MDL 109 Unit 3 Routine Practices PDF

MDL 109 Unit 3 ROUTINE PRACTICES History of the Medical Laboratory Rudimentary examinations of human body fluids date back to 300 BC by the Hippocratics Health consisted in humoral equilibrium. Illness resulted when an excess or a deficiency occurred in one or more or the humors. This theo...

MDL 109 Unit 3 ROUTINE PRACTICES History of the Medical Laboratory Rudimentary examinations of human body fluids date back to 300 BC by the Hippocratics Health consisted in humoral equilibrium. Illness resulted when an excess or a deficiency occurred in one or more or the humors. This theory was believed for 2000 years, until the rise of controlled empirical science in the mid-19th century  The First Clinical Laboratory St. Johns Hopkins Hospital in Baltimore opened in 1896, with the first historically recorded clinical laboratory Most clinicians at the time found lab testing to be an expensive luxury (space and time) It wasn’t until discovering causative agents of epidemics like tuberculosis, diphtheria, and cholera, and developing lab tests to identify them in 1890 that highlighted the importance of lab testing  Clinical Lab Testing MLTs and cMLA/Ts collect and process specimens and perform various types of testing: i. Chemical ii. Biological iii. Hematologic iv. Immunologic v. Microscopic vi. Molecular diagnostics vii. Microbial viii. Histological ix. Cytological etc.  Objective In this unit, we will be discussing routine lab tests performed in each department Each clinical site will have their own policies, but it is up to you to understand these core skills and tests Hundreds of tests are available (www.labtestsonline.org ) but only a small percentage are routinely ordered  1. Blood banking/ Transfusion Med History of Blood banking: William Harvey 1628 Discovers the circulation of blood, and attempts first known blood transfusion Richard Lower 1665 First successful blood transfusion, dog > dog Jean-Baptiste Denis, Richard Lower, Edmund king 1667 Separately report successful transfusions from sheep to humans  1. Blood banking/ Transfusion Med History of Blood banking: Jean-Baptiste Denis kept attempting transfusions less successfully, which resulted in the first recorded transfusion reaction: Pain in kidneys Increased pulse Black urine Nausea Patient death A great legal battle ensued, prohibiting future blood transfusions  1. Blood banking/ Transfusion Med History of Blood banking: Jean-Baptiste Denis kept attempting transfusions less successfully, which resulted in the first recorded transfusion reaction: Pain in kidneys Increased pulse Black urine Nausea Patient death A great legal battle ensued, prohibiting future blood transfusions  1. Blood banking/ Transfusion Med History of Blood banking: During a cholera epidemic in Toronto in 1854, Drs. Edwin Hodder and James Bovell tried transfusing cow’s milk It wasn’t until Karl Landsteiner discovering the ABO blood group in 1900 that changed the way transfusions were performed  ANTIGENS Substance that is capable of causing an immune response Each species of animal has certain antigens unique to that species, present on the red cell membranes Antigens present on RBCs, WBCs and platelets are inherited, following Mendelian laws of inheritance Antigens that exist on a person’s red cells within a particular blood group system represent that person’s type for that system (ABO)  ANTIBODY Protein substance found in plasma, that is formed in the presence of a foreign antigen  ANTIBODY ABO antibodies are naturally produced against the antigens not present on RBCs If RBCs are transfused into a patient who has antibodies against the transfused cells’ antigens, the RBCs will be destroyed (hemolyzed)  ANTIBODY Antibodies that react to antigens from a genetically different individual of the same species are called alloantibodies Antibodies that react to self- antigens are called autoantibodies Patients who receive antigens not present in their RBCs may produce an alloantibody in their plasma Antigen + antibody = agglutination  AGGLUTINATION Clumping of RBCs caused by a reaction of a specific antibody and antigen on the cells Various strengths of clumping occurs depending on the number of antigen sites binding to antibody sites The portion of the antigen molecule that is directly involved in the interaction with the antibody is called the epitope  ABO Phenotypes and Genotypes Phenotypes: The ABO system consists of groups, or phenotypes A, B, AB, and O These groups are defined by the presence or absence of A and B antigens on the red cells Genotypes: Genes on the chromosomes determine the antigens present on the red cell Each person has 2 genes for any trait (one from each parent), which provides these possible allele combinations: AA, AO, AB, BB, BO, OO ABO TYPING When the blood type of an individual needs to be known, the antigens attached to the cells are detected using commercial antibodies This is known as forward typing (what you see is what you have) REVERSE TYPING This involves testing the patient’s plasma for ABO antibodies Meant to confirm the forward type Patient plasma is mixed with commercial cells Antibody Screens Testing the patient’s plasma with commercially purchased RBC to detect abnormal antibodies Blood Bank-Antibody Screen Gel (Ortho MTS) – YouTube Lab 7: Smears and CBCs Hematology: DxH 520 Beckman Coulter’s Compact, Low-volume Hematolo gy Analyzer - YouTube Beckman Coulter Principle The Coulter automated cell counter uses the electronic impedance principle. Based on the detection and measurement of changes in electrical resistance produced by cells as they pass through a small aperture. Cells suspended in an electrically conductive diluent, such as saline, are pulled through an opening (aperture) in a glass tube. Patient results are compared to established reference ranges. Results that fall outside the reference ranges may indicate a disease process like infection, anemia, or leukemia. Results must be evaluated along with other test results, symptoms, and physical examination. One test result can not be used for diagnosis by itself  Automated Cell Counters Electrical resistance or impedance in the current occurs as the cells pass through the aperture. This resistance creates a voltage pulse that is measured. The number of pulses measured is proportional to the number of cells passing through the aperture. The size of the pulse is directly proportional to the size of the cell, thus allowing discrimination and counting of different sized cells ~ red blood cells, white blood cells, and platelets. Some of the red cells are automatically hemolyzed (broken down by destroying the cell membrane) and the hemoglobin (iron content) is measured. A final printout is obtained from the instrument. This is much faster and more accurate than performing manual cell counts and hemoglobin determinations. Blood Smears Examination of a stained, peripheral blood smear is usually part of performing a CBC or complete blood count. A blood smear is prepared by spreading a drop of blood on a microscope slide. The smear is dried, fixed, and stained. The blood components are then viewed microscopically, identified, and evaluated. Careful examination of a well-prepared smear can provide valuable information to the physician to aid in the diagnosis and treatment of many diseases. Hematology: Making a Peripheral Blood Smear – YouTube  Blood Smears Blood Smear Preparation and Staining Practical Lab - YouT ube  Blood Smears Once the smear is dried, it is then fixed and stained using the Wright’s Stain:  Wright’s stain (mixture of methylene blue, giemsa stain in absolute alcohol)  Phosphate buffer (to change the pH)  Rinse (to clean off excess stain)  Review… The function of these cells!  Coagulation Testing Another major component of lab testing in hematology is coagulation: the process of blood clotting after an injury to a blood vessel Hemostasis = the cessation of blood flow from an injured blood vessel The process of hemostasis balances many interdependent coagulation factors that prevent bleeding  Coagulation Hemostasis and coagulation depends on the interactions between blood vessels, platelets, plasma coagulation factors, and inappropriate clotting Primary hemostasis:  Endothelial damage which results in a platelet plug (need adequate numbers and function of PLT)  Immediately vasoconstriction occurs, where the damaged vessel constricts, decreasing the amount of blood flow through the injured area Secondary hemostasis:  Formation of a blood clot caused by coagulation factors present in the blood, forming a fibrin network and a thrombus to stop the bleeding completely  Slow lysis of the thrombus = fibrinolysis which allows for the final step of site repair Coagulation  Blood Clotting When an injury happens in the body, or in vivo, hemostasis and coagulation helps stop bleeding When blood is taken out of the body for testing, or in vitro, it triggers the coagulation cascade In order to test blood that has not clotted, anticoagulants are used  Plasma vs. Serum Plasma Serum Liquid portion of blood after it has been Fluid portion that remains after anticoagulated and centrifuged, or coagulation has occurred in vitro otherwise allowed to settle Does NOT contain fibrinogen (destroyed Contains fibrinogen (clotting factor) in the clotting process)  Some tests require the use of plasma over serum, and vice versa  In hematology, anticoagulated blood is used to test for the clotting function of patients  Coagulation Testing All coagulation testing uses anticoagulated blood to ensure accurate testing of patient’s own clotting system (can’t test for clotting if the sample is already clotted) Sodium citrate is the anticoagulant of choice (light blue) Coagulation tests include: PT/ INR: monitors patients on anticoagulant therapy and to diagnose clotting disorders (extrinsic pathway) aPTT: monitors patients on heparin therapy and clotting factor disorders (intrinsic pathway) D-Dimer: fibrin degradation product found after blood clot is degraded by fibrinolysis (high when blood clot is present) Fibrinogen: investigation of a bleeding disorder or inappropriate blood clot formation (thrombotic episode) like DIC  Clinical Chemistry What happens in the chemistry department? Clinical Chemistry Perform STAT and routine testing on plasma/serum samples- general chemistry, endocrinology, immunology, and toxicology  Blood gases Blood Gases: measures oxygen, carbon dioxide, and pH of blood  Urinalysis Pregnancy tests DOA- Drugs of Abuse LOTS and LOTS of QC  Blood Composition To keep composition as close as possible to the body, good phlebotomy skills are essential. The specimen should be processed and analyzed soon after collection. If the latter is not possible, the specimen should be stored in a manner appropriate to the component being analyzed. (i.e., refrigeration, treated with a preservative, away from light, frozen etc.)  Effects of Temperature Room temperature (18-25◦C) Refrigerator temperature (4-6◦C) Freezer temperature (-17- -20◦C) Generally, the lower the temperature, the greater the stability of most analytes. WHY? 1.Microbial growth is reduced at lower temperatures. 2.Most compound of clinical interest are stable at lower temperatures. 3. Enzymes that may cause deterioration of an analytes are less active at reduced temperatures. Common Blood Tests performed in Chemistry: These tests are typically included as an overall screen of the major systems of the body Called CHEM7, or BMP (basic metabolic panel)… your hospital may have its own term Includes: 1.Glucose: body’s main source of energy 2.Electrolytes (sodium, potassium, CO2, Chloride): electrically charged minerals that help control the balance of fluids and acids/ bases 3.BUN (blood urea nitrogen) and creatinine: waste products removed by the kidneys Comprehensive Metabolic Panel If the physician is suspecting other issues, they will order a CMP (comprehensive metabolic panel) to include liver enzymes (ALT, ALP, AST), total protein, albumin, and calcium  If it exists in the plasma… There is likely a chemistry test that will detect it! https://tests.lifelabs.com/Laboratory_Test_Information/Br owse_By_Test.aspx  Urinalysis- Why Study Urine? 1. To assess kidney function, urinary tract disorders 2. To detect overall body disturbances such as endocrine or metabolic abnormalities in which kidneys function normally but excrete abnormal amounts of metabolic waste products specific to a particular disease  What does routine urinalysis check for? Infection Dysfunction Lesions and cancer Lithiasis or stones Nephrosis  In order for urinalysis to be meaningful: Pre-analytical steps must be followed: Proper patient prep Appropriate specimen collection Proper sample identification Stored refrigerated if not able to test within 2 hours Routine Urinalysis: 3 Main Test Areas Physical Examination Chemical Examination Microscopic Examination 1. Physical Examination of Urine: Physical characteristics include: Colour (shades of yellow, is it brown, red, orange??) Odour (foul or fruity?) Turbidity (clear or cloudy?) Specific gravity (proportion of solids to total volume) Volume (too much? Too little?)  2. Chemical Examination These analytes measured using a dipstick: Glucose Bilirubin Ketones Specific Gravity Blood pH Protein Urobilinogen Nitrite Leukocyte esterase Urinalysis Reference Ranges Chemical Analysis Determines Next Step If certain chemical analytes are POSITIVE, a microscopic examination is ordered. These analytes include: 1.Protein 2.Nitrites 3.Blood 4.Leukocytes 1. Protein in Urine Small amounts of LMW proteins excreted (More sensitive to albumin – seen in renal disease) A negative result does not rule out other significant proteins 2. Nitrites Common UTI causing, gram negative organisms (E. coli, Enterobacter, Citrobacter, Kebsiella and Proteus) produce enzymes that reduce nitrates (normally present in the urine) → nitrite This takes up to 4 hours of ‘incubating’ in the bladder ∴ first morning urine is specimen of choice 3. Blood Myoglobinuria – is rare – crush injuries, beating - rapidly cleared from blood Differentiation: Intact RBCs will make urine appear cloudy, clear supernatant upon centrifugation If intravascular hemolysis is present, serum would be hemolyzed 4. Leukocytes Detects presence of abnormal levels of granulocytic leukocytes, primarily neutrophils in the urine Positive reaction usually reflects a minimum of 5-15 WBCs/hpf Tests are based on the measurement of leukocyte esterase, an enzyme present in granules of granulocytes  Microscopic Analysis Spun sediment observed for significant elements, reported as number seen: May be stained with Sternheimer-Malbin stain (consists of crystal violet and safranin) Urine sediment examined first at low power: 100X Urine sediment then examined at high power: 400X  When in a microscopic exam required? Abnormal findings in the following aspects: Colour Clarity Blood Protein Nitrite Leukocyte Esterase When the physician orders it  Pregnancy Testing Human chorionic gonadotrophin (HCG) is a hormone produced by the developing placenta. This hormone usually can be detected in the urine from 7 days following conception. The appearance of this hormone makes it an excellent marker for the early detection of pregnancy. A first morning urine specimen, which should contain the highest levels of HCG, is recommended for testing. Samples may be collected in a clean dry plastic or glass container and stored refrigerated until testing. The sample should be brought to room temperature before testing.  Pregnancy Testing The immunochromatographic pregnancy test is a qualitative two site sandwich immunoassay for the determination of hCG in urine. A test band is pre-coated with anti-hCG in the test band region. Patient’s urine is added to the test strip and is allowed to react with the coloured monoclonal anti-hCG conjugate, which is pre-dried on the test strip. The mixture moves upward on the test strip by capillary action. If hCG is present in the urine, a coloured band with the specific antibody-hCG coloured conjugate will form on the test strip. If no hCG is present in the urine, no band will form on the test strip. As a procedural control, a coloured band will always appear in the control region, whether hCG is present or not, indicated the test is completed.  Introduction to Microbiology The field of medical or clinical microbiology involves the isolation and identification of infectious organisms, and the development of effective ways to eliminate or control infectious organisms. Medical microbiology is generally divided into the study of bacteria, viruses, fungi, and parasites. The study of bacteria is bacteriology, the study of fungi is mycology, the study of parasites is parasitology, and the study of viruses is virology. Specimens for Microbiological Examination Types of microbiology specimens collected Blood Body fluids Cerebrospinal fluid Inner ear Respiratory Scrapings Stool Sputum Swabs of various anatomical sites Urine Basic Techniques Used in Microbiology Most microbiology laboratory work involves procedures to culture, characterize, and identify various microbes Culture of organisms present in the patient specimens Classification and identification of the isolated organisms Interpretation of organism susceptibility patterns to determine an appropriate antimicrobial agent Inoculating needle or loop Incinerators Solid and liquid media  Streaking Plates Dilution streak technique (streaking for isolation) The inoculating loop is sterilized and allowed to cool between all quadrants. The first streak is continued across approximately one quarter of the plate (first quadrant). The plate is then shifted about a one-quarter turn and streaked again, beginning at the periphery, overlapping the previously inoculated area a few times, and continuing across the second quadrant. The plate is turned once again and streaked a third time, beginning at the periphery, drawing the loop through the second streak a few times, and continuing across the third quadrant. The fourth quadrant is streaked beginning at the periphery, drawing the loop through the third streak a few times, and continuing across the fourth quadrant. Fig. 15.6. Dilution streak technique. (From Tille PM: Bailey and Scott’s diagnostic microbiology, ed 13, St Louis, 2014, Mosby.)  Aseptic Technique Aseptic transfer is required to prevent contamination of a pure culture when working with it. Since many manipulations are required to study the characteristics of a pure culture it is imperative that these manipulations be done using methods known to minimize contamination.  Aseptic Technique  Lids from tubes, plates, and media must be replaced as soon as possible.  Lids and caps are never placed open side down on the bench.  Media plates are placed media side up (lid side down) and the media side is removed from the lid for observation or testing.  Colonies can be quickly observed or touched with loops, wires, or sticks and then placed back into the lid portion of the petri dish.  Never leave an open media plate on the bench. Identification of Bacteria The identification of most bacteria involves microscopic observations (smear preparation and staining), bacterial cultivation, and biochemical tests. Smear preparation and stains used in microbiology Smear preparation Morphology of bacteria Spherical or round bacteria are cocci (singular coccus) Rod-shaped bacteria are bacilli (singular bacillus) Spiral-shaped bacteria are spirochetes and spirilla (singular spirochete and spirillum). Fig. 15.9. Bacterial morphology. Shown are characteristic shapes and cellular arrangements of bacteria. (From Tille PM: Bailey and Scott’s diagnostic microbiology, ed 13, St Louis, 2014, Mosby.) Staining Techniques Simple stains Methylene blue stain Differential stains Gram stain Gram positive vs. gram negative Other stains Gram Stains 1. Flood slide with crystal violet and leave for 30 seconds to 1 minute. 2. Briefly AND GENTLY wash in tap water. 3. Flood the slide with Gram’s iodine. Leave for 30 seconds to 1 minute. 4. Wash off with water and decolourize rapidly by running the acetone alcohol over the slide just until no blue colour runs off (usually about 2 seconds). 5. Wash immediately with tap water. 6. Counter stain with safranin or dilute carbol fuchsin for 30 seconds to 1 minute. 7. Shake off excess water. Stand on end to drain or blot dry with bibulous paper. In the first step, all organisms are stained violet, and all assume a dirty bluish brown color after treatment with the iodine solution. Gram Stains The iodine serves as a dye trapping agent (similar to a mordant) to fix the crystal violet dye in certain types of organisms, so it is not washed out by the decolorized. The decolorization step is critical. Gram positive organisms may easily over decolorize and falsely appear as Gram negative organisms. These over decolorized Gram-positive isolates will appear as a distinctive color which is neither blue- purple of gram positive, nor pink- red of gram negative. Dead organisms or old organisms, both of which have a compromised cell wall structure, will stain gram negative, thus fresh cultures must be stained. The timing of the decolorization step must be adjusted for samples that have a high protein content (sputum, body fluids). The counter stain stains those organisms that have decolorized.  Gram Stains Gram positive organisms are blue or purple in colour because they resist decolorization and retain the crystal violet iodine combination. Gram negative organisms are red because they are decolorized and can then take up the counter stain.  Identification of Bacteria Microscopy techniques used Brightfield microscopy Fluorescent microscopy Bacterial cultivation Primary culture, subculture, and pure culture When culturing specimens, the primary culture plates are inoculated using the dilution streak technique to obtain isolated colonies. If the colonies on the primary plates appear as mixtures of more than one species of bacteria, a subculture (secondary streak plate) must be performed to separate the different types of bacteria. The growth of several colonies originating from a single colony, and thus a single cell, is known as a pure culture. Fig. 15.11. Pure culture of Staphylococcus aureus on a sheep blood agar plate. Each colony began as an individual parent bacterial cell that multiplied many times to become visible. (From Tille PM: Bailey and Scott’s diagnostic microbiology, ed 13, St Louis, 2014, Mosby.)  Types of Culture Media Colony characteristics (appearance) of bacterial cultures Requirements for bacterial cultivation Oxygen requirements: Aerobes and anaerobes Aerobes = grow with oxygen Anaerobes = grow without oxygen Most clinically significant aerobes are really facultative anaerobes, which can grow under either aerobic or anaerobic conditions. Nutrients Identification of Bacteria Various types of media Chocolate agar (Choc) Colistin-nalidixic acid agar with blood (CNA) Hektoen enteric agar (HE) Lysine iron agar (LIA) MacConkey agar (MAC) Phenylethyl alcohol agar (PEA) Selenite broth Sheep blood agar (SBA) Thayer-Martin agar (modified Thayer-Martin agar)  Antimicrobial Susceptibility Tests Susceptibility and resistance Minimum inhibitory concentration and minimum bactericidal concentration Methods for determination of antimicrobial susceptibility Preparation of inoculum McFarland standards Microdilution method Disk diffusion (Kirby-Bauer method)  Antimicrobial Susceptibility Tests Fig. 15.26. Agar disk diffusion. (A) Mueller-Hinton agar has been inoculated with the test organism, and the antibiotic disks have been added to the media. No incubation has yet occurred. (B) After 16 to 18 hours incubation, the zones of inhibition are apparent. The zones of inhibition are measured and compared to a table of values for each antibiotic. (From Tille PM: Bailey and Scott’s diagnostic microbiology, ed 13, St Louis, 2014, Mosby.)  Antimicrobial Susceptibility Tests Selection of media for plating Handling and storage of antibiotic disks Preparation of inoculum Inoculation of Mueller-Hinton agar plate Application of disks Reading of results Quality Control in the Microbiology Laboratory Control of equipment Control media Control of reagents and antisera Control of antimicrobial tests Control of specimens, specimen collection, and specimen rejection  Additional Point-of-Care Rapid Testing Applications Rapid testing for streptococcal infections Rapid testing for pneumococcus and legionellae Rapid testing for human immune deficiency virus (HIV) Rapid for plasmodium falciparum— falciparum malaria Rapid CoVID  Automation Specimen processing Mass spectrophotometry (MALDI-TOF) Today, mass spectrophotometry has standardized microbe identification, and continuous-monitoring blood culture systems, automated microbial identification, and automated antimicrobial susceptibility testing systems are widely used in microbiology laboratories. Total microbiology laboratory automation Who works in the Histology Lab? Pathologists Pathology Assistants Medical Laboratory Technologists/Cytologists Medical Laboratory Technicians/Assistants Clerical staff What defines Histology/Histotechnology? Histology: - the microscopic STUDY of the structure of tissue Histotechnology: - the PREPARATION and treatment of tissue  Accessioning: Specimen arrives in the Histology lab and is documented into the computer system Usually given a unique Histology number Info Doesn’t Match If any of information is in correct on the requisition. i.e The Name, Health Card #, DOB or specimen site don’t match the container. Call the submitting Drs office for correct info…. IRREPLACEABLE!!  Grossing tissue: Macroscopic description of the tissue is recorded All of the tissue or suspicious sections of the tissue are chosen for further processing Histopathology: The study of the microscopic anatomical changes of diseased tissue Purpose of Histopathological Assessment Surgical Biopsy: To conclude or dispute the clinical diagnosis. The Pathologist may order other diagnostic tests after they see the H&E slide How to proceed with treatment of the disease Common Skin Cancers Surgical Biopsy:  Malignant tumours: Ensure there are adequate resection margins To determine the extent of lymphatic involvement or direct spread To stage and classify the disease Autopsies: To determine cause of death Cases are family requested. i.e Mesotheliomas – Lungs only Autopsies are either ordered as a partial. i.e lungs only or complete. Which is brain, lungs and abdominal Introduction to the Histology Laboratory, Safety, VIP Summary of the path a histology sample travels when processed: a) Specimen is removed from body, placed in fixative, and sent to the histology with proper identification and corresponding requisition B) Receive fixed tissue and document into the Histology laboratory system; each sample is given a unique identification number c) Tissue is grossed by pathologist or pathology assistant and placed in a cassette D) Cassette is put in processor (VIP): this is an overnight process  Fixes, dehydrates, clears, and infiltrates with paraffin E) Cassette is removed from processor F) Processed tissue is embedded in paraffin and a tissue block is formed G) The tissue block is cut into sections using a microtome H) Tissue sections floated out on water flotation bath; tissue picked up on slide I) Stain:  Deparaffinize (xylol)  Rehydrate (descending alcohols)  Stain  Dehydrate (ascending alcohols)  Clear (xylol) J) Coverslip K) Slides are sent to pathologist for microscopic assessment of patient’s tissue L) Additional stains may be requested by pathologist to assist in diagnosis Unstained vs. Stained M) Once all tissue samples for the patient have been assessed, a medical transcriptionist creates a Pathology report N) Pathology report sent to all requesting physicians