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Disease Information and Courses from MediaLab, Inc.

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

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Alpha Thalassemia
Defining Thalassemia

Thalassemia is best thought of as a group of disorders rather than a single disease. They demonstrate a hemoglobin synthesis disorder in which there exists a defect in the rate of production of one or more of the globin chains. This defect results from either a heterozygous or homozygous deletion or inactivation of a globin chain gene.Thalassemias are named according to the affected gene or globin chain which is showing reduced or absent synthesis. Globin chain gene loci are found on the following chromosome locations:Chromosome 11 (Beta, Delta, Epsilon, and Gamma)Chromosome 16 (Alpha, and Zeta)

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Alpha Thalassemia States

The Alpha thalassemia genetic defects can be heterozygous or homozygous in inheritance. The heterozygous states of alpha thalassemia express themselves as: silent carrier (one of four gene loci deleted) thalassemia minor (two of four gene loci deleted) hemoglobin H disease (three of four gene loci deleted) The homozygous state (all four gene loci deleted), alpha thalassemia major, is incompatible with life. This is called alpha thalassemia major or hydrops fetalis.

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Alpha Thalassemia Intermedia

Alpha thalassemia intermedia (Hemoglobin H Disease) results from a deletion of three out of four alpha chain gene loci. Infants born with alpha thalassemia intermedia appear normal at birth but often develop anemia and splenomegaly by the end of their first year. Development and life expectancy are usually normal, but some affected individuals may require splenectomy and transfusion therapy.Hepatomegaly is not a common finding and there may be some association with mental retardation. Due to the hemolytic nature of this anemia, there may be an increase in respiratory infections, leg ulcers and gallstones. Skeletal changes are not commonly seen in hemoglobin H disease.Any ethnic group can have occurrences of hemoglobin H disease; but it is most often seen in Southeast Asia, the Middle East and the Mediterranean islands.

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Alpha Thalassemia Minor

Deletion of two out of four alpha chain gene loci results in alpha thalassemia minor. The deletions may be homozygous (two on the same chromosome) or heterozygous (one from each of two chromosomes). Alpha thalassemia minor does not produce a clinical disease but may be discovered upon routine testing.Both the homozygous and heterozygous form are common in Southeast Asians. The homozygous form of alpha thalassemia minor has been shown in African Americans as well.

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Silent Carrier

The silent carrier form of alpha thalassemia results from one alpha chain gene loci deletion. Individuals who are silent carriers show no clinical disease and demonstrate normal results during routine laboratory testing.This form of alpha thalassemia is usually discovered upon genetic familial testing. Silent carrier alpha thalassemia parents can pass on the alpha thalassemia gene and possibly a more serious form of alpha thalassemia if they have children with a partner who also carries thalassemia genes.

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Hemoglobin H disease is found in which ethnic group?View Page
Match the alpha thalassemia variants below with their genotypic notation.View Page
References

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

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Summary

The normal RBC count (4.84 x 1012/L) in this case, together with the decreased hemoglobin (8.4 g/dL) and MCV (59 fl) is an indicator of ineffective erythropoeisis that often points to thalassemia.The RBC morphology shows slight hypochromic microcytosis with codocytes, schistocytes, and basophilic stippling. Schistocytes form by several mechanisms, one being the removal of RBC inclusions.This patient's elevated bilirubin correlates with her presentation of sclera icterus; her splenomegaly is consistent with increased RBC destruction.The Hb electrophoresis demonstrated a normal pattern, initially, but the unstable Hemoglobin H was revealed upon repeat electrophoresis with reduced incubation time. Hemoglobin H is the result of beta globin chain tetramer formation due to the insufficient supply of alpha globin chains in alpha thalassemia intermedia.People with Hemoglobin H disease (alpha thalassemia intermedia) usually have a normal life expectancy without treatment. However, hemolysis may lead to moderate anemia that may be treated with splenectomy.

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Serum Iron

The serum iron for normal adults is about 50-150ug/dL. The iron binding capacity is normally 250-400ug/dL. The transferrin saturation usually falls between 20-50% in healthy individuals.Individuals with alpha thalassemia, especially Hb H disease, may have a slightly increased level of serum iron with a slightly decreased iron binding capacity. The percent of transferrin saturation is usually increased.An iron stain of bone marrow smears usually demonstrate increased levels of hemosiderin. Sideroblasts are present along with an occasional ringed sideroblast, as shown on the right.

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Antibody Detection and Identification (retired 12/6/2013)
Example of Clinically Significant Immune Antibody

The panel below shows reactions in the AHG phase only (clinically significant). Pattern reactivity of sample matches the pattern displayed by C on the panel. Anti-C is a clinically significant antibody that can cause both hemolytic disease of the newborn (HDN) and hemolytic transfusion reaction (HTR).ND= not done

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Course Introduction

Antibody screening and antibody identification are critical components in blood bank testing. Clinically significant antibodies must be identified so that appropriate blood products are selected for transfusion and the risk of adverse reaction is minimized. Clinically significant antibodies are capable of causing transfusion reactions, hemolytic disease of the newborn and in severe cases, death.This course will discuss the techniques that are used by blood bank technologists to detect and identify various types of antibodies.

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Significance of Reactions at Different Phases of Testing

Antibodies have optimum temperatures for reactivity. Reaction readings can be made at different phases: after immediate spin, after incubation at 37°C, and after the addition of antihuman globulin (AHG) and centrifugation. Reactivity in a certain phase will help to determine whether the antibody is cold reacting (IgM) or warm reacting (IgG). It will also help to distinguish between antibodies that are clinically significant and not significant. Clinically significant antibodies that are capable of causing acute and delayed hemolytic transfusion reactions (HTR) or hemolytic disease of the newborn (HDN) are usually IgG and react best in the AHG phase.Readings can be done at all three phases if a tube method is used. If a gel method is used, readings are done only at AHG. Immediate spin: Antibodies reacting in this phase tend to be cold reactive. They are usually IgM class and not clinically significant (with the exception of the A and B antibodies). 37°: Antibodies that react in this phase include strong IgM or IgG antibodies. After incubation, the tubes are examined for the presence of hemolysis. If complement was bound during incubation then hemolysis could be seen. NOTE: This reaction would only occur in serum samples. If EDTA plasma samples are used for testing, the complement cascade has been halted. Magnesium and calcium ions are not available for complement to be activated. AHG:Antibodies reacting in this phase are considered clinically significant. They are usually warm reactive and IgG.

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Antinuclear Antibody Testing: Methods and Pattern Interpretation
Autoimmune Diseases

Autoimmune diseases are a group of disorders where the body's immune system malfunctions and attacks its own tissues. One aspect of these diseases is the formation of antibodies that are directed to self-antigens (autoantibodies). Autoimmune diseases can be divided into two general groups: Organ specific, where the autoantibodies attack a specific organ, and Non-organ specific (or systemic), where the autoantibodies attack multiple organ systems. An example of an organ specific autoimmune disease is Hashimoto thyroiditis where autoantibodies damage the thyroid gland. An example of a systemic autoimmune disease is systemic lupus erythematosus (SLE) where the autoantibodies may attack any organ in the body.

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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.

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Which of these is NOT considered one of the systemic autoimmune rheumatic diseases?View Page
Which of these may contribute to the development of an autoimmune disease? (Choose all that apply)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.

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Cell Morphology

This slide is an illustration of a HEp-2 or HEp-2000® cell with several nuclear and cytoplasmic structures indicated. Antibodies to DNA, histones, centromere, nuclear RNP, cytoplasmic RNP, mitochondria, ribosomes, lysosomes, golgi apparatus, as well as a variety of cytoskeletal proteins such as microfilaments, intermediate filaments, and microtubules, can be detected using the HEp-2 or HEp-2000® cell lines.(Ref10) It is important to note that RNA constitutes the bulk of the nucleic acid in the cells, being 5-10 times more abundant than DNA.(Ref11) Generally we consider 2 compartments in the cell when detecting autoantibodies in systemic rheumatic disease: 1) nucleus, and 2) cytoplasm. Staining of the nucleus includes staining inside the nucleoli.

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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)

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ANA Testing Scheme

Most laboratories use a two-stage approach for ANA testing with the first stage being an initial screen at a 1:40 or 1:80 dilution of the patient sample. Negative samples are reported out as such and positive samples are titered. There are two reasons for titering ANA-positive samples. One is to determine the amount of antibody present and the other is to look for multiple patterns. In the case of ANA testing there is no correlation between titer endpoint and disease activity or severity. However, the higher the titer the higher the likelihood the patient has one of the SARDs. Often more than one ANA pattern present in the sample; titering facilitates the identification of these mixed patterns. (Identification of mixed patterns will be covered later).The titering scheme most frequently used in ANA testing is two-fold dilutions starting at the initial screening dilution. For example: 1:40, 1:80, 1:160... While there is no consensus on how far to titer samples, informal communications with laboratories suggest most stop titering at about the 1:2560 dilution (plus or minus one dilution). By this dilution the samples are often negative and/or no longer contain mixed patterns.For ANA positive results the sample is reported as: ANA positive, the pattern that is indicated along with the titer endpoint. For example: Sample 12345: ANA positive, speckled, titer: 1:640.If more than one pattern is present all patterns are reported along with their respective endpoints. Additionally, many labs will suggest appropriate follow-up testing to identify the antibody(ies) present in the sample. By identifying the specific antibodies present in the sample, the clinician may gain further insight into which of the SARDs the patient has and what other symptoms the patient may develop.

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Antigen Chart

Pattern observed by indirect immuno fluorescence Antigen Disease(s) in which antibodies are seen Routine tests used to confirm specific antibody Homogeneous Double stranded DNA (dsDNA) Characteristic of SLE, lower levels in other rheumatic diseases IFA or CZ using Crithidia luciliae, RIA, ELISA, Addressable Laser Bead Assay (ALBIA) Nucleosome or Chromatin SLE, Drug-induced LE ELISA Histone Drug-induce LE, SLE ELISA, ALBIA Unusual Homogeneous Nuclear Membrane Lupoid hepatitis ELISA for gp-210 Speckled Sm (Smith) Marker antibody for SLE Immunodiffusion (ID), ELISA, ALBIA U1-RNP High levels in MCTD and SLE, low levels in other rheumatic diseases ID, ELISA, ALBIA Speckled (and/or SSA pattern if using HEp-2000®) Can also be ANA negative SS-A/Ro High prevalence in Sjögren syndrome sicca complex, lower prevalence in other rheumatic diseases With HEp-2000 characteristic ANA pattern is confirmatory, others confirm with ID, ELISA, ALBIA Fine speckled or ANA negative Ro52 Sjögren syndrome, myositis, Neonatal Lupus ELISA, ALBIA Fine speckled (sometimes with nucleolar staining as well) SS-B/La High prevalence in Sjögren syndrome sicca complex, lower prevalence in other rheumatic diseases ID, ELISA, ALBIA Fine speckled, Homogeneous, Nucleolar Scl-70 Marker antibody for Scleroderma ID, ELISA, ALBIA Cell Cycle Dependent Speckled PCNA Marker antibody for SLE ID, ELISA, ALBIA Coarse Speckled Nuclear Matrix Seen in some patients with evolving connective tissue disease NONE 3-20 dots NSp I, sp-100, MND, PBC 95 Associated with Primary Biliary Cirrhosis ELISA, ALBIA Cell Cycle Dependent Speckled with speckling in metaphase mitotics NSp II, CENP F Some association with malignancies NONE Staining in cleavage furrow between dividing cells Midbody Unknown Confirm by staining pattern Centromere CENP A, CENP B, CENP C Seen in 57-82% of patients with limited form (CREST) of scleroderma and Raynaud phenomenon Confirmed by staining pattern ELISA, ALBIA Nucleolar Fibrillarin (Clumpy nucleolar) Scleroderma ELISA, ALBIA RNA polymerase I, NOR-90, others? (Speckled nucleolar) Scleroderma and other connective tissue diseases ELISA, ALBIA PM-1 (PM/Scl), others?(Smooth nucleolar) Polymyositis/Scleroderma overlap ELISA, ALBIA

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Why are ANA positive samples titered?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.

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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

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Mitotic Spindle

Anti-mitotic spindles antibodies refers to several different patterns which, in a broad sense, includes CENP-F, Centromere and Midbody. However, in the case of the cytoplasmic patterns it only applies to the staining of the spindle fibers. Mitotic Spindle Antibodies:Several antigens NuMA, HsEg5, othersCytoplasmic pattern Stain spindle fibers in metaphase mitoticReport as Suspect mitotic spindleClinical significance Various autoimmune disease Mycoplasma pneumoniae infections

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Authentic and Spurious Causes of Thrombocytopenia
Increased Splenic Sequestration and Thrombocytopenia

The spleen is an incredibly important organ in the hematopoietic system. Normally, the spleen stores one-third of the platelets that are produced by the bone marrow and the remaining two-thirds of the platelets produced by the bone marrow are in circulation. No platelets are stored in the bone marrow. If a condition causes the spleen to enlarge (splenomegaly), the spleen will function abnormally, sequestering up to 90% of the total platelet mass in the spleen. The result is a decrease in circulating platelets (thrombocytopenia). Some conditions that are associated with splenomegaly and a resulting thrombocytopenia include hepatic cirrhosis, Gaucher's disease, some leukemias, and lymphomas.

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Treatment of TTP

Currently, the most effective treatment for TTP is therapeutic plasma exchange (TPE). Fresh frozen plasma (FFP), preferably cryoprecipitate-poor plasma (that lacks von Willebrand factor), is used as the replacement fluid in the treatment. The exchange takes place over several days until the patient's platelet count stabilizes above 100 x 109/L.The logic of TPE is to rid the circulation of plasma containing ultra-large vonWillebrand factor (vWF) multimers. vWF is a large multimeric protein that is made by megakaryocytes and endothelial cells. It is is a key factor in platelet adhesion and also is responsible for carrying Factor VIII into the circulation. vWF binds glycoproteins Ib, IIb, and IIIa. The largest multimer is called ultra-large vWF and in normal plasma, it is cleaved into smaller fractions (necessary for balanced coagulation activity) by an enzyme processed by the gene, ADAMTS13. In patients with TTP, the enzyme activity is < 5% of normal and therefore, these ultra-large vWF molecules get into circulation, resulting in excessive platelet aggregation and microvascular thrombus formation.Therapeutic plasma exchange has decreased TTP mortality rate from 90% to 15% since the treatment first came into use as the standard primary treatment of TTP in the 1970's. TPE does not cure TTP, but it arrests the manifestations of the disease until spontaneous remission occurs.

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Disseminated Intravascular Coagulation (DIC)

DIC is a condition that is usually secondary to an underlying disease or condition. Some of the activators of DIC are sepsis, placenta abruptio, snake bites, toxin, trauma, graft vs. host disease, and burns.The mechanisms that are involved in DIC include a hyperactivated coagulation system, a hyperactivated fibrinolytic system, or both simultaneously. In most cases the coagulation factors are consumed as soon as they are made and platelets are also consumed in the coagulation process. Clots are made rapidly and then rapidly destroyed as the fibrinolytic system is hyperactivated.

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Basic Tissue Orientation and Paraffin Embedding Technique
A small tubular artery is received in histology to rule out temporal arteritis. When embedding this specimen, how must the sections of the tube must be oriented?View Page

Beta Thalassemia
Delta-Beta Thalassemia

Delta-beta thalassemia exists in both heterozygous and homozygous forms. The symptoms are mild to moderate depending on the severity of the disease and can include mild, hypochromic anemia, slight hepatomegaly and/or splenomegaly and occasional bone changes due to the erythroid hyperplasia. Patients rarely require treatment, but blood transfusions may be necessary in certain cases. In this condition, the body compensates for the lack of beta and delta chain production by increasing the production of gamma globin chains, leading to an increased hemoglobin F level. This form of beta thalassemia can be found in many ethnic groups, but is most common in persons from Greece, Africa, and Italy.

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Blood Banking Question Bank - Review Mode (no CE)
Which BBP is not covered in the OSHA Bloodborne Pathogen Standard?View Page
Which of the following antibodies is the most common cause of hemolytic disease of the newborn:View Page
The use of the direct antiglobulin test is indicated in all the following except:View Page
Which of the following conditions is most frequently associated with anti-I:View Page
Which of the following set of conditions would preclude hemolytic disease of the newborn as a result of ABO incompatibility:View Page
Which one of the following statements about directed donations is true:View Page
To detect the presence of blocking antibodies fixed on the red cells of a newborn infant:View Page
Gamma irradiation of cellular blood components is required in which of the following situations:View Page

Body Fluid Differential Tutorial
Leukemic Fluids

Leukemia and lymphoma cells can be found in cerebrospinal, pleural, peritoneal and pericardial fluids at the time of diagnosis as well as anytime during the course of the disease. They can be found in any of these fluids, even when there are not abnormal cells present in the peripheral blood. Just as there are many morphological variations of leukemia and lymphoma in the peripheral blood and bone marrow, there are as many variations of leukemia/lymphoma in cytospins from fluids.Once the diagnosis is made from the initial tissue samples, there may be frequent spinal taps performed to monitor disease status and to instill intrathecal chemotherapy to prevent the development of central nervous system involvement by the leukemia or lymphoma.Cytospins are made for each sample and must be meticulously scrutinized. If any display abnormalities indicative of a disease state, or if there are any suspicious cells, then they should be sent for hematology or pathology review in compliance with your hospital's policy.

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Central Nervous System (CNS) Toxoplasmosis continued

This is a higher power view of this same smear demonstrating a neutrophil that is filled with Toxopasma gondii tachyzoites (blue arrow).There are a few free organisms in this image well, indicated by the red arrows. Again, the typical morphology for toxoplasmsa organisms is lavender cytoplasm with a red granular cluster in the center of each parasite.This patient was negative for Toxoplasma gondii prior to a transplant but had received 15 units of blood products due to cytopenias.It is believed that a donor for one of the transfused units had been exposed to Toxoplasma gondii either through cats or contaminated food and had transient circulating Toxoplasma gondii in his or her blood when the donation was made. In this case, the recipient was profoundly immunocompromised, which lead to rapidly developing systemic disease.

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Bronchial Lining Cells

Bronchial alveolar lavage (BAL) is a procedure performed to obtain cells from the lungs to evaluate the cause of lung disease. A fiber-optic scope is passed into the section of lung to be examined while a small amount of physiologic saline is instilled and then removed to be sent for examination.Macrophages are frequently found in Bronchial alveolar lavages and can be found singly or in clusters. This photo shows several macrophages (blue arrows).The elongated rectangular cell with the cilia at the end is a bronchial lining cell which is also frequently present in BAL samples (red arrow).

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Bone Marrow Aspiration Part I: Normal Hematopoiesis and Basic Interpretive Procedures
Rules for Bone Marrow Differentials, continued

It is important to note that not all smears will have good areas to perform a differential in the vicinity of the bone marrow fragment. When this occurs, you must keep looking on additional smears. This is one of the reasons that several smears are stained and prepared for possible review. It can take time to recognize from 10x magnification what will be countable on 50x magnification. The best tip is to be patient and do not fail to keep on looking! In fact, sometimes it may be necessary to stain additional smears/slides, if available, to obtain enough readable material. While you are checking the smears on 10x magnification for readable areas, you should take the time to evaluate and record the following: The cellularity of the bone marrow sample Presence and number of megakaryocytes Presence of tumor cells Anything else out of the ordinary, which should be noted on the report (such as evidence of hemophagocytosis, storage disease etc.).Once you have decided where to count the marrow, you will perform the differential count. Usually a 200-cell bone marrow differential is the minimum acceptable count. However, more cells may be required depending on your laboratory/pathology protocol. Remember, unlike peripheral differentials, all nucleated cells are included in the total count, including all maturation stages of the erythroid cell series.Cell counts are performed on 40 - 50x magnification with oil depending on the optics of your scope, moving up to 100x magnification with oil as needed for fine detail. Once oil is added to the smear, move systematically through your chosen area until the morphology/cellularity/stain quality is no longer acceptable, then move back to 10x power to find another good area in the vicinity of the fragment to continue your count. You may need to progress from one slide to the next to accumulate enough cells for your differential. In fact, if there is variability in cell distribution from one smear or fragment/spicule to the next, then the count should be split between more than one smear/fragment to avoid a biased final count.If there are no spicules, then the differential should be performed in any portion of the slide that demonstrates readable morphology. In pull preps and coverslip preps, this will usually be in the thin area near the edge of the smear. If differential-type (wedge) smears are available, then the usual feathered-edge area should be used. On any of these smears, be sure that you are in deep enough from the thin edge so that the numbers of stripped cells are kept at a minimum to avoid skewing the count, as some cell types are more fragile than others.The pathologist is ultimately responsible for the final sign-out and will change/adjust/return smears for recount if there is any disagreement over numbers and cell types.

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Cardiac Biomarkers (retired 12/6/2013)
Atherosclerosis

Atherosclerosis is one of the leading causes of heart disease and its presence is an important risk factor for events leading to acute myocardial infarction (AMI). In the past, atherosclerosis was described as a cholesterol and lipid storage event. Now we know it is a chronic inflammatory disorder of the arterial vessels with lipid components. Atherosclerosis begins with damage to the cells that line the blood vessels. Some possible causes of this cell injury are bacterial infection, hyperlipidemia, hypertension, glycosylated products of diabetes, cytokines from adipose tissue, or exposure to toxins such as pollution and second-hand smoke. Monocytes and lymphocytes adhere to the injured site; macrophages enter and ingest proteins and, along with modified lipoproteins, create foam cells. An inflammatory milieu results as cytokines and other inflammatory molecules become involved; foam cells and white blood cells begin secreting cytokines and metalloproteinases. Myeloperoxidase is also released by degranulated white blood cells and macrophages. As inflammation and accumulation of these products continues, fatty dots and streaks are formed on the vessel lining and the formation of plaque begins. As the atherosclerotic process continues, involved cells proliferate forming a complex extracellular matrix and a fibrous cap. If development continues, possibly over decades, the plaque formations are distributed throughout various vessels, become calcified or collagenized and make the vessel walls rigid. The risk to patients with significant atherosclerosis is that eventually a narrowing of the artery (stenosis) can cause a reduction in oxygen delivery to tissues and plaque rupture can lead to an acute coronary event.

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Fibrous Cap

If the atherosclerotic process continues and other risk factors are involved, the lipid core of the plaque grows and pushes the arterial wall out. The myeloperoxidase and metalloproteinases degrade the cellular matrix, thinning the fibrous cap making it capable of tearing. Once there are tears, platelet aggregation and the coagulation cascade begin; thrombi or blood clots become part of the plaque. Further plaque growth and repair leads to occlusion and necrosis of vessels. Varying degrees of pain, cerebral or pulmonary infarction, and/or ischemic heart disease result.

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Risk Factors for Heart Disease

Those who have hypercholesterolemia, hypertension, poor nutrition and diet, are overweight, smoke, and/or are physically inactive are at a greater risk for heart disease and an AMI. Family history, gender, and lifestyle also contribute to this risk.

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Monitoring Lipids

Most often a lipid panel measures concentrations of total cholesterol, HDL-C, LDL-C, and triglycerides. HDL-C is measurement of the cholesterol in the lipoprotein HDL, and LDL-C, the measurement of cholesterol in LDL.Cardiovascular disease (CVD) is associated with elevations in LDL-C; increased LDL-C in individuals puts them at risk for CVD and is sometimes considered a pre-AMI condition. The opposite is true for HDL-C. One of the functions of this lipoprotein is to remove excess cholesterol, transporting it to the liver for reprocessing or excretion. To prevent cardiac disease, HDL-C levels should remain up and if below recommended range, steps are prescribed to raise the HDL-C concentration.Recommended ranges for lipids from the 2001 National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATP III). These were developed in the US as recommended levels that decrease risk for CHD in adults: Cholesterol <200 mg/dL Triglyceride <150 mg/dL LDL-C <100 mg/dL HDL-C >59 mg/dL

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Terminology Describing Ischemic Heart Disease and Heart Failure

Descriptions of cardiac biomarkers and their use require knowledge of several terms. These terms and their definitions follow.Acute Coronary Syndrome (ACS) Includes all the ischemic events that can occur in the heart. These events range from angina (where there is no cell death or reversible cell injury) to an AMI with large areas of cell necrosis. A continuum of events that are involved in ACS is illustrated on the page that follows this glossary of terms.Acute Myocardial Infarction (AMI) Commonly referred to as a heart attack. A sudden loss of circulating blood and oxygen that causes necrosis of myocardial tissue. It is most often caused by the narrowing of coronary arteries by atherosclerosis, a thrombus, or dislodged plaque material.Angina Chest pain caused by inadequate supply of oxygen to heart myocardium. It is synonymous with angina pectoris, pectoris meaning chest.Congestive Heart Failure (CHF) Usually, a left ventricular dysfunction resulting from aging, hypertension, atherosclerosis or muscle damage from an AMI or repeated AMIs. In CHF, the heart is not able to effectively pump blood through its chambers and to the body. Fluid accumulates in the lungs and tissues causing edema because less blood leaves through the arteries than what entered the heart from the veins. Electrocardiogram (ECG or EKG) The tracings of the electrical current that passes through the myocardium. The heart contractions are stimulated by this current. In areas of myocyte necrosis, the current does not pass and the tracings display abnormal patterns.Infarction An area of tissue death that occurs due to lack of oxygen. Clogging of an artery will cause dead muscle tissue or infarction.IschemiaAn inadequate blood supply that decreases availability of oxygen. Atherosclerosis is the main cause of myocardial ischemia.

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Introduction

Each year in the US alone, over one million individuals are diagnosed with an acute myocardial infarction (AMI) and approximately one half of these have had an AMI in the past. The incidence of congestive heart failure (CHF) is on the rise. It is the leading cause of hospitalization in those age 65 and older. Healthcare costs for cardiovascular disease (CVD), which includes coronary artery disease (CAD) and coronary heart disease (CHD) are more than $400 billion each year. Heart disease is currently the leading cause of death in the US. As many as 1% to 5% of patients with an AMI are misdiagnosed in the emergency department and are discharged. The laboratory's role is especially important in individuals with an AMI who present with AMI symptoms but have a nondiagnostic electrocardiogram (ECG).

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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)

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Optimal Cardiac Biomarkers

An ideal marker for cardiac disease should have these qualitites: Should be specific to myocardial tissue Be in low concentrations in normal peripheral blood Be rapidly released after myocardial injury Should be detected in low quantities with little interference from like compounds Should remain in circulation for a sufficient length of time for detection The plasma concentration of the marker should be directly related to the extent of injury. The test for the biomarker should be easily automated and relatively inexpensive to run, and results should be obtained rapidly.

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Uses of Cardiac Biomarkers

Cardiac markers are measured for diagnosis and monitoring of cardiac disease, most often an AMI or other myocardial injury. They are also used to diagnose CHF. Newer cardiac markers give information on risk of future cardiac disease, further cardiac disease, and assist in predicting the response of heart disease to treatments. Cardiac markers are especially helpful in situations of a suspected AMI where the patient's electrocardiogram (ECG) does not present the typical pattern associated with an AMI.

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Heart Disease and Cardiac Biomarkers

Cardiac biomarkers are important in heart disease where there is damage to the myocardium and myocytes. Interrupted or blocked bloodflow and decreased oxygen cause injury to the myocytes and cell death. As the myocytes become necrotic, their membranes are disrupted and intracellular biomolecules diffuse into cardiac microvasculature and then to lymphatics. From there, these compounds, many of which are biomarkers, enter the peripheral circulation.Cardiac biomarkers are relied upon for the detection and monitoring of two categories of cardiovascular disease: Acute Ischemic Disease -- an AMI is the most serious disease in this category Heart Failure

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History

In the past, an AMI was primarily diagnosed by evaluating symptoms at patient presentation, ECG measurement, and results of enzyme assays that were considered cardiac enzymes. The enzymes, creatine kinase (CK), lactate dehydrogenase (LD), and aspartate aminotransferase (AST) were assayed several times a day often for several days to observe peak concentration and return to normal level for each enzyme. The first assay result was the baseline level or baseline concentration. Isoenzymes of CK and LD were later added for AMI diagnosis. All three of these enzymes are found in other tissues, making the diagnosis difficult and lengthy. In the 1980s, CK isoenzyme, CK-MB, though not totally cardiac specific, became the benchmark marker for an AMI. None of these enzymes are in any of the current recommendations except for CK-MBCurrent diagnosis, monitoring, and screening relating to heart disease includes measurement of lipids, proteins, enzymes, and other biomolecules. Risk stratification for cardiac and vascular disease is an additional role for measurement of these analytes. The physiological changes in the development of heart disease are better understood and the role of the clinical laboratory is greatly expanded.Today's markers are significant because of their location in the myocyte, the kinetics of their release in myocyte damage, and their rate of clearance from peripheral blood.

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CK-MB

Before troponins were used in cardiac disease diagnosis, CK-MB, an isoenzyme of creatine kinase (CK), was the marker of choice for AMI diagnosis. CK-MB is released in circulation 4-6 hours after symptoms of an AMI and usually peaks within 24 hours. Levels of CK-MB are back to normal range in 48-72 hours. The latter is different from the cardiac troponin pattern. Use of CK-MB in diagnosis of an AMI varies. Some institutions have discontinued assaying CK-MB in suspected AMIs; others use CK-MB measurements in conjunction with cTnI or cTnT. Because CK-MB returns to normal much faster than cardiac troponins, CK-MB measurements can be used when a reinfarction is suspected. In reinfarction, CK-MB concentration rises again after the return to baseline levels. Currently, CK-MB results do not predict future adverse cardiac events and do not have any prognostic or risk stratification use.

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Diagnosis of an Acute Myocardial Infarction (AMI)

An ESC/ACC consensus conference in 1999 defined cTnI and cTnT as the cornerstone biomarkers for diagnosis of AMI. If cardiac troponins are not available, then CK-MB should be used as a substitution marker. In 2007, the ESC/ACC/AHA published new criteria for an AMI:Elevated biomarkers and one of the following: Ischemic symptoms ECG changes indicating a new ischemic event Pathological ECG with Q waves (abnormal tracings found in AMI) Imaging evidence of new myocardial damage In 2002, ACC/AHA published practice guidelines for diagnosis of new category of heart disease, ACS. AACC and IFCC continue to improve guidelines in order to improve and clarify diagnosis. The goal is to increase detection of those presenting with an AMI (true positive) and decrease hospitalization of those who present with chest pain and have not experienced an AMI (false positive).

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Select the correct statements regarding troponins.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.

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BNP Versus NT-ProBNP

BNP, as an active protein hormone, has a short biological half-life of 20 minutes. NT-ProBNP, inactive fragment, has a half-life of 1-2 hours. Both are increased in CHF and both levels correlate to the severity of condition. In the future these two hormones may be used as prognostic indicators in ACS also; studies have shown that levels predict cardiac mortality and adverse cardiac events in patients with ACS.Because of its longer half-life, there are advantages to measuring NT-ProBNP over BNP. In CHF patients receiving exogenous and synthetic BNP for treatment, BNP levels may be affected while NT-ProBNP will not be affected. NT-ProBNP is thought to be primarily cleared by kidneys and therefore falsely elevated in severe renal disease.

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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
Risk Stratification

Risk stratification in cardiac disease is the use of biomarker assays and other diagnostic testing of an individual with disease to predict risk of future disease and cardiac events. The results are also utilized in treatment and drug intervention plans. Studies conducted recently use multiple markers for risk stratification for patients with CHF, ACS, and previous AMIs. Multiple markers (BNP, NT-ProBNP, cardiac troponins, and hs-CRP) may be used in near future to predict short-term and long-term risks of cardiac disease and death. Serial troponin levels are currently measured in those with ischemia to determine the risk of AMI. Troponin levels are used to plan medical and surgical treatments.

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Screening Biomarkers

Previously, screening for cardiovascular disease (CVD) focused on hyperlipidemia, obesity, and hypertension. However, approximately one half of AMIs occur in healthy men and women with normal or only slightly elevated plasma lipids. With new insights into cardiac disease and the ACS, novel biomarkers such as inflammatory markers, hormones, and other biomolecules indicating myocardial stress are required. Some new screening markers are in use today and many more are in study and evaluation for future use. New screening markers for CVD and ACS are: Highly Sensitive C-Reactive Protein (hs-CRP) Homocysteine Ischemial Modified Albumin (IMA) Myeloperoxidase (MPO)

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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.

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Homocysteine

Homocysteine is a sulfur-containing amino acid in the blood plasma. Elevation of homocysteine has been linked to a higher risk of cardiovasular disease (CVD). This elevation is significant in those where family history places them at risk for CVD.Folic acid, Vitamin B6, and VitaminB12 help to prevent elevated homocysteine levels. Recent data shows that folate fortification of foods has reduced the average level of homocysteine in the United States. Laboratory testing for plasma homocysteine levels may improve the assessment of cardiac risk, particularly in patients with a personal or family history of CVD, but with no well-established risk factors present such as smoking high cholesterol, or high blood pressure.

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Future Cardiac Biomarkers

There are a large number of other molecules being evaluated and studied to determine their use as reliable cardiac biomarkers. Future research will demonstrate if they provide additional helpful information in ACS and if they are helpful in screening for CVD in asymptomatic individuals. Some future markers under study are lipoprotein(a), oxidized LDL, metalloproteinases, lipoprotein-associated phospholipase A2, pregnancy-associated plasma protein A, and placental growth factor. With advances in molecular diagnostics and proteomics, an individual's plasma proteome, their fingerprint of proteins and peptides, may be a biomarker profile of current and future disease.

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Which biomarkers of cardiac disease risk are inflammatory markers?View Page
Comparisons of Concentrations of CRP and hs-CRP

Laboratory Test Test Result Interpretation CRP 10.2 mg/L Acute inflammation hs-CRP 0.5 mg/L Low risk for cardiac disease hs-CRP 1.5 mg/L Average risk for cardiac disease hs-CRP 3.5 mg/L High risk for cardiac disease

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Case Studies in Clinical Microbiology
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.

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Acute gonorrhea is the most common cause of septic arthritis in patients under 30 years of age.View Page
Clinical History

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

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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
Review 2

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

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Each of the following statements is true concerning Clostridium septicum infections EXCEPT:View Page
Factors predisposing to infections with methicillin resistant Staphylococcus aureus (MRSA) include: (choose all that apply)View Page
Thus, in follow-up to the previous discussion, the reaction shown in the image establishes the identification of a group A, beta hemolytic streptococcus.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.

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Review 2

Low JC. Donachie W.: A review of Listeria monocytogenes and listeriosis. Veterinary Journal. 153:9-29, 1997 Following the initial isolation and description in 1926, Listeria monocytogenes has been shown to be of world-wide prevalence and is associated with serious disease in a wide variety of animals, including man. Our knowledge of this bacterial pathogen and the various forms of listeriosis that it causes has until recently been extremely limited, but recent advances in taxonomy, isolation methods, bacterial typing, molecular biology and cell biology have extended our knowledge. It is an exquisitely adaptable environmental bacterium capable of existing both as an animal pathogen and plant saprophyte with a powerful array of regulated virulence factors. Most cases of listeriosis arise from the ingestion of contaminated food and in the UK the disease is particularly common in ruminants fed on silage. Although a number of forms of listeriosis are easily recognized, such as encephalitis, abortion and septicaemia, the epidemiological aspects and pathogenesis of infection in ruminants remain poorly understood. The invasion of peripheral nerve cells and rapid entry into the brain is postulated as a unique characteristic of its virulence, but relevant and practical disease models are still required to investigate this phenomenon.

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Review 2

Low JC. Donachie W.: A review of Listeria monocytogenes and listeriosis. Veterinary Journal. 153:9-29, 1997 Following the initial isolation and description in 1926, Listeria monocytogenes has been shown to be of world-wide prevalence and is associated with serious disease in a wide variety of animals, including man. Our knowledge of this bacterial pathogen and the various forms of listeriosis that it causes has until recently been extremely limited, but recent advances in taxonomy, isolation methods, bacterial typing, molecular biology and cell biology have extended our knowledge. It is an exquisitely adaptable environmental bacterium capable of existing both as an animal pathogen and plant saprophyte with a powerful array of regulated virulence factors. Most cases of listeriosis arise from the ingestion of contaminated food and in the UK the disease is particularly common in ruminants fed on silage. Although a number of forms of listeriosis are easily recognized, such as encephalitis, abortion and septicaemia, the epidemiological aspects and pathogenesis of infection in ruminants remain poorly understood. The invasion of peripheral nerve cells and rapid entry into the brain is postulated as a unique characteristic of its virulence, but relevant and practical disease models are still required to investigate this phenomenon.

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Cerebrospinal Fluid (retired 7/17/2012)
Clot/Pellicle

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

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Chemical Screening of Urine by Reagent Strip
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.

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The presence of increased levels of protein in the urine may be an early indicator of which of the following conditions?View Page
Examples of conditions resulting in benign proteinuria include: (Choose ALL correct answers)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.

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In hemolytic disease, the urine bilirubin test result is negative and the urine urobilinogen test result is:View Page

Chemical Screening of Urine by Reagent Strip (retired March 2012)
Match the following reagent strip tests to the disease or disorder that would most likely cause a positive test result.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.

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Chemistry / Urinalysis Question Bank - Review Mode (no CE)
Identify the urine sediment elements shown by the arrow:View Page
Identify the urine sediment elements present in this illustration:View Page
Which one of the following crystals is not found in normal urine:View Page
Identify the urine sediment element indicated by the arrow in the illustration:View Page
What type of cast is shown in the illustration:View Page
Identify the urine sediment elements present in this illustration:View Page
Match Increased Analyte with the associated disease:View Page
Which one of the following statements about TSH is true:View Page
Elevation in CSF total protein may be seen in all of the following conditions except:View Page
In a normal CSF the protein concentration as compared to that in the serum is generally:View Page
A spectrophotometric scan of amniotic fluid may be valuable in the determination of which of the following conditions:View Page
Increases in the MB fraction of CK is associated with:View Page
Increases in LD fractions 4 and 5 are indicative of:View Page
The following LDH Isoenzyme pattern would be seen in:View Page
The following LDH Isoenzyme pattern would be seen in:View Page
The following LDH Isoenzyme pattern would be seen in:View Page
The following CK isoenzyme pattern would be seen in:View Page
This SPE scan most likely represents which of the following disease states:View Page
This serum protein electrophoresis scan most likely represents which condition?View Page
Which one of the following are not associated with a polyclonal (broadbased) increase in gamma globulins?View Page
Which of the following conditions is associated with elevated serum uric acid levels:View Page
Which of the following conditions would be suggested by a marked rise in alkaline phosphatase, jaundice, and a moderate rise in ALT:View Page
Which of the following cells when found upon microscopic examination of the urine would be most indicative of kidney disease:View Page

Confirmatory and Secondary Urinalysis Screening Tests
Diseases Associated with Proteinuria

Normal urine contains very little protein, usually less than 10 mg/dL. The majority of the protein that is found in normal urine is albumin. The presence of an increased amount of protein in the urine (proteinuria) can be an indicator of renal disease. The two mechanisms that can lead to proteinuria are glomerular damage or a defect in the reabsorption process of the tubules in the nephron. The concentration of protein in the urine is not necessarily indicative of the severity of renal disease. Severe proteinuria (greater than 3.5 g/day) is characteristically seen in patients with glomerulonephritis, lupus nephritis, lipoid nephrosis, and severe venous congestion of the kidney. Moderate proteinuria (0.5-3.5g/day) is seen in nephrosclerosis, multiple myeloma, diabetes nephropathy, malignant hypertension, and pyelonephritis with hypertension. Mild proteinuria (less than 0.5 g/day) may be seen with polycystic kidneys, chronic pyelonephritis, benign orthostatic proteinuria, and some renal tubular diseases. Transient proteinuria can also be due to physiologic conditions such as stress, exercise, cold exposure, and fever, in the absence of renal disease.

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Diseases Associated with Proteinuria

Severe proteinuria (greater than 3.5 g/day) is characteristically seen in patients with glomerulonephritis, lupus nephritis, lipoid nephrosis, and severe venous congestion of the kidney. Moderate proteinuria (0.5-3.5g/day) is seen in nephrosclerosis, multiple myeloma, diabetes nephropathy, malignant hypertension, and pyelonephritis with hypertension. Mild proteinuria (less than 0.5 g/day) may be seen with polycystic kidneys, chronic pyelonephritis, benign orthostatic proteinuria, and some renal tubular diseases. Transient proteinuria can also be due to physiologic conditions such as stress, exercise, cold exposure, and fever, in the absence of renal disease.

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Microalbumin Test

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

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Bilirubinuria

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

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Urine Glucose

The presence of significant amounts of glucose in the urine is called glycosuria (or glucosuria). The amount of glucose present in urine is dependent upon the blood glucose level, the rate of glomerular filtration, and the degree of tubular reabsorption of the sugar. Usually glucose will not be present in the urine until the blood level exceeds 160-189 mg/dL, which is the normal renal threshold for glucose. The main reason for glycosuria is an elevated blood glucose level (hyperglycemia). Diabetes mellitus is the most common cause of hyperglycemia. However, stress, obesity, brain injury, myocardial infarction, hyperthyroidism, pregnancy, and a lowered renal threshold due to kidney damage can all cause glycosuria.

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Detecting and Evaluating Coagulation Inhibitors and Factor Deficiencies
Elevations in PT or aPTT assays

PT and/or aPTT may be prolonged for a number of reasons. Prolonged PT causes include: Warfarin therapy Liver disease Disseminated intravascular coagulation (DIC) Vitamin K deficiency Liver conditions such as cirrhosis or hepatitis Inadequate level of Factors I, II, V, VII, and/or XProlonged aPTT causes include: Presence of heparin Liver disease, other liver conditions Vitamin K deficiency Hemophilias DIC von Willebrand disease Lupus anticoagulant Inadequate levels of Factors I, II, V, VIII, IX, X, XI, and/or XII

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Summary of Initial Elevations in the PT and aPTT

After confirming that preanaytical factors are not responsible for the prolonged coagulation test(s) and there is no known medical condition, such as liver disease, vitamin K deficiency, or DIC that is causing the prolongation, a mixing study may be ordered to detect the presence of a possible factor deficiency or coagulation inhibitor.Knowledge of the coagulation factors that are monitored by the PT and aPTT tests is necessary to choose the appropriate mixing study(ies) that should be performed.

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When To Perform a Mixing Study

A mixing study should be considered when a patient has a prolonged PT and/or aPTT along with: no history of heparin or warfarin therapy no history of liver disease It is also essential to first verify that a correctly collected sample (free from clots) with a proper blood to anticoagulant ratio has been obtained.

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Introduction: Coagulation Inhibitors

As the name implies, coagulation inhibitors (also called circulating anticoagulants) interfere with normal blood coagulation. Coagulation inhibitors may be congenital or acquired (developing in patients during the course of a disease) and are almost always immunoglobulins, either IgG or IgM. There are two types of inhibitors: those directed toward a coagulation factor (or multiple factors) and the lupus anticoagulant. Lupus anticoagulant is one of the more commonly encountered coagulation inhibitors. It is also known as antiphospholipid antibody because it is directed toward phospholipids. Lupus anticoagulant is usually an IgG antibody. It differs from factor-specific inhibitors in that lupus anticoagulant causes thrombosis and abnormal clotting while factor-specific inhibitors cause serious bleeding.

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Introduction: Factor Deficiencies

A deficiency in one or more coagulation factor will also cause abnormalities in hemostasis. The image to the right depicts the coagulation cascade. Notice how one factor acts upon another to eventually form a stabilized fibrin clot, the end product of the coagulation cascade. Having an abnormally low level, or a complete lack, of a coagulation factor can cause the extrinsic, intrinsic, or common pathways to malfunction, resulting in dangerous hemorrhagic issues including spontaneous bleeding. Two of the most common factor deficiencies are factor VIII (hemophilia A) and factor IX (hemophilia B). Hemophilia A comes in two forms: congenital (inherited) or acquired. Congenital hemophilia A represents the condition where an individual is born defecient (to various degrees) of factor VIII. Acquired hemophilia A is a condition in which an individual spontaneously produced an autoantibody to factor VIII, leaving the body unable to use the factor VIII that may be present. Hemophilia B is an inherited condition where the individual has a mutation of the factor IX gene and is unable to produce adequate levels of this coagulation factor.In some cases, patients have multiple factor deficiencies that are secondary to a primary condition such as vitamin K deficiency, disseminated intravascular coagulation (DIC), and liver disease. With vitamin K deficiency, the liver is unable to produce the coagulation factors that are vitamin K-dependent. During liver disease, the liver may be unable to produce coagulation factors effectively. In DIC, the clotting processes are in overdrive and will consume the coagulation factors that are being produced, leading to low levels of circulating coagulation factors.

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Diabetes and the Current American Diabetes Association Guidelines
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.

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Type 1 Diabetes

Type 1 diabetes is caused by an absolute deficiency of insulin from an autoimmune destruction of pancreatic beta cells or degeneration of these cells. The infiltration of mononuclear cells can be precipitated by environmental factors such as viruses, chemicals, and cow's milk or caused by unknown or idiopathic reactions. Ordinarily the individual has an inherited susceptibility to this autoimmune reaction and diabetes develops suddenly. Most often this onset occurs in childhood or young adult years. Type 1 diabetes encompasses about 10% of diabetes cases.Because of the beta-cell destruction, type 1 diabetic patients require insulin to prevent ketosis and reduce complications of this disease.This class was formerly Type I Insulin Dependent Diabetes Mellitus (IDDM) and referred to as juvenile-onset diabetes. The ADA has abolished using these designations but are noted in this review to correlate previously learned information with new recommendations.

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Type 2 Diabetes Continued

Often with change in environmental factors (diet changes, weight loss, and exercise), a type 2 diabetic can regain acceptable glycemic control. If not, oral hypoglycemic medication is required. An absolute insulin deficiency may develop late in the disease and insulin would then be required.Type 2 diabetes accounts for the majority of those with diabetes, probably 80-90%. Ordinarily insulin resistance and deficiency develop in adult years. Due to poor diet and decreased physical activity, many young adults and school-age children are currently diagnosed with type 2 diabetes in US.Type 2 diabetes was formerly Type II Non-Insulin Dependent Diabetes Mellitus (NIDDM) and referred to as adult-onset diabetes. Again the ADA recommends discontinued use of these designations.

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Type 1 or Type 2

Some patients at diagnosis of diabetes are not clearly a type 1 or type 2 diabetic. A type 2 diabetic may present with ketoacidosis. Some type 1 diabetics develop the disease slowly despite having the autoimmune characteristics of type 1. Often the diagnosis is diabetes but classification as type 1 or type 2 takes time and later in the course of the disease, the type is more obvious.

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Microvascular Damage

Injury to tiny vessels is more often associated with type 1 diabetes but also occurs in other classes of diabetes. Damaged vessels lead to retinopathy, nephropathy, and neuropathy. Diminished eyesight, blindness, renal disease and renal failure can occur in a diabetic patient who does not maintain good carbohydrate control and can occur in a diabetic with good control because of the harm done to the vessel linings. Neuropathy results in pain, numbness, tingling, dizziness, decreased nerve conduction and can progress to cardiac disease and failure.

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Macrovascular Complications

These complications can occur in either type 1 or type 2. Heart disease, stroke, and peripheral vascular disease result from damage to larger vessels. Type 2 diabetic patients often have hyperlipidemia and atherosclerosis leading to a greater risk of heart disease and heart failure.

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Other Complications

Ketoacidosis is always a serious complication for type 1 diabetics. Due to lack of uptake of glucose into cells by insulin, proteins and fats are utilized as energy sources. This results in excess acetyl CoA which is converted to ketone bodies. A serious acidosis results and if untreated or not resolved by the body, coma and death can occur.Most often the acetyl CoA in a type 2 patient is converted to cholesterol and results in hyperlipidemia and heart disease in these patients.The elderly type 2 diabetic is at risk for a hyperosmolar nonketotic coma. The patient becomes dehydrated due to increased urine excretion to lower the blood glucose. If reduced renal or cardiac function is also present, glucose excretion is impaired and blood glucose concentrations can become extremely high. Ketones are not produced in excess, thus the patient remains nonketotic. Insufficient hydration, elevated blood glucose, and decreased renal excretion of waste products result in an increased osmolality and total concentration of all plasma components.

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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.

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Urinary Albumin Excretion

Screening for early occurrence and low amounts of albumin in urine detects microvascular disease before impaired renal function and insufficiency occur. Regular screening of urinary albumin excretion (UAE) is recommended for individuals with both type 1 diabetes and type 2 diabetes as an early indicator of renal disease. It is recommended at the time of initial diagnosis and annually thereafter for patients with type 2 diabetes, and commencing annually 5 years after the initial diagnosis of type 1 diabetes. Control of blood pressure and blood glucose concentrations can slow the rate of renal function decline.

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What is the role of microalbuminuria testing?View Page
Monitoring Diabetic Glycemic Control

A HbA1C that is <7.0% indicates glycemic control for most adults with diabetes.Providers might recommend even lower HbA1c goals than the general goal of <7.0% for some patients ( if this can be achieved without significant hypoglycemia or other adverse effects). This includes patients who have a short duration of diabetes (i.e., gestational diabetes), long life expectancy, and no significant cardiovascular disease.Less stringent HbA1c goals than the general goal of <7.0% may be appropriate for patients with a history of severe hypoglycemia, limited life expectancy, advanced microvascular or macrovascular complications, and those individuals with longstanding diabetes who are not able to consistently achieve the general goal of <7.0%.

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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.

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Emerging Cardiovascular Risk Markers (retired 12/6/2013)
Introduction

We are all aware of the clinical laboratory's role in assessing overall health and we are also aware that measuring a patient's serum lipids will provide some insight into their cardiovascular health. The traditional measurements of low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides are the 'classic' cardiovascular risk markers.Laboratorians, and even the general public are now well-aware that LDL-C ('bad' cholesterol) concentrations should be low while HDL-C ('good' cholesterol) concentrations should be high. Triglycerides should be kept in check as well. Optimal levels are shown in the table below. So what is the risk if these values are not within optimal ranges?Cardiovascular risk can be simply defined as increasing the odds of having a pathology which affects blood flow and/or the heart. The most common cardiovascular pathology is atherosclerosis. Other cardiovascular pathologies whose odds increase as serum lipids and other cardiovascular markers become suboptimal are myocardial infarction (heart attack), stroke, congestive heart disease and coronary artery disease. Other diseases such as diabetes and the metabolic syndrome are also strongly associated with the classic cardiovascular risk markers LDL-C, HDL-C and triglycerides.

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Introduction cont.

The importance of cardiovascular risk markers arises from the fact that cardiovascular disease is the leading cause of death in the United States and that traditional lipid screening protocols often fail to identify high-risk patients. A recent prospective study of nearly 28,000 healthy middle-aged women showed that 77% of cardiovascular events occurred in those with LDL-C values below 160 mg/dL while 46% occurred in those with levels below 130 mg/dL. By using other or additional cardiovascular risk markers we can detect and treat those at risk earlier.

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Risk Markers

We have listed the 'classic' cardiovascular risk markers as LDL-C, HDL-C and triglycerides. But there are many more cardiovascular risk markers as well as cardiovascular risk factors. A cardiovascular risk factor is a condition (not a laboratory analyte) that is associated with an increased risk of developing cardiovascular disease. Examples include: Age Gender (males are at increased risk) Heredity Hypertension Cigarette Smoking Obesity Diabetes StressThere are also negative risk factors, factors which decrease a person's risk of cardiovascular disease. Examples include: Optimal HDL-C concentration Exercise Estrogen Moderate alcohol intakeThis course will not focus on cardiovascular risk factors. Instead we will focus on newer, emerging cardiovascular risk markers. There are well over twenty well-studied cardiovascular risk markers; in this course we will focus on some of the more established markers and the ones which are becoming more commonly measured in the clinical laboratory. These include apolipoprotein A1/apolipoprotein B100, Lp(a), oxidized LDL, LpPLA2, hsCRP and lipoprotein particle size and concentration.It is important to remember that the association between a cardiovascular risk marker and actually having or developing cardiovascular disease is a statistical one. The fact that a patient has a particular risk marker which is abnormal simply increases the probability of developing cardiovascular disease, it does not mean that he or she is certain to develop cardiovascular disease. Conversely, if an individual does not have a particular cardiovascular risk marker present it does not guarantee protection against cardiovascular disease. We must always remember that some percentage of individuals who have heart attacks or strokes will not have abnormal risk markers present.

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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.

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Which of the following statements is true?View Page
Importance of Determining Size and Number of Lipoprotein Particles

In the clinical laboratory, we routinely measure the cholesterol content of high-density lipoprotein and low-density lipoprotein particles and not the apolipoproteins on the particles or the number of particles. Proprietary detergents and reagents are used in assays for HDL-C and LDL-C to separate lipoproteins, allowing the cholesterol content of specific lipoproteins to be measured. For example, HDL-C is commonly measured using a solution of dextran sulfate and magnesium to selectively precipitate HDL from the other lipoproteins present in the sample. Once isolated, the HDL particles are 'dissolved' and the amount of cholesterol in them is determined photometrically using a color-producing enzyme reaction. LDL-C can be measured directly or can be estimated using the HDL-C, triglycerides and total cholesterol (TC) values. The Friedewald formula is often used to calculate LDL: LDL-C = TC - (HDL-C)- (Triglycerides/5). The important point to consider here is that traditional LDL-C and HDL-C measurements only tell us how much cholesterol is associated with each lipoprotein particle class. We are now learning that the number and size of the particles are important as well. The number of LDL particles appears to be more strongly predictive of cardiovascular disease than the LDL-C content, and small dense LDL are known to be more atherogenic than larger, less dense LDL particles.

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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.

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Lp(a)

Lipoprotein (a) is a modified version of LDL containing a unique protein, apolipoprotein (a). It was discovered in 1963 and is well-associated with vascular disease. Do not confuse apolipoprotein (a) with apolipoprotein A that is found on high density lipoprotein particles. Lipoprotein (a) is abbreviated as Lp(a). Lp(a) is an LDL particle whose ApoB molecule has formed a disulfide bond with another protein called Apo(a), see figure. Apo(a) is a protein very similar in structure to plasminogen. Numerous retrospective case control studies and prospective studies have shown Lp(a) to be an independent risk factor for vascular disease. This means that Lp(a) levels alone (not in conjunction with LDL, or patient risk factors) can predict cardiovascular risk. Lp(a) has been called the most atherogenic lipoprotein. Serum concentrations of Lp(a) are related to genetic factors; drugs and diet changes do not typically lower Lp(a) as they do LDL.

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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.

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High Sensitivity-C-Reactive Protein

C-reactive protein (CRP) is a very sensitive acute phase reactant. Serum CRP levels increase following a variety of pro-inflammatory events such as infection, tissue necrosis, trauma, surgery and even malignancy. CRP levels can increase quickly and dramatically (often 100 fold) during inflammation. CRP can activate compliment, bind Fc receptors and can function as an opsonin, enhancing phagocytosis with certain infections. Measurement of CRP is not new, it has been on clinical laboratory testing menus for decades. However, a newer version of the CRP test is now in use to assess cardiovascular risk.High sensitivity-CRP (hs-CRP) assays have been developed that are more sensitive to the more subtle changes that can occur during chronic vascular inflammation. (Recall that atherosclerosis is an inflammatory process.) By measuring hsCRP we can get a glimpse at vascular function. CRP has been shown to be an independent risk factor for atherosclerotic disease and cardiac death. A 2002 prospective study of more than 27,000 patients showed that the CRP concentration is a stronger predictor of cardiovascular events than the LDL-cholesterol level.

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The hs-CRP Test

The traditional CRP test uses immunoassay methods that are sensitive to concentrations of 5-20 mg/L. The hs-CRP test, with its increased sensitivity, is able to detect C-reactive protein in lower levels, 0.5-10.0 mg/L. As with most risk markers, the results of hs-CRP testing are generally interpreted on a relative scale; the higher the value, the higher the risk of a future cardiovascular event.The American Heart Association and Centers for Disease Control and Prevention has defined risk groups with hs-CRP as follows: Low risk: < 1.0 mg/L Average risk: 1.0 to 3.0 mg/L High risk: > 3.0 mg/L It is important to note that hs-CRP assays are measuring the same protein as traditional CRP assays. Thus, in patients with active inflammation (such as chronic, active arthritis; lupus; infection; etc.) hs-CRP values would be expected to be high and would not necessarily implicate cardiovascular risk. If values greater than 10 mg/L are seen in repeated measurements, a non-cardiovascular cause should be considered. Taking anti-inflammatory drugs (NSAIDs, aspirin, etc.) or the statin-class of cholesterol-lowering drugs may reduce CRP levels in patients. This is not an artifact, but is thought to be an effect of treating the underlying inflammatory process.

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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.

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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.

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Size and Number

Although lipoproteins of a particular class are generally within a given size range, there are many biochemical processes that interact with lipoproteins to alter their size, density, and lipid composition. When low-density lipoprotein (LDL) becomes smaller and denser, it is more likely to interact with the arterial wall, leading to deposition of cholesterol and initiating or worsening atherosclerosis. Research has shown that high numbers of smaller, denser LDL are more atherogenic than larger, lighter LDL particles. Small, dense LDL particles are associated with more than a three-fold increase in the risk of coronary heart disease.

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Erythrocyte Inclusions
The presence of erythrocyte inclusions may indicate the presence of disease.View Page
Which of the following is NOT present in normal bone marrow and would be associated with a disease or disorder?View Page

Erythrocyte Inclusions (retired 7/10/2012)
The presence of erythrocyte inclusions may indicate the presence of disease.View Page

Fundamentals of Hemostasis
The product administered to treat Von Willebrands Disease is:View Page
An Introduction to the Fundamentals of Coagulation

As we will discover later in the course, there are other variables which impact the effectiveness of hemostatic mechanisms as well, such as acquired disease states, and inborn metabolic pathway defects.For now, however, our focus will be on the mechanisms, processes, and components which work together to achieve coagulation, or the cessation of blood flow from a damaged vessel.Note: The terms coagulation and hemostasis are used interchangeably throughout this course.

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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.

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Coagulation Disorders

This course began with a discussion on homeostasis, the body's desire to maintain a status of physiological equilibrium. Our inborn system of chemical checks and balances, activators and inhibitors, can be disrupted by numerous factors, two of the more common being acquired disease states and disorders passed on to offspring via inheritance. In regard to coagulation, both disease status and genetics can adversely affect the functionality of many hemostatic processes. Impaired hemostatic mechanisms, whether acquired or inherited, may cause either hemorrhage or thrombosis.Hemorrhage (bleeding external to the vasculature) most often stems from physical vessel trauma, but may also arise from a wide variety of disease states.Thrombosis does not require physical trauma, and is the activation of hemostatic processes at an inappropriate time in an inappropriate place, and may arise from a number of inherited or acquired disease states. The following pages are intended to serve as an introduction to some of the more commonly encountered coagulation disorders.

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Coagulation Disorders - Inherited

Von Willebrands Disease is a platelet disorder. This disorder is characterized by a functional defect in Von Willebrands factor (vWF) itself. This disease often clinically manifests with a concurrent deficiency of factor VIII, but will present with a normal platelet count. As far as genetics and inheritance, both men and women are affected equally. Von Willebrands factor is essential for platelet binding, therefore, a defect in vWF causes impaired platelet adhesion and aggregation. The treatment of Von Willebrands Disease involves the administration cryoprecipitate, as it is rich in vWF.

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Coagulation Disorders - Acquired

Disseminated Intravascular Coagulation (DIC) is best described as a disorder of consumption, because clotting factors are depleted from the blood. Basically, clotting occurs randomly throughout the body, as opposed to just in the localized areas where vascular damage has occurred, consuming clotting factors and other components such as platelets in the process. Symptoms may range from a mild bleed, to severe, profuse bleeding, primarily dependant upon the availability of clotting factors. As more and more coagulation factors and components are consumed, the disorder progresses and symptoms worsen. Most heavily impacted are the levels of factors I, V, and VIII as well as the number of available platelets. Clinically, DIC is detected via an elevated (positive) FDP, positive D-dimer test, a prolonged PT and APTT, plus the manifestation of hemorrhagic episodes. DIC is diagnosed as two primary types, acute and chronic. Acute DIC manifests in a few hours or a few days, has a high mortality rate, and is seen in infections, obstetric complications, liver disease, and tissue injury. Chronic DIC is a secondary condition to some other disease state. Once you treat the primary disease, this type of DIC will go away. Treatment is often factor replacement therapy through the use of fresh frozen plasma and/or cryoprecipitate.

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Coagulation Disorders and Liver Disease

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

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Which of the following statements regarding coagulation disorders is correct?View Page

Fundamentals of Molecular Diagnostics (retired 2/12/2013)
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

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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?"

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Human Genome

Much research has been conducted to identify the alphabet of the human cellular language otherwise known as the human genome. This identification or roadmap of the human genetic material has opened the door to the mainstreaming of molecular diagnostics within the clinical laboratory setting.While the mapping of the human genome project is complete, many times it is not necessary to be able to identify the entire sequence; rather, we can use the specific portion of the code that is unique to the disease or condition in question. These short portions of the genetic molecular sequence or oligonucleotides, can then be used as probes to seek out and detect or amplify the target sequence.

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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).

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General Laboratory Question Bank - Review Mode (no CE)
Which of the following best defines "sensitivity":View Page
Which of the following best defines "specificity":View Page
Which one of the following does not directly regulate clinical laboratories:View Page
Which of the following cells when found upon microscopic examination of the urine would be most indicative of kidney disease:View Page
C-reactive protein:View Page
Which of the following immunoglobulin classes is chiefly responsible for the degranulation of mast cells and basophils:View Page

Hematology / Hemostasis Question Bank - Review Mode (no CE)
The abnormal cells seen in this illustration are indicative of:View Page
The abnormal RBCs seen in this illustration are indicative of:View Page
The abnormal RBC shape seen in this illustration is:View Page
The RBCs indicated by the arrows in this illustration are indicative of:View Page
The predominant cells seen in this CSF are suggestive of:View Page
Which two of the following are associated with macrocytic anemia?View Page
Eosinophilia is commonly found in which of the following disorder(s):View Page
Which of the following morphologic changes is most characteristic of sickle cell disease?View Page
Hypersegmentation of granulocytes is most commonly associated with:View Page
Match the disease conditions on the left with appropriate red cell appearances on the right:View Page
Hemophilia A, hemophilia B, and Von Willebrand's disease together constitute approximately what percentage of all hereditary coagulation disorders:View Page
Hemophilia B or Christmas disease is the result of a hereditary deficiency in which coagulation factor:View Page

Hemoglobinopathies: Hemoglobin S Disorders
Sickle Cell Disorders

Sickle cell anemia is a qualitative hemoglobin synthesis disorder known as a hemoglobinopathy. Sickle hemoglobin (HbS) is a structural disorder caused by valine replacing glutamic acid in the sixth position on the beta chain. The heterozygous state, HbSA, is known as sickle cell trait, while the homozygous state, HbSS, is sickle cell anemia or sickle cell disease. A double heterozygous condition known as Hemoglobin SC disease also exists where one beta chain carries the mutation for HbS and the other beta chain carries the mutation for HbC. In addition, HbS can be present with thalassemia.Sickle cell anemia can also demonstrate hereditary persistance of fetal hemoglobin (HbS/HPFH).Other HbS combinations are very rare and include HbS/HbE, HbS/HbD LosAngeles, HbS/HbG-Philadelphia, and HbS/HbO Arab.

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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.

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Vaso-occlusive Crisis

HbSS blood may contain reticulocytes with an abnormal presence of CD36 on their membranes, allowing platelets to form a bridge between these young sickle cells and endothelial cells in post-capillary venules. This initial slow down of blood flow creates an environment in which cells containing HbSS can easily form sickled cells.The rigid cells, which are formed as a result of the sickling process, can collect in and plug small blood vessels. This can then cause tissue damage and organ infarction. In addition, the polymerized hemoglobin is thought to disrupt the RBC membrane, exposing phosphatidylserine that can trigger hemostasis. Subsequently, white blood cells (WBCs) may adhere to endothelium in response to recruitment in the inflammatory process. This leads to further occlusion as neutrophils capture additional RBCs in the post-capillary venules.Contributing further to vaso-occlusion is the decreased level of L-arginine in patients with sickle cell disease. L-arginine is a substrate needed to produce nitric oxide (NO). NO has vasodilatory properties as well as anti-inflammatory and anti-platelet properties. Thus a decrease in NO may lead to increased cellular adherence to endothelium.

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Aplastic Crisis

The aplastic crisis or phase is caused by the need of the bone marrow to shut down temporarily due to the continual stimulus for production of erythrocytes. During this time, a severe anemia may be present due to the shortened life span of the red blood cells. In addition, infections that sickle cell patients encounter may arrest red blood cell production and also produce a pancytopenia. Parvovirus B19 is the most common causitive agent of aplastic crisis in sickle cell disease.

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Risk Factors

Persons with sickle cell disease (SCD) are prone to crises occurring when they experience abrupt changes in temperature or have a fever, are dehydrated, or hypoxic (including entering high altitudes where oxygen is decreased). Physical exertion, pregnancy, and psychological stresses can also precipitate sickle cell crises.Prognosis in SCD is related to the number of sickle cell crisis episodes. Persons who experience more than three episodes of crisis requiring treatment in a single year, have an increased poor prognosis.The average age of survival for women with sickle cell disease (HbSS) is 48 years, while for men the average age of survival is 42 years.

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Vaso-occlusive Crisis, Continued

This painful event of vaso-occlusive crisis often results in tissue necrosis. Organs affected include the bone marrow, brain, lungs, kidneys, liver, and spleen. Disorders that may result include bone and joint pathology, stroke, acute chest syndrome, nephropathies, and infections. Triggering mechanisms for this crisis include infection, fever, acidosis, dehydration, cold temperatures, anxiety, stress, and depression. Adults may experience acute chest syndrome due to pulmonary infarcts caused by sickling in the pulmonary microvasculature, whereas children with sickle cell disease can experience acute chest syndrome due to infections.

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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.

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Vascular Occlusion

Vascular occulsion seen in sickle cell disease may be a result of cellular interactions. Young sickle cells (reticulocytes that contain HbSS) tend to be more "sticky" and adhere to endothelium. Abnormally present receptors on these cells include CD36, which allows platelets to form a bridge between sickle RBCs and endothelial cells in post-capillary venules. Adding to this occlusive process are leukocytes that respond to tissue damage and inflammation in these vessels.

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Cellular Dehydration

Cells containing hemoglobin S have a decreased capacity to maintain normal levels of potassium (K+). As K+ leaves the cell, water follows. Two mechanisms are responsible for maintaining intracellular ion levels, the Gardos channel and the K+-Cl- channel. Both channels are abnormally activated in patients with sickle cell disease. The resulting loss of water from the cell increases the hemoglobin concentration and the chances for sickling.

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HbS with Other Hemoglobins

Hemoglobin SC occurs as a double heterozygous condition in less than .5% of African Americans but can be as high as 25% in West Africa. Although HbSC disease may produce a less severe anemia, the chance for retinal hemorrhage and renal and bone necrosis is greater due to increased viscosity. Most persons with HbSC disease have splenomegaly, but significant symptoms usually do not show up until the teenage years.The double heterozygote for HbSD is quite rare and produces an anemia midway in severity between sickle cell disease and sickle cell trait.The rare double heterozygote for HbE produces an anemia similar to Hb S beta-thalassemia.

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HbS / Thalassemia

HbS/thalassemia combination Affected populations Severity Comments HbS beta thalassemia North Africa, India, and the Mediterranean region, especially Greece and Turkey. Varies HbS beta-plus thalassemia, type 1 and HbS beta-minus thalassemia need supportive therapy and may have severe anemia HbS beta-plus thalassemia, type 2 requires very little medical attention Hb SA alpha-plus thalassemia Common in persons of African ancestry Usually asymptomatic Less hemoglobin S produced than in persons with Hb S trait Hb SS-alpha thalassemia (either plus or zero) African and Mediterranean ancestry Mild anemia midway in severity between sickle cell disease and trait Produce increased levels of Hb F in proportion to the number of alpha gene deletions present. This acts to retard the sickling process.

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Hb S with Hereditary Persistence of Fetal Hemoglobin

Approximately 1% of persons with homozygous sickle cell disease also demonstrates hereditary persistence of fetal hemoglobin (HPFH). Persons with HbS/HPFH have a milder anemia than individuals with SCD who have none to normal levels of HbF. Increased fetal hemoglobin protects the cell from sickling because of its higher affinity for oxygen. HPFH may also be present in other hemoglobinopaties and thalassemias or in SCD in combination with other hemoglobins (HbSC/HPFH) and thalassemia (HbS/Bthal/HPFH).

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Pharmacotherapy

Any of three major classes of compounds can be used in the management of pain in sickle cell disease. These compounds include opioid, non-opioid, and adjuvants.Opioids include codeine, levophanol, morphine, ocycodone, and pethidine, among others.Non-opioids include salicylates, nonacetylated salicylates, acetic acid derivatives, anthranilic acid derivatives, proprionic acid derivatives, and oxicams.Adjuvants include anti-convulsants, anti-depressants, anti-histamines, and bensodiazepines.

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Supportive Care

Supportive care helps patients to understand their disease, learn how to avoid crisis precipitating situations, and develop coping skills. Supportive care includes educating patients about the genetics and inheritance patterns for sickle cell, their health care rights, and their responsibilities as patients with sickle cell disease.Supportive care also provides patients with support groups and patient advocacy.

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Blood Tranfusions

The use of blood transfusions for stroke prevention in children with sickle cell disease (SCD) has become a standard of care. The goal is to keep the level of HbS under 30%. Transfusions are not normally needed for SCD unless patients develop a sudden worsening of anemia due to splenic sequestration or infection. Of concern is the occurance of iron overload. Iron chelators are helpful in the management of iron overload and include desferrioxamine and deferasirox.Transfusing phenotypically matched blood (especially C, E, and K) is highly recommended to prevent alloimmunization.

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Molecular Therapy

Most sickle cell patients who have increased levels of HbF experience milder forms of the disease than do patients with normal or low levels of HbF. Therefore, the focus of molecular treatments for sickle cell is to increase the level of fetal hemoglobin (HbF). The only drug currently approved by the FDA, which is used to induce increased production of HbF, is hydroxyurea. Hydroxyurea is a myelosuppresent and a ribonucleotide reductase inhibitor. However, the mechanism of hydroxyurea's influence in HbF production is not well understood. Hydroxyurea may also contribute to reduction of vaso-occlusion.

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Natural HbF Inducers

Alternative and natural remedies are under investigation for the use of inducing HbF production in patients with sickle cell disease. One of these natural remedies is resveratrol extract, which is found in red wine and grape skins. Microorganisms that produce various HbF inducers include A. arcangelica, S. hygroscopicus, and Streptomyces species.

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Transplantation and Gene Therapy

Bone marrow transplants may be a cure but currently the risks are too high. Impediments to transplantation include the lack of matched sibling donors and prior transfusions, which have exposed the patient to donor antigens.The best candidates for bone marrow transplants are children less than 16 years old. Two umbilical cord blood transplantations have been performed that reportedly have not remanifested with sickle cell disease.Gene therapy may become an option in the future that may alter the expression of the sickle gene.

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Which of the following is an FDA-approved therapeutic agent for treating sickle cell patients?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.

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Solubility Test

Most hemoglobins are soluble in a high-molarity phosphate buffer; hemoglobin S is not. The buffer is made of of dibasic and monobasic potassium phophates, Saponin and dithionate. Kits are available, which consist of this reagent, pipets and a reading rack. A 1:100 dilution of blood into buffer is made, incubated for 5 minutes, and turbidity is observed against a white background with black lines.A positive result (A below) is indicated by a turbid solution. A negative result (B below) is obtained when lines are visible through the solution.The solubility test should only be used as a screening test as it is not reliable for diagnosing sickle cell disease.

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Hemoglobin Electrophoresis Patterns in Sickle Cell Disorders (Alkaline)

The following list corresponds to this image of an alkaline hemoglobin electrophoresis.Lanes 1 and 2 contain controls ASC and AF (Remember, AF and ASC are labels and do not indicate the order of migration.)Lanes 3 and 4 (patient 1): Homozygous sickle cell disease (HbSS) Hb S is 100%Lanes 5 and 6 (patient 2): Heterozygous sickle cell trait (HbSA)Lanes 7 and 8 (patient 3): Double heterozygous HbSC disease (HbSC)Lanes 9 and 10 (patient 4): Sickle cell hemoglobin with hereditary persistance of fetal hemoglobin.

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Hemoglobin electrophoresis patterns in Sickle Cell Disorders (acid)

The following list corresponds to this image of an acid hemoglobin electrophoresis.Lanes 1 and 2 contain controls ASC and AF (Remember, AF and ASC are labels and do not indicate the order of migration.)Lanes 3 and 4 (patient 1): Heterozygous Sickle Cell Trait (HbSA) Hb S is approximately 30%Lanes 5 and 6 (patient 2): Double Heterozygous HbSC Disease (HbSC)Lanes 7 and 8 (patient 3):Homozygous Sickle Cell Disease (HbSS)Hb S is 100%Lanes 9 and 10 (patient 4): Sickle Cell Hemoglobin with Hereditary Persistance of Fetal Hemoglobin. Hb S is approximately 70%; HbF 30%.

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RBC Morphology in Sickle Cell Disease (HbSS)

Sickle Cell Anemia (HbSS) is a hemolytic anemia, characterized by the presence of drepanocytes (sickle cells) and polychromasia (increased reticulocytes). Nucleated red blood cells (NRBCs) may be seen during episodes of severe hemolysis. The absence of polychromasia may indicate aplastic crisis. The homozygous state of hemoglobin SS causes RBCs to take on the characteristic sickle shape when hemoglobin is in a deoxygenated state. The name "sickle" comes from the tool (seen in the upper image) that is used to manually cut hay. When RBCs sickle they take on the same shape as the blade of the sickle, as seen in the bottom image.

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RBC morphology in Sickle Cell Anemia variant (HbSC)

Codocytes (target cells) are the most common RBC morphologies encountered in the double heterozygous state known as hemoglobin SC disease (HbSC) . HbSC red blood cells are often dehydrated and become xerocytes (cells that have their hemoglobin pooled to one side-half moon cells). Some HbSC red blood cells crystallize as they dehydrate and attempt to take on both the sickle shape and the shape of hemoglobin C crystals (top image). This dual hemoglobin crystal can have many unique shapes. A HbSC crystal is indicated by the arrow in the bottom image on the right.

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CBC and RBC morphology

CBC results for patients with sickle cell disease (HbSS) can reflect a slight, moderate, or severe anemia. The CBC and peripheral blood smear shown below are those of an African-American 12-year-old male patient with HbSS.Reference intervals may vary between facilities and are dependent on patient age and gender. The reference intervals that are shown below are specific to this case. Parameter Patient Result Reference Interval White blood cell count (WBC) 18.9 3.2 - 9.8 x 109/L Red blood cell count (RBC) 3.88 4.50 - 5.70 x 1012/L Hemoglobin 10.0 13.8 - 17.2 g/dL Hematocrit 32 41 - 50% MCV 82 80 - 110 fL RDW 22.7 11 - 14.5 Platelet count 458 140 - 440 x 109/L

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Hemoglobin Electrophoresis

The lanes labeled as "patient" correlate with this Sickle Cell Disease case. The attached PDF can be used as a key to determine the areas of migration.

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CBC and RBC morphology

Patients with double heterozygous Hemoglobin SC Disease (HbSC) may have a slight to moderate anemia. The CBC and peripheral blood smear for an adult patient with HbSC appear below. The arrow in the image of the peripheral blood smear is pointing to a HbSC crystal.Reference intervals may vary between facilities and are dependent on patient age and gender. The reference intervals that are shown below are specific to this case. Parameter Patient Result Reference Interval White blood cell count (WBC) 10.4 4.5 - 11.0 x 109/L Red blood cell count (RBC) 3.98 4.50 - 4.70 x 1012/L Hemoglobin 10.5 11.7 - 16.0 g/dL Hematocrit 33.5 35 - 47% MCV 84 81 - 101 fL RDW 16.0 11 - 14.5 Platelet count 160 150 - 450 x 109/L

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CBC and RBC morphology

CBC results for patients with Sickle Cell Disease who have a hereditary persistence of fetal hemoglobin (HbS/HPFH) may appear normal or demonstrate a mild to moderate anemia. The CBC and peripheral blood smear for a patient with HbS/HPFH appear below.Reference intervals may vary between facilities and are dependent on patient age and gender. The reference intervals that are shown below are specific to this case. Parameter Patient Result Reference Interval White blood cell count (WBC) 6.4 3.8 - 9.8 x 109/L Red blood cell count (RBC) 4.78 4.50 - 5.90 x 1012/L Hemoglobin 11.9 13.8 - 17.2 g/dL Hematocrit 37.7 41 - 50% MCV 81 80 - 100 fL RDW 13.2 12 - 14.6 Platelet count 230 140 - 440 x 109/L

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Hemolytic Disease of the Fetus and Newborn
Foreword

This course is a refresher on current concepts and practices in hemolytic disease of the fetus and newborn (HDFN). As such it is a survey course that provides a broad overview of the field and presents an opportunity to review significant aspects of HDFN and its laboratory investigation and prevention. Because it is a survey course with many topics, not all will be covered in depth. However, Rh immune globulin (RhIg) will be reviewed extensively since it prevents the most severe form of HDFN and is one of the biggest success stories of modern medicine. The course assumes that participants have a basic background knowledge of immunohematology theory and practice. Reading the resources in Further Reading for more information on any topic is encouraged. In brief, the course will: Recap relevant background information on HDFN and its treatment Review the characteristics and uses of Rh immune globulin (RhIg) Discuss typical laboratory findings and their interpretations Examine current best practices in perinatal testing programsThe course is a companion to "Rh negative female with anti-D at delivery: A case study on dealing with the issues" and complements its content.

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Introduction

Although HDFN can be life threatening, in the case of anti-D it is a disease that can be prevented. Regardless of causative antibody, HDFN's serious consequences can be lessened by early laboratory diagnosis and treatment. This course begins with an in-depth review of HDFN and later discusses its prevention in detail. In reviewing HDFN, key questions to be answered include: What are the typical signs and symptoms of severe HDFN? Which serologic tests does the transfusion service laboratory use to diagnose HDFN? How is severe HDFN treated? Which development dramatically changed the incidence of HDFN due to anti-D? Other than the causative antibodies, what are some of the main differences between ABO HDFN and HDFN due to anti-D and other antibodies?

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Prenatal Treatment

Prenatal treatment of severe HDFN due to anti-D consists of in utero transfusions. Because of significant risks, transfusion is indicated only if fetal monitoring suggests significant hemolytic disease. 1. Intrauterine Transfusion (IUT)IUTs are done when fetal monitoring indicates severe HDFN and the fetus is too premature for early delivery. IUTs involve the intraperitoneal infusion of packed red cells. The success of the procedure depends on absorption of the red cells through the subdiaphragmatic lymphatic vessels of the fetus. 2. Intravenous transfusion (IVT)Because there may be erratic and inconsistent absorption of intrauterine transfusions in severely hydropic fetuses, IVTs were developed. IVTs involve transfusing donor RBC directly into the umbilical vein.

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Postnatal Treatment: Exchange Transfusion

Whenever possible, a hallmark of HDFN treatment is to induce labor as early as possible once lung maturity has been attained so that the newborn will be able to survive. Once the infant is born, the main treatment for severe HDFN due to anti-D (and other antibodies causing severe disease) is exchange transfusion. In exchange transfusions, up to 85–90% of the infant's blood can be exchanged with donor blood by a process of removing 5–20 mL of blood at a time, and injecting an equivalent amount until the exchange is complete. An exchange transfusion accomplishes the following: Removes bilirubin and thus helps prevent kernicterus; Removes sensitized red cells that have not been broken down yet; Removes circulating maternal antibody; Provides antigen-negative red cells that will not be destroyed by the maternal antibody, thus will survive and provide oxygen to the tissues.

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ABO HDFN - Etiology and Symptoms

ABO HDFN is the most common type of HDFN, in that anti-A is the antibody most often found bound to the red cells of a newborn. While the disease is usually so mild as to not require treatment, severe HDFN is possible. EtiologyABO HDFN is caused by maternal IgG anti-A or anti-B, which can be produced as a result of prior pregnancy or prior inoculation (some common inoculations contain A or B substances). In Caucasians, most often the mother is group O and the child is group A, although other combinations are possible. Group O people tend to produce IgG ABO antibodies more commonly than other blood groups.Just as in other types of HDFN, maternal IgG antibody crosses the placenta and destroys fetal red cells.SymptomsTypical symptoms of ABO HDFN include mild anemia and especially jaundice appearing in the first 24 hours. In rare severe cases the infant can have the more severe symptoms of Rh HDFN, except that prenatal death is unlikely. Rationales to explain the mild nature of ABO HDFN include Fewer A and B antigens on fetal cells Poorly developed fetal A and B antigens Presence of A and B antigens on cells and tissues other than red cells

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ABO HDFN - Treatment

Prenatal treatment Prenatal management and treatment of ABO HDFN is not routinely done because: Titers of anti-A and anti-B do not correlate well with severity of disease; The risks of fetal monitoring (e.g., amniocentesis, cordocentesis) and fetal transfusion are greater than the risk of ABO HDFN since it is usually mild and subclinical. However, if a woman has a history of infants with moderate to severe ABO HDFN requiring treatment, she may be monitored so that the infant can be treated for possible HDFN as soon as possible. Postnatal TreatmentTreatment of ABO HDFN usually consists of phototherapy in which the newborn is placed under a "blue light" that chemically alters bilirubin in the surface capillaries to a harmless substance.For more severe cases, exchange transfusion may be performed. Donor RBC for exchange transfusion in cases of ABO HDFN must meet these criteria: Group O; Rh compatible with infant; Less than or equal to 7 days old (or fresher); Reconstituted with AB FFP to obtain a prescribed hematocrit; CMV negative (or equivalent, e.g., leukoreduced by filtration); Negative for hemoglobin S to prevent blood from sickling under conditions of reduced oxygen concentration in the newborn; Irradiated to prevent graft-versus-host disease. Exchange transfusion is also discussed later in the course in the section related to HDFN due to anti-D and other antibodies. Red Blood Cells are crossmatched with maternal plasma, although the infant's plasma can be used if a maternal blood specimen is unavailable.

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Choosing Donor RBC for IUT and IVT

Donor RBC for IUTs and IVTs have these criteria: Group O Rh negative*; Crossmatched with maternal serum; Fresh: less than or equal to 7 days (or fresher); High hematocrit, e.g, 85–90% (0.85–0.90) to prevent volume overload; CMV seronegative (or equivalent, e.g., leukoreduced by filtration); Negative for hemoglobin S to prevent blood from hypoxia-induced sickling in the fetal circulation; Irradiated with a minimum dose of 25 Gray (Gy) to prevent graft-versus-host disease.* Some laboratories use red cells that are also K-negative since the K antigen is very immunogenic. This also applies to exchange transfusions.

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Criteria for Transfused Red Blood Cells

The Red Blood Cells (RBC) that are chosen for exchange transfusion must meet these criteria: ABO-compatible with mother and infant (usually group O) and lack antigens to any maternal IgG antibodies; If mother has anti-D, RBCs are group O Rh negative; No greater than 7 days old; Reconstituted with AB Fresh Frozen Plasma (FFP) to obtain a prescribed hematocrit, e.g., 45–60% (0.45–0.60); CMV negative (or equivalent, e.g., leukoreduced by filtration); Negative for hemoglobin S to prevent blood from hypoxia-induced sickling; Irradiated with a minimum dose of 25 Gray (Gy) to prevent graft-versus-host disease.RBC are normally crossmatched with maternal plasma, although the infant's plasma can be used if a maternal blood specimen is unavailable.

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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.

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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.

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Hereditary Hemochromatosis (retired 2/13/2014)
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.

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Overview

Because hereditary hemochromatosis (HH) is a disease of iron overload, a review of the basic principles of iron metabolism is helpful in understanding its pathophysiology. Iron is needed by all body cells and is crucial for oxygen transport, oxidative metabolism, and cell growth and proliferation. To serve these functions, iron must be bound to protein. Iron is potentially harmful when ionized or complexed to inorganic compounds. Iron must be present in amounts sufficient to carry out these normal functions, but not in excessive amounts which may be toxic.Two types of iron-containing compounds are normally found in the body: compounds that serve in metabolic or enzymatic functions and storage compounds. Hemoglobin, myoglobin, cytochromes and other proteins are involved in oxygen transport and utilization. Iron in hemoglobin comprises about 67% of total body iron, thus erythrocytes are rich in iron. Approximately 27% of iron is found in storage compounds. Myoglobin, other tissue iron, and transport iron comprise the remaining 6% of total body iron. (2)

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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.

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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)

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Which of the following does NOT contribute to the under-diagnosis of hereditary hemochromatosis (HH)?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.

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Which of the following is NOT considered to be a cause of secondary iron overload?View Page
Transferrin Saturation

Transferrin saturation (TS) is usually reported along with the serum iron (SI) and total iron binding capacity (TIBC). TS indicates the percent of iron binding sites on transferrin that are carrying iron. TS is derived from a calculation using the formula:TS =(SI/TIBC) x 100TS results are reported as percentages. Typical reference intervals for TS are 20% to 55% for males and 15% to 50% for females. TS is currently considered to be a good test for screening persons for hereditary hemochromatosis (HH) due to its sensitivity and specificity for iron overload. It may be elevated prior to significant deposition of tissue iron. TS levels increase as additional iron is accumulated.A drawback to using the TS is that it is dependent on performing both the SI and TIBC. The unsaturated iron-binding capacity UIBC may be a lower cost alternative.The optimal TS criterion for detecting HH is controversial. Using a TS of >60% for males and >50% for females has been found highly accurate in detecting abnormal iron metabolism in persons with HH. Others studies suggest using lower TS levels, e.g. 45%, as a criterion indicating further testing is warranted. Current guidelines from the American College of Physicians include a TS cutoff level of >55% for identifying iron overload. (11)Patients with initially increased TS should be followed by performing a second TS from a fasting morning specimen. The patient should also be advised not to take vitamins supplemented with iron or oral contraceptives for several days prior to the repeated test. TS levels may be affected by diurnal variation, dietary factors, and co-existing disease states such as inflammation and hepatitis. Patients with HH may have falsely normal TS if chronic blood loss or inflammatory disease is present.

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Serum Ferritin

Serum ferritin (SF) level reflects the amount of storage iron in tissues. An elevated SF combined with elevated TS implies primary iron overload. Patients with hereditary hemochromatosis (HH) generally show increases in SF as adults, but a normal SF does not rule out the diagnosis of the disease. Children and premenopausal females with HFE mutations may have had inadequate time to develop iron overload, but may do so later in life.SF alone is inadequate as the sole screening test for HH because it lacks the necessary sensitivity and specificity. SF is frequently elevated in persons with inflammation, cancer, or infection. SF is often ordered along with the serum iron and TIBC when iron overload is suspected. SF is also important is assessing the efficacy of treatment of HH.Upper limits of reference intervals for SF are 200 ng/mL for premenopausal women and 300 ng/mL for men and postmenopausal women. 40 ng/mL is a typical lower limit for the reference interval.SF is measured in serum using immunochemical methods such as enzyme-linked immunosorbent assay (ELISA), immunoradiometric assay, immunochemiluminescent assay, and immunofluorometry. SF tests are available as automated assays and in kit form.(2)

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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.

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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.

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Definitive Tests for Iron Overload

Measuring the amount of iron deposited in the liver is considered definitive for iron overload. This may be done by liver biopsy, computed tomography (CT), or magnetic resonance imaging (MRI). Demonstrating iron in parenchymal liver cells helps determine disease severity. Liver sections obtained by biopsy are stained with Perls Prussian blue which stains iron present in parenchymal cells. A photomicrograph of this reaction is shown.Although liver biopsy may not be necessary for diagnosing hereditary hemochromatosis (HH), it offers the advantage of detecting liver fibrosis if present. Molecular tests for mutations associated with HH are considered the gold standard of current HH testing. Liver biopsy is not needed for diagnosing all patients suspected of having HH, but may be ordered in some cases.

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Other Treatments

Deferoxamine (DFO), an iron chelating agent, may be used to reduce iron overload in patients for whom phlebotomy is contraindicated or not well tolerated. Examples include patients with sickle cell disease or thalassemia whose anemia would be exacerbated by phlebotomies. DFO is seldom used to treat hereditary hemochromatosis (HH) due to the low cost and efficacy of phlebotomy therapy. DFO is typically administered by intravenous or subcutaneous infusion.Patients with HH may be counseled to avoid alcohol use in order to avoid liver damage. With the exception of iron supplements, dietary restrictions on iron ingestion are rarely advised.

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Which of the following is NOT a cause of death in patients with hereditary hemochromatosis (HH)?View Page

HIPAA Privacy and Security Rules
Case Study: Authorization You are working in a physicians office. The doctor orders laboratory and other diagnostic tests on a patient with suspected Alzheimer's disease. The doctor then asks you to give the patient's name and contact information to the local Alzheimer's support group, without getting permission from the patient or the patient's legal guardian. Does the doctor need authorization from the patient or the patient's legal guardian to do this?View Page

Histology Special Stains: Carbohydrates
Mucopoloysaccharides (continued)

Mucins are generally categorized as Neutral Mucins or Acid Mucins for the purpose of histotechnique. Increased mucin production is indicative of many adenocarcinomas, including cancers of the pancreas, lung, breast, ovary, colon and other tissues. Mucins can also be overexpressed in lung diseases such as asthma, bronchitis, chronic obstructive pulmonary disease (COPD) or cystic fibrosis.

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HIV Safety for Florida
A person commits a misdemeanor of the first degree by:View Page
Risk factors associated with increased HIV infection

The risk factors that increase the risk of an exposure leading to HIV infection are: larger quantity of blood from source person, and blood from source person in terminal stage of HIV disease.

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Confidentiality

A person who receives the results of an HIV test shall maintain the confidentiality of the information received and of the person tested.A person who violates the confidentiality provisions commits a misdemeanor of the first degree.A person who obtains information that identifies an individual who has a sexually transmissible disease and maliciously, or for monetary gain, disseminates this information commits a felony of the third degree.

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Human Papillomavirus (HPV) and Molecular Diagnostic Testing
Introduction

Human papillomavirus (HPV) is a frequently occurring viral sexually transmitted infection (STI). HPV infections often present with mild signs and symptoms or are asymptomatic, and do not always progress to a disease state. HPV infections are especially frequent in adolescents and young adults, between the ages of 15-24 years. About 20 million Americans are currently infected with HPV and there are several million new infections each year in the U.S. Since HPV infections are so common, the CDC believes that most sexually active adults are infected at some point during their lifetime. Though the majority of HPV infections are transient and do not result in serious disease, clinicians are concerned about the HPV infections that cause cervical and other anogenital carcinomas. Diagnosis and management of HPV has changed dramatically with the introduction of DNA methods for diagnosis of HPV infections and vaccines for the prevention of HPV infections. Understanding HPV characteristics and diagnostic testing is important for clinical laboratory scientists.

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Organizations and Agencies

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

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Clinical Diseases Caused by HPV STI Types

Most HPV infections do not cause symptoms or clinical disease. Disease-causing infections develop into lesions that form genital condylomas, precancerous changes on external genitalia, or cervical and other lower genital tract carcinomas.Condyloma acuminata - raised and cauliflower-shaped lesions Condyloma planum - flat and less common lesions High-grade intraepithelial neoplasia - precancerous lesions Cancer - HPV is the causative agent of all cervical cancer and the cause of most vaginal, penile, and anal cancer. The bar graph on the right illustrates the approximate number of HPV-associated cancer cases that occur yearly according to the CDC. This table can be accessed at:http://www.cdc.gov/cancer/hpv/statistics/cases.htm. Accessed March 16, 2011.

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Molecular Testing Basics

Molecular diagnostics or molecular tests are assays that detect nucleic acids, DNA or RNA, in a sample. It is usually impossible to detect a whole strand of DNA or RNA; most often a specific region of DNA or RNA (called a target) that is unique to the organism, mutation, or disease is detected. Because these assays are detecting or quantifying biomolecules that are minute in size and concentration, special techniques are employed. Most assays include a component that is labeled and detection or measurement of the label is used to determine the presence or quantity of target in each sample.Two common techniques used in molecular testing are hybridization and amplification.

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References

Cervical Cancer: Prevention and Early Detection. American Cancer Society. Available at http://www.cancer.org/docroot/CRI/content/CRI_2_6x_cervical_cancer_prevention_and_early_detection_8.asp. Accessed December 1, 2011. Cervista HPV, Cervista HPV – Invader Technology. HOLOGIC. Available at http://www.cervistahpv.com/laboratory/invadertechnology.html. Accessed December 1, 2011.Chin-Hong PV, Klausner JD. Diagnostic tests for HPV infection. Medical Laboratory Observer. October 2004:10-16.Cobo F, Concha A, Ortiz M. Human papillomavirus (HPV) type distribution in females with abnormal cervical cytology. A correlation with histological study. Virology Journal. 2009;3:60-66.Cox JT, Moriarty AT, CastlePE. Commentary on statement on HPV DNA test utilization. American Journal Clinical Pathology. 2009;131:770-773.HPV Vaccine Information for Clinicians. Centers for Disease Control and Prevention. Available at http://cdc.gov/std/hpv/stdfact-hpv-vaccine-hcp.htm. Accessed December 1, 2011.Human Papillomavirus (HPV) Natural History. American Society for Colposcopy and Cytological Pathology. Available at http://www.asccp.org/hpv_history.shtml. Accessed December 1, 2011.Human Papillomavirus (HPV) Vaccines. National Cancer Institute. Available at http://www.cancer.gov/cancertopics/factsheet/prevention/HPV-vaccine. Accessed December 1, 2011.Human papillomaviruses and Cancer: Questions and Answers. National Cancer Institute Fact Sheet. Available at http://www.cancer.gov/cancertopics/factsheet/risk/hpv. Accessed December 1, 2011.Hybrid Capture 2 Technology. QIAGEN - Sample & Assay Technologies. Available at http://www1.qiagen.com/hpv/hc2technology.aspx. Accessed December 1, 2011.Markowitz LE, Sternberg M, Dunne EF, et al. Seroprevalence of human papillomavirus types 6, 11, 16, and 18 in the United States: national health and nutrition examination survey 2003-2004. Infectious Disease. 2009;200:1059-1067.Molecular Diagnostics Fundamentals, Methods, and Clinical Applications. Leal Buckingham and Maribeth L. Flaws. Philadelphia:FA Davis Company, 2007.Schutzbank TE, Jarvis C, Kahmann N, et al. Detection of high-risk papillomavirus DNA with commercial invader-technology-based analyte-specific reagents following automated extraction of DNA from cervical brushings in Thinprep media. Journal of Clinical Microbiology. 2007;45:4067-4069.Solomon D, Papillo JL, Davey DD. Statement on HPV DNA test utilization. American Journal of Clinical Pathology. 2009;131:768-769.Vernick JP, Steigman, CK. The HPV DNA virus hybrid capture assay: what is it—and where do we go from here? Medical Laboratory Observer. Mar 2003:8-13.Voss JS, Kipp BR, Campion MB et al. Comparison of fluorescence in situ hybridization, hybrid capture 2 and polymerase chain reaction for the detection of high-risk human papillomavirus in cervical cytology specimens. Analytical and Quantitative Cytology and Histology. 2009;31:208-216.

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Introduction to Bioterrorism
Category B

Agents in Category B are considered the second highest priority agents and are included in this group because they: Are moderately easy to disseminate Cause moderate morbidity and low mortality Require specific enhancements of Centers for Disease Control and Prevention's (CDC) diagnostic capacity and enhanced disease surveillance

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Agent: Pneumonic plague (Bacterium)

Most likely means of dissemination: AerosolPrimary route of entry: InhalationGeneral signs and symptoms: High fever, chills, headache, coughing up of blood (hemoptysis), and toxemia, progressing rapidly to difficulty in breathing (dyspnea), and bluish discoloration of the skin and mucous membranes (cyanosis).There is another form of the disease called “bubonic plague”. While it is caused by the same organism, it is not transmissible through human contact. Pneumonic plague is transmissible through human contact.

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Agent: Tularemia (bacterium)

Most likely means of dissemination: Solid or aerosolPrimary route of entry: Inhalation, absorption, or ingestionGeneral signs and symptoms: Sudden fever, chills, headaches, muscle aches, joint pain, dry cough, progressive weakness, and pneumonia.The disease is not transmissible through human contact.  When used as a WMD, infection would be acquired by handling infected material, eating or drinking contaminated food or water or by breathing in the bacterium.

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Laboratory Response

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

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What if: Biological Attack

Biological attacks involve bacteria, viruses or natural toxins. The effects of toxins can be immediate but for bacteria and viruses the effects may not be apparent for weeks. A bioterrorist may attack by infecting animals, contaminating food and water, spraying bacteria or viruses into the air. In infections such as smallpox and plague, once a few individuals are infected they can further spread the disease from person to person. An attack could also come from through a building's ventilation system, the mail, or even through exposure to an infected terrorist seeking to spread disease during an infectious stage.

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In Case of a Biological Attack

Listen to the radio for instructions from authorities on whether to evacuate or stay put. If told to stay inside, seek shelter in an internal room or a room with as few doors and windows as possible. Turn off all ventilation and as best as possible seal all openings in windows and doors. Continue to monitor the radio. Some biological attacks may be more immediately apparent than others. Monitor your radio, television, or medical alert for instructions from authorities regarding disease symptoms and how and where to seek medical attention. If you do come in contact with a visible, potentially infectious substance, you should remove and bag your clothes and personal items, wash yourself with warm soapy water immediately, and seek medical assistance.

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Additional Information

Below is additional information that you can obtain off the internet to help you “Be Prepared”.www.ready.gov: This site has emergency preparedness guidance from the United States Dept. of Homeland Security. It also has an excellent training program for kids. Accessed September 25, 2013.www.redcross.org: Preparedness information from the International Red Cross. Accessed September 25, 2013.www.americaswaterwaywatch.org: Prepared by the United States Coast Guard. Discusses what to look for as far as suspicious activities. Accessed September 25, 2013.www.bt.cdc.gov: Discusses agents, disease, and other threats. Accessed September 25, 2013.www.fema.gov/areyouready: FEMA’s most comprehensive source on individual, family, and community preparedness. Accessed September 25, 2013.

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Introduction to Bone Marrow
Collection of Bone Marrow Biopsy

A bone marrow biopsy involves removing a small portion of the bone marrow without destroying the architecture of the marrow. This type of biopsy is necessary when the marrow cannot be aspirated (dry tap) due to a disease process, and also provides additional information complementary to that derived from the aspirate: biopsy specimens are more accurate for assessing cellularity, and infiltrative processes, such as metastatic carcinoma, fibrosis, amyloid, and lymphoma. A biopsy specimen is processed as follows: touch preparation tissue section

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Gaucher Cell

A Gaucher cell is a histiocyte (macrophage) whose cytoplasm is filled with linear or fibrillar material (kerasin). This cell is characteristic of the congenital glycolipid disorder, Gaucher's disease. Gaucher cells may also be seen in the marrow of patients with chronic granulocytic leukemia. When seen in this condition, they are referred to as pseudo-Gaucher cells.

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After Marrow Evaluation

After the marrow is evaluated, the diagnosis is established and extent of the disease is determined. Follow up bone marrow examinations may be needed to monitor changes in the marrow following treatment or when signs and symptoms of relapse occur. To summarize, a bone marrow examination can provide valuable information to aid in the diagnosis of a variety of disorders. Due to the expense involved and the discomfort to the patient, clear indications of need should be present before this examination is undertaken.

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Examples of Conditions

Examples of conditions in which examination of the bone marrow may provide diagnostic information include:Erythrocyte Disordersanemiamegaloblasticsideroblasticiron deficiencyerythrocytosispolycythemia veraLeukocyte DisordersneutropenialeukemialymphomaPlatelet DisordersthrombocytopeniathrombocytosisMiscellaneous Disordersprotein abnormalitiesmultiple myelomaWaldenstrom's Macroglobulinemiadiseases of the RE systemhypersplenismmetastatic carcinomagranulomatous infectionsstorage diseasesGaucher's diseaseNiemann-Pick disease

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Introduction to Flow Cytometry: Blood Cell Identification
Diagnostic Process

In order to work through a white blood cell case, it is first necessary to recognize which cell populations are present and where in the development process such cell populations reside (the focused scope of this introductory course is on chronic / mature processes). It is important to remember that a flow cytometer is just one of the tools used in diagnosis of a disease, and the information that is obtained is almost always considered along with cell morphology, special stains, and other diagnostic indicators.The decision tree below can be used together with the General Cell Types_Surface Markers table to aid in the resolution of the case studies that follow. These case studies and tools are meant to introduce the principles of flow cytometry. There are additional CD markers and processes that need to be considered in more complicated cases. All of the following competencies are needed to interpret a case using the principles of flow cytometry: Knowledge of CD markers and which CD markers are associated with which cell populations. Ability to establish a gated cell population. Skill in reading the cytogram and histogram data.

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Flow Cytometry Course Introduction

Flow cytometry is a laboratory method that allows the simultaneous measurement of multiple physical characteristics of individual cells. A flow cytometer is used to isolate and fluorescently label blood cells based on molecules of interest associated with each cell. Next, the instrument analyzes and stores information about the cells based on the amount of light scatter and the fluorescent light that is emitted.Flow cytometry has many applications including: Immunophenotyping HIV disease assessment Transplant cellular distribution determination and CD3 suppression Leukemia/lymphoma diagnosis, staging, and minimal residual disease testing CD34 quantitation/Stem cell quantitation Fetal hemoglobin detection on red blood cells (RBCs) DNA ploidy determination Research, bacterial identification, population filtering, etc. This course will focus on leukemia/lymphoma assessment and analysis.

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Introduction to the ABO Blood Group System
Immunoglobulin

The predominant immunoglobulin class for the B antibodies produced by individuals with group A phenotype and the A antibodies produced by individuals with group B phenotype is IgM. Small quantities of IgG and IgA may also be present.The ABO antibodies found in the serum of group O individuals include anti-A and anti-B. An antibody designated anti-A,B is also present. Anti-A,B in group O individuals tends to be predominantly IgG, although IgM and IgA components are also present.Infants of group O mothers are at higher risk for hemolytic disease of the fetus and newborn (HDFN) than those born to mothers with group A or B because IgG immunoglobulins readily cross the placenta. IgM molecules do not cross the placenta because of their larger size. However, the HDFN that results is usually mild and often subclinical. Infants generally survive with little or no intervention.It is important to note that immune antibodies are usually IgG. Both naturally occurring and immune ABO antibodies are critically important in transfusion since both sensitize, and usually hemolyze, red cells with the corresponding antigen.

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Laboratory Ergonomics
References

Cornell University. CUErgo. Available at: http://ergo.human.cornell.edu/ Accessed December 6, 2013.National Institute for Occupational Safety and Health. Ergonomics and musculoskeletal disorders. Available at: http://www.cdc.gov/niosh/topics/ergonomics/ Accessed December 6, 2013.UCLA Ergonomics. Musculoskeletal disorders: Anatomy of an injury. Available at: http://ergonomics.ucla.edu/MSD_Anatomy.html. Accessed December 6, 2013.US Department of Labor. Healthcare wide hazards module: Ergonomics. Available at: http://www.osha.gov/SLTC/etools/hospital/hazards/ergo/ergo.html Accessed December 6, 2013.

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Posture and Exercises

Being aware of your posture is important for prevention of MSDs. These are postures that should be avoided:Prolonged or repetitive bending at the waist Prolonged standing or sitting without shifting position Keeping an arm outstretched for a prolonged period of time Holding or turning your head consistently to one side Remaining in an awkward position for a prolonged period of timeThe Centers for Disease Control and Prevention (CDC) recommends several exercises that are beneficial for the prevention of MSDs. These recommendations are included as a resource. A partial representation of the exercises are demonstrated below: Cable Stretch While seated with chin in, stomach in, shoulders relaxed, hands relaxed in lap, and feet flat on the floor, imagine a cable pulling the head upward Hold for 3 seconds and relax Repeat 3 times Sidebend: Neck StretchTilt head to one side (ear towards shoulder) Hold for 15 seconds Relax Repeat 3 times on each side Diagonal Neck StretchTurn head slightly and then look down as if looking in your pocket Hold for 15 seconds Relax Repeat 3 times on each side Shoulder Shrug Slowly bring shoulders up to the ears and hold for approximately 3 seconds Rotate shoulders back and down Repeat 10 times Executive Stretch While sitting, lock hands behind head Bring elbows back as far as possible Inhale deeply while leaning back and stretching Hold for 20 seconds Exhale and relax Repeat Foot Rotation While sitting, slowly rotate each foot from the ankle Rotate 3 times in one direction, then 3 times in the opposite direction Relax Repeat

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Laws and Rules of the Florida Board of Clinical Laboratory Personnel (retired 9/1/2010)
Description of Specialties (3)

Specialists in radioassay use radionuclides to determine the chemical makeup of body fluids such as blood and urine. Specialists in blood gas analysis evaluate lung and breathing function by levels of oxygen, carbon dioxide, pH, and hemoglobin with automated tests. Specialists in histology examine cellular and tissue samples using fixation, dehydration, embedding, microtomy, frozen sectioning, staining, and other similar techniques. Histology specialists licensed as technicians can perform specimen processing, embedding, cutting, staining, and frozen sectioning only under the general supervision of a director, supervisor, or technologist. Specialists in cytology process and interpret samples relating cytopathological disease. Non-gynecological cytology preparations can be screen by a specialist in cytology but final review and interpretation must be done by a physician.

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Description of Specialties (4)

Specialists in cytogenetics detect chromosome abnormalities and genetic disorders. Cytogenetics counseling may only be performed by an individual licenses in the cytogenetics specialty at the director level. Specialists in molecular genetics analyze DNA and RNA to find disease-related genotypes, mutations, and phenotypes in order to detect or predict disease and identify carriers. Specialists in histocompatibility test to determine tissue compatibility, prevent infections, and investigate and post-transplant problems. Techniques include blood typing, HLA typing, HLA antibody screening, disease markers, flow cytometry, crossmatching, HLA antibody identification, lymphocyte immunophenotyping, immunosuppressive drug assays, allogenic, isogeneic and autologous bone marrow processing and storage, mixed lymphocyte culture, stem cell culture, cell mediated assays, and assays for the presence of cytokines. Specialists in andrology and embryology examine gametes and embryos, including production, morphology, number, and motility, to address issues of fertility and infertility.

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Medicare Compliance for Clinical Laboratories
International Classification of Disease (ICD) Coding

ICD codes are used for the classification of diseases and conditions, and for describing signs, symptoms and medical circumstances. These codes are used to indicate the medical necessity of a particular test. All employees who are directly or indirectly responsible for reporting to Medicare must be aware of these guidelines to prevent fraudulent claims:ICD codes can only be supplied by the ordering physician or a representative of that physician. ICD codes cannot be used from a previous laboratory order. If a physician supplies a narrative description instead of an ICD code the laboratory must accurately translate that code using only certified coders. It is against the law to use the wrong ICD code for the purpose of causing or increasing payment for a test.

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Local medical review policies (LMRPs)

Local Medical Review Policies (LMRPs) are published by Medicare for some laboratory tests. They are usually developed for tests that can be used for screening or diagnosis of disease. LMRPs use CPT codes to identify the tests and ICD-9 codes to determine when coverage is allowed. If an LMRP test is ordered by a physician, an ICD-9 code that is included in the LMRP must be given to the laboratory or the Medicare program will not pay for the test. It is against the law for a laboratory to change or add an ICD-9 code submitted by a physician. A laboratory should not submit a claim for an LMRP test that is not accompanied by an acceptable ICD-9 code. The Balanced Budget Act of 1997 made it illegal for physicians to order LMRP tests and not supply an ICD-9CM code with the order.

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Medical Necessity

The Centers for Medicare and Medicaid Services (CMS), the US agency that administers the Medicare program, defines "medical necessity" as services or items reasonable and necessary for the diagnosis or treatment of illness or injury or to improve the functioning of a malformed body member. Medicare will not pay for any tests that CMS determines as unnecessary for diagnosis or treatment of disease.A laboratory may not submit a claim to Medicare or other government payers for any test it suspects is not medically necessary unless: The patient has signed an Advanced Beneficiary Notice (ABN), or A patient has requested the lab to submit such a claim for a determination by Medicare. Medicare does not pay for screening tests or tests that are ordered in the absence of signs or symptoms. Billing department employees are responsible for following all policies and procedures related to the submission of claims to reduce erroneous billings.

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Medicare Compliance for Clinical Laboratories (retired)
Billing and medical necessity

Billing: Highest risk activity a laboratory has. All laboratory activities contribute to the billing process. Many of the risk areas included in this program are components of the billing function. Medical necessity: Medicare is only allowed, by law, to pay for tests that are reasonable and necessary for the diagnosis and treatment of disease. Medical necessity is an underlying principle of the Medicare program. Tests performed for screening or routine exams are not considered medically necessary by the Medicare program.

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Coding

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

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Medical coverage policies (LMRPs)

LMRPs (Local Medical Review Policies) are published by Medicare for some laboratory tests. Developed for tests that can be used for screening or diagnosis of disease. CPT codes describe laboratory tests and ICD-9CM codes determine when coverage is allowed. If an LMRP test is ordered by a physician, an ICD-9CM code that is included in the LMRP must be given to the laboratory or the Medicare program will not pay for the test. It is against the law for laboratory to change or add an ICD-9 code submitted by a physician. The Balanced Budget Act of 1997 made it illegal for physicians to order LMRP tests and not supply an ICD-9CM code with the order.

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Medical necessity

Medical necessity means that Medicare is not allowed by law to pay for any tests that are not necessary for diagnosis or treatment of disease.A laboratory may not submit a claim to Medicare or other government payers for any test it knows is not medically necessary except in certain cases: When the patient has signed an advance notice. When a patient has requested the lab to submit such a claim for a determination by Medicare. Medicare does not pay for screening tests or tests that are ordered in the absence of signs or symptoms.Billing department employees are responsible to follow all policies and procedures related to the submission of claims to reduce erroneous billings.

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ICD-9CM coding

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

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Local medical review policies (LMRPs)

LMRPs (Local Medical Review Policies) are published by Medicare for some laboratory tests. They are usually developed for tests that can be used for screening or diagnosis of disease. LMRPs use CPT codes to identify the tests and ICD-9 codes to determine when coverage is allowed. If an LMRP test is ordered by a physician, an ICD-9 code that is included in the LMRP must be given to the laboratory or the Medicare program will not pay for the test. It is against the law for laboratory to change or add an ICD-9 code submitted by a physician. A laboratory should not submit a claim for an LMRP test that is not accompanied by an acceptable ICD-9 code. The Balanced Budget Act of 1997 made it illegal for physicians to order LMRP tests and not supply an ICD-9CM code with the order.

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Metabolic Syndrome
Adult Treatment Panel III

The National Heart, Lung, and Blood Institute (NHLBI) initiated the National Cholesterol Education Program (NCEP) in 1985. The goal was to reduce the number of Americans with elevated cholesterol and thus reduce illnesses and deaths in the United States due to coronary heart disease. Three adult treatment panels have been published since then with clinical practice guidelines for managing cholesterol levels in adults. The most recent panel, Adult Treatment Panel III (ATP III), was published in 2001 and updated in 2004. The NCEP: ATP III also includes criteria for the diagnosis of metabolic syndrome. This criteria is the most frequently used criteria in the United States.

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At medical examination, a 50-year-old Caucasian male expressed concern regarding diabetes. There is a history of type 2 diabetes, hypertension, and cardiovascular disease in his family. He has gained a few pounds each year and his physician notes abdominal obesity. His physician orders laboratory tests to evalute his risk of cardiovascular disease.Vital Signs and Pertinent Laboratoy Results:Blood Pressure: 128/82 mm Hg Weight: 230 lbsHeight: 5' 11'' Calculated BMI: 32.1Waist Circumference: 45 inchesFasting Blood Glucose: 120 mg/dLTriglycerides: 170 mg/dLHDL-C: 42 mg/dLWhich one of the following statements regarding this patient is true if the physician uses the guidlines of NCEP: ATP III Diagnostic Criteria for metabolic syndrome evaluation?View Page
Treatment

The primary goal in treatment of those with metabolic syndrome is reduction of risk factors for atherosclerotic disease. If the person does not already have type 2 diabetes, prevention of diabetes is another critical goal in management and treatment. Lifestyle changes and medications are utilized to meet these goals.Lifestyle changes that reduce obesity are critical: increase physical activity, reduce the fat in the diet, and decrease calorie intake. Exercise provides benefits beyond just burning calories. Exercise stimulates anabolic metabolism, raises basal metabolism rate, decreases stress, and increases hormonal sensitivity. Cessation of smoking is also important.Often drug therapy is needed to address the patient's hyperlipidemia, hypertension, and/or hyperglycemia. Low-dose aspirin and other antiplatelet agents may be used to prevent thrombosis.

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Analytes and Target Ranges Pertinent to Metabolic Syndrome

Analyte Reference Range Fasting glucose 75-100 mg/dL Total cholesterol < 200 mg/dL HDL-C > 60 mg/dL LDL-C < 100 mg/dL Triglycerides < 150 mg/dL hs-CRP Associated Risk < 1.0 mg/L Low Cardiovascular Disease Risk 1.0-3.0 mg/L Average Risk for Cardivascular Disease >3.0 mg/L High Risk for Future Cardiovascular Disease

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A 45-year-old African American female has been diagnosed and treated for type 2 diabetes for the past five years. She maintains good control of her blood glucose with medication but does not exercise and has gained 12 pounds over the past year.At her next appointment, her physician orders hs-CRP along with blood assays to monitor her diabetes.Laboratory Result:hs-CRP 2.8 mg/LView Page
A physician discusses weight with an overweight 60-year-old female at her yearly physical appointment. The female exercises regularly and eats healthy most of the time. The physician suggests she decrease carbohydrate intake and decrease portion size at meals.Review patient vital signs and laboratory assay results to decide if a diagnosis of metabolic syndrome is appropriate using the NCEP:ATP lll Diagnostic Criteria shown on the right.Height: 5' 7'Weight: 192 lbsBMI: 30.1Waist Circumference: 37 inchesBlood Pressure: 108/70Fasting Blood Glucose: 92 mg/dLTotal Cholesterol: 172 mg/dLLDL-C: 112 mg/dLHDL-C: 46 mg/dLTriglycerides: 70 mg/dLhs-CRP: <1.0 mg/LWhich of these statements regarding this patient is true?View Page
References

Armstrong C. Practice guidelines AHA and NHLBI review diagnosis and management of the metabolic syndrome. Am Fam Physician. 2006;74:891-1062.D'Amore PJ. Evolution of c-reactive protein as a cardiac risk factor. Lab Med. 2005;36:234-238.Devaraj, S, Swarbrick MM, Singh U et al. CRP and adiponectin and its oligomers in the metabolic syndrome evaluation of new laboratory-based biomarkers. Am J Clin Pathol. 2008;129:815-822.Eckel RH, Grundy SM, Zimmet PZ. The metabolic syndrome. Lancet. 2005;365:1415-1428.Expert Panel in Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (authors). Executive summary of the third report of the national cholesterol education program (NCEP) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (adult treatment panel III). JAMA.2001;285:2486-2497.Gade W, Gade J, Collins M et al. Failures of feedback: rush hour along the highway to obesity. Clin Lab Sci. 2010;23:39-50.Gade W, Gade J, Collins M et al. Beyond obesity: the diagnosis and pathophysiology of metabolic syndrome. Clin Lab Sci. 2010;23:51-61.Grundy SM. Does a diagnosis of metabolic syndrome have value in clinical practice? Am J Clin Nutr. 2006;83:1248-1251.Grundy SM, Brewer HB, Cleeman JI, et al. Definition of metabolic syndrome: report of the national heart, lung, and blood institute/american heart association conference on scientific issues related to definition. Circulation. 2004;109:433-438.Grundy SM, Cleeman JI, Daniels SR, et al. Diagnosis and management of the metabolic syndrome: An American Heart Association/National Heart, Lung, and Blood Institute scientific statement. Circulation. 2005;112:2735-2752.Grundy SM. Obesity, metabolic syndrome, and cardiovascular disease. J Clin Endocrinol Metab. 2004;89:2595-2600.Mathew B, Francis L, Kayalar A, et al. Obesity: effects on cardiovascular disease and its diagnosis. J Am Board Fam Med. 2008;21:562-568.Metabolic Syndrome. National Heart Lung and Blood Institute. Diseases and Conditions Index. Available at http://www.nhlbi.nih.gov/health/dci/Diseases/ms/ms_whatis.html. Accessed December 5, 2011.Mittal S. The Metabolic Syndrome in Clinical Practice. London, England. Springer-Verlag Springer Science; 2008.Molinaro RJ. Metabolic syndrome: an update on prevalence, criteria, and laboratory testing. MLO. 2007;39:24-27.Ronti T, Lupattelli G, Mannarino E. The endocrine function of adipose tissue: an update. Clin Endocrinol. 2006;64:355-365.

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Metabolic Syndrome

Metabolic syndrome is a designation for metabolic risk factors that, if occurring together, increase a person's risk for developing heart disease, stroke, and/or diabetes. An individual diagnosed with metabolic syndrome has an increased risk of diabetes that is five times greater than an individual without these metabolic factors and an increased risk of cardiovascular disease that is two times greater.

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Medical Complications of Metabolic Syndrome

In addition to cardiovascular disease, stroke, and diabetes, that are closely associated with metabolic syndrome, there are several medical complications and conditions that could also potentially occur. An individual with metabolic syndrome is susceptible to: Fatty liver disease Other liver diseases Cholesterol gallstones Asthma Sleep apnea Osteoarthritis Pulmonary disease Renal disease Ocular complications Polycystic ovary syndrome Colon, endometrial, and breast cancers

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The Importance of Diagnosing Metabolic Syndrome

Diagnosis of metabolic syndrome does not necessarily correlate directly to a diagnosis of a specific disease. This diagnosis links several health risks with obesity and the other syndrome criteria. It is not a prediction of a short-term risk for cardiovascular disease and diabetes. Those with this syndrome have a high lifetime risk for these diseases. The American Heart Association reports that the individual's short-term risk (approximately 10 years) is in the low to moderate range. The metabolic syndrome diagnosis urges patients to work with their physicians and other professionals to take steps to decrease their risk for cardiovascular disease and type 2 diabetes.

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Other Names for Metabolic Syndrome

In scientific literature other names are sometimes used for this syndrome: Reaven's Syndrome - Gerald Reaven, MD, first scientist to cluster risks factors for atherosclerosis and cardiovascular disease Insulin Resistance Syndrome Syndrome X Deadly Quartet - abdominal obesity, glucose intolerance, hypertension, hyperlipidemia Obesity-Dyslipidemia Syndrome Dysmetabolic Syndrome Obesity Syndrome Hypertriglyceridemic Waist

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An individual diagnosed with metabolic syndrome has an increased risk for developing cardiovascular disease and/or type 2 diabetes.View Page
Which of the following are possible medical complications for an individual diagnosed with metabolic syndrome?View Page
Adipokines and Atherosclerotic Inflammatory Process

Increased TNF-a, IL-6, PAI-1, leptin, resistin and decreased adiponectin promote insulin resistance leading to impaired glucose management and diabetes. Some of these adipokines also affect endothelial function and the coagulation system, promoting atherosclerosis. The low-grade inflammatory state created by abnormal adipokine levels is likely an important connection between metabolic syndrome and cardiovascular disease.Atherosclerosis is an inflammatory process in arterial walls. It probably begins when monocytes adhere to damaged endothelium, move to subendothelial places and transform into foam cell while incorporating lipids. Gradually atherosclerotic plaque is formed. The vessell wall shape and integrity is distorted also. Metalloproteinases produced by the foam cells digest the plaque. The plaque fibrous cap and/or portions of the plaque can then rupture and be released into circulation. Myocardial infarction and stroke can result

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Endothelial Dysfunction

The endothelium is the thin layer of cells at the inner lining of blood vessels. Endothelial dysfunction is a pathological state where the balance of vasodilating and vasoconstricting is lost. Endothelial dysfunction is also a preclinical stage of atherosclerosis and precursor of future cardiovascular disease. Inflammation from increased levels of inflammatory adipokines are one factor in the development of endothelial dysfunction.

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Dyslipidemia and Metabolic Syndrome

Those diagnosed with metabolic syndrome are at risk for atherogenic dyslipidemia, a state of abnormal lipids and lipid levels. A state of atherogenic dyslipidemia also enhances the development of atherosclerosis and cardiovascular disease. The increased release of NEFAs and their infiltration of the liver initiate the dyslipidemia process. Increased NEFAs in the liver result in a fatty liver and increased liver triglyceride synthesis. Increased liver synthesis and secretion of very low density lipoprotein (VLDL) follow. VLDL is the lipoprotein that transports triglycerides in circulation. Blood triglyceride concentration then increases.A fatty liver also increases low density lipoprotein-cholesterol (LDL-C) circulating in blood. The predominant lipid in LDL molecules is cholesterol. LDL molecules in the dyslipidemia state are described as small dense LDLs. The increased triglyceride presence causes depletion of the cholesterol and phospholipid content in LDL, making the molecules smaller and denser.Decreased high density lipoprotein-cholesterol (HDL-C) also results. Most researchers believe this is also caused by the increased production of triglyceride-rich VLDL. Decreasing the concentration of HDL molecules is atherogenic in that HDL is the helpful lipoprotein transporting excess cholesterol to the liver and decreasing total blood cholesterol. Higher levels of HDL-C aid in preventing atherosclerosis and cardiovascular disease.

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C-Reactive Protein (CRP)

CRP is an important acute phase reactant protein synthesized and released by the liver. In obesity, increased secretion of cytokines results in increased stimulation of the liver and increased CRP. Elevated CRP levels are a marker of inflammation. In metabolic syndrome and obesity, CRP levels can be used to identify a proinflammatory state. Increased CRP levels are correlated with an increased risk for cardiovascular disease, particularly myocardial infarction and stroke.

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CRP Measurement

Elevated CRP levels were a marker for non-specific inflammation and were used to monitor acute inflammatory diseases. Recently, highly sensitive measurements of CRP have been developed that detect this protein in lower levels. This measurement known as high sensitivity-CRP (hs-CRP) detects levels seen in chronic and non-acute inflammation. Hs-CRP levels are reported as low, moderate, or high risk for future cardiovascular disease. Hs-CRP concentrations > 3 mg/L indicate a proinflammatory state.

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Microbiology / Serology Question Bank - Review Mode (no CE)
Koch's postulates include all of the following except:View Page
Which of the following specimens is the most sensitive for detecting active CMV infection:View Page
Match organism on right to common name on the left.View Page
Match the hepatitis B test with the appropriate disease phaseView Page
Match the virus with its associated disease:View Page
Match the virus with its disease:View Page
Match the virus with its disease:View Page

Molecular Methods in Clinical Microbiology
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.

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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.

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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.

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2009 - Swine Flu

The 2009 H1N1 influenza virus was first detected in the United States on April 15, 2009.The virus was a unique combination of influenza virus genes never previously identified in either animals or people; they were most closely related to swine-lineage H1N1 viruses (hence the designation of "swine influenza"). However, epidemiological investigations of initial human cases did not identify exposures to pigs and it became apparent that this new virus was circulating among humans and not among U.S. pig herds.By April 21, 2009, the Centers for Disease Control and Prevention (CDC) began working on development of a new vaccine effective against this new strain. On April 24, 2009, the CDC uploaded complete gene sequences of the 2009 H1N1 virus to a publicly accessible international influenza database. At the same time vaccine development was occurring, work was also being done at CDC to help laboratories more quickly identify the 2009 H1N1 virus in patient samples. A real time PCR assay developed by the CDC was cleared for use by the Food and Drug Administration (FDA) under an Emergency Use Authorization (EUA) on April 28, 2009.The development of an effective, rapidly performed molecular assay was critical, because a CDC evaluation of non-molecular rapid influenza assays indicated that while these tests were capable of detecting the novel H1N1 strain when present in high concentrations, the overall sensitivity was low. Positive results with these assays were useful, but negative results did not rule out infection with influenza.

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Improvements for Influenza Testing

Public health laboratories were the first provided with the reagents and procedures for the reverse transcriptase-polymerase chain reaction (RT-PCR) assay developed by the Centers for Disease Control (CDC) under the Emergency Use Authorization (EUA). As information was shared between laboratories, other facilities implemented RT-PCR procedures that provided for the detection and differentiation of the H1N1 "swine" strain from previously encountered seasonal strains. Although many facilities utilized laboratory developed procedures, the FDA did grant emergency approval to a handful of commercially developed methods. One example was Prodesse's ProFlu-ST™ assay which became available in October 2009. Employing real time methodology, the kit was also optimized for use with automated extraction platforms, such as Roche's MagNA Pure Systems and Biomerieux's NucliSENS® easyMAG®.The ProFlu-ST™ assay is a multiplex RT-PCR assay utilizing fluorogenic hydrolysis (Taqman) probes for use on the SmartCycler platform. As a multiplex assay, it includes primers and probes for seasonal H1, seasonal H3, and 2009 H1 strains of influenza A. Targets are as follows:Seasonal H1: conserved area of A/H1 hemagglutinin (HA) geneSeasonal H3: conserved area of A/H3 hemagglutinin (HA) gene2009 H1/N1: conserved area of the 2009 nucleoprotein (NP) geneExtraction of RNA from patient samples is followed by a one-step multiplex reverse transcription of RNA targets into complementary DNA (cDNA), which is subsequently amplified in a real time thermocycler. In this process, the probe anneals specifically to the template, followed by primer extension and amplification. The assay utilizes the 5' - 3' exonuclease activity of the Taq polymerase, which cleaves the probe, thus separating the reporter dye of the fluorogenic probe from the quencher. This generates an increase in fluorescent signal. With each cycle, additional reporter dye molecules are cleaved from their respective probes, further increasing the fluorescent signal.

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Which statements are TRUE about the molecular methods made available under the Emergency Use Authorization (EUA)? (Choose all that apply.)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.

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Previous Methodologies: Antigenic Detection of Toxin and Glutamate Dehydrogenase (GDH)

Toxin assaysThe most common laboratory tests for the detection of C. difficile are enzyme immunoassays (EIA) for the detection of C. difficile toxin A and toxin B. The immunoassays are simple to perform, provide rapid results, and are easily incorporated into the workflow of most laboratories. Sensitivities of these tests do NOT compare favorably to culture, cell cytotoxicity neutralization assay (CCNA), or molecular methods. There are many test kits commercially available for detection of C. difficile toxins. Results are available in 15 minutes to 2 hours, depending on the assay. Initially, toxin A was thought to be the toxin responsible for the majority of the effects of C. difficile disease, so most early kits only detected toxin A. With the realization that there are strains that produce aberrant or no toxin A (A-) that are known to produce infection, and more recently toxin B negative (B-) strains, it is now recommended to use kits detecting BOTH toxins.Glutamate Dehydrogenase (GDH) assaysPublished studies have indicated that toxin immunoassays, by themselves, may not provide adequate sensitivity of detection. GDH assays initially attracted attention as a possible means to provide a rapid but more sensitive means for screening for C. difficile.GDH is an enzyme produced by C. difficile. EIAs negative for the GDH antigen have been associated with high negative predictive values. However, positive results are not necessarily associated with a toxin producing strain. A second assay on GDH positive samples is required to confirm the presence of a toxigenic strain. Initially, CCNA assays were recommended as the confirmatory method of choice; molecular methods (PCR for the toxin gene) were subsequently explored for this purpose.

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Multi-drug Resistant Organisms: MRSA, VRE, and Clostridium difficile
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 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.

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Enzyme Immunoassay Methods

The most common laboratory tests for detection of C. difficile are enzyme immunoassays (EIA) for detection of C. difficile Toxin A and Toxin B. The immunoassays are simple to perform and provide rapid results. However the sensitivities of these tests are not as good as culture, CCNA, or molecular methods. Only liquid stool samples should be processed. Due to the fact that the colonization rate is high, a positive result with a normal stool sample proves that the patient is colonized with C. difficile but not necessarily infected. There are many test kits available commercially for detection of C. difficile toxins. Results are available in 15 minutes to 2 hours, depending on assay. Initially Toxin A was thought to be the toxin responsible for the majority of the effects of C.difficile disease. Therefore, most early test kits only detected Toxin A, based on monoclonal anti-Toxin A antibodies. With the discovery that there are strains that cause infection and produce aberrant or no Toxin A (A-), it is now recommended that a kit is used that detects both toxins.

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Various methods have been employed for detection of C. difficile disease: cultivation of the organism, cell cytotoxin neutralization assays, and enzyme immunoassays have all been among the staples of diagnostic approaches. Which statements are accurate characterizations of these assays?View Page
Treatment of Clostridium difficile Infection (CDI) and C. difficile Associated Disease (CDAD)

The first step in treating patients with CDAD is to discontinue the causative agent wherever possible. The choice for initial antibiotic therapy depends on the severity of disease. Oral vancomycin or metronidazole remain the mainstays of therapy for CDI, with vancomycin reserved for patients with more severe disease and/or those who have not responded to metronidazole. Metronidazole is currently favored in guidelines from the Centers for Disease Control and Prevention (CDC) on the basis of cost and concern that oral vancomycin promotes colonization with vancomycin-resistant Enterococcus. Oral fluids (water and electrolytes) may be necessary to counteract fluid loss as a result of excessive diarrhea, which can quickly lead to dehydration. Patients with fulminant disease and toxic megacolon may require colectomy. Recurrence of CDI is becoming an increasing problem. Most recurrences happen 7-14 days after completion of therapy, suggesting relapse rather than re-infection. If a patient develops a second episode of CDI following initial successful treatment, it is recommended that if possible, the same drug be used to treat the second episode. Contributing factors to recurrent CDI include:Continuing exposure to organisms either through re-infection (via contaminated environment or poor hand hygiene) or an endogenous source, such as C. difficile spores in GI tract. An inability to mount an adequate anti-Toxin A IgM and/or IgG antibody response (i.e., poor host immune response); a likely reason why CDI affects an increasingly elderly population. Unfortunately a vicious cycle can arise whereby the initial treatment prescribed, vancomycin or metronidazole, significally disrupts normal colonic flora reducing colonization resistance and leaving the patient vulnerable to the next recurrent episode.Other treatments, including the use of probiotics or anion-exchange resins to absorb toxins, may work in some cases but none work in every case.The goal of all treatment is to reestablish normal colonic flora so as to control C. difficile (over)growth.

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Future perspectives - CDI/CDAD

There is little doubt that antimicrobial use increases the risks for CDAD and certain compounds or classes of compounds are associated with increased risk. However the exact role (risk) of each compound is still to be elucidated. With all pharmaceutical products, use is based on a risk-benefit ratio; that is, if the patient will benefit to the extent that using the particular antimicrobial is warranted, risks associated with its use are accepted as a part of patient management. There are a number of new antibiotics in various stages of development eg, nitazoxanide and ramoplanin, though none to date have FDA approval for treatment of CDI.Little is currently known about the relationship between strain virulence, disease severity, and transmission. Also while the role(s) of Toxins A and B in CDI are well established, the role of the Binary Toxin is not well understood and research is necessary to assess its role in CDAD.Monoclonal antibodies against C. difficile toxins are under development as a form of treatment to induce passive immunity in patients.Anti-Clostridium difficile vaccines are also being researched.

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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

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Clostridium difficile

Most Clostridium infections arise from endogenous sources. That is, many of the Clostridium species that are associated with disease in humans are part of the normal intestinal microflora, which is true of Clostridium difficile.The organism was originally isolated in 1935 as a component of the normal intestinal flora of healthy newborns. It was dubbed difficile because the organism grows slowly and is difficult to culture. Early investigators also noted that the organism produced a potent toxin, but the relationship between C. difficile antibiotic-associated diarrhea (AAD) and pseudomembranous colitis (PMC) was not elucidated until the 1970's. PMC is an inflammatory disease of the colon caused by toxins of Clostridium difficile. Normal intestinal flora is an important factor in host response to an infectious microorganism. Resistance to intestinal infection is significantly reduced when there is a reduction in the normal flora as a result of antibiotic treatment. The most common manifestation of this decreased host resistance is the development of PMC.

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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.

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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).

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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.

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Risk factors for Clostridium difficile Infection

The incidence of C. difficile infection varies considerably but is increasing worldwide, largely due to widespread use of broad-spectrum antibiotics. The risk factors associated with C. difficile infection and colitis are: Antimicrobial use length of course multiple antibiotics Hospitalization length of stay illness & weakness presence of spores in hospitals and long-term care facilities(LTCF) Age Advanced age > 65 (weakened immune systems Young children (immature immune systems) Underlying disease (weakened immune system) Use of proton pump inhibitors, gastric acid suppressants, or anti-ulcer medications that decrease acidity levels in stomach/GI tract, which can alter normal flora and allow C. difficile to proliferate Chemotherapeutic drugs (weakened immune system) Laxative use Gastrointestinal (GI) surgery or non-surgical invasive procedures such as intubation

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Clostridium difficile-associated Diarrhea

Clostridium difficile-associated diarrhea (CDAD) is a unique hospital infection that occurs almost entirely in patients who have received previous antimicrobial treatment. Anaerobic gut flora are crucial to colonization resistance, so any disruption of the normal colonic flora (through illness, therapeutic procedures or, most commonly, antibiotic use) is essential to the pathogenesis of C. difficile infection. The association of CDAD with antibiotic use is significant. Early attention (1970s) focused on clindamycin but later on (1980s,1990s & continuing today) the cephalosporins, especially third generation, and broad spectrum penicillins (e.g., amoxycillin/ampicillin) were also implicated. The risk of CDAD is increased if C. difficile is resistant to the particular antimicrobial. In the case of clindamycin, C. difficile resistance is variable. Risk of infection due to a clindamycin-resistant strain increases with use of the drug. For the third generation cephalosporins, C. difficile is universally resistant; thus, any toxigenic strain is capable of causing CDAD during cephalosporin use. Other less commonly implicated antibiotics are the macrolides, e.g., erythromycin, azithromycin, clarithromycin. However, prolonged courses of any antibiotics will increase the risk of disease. Even those antibiotics used to treat colitis (metronidazole, for example) have sometimes been reported to cause CDAD.The fluoroquinolones have been in use since the 1980s. Ciprofloxacin was approved in 1987, but it is only in recent years with the emergence of the epidemic strain 027/NAP1/BI, which is resistant to the fluoroquinolones, that this class of drugs has been implicated in Clostridium difficile disease. The fluoroquinolones were initially considered to be low risk but their use has been increasing, both with hospital inpatients and in the community, and fluoroquinolones are now implicated as a risk factor for C. difficile infection. The newer fluoroquinolones, e.g., gatifloxacin, moxifloxacin, have better activity against anaerobes, but poor in vitro activity against C. difficile, thus increasing the likelihood of CDAD. The CDC now recommends that all fluoroquinolones, as a class, be used sparingly as each poses an increased risk for CDAD.

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C. difficile disease is more likely to occur when:View Page
Risk Factors and Resistance

Enterococci are largely commensal organisms that are opportunistic pathogens. Underlying disease, an immunocompromised state, age, lengthy hospital stays or long term care, invasive treatments, and/or prior antimicrobial therapy are factors that are associated with significant infections with these species. As noted previously, enterococci are intrinsically resistant to many antibiotics. Intrinsic resistance affects not only beta lactams (including a broad range of cepahlosporins) and aminoglycosides, but also clindamycin and trimethoprim/sulfamethoxazole. The standard recommended therapy for systemic infections is a combination of either penicillin or vancomycin and an aminoglycoside (gentamicin or streptomycin). The goal of combination therapy is to achieve a synergistic bacteriocidal effect between the cell wall agent and the aminoglycoside.In recent decades, increasing resistance to other antibiotics through acquired resistance mechanisms has become a growing therapeutic and infection control problem. Of key concern are high level resistance (HLR) to aminoglycosides and increasing resistance to glycopeptides such as vancomycin.

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Mycology: Hyaline and Dematiaceous Fungi
Match each of the names of the fungi listed in the left column with its most likely associated disease listed in the right column.View Page
The fungus illustrated in this image was recovered from an induced sputum specimen from a 74-year-old man with chronic obstructive pulmonary disease. This isolate is most likely:View Page
Match the name of each dematiaceous fungus listed in the drop-down box with its most likely disease.View Page
The disease with which the dematiaceous fungus illustrated in this image is most likely associated is:View Page

Mycology: Yeasts and Dimorphic Pathogens (retired 2/12/2013)
Match each of the names of the dimorphic fungi listed with the names of the animals that most commonly may be related to transmission of disease to humans.View Page
Match the names of each of the diseases listed with its appropriate situation:View Page
Each of the following dimorphic fungal infections have been observed in animals living in their natural environment except:View Page
Which of the following fungal infections was once known as "Chicago disease" because so many cases had occurred in the Chicago area?View Page
Match the complications that are most likely to be associated with each of the two yeast diseases that are listed in the drop-down box:View Page
The growth of the yeast-like colonies shown in the upper image was obtained on blood agar from a skin culture only in the area overlaid by virgin olive oil. The lower image is a photomicrograph of a lactophenol blue mount made from a portion of the colony. The disease associated with this fungus is:View Page
Although only a few human cases have been reported, brewers and bakers may in particular be at increased risk for developing infections with:View Page
Of the following responses, the one observation that would rule out cryptococcosis as the cause of meningoencephalitis is:View Page

Normal Peripheral Blood Cells (retired 6/20/2012)
Definition of a Segmented Cell continued.

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

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OSHA Bloodborne Pathogens
Standard Precautions

Standard precautions mean that all blood and body fluids should be handled as if they are infectious and capable of transmitting disease. Standard precautions apply to: BloodBody fluidsSecretions (except sweat)ExcretionsNon-intact skinMucous membranes

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What Happens After HIV Infection?

Days to weeks after exposure, the patient may begin to complain of fever, headache, and fatigue. This may also be accompanied by a rash.For the first several months after the infection, the exposed individual may be HIV-antibody negative and the disease may not be detected. However, the individual is still infective and can transmit the disease during this period.The disease may remain silent in the patient for months to years, even with no treatment.When the immune system is weakened enough, the patient will develop opportunistic infections and be classified as having acquired immunodeficiency syndrome (AIDS).

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HCV Infection

Like HBV infection, HCV infection results in damage to the liver. Approximately 75 to 85% of individuals who are infected with Hepatitis C and develop antibodies in their serum (seroconvert) progress to a chronic form of the disease and about 70% of the chronically-infected individuals will develop active liver disease.

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Information From The Centers for Disease Control and Prevention (CDC) Regarding Hepatitis C

According to the CDC, persons born between 1945-1965 account for three-fourths of the cases of HCV infection. This group is also at greatest risk for hepatocellular carcinoma and other HCV-related liver diseases. The CDC recommends a one-time HCV testing for all persons born between 1945-1965.Although there is still no vaccine for HCV, there are therapies that can halt progression of the disease and provide sustained clearance of the virus following treatment. For this reason, it is critical to detect infection as soon as possible. If an infected person progresses to liver failure, a liver transplant may be required.Health care workers must be diligent in their adherence to standard precautions in order to prevent occupationally-acquired HCV infection.

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Packaging and Shipping Infectious Materials (retired July 2013)
Classifications of Hazardous Materials

The US Department of Transportation (DOT) classifies hazardous materials according to the risks that they pose. There are nine hazard classes: Class 1: Explosives Class 2: Gases Class 3: Flammable liquids Class 4: Flammable solids Class 5: Oxidizers/organic peroxides Class 6: Toxic and infectious substances Class 7: Radioactive material Class 8: Corrosives Class 9: Miscellaneous hazardous materials Within class 6 are two divisions: Division 6.1- poisonous material Division 6.2- infectious substanceA division 6.2 infectious substance is defined as a material known or reasonably expected to contain a pathogen. A pathogen is a microorganism or other agent (e.g., a prion) that can cause disease in humans or animals. The regulations that govern packaging and shipping a class 9, miscellaneous hazardous material, may also need to be reviewed by those who package and ship laboratory specimens. Dry ice is a class 9 hazardous material and, if used, requires special packaging, and specific labeling and marking on the outer package.

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Categories of Division 6.2 Infectious Substances

Hazardous material classifications are consistent across all agencies who regulate commercial shipping and are based on criteria developed by the United Nations (UN) Committee of Experts working with the World Health Organization (WHO), the Centers for Disease Control and Prevention (CDC), medical professionals, microbiologists, transportation professionals, and packaging technical experts. These requirements can be found in the 13th and 14th editions of the United Nations Recommendations for the Transport of Dangerous Goods, the 2005 - 2006 edition of the International Civil Aviation Organization Technical Instructions for the Safe Transport of Dangerous Goods by Air (ICAO Technical Instructions), and the International Maritime Organization (IMO) Dangerous Goods Code. The classification system for Division 6.2 Infectious Substances includes two catergories, known simply as Category A and Category B.

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Category A Definition and Examples

A category A infectious substance is in a form that is capable of causing permanent disability or life-threatening or fatal disease in otherwise healthy humans or animals when exposure to it occurs. Exposure would occur if the substance were released from its protective packaging and a human or animal came into contact with it. Some examples of category A infectious substances are listed below. A more comprehensive list is included as a PDF attachment on this page.Bacillus anthracis (cultures only) Brucella abortus (cultures only) Brucella melitensis (cultures only) Burkholderia mallei (cultures only) Clostridium botulinum (cultures only) Creutzfeldt-Jakob disease (CJD) brain tissue specimens Dengue virus (cultures only) Escherichia coli, verotoxigenic (cultures only) Ebola virus Francisella tularensis (cultures only) Hantaviruses causing hemorrhagic fever with renal syndrome Herpes B virus (cultures only) Human immunodeficiency virus (cultures only) Lassa virus Mycobacterium tuberculosis (cultures only) Poliovirus (cultures only) Rabies and other lyssaviruses (culture only) Shigella dysenteriae type I (cultures only) West Nile virus (cultures only) Yersinia pestis (cultures only)New and emerging pathogens should also be classified as category A until or unless additional information is received to move them to category B. For example, in 2009, shipments of Influenza A 2009 H1N1 subtype specimens were initially placed into category A until sufficient information allowed them to be moved to category B. This is not an exhaustive list. Sometimes, deciding on the classification of an infectious substance requires professional judgement and involves knowing the medical history or symptoms of the source patient or animal and/or knowing the local epidemiological conditions at the time the patient specimen or culture was obtained. If there is doubt as to whether or not a substance meets the criteria of category A, it must be treated as a category A substance for shipping.

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Category B Definition, Shipping Name, and Identification Number

A category B infectious substance is not in a form generally capable of causing permanent disability or life-threatening or fatal disease in otherwise healthy humans or animals when exposure to it occurs. The proper shipping name and Identification number is:Biological substance, Category B, UN 3373

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IATA and US Postal Service Exempt Specimens

Laboratory specimens that are unlikely to cause disease and do not meet the criteria for category A or B substances are not subject to Division 6.2 regulations. Specimens for which the hazardous materials regulation (HMR) does not apply include human or animal samples (including, but not limited to, secreta, excreta, blood and its components, tissue and tissue fluids, and body parts) being transported for routine testing not related to the diagnosis of an infectious disease. This includes specimens that are being sent for:drug or alcohol testing cholesterol testing blood glucose level testing prostate specific antibody (PSA) testing testing to monitor kidney or liver function pregnancy testing tests for diagnosis of non-infectious diseases such as cancer biopsies The US Department of Transportation (DOT) has no "Exempt Specimen" classification and there are no DOT guidelines for packaging non-regulated specimens.* According to the DOT, in the U.S., if a package is marked as "Exempt Human/Animal Specimen" the understanding is that it contains no infectious substance. However, both IATA and the US Postal Service (USPS) have these requirements for packaging exempt specimens: Packaging IssueIATAUSPSType of packaging requiredTriple packagingTriple packagingOuter containerOne dimension must be a minimum of 100 mm X 100 mm (approximately 4 x 4 inches) Must be able to survive a drop test of 4 feet One dimension must be a minimum of 100 mm X 100 mm (approximately 4 x 4 inches) Must be able to survive a drop test of 4 feet Quantity limits: outer containerNone NoneQuantity limits: Primary receptacleNone500 mLQuantity limits: secondary packagingNone500 mL* Non-regulated specimens may become regulated because of preservatives, such as 10% formaldehyde (class 9) or 25% formaldehyde (class 8); or 25% ethanol (class 3).

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Definitions

Before further discussion of Category A and Category B, it is important to define two additional terms that are used in the classification process. CultureAn infectious substance containing a pathogen that is intentionally propagated, for example a bacterium grown on bacteriological medium as seen in the image below. Culture does not include a human or animal patient specimen.Patient specimenHuman or animal materials collected directly from humans or animals and transported for research, diagnosis, investigational acitivities, or disease treatment or prevention. Patient specimen includes excreta, secreta, blood and its components, tissue and tissue swabs, body parts, and specimens in transport media (e.g., transwabs, culture media, and blood culture bottles).* *It is important to note that this means specimens that have been collected into these transport media, but have not yet been incubated and are not actively growing in the media.

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Security Awareness

A category A infectious substance is in a form that is capable of causing permanent disability or life-threatening or fatal disease in otherwise healthy humans or animals when exposure to it occurs. Exposure would occur if the substance were released from its protective packaging and a human or animal came into contact with it. Therefore, it is critical that a category A infectious substance does not end up in the hands of an unauthorized individual who may purposely or unknowingly release the substance from its protective packaging and endanger humans or animals. Being aware of the people that you interact with in the process of packaging and sending category A substances is vital to the safety of the transport and prevention of a health disaster. An outsider with limited access and system knowledge could constitute a threat, but be aware that insiders could also be a threat, e.g., a disgruntled employee or a person who is angry with his or her supervisor or job or the government. Anyone desiring to do harm could potentially seize the opportunity to steal a hazardous material.Follow these precautionary procedures: When you are questioned about an infectious substance that you are packaging for shipment, it is important that you know the person that is asking AND that he or she has a need to know. If you do not know the person and if you are not aware that the person needs to know about the substance that is being shipped, do not answer the questions. You could refer him or her to your supervisor. Watch for unusual behavior. Secure the package until it is picked up. Check the identification of the courier who will be picking up the package. Use an intralaboratory chain of custody procedure if the specimens are tranferred within the facility or system. Track the package once it has been sent to be sure it arrives safely. Notify the Responsible Official or federal authority if the package does not arrive at its destination.

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Security Plan for Category A Infectious Substances

Each facility that stores and transports hazardous materials must have a written, detailed security plan. The Select Agents and Toxins Security Information Document that was prepared by the Centers for Disease Control and Prevention (CDC) and the U.S. Department of Agriculture, Animal and Plant Health Inspection Service (APHIS) is an excellent resource to use for developing a security plan that would apply to category A infectious substances. This document can be found at http://www.selectagents.gov/resources%5CSecurity%20Information%20Document.pdfThe current version, dated March 8, 2007, is available in this course as a resource. However, because the document does undergo revisions, it is recommended that the URL given above be checked periodically for document updates.

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Parasitology Question Bank - Review Mode (no CE)
A 32 year old male was seen in the emergency room with gastrointestinal discomfort. Upon questioning the patient it was learned that he first began feeling ill after spending a day at the park where he swam and played volleyball barefooted. He first noticed a lesion on his foot. Later, he developed vague respiratory symptoms. Now his largest complaint is severe abdominal pain along with occasional vomiting. This patient is most likely suffering from:View Page
This stool parasite measures 55 µm by 50 µm and is the causative agent of:View Page
The suspicious form pictured here is responsible for which of the following conditions?View Page
This suspicious form, recovered in stool, measures 12 µm in length. Which of the following conditions is this form responsible for causing when present?View Page
A 68-year-old female, who recently vacationed in Brazil, presented to her physician exhibiting overall weakness, fever, and enlarged lymph nodes. Blood was collected for culture and parasitic examination. The culture was negative. This suspicious form was recovered upon examining the Giemsa-stained preparation. This patient is most likely suffering from:View Page
Match each pictured parasite with its corresponding associated condition:View Page
Protozoal parasites that typically do not produce disease in humans are referred to as being:View Page
A 31 year old male missionary worker recently returned from Africa where he helped a small rural community update their sanitation practices. He presented to his physician weak and complained of recent weight loss, abdominal pain, and diarrhea that was often bloody. The doctor ordered a battery of tests including a complete blood count (CBC) and stool for parasite examination. The CBC revealed eosinophilia and anemia. This suspicious form was seen on the wet preparations. It measured 52 µm by 27 µm. What parasite is mostly likely present?View Page
A 58 year old male, who recently returned from an extensive overseas business trip to Africa, presented to the local clinic complaining of nausea, vomiting, and an achy feeling all over his body. At first he thought it was just the flu, but it persisted. The doctor ordered a battery of tests including blood smears for parasitic study. This suspicious form was recovered. The patient is most likely suffering from:View Page
A 45 year old mother of two went to her physician because her children were recently diagnosed with ascariasis and she was concerned that she had also contracted the disease. Other than complaining of recent sporadic diarrhea, she was in overall good health. The doctor ordered a stool for ova & parasite examination. This suspicious form, measuring 55 µm was seen throughout the sample. This form is most likely:View Page
Xenodiagnosis has historically been used to identify:View Page
What term is defined as the presence of arthropods in or upon a human host:View Page

Pharmacology in the Clinical Lab: Therapeutic Drug Monitoring and Pharmacogenomics (retired 10/15/2012)
Unexpected Concentrations

TDM provides a quantitative measure of the circulating concentration of a drug. The physician determines if the dosage of the drug needs to be adjusted based on this information.If a drug concentration is determined to be outside the therapeutic range, it may be for one of the reasons listed in the table below. Reason Discussion Noncompliance Patients may (intentionally or unintentionally) not take the drug. TDM can thus help monitor compliance. Dosing errors The dose may have been erroneous or inappropriate given the patient's condition. Malabsorption The TDM result will reveal if the drug cannot be absorbed well through the gut and an alternative route of administration will be needed. Drug interactions Many drugs interfere with the absorption or metabolism of other drugs. These interactions will be revealed by TDM. Kidney or liver disease Any pathology that affects elimination will cause an elevation in a drug level that will be unmasked by TDM. Altered protein binding Changes in serum proteins can lead to big changes in the amount of free drug in serum. Variations in the genetics of drug-metabolizing enzymes can also affect drug concentrations in the body. This is the field of pharmacogenomics that will be discussed later in the course.

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TDM for Theophylline

Theophylline is used as a bronchodilator for treatment of moderate to severe asthma and chronic obstructive pulmonary disease (COPD). TDM is needed for theophylline because the kinetics of the drug are highly variable. It has a narrow therapeutic window, and overdose can result in elevated heart rate, arrhythmia, and CNS excitability. Clearance of the drug is increased in children, smokers, persons with cystic fibrosis, and persons with hyperthyroidism. Elimination is slowed in congestive heart failure and in the elderly.

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Protein Availability and Drug Dosing

Drug-binding proteins in serum can fluctuate in disease states. For example, if albumin levels fall, as can occur in liver failure or nephrotic syndrome, less albumin will be available for drug binding; a subsequent dose may produce a toxic concentration of free drug.The image on the right illustrates the loss of equilibrium between a protein-bound drug and a free drug when drug-binding proteins are diminished.Doses of drugs that are highly protein-bound may need to be adjusted in patients with lower drug-binding protein levels. Examples of some common drugs that are highly protein-bound include thyroxine, warfarin, diazepam, heparin, imipramine and phenytoin.

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Warfarin Metabolism, continued

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

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Phlebotomy
Plasma lipids

Lipids are fats dispersed in plasma. They include: Triglycerides Cholesterol Lipoproteins The amount and ratios of various lipids in the blood will determine a person's risk of getting coronary artery disease.

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Preliminary Identification of the Primary Select Agents of Bioterrorism
Development of the Laboratory Response Network (LRN)

The Laboratory Response Network (LRN) was created under presidential directive as part of the government bioterrorism response program. The initial partners included the Centers for Disease Control (CDC), Federal Bureau of Investigation (FBI), and the Association of Public Health Laboratories (APHL). The LRN became operational in 1999. Due to limited ability to respond to a bioterrorism event, the initial objective was to improve the nation's public health laboratory infrastructure.

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Role of Reference and National Laboratories Within the Laboratory Response Network (LRN)

A reference laboratory within the LRN performs tests to detect and confirm (rule-in) the presence of a threat agent. These labs ensure a timely local response in the event of a terrorist incident. Rather than having to rely on confirmation from laboratories at the Centers for Disease Control (CDC), reference laboratories are capable of producing conclusive results. This allows local authorities to respond quickly to emergencies. A national laboratory is the highest level within the LRN. Examples would include those operated by CDC, the United States Army Medical Research Institute for Infectious Diseases, and the Naval Medical Research Center. These laboratories have very unique resources to handle highly infectious agents and the ability to identify specific agent strains.

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Protocols

The American Society for Microbiology (ASM) has developed standardized guidelines in coordination with the Centers for Disease Control (CDC) and the Association of Public Health Laboratories (APHL). These protocols should be integrated into the standard operating procedures of any sentinel laboratory. The purpose is to provide the algorithms used to rule out suspected critical agents of bioterrorism and to refer the specimens to public health laboratories for confirmation. The protocols are available to sentinel laboratories at: http://www.asm.org/index.php/policy/sentinel-level-clinical-microbiology-laboratory-guidelines.html

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Additional Resources

Additional resources are available at:American Society for Microbiology (ASM) www.asm.orgCenters for Disease Control (CDC)www.bt.cdc.gov/lrn/

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Where can sentinel laboratory staff find the standardized testing protocols?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

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Location Where Organisms Naturally Occur, Disease Produced, and Mode of Transmission

These organisms can be encountered outside of a bioterrorism event and produce human disease. It's important to be familiar with the geographic areas where these organisms naturally occur and the how disease is transmitted.Bacillus anthracis: Bacillus species inhabit the soil, water, and airborne dust. Anthrax is the disease produced, which is transmitted to humans via direct contact with infected herbivorous animals. This is where the disease is primarily encountered. Anthrax is controlled in animals in the United States, so the disease is rare. In humans, most cases are cutaneous infections found in people that handle animals and animal products, including veterinarians and agricultural workers. Anthrax is consistently present in the animal population of some geographical regions, such as Iran and Pakistan, but only small numbers of animals experience the disease at any given time. Yersinia pestis: Y. pestis is found primarily in rodents, but can also be found in several animal species, such as cats, rabbits, camels, squirrels. Animal to human transmission most commonly occurs via a flea bite, causing the most common form of the disease known as the bubonic plague. Human-to-human transmission occurs by either flea bite or respiratory droplets. This causes an overwhelming disease known as pneumonic plague, which is the most likely form that would be implicated in the event of a bioterrorist attack. Human cases of the plague continue to occur in many countries, including Africa, the southwestern United States, parts of Asia, and the former Soviet Union. Francisella tularensis: Many animals, including rodents, rabbits, deer, and raccoons act as host for this organism. Humans and domesticated animals, such as horses, cattle, cats, and dogs can become infected. The infection is transmitted to domesticated animals by ticks and biting flies. Humans are most commonly infected from the bite of an infected tick or fly. Other means of infection include direct contact with the blood of infected animals when skinning game, eating contaminated meat, drinking contaminated water, or inhaling the organisms produced by aerosols. F. tularensis carries a high risk of laboratory acquired infection and documented cases of infection have occurred. Most cases of tularemia are reported in the southern and south-central United States.

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Location Where Organisms Naturally Occur, Disease Produced, and Mode of Transmission, continued:

Brucella species: Brucella is distributed in nature worldwide and found in domesticated and wild animals, such as cattle, sheep, and pigs. Infection with Brucella species, known as brucellosis, is caused in humans by exposure to infected animal fluids or food products. This includes ingesting non-pasteurized dairy products, such as milk or cheese, inhaling aerosols, and skin contact with the fluids of infected animals. Brucellosis poses an increased risk of occupational exposure to laboratory, veterinary, and slaughterhouse workers. Brucella is the most commonly reported laboratory-associated bacterial infection.Burkholderia mallei and B. pseudomallei: Most Burkholderia are found in soil, but B. mallei is only found in mammals. B.mallei is the causative agent for Glanders which primarily affects animals such as donkeys, mules, and horses. Horses, the organism's natural host, are highly susceptible to infection. Human infection is rare and usually occurs in people working with infected animals or laboratory workers handling the organism. The organism is endemic in Africa, Asia, the Middle East, and Central and South America, and usually enters via the eyes, nose, mouth, abrasions or cuts in the skin, or through inhalation. B. pseudomallei is found in soil and water and can accidentally infect animals, plants, and rarely humans. It is the causative agent of melioidosis, which is endemic in areas of southeast Asia, Taiwan, and northern Australia. The organism generally enters through cuts in the skin, ingestion of contaminated water, or by inhalation of an aerosol.

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Match the organism to the disease produced outside a bioterrorism event.View Page
Category A Agents: Reasons Why They May be Used to Create Public Health Emergencies

Anthrax (B. anthracis): Inhalation of anthrax spores is virtually 100% fatal Spores can remain infectious for decadesBotulism: Most lethal toxic agent known Toxin could be used to contaminate food supplies Can be aerosolized in enclosed areasPneumonic Plague (Y. pestis): Aerosolized in large amounts Short incubation period, usually in less than three days, and invariably fatal without early and effective antimicrobial therapy Untreated, fatality rate exceeds 90% Disease is spread from direct exposure to respiratory droplets of infected humansSmallpox: Highly contagious and deliberate spread by aerosol is extremely infectious Mass panic would be createdTularemia (F. tularensis): Highly contagious and easily spread An aerosol containing as few as 25 organisms can cause infection Easily penetrates the smallest breaks in the skinViral Hemorrhagic Fever: Causes internal and external bleeding and would likely cause great panic and easily spread by direct contact with body fluids or respiratory droplets Outbreak due to bioterrorist attack could lead to mass illness and death

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Categories of Biological Agents

The federal government has defined the pathogens or toxins that have the potential to pose a severe threat to public health or safety.The Centers for Disease Control (CDC) categorized these critical agents into three groups: Category A agents (highest priority) Category B agents (second highest priority) Category C agents (third highest priority)The biological agents are placed in one of the three groups depending on the level of risk each poses to national security.

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Category B Agents

Agents in Category B are considered the second highest priority agents and are included in this group because they: Are moderately easy to disseminate Cause moderate morbidity and low mortality Require specific enhancements of Centers for Disease Control (CDC) and Prevention’s diagnostic capacity and enhanced disease surveillance

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Which of the agent categories is the easiest to spread, has the ability to cause major harm, panic, and death, and carries the highest level of risk with regard to national security?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.

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Quality Control
Calculating Sensitivity

Sensitivity is the ability of a test to correctly identify individuals who do have a particular disease or disorder. To determine sensitivity, we use the following equation: True Positives (TP) Divided by True Positives (TP) plus False Negatives (FN) Times 100 or (TP ÷ (TP + FN)) x 100 The result will be a percentage.

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Specificity versus Sensitivity

Specificity is "disease focused." The more specific a test is, the fewer false-positive results will occur. Remember that a false-positive result can possibly lead to a misdiagnosis with the possible consequence of unnecessary diagnostic procedures and therapies. Sensitivity, on the other hand, is "wellness or normal focused." The more sensitive a test, the fewer false-negative results it produces.

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Verification of Performance Specifications for Nonwaived Testing

On April 24, 2003, the Clinical Laboratory Improvement Amendments (CLIA) Final Rules went into effect.As of that date, each laboratory that introduces a nonwaived, unmodified, FDA-cleared or approved test system must do the following before reporting patient test results: Demonstrate that it can obtain performance specifications comparable to those established by the manufacturer for the following performance characteristics: Accuracy. Precision. Reportable range of test results for the test system. Verify that the manufacturer's reference intervals (normal values) are appropriate for the laboratory's patient population.

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Reading Gram Stained Direct Smears
Cellular elements

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

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Real-Time PCR
Laboratory Applications

There are numerous applications for real-time PCR in the laboratory for both diagnostic and research purposes. Diagnostic applicationsReal-time PCR can rapidly detect nucleic acids that are diagnostic of infectious diseases, cancers, and genetic abnormalities. Real-time PCR has allowed for viral quantitation of infectious and newly emerging diseases such as influenza A H1N1 subtype. In malignant diseases, real-time PCR can be performed directly on genomic DNA to detect translocation-specific malignant cells. For RNA samples, real-time PCR has become extremely important for the detection and monitoring of HIV, hepatitis C and CMV. Real-time PCR can also be used for array verification and drug therapy efficacy. Research applicationsIn a research setting, real-time PCR is primarily used to measure gene transcription. The technology is commonly used to determine genetic expression of a particular gene over time in response to different pharmacologic agents or environmental conditions and can also be used to compare gene expression in exposed and unexposed individuals. The use of real-time PCR in this manner can help researchers find and detect diagnostic or prognostic indicators to increase the understanding of disease pathogenesis.

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References

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

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Red Cell Disorders: Peripheral Blood Clues to Nonneoplastic Conditions
The erythrocyte at the tip of the arrow in the image to the right is an echinocycte (burr cell).View Page
Sickle cells along with target cells, as shown in this image, confirm a diagnosis of sickle cell disease (HbSS).View Page
A peripheral smear with red blood cells photographed in a typical field was submitted for review. Which of the following conditions might be eliminated because of the red blood cell population found here?View Page
Hemoglobin E (Hb E) and HbE/Beta Thalassemia

Homozygous Hb E is common in Southeast Asia and presents with very mild anemia and seldom requires transfusion. Over 30 million people in the world are HbE carriers, making this abnormal hemoglobin almost as common as HbS. Hb E is uncommon in North America and in Europe, but with changing immigration patterns, Hb E and related diseases cannot be ignored. Peripheral blood smear findings of target cells, microspherocytes, red cell hypochromia, red blood cell fragments, and nucleated red blood cells may be noted. Evidence from hemoglobin electrophoresis is required to establish a diagnosis.Clinically, a very important and severe disease is Hb E/beta thalassemia in which there is hemolysis requiring repeated transfusions. Severe anemia, low MCV, and elevated RBC are characteristic of Hb E/beta thalassemia.

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The complete blood count was obtained from a patient recently admitted to the emergency room. The red blood cell indices obtained revealed an MCV of 115 femtoliters (fL) (normal range 80 - 90 fL). The patient met the criteria for a peripheral blood smear examination. A representative field is shown on the right.Which of the following conditions may be indicated by the results seen on this peripheral blood smear?View Page
Criteria for Peripheral Blood Smear Review

When an Initial analysis of red blood cells (RBCs) from an automated instrument are found to be abnormal, many laboratories will microscopically evaluate the peripheral blood morphology of the RBCs. This important step can help to establish which, if any, abnormalities are present as well as correlate possible disease states or conditions associated with the findings. Most laboratories will employ guidelines for review of the peripheral blood smear for RBC morphology. Though each laboratory will create their own guidelines, the following are a few examples that could trigger a manual, microscopic peripheral blood smear review:Hemoglobin: < 8 or >18 g/dL (<10 or > 21g/dL in a newborn)Hematocrit: <20% or > 60% in adults (<40% or >65% in a newborn)MCHC: <29 g/dLMCV: <69 femtoliters (fl) or >110flFlags generated by the hematology analyzer that indicate possible red cell abnormalities or spurious results In most laboratories, when these findings are noted, they should be followed up with a peripheral blood smear review for RBC morphology.

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Normal Red Blood Cell (RBC) Morphology

In order to properly understand and identify the abnormal red blood cell morphologies associated with RBC disorders, the laboratorian must first become competent in normal RBC characteristics.Normally, RBCs will display a defined morphology in the peripheral blood. Mature RBCs, under normal circumstances, are round, biconcave disc-shaped, anuclear cells measuring approximately 7-8 microns in diameter with an internal volume of 80-100 fL. The term used to express RBCs of normal size is normocytic. Mature red blood cells, under normal circumstances, will also have an appropriate hemoglobin content (a normal MCH and MCHC), giving them a red-orange appearance on Wright-stained smears. These cells will display a central pallor (lighter area inside of the cell) no larger than 3 microns in diameter. This normal morphology is indicated by the term normochromic. It is paramount for RBCs to contain an adequate amount of hemoglobin for the purpose of transporting oxygen to the tissues and carbon dioxide back to the lungs. An example of a normocytic, normochromic peripheral blood picture is shown on the right.In addition, the RBC membrane plays a key role in allowing the deformability of the cell to take place in order to travel through smaller vessels. Normally functioning RBCs survive for approximately 120 days in the peripheral blood circulation before being removed by the liver or spleen. Under normal circumstances, the body produces enough RBCs each day to account for the senescent (old) cells that are removed. Certain disease states can alter the normal RBC characteristics described above. This course will illustrate and correlate various RBC morphologic changes with the specific disease states or conditions, which they are associated.

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Anisocytosis Reference Table

Cell TypeImageCellular DescriptionAssociated Diseases and ConditionsNormocyteMCV 80-100 fL (approximately the same size as the nucleus of a small lymphocyte) MCHC 32-36 g/dL or 32%-36%N/A; cell is normalMicrocyteRed blood cells are smaller than normalMCV < 80 fLIron deficiency anemiaSideroblastic anemia Certain thalassemias Lead poisoning Certain hemoglobinopathiesMacrocyteRed blood cells are larger than normalMCV > 100 fLMegaloblastic Anemia (Vitamin B12/Folate deficiency)MyelodysplasiaLiver disease

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Introduction to Red Blood Cell Nonneoplastic Conditions Case Studies

Now that you have reviewed the red blood cell morphology tables, we will incorporate disease state correlations with the help of case studies and questions with answers. The following case studies and questions will be presented in groups of related morphologic findings on the peripheral blood smear. The case studies and questions will contain multiple choice and true/false questions for you to answer. There will be helpful information regarding the morphologies noted and disease states present displayed in the feedback sections for each question- so be sure to review the explanations after answering the questions. Enjoy!

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Red Cell Morphology Reference Tables

In the next few slides, red blood cell morphology reference tables will be available for your review. These summary tables include images with descriptors and disease state associations. The information found in these tables may be of assistance in the case study sections of this course. PDF copies of the pages are also included as printable resources.

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Poikilocytosis Review Table

Cell TypeImageCellular DescriptionAssociated Diseases and ConditionsTeardrop cellRed blood cells (RBCs) are shaped like a teardrop with a projection extending from one end.Myelofibrosis with myeloid metaplasia (MMM)SpherocyteRBCs smaller than normalNo central pallorRound rather than disc-shapedHereditary spherocytosisCertain hemolytic anemiasSevere burnsTarget cellRBCs with characteristic bull's-eye morphology due to hemoglobin distribution.Hemoglobinopathies (e.g., sickle cell disease)Certain thalassemiasIron deficiency anemiaSplenectomySevere liver diseaseSickle cellRBCs contain hemoglobin S.Thorn or crescent-shapedSickle cell anemiaStomatocyteRBCs with thin, elongated area of central pallor (slit-like, or coffee-bean-shaped on peripheral blood smears).Three-dimensionally, RBCs are cup-shaped.Hereditary stomatocytosisAlcohol-related diseaseLiver diseaseRh null phenotypeArtifactSchistocyte (fragmented red cells)RBC blood cell fragments or piecesVary widely in size and shapeSevere burnsHemolytic uremic syndrome (HUS)Microangiopathic hemolytic anemia (MAHA)Disseminated intravascular coagulation (DIC)Thrombotic thrombocytopenic purpura (TTP)Ovalocyte (elliptocyte)RBCs are elongated-oval, cigar, or pencil-shapedHereditary elliptocytosisMegaloblastic anemiaMyelophthisic anemiaCertain thalassemiasSevere iron deficiency Acanthocyte (Spur cell)RBCs demonstrating irregularly-spaced, spiny projections that vary in size and numberNo central pallor.AbetalipoproteinemiaSevere hepatic diseaseMyeloproliferative disordersMAHANeuroacanthocytosissyndromesEchinocyte (Burr cell)RBCs have short and evenly-spaced, rounded projections surrounding the cellCentral pallor presentUremiaHeart diseasePyruvate kinase deficiencyStomach cancersBleeding peptic ulcersBite cellRed cells that appear to have bites taken out of them (Image A)Supravital stain reveals the presence of Heinz bodies--precipitated denatured masses of hemoglobin (Image B) Disorders associated with Heinz body formation:Unstable hemoglobinsChemical poisoningG-6PDHemolytic anemia associated with severe alcoholic liver disease

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A known case of hemolytic disease of the newborn (HDN) is presented in the image on the right. Many different cellular morophologies are present. Apart from the obvious anisocytosis (microcytes and macrocytes), which additional red blood cell morphologies are worth reporting?View Page

Red Cell Morphology
Stomatocytes, continued

Stomatocytes are indicated by the arrows in the image on the right. Conditions in which a significant number of in vivo stomatocytes can be seen include hereditary stomatocytosis, neoplastic disorders, liver disease and Rh null disease. The largest numbers of in vivo stomatocytes are seen in hereditary stomatocytosis and their identification is necessary to make the diagnosis.

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Target Cells, continued

Another example of a target cell (or codocyte) is seen in the center of this slide. Notice that the hemoglobin in the center of this cell is lighter in appearance than in the previous slide. Target cells appear in conditions that cause the surface of the red cell to increase disproportionately to its volume. This may result from a decrease in hemoglobin, as in iron deficiency anemia, or an increase in cell membrane. Target cells have excess membrane cholesterol and phospholipid and decreased cellular hemoglobin. Examples of other conditions in which target cells may be present include thalassemias, Hemoglobin C disease, post splenectomy, and obstructive jaundice.

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Rh negative female with anti-D at delivery: A case study
Introduction

This case concerns a common scenario in the transfusion service (TS) laboratory, the detection of anti-D at delivery in a female who has received Rh immune globulin (RhIg) during pregnancy.Distinguishing between passive and immune anti-D is important clinically: If passive anti-D is misinterpreted as immune, RhIg prophylaxis may be omitted leading to D sensitization. If immune anti-D is misinterpreted as passive, appropriate follow-up of the antibody may be curtailed putting the fetus at risk.Unfortunately, differentiating between immune and passive anti-D is often impossible. This case study presents an opportunity to review perinatal testing programs and the crucial role of RhIg in preventing hemolytic disease of the fetus and newborn (HDFN) due to anti-D. The case also examines practical aspects of routine serologic testing involving neonates and women who have received RhIg during pregnancy. The case is a companion to "Hemolytic Disease of the Fetus and Newborn" and complements its content.In brief, the case will: Guide participants through laboratory findings that need to be interpreted and resolved; Examine current best practices in perinatal testing programs; Review the characteristics of RhIg and its use in pregnancy; Review and investigate key issues associated with detection of anti-D in women who have received antenatal RhIg; Discuss crossmatch and LIS policies related to RhIg-derived passive anti-D.

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The positive DAT on the newborn means that the infant probably has clinically significant hemolysis.View Page
Routine Serologic Tests - Mother

Tests done routinely as part of perinatal testing programs vary from country to country and within countries. Below is one example of serologic tests typically done when pregnant females lack clinically significant antibodies. Other test protocols exist.Mother ABO, Rh, and antibody screen at first prenatal visit; Optional (not mandated by blood safety standards): Test for weak D, if initial Rh typing appears to be D-negative; D-negative females: Tested again (ABO, Rh, and antibody screen) at ~ 28 weeks weeks gestation prior to administration of RhIg (depending on the country) and again at delivery. Note: The application of DNA analysis to typing blood group antigens started in the early 1990s but is not yet widely available. When available, the mother can be typed for D using molecular methods, but this is usually not done unless she is weak D. The purpose is to determine using molecular methods which D variant the mother has, weak D or partial D, since the latter can produce anti-D. (see Further Reading) Molecular typing is reviewed more fully in Refresher on Hemolytic Disease of the Fetus and Newborn and Its Prevention, a companion course that complements this one.

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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.

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Risk Management in the Clinical Laboratory
Clinical Laboratory Improvement Amendments of 1988 (CLIA)

In 1988, Congress passed The Clinical Laboratory Improvement Amendments in order to establish quality standards for all laboratory testing to ensure accuracy, reliability and timeliness of laboratory results regardless of where the patient's specimen was tested. The CLIA regulations are based on the complexity of the test method. The more complicated the method, the more stringent the requirements. Three categories of tests have been established: waived, moderate (which includes the sub-category of provider-performed microscopy), and high complexity. CLIA stipulates the quality standards for proficiency testing, patient management, quality control, personnel qualifications, and quality assurance for those laboratories performing moderate and/or high complexity testing. Those laboratories performing only waived testing must enroll in CLIA and are required to follow the manufacturer's instructions for those testing methods performed. The Centers for Medicare and Medicaid Services is charged with laboratory registration, fee collection, surveys, surveyor guidelines and training, enforcement, approval of proficiency testing providers as well as accrediting organizations and exempt states. The Centers for Disease Control and Prevention is responsible for CLIA studies, convening the CLIA Committee, and providing scientific and technical support and consultation to the Centers for Medicare and Medicaid Services. It is the responsibility of the Food and Drug Administration to categorize new test methodologies.

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The Disappearing Antibody: A Case Study
Risks of transfusing unmatched RBC

We often "get away" with transfusing unmatched RBC because the incidence of unexpected antibodies in patients experiencing medical emergencies is thought to be relatively low ( ~3-5% is sometimes cited, but with little solid evidence).Antibody incidence may vary according to several factors: Genetic disposition Patient's underlying disease Number of prior transfusions Gender (females may get exposed to foreign antigens via fetomaternal bleeds as well as transfusion) Concordance of antigen phenotypes of patients vs blood donors in a given locale.In general, antibody incidence increases with the number of transfusions that are given, although most antibody producers will respond within the first 3 - 4 transfusions. Antibody incidence in transfusion-dependent patients, such as those with sickle cell anemia or thalassemia, is very high. Regardless of likelihood, transfusing uncrossmatched blood to a patient with unexpected antibodies can result in a serious hemolytic transfusion reaction.

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The Influenza A Virus: 2009 H1N1 Subtype
How 2009 H1N1 Differs from Seasonal Flu Viruses

The 2009 Influenza A H1N1 virus is both similar and different from previous influenza A viruses that have caused seasonal flu in recent years. The symptoms associated with the H1N1 virus are very similar to those of other influenza A viruses causing seasonal flu. One difference between the influenza A 2009 H1N1 virus and seasonal influenza A viruses is the time of year in which the infection is at its peak. The influenza 2009 H1N1 virus was prevalent during warmer weather months in 2009, whereas the peak time for seasonal influenza viruses is winter months.Another way in which the H1N1 virus differs from seasonal flu viruses is the age group that is most affected. The number of cases and severity of the disease has been greater in the younger population, whereas most seasonal influenza virus-related deaths have been reported in those age 65 and older.

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How Severe is the Illness?

•Influenza A 2009 H1N1 virus-related symptoms range from mild to severe. Many infected individuals are able to recover without medical treatment. Occasionally, some individuals require hospitalization, and these patients receive supportive care and antiviral treatment. Serious infections from the 2009 H1N1 virus have resulted in some patient fatalities, usually due to secondary bacterial pneumonia or other respiratory complications.It is important to note that approximately 70% of the individuals that require hospitalization due to H1N1 infection, have also had one or more previously recognized underlying medical condition that may compromise an effective immune response. These conditions include, but are not limited to: diabetes heart disease asthma kidney disease neurocognitive diseases pregnancy

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Impact of the 2009 H1N1 Virus

In September 2009, the CDC reported that more than 99% of circulating influenza viruses at that time in the United States were the Influenza A 2009 H1N1 strain.In a press briefing December 10, 2009, Thomas Frieden, the Director of the Centers for Disease Control and Prevention (CDC) stated that people under the age of 65 are most impacted by the H1N1 virus. The CDC estimated at the time of the briefing that more than 50 million people in the United States alone had been infected with the H1N1 virus (one in six people). Of this number, more than 200,000 had been hospitalized. An estimated 10,000 deaths could be attributed to the virus, the majority being children and younger adults.The transcript of this press briefing can be accessed at: http://www.cdc.gov/media/transcripts/2009/t091210.htm.Accessed January 17, 2010.On January 14, 2010, Dr Keiji Fukuda, Special Adviser to the Director-Generalon Pandemic Influenza, World Health Organization stated in a press conference that conservatively, about 13,000 people worldwide have died as a result of 2009 H1N1 virus. The transcript of this press conference can be accessed at: http://www.who.int/mediacentre/vpc_transcript_14_january_10_fukuda.pdfAccessed January 17, 2010.

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According to the Centers for Disease Control and Prevention (CDC), which group of individuals have been most impacted by the 2009 H1N1 virus?View Page
Treatment Options for H1N1 Infection

Most patients who have suspected or confirmed cases of H1N1 infection have a mild, uncomplicated, self-limited illness that may not require antiviral treatment. If infected individuals have a normal immune system, they should be able to recover from the infection with symptomatic treatment only and without antiviral therapy. However, it is the decision of the patient's physician whether to treat or not to treat. The CDC provides this decision tree as a guideline if the illness is mild and uncomplicated:The CDC suggests that patients with suspected or confirmed influenza should be treated if: They are hospitalized as a result of the illness They are at risk for severe disease including these patients: Patients that have certain medical conditions, such as asthma, diabetes, heart disease, or patients with weakened immune systems that may exacerbate the infection. Children younger than 2 years old Adults 65 years or older Pregnant women or women up to 2 weeks post-partum They have a progressive or complicated illness characterized by signs of: lower respiratory tract disease such as hypoxia or abnormal chest x-ray CNS complications such as encephalitis Complications of low blood pressure including shock or organ failure Myocarditis Invasive secondary bacterial infection The treatment options indicated for the 2009 H1N1 infection include oseltamivir (brand name Tamiflu®), an oral tablet, and zanamivir (brand name Relenza®), an inhaled antiviral agent.Reference: Centers for Disease Control and Prevention. Updated interim recommendations for the use of antiviral medications in the treatment and prevention of influenza for the 2009-2010 season. December 7, 2009. Available at: http://www.cdc.gov/h1n1flu/recommendations.htm. Accessed January 18, 2010.

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WHO Surveillance

The World Health Organization's (WHO) is the directing and coordinating authority for health within the United Nations. WHO has been actively monitoring the progress of the H1N1 virus globally and provides updates on its website:http://www.who.int/csr/disease/swineflu/action/en/index.html. Accessed June 5, 2012.

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The Urine Microscopic: Microscopic Analysis of Urine Sediment
Match the following casts with their associated conditions/descriptions.View Page
Formation and Significance of Casts

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

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Waxy Casts

Waxy casts appear as cylinders of smooth, highly refractive material. They are yellow, homogeneous, and their ends may be square or broken off. Cracks may occur within the cast, giving it a segmented appearance. Waxy casts are believed by some to be the final stage of degeneration of the fine granules of granular casts. Since the granules need time to degrade, this finding implies localized nephron obstruction. Waxy casts are seen in chronic renal failure, and acute and chronic renal allograft rejection. Unusually broad waxy casts are known as renal failure casts. These very broad casts are created in the dilated tubules seen in end-stage renal disease.

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Fatty Cast

A fourth type of cast is the fatty cast. Fatty casts are clear cylinders containing droplets of fat which are highly refractile. These casts originate from the breakdown of the tubular epithelium containing oval fat bodies. Fatty casts are characteristic of degenerative tubular disease and are frequently seen with heavy proteinuria.

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Tyrosine Crystals

Tyrosine crystals appear as fine silky needles arranged in sheaves or bundles in acid urine. They are rarely present and may appear together with leucine crystals in liver disease. Do not confuse tyrosine with crystals caused by x-ray dye. X-ray dyes will cause the urine specific gravity to be greatly increased (1.040), Tyrosine crystals are soluble in alkali or dilute mineral acid.

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Which of the following abnormal crystals may indicate liver disease?View Page
Cholesterol Crystals

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

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Theoretical and Practical Aspects of Routine H&E Staining
Which of the following statements about histology are TRUE? (Choose all that apply.)View Page
Clinical Significance and Correlation of Histology

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

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How is Histology Useful to the Pathologist?

The pathologist relies on the histological preparations to diagnose disease by studying the: Etiology or cause Mechanism by which it develops Structural alterations of the cells Consequences of the cells' alterations

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Healthy Versus Diseased Tissue with H&E Stain

Histology involves looking at the structure of body tissues. Because structure is closely related to function, different tissue types are distinguishable from one another by examination of their component parts. Knowledge of the normal histology of all of the multitude of tissue types within the body is necessary for the recognition and understanding of disease.The following pages show microscopic photos of some select human tissue stained with the hematoxylin and eosin (H & E) stain.

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Transfusion Reactions
Iron Overload

A unit of Red Blood Cells contained 250 mg of iron as part of the hemoglobin molecule. A long-term complication of red cell transfusion is iron overload, or hemosiderosis. As red cells are destroyed, they release iron. The iron cannot be excreted and is stored as hemosiderin and ferritin. Iron accumulates in the liver, heart, spleen, and endocrine organs. Tissue damage, heart failure, liver failure, diabetes, and hypothyroidism can occur. Patients who are transfused frequently are at the greatest risk for iron overload. Diseases such as sickle cell disease, thalessemia, aplastic anemia, and other chronic anemias usually require frequent transfusions. Signs and symptoms of hemosiderosis include muscle weakness, fatigue, weight loss, mild jaundice, anemia, and cardiac arrhythmia. Ferritin levels and other iron studies should be assessed. Specific stains may be used to detect iron in tissue biopsies. Iron chelation may be used to treat and prevent iron overload. Chelation works by using an agent that binds to iron and helps remove it through the urine or feces.

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Disease Transmission

Even though blood components are tested rigorously for certain infectious diseases, bacterial, viral, parasitic, and prion pathogens continue to evolve. If they are not detected, they can cause harm to the patient and even death. Donors must be screen to determine eligibility. Their blood samples are also tested for hepatitis B and C, human immunodeficiency virus (HIV) 1 and 2, human T-cell lymphotrophic virus (HTLV) I and II, West Nile virus and syphilis. The table to the right describes the screening tests performed on all blood donors in the United States.It is not yet possible to eliminate the risk of infectious disease transmission through transfusions. There are many other organisms that may be transmitted through transfused blood, which are not routinely tested for in the blood supply. These organisms include the Epstein-Barr virus, cytomegalovirus (CMV), bacteria, and parasites such as malaria, Babesia microti, and Trypanosoma cruzi, which is responsible for Chagas disease, and prions such as variant Creutzfeldt-Jakob disease (vCJD).Selection of eligible donors is a critical part of ensuring the safety of the blood supply. Donors with certain lifestyles, medical conditions, travel histories, immigration backgrounds, or specific physical findings are deferred, either for a specific period of time or indefinitely. This minimizes the risk that a transmittable agent will be present in the donors blood. Click here to learn more about donor eligibility criteria from the American Red Cross.Click here to learn more about Babesia microti. Click here to learn more about Chagas Disease. Click here to learn more about vCJD.Click here to learn more about malaria.

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References

AABB. Standards for Blood Banks and Transfusion Services. 28th ed. Bethesda, MD: AABB; 2012. Harmening, DM. Modern Blood Banking and Transfusion Practices. 5th ed.Philadelphia, PA: FA Davis; 2005.Hillyer CD, Silberstein LE, Ness PM, Anderson, KC, Roback, JR. Blood Banking and Transfusion Medicine: Basic Principles and Practice. 2nd ed. Philadelphia, PA: Churchill Livingstone; 2007.Roback JD, Grossman BJ, Harris T, Hillyer CD, eds. Technical Manual. 17th ed. Bethesda, MD: AABB; 2011.Rudman, SV. Textbook of Blood Banking and Transfusion Medicine. 2nd ed. Philadelphia, PA: Elsevier Saunders; 2005. U.S. Food and Drug Administration. Infectious Disease Tests. Available at: http://www.fda.gov/BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/LicensedProductsBLAs/BloodDonorScreening/InfectiousDisease/default.htm. Accessed November 18, 2013.

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Bacteria Implicated in Contamination

Yersinia enterocolitica is most likely responsible for septic reactions in transfusions of Red Blood Cells. This organism is usually acquired by ingestion of contaminated food and causes mild symptoms of abdominal pain and diarrhea. Growth of Y. enterocolitica is enhanced in iron-rich environments such as red blood cells. Other organisms reportedly found in Red Blood Cell units are Campylobacter species, Serratia species, Pseudomonas species, Enterobacter species, and Escherichia coli. These bacteria can produce endotoxins, which cause a reaction in the patient. The majority of organisms associated with Platelet transfusions are normal skin flora. Staphylococcus aureus, coagulase-negative staphylococci, aerobic and anaerobic diptheroid bacilli, streptococci, and gram-positive bacilli are frequently isolated. Some transfused organisms have been implicated in a delayed post-transfusion illness. Pseudomonas aeruginosa and Burkholderia cepacia have been isolated in CRYO and FFP. These organisms grow optimally at 30°C and have been found in water baths, accentuating the importance of overwrapping components that are thawed in a water bath. Rickettsia species are intracellular bacteria that are transmitted by ticks or insects. These bacteria are the causes of Rocky Mountain Spotted Fever, Ehrlichiosis, and scrub typhus. These organisms may also be transmitted through transfusion.

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Reducing Transfusion-Associated Septic Reactions

Measures taken to reduce bacterial contamination of blood components include donor screening, improved skin disinfection, diversion of the first aliquot of blood, and pretransfusion bacterial detection. Screening of donors is done by questioning them about fever occurrence and dental or medical procedures that occurred days before donation. Donors who develop symptoms of an infection may be asked to notify the blood bank. Complete skin disinfection is not possible because of organisms living in places that are inaccessible, such as sebaceous glands and hair follicles. Factors affecting skin disinfection are the type and concentration of antiseptic, use or single or multiple antiseptics, method and steps of application, and contact time. Studies have shown that a two-stage method using a sponge scrub and ampule with tincture of iodine is the most effective method. The AABB recommends an initial 30-second scrub with a 0.7% iodophor solution followed by the application of a 10% iodophor compound, which must be allowed to dry for 30 seconds. To avoid normal flora contamination, blood may be diverted into a satellite bag at the beginning of donation. These bags are developed so that backflow is prevented. Blood contained in the satellite bag is used for blood grouping and infectious disease testing. Blood diversion is not a mandatory practice in the United States. The AABB requires that the transfusion service have a method to detect bacteria in all platelet components. Culture-based methods are used at blood collecting facilities near the time of collection. Hospital-based transfusion services use other less costly non-culture based methods such as gram staining or pH and glucose analysis prior to releasing the product for transfusion. Recently, a qualitative immunoassay for the detection of bacteria in platelets has been developed. This test detects antigens on the cell walls of the bacteria. It has been documented to be more sensitive than other non-culture based methods.

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Definition and Incidence

Transfusion-associated graft versus host disease (TA-GVHD) is a rare but highly lethal adverse reaction. The disease has a 90% mortality rate. It is caused by the transfusion of donor lymphocytes to a recipient who is immunocompromised. The donor lymphocytes engraft and escalate an immune response against the host's tissues including organs such as the lungs, skin, intestines, and liver. The recipient is unable to destroy the foreign lymphocytes and the cells proliferate and respond to incompatible antigens in the host. Certain recipients have increased risk for developing TA-GVHD. They are: Neonates less than 4 months of age Fetuses Recipients with a congenital or acquired immunodeficiency Recipients of donor units from a blood relative

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Therapy and Prevention

Transfusion-associated graft versus host disease (TA-GVHD) is generally unresponsive to medical treatment. Hematopoetic stem cell transplantation has been successful in rare instances. Gamma-irradiation of blood components containing viable lymphocytes is effective in preventing TA-GVHD. Irradiation is recommended for all Whole Blood, Red Blood Cell, Platelet, and Granulocyte transfusions to patients at risk. Patients at risk include neonates less than four months, patients with an acquired or congenital immunodeficiency, or patients receiving a directed donation from a family member. Irradiation prevents proliferation of donor lymphocytes with a required dose of 25 Gy to the mid plane of the blood container and a minimum of 15 Gy elsewhere. The dosage must not exceed 50 Gy to prevent harm to the patient from irradiation. Irradiation of blood can result in a decreased survival of red cells and a leakage of potassium from intracellular stores. Because of this, red cell units may only be stored for up to 28 days following irradiation. No reduction in storage time is required for platelets. Because Fresh Frozen Plasma (FFP) and Cryoprecipitate do not contain cells, irradiation is not required to prevent TA-GVHD in patients at risk.

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Which of the following patients are at risk for transfusion-associated graft versus host disease (TA-GVHD) and require irradiated cellular blood products? (Choose all that apply)View Page

Tuberculosis Awareness for Health Care Workers
Trends in Tuberculosis (TB) Incidence

The TB incidence rate in 2010 was the lowest recorded since national reporting began in 1953. However, the national goal of TB elimination (defined as <0.1 case per 100,000 individuals) by 2010 was not met. The inability to reach this goal can be attributed in part to the impact of the HIV/AIDS epidemic, which caused a TB resurgence in the early 1990s. Another factor is the worldwide increase in TB that has affected foreign-born persons in the U.S.In 2009 (the most recent year for which complete drug-susceptibility data is available), 113 cases of multidrug-resistant TB were reported.There is persistent disparity in the incidence of tuberculosis between different ethnic groups and also between foreign-born persons and U.S.-born persons. Reference: Pratt R, Robison V, Navin T. Trends in tuberculosis. MMWR/60(11);333-337; Centers for Disease Control and Prevention: March 25, 2011. Available at: http://www.cdc.gov/mmwr/pdf/wk/mm6011.pdf. Accessed October 28, 2011.

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High-Risk Progression Groups

The following persons are at high risk for progression from LTBI to TB disease: Persons infected with HIVPersons infected with Mycobacterium tuberculosiswithin the past two yearsPersons with untreated or inadequately treated TB diseaseInfants and children <4 years of agePersons with chronic medical conditions or immunocompromising conditions

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The descriptions listed below all relate to tuberculosis (TB). Match each of the descriptions with the item in the drop-down box that it describes.View Page
Latent Tuberculosis Infection (LTBI) Detection Methods

It is important to identify and treat persons with LTBI to prevent progression to active disease. The tuberculin skin test (TST) is traditionally used for the identification of prior exposure to Mycobacterium tuberculosis. However, specificity is reduced if an individual has had the vaccination Bacille Calmette-Guerin (BCG), which is intended to prevent TB, or has been infected with nontuberculous mycobacterium (environmental mycobacterial species). In these cases, newer blood tests, collectively known as interferon-gamma release assays (IGRA), may be more effective in identifying LTBI. IGRAs are more sensitive and specific, do not require the individual to return for test interpretation, and are not subject to possible inaccuracies and bias in reading the test, as may be the case with the TST. The turnaround time is usually less than 24 hours. However, the blood sample must be processed within 8-30 hours after collection while white blood cells are still viable. IGRA tests are more expensive to perform than the TST and not as widely available. There is also limited data on the ability of IGRAs to predict the risk for progressing to tuberculosis disease.

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CDC Guidelines

The Centers for Disease Control and Prevention (CDC) issued Guidelines for Prevention of Tuberculosis in Healthcare Settings in 2005.These guidelines have broader applications than the Guidelines for Prevention of Tuberculosis in Healthcare Facilities issued by CDC in 1994.

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Centers for Disease Control and Prevention (CDC) Risk Categories

Workers in health care facilities must recognize that the transmission of Mycobacterium tuberculosis is a risk. The risk varies according to the patient population; the prevalence of TB in the community; the number of patients known to be infected with TB who are present in the facility; whether the workers come into contact with TB patients in the performance of their jobs or performs procedures on specimens containing M. tuberculosis organisms where aerosols may be released; and the effectiveness of a tuberculosis control plan for the facility.CDC has identified three risk categories in health care settings:A low-risk health care setting is one in which health care workers (HCW) will most likely not be exposed to persons with TB disease or to clinical specimens that might contain Mycobacterium tuberculosis. A medium-risk health care setting is one in which the HCW will or might possibly be exposed to persons with TB disease or to clinical specimens that might contain M. tuberculosis. A potential ongoing transmission health care setting is temporarily applied to any setting if there is evidence of person-to-person transmission of M. tuberculosis in the past year.

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Three Levels of TB Infection Control

Administrative controls reduce the risk of exposure to persons who might have TB disease.Environmental controls prevent the spread and reduce the concentration of infectious droplet nuclei in ambient air.Respiratory protection controls are for situations that pose a high risk of exposure. These controls further reduce risk of occupational exposure to infectious droplet nuclei.

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References

Biosafety. The Centers for Disease Control and Prevention website. Available at: http://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf. Accessed November 7, 2013.Clinical and Laboratory Standards Institute (CLSI). Protection of Laboratory Workers From Occupationally Acquired Infections; Approved Guideline. 3rd ed. CLSI document M29-A3. CLSI. Wayne, PA: 2005.Guidelines for preventing the transmission of Mycobacterium tuberculosis in health-care settings, 2005. The CDC website. Available at: http://www.cdc.gov/mmwr/pdf/rr/rr5417.pdf. Accessed November 7, 2013.Tuberculosis (TB). The Centers for Disease Control and Prevention website. Available at: http://www.cdc.gov/tb/pubs/slidesets/InfectionGuidelines/program.htm. Accessed November 7, 2013. Updated guidelines for using interferon gamma release assays to detect Mycobacterium tuburculosis infection-- United States, 2010. CDC website. Available at: http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5905a1.htm?s_cid=rr5905a1_e. Accessed November 7, 2013.

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Biosafety Levels

Laboratory workers who handle infectious materials in the microbiology laboratory should be aware of the work practices, safety equipment, and barriers that will protect them, and others in the area, from infectious agents. The Centers for Disease Control and Prevention (CDC) and the National Institutes of Health (NIH) created guidelines to assist laboratories in developing safe practices based on the infectious agents that are handled. These guidelines are referred to as Biosafety Levels 1 through 4. Each increasing number represents increased risk, requiring more stringent work practice and increasingly protective safety equipment and barriers. A copy of the Guidelines can be obtained from the CDC or accessed online on the CDC website:http://www.cdc.gov/biosafety/publications/bmbl5/index.htm. Accessed November 7, 2013.

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Variations in White Cell Morphology -- Granulocytes
Importance of Recognition

It is important to recognize the presence of these morphology changes and to accurately identify them for several reasons: If the changes are pathological, their identification may aid the physician in diagnosing a specific condition. If the changes are not pathological, their identification alerts the physician to the fact that the changes are present, thus avoiding a possible misdiagnosis. If reactive, it indicates that although the cells are functioning normally, they are reacting to a stimulus. Indicating the presence of such cells may aid in determining the diagnosis or monitoring the course of disease once a diagnosis has been made.

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Variations in Morphology

Many variations in morphology may be seen when examining Wright stained peripheral blood smears. One method of classifying these variations in white cell morphology is based on the way the body responds to a stimulus, deficiency, or the presence of an inherited defect. This classification falls into three groups:Pathological: Cells may show abnormalities in appearance and/or function. The body is responding abnormally to a stimulus or inherited defect, resulting in physiological impairment in the patient. Nonpathological: Cells may show variation in morphology but their function is normal. Their presence does not cause physiological impairment. Reactive: Cells show variation in morphology but are functioning normally in response to a specific stimulus, such as a virus or bacteria. There is a disease process in progress to which the cells are responding. Although the morphology has varied from normal and their presence is significant, the body is responding normally to a stimulus.

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White Cell and Platelet Disorders: Peripheral Blood Clues to Nonneoplastic Conditions
A most useful follow-up test to consider when faced with hypersegmented neutrophils and oval macrocytes (see image) in a peripheral blood smear 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


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