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The antibody screen can provide sufficient data to make initial hypotheses regarding the likely antibody specificities and may be useful to presumptively rule-out some antibody specificities. When analyzing the antibody screen data, the strength/characteristics of reactions (for example, mixed-field or weak versus strong), the phase of testing (for example, room temperature versus AHG), and the pattern of reactivity (which cells react and which do not) are all important factors that will provide clues about the possible identification of the antibody(ies). Many antibodies exhibit dosage, that is, they react more strongly with homozygous cells than with heterozygous cells. If a tube method is used, reactions are usually read at immediate spin and AHG phase. If a gel method is used, reaction readings are done only at AHG phase. Reactions occurring only at immediate spin phase could indicate a possible IgM antibody, cold agglutinin, or rouleaux. Reactions occurring in the AHG phase could indicate a possible IgG antibody. Reactions occurring in both reaction phases could indicate a combination of both IgM and IgG antibodies or a strong IgM antibody that carries through to AHG phase. The presence of multiple antibodies should be considered if reactions vary in strength or there are two separate reaction patterns in the IS and AHG phases.

The stages of mitosis include interphase, prophase, metaphase, anaphase and telophase. When we view mitotic cells we are generally interested in the metaphase mitotic cells. This is the only stage in the entire cell cycle in which the nuclear lamins and nuclear envelope are depolymerized and a major redistribution of nonchromosomal nuclear antigens occurs. Distinct differences in the staining characteristics of the metaphase mitotic cells can be observed with various autoimmmune sera.(Ref12)When reading ANA results it's important to be familiar with the different stages of mitosis. To be considered positive there must be a clearly discernible pattern staining the nucleus of the interphase cell. When a pattern is present in the nucleus of the interphase cell then the metaphase mitotic cells are examined to assist in identifying the pattern(s).If there is no staining in the nucleus of the cell but there is a discernible pattern in the cytoplasm of the cell, the sample is reported as ANA negative. However, the presence of a cytoplasmic pattern should be noted.

Histology is literally, "the study of tissue." Tissue sections cannot be produced which are completely accurate and representative "snap shots" of the tissue in its' living state. But instead, histologists seek to preserve the tissue in the most life-like state possible. In reality, what we are able to achieve is a somewhat distorted, yet permanent, representation of the tissue's living condition.Histologists must learn to be able to visualize how specimen handling, including gross dissection and tissue processing, affect the appearance of tissues at both the macroscopic and microscopic levels.A clear understanding of anatomy and the ability to spatially visualize the specimen in three dimensions is extremely helpful. The histologist must use this background knowledge and understanding to visual identify any alterations in the tissue appearance caused during gross dissection and/or tissue processing; Apply this insight for correct specimen orientation during embedding.In visually assessing and identifying tissue samples, note that: Some sections cut through tubes or invaginations are not large enough to show the entire lumen. Many cells and tissues are organized into partitions, which divide the structure into numerous smaller areas which may be transected in any given specimen.Many submitted specimens may not show all the structural details that are depicted in images in histology atlases or anatomy textbooks. You may become quite skillful at visually identifying many specimen types, but you MUST still verify the specimen type using an electronic or paper-based worksheet.

The medical laboratory industry has become more competitive in recent years due to cuts in reimbursement, more restrictive regulatory requirements, increased competition in outreach, and increased cost of labor and materials. This has significantly affected the bottom line of both hospital and reference laboratories. In order to stay competitive, laboratories must be able to meet the needs of their customers at a reasonable cost. This transition has led to more laboratories utilizing Lean and Six Sigma to achieve customer satisfaction and improve finances.The National Institute of Standards and Technology (NIST) Manufacturing Extension Partnership defines Lean as:"A systematic approach to identifying and eliminating waste (non-value added activities) through continuous improvement by having the product flow towards the pull of the customer in pursuit of perfection." *Six Sigma is a quality improvement method that strives to prevent defects. Six Sigma quality performance means no more than 3.4 defects per million opportunities. The methodology emphasizes the DMAIC approach to quality improvement and problem solving.Lean and Six Sigma are complementary concepts. Both Lean and Six Sigma focus on process improvement. While Lean focuses on reducing waste, Six Sigma focuses on reducing variation. By eliminating both waste and variation, you will approach a defect-free process.*Reference:Czarnecki H, Loyd N. (2002). Simulation of lean assembly line for high volume manufacturing. Center for Automation and Robotics, University of Alabama in Huntsville. Available at: www.scs.org/confernc/hsc/hsc02/hsc/papers/hsc037.pdf. Accessed January 30, 2012.

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.

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.

All white cells present in a cerebrospinal fluid must be identified. If more than 10 cells/mm3 are present or there is difficulty identifying the few cells that are present, make a cytospin, a filtration, or a sedimentation preparation, stain with Wright-Giemsa, and perform differential count. Cytospins made with a cytocentrifuge are preferred since they are easiest to make and interpret, but filtration and sedimentation methods can also be used to prepare a slide for subsequent staining.

In addition to hereditary hemochromatosis (HH), there are other conditions of iron overload that must be considered in a differential diagnosis. Disorders such as sickle cell disease, thalassemia, sideroblastic anemia, congenital dyserythropoietic anemia, and liver disease may also cause iron overload. Transfusion-dependant patients and persons who abuse iron-containing vitamin supplements are also at risk. These conditions are usually described as secondary iron overload, in contrast to the primary iron overload of HH.Patient history, clinical signs and symptoms, biochemical and hematologic laboratory analyses, and possibly results of a liver biopsy may be needed to establish a diagnosis of a condition causing secondary iron overload. DNA tests for common HFE mutations are very likely the most important diagnostic tool for identifying HH as the cause of iron overload. In some patients, both secondary causes and HH may be contributing to iron overload. Differentiating the secondary causes of iron overload from HH is heavily dependent on the results of laboratory assays, but a complete discussion is beyond the scope of this course.

The following basic associations can be made between certain surface markers and cell types. Please note that only the most basic associated cell types are addressed as this is an introductory flow cytometry course. A PDF file of this table is also available on this page and can be printed to use as a reference for case studies presented later in this course. Surface Marker Associated Cell Type CD2 Pan (across all) T cells; natural killer cells CD3 Pan T cells CD4 T-helper cells CD5 Pan T cells and B cell abnormalities (e.g., B-chronic lymphocytic leukemia (B CLL) and mantle cells) CD7 Pan T cells (earliest marker) CD8 T-suppressor cells (cytotoxic T cells) CD10 also known as common acute lymphocytic leukemia antigen (CALLA) Early T and B cells, mature follicular cells CD19 Pan B cells (earliest marker) HLA-DR B cells (also present on activated T cells) CD20 B cells CD23 B cells (present in B CLL and not present in mantle cells) Kappa or Lambda light chain immunoglobulin (not antigen) B cells --A mature B cell should express either one or the other and, across a B-cell population, there should be a good mix of both Kappa and Lambda. In a clonal (cancerous) population, one cell line will proliferate and that line will exhibit one of these light chains. This will indicate clonality. CD45 also known as the leukocyte common antigen Pan white blood cells (May vary in staining intensity between mature and immature white blood cells).

Introducing molecular methods into a clinical microbiology laboratory is not a process without challenges. Some of these may include:Space requirements for both workflow and instrumentation Identifying separate areas for key aspects of the molecular process Familiarizing staff with new workflow requirements Instilling potentially new skill sets Cost of equipment and reagentsSpace requirementsIntroducing molecular methods will necessarily entail the introduction of new equipment. In many laboratories, space is at a premium; identifying space for new and additional equipment can be challenging. Some platforms are quite large; others have a significantly smaller footprint. The necessary separation of certain activities in the workflow will also impact space planning.

All three types of peripheral blood cells have different characteristics. In order to accurately identify each type of cell, a peripheral blood film must be made, preferably from blood anticoagulated with EDTA (Ethylenediaminotetracetic Acid) or from capillary blood. EDTA, in contrast to many other anticoagulants, preserves cellular morphology. The individual characteristics of each cell type are made visible by staining the blood films with Wright's stain, and observing them under the microscope. Most laboratories utilize high powered, oil magnification for the morphologic identification of peripheral blood cells.

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.

Bobby Jones, a phlebotomist at Georgetown Hospital, entered the room of Mrs. Mary Grayson with a physician's order to draw some blood work. After properly greeting Mrs. Grayson, identifying himself and checking her armband, Bobby prepared for the venipuncture. He suddenly notice a sign posted above the bed that read: "Restricted left arm usage. Previous mastectomy - Do no use left arm for venipuncture." Bobby set up his equipment to use her right arm and noticed an IV line in Mrs. Grayson's right arm positioned in a vein slightly above her wrist on the dorsum (top) of her forearm.

Single nucleotide polymorphisms (SNP) genotype analysis looks for small changes in sequences instead of simply identifying the amplicon. One of the best ways to detect SNPs is to compare melting curves. The use of real-time PCR to create melting curves can detect differences as small as one base pair. Melting curve analysis is an assessment of the disassociation characteristics of double stranded DNA when exposed to heat. The strength of the hydrogen bond of each piece of DNA is dependent upon several different factors: the length of the DNA segment, the amount of guanine and cytosine pairs, and the degree of compliment. In real-time PCR the fluorescence that is given off by the probes will decrease once the strand of DNA disassociates. After the DNA or RNA is amplified, the temperature of the sample is slowly increased while the fluorescence is recorded. The melting points should show up as peaks when plotting temperature against fluorescence. These peaks allow one to differentiate between homozygous wild type, heterozygous, and homozygous mutant alleles. One clinical use of this method is to detect both HIV-1 and HIV-2 in samples.

Recognition and identification of RBC morphologic abnormalites can be an invaluable aid in the diagnosis of a variety of disorders. Observation of RBC morphology from a properly prepared smear will also confirm the red cell indices reported by the hematology analyzer.RBC morphology can be visualized by examining erythrocytes on a Wright, or Wright-Giemsa stained peripheral blood smear. Evaluation of red cell morphology involves differentiating normal morphology from abnormal and artifactual morphology.

Errors occur much less frequently in the analytic phase of laboratory testing than in either the preanalytic or postanalytic phases. The supposition of published studies on the error rate in this category is that the percentages remain low primarily because of: The qualifications of testing personnel Effectiveness of internal quality control programs and external assessment practices which assist in identifying analytical errors and detecting possible sources.Following is a list of examples of errors that may be encountered during the analytic testing activities. The list includes both human and instrumentation errors. While random errors (those that occur independently of the operator) may be encountered during the analytic phase, primarily listed are systematic errors. That is, errors that bias the measurement resulting from either instrument malfunctions or human mistakes. Errors in quality control and verification of performance specifications. Instrument malfunctions. Calibration errors causing a direction of bias in results. Manual pipetting errors. Reagent errors. Test interference caused by unsuspected antibodies. Specimen interference i.e. failing to visually see sample was lipemic. Math errors. Staff errors in testing preparation and processing. Inadequate staffing which may precipitate errors caused by fatigue.

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

Root cause analysis is a process for identifying factors that cause risks. It focuses primarily on systems and processes, not on individual performance. The process progresses from identifying causes to identifying potential strategies that could possibly be implemented to improve the system or activity (cause and effect). In other words, after determining what caused the risk in the first place the next step is to determine what can be done to stop the risk from happening in the future. This is referred to as loss prevention. Some examples of risk control treatments in loss prevention might include: Staff education Procedure revisions Policy review

In order to prevent errors that affect specimen quality, the phlebotomist must pay close attention to detail during the entire venipuncture process. All steps of the phlebotomy procedure must be included for every venipuncture. This will help to maintain specimen integrity during the collection, transport, and handling of blood specimensProperly identify the patient every timeThe phlebotomist is responsible for correctly identifying the patient using two unique patient identifiers that include the patient's complete first and last name, medical record or hospital number, and/or date of birth. The patient location or room number, bed tag and chart are not reliable forms of identification and should not be used for patient identification. Every patient must verbalize his/her name to the phlebotomist, if able to do so. It is unacceptable for the phlebotomist to ask the patient to confirm his/her name that was verbalized by the phlebotomist. For example, the phlebotomist should say, "Would you please tell me (or spell) your name and birthdate. " The phlebotomist should NOT say, "Are you Sally Brown, and is your birthdate June 1, 1925?" If this is a hospital inpatient, check the information on the patient's wristband and confirm that the name and hospital number or medical record number matches the patient information on the test order. Never rely on identification attached to a bed, chart or door. NEVER draw a patient whose identity is not established or is in conflict. If there is a discrepancy, the phlebotomist must STOP and seek assistance to have the discrepancy resolved before proceeding with the venipuncture. If this is an outpatient that does not have a wristband, ask the patient (or guardian/caregiver) to state the patient's date of birth. A picture ID, such as a driver's license, can also be used for positive patient identification.

There are some commercial products available that are designed to alleviate pain from venipuncture.Cream: A topical cream can be applied to numb the venipuncture site. Apply well in advance to be effective. Always refer to manufacturer's instructions before use on patients. Be certain to determine that no allergy exists before using the product on a child.Mechanical device: A mechanical device can be used to stimulate nerves surrounding the venipuncture site to numb the site. This device must be used according to the manufacturer's instructions.Vein Viewer: This device enables the phlebotomist to determine the flow of blood thereby identifying the presence and direction of a vein. This device does not aid during palpation of the vein to determine vein health, diameter or depth.

To improve the quality of conclusions when identifying antibodies, a checklist is a simple quality control tool to increase transfusion safety. If a specific antibody pattern cannot be identified with acceptable confidence, or if significant serologic or non-serologic data are inconsistent and cannot be rationalized, further testing will be required.Before concluding that the investigation is complete, unless not applicable, mentally reply to each question in the checklist. If any answer is no, has it been resolved? Antibody Identification Checklist Yes/No/NA 1. For a single antibody, does the reaction pattern fit only one antibody specificity? 2. Is antibody specificity consistent with the results of the initial antibody screen? 3. Are reaction phases consistent with antibody specificity? 4. If multiple antibodies are present, can all reactions be explained by the antibody combination? 5. If the autocontrol is negative, are patient red cells negative for the corresponding antigen(s)? 6. Have additional possible antibodies been excluded by selected red cells? 7. Can all variable reaction strengths be explained? 8. If tested, are antigen-negative donor cells compatible by antiglobulin crossmatch? 9. If there are data that do not fit antibody specificity or if there are results that are improbable, are they explainable? 10. Have all results and conclusions been systematically evaluated for consistency?

If a portion of fallopian tube from a sterilization procedure is submitted for pathology, it will be examined to ensure that it is indeed a portion of fallopian tube. Microscopically the pathologist will look for the identifying cilia lined lumen.

The first component of therapy is to stop the transfusion immediately. Vital signs must be closely monitored. Management involves treatment of hypotension and disseminated intravascular coagulation (DIC). It is essential to maintain blood volume and adequate renal blood flow. Diuretics, substances that increase urine output, may be administered. If the patient enters renal failure, dialysis must be initiated rapidly. It is impossible to prevent all hemolytic transfusion reactions. The purpose of pre-transfusion compatibility testing is to decrease the probability of a hemolytic transfusion reaction by performing ABO/Rh testing, detecting and identifying alloantibodies, and crossmatching compatible blood. Human error, the most common cause of hemolytic transfusion reactions, cannot be completely eliminated. Steps must be taken to reduce the possibility of human error in identification of patient samples, donor units, and recipients. Each person involved in the transfusion process, from collection of the blood sample to administration of the donor unit, must carefully adhere to each step outlined in the standard operating procedures. All appropriate protocols must be followed. Some examples are: Technologist checks blood sample to ensure proper labeling. Patient's previous transfusion records are examined and all transfusion testing is performed correctly and accurately. Technologist ensures correct unit is released from the blood bank. Transfusionist ensures the recipient is correctly identified.There must be a mechanism in place to train and assess all personnel involved in the transfusion process.