HBV and HCV received approval by 2003. Although by 2005, molecular diagnostics had begun to find its way into the routines of clinical diagnostic laboratories, for the most part, areas of testing were confined to specific applications that had been well developed and were available in FDA approved formats: sexually transmitted diseases, hepatitis testing, HIV monitoring, and in some labs, Mycobacterium detection and/or identification. Until this point, for other more easily maintained, cultivated, and identified pathogens, molecular methods, if available, had not found broad based acceptance and utilization. | View Page |
| Initially, why were molecular methods difficult to perform in routine clinical laboratories? (Choose all that apply.) | View Page |
| The Key Benefits: Reduced Turnaround Time Organisms that are either present in very low numbers, or that possess a characteristically slow growth rate, may require an extended incubation before they are detected in culture. Amplification and/or detection of unique sequences of either DNA or RNA provide for a more timely identification. This is true whether they are applied to the specimen for direct detection, or in some cases, to culture positive specimens for culture confirmation/identification.Even for organisms that are easy to recover and identify (example: Staphylococcus aureus) various molecular methods offer the ability for either direct detection in clinical material, or more rapid identification that would greatly aid in treatment and/or clinical management decisions. | View Page |
| Why can molecular methods offer improved turnaround times over cultivation methods? (Choose all that apply.) | View Page |
| Challenges for Implementation: Space Requirements Introducing molecular methods into a clinical microbiology laboratory is not a process without challenges. Some of these may include:Space requirements for both workflow and instrumentation Identifying separate areas for key aspects of the molecular process Familiarizing staff with new workflow requirements Instilling potentially new skill sets Cost of equipment and reagentsSpace requirementsIntroducing molecular methods will necessarily entail the introduction of new equipment. In many laboratories, space is at a premium; identifying space for new and additional equipment can be challenging. Some platforms are quite large; others have a significantly smaller footprint. The necessary separation of certain activities in the workflow will also impact space planning. | View Page |
| Challenges for Implementation: Cost 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. | View Page |
| Detection and Identification of Polymerase Chain Reaction (PCR) Products: Advantages of Real-Time PCR In real-time PCR, amplification and detection occur simultaneously, within the same reaction tube. Amplification occurs in the presence of a reporter: a probe specific for the target to be amplified, which is typically bound to some type of fluorogenic compound. Although the principles of real time reactions vary depending on the type of probe utilized, the basic premise is that the instrument platform combines a thermocycler with a fluorimeter, and utilizes light of specific wavelengths to excite the reporter molecules. If the target is present, probes bind to the target during each cycle of amplification. A fluorescent signal of increasing intensity is generated as the target is amplified, which is measured by the real time instrumentation. This signal intensity is directly proportional to the amount of amplified nucleic acid. When the signal exceeds a threshold, amplification of the target can be demonstrated.Once the sample is prepared and placed on the instrument, results are available in approximately 1 to 2 hours, depending on the assay and platform. In addition to reduced turnaround times, real time methodologies are more adaptable to automation and require significantly reduced handling of amplicons, and reduced risk of cross contamination. In some ways, the introduction of real time PCR accelerated the integration of molecular methods into the routine clinical diagnostic laboratory setting. Melt curve analysis Real time PCR methods can also specifically identify amplified products through melt curve analysis. The melting temperature of double stranded DNA depends on its base composition and length. All PCR products for a specific primer pair should have the same melting temperature, unless there is contamination, primer-dimer pairs, or some other problem. Real time instruments can be programmed to perform an analysis of the melting temperature of the PCR product. When hybridization probes are utilized, after the last PCR cycle, the samples are denatured, and then cooled to a temperature approximately 10o C below the expected melt temperature (Tm), as determined by previous testing of known samples. (This cooling protocol maximizes the formation of probe-target duplexes.) Then the temperature is incrementally raised while the fluorescence is continually monitored. At the melting point, the probe separates from the target strand, and the fluorescence rapidly decreases. The instrumentation software plots the rate of change in fluorescence with time on the Y axis, versus temperature on the X axis. The temperature at which the peak rate of change occurs is the Tm, and can be compared to known controls or established ranges to identify the PCR product. | View Page |
| Polymerase chain reaction (PCR) copies DNA through repeated cycles of three basic steps. What is the correct order of these steps? (Choose the BEST answer) | View Page |
| Detection and Identification of Methicillin-resistant Staphylococcus aureus (MRSA) by Polymerase Chain Reaction (PCR) MRSA presents both clinical and infection control challenges. Because of the increasing incidence of MRSA strains, empiric treatment for serious staph infections is usually vancomycin in the hospital setting. Although PNA-FISH can identify Staphyloccocus aureus more rapidly, it cannot yet differentiate MRSA from methicillin-susceptible S. aureus (MSSA) strains. Early differentiation of MRSA from non-MRSA strains could allow for adjustment from broad spectrum antimicrobial therapy, and reduced risk of development of resistance. Hospital acquired infections have garnered increasing attention from many quarters; MRSA is one of several drug resistant organisms that are of concern. Many institutions have implemented active surveillance culture (ASC) protocols to identify carriers of MRSA, both upon admission, and throughout the hospital stay. Identified carriers are placed under precaution protocols, to minimize risk of transmission to other patients during the hospital stay. MRSA status is also an important consideration for those patients who are being discharged to another facility (long term care or rehabilitation centers). Identifying carriers sooner rather than later can reduce the risk of transmission by earlier implementation of precaution protocols and reduce delays in discharge (and length of hospital stay) in situations where carrier status is needed prior to discharge. PCR methodologies offer the prospect of providing screening results 24 to 40 hours sooner than culture methodologies, depending on the culture medium employed. | View Page |
| References 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. | View Page |
| Prior Traditional Methods and the Need for Change Rapid detection of influenza was a key focus for method development for many years. Traditional viral culture methods require special transport mediums, appropriate cell culture lines, and staff well versed in the manipulation of these cultures. Although the introduction of shell vial cultures and monoclonal fluorescent staining provided some improvement, the availability of results did not always meet the clinical need.Direct fluorescent antibody (DFA) staining of specimen smears can provide more immediately available results; however the availability of trained staff to interpret these smears is an obstacle for many laboratories. Antigen detection kits employing enzyme immunoassay (EIA) or immunochromatographic membrane principles did provide easily performed alternatives that fit well in most laboratory settings and provided more immediate results. Despite the fact that published studies demonstrated less than desirable sensitivity, these assays had found a niche and remained in place, even as molecular methods began to target these viruses. | View Page |
| Clinical Significance 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. | View Page |
| Molecular Methods A 2009 evaluation and comparison of a variety of commercially available toxin detection assays, glutamate dehydrogenase (GDH) assays, the cytotoxin assay, cytotoxigenic culture, and real time PCR for the C. difficile tcdB gene revealed that ALL methods demonstrated a relatively low positive predictive value, which compromised the utility of a single test for laboratory diagnosis of C. difficile. However, of all methods, PCR had the highest negative predictive value, and was considered the optimum rapid single test.Molecular methods for C. difficile are based on the detection of the tcd gene. With the application of real time methodology, results can be available within 2 to 3 hours. These methods are highly sensitive and demonstrate good sensitivity, in comparison to all methods with the exception of toxigenic culture. As the methodologies and instrumentation are developed and improved, they are increasingly adaptable to the environment of a busy clinical diagnostic setting. The BD GeneOhm™ and Meridian illumigene® assays are examples of currently available molecular assays for C. difficile. | View Page |
| Several methods of detection are available for the detection of Clostridium difficile in clinical samples. Which methods have the capability for detection in less than 48 hours? (Choose all that apply.) | View Page |
| Clinical significance of Staphylococcus aureus 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. | View Page |
| Healthcare (Hospital)-Associated MRSA versus Community-Associated MRSA As mentioned in the course introduction, MRSA infections fall into two general types: Healthcare-associated MRSA (HA-MRSA) Infections that occur in people who are, or have recently been, hospitalized. Community-acquired MRSA (CA-MRSA) Infections that are apparently acquired in the community There are a number of factors that distinguish HA-MRSA from CA-MRSA isolates. These factors are summarized in the table below. Factor HA-MRSA CA-MRSA Origin of strains Nosocomial infections Five isolates associated with healthcare settings: USA100, -200, -500, -600, -800 USA100 is the predominant isolate while USA 200 is the second most common isolate. USA700 has been isolated in both healthcare and community settings. Evolved from endemic methicillin susceptible S. aureus (MSSA) strains Two clones, USA300 and USA400, are associated with the majority of CA-MRSA infections in the United States. USA300 has emerged as the most prominent clone and is not found among hospital strains. Genetic lineage Isolates usually carry large SCCmec types I, II or III (34-67 kb) The larger size of SCCmecII and III permits the inclusion of other non-beta lactam resistance genes so that HA-MRSA strains tend to be multi-drug resistant Isolates carry a smaller SCCmec variant, predominantly type IV (24 kb), less often type V or variant VT. SCCmecIV (except for mecA) does not permit the inclusion of other non-beta lactam resistance genes so that CA-MRSA isolates exhibit resistance to only methicillin and erythromycin and are more often susceptible to other non-beta lactam antibiotics (eg., trimethoprim/sulfamethoxazole (SXT) and clindamycin). Affected population Largely affects older adults and people with weakened immune systems; those who have undergone surgical procedures are at increased risk. Healthy persons in the general population without established risk factors for MRSA acquisition Clinical syndromes Found at multiple sites, most commonly bloodstream infections, urinary tract infections (UTI) and respiratory tract infections Predominantly skin and soft tissue infections (SSTIs), such as abscesses, cellulitis, folliculitis and impetigo and a serious form of pneumonia Genes for Panton-Valentine Leukocidin (PVL) are associated with SCCmecIV; the clinical spectrum of infections caused by CA-MRSA is directly related to the presence of PVL genes, coding for the production of a cytotoxin that causes tissue necrosis and leukocyte destruction. | View Page |
| Laboratory Identification of Staphylococcus aureus 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. | View Page |
| Future Perspectives (continued) Important epidemiological and microbiological differences exist between CA- and HA-MRSA strains so strategies to prevent and treat these infections should also differ. To prevent clinical complications from CA-MRSA, it is recommended that culturing and susceptibility testing of S. aureus clinical isolates become routine practice along with more careful selection and use of antimicrobials when treatment is indicated. MRSA isolates are NOT susceptible to beta-lactam antibiotics, however CA-MRSA infections are susceptible to some currently available non- beta-lactam antibiotics such as clindamycin and trimethoprim/sulfamethoxazole. | View Page |
| Future Perspectives (continued) 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. | View Page |
| Laboratory Detection of Clostridium difficile Several laboratory methods are currently available to aid in the detection of C. difficile including culture for toxigenic C. difficile (considered the "gold standard" for viable C. difficile detection), detection of Toxin A, B, or both, and molecular detection methods. These methods differ in their sensitivity and specificity and should always be used in conjunction with clinical considerations. To make the diagnosis, it is usually only necessary to submit 1-2 diarrheic (non-formed) stools per episode. Once positive for C. difficile by any laboratory method, there is no need for follow-up assays to make sure the organism or toxins are absent from the initial episode. If assays are performed for subsequent episodes, culture or tissue culture assay for Toxin B are probably most appropriate to avoid the possibility of detecting the initial antigen, toxin, or gene. | View Page |
| Clostridium Species 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 | View Page |
| References Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing, Twentieth Informational Supplement. CLSI document M100-S20. CLSI. Wayne, PA: 2010.Fenner L, Widmer AF, Goy G, Rudin S, Frei R . Rapid and reliable diagnostic algorithm for detection of Clostridium difficile. JCM, 2008; 46(1): 328-330.Forbes BA, Sahm DF, Weissfeld AS, eds.Bailey & Scott's Diagnostic Microbiology. 11th ed. Mosby; 2002.Gillespie SH, Hawkey PM, eds. Principles and Practice of Clinical Bacteriology. 2nd ed. West Sussex, England: John Wiley & Sons Ltd; 2006.Isenberg HD. Clinical Microbiology Procedures Handbook. 2nd ed. Washington, DC: ASM Press; 2004.MDRO Guidelines. CDC website. Available at: http://www.cdc.gov/ncidod/dhqp/pdf/ar/MDROguideline2006.pdf. Accessed July 9, 2010.Vancomycin Resistant Enterococci and the Clinical Laboratory. CDC website. Available at: http://www.cdc.gov/ncidod/dbmd/diseaseinfo/drugresisstreppneum_t.htm Accessed July 9, 2010. | View Page |
| C. difficile Toxin A and Toxin B 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. | View Page |
| Pathogenisis of C. Difficile-Associated Diarrhea Clostridium difficile is the leading cause of hospital-acquired diarrhea in the United States, with the number of cases rising annually over the last three decades. This is largely due to the increased frequency of antibiotic usage, the development of better detection methods, and the fact that hospital environments are increasingly contaminated with spores of C. difficile. The definition of C. difficile diarrhea includes > 6 episodes of non-formed diarrheic stool per 24 hours, along with prior antibiotic treatment. At least three events must occur in the pathogenesis of C. difficile-associated diarrhea (CDAD): Alteration of the normal fecal flora Colonic colonization with toxigenic C. difficile Growth of the organism with elaboration of its toxins"Colonization resistance" is the term used to describe the mechanism by which indigenous flora control overgrowth of C. difficile. This resistance may be compromised by the use of antimicrobial compounds, underlying illness, or therapeutic procedures. Infection begins with the ingestion of either the organism itself or spores, usually via the fecal-oral route. Spores in particular are able to survive the acidity of the stomach and germinate in the colon to produce vegetative organisms. Toxinogenic strains subsequently produce Toxin A, Toxin B, and/or the Binary Toxin leading to colitis, pseudomembrane formation, and watery diarrhea. Significant complications of the clinical disease associated with infection are hypoalbuminemia, toxic megacolon (acute toxic colitis with dilatation of colon), and pseudomembranous colitis (PMC). | View Page |
| Strain BI/NAP1/027 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. | View Page |
| Identification of Enterococcus species from clinical cultures Gram stain: gram-positive cocci in singles, pairs, or chains; cells can be ovoid to coccobacillaryColony morphology: on blood agar after 24 hours of incubation, colonies are nonhemolytic or alpha hemolytic (rare strains may be beta hemolytic), and approximately 1 to 2 mm in diameter.Catalase: negativePresumptive identification: Growth on bile esculin agar and in 6.5% salt broth are two characteristics that have commonly been used to identify Enterococcus to the genus level. A positive esculin in combination with a positive PYR reaction is another approach to presumptive identification.Species identification: E. faecalis and E. faecium are usually easily identified by most commercial systems. Successful identification of the other species on these systems may vary. With respect to vancomycin intermediate or resistant strains, two key characteristics are motility and pigment. E. casseliflavus is both motile and possesses a yellow pigment; E. gallinarum is also motile but non-pigmented. E. faecalis and E. faecium demonstrate neither characteristic. | View Page |
| HLR screening 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. | View Page |
| Susceptibility Testing When a clinical isolate is presumptively identified as S. aureus, susceptibility testing will be performed by either the Standardized Disk Diffusion (Kirby-Bauer) or Broth Dilution (MIC) methods, using the following testing conditions as recommended by the Clinical and Laboratory Standards Institute (CLSI): Medium: MHA for disk diffusion; CAMHB + 2% NaCL for oxacillin, methicillin, and nafcillin; CAMHB supplemented up to 50 ug/ml calcium for daptomycin Inoculum: Direct colony suspension (0.5 McFarland Standard) Incubation: 35° C (Testing at temperatures above 35° C may not detect MRSA); ambient air; disk diffusion; 16to 18 hours; dilution methods; 16 to 20 hours. All methods: 24 hrs for oxacillin, methicillin, nafcillin, and vancomycin. | View Page |
| Basic Pharmacokinetics In order to discuss TDM and PGx we need to also introduce the concept of pharmacokinetics. Pharmacokinetics is the study of drug disposition in the body: how and when drugs enter the circulation, how long they remain in the blood, and how they are eliminated. TDM is the clinical assessment of a drug's pharmacokinetic properties. Physicians and pharmacists need to establish that a drug is present at an effective concentration but not at a toxic concentration. The next few pages will describe some of the factors that determine a drug's disposition in the body. These factors ultimately decide the need for therapeutic drug monitoring. | View Page |
| Why TDM? Pharmacologists determine a drug's pharmacokinetic characteristics empirically during clinical drug trials. From these studies, they are able to determine the solubility and distribution, the average half-life, the levels of protein binding, and the effective concentrations needed for treatment. | View Page |
| Laboratory Methods Immunoassay is the most common technique used by clinical laboratories for therapeutic drug monitoring. Antibodies that recognize drugs can be developed. Although most drugs are much too small to evoke an immune response, scientists can conjugate drugs to immunogenic proteins to produce antibodies that recognize drug-specific epitopes. There are several methods that utilize the principals of immunoassay for detection and quantification of therapeutic drugs in serum. Some of these methods are: Particle-enhanced turbidimetric inhibition immunoassay (PETINIA) Fluorescence Polarization Immunoassay (FPIA) Chemiluminescent assays | View Page |
| Therapeutic Drug Monitoring Definition Therapeutic Drug Monitoring (TDM) is a branch of clinical chemistry that specializes in the measurement of medication levels in serum. TDM requires quantitative measurements of drugs and/or their metabolites. | View Page |
| Enzyme Abnormalities and Drugs The following is a list of enzymes for which known mutations have been associated with clinical effects. Enzymes Substrates (Drugs) Acetylaldehyde dehydrogenase Alcohol Acetylcholinesterase Succinylcholine Alcohol dehydrogenase Alcohol Dihydropyrimidine dehydrogenase Fluorouracil CYP2C9 Warfarin, phenytoin, losartan CYP2C19 Diazepam, omeprazole (Prilosec) CYP2D6 Many antidepressants, opioids, antiarrhythmics Glucose-6-phosphate dehydrogenase Aspirin, quinidine N-acetyltransferase Procainamide, isoniazid Thioprine methyltransferase 6-mercaptopurine UDP-glucuronosyl transferase Acetaminophen, tolbutamide, irinotecan | View Page |
| Clinical Utility The ultimate goal in measuring CYP450 function or identifying polymorphisms is to predict effective therapeutic doses and responses in patients.Polymorphisms are identified using molecular techniques (allele-specific PCR, restriction digests, sequencing, hybridization assays, bead-based systems, microarrays, pyrosequencing, et al).Although most clinical labs do not offer PGx testing, reference labs are beginning to market these tests. For example, one reference laboratory in the Midwest that offers CYP2D6 profiling measures about one dozen of the most common and significant mutation sites on this enzyme. This allows for detection of approximately 98% of the known CYP2D6 polymorphisms. The laboratory then generates a report which will advise the physician on the patient's drug-metabolizing status.Estimates show that 6-10% of the general population have a complete deficiency of CYP2D6, with the prevalence of mutations varying from <1% to as much as 21% within a given population. | View Page |
| Metabolizers When discussing PGx, we classify a person according to his/her phenotype (metabolic capacity for a given enzyme).A poor metabolizer (PM) is a person who lacks the functional enzyme and therefore exhibits decreased metabolism of drugs. This person would require lower doses of a drug that is metabolized by that enzyme. A PM who receives a standard dose is more likely to experience unwanted side effects or toxicity. A PM can also experience diminished effects with drugs that need to be metabolized to active compounds by the enzyme in question.An ultrarapid metabolizer (UM) will require a higher dose than usual since he/she will eliminate the drug more quickly. A UM may be resistant to standard treatments, and it may take some time to adjust the dosage before therapy is achieved.An intermediate metabolizer (IM) has one wild-type (normal) copy of the gene and one absent or dysfunctional copy. The IM group is very heterogeneous.A person with normal enzyme activity is referred to as an extensive metabolizer (EM). This person should respond to standard dosages of a drug. Most people are EM's. This is the population in which most dosing regimens have been worked out in clinical trials. | View Page |
| The Bottom Line By knowing a patient's disposition to specific drugs, the physician should be able to start the patient on an appropriate regimen rather than perfecting treatment based on trial and error. Drugs whose metabolism may prove to be problematic can be avoided, and second-line therapies that are metabolized by different, unaffected enzymes can be chosen. Clinical chemists, pharmacologists, and physicians need to translate knowledge of CYP450 polymorphisms into clinically-validated treatment algorithms. Dosing recommendations for PM, EM, IM and UM patients are beginning to appear in the literature for various classes of drugs, and the FDA is encouraging the incorporation of pharmacogenomic testing in the development process for new drugs. | View Page |
| CYP450 Induction and Inhibition CYP450 Inhibitor Inducer CYP1A2 Amiodarone Cimetidine Ciprofloxacin Tobacco CYP2C9 Amiodarone Fluvastatin Isoniazid Fluconazole Rifampin Secobarbital CYP2C19 Cimetidine Indomethacin Ketokonazole Prednisone CYP2D6 Celecoxib Cimetidine Cocaine Methadone Pentazocine Imipramine Desipramine Amitriptyline CYP2E1 Disulfiram Fluoxetine Ethanol Isoniazid CYP3A Midazolam Erythromycin Methadone Phenobarbital Dexamethasone Note: This is not an exhaustive listing of inducers and inhibitors.Reference: Tanaka E, Terada M, Misawa S. Cytochrome P450 2E1: it's clinical and toxicological role. J Clin Pharm Ther. 2000 Jun;25(3):165-75. | View Page |
| Genotype versus Phenotype Phenotyping involves measuring the metabolism of a probe drug. For example, with CYP2D6, dextromethorphan or debrisoquine can be given to a patient to see how well the drug is metabolized. Both these drugs are safe and extensively metabolized by CYP2D6. By measuring the parent drug and the metabolite in urine, the metabolic capacity of a CYP450 enzyme can be estimated. Such testing is complex and tedious, however, and has not become routine in clinical laboratories. Therefore, genotyping is likely to be the main tool that is used for assessing the PGx of a patient. | View Page |
| References Clinical Chemistry: Theory, Analysis, Correlation, 4th Edition. Lawrence A. Kaplan, Amadeo Pesce, Steven Kazmierczak. New York: Mosby, 2002.FDA Clears Genetic Lab Test for Warfarin Sensitivity. FDA News. U.S. Food and Drug Administration. Available at http://www.fda.gov/bbs/topics/NEWS/2007/NEW01701.html. Accessed June 3, 2008.Goodman & Gilman's The Pharmacological Basis of Therapeutics, 11th Edition. Laurence Brunton, John Lazo, Keith Parker. McGraw-Hill, 2005.Tanaka E, Terada M, Misawa S. Cytochrome P450 2E1: it's clinical and toxicological role. J Clin Pharm Ther. 2000 Jun;25(3):165-75.The Chemistry of Mind-Altering Drugs: History, Pharmacology, and Cultural Context. Daniel Perrine, American Chemical Society Publication, 1996.Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 4th Edition. Carl A. Burtis and Edward R. Ashwood, eds. Philadelphia: WB Saunders, 2005. | View Page |
| Bacillus anthracis 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! | View Page |
| Yersinia pestis Clinical specimens where organism may be encountered: Blood Lymph node aspirate Respiratory secretionsGram stain morphology: Gram-negative rod Resembles other Enterobacteriaceae Can form short chains Gram stains performed from blood culture or other liquid media may show bipolar staining (displayed by the arrows)Note: Use of Wright-Giemsa staining on direct specimen may enhance demonstration of characteristic bipolar staining, also referred to as "safety-pin" morphology. Use of this staining is of limited value, as the method is not very sensitive or specific. | View Page |
| Francisella tularensis 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 | View Page |
| Brucella species Clinical specimens where organism may be encountered: Blood Bone marrow TissueGram stain morphology: Very small, gram-negative coccobacilli Stains very faintly and tends to retain crystal violet, especially in blood cultures May initially be identified as gram-positive Organism is larger than F.tularensis Individual cells are evident | View Page |
| Burkholderia species 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 | View Page |
| Gram stains are performed on positive blood culture bottles. Match the organism that MOST closely resembles the description of the Gram stain morphology provided. | View Page |
| Toxins 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 | View Page |
| Role of a Sentinel Laboratory Regarding Toxins and Viruses Always follow your facilities procedures and contact your Laboratory Response Network (LRN) reference laboratory for guidance with regard to the toxins and viruses that may be used as bioterrorism agents. A sentinel laboratory should NOT attempt to: Accept or process environmental or animal samples Culture from clinical specimens or detect for these agents Collect specimens for suspect viruses unless directed by a public health officialThe laboratory testing listed on the following pages is intended ONLY as information. For suspect specimen collection, contact your LRN reference laboratory for guidance before collecting or referring specimens. | View Page |
| A sentinel laboratory should NOT accept orprocess environmental or animal samples or culture clinical specimens for viruses or toxins that are potential agents of bioterrorism. | View Page |
| References American Society for Microbiology. Sentinel Level Clinical Microbiology Laboratory Guidelines. Available at: http://www.asm.org/index.php/guidelines/sentinel-guidelines. Accessed on April 12, 2013.Association of Public Health Laboratories web site. LRN Sentinel Laboratories Clinical. Available at: http://www.aphl.org/aphlprograms/preparedness-and-response/Documents/LRN_Sentinel_Clinical.pdf. Accessed on October 29, 2012.Centers for Disease Control and Prevention. Laboratory Information for Bioterrorism Emergencies. Available at: http://emergency.cdc.gov/bioterrorism/lab.asp. Accessed August 16, 2011.Centers for Disease Control and Prevention. Laboratory Network for Biological Terrorism. Available at: http://emergency.cdc.gov/lrn/biological.asp. Accessed August 16, 2011.Klietmann WF, Ruoff KL. Clincal Microbiology Reviews: Bioterrorism: Implications for the Clinical Microbiologist. American Society for Microbiology; April 2001 p. 364-381, Vol. 14, No. 2. Available at: http://cmr.asm.org/cgi/content/full/14/2/364. Accessed August 16, 2011.Snyder JW. Role of the Hospital-Based Microbiology Laboratory in Preparation for and Response to a Bioterrorism Event. J Clin Microbiol; 2003 41(1): 1–4. Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC149646/. Accessed August 16, 2011. Winn WC Jr, Allen SD, Janda WM, Koneman EW, Procop G, Schreckenberger PC, Woods G. Koneman's Color Atlas and Textbook of Diagnostic Microbiology. 6th ed. Philadelphia, PA: Lippincott Williams and Wilkins; 2006. | View Page |
| References Glassy, Eric F.,(Ed). Color Atlas of Hematology: An Illustrated Field Guide Based on Proficiency Testing. 1998. College of American Pathologists Hematology and Cliical Microbiology Research Committee. College of American Pathologists, Northfield, IL 60093-2750.Hookey,L., Dexter, D., Lee,D. H. The Use and Interpretation Of Quantative Terminology In Reporting Red Blood Cell Morphology. Laboratory Hematology 7:85-88, 2001.Peterson P, Blomberg DJ, Rabinovitch A, Cornbleet PJ. Physician Review of the Peripheral Blood Smear: When and Why. For the Hematology and Clinical Microscopy Resource Committee of the College of American Pathologists. Laboratory Hematology 7:175-179, 2001 | View Page |
| An isolated acanthocyte is most likely of little importance on an otherwise normochromic, normocytic peripheral blood smear. | View Page |
| A blood smear represented by the photograph was submitted for hematologic review. Based on the erythrocyte morphology and the accompanying histogram, which of the following choices is the most likely situation or condition? | View Page |
| Stomatocytes Stomatocytes are erythrocytes with a slit-like central pallor, given them the appearance of "coffee beans" or "kissing lips". In three dimensions, the stomatocyte is actually the shape of a bowl, as the cell has lost its biconcave morphology due to a membrane defect. Most cases of stomatocytosis are due to alteration in permeability, leading to an increase in red cell volume. Stomatocytes form at a low blood acidic pH as seen in exposure to cationic detergents, and in patients receiving phenolthiazine or chlorpromazine. Stomatocytosis can be an inherited or acquired condition.In hereditary stomatocytosis, mild anemia and findings of on-going hemolysis may be evident if the condition presents as a clinical problem at all. Individuals who possess the Rh null phenotype have osmotically fragile red cells, which take the form of stomatocytes. Individuals with this phenotype tend to experience varying degrees of chronic hemolytic anemia. Note: Unless 10% or more of the RBC's are stomatocytes, their presence is probably artifactual. | View Page |
| Conditions suggested by the macrocytes and the neutrophil in the photograph to the right include which of the following? | View Page |
| Introduction to Red Blood Cell Morphology Reporting After an automated complete blood count (CBC) analysis has determined that abnormal RBC morphology may be present, a well-made and well-stained peripheral blood smear should be prepared. When a peripheral blood smear is made for the purpose of evaluating RBC morphology, accurate recognition and identification of RBC morphologic abnormalities can be an invaluable aid in the diagnosis of a variety of disorders. It is important to understand that red blood cell morphology report formats tend to vary widely among laboratories. Despite the standardization of many laboratory technologies and test result formats, there are still various protocols in use in the area of red cell morphology reporting. Current methods of reporting and quantifying red cell morphology include descriptive terms such as 'rare,' 'occasional,' 'many,' 'slight,' or 'moderate,' as well as numerical gradings of 1+, 2+, 3+, etc. Regardless of the terminology used, consistency is of greater importance. There must be a defined, semi-quantitative scheme that dictates how many cells with a specific morphologic abnormality qualify as "rare" or "many," and so on. The report format must be clear and useful to the physician. Some morphologic abnormalities are quite specific and diagnostic, but others are ambiguous and of little diagnostic significance.A well-defined, semi-quantitative report format for RBC morphology should be based on clinical significance. Some morphologic abnormalities are significant, even when they occur in very low numbers. These include:SchistocytesSickle cellsAcanthocytesSpherocytesTeardrop cellsPolychromatophilic cells Other morphologic abnormalities are significant only when seen in considerable numbers. These include:MacrocytesMicrocytesOvalocytesBurr cells (echinocytes)Target cellsStomatocytesHypochromic cells A final category includes morphologic abnormalities that do not need to be quantified as it serves no purpose; these findings can be noted as "present." These include RBC agglutinationRouleauxDimorphic or double red cell populationAn example of a standardized reporting format is given on the following page. | View Page |
| Dimorphic (Double Cell) Population 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. | View Page |
| Clinical Relevance of D Phenotypes 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 | View Page |
| Newborn's Clinical Status The newborn showed no clinical evidence of HDFN or early newborn hyperbilirubinemia, with related laboratory tests as follows (laboratory's reference ranges for newborns in brackets): Test USA SI Hemoglobin 16 g/dL (13.5 - 21.0 g/dL) 160 g/L(130.5 - 226.0 g/L)* Hematocrit 52% (43-62%) 0.52 (0.43-0.62) Total bilirubin (cord blood) 2.1 mg/dL (<2.5 mg/dL) 35.9 µmol/L (<43 µmol/L) *Most countries that adopted SI do not use the official SI unit for Hb (mmol/L), but rather use g/L. | View Page |
| Routine Serologic Tests - Newborn Protocols 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. | View Page |
| A group A Rh positive mother is about to deliver her infant. Is it acceptable good practice not to test the newborn (ABO, Rh, DAT)?Answer Y (for yes) and N (for no) | View Page |
| Main Learning Goals 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. | View Page |
| Literature and Online Resources 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. *Wagle S, Deshpande PG. Hemolytic disease of the newborn. eMedicine / WebMD. Updated Apr. 9, 2010. | View Page |
| Federal Regulations for Risk Management 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 | View Page |
| References and Recommended Reading Astion ML, Shojania KG, Hamill TR, Kim S, Ng VL. Classifying laboratory incident reports to identify problems that jeopardize patient safety. Am J Clin Path. 2003;120:18.Bersch C, Clemons K. The wheel of misfortune prepare to win if disaster strikes. MLO. 2008;40:12. Berte L. Laboratory quality management: A road map. Clin Lab Med. 2007;27:771.Bonini P, Plebani M, Ceriotti F, Rubboli F. Errors in laboratory medicine. Clin Chem. 2002;48:691-698. Available at http://www.clinchem.org/cgi/content/full/48/5/691#T2B Accessed February 2, 2012.Carraro P, Plebani M. Errors in a stat laboratory: Types and frequencies 10 years later. Clin Chem. 2007;53:1338-1342.Carroll R ed. Risk Management Handbook for Health Care Organizations. 5th ed. San Francisco: Jossey-Bass; 2006.Howanitz PJ. Errors in laboratory medicine: Practical lessons to improve patient safety. Arch Pathol Lab Med. 2005; 129:1252 - 1261.Kalra J. Medical errors: Impact on clinical laboratories and other critical areas. Clin Biochem. 2004;37:1052-1062. Kohn LT, Corrigan JM, Donaldson MS, eds. To Err Is Human: Building a Safer Health System. Washington, DC: National Academies Press; 2000.Lippi G, Guidi G. Risk management in the preanalytical phase of laboratory testing. Clin Chem & Lab Med. 2007;45:720. Malone B. Risk management for clinical labs. Clinical Laboratory News. 2008;34:1.Pierangelo B, Plebani M, Ceriotti F, Rubboli F. Errors in laboratory medicine. Clin Chem. 2002;48:691-698.Sazama K. Legal implications of laboratory errors. Lab Med. 2005;36:213.Smith TJ. Strategies for error reduction, Advance Magazine. 2009;18:25.Vidal Y. 101 Ways to Prevent Medical Errors: A 24-year Odyssey. Andrews, TX: Lara Publishing; 2002. | View Page |
| The Fishbone Diagram 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. | View Page |
| Which of these methods should be used when an adverse event occurs that results in patient injury (negative patient outcomes) to prevent a recurrence? | View Page |
| Risk Management Accrediting Organizations: Joint Commission 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. | View Page |
| Risk Management Accrediting Organizations: College of American Pathologists The College of American Pathologists (CAP) is a private, non-profit accrediting organization that has been granted "deeming" authority by CMS. The goal of the CAP laboratory accreditation program is to "Improve patient safety by advancing the quality of pathology and laboratory services through education, standard setting, and ensuring laboratories meet or exceed regulatory requirements." Though CAP inspects only clinical laboratories, there are various types such as: reference laboratories, clinics, military installations, hospital laboratories, etc. which may be accredited through this organization.Surveys are unannounced and the program uses volunteer teams of practicing laboratory professionals as inspectors, or CAP inspection specialists. Inspection specialists are CAP employees who have extensive experience in laboratory medicine. If a hospital is inspected by the Joint Commission and the laboratory chooses to seek CAP accreditation, the CAP inspection is recognized by the Joint Commission so that the laboratory will not be inspected again by the Joint Commission during the hospital inspection. In the fall of 2008 CAP launched a laboratory accreditation program based on the International Organization for Standardization (ISO) 15189:2007. This program utilizes criteria and procedures specifically developed to determine technical competence and account for quality management systems. It does not replace the CAP Laboratory Accreditation Program but is an addition to it. Participation is strictly voluntary and there is no tie to CMS for Medicare reimbursement.The table to the right depicts factors involved in determining technical competence. | View Page |
| Transfusion Service Laboratory 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. | View Page |
| Using p values in medical research Let's briefly review using p values in medical research. A simple example would be a randomized clinical trial to assess whether a new drug decreases levels of low-density lipoprotein (LDL) more than an established drug. Data are collected from subjects treated with new Drug A and established Drug B. Let's suppose that the mean LDL of Drug A is lower than that of Drug B. We want to know whether the difference is due to an effect of Drug A or if the difference is due to chance. There is no way we can ever be certain whether the observed difference reflects a true difference (Drug A is more effective in lowering LDL) or is just a coincidence of random sampling. All we can do is calculate probabilities (the p value) based on a null hypothesis. A null hypothesis states that there is no difference between the drugs. The p value is the probability of observing a difference as large or larger than was observed in the study, if the null hypothesis of no difference were true. | View Page |
| The p value in this case 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. | View Page |
| Balancing the risks Life-Threatening HemorrhageDespite potential risk, sometimes immediate transfusion is necessary, even for patients with red cell antibodies. In such cases transfusion service staff should alert the medical director, who can discuss options with clinical staff.The medical director will generally talk to the staff attending the patient and indicate that, if possible, they should hold off transfusion. But if it is a case of massive bleeding where exsanguinating hemorrhage is likely, it is better to give some blood and monitor for a delayed hemolytic transfusion reaction than to let the patient bleed to death.Transfusing when bleeding is brisk will result in much of the autologous and incompatible blood bleeding out, with the possibility of a delayed hemolytic reaction once the patient's antibody rebounds and destroys still present antigen-positive donor red cells.Some transfusion services also try to minimize the risk of unmatched blood by typing their emergency supply of O Rh negative RBCs for the K antigen, since anti-K is a relatively common clinically significant antibody. See Resources for two papers that discuss the risks of transfusing un-crossmatched emergency blood. | View Page |
| Think about your responses to each of the following questions, then click on the questions. | View Page |
| Literature and online resources LiteratureDutton RP, Shih D, Edelman BB, Hess J, Scalea TM. [abstract]. Available at: Safety of uncrossmatched type-O red cells for resuscitation from hemorrhagic shock.J Trauma. 2005 Dec;59(6):1445-9. Accessed November 5, 2012.Johnson ST, Rudmann SV,Wilson, SM. Serologic problem solving strategies:a systematic approach. Bethesda, MD: AABB, 1996.Online resourcesThe following are online examples of good practice. The information 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.Transfusion reactions: Transfusion complications (Canadian Blood Services)Education website for CBS's hospital customersREACT (Sunnybrook HSC, Toronto, ON, Canada) Pocket reference card for nurseson signs and symptoms of transfusion reactionsQuick cals (online calculator of p values for Fisher's exact test) Use a one-tailed test (since we would expect an antibody to react with red cells that are positive for the corresponding antigen) | View Page |
| The antibody screen is positive but the transfusion of the O Rh-negative RBCs is already in progress. What are the transfusion service (TS) laboratory's priorities in this case?Place the following procedures that will be followed by the TS in the appropriate order of priority. | View Page |
| Follow-up with clinical staff The patient's physician was notified that compatible blood was unavailable and that the patient's antibody was still being investigated.When asked whether or not the patient was experiencing a transfusion reaction due to the transfusion of the two unmatched and incompatible O Rh negative RBC, the nurse in the OR stated that the patient was undergoing surgery and completely sedated. A transfusion reaction was not apparent but they would investigate and closely monitor.Hemolytic Transfusion Reactions (HTR)Before proceeding to the next page, make a short list of signs and symptoms associated with immediate hemolytic transfusions reaction and another list associated with delayed hemolytic transfusion reactions. | View Page |
| Signs and symptoms - Job Aids 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). | View Page |
| Clinical Signs and Symptoms 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. | View Page |
| Clinical Presentation and Laboratory Findings Symptoms begin within 6 hours of transfusion and include acute respiratory distress, severe hypoxemia, hypotension, fever and bilateral fluffy infiltrates on chest radiograph. Respiratory distress is due to noncardiongenic pulmonary edema. Patient may have shortness of breath. Signs and symptoms may be mild, and resolve after a few days, or they may be severe and result in pulmonary failure. Laboratory findings include leukopenia and hypocomplementemia. | View Page |
| Diagnosis, Treatment, and Prognosis 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. | View Page |
| Evaluation of Donors Associated with Transfusion-Related Acute Lung Injury (TRALI) The AABB published an interim standard in 2005 that states, "Donors implicated in TRALI or associated with multiple events of TRALI shall be evaluated regarding their continued eligibility to donate." A donor is associated with TRALI when one of his/her donor units is transfused 6 hours before the clinical presentation of TRALI in a patient. A donor is implicated in TRALI if he/she is found to have an antibody to an HLA class I or II antigen and the antibody is specific for an antigen on the recipient's leukocytes or a positive crossmatch is obtained.*It is suggested that donors at greatest risk of developing HLA antibodies be tested, such as multiparous women. It has also been suggested that donors that present with demonstrable antibodies and have been implicated in TRALI be permanently deferred from donating. Studies have shown that donors implicated in TRALI reactions may present a future danger to transfusion recipients. Although, there are some instances where donors with HLA antibodies have not caused TRALI reactions. Another option would be to wash all red cell products from these donors in special circumstances such as rare donors. Reference: Association bulletin #05-09. AABB; August 2005. Available at: http://www.aabb.org/resources/publications/bulletins/Pages/ab05-09.aspx. Accessed November 12, 2010. | View Page |
| Definition and Incidence 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. | View Page |
| Diagnosis The symptom most commonly associated with a delayed hemolytic transfusion reaction (DHTR) is unexplained decrease in hemoglobin and hematocrit. Patients may also present with fever and jaundice. Hemolysis occurs slowly and is primarily extravascular. Unlike an acute hemolytic transfusion reaction (AHTR), hemoglobinuria, acute renal failure, and disseminated intravascular coagulation (DIC) are not generally seen. On some occasions, patient's may not present with any symptoms. Serologic findings include a positive direct antiglobulin test (DAT) and/or a positive antibody screen in post-transfusion testing. In many cases, the physician will send a request for an additional transfusion because of the decreased hemoglobin levels, and not suspect a DHTR. The positive antibody screen will trigger an investigation including antibody identification. The DAT may have a mixed field appearance because of the antibody-sensitized transfused red cells and the non-sensitized patient red cells. Segments from the donor unit can be tested for the offending antigen once the antibody has been identified.Antibodies that are most often reported as the cause of DHTR are anti-Jka and anti- Jkb. Other antibodies that are also commonly implicated in a DHTR include Kell, Rh, and Duffy system antibodies.The patient's physician should be notified so that additional clinical and laboratory evidence can be evaluated. | View Page |
| Severe Delayed Hemolytic Transfusion Reactions (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. | View Page |
| Clinical Presentation and Diagnosis Patients present with fever, a characteristic red rash from trunk or face to the extremities, watery diarrhea, nausea, vomiting, and hepatitis within seven to ten days following the transfusion. The rash may progress to blister-like lesions and erythroderma. Pancytopenia will develop due to the immune destruction of the recipient's bone marrow. The low platelet count causes hemorrhaging while a low white blood cell count can lead to infection. Most patients die within one to three weeks after the onset of symptoms. The diagnosis is often missed and is usually made too late or after death. Routine laboratory studies are not helpful. The only definitive method is the identification of donor lymphocytes in the circulation or tissues of the recipient which is accomplished through human leukocyte angtien (HLA) typing or cytogenic analysis. | View Page |
| Match the letters representing the peripheral white blood cells with the most likely clinical conditions in which the cell would be present in increased numbers. | View Page |
| Assume that several other lymphocytes similar to the one in the center of the photograph, are found on review of the peripheral smear. A work up for leukemia should be recommended. | View Page |
| Smudge Cells Smudge cells are remnants of cells that lack any identifiable cytoplasmic membrane or nuclear structure. Smudge cells, also called basket cells, are most often associated with abnormally fragile lymphocytes in disorders such as chronic lymphocytic leukemia (CLL). However, they can also be seen in degenerating samples; in which case, their origin may not be lymphocytic. Smudge cells are indicated by the arrows in this image. In some laboratories, a semi-quantitative estimate of the number of smudge cells may be made; in others, a report of "smudge cells present" may suffice. This reporting scheme must be understood by the physician in order to maximize patient care outcomes through his/her decision making process. For example, in the context of this exercise, does it make any difference to the physician if you report few or many smudge cells; or, is a report of smudge cells present sufficient? The answer to this question applies not only to smudge cells, but to the reporting of any other atypical white cells as well. An agreement must be reached between the hematology laboratory and clinical services as to how semi-quantitative estimates will impact the need for further testing in view of patient care outcomes. | View Page |
| Evaluation Criteria: White Blood Cells and Platelets 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. | View Page |
| A representative field from a peripheral blood smear, seen on the right, was held for morophological and clinical review as the total platelet count was 10.0 X 109/L. Conditions fitting this picture include: | View Page |
| Cells that appeared similar to those illustrated in this image were repeatedly encountered as the smear was reviewed. The peripheral white blood cell count was 51.0 X 109/L with an orderly maturation sequence. The comment "leukemoid reaction" may properly be appended to the report. | View Page |
| Familial disorders: summary Several additional familial and congenital disorders associated with atypical inclusions in WBCs are now recorded. These individual syndromes carry the following names: Fechtner, Alport, Epstein, Sebastian, and Paris-Trousseau.Fechtner syndrome( Peterson etal,Blood 65:397-406,1985)was described with 8 family members spanning 4 generations presenting with varying degrees of nephritis, deafness,and congenital cataracts. The syndrome is likely a variant of Alport syndrome with the addition of leukocyte inclusions and macrocytothemia. Several more cases involving other families have been reported. The inclusions resemble toxic Doehle bodies or those of the May-Hegglin anomaly by light microscopy, but are ultrastructurally unique.Alport syndrome is autosomal dominant, X-linked , hereditary and characterized by sensorineural deafness and hereditary nephritis. It is believed to result from abnormal glycopeptide synthesis in renal basement membranes. Recurrent hematuria and slowly progressive renal insufficiency are clinical findings. Cataracts and platelet abnormalities may be added features.Epstein syndrome is essentially Alport syndrome with the addition of macrothrombocytopenia (Seri, et al. Hum Genet 110:182-186, 2002). Neutrophil inclusions are absent in this disorder; neutrophilic inclusions are considered part of the Fechtner syndrome.The Sebastian platelet syndrome is a variant of hereditary macrothrombocytopenia combined with neutrophil inclusions that differ from Doehle bodies, but are similar to those inclusions in Fechtner syndrome. (Greinacher, et al, Blut 61:282-288, 1990).Paris-Trousseau syndrome includes large platelets containing giant alpha granules identifiable in the peripheral blood.(Breton-Gorius, Blood 85:1805,1995) | View Page |
| Atypical neutrophilic intra-cytoplasmic inclusions, as noted in the image, are present in a peripheral blood smear when one or more of the following underlying conditions are present: | View Page |
| WBC inclusions: Summary The presence of atypical inclusions within the cytoplasm of neutrophils and other leukocytes should lead to a clinical investigation of the setting for these findings. Atypical neutrophil inclusions may be seen in the following disorders: Chediak-Higashi syndrome, May-Hegglin anomaly, Alder-Reilly anomaly, Fechtner , Sebastian, Epstein and Alport-like syndromes and in infectious and toxic conditions (in the form of Dohle bodies).Although a specific entity may not be evident from examination of the peripheral blood alone, it is important that hematology technologists include a comment reporting on the presence of these inclusions or granules. A clinical investigation with further hematologic and genetic studies may then appropriately be considered. Many of the disorders with atypical neutrophil cytoplasmic granules are also associated with platelet abnormalities, particularly giant platelets (lower image). Therefore, when atypical granules are recognized, scanning of the peripheral blood smear for atypical platelets may be revealing. These observations serve as readily identifiable markers for acquired and genetic human maladies, and as a guide for unraveling the reasons for a patient's suffering and impaired health. | View Page |
| Dohle Bodies: Review | View Page |
| A peripheral blood smear was reviewed and a representative field is shown on the right. Which of the following conditions may produce the results seen in this image? | View Page |
| The neutrophils seen in two fields in the upper and lower images are representative of a majority of the left shift neutrophils found in this peripheral blood smear. The diagnosis of Pelger-Huet anomaly can be made. | View Page |
| Case One Follow-up The blood count alone might be interpreted as reflecting infection, possibly supporting a diagnosis of acute appendicitis. However, the technologist performing the differential noted that more than 70% of the segmented neutrophils had bi-lobed or mono-lobed nuclei, strongly suggesting Pelger-Huet anomaly. Since the peripheral blood smear did not support the diagnosis of appendicitis in this patient, and since abdominal pain localized to the right lower quadrant never developed, the boy was hydrated with intravenous fluid and observed. After hydration, his constitutional symptoms improved and the abdominal pain subsided. People entering high altitude where the humidity may be very low are susceptible to dehydration and may experience symptoms related to mountain sickness. | View Page |
| The cell in this image is known as a MOTT cell. The condition in which these cells are associated is: | View Page |
| A peripheral smear was submitted for review due to increased monocytes on the automated differential. The images on the right are representative fields from the Wright-Giemsa stained blood smear (1000X magnification). The increased monocytes and peripheral picture are consistent with each of the following conditions EXCEPT: | View Page |
| The peripheral smear on the right was submitted for morphologic/clinical examination. The predominant cells comprised 70% of the total white blood cells and are consistent with lymphocytes in a 4-month-old infant. | View Page |
| Multiple Myeloma Plasma cells are uncommonly observed in the peripheral blood smear. They are normal constituents of lymph nodes, spleen, connective tissue and bone marrow. The presence of plasma cells in the peripheral blood is indicative of a large number of conditions, mostly related to infections , immune disorders, malignancies, toxic exposures, hypersensitivity reactions and their responses.Although mature plasma cells have a distinct appearance, they still may be confused morphologically with immature plasma cells and other cells with inclusions, reactive changes or nucleated red bloods cell with altered identities. In the image to the right, a plasma cell is present. The plasma cell has an eccentric immature nucleus with a muddy chromatin pattern. Note also clumping and stacking of the erythrocytes, typical of rouleaux formation, implicating an increase in plasma gamma globulin. Further studies are in order, including a bone marrow examination, where at least 30% of bone marrow cells should be variations of mature and immature plasma cells. Serum protein electrophoresis will reveal a monoclonal globulin spike, and light chains in excess of 1.0 gm/24 hours may be seen in the urine. The presence of lytic bone lesions is a convincing clinical clue. With these findings in combination, a diagnosis of myeloma can be made with assurance. | View Page |