Laboratory Information and Courses from MediaLab, Inc.

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.

Alba MA. Clinical Immunohematology Laboratory Manual. Albuquerque, NM: UNM Health Sciences Center; 2008.Brecher MF, Leger RM, Linden JV, Roseff SD, eds. Technical Manual 15th ed. Bethesda, Md. AABB; 2005.Harmening DM. Modern Blood Banking and Transfusion Practices. 5th ed. Philadelphia, Pa: F.A. Davis Company; 2005.

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.

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.

Thrombocytopenia is a decrease in the number of platelets in the peripheral blood. Most laboratories use a reference range for platelets that is approximately 150 - 450 x 109/L. Thrombocytopenia occurs when the platelet count falls below the lower limit of this range. The image shows how a normal platelet count would appear on a peripheral blood smear (Wright-Giemsa stain; 1000x original magnification). Representative platelets are indicated by arrows.

Laboratory findings include: Thrombocytopenia Prolonged PT, aPTT, thrombin time Decreased fibrinogen Elevated D-dimers Schistocytes on the peripheral blood smearThrombocytopenia and schistocytes are also associated with TTP and HUS; it is the abnormal coagulation tests that distinguish DIC from these other conditions.

The focus of this course will be on the technical process of producing human tissue blocks embedded in paraffin wax. Other embedding techniques using various media such as gelatin, ester wax, polyethylene glycol, and epoxy resin are also used in some histological techniques. However, in this course we will be discussing the method of embedding human tissue samples in molten, or melted, paraffin wax. This is the most commonly utilized method for routine tissue embedding and is the method most utilized in nearly all hospital histopathology laboratories for processing human tissue samples for diagnostic interpretation.In the order of events (chronology) of the total histology process, paraffin embedding takes place following tissue processing and prior to and in preparation for microtomy. For proficiency in paraffin embedding, the histologist needs:An understanding of basic anatomy for use in tissue type orientationKnowledge of basic tissue sampling methods used in gross dissectionTo develop manual dexterity and spatial reasoning in order to correctly orient the specimen in the tissue block for microtomyThis course will introduce and review some of the essential background information needed for correct embedding technique. Also discussed in this course will be guidelines for orientation of common histology specimens. Mastery of this information facilitates practice and application of these concepts during execution, to increase the histologist's technical proficiency at paraffin embedding in the histology laboratory.

Whatever paraffin formula has been selected for use in your laboratory, its' ultimate performance will be mostly dependent on the melting point and polymerization that occurs as it solidifies through the process of crystallization. Most manufacturer will provide a list of the ingredients and additives as well as the product melting point on the paraffin media bag or packaging. For example: Melting point: 133-136° F (56-58° C)Working temperature: 140° F (60° C)Water bath temperature: 113-122° F (45-50° C)Store below: 86° F (30° C)A "rule of thumb" however, is that pure paraffin appears almost clear when solid. It is a "softer" paraffin and would be more ideal for sections that are 5 microns (μ) thick. In contrast, paraffin which contains a greater amount of additives or stabilizers will be "harder" and better suited to sections of 2 to 3 µ. However, any paraffin formula performance can be compromised by failing to monitor and control temperatures during the infiltration stage of tissue processing or during the embedding process.

Many different tissue specimens may be submitted to the typical histology laboratory. Identification of each tissue type is very important for proper embedding orientation. An understanding of the surgical techniques and grossing methods for common specimens will allow visual recognition of the more common tissue types.

The following steps can be taken if you open a cassette and it appears that NO specimen is within:Look carefully in the cassette lid and interior corners and crevices. If you still find no visible tissue, ALWAYS have a second (or even third person) verify and help you check for any tiny fragments that you might have missed.Check the gross description, it may indicate that the specimen was very minute and was not expected to survive processing. If the specimen has been submitted in lens paper or a mesh biopsy bag, scrape all surfaces with a warmed, dull knife and transfer ANY, perhaps unseen, cells or particles to a mold where you have placed a small amount of molten paraffin. Anything microscopic that might be present will be likely to "float" off the knife, and can then be transferred to the block where it may be visible in the final section. Retain the lens paper or mesh bag in the cassette lid to document that "no tissue was seen."Individual laboratory procedure should be followed when documenting specimen loss on the accession log and/or laboratory information system (LIS).Missing fragments (less than dictated) that have been inadvertently dropped, can sometimes be retrieved, salvaged, and identified by retaining cassette lids in a separate bag for 24 to 48 hours (or until the case is signed out).

Beside batch size reduction, cross-training staff is also a key to achieving a Lean laboratory operation. Not only is cross-training essential for Lean operation but it also makes sense, based on the current shortage of laboratory professionals. In a traditional laboratory, every section is separated and has its own staff and space. Daily workload is usually not evenly distributed, since it is based on the workload of a particular section. In a Lean laboratory work cell, automated analyzers (hematology, chemistry, immunoassay and urinalysis) are place together in a U shape. These automated/high volume analyzers are placed as close to the central processing area as possible to decrease excess motion, while manual/low volume testing is located in an area that is further away from specimen processing. The images on the right illustrate the traditional and work cell layouts. A work cell provides several advantages: Less staff is required to work in a work cell designEase of coverage during break timeLess wasted motionEnhanced team work

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

Lean and Six Sigma are very powerful quality improvement tools, but they will only work if they are implemented appropriately. Certain factors will determine the outcome of Lean Six Sigma efforts.Support and commitment of the executive management team for Lean and Six Sigma effortsUnderstanding what resources are available prior to the start of the projectThe amount of training received by the staffStaff acceptance of Six Sigma and Lean conceptsThe size and scope of the projectsAbility for management to communicate the importance of projects to staffWithout support from senior members of the management team, most of the effort will eventually lose traction. It is important for management to understand what resources (financial and human) must be available before starting any project. Trying to complete multiple projects simultaneously when a laboratory barely has enough staff to complete the normal day-to-day work, or taking on an inter-departmental project as the first project will likely result in failure and will lead to staff frustration and resentment over future projects. There is no need to train the entire department to become Six Sigma Black Belts, but they should be trained on the basic principles before they begin participating in improvement teams. Management should include the staff in project selection as much as possible, since it is difficult for the team to perform if they do not understand the importance of the project. Lean and Six Sigma involve commitment from everyone in the organization, from the CEO to the employees. Only when an entire organization is committed will Lean Six Sigma become an organization's philosophical approach to improvement.

The core of the Six Sigma quality management approach is measurement of defects in order to get as close to zero defects as possible. Sigma is a statistical term that measures how far a process deviates from total accuracy or perfection. The process sigma, which is also known as the sigma level, is a measure of process capability. The higher the process sigma, the more capable the process is. A Six Sigma process has a short-term (DPMO) process sigma of 6. When determining the long-term process sigma, 1.5 is subtracted from the short-term metric, so that the long-term process sigma for a Six Sigma process is 4.5. Six Sigma is often wrongly defined as "3.4 defects per million products," when in fact, Six Sigma is actually defined as 3.4 defects per million opportunities (DPMO). Six Sigma's goal is to improve all processes to that level or better.To determine the number of opportunities a process contains, one should think of the number of opportunities in which a defect may occur. For example, if you are measuring emergency department (ED) stat turnaround times from order to completion, a defect would be any result not reported within the specified turnaround time. Opportunities for defects (delays) can occur in the three phases of laboratory testing (pre-analytical, analytical, and post-analytical phases). An example of DPMO and process sigma (sigma level) measurement is given on the following page.

During the measure phase, the team will: Observe and review the current process.Measure the current process to established a baseline.The observation part is critical even for those that are involved in the process daily, since observation can often uncover errors or variations that are not obvious. Observation is also useful to eliminate bias. For example, management's perception of the workload is likely different from the perception of the employees. Observing and reviewing the process together in real-life as a team can eliminate those biases. After the team has had a chance to observe the process, it can then decide on the appropriate data to measure. Data can be either discrete or continuous. Discrete data contain distinct values. Discrete data are usually the result of counting something using whole numbers. Continuous data are data where the values can be any numeric values and not just whole numbers (eg, time, height, and temperature measurements). Here are some examples of continuous vs. discrete data that are used in the laboratory.ContinuousDiscreteAverage time from receipt to completion for stat samplesNumber of stat samples not completed within 1 hourTime between completion of stat test by the analyzer and LIS release Number of stat samples not released in LIS within 5 minutes after the test completed by analyzerTotal LIS downtimeIncidences of LIS downtime in one monthAverage wait time for outpatients prior to specimen collectionNumber of outpatients not drawn within 15 minutesAverage response time for physician to return page for critical resultThe number of critical result pages not return by the physician within one hour

Burtis CA, Ashwood ER. Tietz Textbook of Clinical Chemistry 2nd ed. WB Saunders; 1994.Doig, K. Rodak's Diagnostic Hematology. 3rd ed. WB Saunders Co; 2007.Harmening DM. Clinical Hematology and Fundamentals of Hemostatis. 5th ed. FA Davis; 2008Hoffman R, Benz EJ Jr., Shattil SJ, Furie B, Cohen HJ, Silberstein LE. Hematology Basic Principles and Practice, 2nd ed. Churchill Livingstone; 1995.McKenzie SB. Textbook of Hematology, 2nd ed. Williams and Wilkins; 1996.McKenzie SB. Clinical Laboratory Hematology, 2nd ed. Pearson; 2010.Miale JB, Laboratory Medicine Hematology, 6th ed. Mosby; 1982.Stiene-Martin EA, Lotspeich-Steininger CA, Koepke JA, Clinical Hematology Principles, Procedures, Correlations, 2nd ed. Lippincott; 1998.

The laboratory findings in this case represent classic findings seen in beta thalassemia minor including: erythrocytosis, decreased hemoglobin, normal hematocrit, normal RDW, and the presence of codocytes (target cells). This patient does have a mild anemia, but some patients with beta thalassemia minor have no anemia. Hemoglobin electrophoresis confirms this diagnosis, showing an increased Hb A2 level and decreased Hb A.In addition, the slightly increased iron and slightly decreased TIBC contradict a suspicion of iron deficiency. These chemistry results are typical for beta thalassemia, even though the red blood cells are microcytic and hypochromic.

Medulloblastoma is a malignant brain tumor that originates in the cerebellum. It is included in the family of primitive neuroectodermal tumors, or PNETs. It is a highly invasive brain tumor which is known to spread through the brain and cerebrospinal fluid.Patients with meduloblastoma will typically have spinal taps performed after resection to evaluate for the presence of metastasis. These samples are then evaluated in the laboratory.This cytospin shows multiple large bizarre tumor clumps. Note that there are also several tumor cells that appear to have ingested other tumor cells (see arrows).

Bone marrow aspiration and biopsy are standard tools used in the hematology laboratory to aid in the evaluation and diagnosis of peripheral blood abnormalities. Some of these abnormalities include: cytopenias (such as neutropenia), thrombocytopenia and anemias. Bone marrow aspiration and biopsies are also used by hematology/oncology specialists in the diagnosis of leukemias, dysplastic syndromes, and proliferative syndromes. A bone marrow aspiration and biopsy may also be part of the evaluation of fever of unknown origin (FUO), failure to thrive(FTT) in the pediatric setting, as well as some metabolic and genetic disorders.A bone marrow aspirate sample is obtained by inserting a needle into the bone marrow space and withdrawing 5- 10 milliliters (mL) of marrow in several different syringes. These samples are then transferred to evacuated blood collection tubes containing the anticoagulants required for the types of assays desired. A portion of this liquid marrow is smeared for staining and evaluation under light microscopy. It can be sent for various types of laboratory assessment including : immunophenotyping, cytogenetic evaluation, and molecular analysis.While bone marrow aspirations and biopsies are usually obtained by the hematologist or oncologist, they are evaluated and interpreted by a hematopathologist with the assistance of the laboratory technologists who prepare and stain the smears. In many laboratory settings the technologists also perform the bone marrow differentials.

Bone marrow differentials have significant differences from peripheral blood differentials that need to be considered as they are reviewed and counted.One of the most important facts to consider is the large variability in cellularity and cell distribution depending on the type of preparation that is used. Choosing where to count and when to use which of the smear types available to you, takes time and experience and can be directed by a pathologist's preference.Regardless of how many, or what types of smears you have available to choose from, you will always start with a simple visual inspection of your smears. Begin by recording the patient identification information as well as date of sample, and any other mandatory patient identifying information necessary for your laboratory. Record aspiration site information when provided. Many patients will have bilateral bone marrow aspirates performed as part of a diagnostic or staging workup. Standard aspiration sites are: posterior iliac crest (PIC), anterior iliac Crest (ANT), sternum (S), spinous process (SP) and sometimes in very young children, bone marrow is obtained from the tibia (T). Be aware, that while a bilateral bone marrow aspirate usually involves an aspirate of the same site from opposite sides of the body, e.g., L-PIC and R-PIC, in some situations, a bilateral staging aspirate will be from two different compartments on the same side, e.g. R-AIC, R-PIC. Observe the appearance of the bone marrow smears. Do any have feather edges? Are there fragments or spicules present on any of the smears available? If so, they should be your first choice to view, since they are more representative of what the biopsy will show if one was obtained. Once you select your smears, scan using 10X magnification on the microscope. Are some of the fragments/smears so thick that you cannot see good morphology? If so, reject these areas/slides. Are some of the fragments/smears so thin that everything is smashed? These areas/smears cannot be used either. Are there areas in the vicinity of any of the fragments that have good staining characteristics as well as readable morphology? This is where you should begin your differential.

Depending on the laboratory protocols and the hematopathologist's preference, the technologist's responsibilities in bone marrow aspirate processing and reviewing can vary from simply staining slides to complex tasks such as smearing, staining, counting and distributing samples based on laboratory standard protocols. While laboratory professionals may perform bone marrow differentials, it is the hematopathologist's responsibility to review, interpret, and verify those counts after evaluating all of the samples that were provided. This includes both the bone marrow aspirate and biopsy smears, as well as the flow cytometry data, if required. When differentiating and counting samples with increased numbers of blasts, the technologist may make a determination of cell line for the blasts, according to observations on a Wright or Wright-Giemsa stained slide, but the hematopathologist may modify this placement based on flow cytometry data and additional special stains.The laboratory technologist can assess cellularity, presence of megakaryocytes, and possible presence of tumor cells on the bone marrow smears. Ultimately, the hematopatholgist is responsible for interpreting the bone marrow differential results after meticulous review of all smears stained, as well as any biopsy sections available. It is also the hematopatholgist's responsibility to identify tumor cells that may be present.In laboratories that use a standard order set with sample drop off directly from bedside, it is the technologist's responsibility to split and distribute the marrow based on laboratory protocol. It is hematopathologist's responsibility to verify that standard order testing was actually sent and to add on any additional testing deemed necessary, based on clinical history and lab findings. Once you become comfortable with bone marrow morphology and more familiar with your laboratory's protocols, performing marrow differentials will become less intimidating and more of a collaboration between technologist and hematopathologist.

Bone marrow smears can be very cellular and it can be difficult to keep track of where you are on the smear while keeping your correct hand position on the keyboard . Having a good strategy to use when counting cells and performing differentials can make this less difficult. On peripheral blood differentials, it is easy to observe and count each cell individually as the stage is moved to bring the next field into view. However, with bone marrows, the total number of keys that need to be used on the differential counter is greater than the number that need to be used with a peripheral blood smear and the number of cells per field is also increased dramatically, making it easy to lose track of the cells on the smear or one's hand/keyboard placement. It can be simpler and less stressful to work on the quadrant system. There are two different ways to do this: Divide the field into quadrants. Count the individual cells in each quadrant separately. This decreases the number of cells into more manageable bites. However, you still have the increased number of cell types to deal with and possible keyboard frame-shifts. Divide your keyboard into quadrants. Search your field for a limited number of cell types and tally all you see before moving on to the next grouping of cell types. Once you tally all your groups then move on to the next field (e.g., lymphocytes, monocytes, macrophages, eosinophils, basophils, plasma cells, erythroids, segmented neutrophils, bands, etc). You can make these small groupings for any cells as long as you cover the entire list of cell types that your laboratory reports in its bone marrow differential protocol. Remember that blasts are identified by cell type and there will usually be a separate key for pronormoblasts, myeloblasts, lymphoblasts, and possibly monoblasts and plasmablasts. It is possible to combine both methods, using the keyboard quadrant technique with a restricted portion of the total microscope field. This is useful when you are getting close to your total tally and do not want to alter the balance by only counting one cell type for the last few cells.

While smears from the bone marrow aspirate are the most common preparations, touch preparations (touch preps) made from the bone marrow biopsy core may also be useful or necessary. When aspirates are difficult or a dry tap occurs, the only sample available to be evaluated in the hematology laboratory may be the bone marrow biopsy. To create a fresh biopsy touch preparation, the fresh bone marrow core is gently rolled between two slides, then gently rolled between five or six pairs of coverslips. There should be enough cellular elements present when using this method for the laboratory professional to evaluate. The imprints will be wet and cellular at first but as the surface dries it will eventually become less cellular. At this point the core is placed in fixative and sent to pathology for evaluation. The number of touch preps you can make is dependent on how wet/ bloody or fibrotic the core is to begin with, but even one set can be enough to aid in diagnosis.While it is not practical to practice making touch preps from a real biopsy core, it is possible to practice the technique by using a length of applicator stick soaked in either blood or stain to simulate a real biopsy core. To do this, simply break off a short, 0.5 inch piece of a standard plain or cotton tipped applicator stick and soak it in the fluid of your choice. As you roll it between slides or coverslips you will see the pattern it leaves behind. Think of the motion of a teeter-totter (seesaw) as you roll. There should be very little downward force on the core as you coax it to roll. If the core will not roll then you can just touch the slides or coverslips to the surface of the core few times on each slide.Note: If the biopsy is placed directly into fixative and sent to pathology, it must first be decalcified before it can be sectioned and stained. This process will take at least 24 hours depending on the lab and if additional stains are required, it may be at least 48 hours before a result is released.

Most bone marrow slides are made simply by placing a drop of bone marrow on a slide and using a smear preparation technique. However, in order to obtain consistently high quality smears, it is necessary to select or concentrate the fragments on these smears. Selecting or concentrating fragments can be performed with different methodologies. At the patient bedside, some clinicians will use the touch-preparation or pull-preparation method, while tilting the slide to allow excess blood to roll off. This leaves more of the bone marrow spicules on the slide. This can be wasteful and rather messy but does not require a high level of skill.A less wasteful method is to pour a portion of the marrow aspirate into a small petri dish and swirl it about, then tilt the dish to reveal the marrow spicules. These can then be extracted using a capillary pipette with a micro-pipette bulb and transferred to the slide for use in making smears. This technique allows the laboratory professional to make numerous smears containing fragments rather than relying on the random luck of the drop. Any excess marrow can be saved and returned to the EDTA tube for further testing. This capillary pipette concentration technique can be coupled with any of the smear preparation techniques but does require practice to perfect and maintain proficiency. When coupled with the coverslip method, it is possible to make 2-3 dozen quality smears from as little as a 0.25 - 0.50 mL of marrow aspirate, making it ideal in small sample volume situations.

In some institutions, the laboratory technologist does not assist the clinician at the bedside with the bone marrow aspiration procedure. Instead the clinician delivers the bone marrow sample to the laboratory, similarly to other laboratory specimens. When this is the case, the bone marrow sample may be delivered in one of two manners with the laboratory's responsibilities dependent on which method is used. A clinician may deliver to the laboratory a specified number of smears, made at bedside, along with the bone marrow sample. Samples may also be designated for flow cytometry, cytogenetics, or molecular diagnostics. A clinician may deliver a standard package of bone marrow aspirate to the laboratory in various evacuted blood collection tubes. In this situation the laboratory will usually have a standard order set that directs the distribution of the marrow samples based on diagnosis. The hematology laboratory will use these samples to prepare the bone marrow smears, while the other tubes would be distributed for flow cytometry, cytogenetics, molecular diagnostics, etc. based on the direction of the hematopathologist.

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).

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.

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

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

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

Vancomycin and ampicillin resistance among Enterococcus species, particularly E. faecium have been on a steady increase. The disk diffusion screening test is used in many laboratories to detect vancomycin resistant strains. Note in the upper image that no zone of inhibition is seen around either the vancomycin or the ampicillin disk, indicating resistance to both drugs. Vancomycin-resistant Enterococci (VRE) have been divided into three phenotypes--Van A, Van B, and Van C. Vancomycin-resistant strains of E. faecalis and E. faecium are commonly of the Van A phenotype, demonstrating high level resistance (MIC's higher than 64 ug/mL), as illustrated by total resistance of the test strain in the E test and the VA disk, as illustrated in the lower image. The strain shown in the lower image, however, is ampicillin susceptible at the level of 1 ug/mL (see lower set of yellow arrows), indicating that this drug may be effective in treating the urinary tract infection.

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

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

The cerebrospinal fluid sample should be properly labeled with the tube number, patient's name and hospital number or other unique identifier. The samples should be transported to the laboratory immediately.

Normal cerebrospinal fluid has the following characteristics:colorlessclearno clot presentspecific gravity of 1.006 - 1.008pH 7.3When the specimen is received in the laboratory, the macroscopic examination is performed immediately. The specific gravity examination may be optional in some laboratories.

The cytospin technique uses a high speed centrifuge to concentrate the cells on a slide in a uniform monolayer 6 mm in diameter. The monolayer distribution enhances the morphological appearance of the cells present.Allow the slides to dry in air for several minutes and then stain them with Wright-Giemsa stain. Cytospin slides may be placed in an automatic stainer or stained manually.Perform a 100 or 200 cell differential and record the number of neutrophils, eosinophils, basophils, lymphocytes, monocytes, macrophages, and blasts cells.Pathologists must review any slide which has tumor cells, unidentified cells, or immature stages of cells, such as blasts.Since criteria for review may vary from one laboratory to another, be sure to check the requirements in your laboratory before reporting the differential.

It is important for a quality urine sample to be sent to the laboratory in order for an accurate set of results to be produced. There are various types of urine samples that may be acceptable for urinalysis in the laboratory. The most common urine collection types are:Random Specimen: This is the specimen type which is sent to the laboratory for analysis most commonly. This type of urine sample is easy to obtain and is also readily available. As the name implies, the random specimen can be collected at any time. Patients should be careful not to touch the inside of the cup or cup lid to avoid any contamination.First Morning Specimen: This type of urine specimen is collected when the patient first wakes up in the morning. This is also occasionally called an 8-hour urine specimen.Midstream Clean Catch Specimen: This type of urine specimen concentrates on the reduction of contaminants in the urine sample by requiring special cleansing protocols. The urine midstream is then collected into a clean container.Other, less common types of urine specimens include:Timed Collection SpecimenCatheter Collection SpecimenSuprapubic Aspiration SpecimenPediatric SpecimenThe general procedure for using a reagent strip is outlined in this exercise. Each test on the strip will be discussed in detail in the remaining exercises.

False Positives:False positive results can be attributed to strong oxidizing agents such as hydrogen peroxide (H2O2) or bleach (hypochlorite). False Negatives:False negative results occur when elements present in the urine interfere with either the enzymatic reaction or prevent the oxidation of potassium iodide. Examples of some substances that may produce false negative results, depending on the reagent strip that is used, include: large quantities of ketonesaspirinascorbic acid > 50 mg/dL levadopa5-hydroxyindoleacetic acidhomogentisic acidsodium fluoride ( a preservative) A specific gravity higher than 1.020 may lower glucose reagent sensitivity, especially in the presence of a high urine pH. Exposing reagent strips to excess humidity may also reduce glucose reagent reactivity.NOTE: Check the package insert of the reagent strips used in your laboratory for interfering substances that may affect glucose results.

Although the procedure is simple to perform, accurate results depend on careful adherence to manufacturer's directions and adequate quality control. Normal and abnormal controls should be tested whenever a new lot of strips is opened, and at the frequency defined by the laboratory's procedure. If quality control results do not correspond to the published control values, the problem must be resolved before patient samples are tested. High levels of ascorbic acid (Vitamin C) in the urine may inhibit some reagent strip reactions, such as glucose, blood, bilirubin, nitrate and leukocyte esterase. The urine dipstick's package insert will provide information about potential interfering substances, including ascorbic acid. Intensely colored urine may make it difficult to correctly interpret color reactions on the dipstick, as demonstrated in the image on the right. The affected tests should not be reported from the dipstick. It would be necessary to use an alternative method of testing if available.

False negative results occur when elements present in the urine interfere with either the enzymatic reaction or prevent the oxidation of potassium iodide. Examples of such substances include: large quantities of ketones aspirin ascorbic acid > 50 mg/dL with some reagent strips levadopa 5-hydroxyindoleacetic acid homogentisic acid sodium fluoride ( a preservative)A specific gravity higher than 1.020 may lower glucose reagent sensitivity, especially in the presence of a high urine pH. Exposing reagent strips to excess humidity may also reduce glucose reagent reactivity.Check the package insert of the reagent strips used in your laboratory for interfering substances that may affect glucose results.

Several manufacturers offer semi-automated instruments (dipstick readers) for reading reagent strips. Use of an instrument removes the subjectivity of visually interpreting color changes on reagent strips, and assures that tests will be read at the correct time. Transcription errors will also be avoided if the instrument is interfaced with the laboratory information system. The technology employed is based on the principle of reflectance, with the amount of light reflected being inversely related to the concentration of substances present. An example of reflectance is the light which is scattered after light strikes an unpolished surface. Since each component on the dipstick produces a different color reaction, the light source for each test must be at the appropriate wavelength. This is accomplished either by using filters or monochromatic light sources. The percent reflectance is determined by dividing the test reflectance by the calibration reflectance and multiplying by 100. Algorithms are used to change the results obtained into a linear relationship with concentration of analyte.

The reagent strip protein method is based on the principle of "protein-error-of-indicators." It produces a visible colorimetric reaction that is capable of detecting most instances of proteinuria. Falsely elevated results can occur if the urine sample is visibly bloody.At one time, sulfosalicylic acid (SSA) was used to confirm all positive protein reagent strip results. However, this no longer routinely done. SSA is a precipitation method that reacts with all forms of protein. False-positives can occur. Any substance that is precipitated by acid will produce false-positive SSA results, including radiographic dyes, cephalosporins, penicillins, and sulfonamides. SSA may be used as a secondary protein detection method if the urine is highly colored so that the colorimetric reaction is masked on the reagent strip.

The product profile for Ictotest® points out that bilirubin is very light sensitive, so urine specimens should be protected from excessive light exposure and examined as soon as possible after specimen collection. On standing, bilirubin, which has a goldish color, is oxidized to biliverdin, which is a green color. Many of the procedures used to detect bilirubin will not react with biliverdin, so false-negative results may occur if urine is not fresh when tested.

Record the test result that is associated with the color block that most closely matches the color of the test in the tube. Remember that the final color may not be the test result if the "pass through" phenomenon occurred. Test results should be recorded according to your laboratory's procedure. Laboratories may choose to record results as 1%, 2%, 3%, etc; 1+, 2+, 3+, etc; or 100mg/dL, 200mg/dL, 300mg/dL, etc. The result of the test in the top image is negative, and the result of the test in the bottom image, reported as a percentage, is 2%. (Note: Colors in the photograph may vary slightly from actual test colors.)

While the Federally Regulated CCF must be used exclusively for DOT drug screen collections, there are extenuating circumstances when another form of CCF may be used. For example, where a post-accident collection must be made and the collector does not have time to obtain a Federally Regulated CCF. In this situation, a Non-Federally Regulated CCF may be used. The collector should note in the "Remarks" section why the Non-Regulated CCF was used.The use of a Non-Regulated CCF for a Federally Regulated collection is not a reason for the laboratory to refuse to test the specimens nor the Medical Review Officer (MRO) to release the results. The use of a Non-Regulated CCF for a Federally Regulated drug screen is a "correctable flaw," which may include the collector detailing the reason for using a Non-Federally Regulated CCF in a "Memorandum for Record."

Scenario 4: The donor returns from the restroom with a sufficient specimen. It is very warm to the touch. The collector is unable to obtain a reading from the temperature strip. Collector's response: The collector completes the collection and prepares the specimen for shipment. The collector explains the situation with a supervisor. If the supervisor concurs that an observed collection is in order, the collector next tells the donor that a new collection will be conducted under direct observation. The collector explains that because the temperature of the specimen was not within the acceptable range (90-100° F/32-38° C) there is suspicion of substitution or adulteration. A new CCF is initiated. The collector marks on the CCF that the collection is observed and notes under Remarks why it is observed. The collector also notes the control number of the suspect collection. The observed specimen along with the suspect specimen are both shipped to the laboratory in separate plastic tamper-resistant bags.

Scenario 5: The collector notices that the urine the donor just handed to her has a very strong smell like that of a cleaning product such as bleach. Collector's response: The collector completes the collection in the usual manner and prepares the specimen for shipment. The collector explains the situation to a supervisor. If the supervisor concurs that an observed specimen should be collected, the collector explains to the donor that because of the strong, unusual smell, the first specimen is suspect for adulteration and that a directly observed collection will be done. A new CCF is initiated. The collector marks on the CCF that the collection is observed and notes under Remarks why it is observed. The collector also notes the control number of the suspect collection. The observed specimen along with the suspect specimen are both shipped to the laboratory in separate plastic tamper-resistant bags. In addition to an unusual smell, other indications of adulteration might be an unusual color that cannot be explained by medication, particles or debris in the urine, and a heavy or thick foam that is inconsistent with urine.

Procedural Step Comment Caution Greet and positively identify patient Always use at least two patient identifiers to ensure positive patient identification. Never rely on name placards that are placed on or near the patient's bed or crib to identify the patient. If there is a discrepancy in identification, do not proceed until the discrepancy is resolved. Explain the procedure If the patient is a small child, be at eye level when explaining the procedure. Also explain the procedure to the parent(s). If the patient is aware of what will be happening there is less chance of the patient suddenly jerking away his/her hand when the puncture occurs. Position patient appropriately An outpatient who is a small child should sit on the parent's lap. If necessary, seek assistance for finger puncture if the patient is a small child. Cleanse hands and put on gloves Use soap and water or alcohol-based gel to cleanse hands. Cleanse hands before donning gloves and after removing gloves. Warm puncture site if needed Use the method that is approved by the laboratory for prewarming the puncture site. Never use a moist cloth that has been heated in a microwave as this may cause injury to the patient. Gather appropriate equipment Only have needed equipment at hand. Keep track of ALL equipment to prevent patient injury. Cleanse the puncture site Use 70% isopropanol unless the patient is sensitive to alcohol. Allow the site to air dry. Performing the puncture before the alcohol has dried may hemolyze the blood specimen. Securely grasp and puncture finger Puncture the side edge of the fleshy pad of fingertip. Avoid extreme side and tip of finger. Discard puncture device into appropriate container Puncture device should be discarded into a sharps container that is puncture-proof, has rigid sides, and has a lid Do not discard puncture devices into regular trash or biohazard bags. Injury to personnel who handle these bags may occur. Wipe away the first drop of blood Use slight pressure to facilitate blood flow. The first drop of blood contains tissure fluid that may contaminate or dilute the blood specimen and affect test results. Collect blood into container Allow blood to flow freely into the collection device. Tap the container gently on a hard surface to move blood further down into the tube if necessary. Do not "milk" the finger or scrape the collection device across the finger to obtain specimen; both actions may cause the specimen to hemolyze. Mix specimen immediately upon completion of the collection. Apply pressure to the puncture site to stop the bleeding. Use gauze to apply pressure to the puncture site. It is not advisable to apply an adhesive bandage over the skin puncture site if the child is less than two years old as the child may place the bandage in his/her mouth. Label specimen Specimen must be labeled in the presence of the patient. Unlabeled specimens will be rejected by the laboratory.

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

In some situations, the phlebotomist will make the decision if a blood specimen will be obtained by dermal puncture or venipuncture. The patient's condition, the age of the patient, the amount of blood needed for testing, and the risks associated with the procedure will help the phlebotomist determine the best method for collection.A dermal puncture requires less precision, therefore it is less critical for the patient to be still or immobilized. However, if the puncture is not performed correctly, or an approved site is not used, the puncture may cause more discomfort, or even injury to the patient.The risk of accidental needlestick injury to the patient and phlebotomist is minimal since the puncture device is designed to retract the needle once the puncture is made. The puncture is quick and standardized for puncture depth. However, the procedure takes longer to complete. This delay in collection of the blood specimen could result in hemolysis or clotting of the blood or tissue fluid contamination of the specimen and specimen rejection by the laboratory.The dermal puncture minimizes the amount of blood taken from the patient. This will be important to consider, especially with infants in an intensive care nursery. However, some laboratory tests require larger amounts of blood for testing; in these cases, capillary collection is not an option.If a patient is dehydrated or has poor peripheral circulation, an adequate blood collection from a dermal puncture may not be possible.

On occasion, blood may stop flowing from the punctured site before the required amount of blood is obtained. When this happens, it is not recommended to squeeze harder. This only serves to cut off the supply of blood to the capillary bed. Additionally, squeezing with too much force, especially on the heel of an infant, may cause injury to the patient. The phlebotomist should never scrape the skin with the collection device in an attempt to scoop up the blood that is laying on the surface of the finger or heel. This could cause the blood specimen to hemolyze, making the specimen unacceptable for some laboratory tests. Always allow the drop to flow freely into the collection tube.If a clot has formed, an attempt could be made to dislodge it and re-establish blood flow by wiping the puncture site again with a new alcohol pad, massaging the finger or heel gently, and attempting to recollect the specimen once the alcohol has dried. If blood is not flowing freely from the initial puncture, it may be necessary to perform a second puncture to obtain enough blood for the testing required. If a second puncture must be performed, do not repuncture the same site.

In some instances, the healthcare provider may request an analysis of the capillary blood for blood gases. This is most often requested on infants. Collection of this specimen requires a skilled phlebotomist and specialized equipment. The patient must be positively identified. All appropriate PPE must be used. The procedure for site selection, preparation and puncture is identical to other infant dermal punctures, however, capillary blood gases are always drawn first if other capillary blood specimens will be collected.Blood specimens for capillary gases are always collected in long, large-bore heparinized glass tubes. Blood should be drawn into the tube using capillary action. The phlebotomist should start filling the tube using a large well-formed drop of blood, drawing continuously as the blood flows. Each tube must be filled completely end to end as shown in the image on the right. Every effort must be made to avoid drawing air bubbles or air gaps into the tubes as these could adversely affect the results of the test. Before sealing both ends of the tube, the phlebotomist will insert a tiny metal "flea" into the blood-filled tube and slide a magnet lengthwise back and forth on the outside of the tube. The magnet will cause the flea to move back and forth inside the tube mixing the specimen with the anticoagulant coated on the inside of the tube. This technique should also prevent the blood from clotting, which could result in specimen rejection by the laboratory.The properly filled glass tubes must be delivered to the analyzing laboratory in a timely manner. Delay in specimen delivery may adversely affect the quality of the patient results.

Improper collection of the blood specimen that is used for testing can cause false prolongation of PT or aPTT results. The following table covers several preanalytical variables that may affect PT or aPTT test results Preanalytical Variable Cause of False Elevation of PT and or aPTT Test Result Corrective Action Blood collection tube is inadequately filled. Improper ratio of blood to anticoagulant. Excess anticoagulant causes prolonged PT or aPTT result. Recollect specimen ensuring proper fill to achieve a blood to anticoagulant ratio of 9:1. Patient has a hematocrit level above 55% Improper ratio of blood to anticoagulant. Excess anticoagulant causes prolonged PT or aPTT result. Prepare a specimen collection tube that contains less anticoagulant. Refer to your laboratory's procedure for the proper amount of anticoagulant. Specimen is clotted. Coagulation factors have been activated; insufficient levels left in the plasma. PT and aPTT results will be affected. Recollect the specimen. Specimen collected from an arm with a heparin lock or from a heparinized vascular access device (VAD). Heparin contamination will prolong the aPTT. Collect the blood from a vein rather than a VAD. If blood must be drawn from the VAD, flush it first with 5 mL of saline, and discard the first 5 mL of blood before collecting the specimen. Patient is receiving heparin therapy. Heparin will prolong the aPTT If the patient is being evaluated for possible factor deficiencies or coagulation inhibitors, use a heparin digesting enzyme as a pretreatment before testing the PT or aPTT. .

Step TwoThe next step is an immediate re-run of the PT and/or aPTT test with the newly created sample mixture. The results should be documented on a worksheet to compare to the original PT and/or aPTT tests.Step ThreeThe sample that has been made for the mixing study consisting of the pooled normal plasma and the patient plasma should also be incubated to rule-out any slow reacting inhibitors. To do this the "mixed" specimen is incubated at 37°C for 1 - 2 hours or as long as required by your laboratory's procedure. A set of control tubes should also be incubated at the same time as the mixed sample tube. A pure patient plasma sample and a pure pooled normal plasma sample will serve as the controls in this procedure. You may incubate all 3 tubes together in a water bath or heat block. Refer to the image to the right. The incubation of these controls will account for the heat-labile state of some coagulation factors which will be discussed again.After the incubation phase, the PT and/or aPTT tests should then be repeated once more. If any coagulation inhibitors were present in the patient sample, the incubation phase would have given the ideal temperature and time for the antibody-coagulation factor reaction to take place. This is especially helpful in the case of anti-factor VIII inhibitors since they are often slow acting or weak inhibitors.

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

Case StudyA young patient is admitted from the emergency room with petechial bruising. The attending physician orders a battery of tests including a PT and aPTT. The laboratory performs the requested testing and the result of the aPTT is normal; however the PT is prolonged:PT: 38 seconds (normal range 11-13 seconds)aPTT: 32 seconds (normal range 21-34 seconds)The physician then decides to order a mixing study for further analysis since the patient is not taking any oral anticoagulants, nor does she meet the known patient histories associated with prolonged PT results.

A 50-year-old male with a family history of diabetes visits his physician for routine physical. He reports that he feels his health is excellent. He exercises regularly, but often his diet is high in calories and fat.Physical Examination: Slightly overweight; blood pressure and pulse normal.A basic metabolic panel and PSA are ordered. All results are within reference range except the plasma glucose. The patient's physician orders a hemoglobin A1C (HbA1C) the following week.Laboratory results:Fasting plasma glucose (FPG)= 110 mg/dL (Reference interval 75 - 100 mg/dL)One Week Later:Hb A1C= 6.0% (Reference interval 4 - 6%)

The following describes three patients with a history relating to diabetes and pertinent laboratory results. As this study proceeds, you will be asked if they meet the criteria for diagnosis of diabetes, are at risk for diagnosis of diabetes, and/or whether they are a type 1 or type 2 diabetic.

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

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.

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

Clinical Chemistry Concepts and Applications. Shauna C. Anderson and Susan Cockayne. Long Grove, Illinois: Waveland Press, Inc, 2003.Clinical Laboratory Instrumentation and Automation Principles, Applications, and Selection. Kory M. Ward, Craig A. Lehmann, Alan M. Leiken. Philadelphia: WB Saunders Company, 1994.Laboratory Instrumentation, 4th Edition. Mary C. Haven, Gregory A. Tetrault, Jerald R. Schenken, eds. New York: Van Nostrand Reinhold, 1995.Molecular Diagnostics Fundamentals, Methods, and Clinical Applications. Lela Buckingham and Maribeth L. Flaws. Philadelphia: FA Davis Company, 2007.Principles of Gel Electrophoresis. Available at http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/principles.html accessed 9/29/08.Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 4th Edition. Carl A. Burtis, Edward R. Ashwood, David E. Burns, eds. Philadelphia: Elsevier Saunders, 2005.

An agarose gel electrophoresis first separates the proteins in a serum sample. Antiserum against the protein of interest is spread directly on the gel. The protein of interest precipitates in the gel matrix. After a wash step to remove other proteins, the precipitated protein is stained. This method is qualitative and is used to identify proteins found in multiple myeloma.Below is the immunofixation electrophoresis gel from a serum sample analyzed on SPIFE 3000, Helena Laboratories. After electrophoresis, the precipitated proteins are stained with Acid Violet, a stain developed and used by Helena Laboratories. The SP lane represents a routine serum protein electrophoresis of this specimen. On the next three protein separations, antiserum against IgG, IgA, and IgM were applied to the G, A, M lanes respectively. Antiserum to kappa light chain was added to the next protein separation and antiserum to lambda light chain to the last protein separation.

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.

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

Atherosclerosis. U.S. Department of Health & Human Services National Institutes of Health. Available at http://www.nhlbi.nih.gov/health/dci/Diseases/Atherosclerosis/Atherosclerosis_WhatIs.html Accessed March 25, 2013.Daniels LB, Barrett-Connor E, Sarno M, Laughlin GA,Bettencourt R, Wolfert RL. Lipoprotein-associated phospholipase A2 (Lp-PLA2) independently predicts incident coronary heart disease (CHD) in an apparently healthy older population: The Rancho Bernardo study. J Am Coll Cardiol. 2008;51:913-919.Executive Summary of the third report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA. 2001; 285:2486-2497. Frostegard, J, Wu R, Lemne C, Thulin T, Witztum JL and de Faire U. Circulating oxidized low-density lipoprotein is increased in hypertension, Clin Sci 2003; 105, 615.Garza CA, Montoir VM, McConnell JP, et al. Association between lipoprotein-associated phospholipase A2 and cardiovascular disease: a systematic review. Mayo Clin Proc. 2007;82(2):159-165.Interpretive Handbook, (MC0440rev0407) Mayo Clinic, RochesterMN;2007. Maksimowicz-McKinnon K, Bhatt DL, Calabrese LH: Recent advances in vascular inflammation: C-reactive protein and other inflammatory biomarkers. Curr Opin Rheumatol. 2004;16:18-24.Mora S, Szklo M, Otvos JD, et al. LDL particle subclasses, LDL particle size, and carotid atherosclerosis in the multi-ethnic study of atherosclerosis. Atherosclerosis. 2007;192:211-217.NACB Laboratory Medicine Practice Guidelines. Emerging biomarkers of cardiovascular disease and stroke. NationalAcademy of Clinical Biochemistry Laboratory Medicine Practice Guidelines. 2006.PLACtest animation, diaDexus. http://www.plactest.com/laboratorians/action.php Accessed March 25, 2013.Rifai N, Warnick GR. Lipids, lipoproteins, apolipoproteins, and other cardiovascular risk factors. In: BurtisCA, Ashwood ER. BrunsDE. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 4th ed. St. Louis, MO: Elsevier Saunders: 2006; chap. 26.Ridker PM, Rifai N, Rose L, et al. Comparison of C-reactive protein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular events. N Engl J Med. 2002;347:1557-1565.Sniderman AD. Differential response of cholesterol and particle measures of atherogenic lipoproteins to LDL-lowering therapy: Implications for clinical practice. J Clin Lipidol 2008;2:36-42.Tsimikas, S, Brilakis ES, Miller ER, et al. Oxidized phospholipids, Lp(a) lipoprotein, and coronary artery disease, N Engl J Med: 2005;353:46.Tsimikas S, Bergmark C, Beyer RW, et al. Temporal increases in plasma markers of oxidized low-density lipoprotein strongly reflect the presence of acute coronary syndromes. J Am Coll Cardiol. 2003; 41: 360.Tsimikas, S, Lau HK, Han KR, et al. Percutaneous coronary intervention results in acute increases in oxidized phospholipids and lipoprotein(a): Short-term and long-term immunologic responses to oxidized low-density lipoprotein. Circulation. 2004;109, 3164.Tsimikas S, Witztum JL, Miller ER, Sasiela WJ, et al. High-dose atorvastatin reduces total plasma levels of oxidized phospholipids and immune complexes present on apolipoprotein B-100 in patients with acute coronary syndromes in the MIRACL trial, Circulation: 2004;110, 1406. Walldius G, Jungner I, Holme I, et al. High apolipoprotein B, low apolipoprotein A-I, and improvement in the prediction of fatal myocardial infarction (AMORIS study): a prospective study. Lancet. 2001;358:2026-2033.Yusuf S, Hawken S, Ounpuu S, et al. Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study. Lancet. 2004;364:937-952.

This program has provided some basic information about emergency situations that can occur in a laboratory.However, it is not a substitute for a hands-on first aid course, such as those that may be given at a local community center.The more you know about first aid, the more prepared you will be to act calmly and effectively in emergency situations, possibly saving lives.

Specimen rejection criteria established by your laboratory should be followed at all times, as improperly collected or processed coagulation specimens could adversely affect patient results. Blue-top tubes that are not filled adequately must not be used for coagulation testing as test results may be inaccurate.Hemolyzed specimens should not be used in coagulation testing because of possible clotting factor activation, which may also produce inaccurate results.Grossly lipemic specimens may cause erroneous results or a clot may not be detected if a photo-optical coagulation system is used. An alternative method that is not affected by lipemia, such as an electromechanical method, may be required One way to avoid a grossly lipemic specimen is to ask the patient to fast prior to specimen collection.

The INR component of the laboratory PT/INR result is a calculated value that is used by the clinician to monitor warfarin therapy and adjust dosage as dictated by clinical status. An INR of 2.0 - 3.0 is often desired as the therapeutic range. The following formula is used by the clinical laboratory to derive an INR value. The INR must be adjusted for every new lot of PT reagent (thromboplastin).INR= (PT of patient/PT of geometric mean of the normal population)ISIThe International Sensitivity Index (ISI) value, is provided by the reagent manufacturer as the relative sensitivity of the reagent itself. When opening a new lot of PT reagent (thromboplastin), it is essential to verify that the ISI value provided by the reagent manufacturer is being used with that reagent lot to prevent the reporting of erroneous INR results, which may have serious consequences for the patient.The INR is used to standardize PT results, and in turn, anticoagulant therapy, across laboratory instrumentation, methodologies, and locale.

Molecular diagnostics have begun to play an integral part in clinical laboratory diagnostic testing. Traditionally, molecular diagnostics have been utilized in three major clinical areas: Infectious diseases Genetics Tumor markers These molecular based diagnostic tests, while historically reserved for specialty/reference labs, have recently seen expansion of their utility within the scope of routine clinical laboratories. Molecular based diagnostics can be utilized by small labs as well as large ones, and can be found in virtually every department of the clinical laboratory.

Many clinical laboratories utilize reference laboratories for molecular methodology testing. Transport and shipping of biological specimens must follow laws and regulations governing these types of specimens. Consult your laboratory's accrediting agency and reference laboratory for specific polices and procedures.

Molecular methodologies can be useful in the detection of a variety of diseases that are important public health issues such as:Chlamydia trachomatis (CT) Neisseria gonorrhoeae (GC)Human papillomavirus (HPV)Human immunodeficiency virus(HIV)Herpes simplex virus(HSV)Cytomegalovirus(CMV)In many clinical laboratories, traditional methods have been replaced by molecular methodologies because testing can occur for several pathogens in a single specimen. This is termed multiplex testing.

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.

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

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?

Before ABO HDFN is considered as a possible cause of jaundice and anemia in the newborn, other causes should be considered, for example, erythrocyte membrane defects or red cell enzyme deficiencies. The diagnosis of ABO HDFN in the laboratory differs from diagnosing Rh and other types of HDFN in which clinically significant antibodies must be identified. Diagnosis may be difficult, because the DAT on the newborn's red cells is unreliable. Indeed, many labs do not routinely do a DAT on infants born to Rh positive females, since many will be positive in the absence of clinically significant hemolysis. Cord blood is often retained (e.g., for 7 days) should the infant develop signs of HDFN and required testing.If ABO HDFN is possible, based on incompatible ABO blood groups and a positive DAT, and the mother's antibody screen is negative, many laboratories do not investigate the positive DAT as would be done for unexpected antibodies like anti-D or anti-K (the laboratory does not perform an elution on the newborn's red cells). Instead, the infant's plasma is tested against group A1 (or B cells) and group O screen cells using the indirect antiglobulin test (IAT). A positive reaction with A1 or B cells, but not group O cells, would suffice to report a case of possible ABO HDFN.

Numerous studies have shown that, if administered correctly, RhIg is effective at preventing D immunization. To work, RhIg must be given in sufficient dose, and it must be given before Rh immunization has begun.Unfortunately, despite RhIg's proven efficacy, some women continue to make anti-D in the perinatal period. Such 'failures' are mainly (but not totally) due to human error. Examples of how women may still produce anti-D some 40+ years after the implementation of RhIg prophylaxis: Immunization to D occurred before the administration of RhIg, e.g., before 28 weeks gestation*; Immunization to D occurred after the administration of RhIg at 28 weeks and before delivery because an antenatal fetomaternal hemorrhage (FMH) occurred that was too large for residual passive anti-D to give protection; Female was already immunized from a prior pregnancy but anti-D was too weak to be detected in antibody screen tests prior to RhIg administration; RhIg dosage was insufficient to clear a larger fetal bleed at delivery (e.g., FMH screen was not done or a false negative occurred); Incorrect calculation of RhIg dosage; RhIg administered too late , e.g., well after 72 hours of delivery; Antenatal RhIg not given, e.g., mother had no, or limited, access to prenatal care, or did not seek it, and a FMH occurred during pregnancy; Failure of physician to carry out prenatal blood testing; RhIg not given due to laboratory clerical or technical error in Rh typing the mother or child; RhIg not given in cases such as abortions, ectopic pregnancies, and trauma (e.g., car accidents).* Because anti-D production before 28 weeks is rare (the order of 0.24% to 0.31%), RhIg's use earlier in pregnancy is not recommended. It is not cost effective and would expose most women to an unneeded blood product.

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

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

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

The diagnosis of hereditary hemochromatosis (HH) is made through a combination of laboratory tests and medical evaluation of a patient's signs and symptoms. Iron overload is identified by tests that evaluate iron metabolism, while molecular assays are needed to document mutations in the HFE gene or others such as hepcidin, hemojuvelin, or transferrin receptor. Individuals with documented iron overload who exhibit signs and symptoms consistent with HH and who possess HFE or other mutations are considered to have HH. Other causes of secondary iron overload may need to be ruled out.An example of a testing algorithm is shown.

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

The privacy regulations give covered entities permission to use and disclose PHI for treatment, payment, and health care operations (TPO), without obtaining specific authorization.A covered entity may disclose PHI to other covered entities such as reference laboratories, and home care services, which are providing services to the primary covered entity.The service that the other covered entity is providing must fall within treatment, payment, or health care operations (TPO).If the service being provided does not fall within TPO, an authorization is generally required.An authorization form must state the specific disclosures of PHI to be made, what the information will be used for, and must be signed and dated by the patient.

You will have many opportunities to avoid disclosing protected health information. Here is one simple example: Your best friend asks you to look up her mother's laboratory results. Knowing the HIPAA privacy regulation and your own departmental policies and procedures, you do not disclose the protected health information which she is requesting. You politely tell your friend that you are not allowed to give her the laboratory results.

The simplest form of a carbohydrate is a Monosaccharide, which is the building block of larger and more complex carbohydrates, namely polysaccharides and and glycoconjugates like mucopolysaccharides. Monosaccharidesare not easily demonstrated using histology techniques due to the high number of hydroxyl (OH) groups which render the molecule extremely water soluble. Most monosaccharides that are present within tissue specimens will be lost during the fixation and standard tissue processing. Glucose, is the most commonly demonstrated monosaccharide in the histopathology laboratory.

Alcian Blue is used in the clinical histology laboratory to demonstrate acid mucins which can be secreted by various connective and epithelial tissue tumors. It is most commonly used on tissue samples obtained from the gastrointestinal tract. The detection of mucins in certain tissue samples may also be indicative of collagen diseases and atherosclerosis. Alcian Blue may also be used in combination with the PAS staining procedure so that both acid and neutral mucins can be demonstrated in the same tissue sample. Alcian Blue will stain acidic mucins blue and PAS will stain neutral mucins rose red. This technique is particularly useful in diagnosing diseases of the gastrointestinal (GI) tract.

Microwave ovens are commonly used in the histopathology laboratory to help expedite special staining procedures and improve diagnostic turnaround times. Staining solutions (with or without slides) are often heated in the microwave to increase the rate at which the dye or other reagent is diffused throughout the tissue sample.

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

Microwave ovens are commonly used in the histopathology laboratory to help expedite special staining procedures and improve diagnostic turnaround times. Staining solutions (with or without slides) are often heated in the microwave to increase the rate at which the dye or other reagent is diffused throughout the tissue sample.

Prevention of HIV exposure is the best line of defense to prevent occupational transmission of HIV as there is no vaccine available to develop specific immunity and the postexposure prophylaxis is toxic. Following appropriate workplace practices in the laboratory focus on preventing needlesticks or other sharps injuries and exposure of mucous membranes and abraded skin to HIV-infected blood or body fluids.

Papillomaviruses are small DNA viruses that measure approximately 55 nm in diameter and belong to the family Papovaviridae. HPV is a non-enveloped virus and is composed of an icosahedral-shaped protein capsid enfolding a circular, double-stranded DNA genome of approximately 7,900 base pairs.Until recently, diagnostic laboratory testing for HPV was impossible since the virus does not grow in tissue cultures or in laboratory animals. Currently, with the recent technologic advancements in molecular biology techniques for HPV testing, scientists have isolated more than 100 different HPV types.

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.

They are easily available.Biological pathogens can be obtained from nature, hospital laboratories, university research facilities, etc.They can be hard to detect.Small quantities can have potentially deadly or incapacitating effects on a susceptible population.They can be used covertly.They can be spread throughout large areas by natural convection, air or water currents.They can be easily spread.Ventilation systems in buildings is one way biological agents may be spread. In addition, transportation facilities could become part of the dissemination system by carrying biological agents far from their initial source.

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

At the highest level are the "national" laboratories. 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.

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

At present, 5 laboratories participate in Level 1 activities. At this level, technical personnel are trained to detect exposure to an expanded number of chemicals in human blood and urine. This includes all Level 3 and 2 laboratory analyses, plus analyses for mustard agents, nerve agents, and other toxic chemicals.

Recent events, including the terrorist attacks on September 11, 2001 and the subsequent bioterrorist releases of anthrax, have been a harsh awakening that the nation’s workplaces could be terrorist targets.Traditionally laboratory safety guidelines have emphasized use of optimal work practices, appropriate containment equipment, well-designed facilities, and administrative controls to minimize risks of unintentional infection or injury for laboratory workers. Today, in addition to the above, laboratories must make a risk and threat assessment, secure data and electronic technology systems, plus develop policies regarding specimen accountability, facility security, and emergency response.The next few pages will cover a number of things that you can do to assist in making your laboratory more risk free to a terrorist attack and some things you can do in case that security is breached. You too have a role in the security of your workplace!

In some laboratories the anticoagulated sample is used to prepare concentrated smears. Placing the fluid in a Wintrobe tube and centrifuging it separates the sample into four layers:fat and perivascular cellsplasmabuffy layer - myeloid and nucleated erythroid cellserythrocytesThe volume of each layer is measured using the scale on the Wintrobe tube and then the percentage of each layer is calculated. Next the plasma is removed and a smear is made from the buffy coat and top of the red cell layer. Either the manual push method or cytospin technique may be used to make the smears. They may be stained with a variety of cytochemical stains. Concentrated smears are used to examine cell morphology and demonstrate the presence of abnormal cells when the marrow is hypocellular. The smears cannot be used for differential counts or evaluation of cellularity.

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.

Quality control comprises both accuracy and precision.Accuracy: refers to the closeness of the measured quantitative value to the reference value.Systematic errors will affect a system's accuracy.Bias is an estimate of systematic error.Precision refers to the agreement of results upon replicate testing of a material or similar material under specified conditions.Random errors will affect a test system's precisionStandard deviation and coefficient of variation are measures of imprecision.

QC testing is performed within a laboratory to monitor and ensure the reliability of test results produced by the laboratory. Control materials (usually liquid controls) are used to monitor the test system and verify that quality patient test results have been attained. A control is a stabilized sample with a predetermined range of result values that simulates a patient sample.Control samples are tested in the same way as patient samples. If the results from testing a control sample are not within the acceptable ranges, we assume there has been a problem in the test procedure, equipment, or the samples themselves. There are many criteria for rejecting a test based on the control samples measurements; these criteria will be detailed further in a later section. Patient results are not reported until the cause of the problem has been found, the problem resolved, and the controls retested to verify that everything is working normally.

Depending on the type of testing that is being performed, many other factors may need to be monitored to ensure reliable test results. These include:Water quality Analytic balance calibration Glassware calibration Centrifuge calibration Thermometer calibrationIncubator temperatures Refrigerator temperatures Freezer temperatures Expiration of reagents, standards, and controls Instrument maintenance Procedure manuals, including written step-by-step procedures of all tests performed by laboratory Method selection, based on local population and clinician needs Normal range verification based on the local population Technical competencies of laboratory staff members

External quality assessment or proficiency testing (PT) is performed to ensure the reliability of test results by comparing results to other laboratories that use the same method system and/or to an assigned value.The CLIA standards for handling proficiency testing specimens are as follows:PT samples must be tested with the laboratory's regular patient load. PT samples must be tested the same number of times that patients' samples are tested routinely. Laboratories participating in PT programs must not engage in interlaboratory comparison of PT sample results. Laboratories may not send PT samples to another laboratory for analysis. Laboratories must document all steps of processing for PT samples. PT is required for only the primary method used for testing of analytes in patients' samples during the period covered by the PT event.In return for their participation, the laboratory will receive the following information:Results for each analyte sample Mean result for each analyte Standard deviation of results by the comparative method Number of laboratories using the same method Standard deviation index (SDI) Lower and upper limits of acceptability of resultsPT results that are between the lower and upper limits of acceptability are considered satisfactory.External quality control serves several purposes, including: Evaluates the internal quality control programDetects errors in a lab's methods, including technical errorsProvides a comparison of testing methods, which is useful in selecting new methods

Both the employer and the employee should share the responsibility for assessing and improving ergonomics in the laboratory. A three-step ergonomic program includes: Finding the hazard Determining what improvements / changes should be made Taking action to improve the workplaceEmployers should: Provide ergonomics education Provide ergonomically designed tools and equipment Allow frequent stretch breaks If possible, adjust work schedules to prevent employees from performing repetitious tasks for prolonged periods of time. As an employee, you should evaluate the ergonomic practices in your work area.Employees should: Understand the risk of injury Apply ergonomic principles to the performance of tasks Look for ergonomic hazards and improve the workplace whenever possible Recognize and report early signs of musculoskeletal disorders (MSDs)

Cornell University. CUErgo. Available at: http://ergo.human.cornell.edu/ Accessed August 4, 2011.National Institute for Occupational Safety and Health. Ergonomics and musculoskeletal disorders. Available at: http://www.cdc.gov/niosh/topics/ergonomics/ Accessed August 4, 2011.UCLA Ergonomics. Musculoskeletal disorders: Anatomy of an injury. Available at: http://ergonomics.ucla.edu/MSD_Anatomy.html. Accessed August 4, 2011.US Department of Labor. Healthcare wide hazards module: Ergonomics. Available at: http://www.osha.gov/SLTC/etools/hospital/hazards/ergo/ergo.html Accessed August 4, 2011.

The primary goal of ergonomics is the prevention of musculoskeletal disorders (MSDs). There are many potential causes of MSDs. Injury can occur from a single event (strain, sprain, slip, or fall) or result from build-up of tissue damage from many small injuries. An MSD can develop over time if a motion is repeated consistently so that the constant trauma causes damage to a muscle, tendon, bone, or bursa of a joint. Force, vibration, or maintenance of an awkward position for a prolonged period of time can cause MSDs.Some specific causes of MSDs that are related to laboratory tasks are: Prolonged use of a keyboard or mouse Prolonged sitting at a microscope Pipetting Screwing and unscrewing vial caps Standing at a laboratory instrument for a prolonged period of time Lack of rest - intensive hours at the workstation with few breaks Sustained awkward position

As a clinical laboratory worker, your role is vital in the health care process. You provide information to doctors, nurses, and healthcare organizations that is vital to proper patient care. Because your role is so important, you must be properly qualified, trained, and licensed for your position. You must also keep up with the latest laboratory techniques and developments by fulfilling continuing education requirements; and you are bound by a code of ethics, which ensures that patient results are accurate, reliable, and free from error and bias.

To practice as a clinical laboratory scientist in the state of Florida, you must have an appropriate Florida license. Without a license, you cannot conduct clinical laboratory examinations or report test results. You do not need a Florida license to work in: Laboratories run by the federal government. Labs that perform only waived testing. Labs run exclusively for research and teaching purposes that do not report patient results.These laboratories may have other licensing and training requirements.

Effective date March 17, 2008All applicants for a Director license must meet the qualifications for a high complexity laboratory director that are defined in 42 CFR 493.1443 as published on October 1, 2007.A licensed physician may direct a clinical laboratory without a separate laboratory director's license if he / she is certified in clinical pathology by the American Board of Pathology (ABP) or the American Osteopathic Board of Pathology (AOBP); is board-certified in the pertinent laboratory speciality; and/or has four years of pertinent clinical laboratory experience (post-graduate) with two years experience in the speciality that will he/she will direct.A non-physician may obtain a director's license for a specialty area if he / she: Holds an earned doctoral degree in a chemical, biological, or clinical laboratory science Is certified in the pertinent laboratory specialty by an approved national board A director can oversee up to five laboratories.

A supervisor is the laboratory director's designee for monitoring compliance with all applicable regulations of the board and the department. Other duties include: Performing the duties of a technologist, if needed, in the specialty area(s) where licensure is held Assigning direct supervision responsibilities to licensed technologists if needed, while ensuring that direct supervision of technicians is properly performed. Evaluating technologists' and technicians' competency in running tests and reporting results Being available to all personnel to answer questions and resolve problems Providing day-to-day supervision of test performance, including on-site direct supervision of testing that is performed by technicians Ensuring that quality control is performed and corrective action taken if necessary Ensuring that no patient testing is reported until corrective action has been taken and the test system is properly functioning Providing orientation to all testing personnel Implementing a quality maintenance program

Technicians perform laboratory testing under direct and general supervision, as required by the test and the conditions of the technician's license. Other duties include:Performing tests only as authorized by the director and the technician's licensed specialty.Following the laboratory's procedure for specimen handling and running testsParticipating in proficiency testing and demonstrating that proficiency samples are tested in the same manner as patient samplesFollowing quality control and instrument calibration policiesDocumenting corrective action taken when results exceed the laboratory's acceptable performance valuesIdentifying potential problems with tests or report resultsNotifying a technologist or supervisor if results are outside the laboratory's acceptable performance levels

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

Laboratory professionals can learn about medical errors and prevention by maintaining their awareness of laboratory-related news and events. They can read print and online news sources, magazines, and professional journals to stay informed. They can also learn what is happening through televised and other media reports. Staying up to date about current trends and progress in error prevention enables professional to learn from mistakes and prevent new ones.

Laboratory discussion meetings help to prevent medical errors. The staff can meet periodically to discuss recent averted adverse events and ones that might have been averted.Discussion should not be about blame. Privacy must be protected, so real names should not be identified. Management can provide guidelines for discussion and analysis.A suggested format for discussion:1. Briefly describe each adverse event.2. Identify its possible causes.3. Discuss relevant guidelines.4. Suggest possible preventive actions.Discussion can include actions that do and do not work to prevent medical errors.

Medical errors are possible at any phase of patient care. Preanalytic medical errors begin with the patient and the places he or she receives medical care--the bedside, chair-side, hospital, clinic-- wherever the patient is located. The possibility for these errors continues through the ordering processes for medical tests or procedures. Preanalytic medical errors also happen with the systems, processes, and procedures involved in the collection of test samples from patients. These medical errors occur during the time before the laboratory is directly involved in assaying and analyzing test samples. Examples of preanalytic medical errors: Wrong patient Wrong test Wrong timing Wrong collection procedure Wrong tube, container, additive

Errors also occur after analyses are completed and reported. Postanalytic errors begin with the medical professionals who receive test results, and they include interpretation of the results. These errors can occur at--the bedside, chair-side, hospital, clinic-- wherever the patient and the medical professional are located. The possibility for postanalytic medical error continues through diagnosis and treatment procedures and processes. These medical errors occur during the time after the laboratory reports test results. Examples: Wrong test value associated with patient Wrong test interpretation Wrong diagnosis Wrong treatmentLaboratory professionals might believe they are not associated with postanalytic medical errors, but they can be. One deadly example is fatal hemolytic transfusion reaction involving laboratory error.

Laboratory administration has these responsibilities: Communicate compliance policies, procedures, standards of conduct to all employees and the consequences of non-compliance. Act on reported problems and suspect activities. Resolve problems and ensure they don't recur. Take appropriate and fair disciplinary actions against all involved. Report to the appropriate government agency when necessary. Promptly return money received from the government to which the laboratory is not entitled. Audit the laboratory's policies and procedures to ensure they are being followed and that they work.

All health care providers, including laboratories, could potentially submit erroneous claims for Medicare reimbursement. These billing errors can trigger an investigation. The creation of a voluntary compliance program, using the guidelines provided by the Office of Inspector General (OIG), can assist health care institutions and laboratories to audit themselves, thereby preventing submission of erroneous claims and a possible fraud and abuse investigation.

The laboratory has written and distributed to all affected employees, standards, policies, and procedures that instruct employees in the proper legal and ethical conduct expected in all areas of business the laboratory conducts. These policies must be adhered to by all employees regardless of status or position in the company.

The laboratory has established a comprehensive training and education program about the laws and regulations that govern the laboratory and the standards, policies and procedures of the compliance program. Mandatory for all employees regardless of status or position in the company. There is additional training for those employees who work in higher compliance risk areas of the laboratory such as billing, marketing, and sales. This interactive software is a component of that training program.

The laboratory monitors and regularly audits compliance activities, policies, and procedures to ensure they are being followed. If a problem is detected through the audit or monitoring process, it should be reported to the appropriate supervisor, manager, or the CO.

The laboratory can only perform tests that are ordered by individuals authorized to order tests. If an employee knows that a test has been ordered by someone other then an authorized individual, the employee should report it to their supervisor or the compliance officer (CO). The laboratory must have a system in place to detect tests that are not performed due to a laboratory error and stop or credit the billing for these tests. The laboratory cannot bill for tests that are not performed. An employee who is aware that a test has been canceled or has not been performed for some reason must follow the policies and procedures associated with correcting the billing. Release of test results Laboratory employees should release test results only to a person who is authorized to receive the test results. The Clinical Laboratory Improvement Amendments (CLIA) define an authorized person as: "an individual authorized under State law to order tests or receive test results, or both." Patients may be considered "authorized persons," if State law defines them as such. Refer to your own State laws regarding release of laboratory results directly to patients.

Panels and Profiles: It is not against the law for a laboratory to allow the use of panels, profiles, and custom panels. However, all applicable CPT codes must be included. The laboratory must ensure that the ordering doctor knows what tests are included in a panel or profile and what CPT codes will be billed to the Medicare program. The laboratory notifies doctors about panels and profiles through a written notice and the requisition. Employees should not permit the order of any panel or profile not authorized by the laboratory.

The laboratory should not offer or provide free testing to any individual in a position to make or control referral for laboratory services: The laboratory may write off charges only when laboratory errors in billing or testing occur. Sales and marketing personnel cannot offer free testing in any form unless approved by the compliance officer. Free testing for indigent patients must be approved by the Compliance Officer. Sales and marketing personnel cannot offer or give anything of value to a customer or potential customer beyond the usual promotional items. If a client solicits special consideration, it should be reported to the sales manager or compliance officer.

A laboratory that receives referrals from a nursing home or skilled nursing facility (SNF) should have a written agreement with that facility: The agreement should define billing and documentation responsibilities. The facility should be responsible for determining the payment status of its patients and is liable for submitting incorrect payment information to the laboratory. Fees should be consistent with other similar customers. A laboratory that provides services to a home health agency treating Medicare/Medicaid beneficiaries should have a written agreement with that agency: The agreement should define billing and documentation responsibilities. The agreement should place the responsibility on the home health agency to establish that all patients receiving laboratory services are "homebound" as defined by Medicare. Fees should be consistent with other similar customers.

The setting is the cafeteria in a hospital or the lounge in an independent laboratory. Two employees, a billing clerk and a medical technologist, from different departments are having lunch together. The billing clerk mentions that she saw a bill go through the system for one of her coworkers for a biopsy. She asks the medical technologist if she has the necessary security level access to see pathology test results because she is concerned about the welfare of the coworker. The medical technologist does have the necessary security clearance to see the results. She should:Correct Answer: Refuse to look up the results for the clerk and remind the clerk that it is a violation of compliance policies to do so, or to ask another to do so. Discussion: The medical technologist has a responsibility to report violations of compliance policies and the friend has put her in a difficult position. For that reason, it is not enough to just refuse the clerk's request. If the medical technologist does not take the responsibility to inform the employee of the policy then there is a possibility that the employee would ask some other employee to do it for her.

The Client Services department is a crowded room divided into cubicles which contain desks separated only by thin moveable dividers. Lots of activity is present including phones ringing, multiple conversations going on at once, etc. A client service representative receives a call from a large client office that she speaks with every day for a variety of reasons. Today the client is requesting the laboratory to write off the charges for a test that the office person ordered by mistake, even though the laboratory has already done the test and reported the results back to the office. Since this service representative works with this office frequently, she believes that this is a rare request. Actions that the client service representative may take are:Correct Answers: Refuse to write off the charges for the test and inform the client that it could be considered an inducement if the laboratory does that, which would make both the laboratory and the office liable should it ever come to light. Offer whatever billing options are available according to lab policies. or Refuse to write off the charges and explain to the client that approval must be obtained from the department manager or the laboratory compliance officer before any action can be taken because writing the test off could be considered an inducement.Discussion: Tests should never be written off by the laboratory automatically. There should always be an approval process involved or a policy that strictly forbids any write off except in the case of an error on the part of the laboratory where documentation exists to support it.

A courier is making a routine stop in a client's office and is approached by the office manager with whom he is very friendly because he has been going to this office for years. The office manager asks the courier if Dr. John Smith is a regular stop on his route and the courier answers yes. She then asks the courier if he would do her a little favor since he stops at Dr Smiths office regularly anyway and drop off an x-ray for her so she won't have to call a courier service. The courier knows that this is a big account for the laboratory and customer service is a high priority for the laboratory. This courier should:Correct Answer: Refuse to do it for the customer and explain to the customer that the laboratory has a policy that says he must only provide courier services related to laboratory.Discussion: Even though this is a single incident, by doing this favor the courier is giving the office manager license to ask these kinds of favors in the future. Since the provision of this free courier service is a form of inducement or kickback to the client, both the client and the laboratory would be involved if the such a practice were to go on regularly and be discovered by the government or by the laboratory. Hiding the incident and asking the office manager to conspire with him to do this will only make it worse for the employee and would lead to serious disciplinary action up to termination.

A busy laboratory is located in a 350 bed urban hospital that provides laboratory testing for the hospital and for the hospital's outreach testing program. A medical technologist in the microbiology department receives a call from a friend who works in a laboratory in a physician office. The physician is not a regular client of the laboratory currently but uses another laboratory for most of their work. The microbiologist knows that the sales department would like to get this account. The friend explains to her that she is doing a quality control check on her in-office microbiology testing and her regular laboratory will do it but is going to charge her for it. She asks the microbiologist if she will do it for free since it is quality control, not Medicare, and is not going to be billed to anyone.She tells the microbiologist that she would like to use the hospital lab for everything but her doctor insists on using the competitor. She indicates that the favor might help get the doctor to try the hospital laboratory for other tests. How should the microbiologist respond to this request?Correct Answer: Explain to her friend that if the hospital does the tests for no charge on the promise of other referrals, both the physician office and the hospital could be liable for violations of the antikickback statute.Discussion: The antikickback statute is implicated in this scenario because the free testing is solicited on the condition that other referrals may occur as a result of providing the favor. In fact, the solicitation itself is a violation of the law. The fact that Medicare patients are not specifically mentioned in the scenario is not sufficient to remove the risk. The technologist should also report the incident to the Compliance Officer and seek advise about what documentation, if any, should be kept concerning the incident.

An Advance Beneficiary Notice (ABN) is given to a Medicare beneficiary to inform him/her (or that person's representative) that Medicare may not provide coverage in a specific case (e.g., for a specific laboratory test). Entities who issue ABNs are known as "notifiers." "Notifiers" can include physicians, practitioners, laboratories, and other suppliers of services that are paid under Medicare Part B. The "notifier" (e.g., the laboratory) must complete the ABN and deliver the notice to the affected beneficiary or his/her representative before providing the items or services that are the subject of the notice. The ABN must be verbally reviewed with the beneficiary or his/her representative and any questions raised during that review must be answered before it is signed. The ABN must be delivered far enough in advance that the beneficiary or representative has time to consider the options and make an informed choice. Once all blanks are completed and the form is signed, a copy is given to the beneficiary or representative. In all cases, the notifier must retain the original notice on file. Note: ABNs are never required in emergency or urgent care situations.

The Healthcare Common Procedure Coding System (HCPCS) and the Current Procedural Terminology (CPT) codes are used to describe specific tests or services. The amount of payment for a test is dependent on the HCPCS or CPT code. HCPCS or CPT codes should be assigned under the supervision of the laboratory technical staff. Billing department employees should never change a HCPCS or CPT code without the approval of a manager or compliance officer. If a billing department clerk notices that a particular HCPCS or CPT code is being rejected by a payer they should report it to their manager. It is against the law to use the wrong HCPCS or CPT code for the purpose of causing or increasing payment for a test.

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.

All employees have a responsibility to maintain the confidentiality of medical information. Medical information should never be discussed outside of the laboratory. Do not leave test orders or test results in areas where they can be viewed by patients. Do not discuss test results or any patient information in areas where patients can overhear the conversation. Be careful not to discuss confidential information on the telephone where patients can overhear the conversation. Employees should verify the identity of an individual requesting patient information.

Laboratories may place phlebotomists or other employees in a physician office if all of the following are done: Employee only performs laboratory related tasks. There is a written understanding given to the physician about what the employee can and cannot do. Periodic audits are done to ensure the employee is following these policies. Laboratories may place printers, computers, fax machines or other equipment or products in client offices as long as they ensure that: The physician understands the equipment belongs to the laboratory. It is used for laboratory purposes like receiving reports or ordering tests. Periodic audits are done to ensure that the client is using the equipment only for laboratory related activities.

The laboratory must ensure that its sales and marketing materials are clear and not deceptive: They should fully inform the physician about the appropriate use of laboratory tests and services. They should not be designed to induce unnecessary testing. The laboratory must have in place a document control system and record retention policy that ensures records are accessible in case of an audit or investigation. Records should be retrievable. Related documents should be linked.

Billing is the highest risk area for the laboratory because it generates the claims that are sent to Medicare and other government payers. Payment and payment errors are the focus of the OIG compliance guidance for the laboratory because of the revenue involved. Fraud and abuse is often perpetrated as a billing scheme. Nearly all laboratory functions can affect the billing of laboratory tests.

A laboratory may not bill a Medicare beneficiary for a test unless it notifies the patient in writing before the testing is done that Medicare is not going to pay for the test. This notice is called an ABN. The ABN must contain the specific name of the test and give a specific reason the laboratory thinks payment for the test will be denied. The beneficiary should sign the ABN and a copy should be sent to the laboratory and one given to the beneficiary. The billing department must have evidence that the ABN has been signed before it bills a patient. A laboratory may bill Medicare even though it knows it will not be paid when it has evidence an ABN has been signed.

The government believes that fraud, abuse and waste exist in the healthcare industry today because of cases it has settled and prosecuted.All healthcare providers, including laboratories, make billing errors.The Office of Inspector General (OIG) believes that honest members of the healthcare community can police themselves if they receive guidance.The OIG has published Compliance Program Guidance documents for health care providers, including laboratories.

Element 7: The laboratory has a duty to respond to problems it detects either through its auditing or monitoring system or as reported by employees.It is required to take corrective action and review policies and procedures to ensure the problem does not occur again.It is required to discipline or retrain employees if necessary.The laboratory will report problems to appropriate government agencies and return any money to which it is not entitled.

Element 2: The laboratory has appointed a Compliance Officer (CO) and a Compliance Committee to serve as the focal point for all compliance activities and decision making.The CO and any member of the compliance committee is accessible to all employees in the laboratory.

Element 4: The laboratory has established a system of communication so that employees can and are encouraged to report problems and suspect activities they might observe or discover, without fear of retribution, including an anonymous option.The anonymous system instructions are posted throughout the laboratory.

Social Security Act: Medicare and Medicaid laws are in this act. Medicare rules and regulations come under this act. Antikickback laws: Provide criminal penalties for individuals or entities that knowingly and willfully offer, pay, solicit or receive money or favors for referrals of tests or services that will be paid for by the Medicare or Medicaid programs. False Claims Act: Provides criminal penalties for knowingly or willingly filing a false claim to a government program. Self Referral (Stark) laws and regulations: Identify financial relationships that have the potential to result in directed referral to one or both of the individuals or entities involved. Prohibit the referral of patients or tests between related entities unless certain conditions are met. Health Insurance Portability and Accountability Act (HIPAA) Prohibits health care providers and payers from improper or inappropriate use of a patient's confidential health information Requires health care providers to insure that a patient's confidential information is kept secure Provides for standardized electronic formats for all health care transactions

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.

Advance Beneficiary Notices (ABNs) allow laboratories to bill Medicare patients directly for specific tests that are not covered by Medicare. A laboratory cannot bill a Medicare Beneficiary for a laboratory test unless it notifies the patient in writing that Medicare is not going to pay for the test. This notice is called an ABN. The beneficiary can choose not have the test performed if they do not want to pay for it. Laboratories cannot make all Medicare beneficiaries sign ABNs. The ABN must contain the specific name of the test. The ABN must give a specific reason the laboratory thinks payment for the test will be denied. The beneficiary should sign the ABN and be given a copy.

Requisitions must be designed to ensure that ordering physicians can choose tests that are medically necessary for their patients. Requisitions should contain reminders about Medicare rules of medical necessity and list the contents of panels and profiles. Requisitions must provide a place for the physician to include diagnosis (ICD9-CM) codes. Physicians should be encouraged to use only the requisitions supplied by the laboratory to order tests. Ambiguous or unclear test orders When the orders for a test are not absolutely clear, the laboratory must contact the ordering physician to clarify the orders before performing and billing for the test. The laboratory cannot guess at the order. The laboratory cannot perform and bill for tests that are not specifically ordered. The laboratory cannot change a physician order without contacting the physician. In any case where specimen integrity or patient care will be compromised by a delay in testing follow the policies the laboratory has established for such cases.

Panels and Profiles: It is not against the law for a laboratory to allow the use of panels, profiles and custom panels. The laboratory must ensure that the ordering doctor knows what tests are included in a panel or profile and what CPT codes will be billed to the Medicare program. The laboratory notifies doctors about panels and profiles through a written notice and the requisition. Employees should not permit the order of any panel or profile not authorized by the laboratory.

It is against the law to offer or ask for money or favors to get a physician to order tests from a laboratory. This is known as an "inducement" or a "kickback." Laboratories should only give supplies to a physician for the drawing, processing, storing or transporting of specimens to the laboratory. The laboratory cannot provide supplies physicians use for their own purposes. The laboratory must monitor the amount of supplies provided to ensure that it matches the number of tests sent to the laboratory. The lab cannot give free tests except in the event of laboratory error. The lab cannot give free education to clients unless it is about the laboratory's services or policies. The lab cannot give excessive or expensive gifts or entertainment to physicians The lab can give discounts but the price must be above cost and at "fair market value."

The laboratory's couriers may not transport items except those related to the testing services offered by the laboratory. Couriers must follow all OSHA standards for the handling and transport of specimens. The laboratory is responsible for all tests it refers to other laboratories. Laboratory should not change CPT codes supplied by a reference laboratory without contacting the reference laboratory. The laboratory is responsible for all tests it bills to Medicare/Medicaid even if the test was performed by a reference laboratory.

Laboratories must not induce physicians to order unnecessary tests through their marketing or education activities: They must monitor the use of laboratory services by their clients. They must correct any situation where something they did caused an unnecessary increase in test utilization. Cost Reports Hospitals laboratories must ensure that information used in hospital Medicare cost reports is accurate and includes only those costs which are appropriate. Laboratories must follow all CLIA and OSHA regulations: failure to do so may result in a False Claims Act violation.

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.

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.

It is important that billing department employees are clear and accurate when communicating with physician office personnel and patients. Never guess at the answer to a question; ask if you are unsure. Do not speculate or express personal opinions. When requesting diagnosis information from the physician office staff be careful to not lead them to give a billable code: The code must come from the patient's medical record. There is an incentive program for patients to find and report fraud and abuse by health care providers, including laboratories, so: Billing department employees must accurately state laboratory policies and procedures, or forward the call to a supervisor to avoid misstatements and misunderstandings.

The laboratory should not offer or provide free testing to any individual in a position to make or control referral for laboratory services: The laboratory may write off charges only when laboratory errors in billing or testing occur. Sales and marketing personnel cannot offer free testing in any form unless approved by the compliance officer. Free testing for indigent patients must be approved by the compliance officer. Sales and marketing personnel cannot offer or give anything of value to a customer or potential customer beyond the usual promotional items. If a client solicits a questionable or illegal item or special consideration, it should be reported to the sales manager or compliance officer.

Laboratories may only lease space from physicians who refer Medicare patients to them under certain circumstances: There must be a written lease for at least one year. Lease price must be at "fair market value." All leases must be reviewed by legal counsel to ensure compliance with antikickback and Stark laws.When leasing or renting equipment to a physician or from a physician the same basic rules apply as for space.If the laboratory is located in a hospital, the relationship between the hospital and a physician who refers to the lab may have antikickback or Stark implications.

Never use sign and symptom information received from a patient for laboratory billing purposes.Never use ICD-9 codes from previous visits for a new visit even if the request is for the same test and the patient assures you that it is for the same reason. (Standing orders are an exception.)ICD-9 codes should be requested when setting up standing orders and will then apply to all subsequent visits. It is not necessary in this case to get a new ICD-9 code for each visit.If the patient refuses to sign an ABN but demands to have the test done: Have the fact that they were given notice (ABN) witnessed by a second person. (By phone if you are located in single-person drawing site). Ensure that documentation is complete.

When communicating directly with patients concerning their laboratory tests or orders be careful not to discuss the following: Why a test has been ordered by a physician. What the test might indicate or what the test results mean. Any opinions about their doctor. Any information about internal laboratory issues. Refer the patient to their doctor for information about the tests and the results.If the laboratory provides any printed information about tests that are designed for patients, give the patient that information.

The setting is automated chemistry department, night shift, busy core laboratory for a hospital based outreach laboratory. A medical technologist who operates the automated chemistry analyzer on third shift encounters short samples a couple of times a night. When this happens, he runs as many of the ordered tests as he can and fills in the blank results with a comment indicating that a short sample occurred. As far as he knows there isn't a policy that addresses this problem directly.The test reports out with the results and the comments. The technologist does not have to change the physician order in any way and is providing the maximum results that can be reported for the specimen in a timely fashion. This is done as a matter of patient care and quality service. There has not ever been a complaint about this practice as far as he knows. Are there any additional steps this technologist should be taking?Correct Answer: The technologist should follow the procedures that the laboratory has in place for testing and billing samples for which there is no order or for ambiguous orders. If the policies do not seem to address his particular situation, he thinks there should be a separate policy to cover this situation or has a question about it, he should talk to his supervisor or to the laboratory compliance officerDiscussion: This choice addresses the problem in the most complete manner, in that the employee fulfills his responsibility to take action when he thinks there is a problem.

The setting is the cafeteria in a hospital or the lounge in an independent laboratory. Two employees from different departments are old friends are having lunch together. A billing clerk and a medical technologist are friends and are having lunch together. The billing clerk mentions that she saw a bill go through the system for one of her coworkers for a biopsy. She asks the medical technologist if she has the necessary security level access to see pathology test results because she is concerned about the welfare of the coworker. The medical technologist does have the necessary security clearance to see the results. She should:Correct Answer: Refuse to look up the results for the clerk and remind the clerk that it is a violation of compliance policies to do so, or to ask another to do so. Remind her of the requirement for each employee to report any violations of policy. Discussion: The Medical technologist has a responsibility to report violations of compliance policies and the friend has put her in a difficult position. For that reason, it is not enough to just refuse the clerk's request. If the medical technologist does not take the responsibility to inform the employee of the policy then there is a possibility that the employee would ask some other employee to do it for her.

The Client Services department which is a crowded room divided into cubicles which contain desks separated only by thin moveable dividers. Lots of activity, phones ringing, multiple conversations going on at once etc. A client service representative receives a call from a large client office that she speaks with every day for a variety of reasons. Today the client is requesting the laboratory to write off the charges for a test that the office person ordered incorrectly by mistake even though the laboratory has already done the test and reported the results back to the office. Since this service representative works with this office frequently she believes that this is a rare request. Actions that the client service representative may take are:Correct Answer: Refuse to write off the charges for the test and inform the client that it could be considered an inducement if the laboratory does that, which would make both the laboratory and the office liable should it ever come to light. Offer whatever billing options are available according to lab policies. or Refuse to write off the charges and explain to the client that approval must be obtained from the department manager or the laboratory compliance officer before any action can be taken because writing the test off could be considered an inducement.Discussion: The primary reason is that the test is not written off simply because the client asks for it to be done. Tests should never be written off by the laboratory automatically. There should always be an approval process involved or a policy that strictly forbids any write off except in the case an error on the part of the laboratory where documentation exists to support it.

A courier is making a routine stop in a client's office and is approached by the office manager with whom he is very friendly because he has been going to this office for years. The office manager asks the courier if Dr. John Smith is a regular stop on his route and the courier answers yes. She then asks the courier if he would do her a little favor since he stops at Dr Smiths office regularly anyway and drop off an x-ray for her so she won't have to call a courier service. The courier knows that this is a big account for the laboratory and customer service is a high priority for the laboratory. This courier should:Correct Answer: Refuse to do it for the customer and explain to the customer that the laboratory has a policy that says he must only provide courier services related to laboratory.Discussion: Even though this is a single incident, by doing this favor the courier is giving the office manager license to ask these kinds of favors in the future. Since the provision of this free courier service is a form of inducement or kickback to the client, both the client and the laboratory would be involved if the such a practice were to go on regularly and be discovered by the government or by the laboratory. Hiding the incident and asking the office manager to conspire with him to do this will only make it worse for the employee and would lead to serious discipline action up to termination. The courier's best course of action, for the protection of his friend the office manager and himself, the physician practice and the laboratory is to not do this and explain the reason to the office manager so she is aware of the consequences of asking this favor.

Busy hospital laboratory in a 350 bed urban hospital that provides laboratory testing for the hospital and for the hospital's outreach testing laboratory. A medical technologist in the microbiology department receives a call from a friend who works in a laboratory in a physician office. The physician is not a regular client of the laboratory currently but uses another laboratory for most of their work. The microbiologist knows that the sales department would like to get this account. The friend explains to her that she is doing a quality control check on her in-office microbiology testing and her regular laboratory will do it but is going to charge her for it. She asks the microbiologist if she will do it for free since it is quality control, not Medicare and is not going to be billed to anyone.She tells the microbiologist that she would like to use the hospital lab for everything but her doctor insists on using the competitor. She indicates that the favor might help get the doctor to try the hospital laboratory for other tests. The microbiologist should:Correct Answer: Explain to her friend that if the hospital does the tests for no charge on the promise of other referrals, both the physician office and the hospital could be liable for violations of the antikickback statute.Discussion: The antikickback statute is implicated in this scenario because the free testing is solicited on the condition that other referrals may occur as a result of providing the favor. In fact, the solicitation itself is a violation of the law. The fact that Medicare patients are not specifically mentioned in the scenario is not sufficient to remove the risk. The technologist should also report the incident to the Compliance Officer and seek advise about what documentation, if any, should be kept concerning the incident.

The company also carries certain responsibilities: The laboratory has a responsibility to communicate to all its employees its compliance policies, procedures, standards of conduct and the consequences of non-compliance. All employees must be able to understand the policies and standards. The laboratory has a responsibility to act on reported problems and suspect activities. The laboratory must resolve problems and ensure they don't recur. The laboratory must take appropriate and fair disciplinary actions against all involved. The laboratory must report to the appropriate government agency when necessary. The laboratory must promptly return money received from the government to which it is not entitled. The laboratory must audit its policies and procedures to ensure they are being followed and that they work.

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

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

There are three key benefits that molecular methods can offer, in contrast to traditional culture methods:Improved sensitivity of detectionImproved specificity of identificationReduced turnaround timeImproved sensitivity of detectionSuccessful cultivation or detection of an organism depends on many factors, including the:Ability of the organism to survive transport Fastidious nature of the organism/its ability to grow in available culture media/systems Number of organisms present in a specimen Ability of a staining/culture system to visualize/recover low numbers of organisms Sensitivity of non-culture (antigen detection) methodsOrganisms that have been shown to be very labile are difficult to cultivate even when they are present in significant numbers. The immediacy of transport, plating, and incubation are critical factors that frequently cannot be controlled in a positive direction.Even under the ideal transport conditions, some organisms may require culture media and conditions that are not routinely available in every laboratory, which reduces the likelihood of successful cultivation.

Prevention of contamination is a very key consideration when introducing molecular methods into the routine diagnostic laboratory setting. Because amplification methods are so sensitive, the incidental introduction of even a few copies of exogenous nucleic acid can lead to false positive results. Both physical design and process controls are key aspects of preventing erroneous results.Significant potential sources of contamination are the large quantities of target molecules from previously amplified materials. Amplicons may contaminate work surfaces, air space, pipettes, and reagents. Scrupulous technique is one aspect of preventing contamination; another is the physical separation of specific steps of the process. Ideally, all molecular work will take place in distinct areas designated for key aspects of the workflow, with each area having its own dedicated equipment (especially pipetting equipment). The three designated areas are:Clean area: Where master mixes and other reagents are prepared in the absence of any specimen material. In this area, protection from aerosol contamination is a key consideration. Use of a dead air box with ultraviolet (UV) lighting for decontamination, or a separated area with controlled airflow, are two ways to address this need.Specimen preparation and extraction area: Separated from the area where amplification and detection of amplified product occurs, in order to prevent contamination of samples with amplicons of previously processed specimens.Amplification, detection, and identification of the amplified product: Ideally, this area would be both separated and enclosed.To some extent, the introduction of platforms that utilize automated specimen processing equipment and/or closed amplification and detection systems mitigates stringent separation and space requirements. Good practice, however, would always include designated spaces for each activity, as well as a defined workflow.

Traditional culture methods for the detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) employing mannitol salt and/or blood agar for cultivation, can take up to 72 hours for isolation and identification, depending on the identification procedures utilized. Concurrent with the development of molecular assays, improvements in culture methods have also been achieved. CHROmagar™ media, specific for MRSA, are employed by many laboratories. These media are both selective and differential, containing chromogenic substrates. MRSA strains utilize the substrates to produce colonies of a specific and characteristic color, minimizing the need for additional identification procedures.Initially these agars required 48 hours of incubation; newer formulations require only 24 hours incubation.Given the reduced incubation and identification requirements, what are the pros and cons of the molecular assays? Cost per test will be greater with the molecular assays as compared to culture methods. Will molecular methods provide for a more efficient workflow and significant improvement in availability of results? To some extent, this will depend on how they can be implemented within each different laboratory setting. Both of the previously described molecular assays require manual specimen preparation and extraction before the sample is placed into the instrument. This hands-on work may actually be greater than the effort expended in swabbing and streaking a culture plate. How much an obstacle this is for implementation will depend on both the volume of testing and the staff available. In a high volume setting, this will be a greater factor.Will tests be performed as specimens come in, or will specimens be accumulated and batched? If controls are required with each run, batching is desirable to reduce this cost. If testing will occur in batches, how many batches can be performed in one day? This will be heavily influenced by the capacity of the instrument. (For example, a single Smart Cycler unit can run up to 16 samples; multiple units would be needed in a high volume lab.) Can they be set up on more than one shift? The greater the number and frequency of batches that can be run, the greater improvement in turnaround time can be realized. Given these variables, implementation of a molecular assay for MRSA is not a given in each laboratory.

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.

A 2009 evaluation and comparison of a variety of commercially available toxin detection assays, glutamate dehydrogenase (GDH) assays, the cytotoxin assay, cytotoxigenic culture, and real time PCR for the C. difficile tcdB gene revealed that ALL methods demonstrated a relatively low positive predictive value, which compromised the utility of a single test for laboratory diagnosis of C. difficile. However, of all methods, PCR had the highest negative predictive value, and was considered the optimum rapid single test.Molecular methods for C. difficile are based on the detection of the tcd gene. With the application of real time methodology, results can be available within 2 to 3 hours. These methods are highly sensitive and demonstrate good sensitivity, in comparison to all methods with the exception of toxigenic culture. As the methodologies and instrumentation are developed and improved, they are increasingly adaptable to the environment of a busy clinical diagnostic setting. The BD GeneOhm™ and Meridian illumigene® assays are examples of currently available molecular assays for C. difficile.

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

Several laboratory methods are currently available to aid in the detection of C. difficile including culture for toxigenic C. difficile (considered the "gold standard" for viable C. difficile detection), detection of Toxin A, B, or both, and molecular detection methods. These methods differ in their sensitivity and specificity and should always be used in conjunction with clinical considerations. To make the diagnosis, it is usually only necessary to submit 1-2 diarrheic (non-formed) stools per episode. Once positive for C. difficile by any laboratory method, there is no need for follow-up assays to make sure the organism or toxins are absent from the initial episode. If assays are performed for subsequent episodes, culture or tissue culture assay for Toxin B are probably most appropriate to avoid the possibility of detecting the initial antigen, toxin, or gene.

Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing, Twentieth Informational Supplement. CLSI document M100-S20. CLSI. Wayne, PA: 2010.Fenner L, Widmer AF, Goy G, Rudin S, Frei R . Rapid and reliable diagnostic algorithm for detection of Clostridium difficile. JCM, 2008; 46(1): 328-330.Forbes BA, Sahm DF, Weissfeld AS, eds.Bailey & Scott's Diagnostic Microbiology. 11th ed. Mosby; 2002.Gillespie SH, Hawkey PM, eds. Principles and Practice of Clinical Bacteriology. 2nd ed. West Sussex, England: John Wiley & Sons Ltd; 2006.Isenberg HD. Clinical Microbiology Procedures Handbook. 2nd ed. Washington, DC: ASM Press; 2004.MDRO Guidelines. CDC website. Available at: http://www.cdc.gov/ncidod/dhqp/pdf/ar/MDROguideline2006.pdf. Accessed July 9, 2010.Vancomycin Resistant Enterococci and the Clinical Laboratory. CDC website. Available at: http://www.cdc.gov/ncidod/dbmd/diseaseinfo/drugresisstreppneum_t.htm Accessed July 9, 2010.

When a clinical isolate is presumptively identified as S. aureus, susceptibility testing will be performed by either the Standardized Disk Diffusion (Kirby-Bauer) or Broth Dilution (MIC) methods, using the following testing conditions as recommended by the Clinical and Laboratory Standards Institute (CLSI): Medium: MHA for disk diffusion; CAMHB + 2% NaCL for oxacillin, methicillin, and nafcillin; CAMHB supplemented up to 50 ug/ml calcium for daptomycin Inoculum: Direct colony suspension (0.5 McFarland Standard) Incubation: 35° C (Testing at temperatures above 35° C may not detect MRSA); ambient air; disk diffusion; 16to 18 hours; dilution methods; 16 to 20 hours. All methods: 24 hrs for oxacillin, methicillin, nafcillin, and vancomycin.

A ground is a conducting connection between an electrical circuit or equipment and the earth, or between an electrical circuit and some conducting body that serves in place of the earth.The purpose of a ground is to prevent the buildup of voltages that may result in a hazardous situation for the connected equipment and/or for the person operating the equipment.All electrical equipment in the laboratory that is not clearly marked as "double-insulated" must be grounded by using a three-pronged power cord.

All laboratory instruments and appliances should be checked for ground integrity and current leakage before initial use, after repair or modification, and any time a problem is suspected.Periodic checks should be made on all electrical wires. Frayed cords are the most common cause of laboratory fires. If frayed cords or wires are found, the equipment should be immediately removed from use and repaired.Report to your supervisor any shocks or tingling received from electrical equipment.

1987 Haz-Com Standard (29 CFR 1910.1200) is designed to help control employee exposure to chemicals on the job. 1990 Chemical Hygiene Standard (29 CFR 1910.1450) is specifically designed to meet the needs of laboratories with large varieties of chemicals, and to require specific training for laboratory employees. 1992 Formaldehyde Standard (29-CFR 1910.1048) is designed specifically for employees who work with formaldehyde. The goal of this standard is to reduce the risk of formaldehyde overexposure by establishing safe exposure limits.

Small spills (as defined by your facility) may in most instances be handled by laboratory or other employees. However, if you experience symptoms of overexposure during the clean up, such as burning eyes, or throat irritation, immediately leave the cleanup area and get help from your institution's Spill Response team or other designated persons.Major spills (as defined by your facility) will usually require immediate assistance from the Spill Response team or other designees. There are several ways to clean up small spills, two of which are described below:1. Dike up the formaldehyde with absorbent pillows. Then dispose of these pillows in a sealed, formaldehyde-labeled container. 2. A chemical that reacts with and neutralizes formalin may be used to treat the spill.Your supervisor will show you the location of these emergency spill clean-up materials or discuss alternative procedures. Be sure to follow your own institution's policies and procedures in regard to formalin spills.

The appropriate SDS (Safety data sheet) for formalin can be found in your laboratory's SDS book or online. You should know where to access the SDS and be familiar with its content. Direct any questions you may have to your supervisor.

Engineering controls must be established to reduce formalin exposure to the lowest possible level. In most cases, chemical fume hoods or/and ventilated grossing stations serve as the primary engineering controls to reduce formaldehyde vapors. Rooms in which formalin is used may also require special direct exhaust ventilation. Formaldehyde should be dispensed or used in a chemical fume hood or other appropriately ventilated and approved work area. Check your laboratory's policies and procedures to be sure you use the engineering controls provided, as well as the required personal protective equipment.

1987 Haz-Com Standard is designed to help control employee exposure to chemicals on the job.1990 Chemical Hygiene Standard is specifically designed to meet the needs of laboratories with large varieties of chemicals, and to require specific training for laboratory employees.1992 Formaldehyde Standard is specifically for employees that work with formaldehyde. The goal was to reduce the risk of formaldehyde overexposure by establishing safe exposure limits.

Formaldehyde solution is a colorless, aqueous solution containing not less than 37% of formaldehyde or CH2O. It is usually supplied in 55 gallon drums.Formalin is a 10% formaldehyde solution that is commonly used in the laboratory.

Your laboratory is committed to providing you with a safe working environment. It also expects you to do your part : Be a responsible member of the laboratory team. Use safe work practices.

The portion of the OSHA Standard that is specific to laboratories is 29CFR1910.1450, which is titled "Occupational exposure to hazardous chemicals in laboratories." Part of this regulation requires laboratory administration to develop a documented chemical hygiene plan (CHP). The CHP defines provisions for procedures, special equipment, personal protective equipment (PPE), and work practices that, if used correctly, will protect employees from the hazards associated with chemical exposure. Within the CHP, you will find:Provisions for worker training in the elements of the CHPThe laboratory's policy regarding chemical exposure monitoring, where appropriateSpecific information regarding medical consultation when exposure occursThe laboratory's criteria for the use of personal protective equipment (PPE) and engineering controlsSpecial precautions for particularly hazardous substancesThe person who is designated as the Chemical Hygiene Officer (the person responsible for implementation of the CHP)

Section seven includes handling and storage information. Section eight contains exposure controls/personal protection information including:Occupational exposure limit values or biological limit valuesAppropriate engineering controlsIndividual protection measures, such as personal protective equipment

Laboratory safety includes a number of precautions designed to protect you and your coworkers. Remember that: eating drinking smoking applying cosmetics or lip balm are forbidden in areas where chemicals are present.

Chemicals that are flammable or combustible should be stored in a fire-resistant cabinet; only a minimal amount should be stored on open shelving in the technical work areas. Be conscientious about storing flammable and combustible chemicals far from sources of ignition. Conduct work involving flammable and combustible chemicals within a chemical fume hood if possible.

Also remember to: Learn basic first aid measures.Read chemical labels.Read safety data sheets (SDSs).Follow warnings and instructions.Use the correct engineering controls and personal protective equipment.Practice sensible, safe work habits.Be knowledgeable about your laboratory's chemical hygiene plan and the location in your laboratory of all reference materials on the hazards, safe handling, storage, and disposal of hazardous chemicals, including the location of SDSs.Know the location of the eyewash stations that are closest to the areas where chemicals are handled and know how to use the eyewash correctly.Know the location of spill kits.

Training records must include:Employee name Most recent date trained Description of training Description, copy, or location of training materials Name and address of trainerThese records must be maintained throughout employment and 90 days thereafter, according to the US Department of Transportation (DOT). IATA requires repeat training every two years. DOT requires training every three years. Laboratory accrediting agencies require training at the frequency appropriate to the specimen types and distance transported. You will be able to print a certificate when you have completed this course that will certify your completion of training for packaging and shipping Division 6.2 (infectious) materials.

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).

A blood specimen is collected from a patient that is suspected of having Hepatitis B. The specimen will be sent via commercial carrier (e.g., UPS) to a reference laboratory for further testing. What classification should be used for appropriate packaging and labeling? Work through the Classification Decision Tree.

A blood sample collected from an outpatient, will be sent by ground using a carrier who follows DOT Regulations for classification of dangerous goods. The specimen is being sent to a reference laboratory for cholesterol screening. What classification should be used for appropriate packaging and labeling?Work through the Classification Decision Tree.

Several things need to be considered when you are determining how to package a laboratory specimen. These considerations include: Type of specimen Solid Liquid Classification Category A Category B Exempt Size of the specimen Temperature at which the specimen must be held during shipping Will dry ice be included in the package? The specimen components Does the specimen contain a preservative, such as formalin, that may be regulated? Mode of transportation Commercial ground Passenger air Cargo air Postal service Private or contract carrier using exclusive use motor vehicle

International Air Transport Association. Guidance document: Dangerous Goods Regulations (DGR). 52nd ed. 2011.National Laboratory Training Network. Packaging and shipping Division 6.2 materials. Georgia Public Health Laboratory; 2011. Sentinel laboratory guidelines for suspected agents of bioterrorism: Clinical laboratory bioterrorism readiness plan. Available at: http://stanfordhospital.org/PDF/bioterrorism/labGuidelinesSuspectedAgentsBT.pdf. Accessed January 31, 2011.US Department of Transportation Pipeline and Hazardous Materials Safety Administration. Transporting Infectious Substances Safely. Available at http://www.phmsa.dot.gov/staticfiles/PHMSA/DownloadableFiles/Files/Transporting_Infectious_Substances_brochure.pdf.

Therapeutic drug monitoring and pharmacogenomics are both pharmacy-related areas within the clinical laboratory. Although each is considered a sub-discipline within laboratory medicine, the two fields overlap significantly. In this course, we will provide an overview of each of these laboratory sub-disciplines and discuss the utility, rationale, and practice of each one.

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

James Brown, a phlebotomist from the laboratory went to the second floor of Memorial Hospital to draw a STAT BMP (chem-8), CBC, and PT on a patient. The patient was in critical condition so the lab results were crucial for treatment. James quickened his pace in order to speed up the result time. He collected the specimens and took them back to the lab. However, the technologist in hematology and coagulation notified him that he would need to recollect the specimen because the CBC and PT were clotted.

Poor and unsatisfactory specimens pose significant problems : They can cause misleading laboratory results.Unsatisfactory specimens must be rejected by the laboratory. The patient must then undergo another venipuncture to get a better specimen. It costs time & money to redraw the specimen.The credibility of the laboratory is reduced if too many unsatisfactory specimens are drawn.

The phlebotomist collects blood & other specimens which ultimately provide doctors and nurses with laboratory test information critical to patient care.He or she therefore plays a vital role in any health care system.

Phlebotomist positively identifies patient. Phlebotomist draws and labels blood specimen. Specimen is transported to laboratory. Specimen is accessioned and processed in lab.

As a professional phlebotomist, you have a critical role in this basic work-flow cycle. The rest of this program contains the information you need to begin training in this important profession.

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.

Laboratories within the LRN are divided into 3 levels: Sentinel labs Reference labs National labsAny laboratory that performs microbial analysis is by default a sentinel laboratory. A laboratory that refers specimens that may contain microbial agents or toxins is also a sentinel laboratory. These laboratories are the first responders in the line of defense. Based on the extent of service provided, sentinel laboratories are designated as basic or advanced.

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.

A basic sentinel laboratory has the following characteristics: Certified by the Centers for Medicare & Medicaid Services (CMS) for subspecialty within the specialty of microbiology Accredited by Clinical Laboratory Improvement Amendments of 1988 (CLIA) or a deemed status agency Has policies and procedures in place for the referral of diagnostic specimens to an advanced sentinel laboratory Has policies and procedures in place for the direct referral of suspicious isolates to an appropriate LRN reference laboratory

These agents are dangerous, highly virulent organisms that should NEVER be manipulated on an open bench! Laboratory infections can occur and the use of a class II, or higher, biological safety cabinets (BSC) is critical when aerosols are likely. The importance of following facility specific safety protocols and standard microbiology practices at ALL times cannot be understated. Agent Biosafety Level Laboratory Exposure Risk B. anthracis BSL-2 Low Y. pestis BSL-2 Medium F. tularensis BSL-2/3 High Brucella species BSL-2/3 High Burkholderia species BSL-2/3 High

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

Any isolate with the following features should be immediately referred to your LRN reference laboratory: Gram stain shows large, gram-positive rods with sub-terminal or central spores (if present) Gray colonies with a ground glass appearance Non-hemolytic on sheep blood agar (SBA) Tenacious or "sticky" colonies like petroleum jelly Catalase positive Non-motile

Any isolate with the following features should be immediately referred to your LRN reference laboratory: Gram stain shows tiny, weak staining, gram-negative coccobacilli Gray-white, opaque colonies on sheep blood agar (SBA) and chocolate (CHOC) agar a at 48 hours Slow growth in broth (up to three weeks) Oxidase negative Urea negative

Always follow your facilities procedures and contact your Laboratory Response Network (LRN) reference laboratory for guidance with regard to the toxins and viruses that may be used as bioterrorism agents. A sentinel laboratory should NOT attempt to: Accept or process environmental or animal samples Culture from clinical specimens or detect for these agents Collect specimens for suspect viruses unless directed by a public health officialThe laboratory testing listed on the following pages is intended ONLY as information. For suspect specimen collection, contact your LRN reference laboratory for guidance before collecting or referring specimens.

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.

It is important to consider that commercial identification systems may misidentify the bioterrorism threat agents. There is an increased risk of incorrect identification for organisms that are: Slow growing Fastidious Limited in the number of strains available in the database It is also important to note that automated systems can create aerosols, which increases the possibility of laboratory exposure to these pathogens.

Assayed controls have been analyzed by the manufacturer so that the range of values for the analytes they contain is known. Unassayed controls are unknowns. The laboratory purchasing the controls must determine the concentration of each analyte.

External quality control (also called proficiency testing or PT) evaluates a laboratory's testing results by comparing them to those of similar laboratories. Specially prepared specimens are obtained by multiple laboratories participating in the proficiency testing program.These "unknowns" are then tested by the participating laboratories and results are returned to the proficiency testing program. For external quality control, the laboratory must use its routine testing methods.

Daily documentation and evaluation of quality control is vital to detection of errors. One of the most commonly used methods for documentation is the Levey-Jennings control chart (L-J chart). In 1931, Dr. Walter Shewhart, a scientist at the Bell Telephone Laboratories, proposed the application of statistical-based control charts to monitor industrial manufacturing processes. In 1950, S. Levey and E.R. Jennings applied Dr. Shewhart's control charts to the clinical laboratory.

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.

Most bacteria that are encountered in the clinical microbiology laboratory will be gram-negative bacilli. Gram-negative bacilli include the Enterobacteriaceae, nonfermentative bacilli, Haemophilus species, and several fastidious species. Gram-negative bacilli can be observed in this field.

Polymerase chain reaction (PCR) is a molecular diagnostic tool that allows for in vitro amplification of DNA at a rapid pace. The steps of PCR are: denaturation, annealing, and extension. DNA doubles during each PCR cycle, resulting in exponential accumulation of the targeted DNA fragment. The ability to rapidly amplify a specific nucleic acid sequence in a short period of time has revolutionized molecular diagnostics. A major advantage of PCR is that only a small amount of initial intact genomic DNA is required. PCR can be effective with only a single strand of intact DNA. This is often critical for forensic analysis where only trace amounts of DNA are available. PCR has also been used to amplify and analyze ancient DNA for anthropologic and archeological purposes. PCR augments many common laboratory methods such as Southern and northern blotting. These techniques require a large amount of DNA. PCR can be used to supply these techniques with the needed amounts of specific DNA. PCR can also be incorporated into DNA sequencing and genetic fingerprinting.

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

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.

The invention of PCR and real-time PCR has lead to many major scientific advances. Though both methods are still regularly used in laboratories, real-time PCR is gaining popularity and quickly becoming the most cost- and time-effective method for analyzing DNA products.The use of real-time PCR expands to many areas of the clinical laboratory including genetics, virology, and microbiology. The uses mentioned throughout this course are only a small sampling of the many different applications in which this technology is used. With more real-time PCR platforms and practices being created, the growth and potential of this technology is just beginning. However, the concept and process will stay the same and it is important for laboratory professionals to understand and learn about this technology.

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

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.

Dimorphic is a term used to describe two circulating red cell populations. One is the patient's basic red cell population while the other is a second population with distinct morphological features. The distinct populations can be observed in the top image on the right. The bottom image on the right illustrates the two distinct peaks that are observed on the RBC histogram from the automated hematology analyzer.Dimorphic red blood cell populations can be found in conditions/situations such as: red blood cell transfusions, myelodysplasia, refractory anemia with ringed sideroblasts, hemolytic processes involving a reticulocyte response, and when patients are given erythropoietin therapy.It is important to recognize when a population of cells in the peripheral smear is not in context with anticipated laboratory findings and the clinical situation.

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

Patient A.D., a 30 year old female, was admitted to the hospital in active labor to deliver at 37 weeks gestation. Transfusion service (TS) records showed A.D. to be group O Rh negative with no record of unexpected red cell antibodies.Maternal history showed two prior pregnancies. Her first pregnancy four years ago ended in spontaneous abortion at 9 weeks gestation and she received a mini-dose (50 µg) of RhIg.In the second pregnancy, two years ago, the infant typed as Group A Rh positive, DAT negative. Patient A.D. was injected with RhIg within 72 hours of delivery. The laboratory also confirmed that in the current pregnancy RhIg was administered at approximately 28 weeks gestation subsequent to a negative antibody screen.After many hours of non-productive labor, the physician considered that labor had stalled and decided to do a cesarian section (C-section). According to hospital policy for C-sections, a type and screen was ordered.

As applied to pregnancy, RhIg's purpose is to prevent immunization to the D antigen in the perinatal period and thus prevent HDFN due to anti-D. If the mother has already produced anti-D, RhIg is of no use.Accordingly, RhIg is routinely administered to Rh negative women* not previously sensitized to the D antigen under the following circumstances:1, Antenatal. Antepartum prophylaxis of 300 µg (1500 IU) at about 28 weeks gestation in the USA and Canada, which could be weeks later, depending on how physician appointments are scheduled. To illustrate variation in antenatal international practice, in the UK smaller doses of RhIg (e.g., 500 IU) may be given at 28 weeks and 34 weeks, although many UK facilities issue a 1500 IU dose at 28–30 weeks. With antenatal administration, the Rh of the fetus is usually unknown. Some transfusion services recommend a further antenatal dose if the infant is undelivered after 40 weeks.2. Postnatal. Prophylaxis of 300 µg (1500 IU) at delivery of an Rh positive or weak D infant, preferably within 72 hours of delivery but can be given up to 28 days later if administration is delayed. If RhIg administration is delayed beyond 72 hours, laboratory policies differ as to when it would no longer be administered.* Policies related to women who are weak D (formerly Du) are discussed later.Note: Because RhIg contains IgG anti-D, when given antenatally, it can cross the placenta and sensitize fetal D-positive red cells. Occasionally the fetus may be born with a weakly positive DAT, but significant hemolysis does not occur.

As noted, reaction strength can suggest whether anti-D at delivery is likely immune or passive, however, several factors affect RhIg's reaction strength in laboratory tests.Before proceeding, take a moment to think about the following questions. Which reaction strengths are typically seen at delivery from RhIg-derived passive anti-D? Which variables can affect RhIg-derived passive anti-D reaction strengths?

Based on the results of the mini-panel, the laboratory concluded that only anti-D was present and that it was consistent with administration of RhIg at 28 weeks.Patient A.D. delivered a 5 lb 13 oz female by C. section with serologic test results on cord blood as follows. Well washed cord red cells were used for ABO and Rh(D) typing to remove possible Wharton's jelly.Before proceeding to the next page, evaluate if the infant's ABO and Rh(D) types are valid. You will be asked questions that assess basic knowledge of blood grouping practices and test results for newborns. ABO Forward Group ABO Reverse Group Rh anti-A anti-B A1 cells B cells anti-D* 0 0 NT NT 3+ NT = not tested / * monoclonal IgM anti-D DAT Reagent DAT CC Polyspecific AHG w+ 2+ W+ = microscopic positiveAHG = antihuman globulin serum CC = IgG sensitized cells Note: It is the lab's policy to add IgG sensitized cells to weak antiglobulin test results.

The newborn showed no clinical evidence of HDFN or early newborn hyperbilirubinemia, with related laboratory tests as follows (laboratory's reference ranges for newborns in brackets): Test USA SI Hemoglobin 16 g/dL (13.5 - 21.0 g/dL) 160 g/L(130.5 - 226.0 g/L)* Hematocrit 52% (43-62%) 0.52 (0.43-0.62) Total bilirubin (cord blood) 2.1 mg/dL (<2.5 mg/dL) 35.9 µmol/L (<43 µmol/L) *Most countries that adopted SI do not use the official SI unit for Hb (mmol/L), but rather use g/L.

Once again, policies vary from laboratory to laboratory since the issue is not directly addressed by blood safety standards. For example, AABB and other standards require a version of the following: When clinically significant red cell antibodies are detected or the recipient has a history of such antibodies, RBC components shall be prepared for transfusion that lack the corresponding antigen and are serologically crossmatch-compatible, where serologically is taken to be an IAT at 37oC. If no clinically significant antibodies were detected in antibody screen tests and the patient has no record of such antibodies, detection of ABO incompatibility is required, which can be accomplished by immediate spin crossmatch or an electronic crossmatch. The key issues are whether detectable passive anti-D from RhIg or a record of passive anti-D from RhIg should be considered clinically significant for crossmatch purposes. Because standards do not directly address these issues, TS laboratories are left to interpret what is required to meet the standards. Practices may be further complicated because of the transfusion service's laboratory information system (LIS).

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

A risk is a future event that may result in loss or injury. In healthcare, the future event specifically refers to injury to a patient (negative patient outcomes). Risk management involves the creation of policies and procedures and the implementation of practices that are aimed at the prevention of future events that could result in negative patient outcomes. Basically, risk management is the process of making and carrying out decisions that will assist in the prevention of detrimental events. It also describes techniques used in the prevention of adverse consequences or losses if an unintended or unexpected event does occur. It is important to demonstrate how to analyze risks and what options a laboratory has in deciding how to deal with risks. Often, a critical incident that affects patient care is the result of multiple errors and system flaws that coincide.

Every director and manager knows that at any given time, unexpected events arise in the laboratory. Unfortunately, the negative unexpected events can have a serious impact on the effectiveness as well as the financial welfare of the laboratory. Some of these negative events can have such devastating consequences that the sheer risk of them occuring cannot be left to chance. Such impacts can be avoided or dealt with systematically through the process of risk management. The challenge is to assess and control risk in an appropriate and cost-effective manner. By using risk management strategies, the laboratory can approach risk in a structured and calculated manner.

The Health Insurance Portability and Accountability Act (HIPAA) was enacted by Congress in 1996. There are two titles within the Act that are of importance in the clinical laboratory. Title I of the act protects insurance coverage for workers and their families if they change or lose their jobs. Title II of the act requires the establishment of national standards for electronic health care transactions as well as national identifiers for providers, health insurance plans, and employers. A very important feature of Title II is that it addresses the security and confidentiality of health data. It also entitles patients to have access to their medical records within a reasonable amount of time. Another concept worth noting within HIPAA rules is the term "covered entity". A covered entity is a term applied to a health care provider who transmits any health information in connection with a HIPAA transaction. Since substantial fines can be levied against any covered entity that does not comply with the Privacy Rule, it is important that all employees in the laboratory are fully aware of and compliant with both HIPAA and state confidentiality requirements. This should be enforced with both initial and follow-up training. The privacy of electronically transmitted personal health information (PHI) was further expanded and strengthened with the passage of the American Recovery and Reinvestment Act of 2009 (ARRA).

The Safe Medical Devices Act of 1990 requires user facilities (e.g., hospitals, nursing homes) to report suspected medical device-related deaths to both the FDA and the manufacturers. Medical device-related serious injuries must be reported to the manufacturer. However, if the medical device manufacturer is unknown, the serious injury is reported by the facility to the FDA. Laboratory personnel should familiarize themselves with their institution's procedures for reporting adverse events to the FDA.Medical devices that are included in this reporting requirement (if they may have caused serious injury to a patient or patient death) are laboratory instruments, reagents, or devices used during phlebotomy procedures. If it appears that a device has caused injury, it is important that the device and packaging be saved and any serial or lot numbers noted. An incident report should be completed within 24 hours. The incident report must then be handled by the institution's "Risk Management" department (if applicable), who will file the necessary reports.

A study that was published in 2002 concluded that 68 - 87% of laboratory errors occur in the preanalytic and postanalytic stages of the testing process with the majority occurring in the preanalytic phase.* Steps in the pre-analytic phase occur both inside and outside the laboratory, and are performed by both laboratory and non-laboratory personnel. While the following list is not exhaustive, some of the most common sources of error in the preanalytic phase include:Patient preparation Patient not told to be fasting; improper or no instruction to patient on proper collection of specimen such as clean catch urine. Patient injured during phlebotomy Development of hematoma resulting in no specimen obtained for testing. Requisition errors Patient information missing, illegible, or on wrong patient; wrong tests ordered. Patient identification Patient incorrectly identified. Labeling of specimen Specimen not labeled or incorrectly labeled. Preparation of specimen Specimen centrifuged too long or not long enough; specimen placed in improper preservative.Storage of specimen Specimen not refrigerated or frozen as required or refrigerated when it should be at ambient temperature. Shipment of specimen Shipped at ambient temperature when it should have been shipped frozen; delay in shipment. Accessioning process including preparation for analysis Sorted into wrong batch; incorrect labeling. Order entry Incorrect data entered during manual entry of a test requisition. Specimen sub-optimal Not enough specimen for testing; visible hemolysis. Contamination Inadequate cleansing of venipuncture site resulting in contamination during blood culture collection. *Reference: Bonini P, Plebani M, Ceriotti F, Rubboli F. Errors in laboratory medicine. Clin Chem. 2002;48:691-698. Available at http://www.clinchem.org/cgi/content/full/48/5/691#T2BAccessed June 23, 2010.

Recently, significant attention has been focused on errors made during the postanalytic phase of laboratory testing and the impact errors made during this phase have on laboratory-related patient outcomes. Similar to the preanalytic phase, the postanalytic phase can be subdivided into those procedures that are within the laboratory, and those outside the laboratory; where the physician receives, interprets, and acts on the laboratory results. The examples listed below are limited to possible postanalytic errors that may occur within the laboratory and over which the laboratory has more control: Laboratory results not verified before being reported. Improper data entry or typing mistakes causing erroneous information to be reported. Critical values not reported, or not reported in a timely manner. Laboratory tests not reported or reported to the wrong health provider.(For example, poor communication to a patient's physician of the results of laboratory tests that are pending at the time of a patient's discharge.) Lack of timeliness of reporting laboratory results (slow turnaround time). Misinterpretation of an alphabetic flag in the result field (i.e. lower case "l" interpreted as the number "1". Oral results misunderstood by receiving party- no "read back" requested to confirm that data was correctly received.

Another area where risk management is vitally important is that of employment practices. The laboratory can also minimize its potential liability by being vigilant during the pre-employment process; ensuring that employment guidelines (and if required, termination guidelines) are followed consistently. A resource that discusses major statutes related to employees is included on this page.

Astion ML, Shojania KG, Hamill TR, Kim S, Ng VL. Classifying laboratory incident reports to identify problems that jeopardize patient safety. Am J Clin Path. 2003;120:18.Bersch C, Clemons K. The wheel of misfortune prepare to win if disaster strikes. MLO. 2008;40:12. Berte L. Laboratory quality management: A road map. Clin Lab Med. 2007;27:771.Bonini P, Plebani M, Ceriotti F, Rubboli F. Errors in laboratory medicine. Clin Chem. 2002;48:691-698. Available at http://www.clinchem.org/cgi/content/full/48/5/691#T2B Accessed February 2, 2012.Carraro P, Plebani M. Errors in a stat laboratory: Types and frequencies 10 years later. Clin Chem. 2007;53:1338-1342.Carroll R ed. Risk Management Handbook for Health Care Organizations. 5th ed. San Francisco: Jossey-Bass; 2006.Howanitz PJ. Errors in laboratory medicine: Practical lessons to improve patient safety. Arch Pathol Lab Med. 2005; 129:1252 - 1261.Kalra J. Medical errors: Impact on clinical laboratories and other critical areas. Clin Biochem. 2004;37:1052-1062. Kohn LT, Corrigan JM, Donaldson MS, eds. To Err Is Human: Building a Safer Health System. Washington, DC: National Academies Press; 2000.Lippi G, Guidi G. Risk management in the preanalytical phase of laboratory testing. Clin Chem & Lab Med. 2007;45:720. Malone B. Risk management for clinical labs. Clinical Laboratory News. 2008;34:1.Pierangelo B, Plebani M, Ceriotti F, Rubboli F. Errors in laboratory medicine. Clin Chem. 2002;48:691-698.Sazama K. Legal implications of laboratory errors. Lab Med. 2005;36:213.Smith TJ. Strategies for error reduction, Advance Magazine. 2009;18:25.Vidal Y. 101 Ways to Prevent Medical Errors: A 24-year Odyssey. Andrews, TX: Lara Publishing; 2002.

Risk management is the process by which the laboratory becomes aware of risk that can cause potential loss exposure. Loss could result from a variety of circumstances and, depending upon the circumstances, can be insignificant to catastrophic. Therefore, in addition to identifying the risk, it is also important to establish the severity of the risk by determining the probability of loss if the risk is not controlled. There are a variety of ways the laboratory can identify potential risks. The following methods are generally considered among the more effective methods. Evaluating complaints from patients and clients Reviewing incident reports Evaluating deficiencies cited by accreditation or governmental inspections (external assessments) Reviewing proficiency testing results However, it is important to be sensitive to events or trends that may alert you to risk potential. For example, although continually monitoring the number of phlebotomies that are performed in a day may not be a normally effective method for evaluating risk, if there is a sudden staff shortage of phlebotomists or a sudden increase in hospital census, it may be worth evaluating the number of phlebotomies that are done because the risk potential may increase if the phlebotomy staff is overworked.

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

The Commission on Laboratory Accreditation (COLA) was founded in 1988 and in 1993 was granted deeming authority by CMS. Although COLA was initially created to provide voluntary inspections of physicians' offices, COLA now accredits not only laboratories in physicians' offices but also laboratories associated with hospitals, mobile clinics, Veterans Administration, Department of Defense and independent laboratories. Its laboratory accrediting program is also recognized by the Joint Commission. COLA is approved by CMS to accredit the following specialties: Chemistry Hematology Microbiology Immunology Immunohematology/transfusion services Pathology, including cytology, histopathology, and oral pathology

Venipuncture is the collection of blood from a vein. The person having the responsibility for the performance of the venipuncture may be a phlebotomist who is a part of the laboratory staff, or he/she may be another healthcare professional that has been trained to perform this duty. In this course, we will refer to the person performing the venipuncture as the phlebotomist.

Hidden errors are those that cannot be detected or corrected by the laboratory analyst prior to testing. Most often these errors can be prevented by the phlebotomist following correct venipuncture procedure for every procedure, every time.Hidden errors include hemoconcentration, incorrect order of draw, and (the most serious of all errors) misidentification of patient or specimens. Because these errors often are unknown, the analyst may inadvertently report erroneous patient results which could be harmful to the safety and well-being of the patient. Condition What is it? How does it happen? What is the Result? Hemoconcentration Blood pools at site of venipuncture Tourniquet is applied for a prolonged period of time Test results may be inaccurate because blood components move between blood and tissues Pouring Blood between tubes Mixing contents of two or more tubes Removing top of tube to combine contents of one tube with another Inaccurate test results due to over or under dilution or incorrect anticoagulant Clots form due to lack of mixing Patient may have to be redrawn Incorrect patient identification and incorrect specimen labeling Using the wrong name to label a specimen Failure to positively identify EVERY patient using 2 unique identifiers BEFORE beginning venipuncture Failure to label EVERY specimen in the presence of the patient Failure to concentrate fully on the task Results reported to caregiver for wrong patient Compromises patient care; may be life-threatening

Fill blood collection tubes completely (until vacuum is exhausted) to ensure the correct blood to anticoagulant ratio necessary for accurate patient results. Specimens may be rejected by the laboratory if the tube is short-filled or over-filled. To avoid short-filling of tubes, the phlebotomist must ensure that the blood flow stops completely before removing the tube from the needle. When using a winged device (butterfly) to collect blood for coagulation studies (e.g., protime, aPTT), the phlebotomist must draw a light blue top "waste" tube before attaching another light blue top tube for testing. If the air in the tubing of the winged device is not displaced into a waste tube and is drawn into the tube used for testing, the tube used for testing will short-fill. The laboratory may reject the specimen because of invalid blood to anticoagulant ratio.

Clinical and Laboratory Standards Institute (CLSI). Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays; Approved Guideline. Fourth ed. CLSI document H21-A4. NCCLS. Wayne, PA: 2003.Clinical and Laboratory Standards Institute (CLSI). Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture; Approved Standard. Sixth ed. CLSI document H3-A6. NCCLS. Wayne, PA: 2007.Clinical and Laboratory Standards Institute (CLSI). Procedures for the Handling and Processing of Blood Specimens; Approved Guideline. Third Edition. CLSI document H18-A3. NCCLS.Wayne, PA: 2004.Ernst DJ. Applied Phlebotomy. Baltimore, MD: Lippincott Williams & Wilkins: 2005.Lowe B. Reinforcing safety sticklers. Advance for Medical Laboratory Professionals. May 2004; 16:2A-3A.The Joint Commission. National Patient Safety Goals. Available at: http://www.jointcommission.org/standards_information/npsgs.aspx. Accessed July 2, 2012.

When the results on Mr. John Ready were called to the nurse, she was very surprised that the result of his CBC was normal. The nurse explained to the laboratory technologist that Mr. John Ready had a known diagnosis of lower GI bleeding. His hemoglobin had been very low for the past 24 hours because of the internal bleeding, and she thought it was very surprising that his hemoglobin had normalized so quickly without having received a blood transfusion. Mr. Ready's doctor decided the patient should be redrawn to ensure a correct result. The nurse further questioned if the phlebotomist could possibly have drawn the wrong patient because earlier that day Mr. Ready had been moved to room 831, and room 825 was presently occupied by a patient named Walter Redding. If Julie had properly identified the patient by asking him to state his name and then checking the name and identification number on the wristband, she would have realized that the patient in 825 was the wrong patient.

Round cells in semen are of two types: immature sperm (germ cells) and white blood cells (WBCs). These cells can be differentiated by examining a stained smear at 1000X magnification. A more precise identification can be achieved by detecting peroxidase activity. The presence of immature germ cells could indicate testicular damage; increased numbers of WBCs may indicate inflammation of the accessory glands.

This presentation will describe basic aspects of WHO III assessment. For information on details of some of the morphological assessments you will need to review information presented in the 1992 WHO III manual.Learning to do strict morphology assessments is more complicated than learning WHO III and generally requires that the technician take one of the many hands-on laboratory courses offered periodically around the country. Details of strict morphology assessment are presented in the 1999 WHO IV manual.

Liquefaction should be complete before viscosity is assessed. Semen viscosity can be estimated by aspirating the sample into a wide-bore plastic disposable pipette, allowing the semen to drop by gravity and observing the length of the thread that is formed. A normal sample leaves the pipette in small drops with very little trailing thread. A semen sample that is abnormally viscous will form a thread more than 2 cm long.A specimen that is more viscous than normal after liquefaction may have reduced sperm motility. During sexual intercourse, hyperviscosity can prevent the sperm from reaching the cervix.

Other aspects of specimen collection that must be considered are the temperature of the specimen and the time needed to transport it to the laboratory.Ideally, the specimen should be collected in a room at the testing site.If onsite collection is not possible:The specimen should be kept between 20 -37°C (room temperature to body temperature) from the time of collection until it arrives at the laboratory. This can be facilitated by holding the container close to the body, for example by carrying it in an inside pocket.Semen should arrive at the laboratory as soon as possible after collection, preferably within one hour.The man should record the time of semen production.The report should note that the sample was collected at a location outside the laboratory.

Initial microscopic examination of semen using 100X magnification includes assessment of: Mucus strand formationSperm aggregation or agglutinationCellular elements other than spermatozoa, for example round cells, including leukocytes and immature germ cells, and isolated sperm heads or tailsAgglutination should be noted in the report. The WHO 5th edition recommends grading agglutination as:Grade 1 - isolated: <10 spermatozoa per agglutinate, many free spermatozoaGrade 2- moderate: 10-50 sperm per agglutinate, free spermatozoaGrade 3- large: >50 sperm per agglutinate, some spermatozoa still freeGrade 4-gross: All spermatozoa agglutinated and agglutinates interconnectedTo assess motility and estimate sperm concentration, switch to 400X magnification. An estimate of sperm concentration may be needed to determine an appropriate dilution for performance of the sperm count.A phase contrast microscope is recommended for initial microscopic semen examination, motility evaluation, and sperm count.Thoroughly mix the semen specimen in order to ensure as much as possible that the sample you are examining is representative of the whole specimen. One recommended mixing method is to aspirate the sample 10 times into a wide-bore disposable plastic pipette, gently so as not to create air bubbles. Obtain the sample to be examined immediately after mixing the contents in the original container. If additional aliquots are taken, mix the specimen in the original container again before taking those samples.

Sperm motility is important because sperm must be moving in order to penetrate the cervical mucus, travel to the fallopian tube, and fertilize ova.Accurate motility evaluation requires standardization of temperature, sample size, and depth of chamber for viewing. Some laboratories read motility at 37°C while others routinely report motility at room temperature. The temperature of the assessment should be specified in the final report and all laboratory personnel must perform the analysis at the temperature stated in the laboratory's procedure.The laboratory's procedure should also state the volume of semen to be used for viewing motility, e.g., 10 mL. The coverslip that is used for the stated semen volume should provide a chamber approximately 20 µm in depth. A chamber depth that is less than 20 µm may constrict the movement of sperm and a chamber that is too deep will not display the spermatozoa in a single plane, making it difficult to assess motility.The use of an eyepiece reticle with grid, as shown in the image on the right, is recommended for more accurate assessment within a defined area of the microscopic field. If the sperm concentration is high, score only the top row of the grid. If the concentration is low, score the entire grid. Do NOT choose fields to view based on observation of highest motility. This will cause inaccurate results to be reported.

Automated semen analysis instruments are available that are able to evaluate sperm motility more accurately than manual microscopic methods and can add specific information about motility parameters. In addition to determining percent motility, some instruments may calculate the speed at which the sperm are swimming in microns/second.

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

Some laboratory tests are so specialized that they require the use of special collection tubes to prevent erroneous results.Some examples of special requirements include:Laboratory TestConsiderationsHeavy metalsBlood collection container material must be free of heavy metalsTissue typingSample may need to be collected in preservative solutionBlood culturesSpecimens must be collected in bottles containing nutrient media to promote growth of bacteria within the bottle. Genetic studiesSpecial tubes may be needed to preserve DNA and/or RNA It is the responsibility of the phlebotomist to be aware of special requirements for certain tests. If the correct collection tube is not known then the phlebotomist MUST refer to the specimen collection manual or ask the appropriate laboratory worker to obtain the information. It is also imperative that the phlebotomist obtain and use only the correct equipment and not substitute something that is "close". This could affect the test results and the safety of the patient.

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

The laboratory obtained post-transfusion blood specimens in order to perform a serological investigation. Pretransfusion and post-transfusion DATs were performed. Patient cells DAT CC Pretransfusion 0 2+ DAT = direct antiglobulin test with polyspecific antiglobulin serumCC = IgG sensitized RBC

The influenza A H1N1 2009 virus is regarded as being genetically different from the normally prevelent seasonal influenza A viruses. Novel influenza A virus was originally referred to as "swine flu" because laboratory testing showed that many of the genes in this new virus were very similar to influenza viruses that normally occur in pigs in North America. However, influenza A, subtype H1N1 virus actually has genetic components of human, avian, and swine influenza A combined into one virus; a process called antigenic shift. This virus contains: Two genes from flu viruses from pigs in Europe and Asia One avian, or bird, gene One human geneThis type of virus is called a "quadruple reassortment" virus. Pigs serve as intermediate hosts for influenza A viruses since the respiratory tract of pigs contains receptors for not only swine influenza A viruses, but also avian and human influenza A viruses. Since pigs are also susceptible to avian influenza A viruses from turkeys, ducks, and wild waterfowl, the genetic mixing of swine and avian viruses has been documented over the past 15 years. An additional reassortment may occur with the inclusion of human influenza A genes when pigs are exposed to farmers with respiratory tract infections. The current 2009 H1N1 virus thus represents the most recent example of swine influenza A virus reassortment that contains both avian and human influenza A genes.Reference 1

The manufacturer's instructions (package insert) lists the acceptable respiratory specimen types for testing in each rapid influenza A/B test kit. The following specimen types are preferred for influenza virus diagnostic testing: nasopharyngeal swabs, washes and aspirates; endotracheal aspirates, and bronchoalveolar lavage (BAL). The CDC recommends nasopharyngeal washes for patients less than 3 years old and nasopharyngeal swabs for patients over 3 years of age. The image on the right illustrates the correct area to swab when collecting a nasopharyngeal specimen. Two swabs or washes would facilitate both rapid influenza A/B testing and a second specimen that could be sent for confirmatory H1N1 testing by RT-PCR if needed. Nylon fiber flocked swabs have been shown to be more effective than dacron or other woven fiber swabs for the collection of virus from the nasopharynx. The soft brush-like nylon fiber swabs are more efficient at collecting cells and viral particles and subsequently releasing virus when swabs are placed into viral transport media due to the capillary action of the flocked swab. It is suggested that the ill patient should have specimens collected as soon as possible after the onset of symptoms. For certain patients, more than one specimen may be necessary to confirm the presence of the virus. Nasopharyngeal swabs should be placed into sterile universal transport medium (UTM) or comparable viral transport media and promptly delivered to the laboratory within 2 hours. If processing is delayed longer than 2 hours, respiratory specimens should be placed in a refrigerator (held at 40C). Viruses usually remain stable for 2 to 3 days when held at this temperature.

Influenza A viruses, including the 2009 H1N1 strain, are able to survive and maintain infectivity on surfaces for extended lengths of time. Influenza A viruses typically remain infectious for 12 - 48 hours on non-porous surfaces, for 8 - 12 hours on cloth or paper, and for 5 minutes on hands.To reduce spread of the virus from person to person, it is important to discard contaminated items such as tissues and laboratory testing supplies and to wash hands frequently. To eliminate viruses from contaminated surfaces, a number of disinfectants can be used, such as chlorine, hydrogen peroxide, detergents, iodophores, and alcohols. Influenza viruses also can be rapidly inactivated with heat from 167 - 212°F.

The microscopic examination was traditionally performed on all urine specimens. Today, many laboratories perform a urine microscopic only if preliminary evaluation indicates the need for microscopic examination. Such laboratories must have criteria determining the specimens on which urine microscopic examinations will be performed. The microscopic exam is often important in detecting and evaluating renal and urinary tract disorders as well as other systemic diseases.

When the laboratory receives notification of a transfusion reaction, the first step is a clerical check. The clerical check should be performed as soon as possible to identify any possible ABO incompatibility. The technologist will compare the component bag, label, paperwork, and patient sample and look for errors. If an error is found, the physician must be notified. Once the post-transfusion sample is received, the sample should be examined for the presence of hemolysis. Both the pre-transfusion sample and post-transfusion sample can be compared. Destruction of red cells and release of free hemoglobin will result in a pink to red supernatant. Pink or red colored serum may indicate intravascular hemolysis. The patient's serum may appear icteric if the hemolytic process is extravascular. The ABO testing must be repeated on the post-transfusion specimen as well. Examination of a post-reaction urine sample made aid in the diagnosis of acute hemolysis. Free hemoglobin in the urine indicates intravascular hemolysis. A direct antiglobulin test (DAT) must be performed on the post-transfusion sample. An EDTA lavender top tube is the required specimen type. If the DAT is positive on the post-transfusion sample, then one should be performed on the pre-transfusion sample. If the pre-transfusion DAT is negative and the post-transfusion is positive, the presence of incompatible red cells should be suspected. All findings must be reported to the supervisor or medical director, who may request additional tests.

Adverse reactions after transfusion of blood components must be evaluated promptly. Most serious reactions occur within the first 15 minutes of starting a transfusion. Continuous monitoring allows reactions to be discovered in a timely manner. The transfusionist must be able to recognize the symptoms of a transfusion reaction and know the appropriate steps to take when one occurs. The first critical step is to stop the transfusion immediately, but keep the patient's line open with saline. The physician should be contacted immediately for instructions regarding patient care. The transfusion service must be notified of the reaction. They will usually provide instructions on proper documentation of the reaction, and the return of any remaining component and/or tubing. The appropriate patient samples are to be sent to the laboratory and usually include blood and urine. The transfusionist must be sure to follow all hospital policies.

Symptoms begin within 6 hours of transfusion and include acute respiratory distress, severe hypoxemia, hypotension, fever and bilateral fluffy infiltrates on chest radiograph. Respiratory distress is due to noncardiongenic pulmonary edema. Patient may have shortness of breath. Signs and symptoms may be mild, and resolve after a few days, or they may be severe and result in pulmonary failure. Laboratory findings include leukopenia and hypocomplementemia.

The symptom most commonly associated with a delayed hemolytic transfusion reaction (DHTR) is unexplained decrease in hemoglobin and hematocrit. Patients may also present with fever and jaundice. Hemolysis occurs slowly and is primarily extravascular. Unlike an acute hemolytic transfusion reaction (AHTR), hemoglobinuria, acute renal failure, and disseminated intravascular coagulation (DIC) are not generally seen. On some occasions, patient's may not present with any symptoms. Serologic findings include a positive direct antiglobulin test (DAT) and/or a positive antibody screen in post-transfusion testing. In many cases, the physician will send a request for an additional transfusion because of the decreased hemoglobin levels, and not suspect a DHTR. The positive antibody screen will trigger an investigation including antibody identification. The DAT may have a mixed field appearance because of the antibody-sensitized transfused red cells and the non-sensitized patient red cells. Segments from the donor unit can be tested for the offending antigen once the antibody has been identified.Antibodies that are most often reported as the cause of DHTR are anti-Jka and anti- Jkb. Other antibodies that are also commonly implicated in a DHTR include Kell, Rh, and Duffy system antibodies.The patient's physician should be notified so that additional clinical and laboratory evidence can be evaluated.

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

All specimens suspected of containing M. tuberculosis (including specimens processed for other microorganisms) should be handled in a Class I or II biological safety cabinet (BSC). Appropriate personal protective equipment (PPE) must be used. At a minimum, this includes gloves and fluid-resistant laboratory coat or gown. Non-aerosol-producing manipulations (eg, preparing direct smears for acid-fast staining when done in conjunction with training and periodic checking of competency) can be performed using biosafety level-2 (BSL-2) practices and procedures, containment equipment, and facilities. BSL-3 practices, safety equipment, and facility design and construction are applicable to microbiology laboratories that work with indigenous or exotic agents with a potential for respiratory transmission, and which may cause serious and potentially lethal infection. If the laboratory is propagating and manipulating cultures for M. tuberculosis, BSL-3 practices, containment equipment, and facilities are required. Barriers include controlled access to the laboratory and ventilation requirements that minimize the release of infectious aerosols from the laboratory. Secondary barriers should include self-closing double-door access and negative airflow into the laboratory. Exhausted air must not be recirculated. Work surfaces must be decontaminated, using the laboratory-approved disinfectant, upon completion of procedures, immediately following a spill, and at the end of the work shift, if the surface was recontaminated since the last cleaning. Laboratory equipment should be routinely decontaminated.Hands must be washed upon completion of work with potentially infectious materials and before leaving the laboratory.

The findings in the image to the right (peripheral blood smear) would elicit a report comment of "increased platelets" of a high magnitude, such as "marked" or "4+." Estimates of platelet counts from review of a peripheral blood should be made on each smear examined. This provides a simple estimate of "high", "low", or "normal" which usually corroborates the value generated from an automated cell counter. A formula for estimating platelet counts must be established for each laboratory. One guideline for the estimation of platelets is as follows: Count platelets on 5 fields using 1000X magnification (care should be taken to ensure the fields used for counting are not too thick or too thin) Average the platelet counts obtained Multiply by 15 X 109/L to obtain estimated platelet count (some laboratories prefer a 20 X 109 multiplier in this step if capillary blood is used)Such a counting scheme for platelets when clustered, as in the image, is probably not needed, as there are more than 100 platelets in the field. This translates into a platelet count of 1500 X 109/L or more.

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