In-Depth Clinical Laboratory Science Study Guide PDF

In-Depth Clinical Laboratory Science Study Guide 1. Clinical Lab Professions (8%) Overview: Focuses on understanding the roles and qualifications of clinical laboratory professionals. Key Topics: Phlebotomist: ○ Collects blood samples for testing. ○ Requires a high sc...

In-Depth Clinical Laboratory Science Study Guide 1. Clinical Lab Professions (8%) Overview: Focuses on understanding the roles and qualifications of clinical laboratory professionals. Key Topics: Phlebotomist: ○ Collects blood samples for testing. ○ Requires a high school diploma, formal training, and certification (e.g., ASCP Phlebotomy Technician Certification). Medical Laboratory Technician (MLT): ○ Conducts routine lab tests under supervision. ○ Requires an associate degree and certification (e.g., ASCP or AMT). Clinical Laboratory Scientist (CLS): ○ Performs advanced testing, interprets results, and supervises lab staff. ○ Requires a bachelor’s degree and certification. Pathologist: ○ A medical doctor specializing in diagnosing diseases through lab tests. Professional Organizations/Agencies: ASCP: Certifies lab professionals and sets standards. NAACLS (National Committee for Clinical Laboratory Standards): Accredits educational programs for lab professions. CLSI (Clinical and Laboratory Standards Institute) FORMERLY KNOWN AS NAACLS: develops standards of current best practice for clinical laboratory procedures CLIA (Clinical Laboratory Improvement Amendments of 1988): revision of CLIA 1967, specifies minimum performance standards for all clinical laboratories ○ Labs can perform waived tests, moderate and high complexity tests, and PPMP (provider-performed microscopy procedures) 2. Biological Safety (12%) Overview: Covers procedures to maintain safety and prevent contamination. Key Topics: Personal Protective Equipment (PPE): ○ Gloves, goggles, lab coats, face shields, and respirators. ○ Protects against biological and chemical hazards. Disinfection and Sterilization: ○ Disinfection: Removes most microbes (e.g., using 10% bleach). ○ Sterilization: Eliminates all microbes (e.g., autoclaving). Chemical Name Organisms Effective Against Use Alcohols 70-90% Bacteria, Mycobacterium, some Skin and (isopropanol, ethanol) viruses surfaces Iodine Bacteria, fungi, viruses, protozoa Skin 10% chlorine bleach Especially good for viruses Surfaces Phenolics (Amphyl) Most bacteria and viruses, Surfaces mycobacterium Quaternary ammonium Bacteria, some fungi Surfaces salts (QUATS) Bloodborne Pathogens: ○ Infectious microorganisms in blood (e.g., HIV, Hepatitis B). Autoclave: use steam under pressure to sterilize items such as dental and surgical instruments, solutions, and materials to be used in microbiology OSHA Laboratory Safety Guidelines OSHA's regulations are designed to maintain a safe work environment in laboratories. Key components include the following: 1. Hazard Communication Standard (HCS) Purpose: Ensures that employees are informed about chemical hazards in the workplace. Key Requirements: ○ Labeling: Containers must be labeled with hazard warnings.  ○ Safety Data Sheets (SDS): Provide detailed information on chemical properties, handling, and emergency measures. ○ Training: Workers must be trained on understanding labels and SDS. 2. Bloodborne Pathogens Standard Purpose: Protects employees from exposure to infectious materials. Key Requirements: ○ Exposure Control Plan: Written plan detailing procedures to minimize exposure. ○ Universal Precautions: Treat all blood and bodily fluids as potentially infectious. ○ Sharps Safety: Use needleless systems or sharps with engineered safety features. ○ Hepatitis B Vaccination: Offered to employees with potential exposure. 3. Chemical Hygiene Plan (CHP) Purpose: Promotes safe handling and use of chemicals in laboratories. Key Requirements: ○ Standard Operating Procedures (SOPs): Written procedures for safe chemical use. ○ Personal Protective Equipment (PPE): Gloves, goggles, lab coats, and respiratory protection. ○ Engineering Controls: Fume hoods, ventilation systems. 4. Personal Protective Equipment (PPE) Requirements: ○ Conduct hazard assessments to determine required PPE. ○ Ensure PPE is appropriate, maintained, and worn correctly. 5. Emergency Action Plans Requirements: ○ Clear evacuation procedures in case of fire, chemical spills, or other emergencies. ○ Access to emergency showers, eyewash stations, and fire extinguishers. CDC Laboratory Safety Guidelines The CDC emphasizes biosafety in handling infectious agents and biological materials. Key areas include: 1. Biosafety Levels (BSLs)  Laboratories are categorized into four levels based on the agents handled: ○ BSL-1: Low risk; standard microbiological practices (e.g., handwashing). ○ BSL-2: Moderate risk; use of PPE and restricted access. ○ BSL-3: High risk; requires controlled access, respiratory protection, and specialized ventilation. ○ BSL-4: Maximum risk; work with life-threatening agents (e.g., Ebola). 2. Biological Risk Assessment Evaluate hazards associated with infectious agents, toxins, and equipment. Implement control measures to minimize risks. 3. Standard Microbiological Practices Ensure proper training for laboratory personnel. Prohibit eating, drinking, or applying cosmetics in the lab. Decontaminate work surfaces after procedures and immediately after spills. 4. Handling Sharps Use puncture-resistant containers for disposal. Avoid recapping needles unless using a mechanical device. 5. Decontamination Procedures Use appropriate disinfectants, such as: ○ 10% chlorine bleach solution: Effective for surfaces. ○ Alcohol (70%): Suitable for quick disinfection. Autoclaving: Sterilizes reusable equipment. 6. Hand Hygiene Wash hands after removing gloves, handling infectious materials, and before leaving the lab. Use alcohol-based hand sanitizers if soap and water are unavailable. Steps for Spill Cleanup and PPE Donning/Doffing 1. Spill Cleanup Procedure When dealing with a chemical, biological, or infectious material spill in the lab, follow these steps to ensure safety: A. Small Spill Cleanup (e.g., less than 100 mL) 1. Alert and Secure the Area: ○ Notify others of the spill. ○ Restrict access to the spill area. 2. Wear PPE: ○ Put on gloves, goggles, lab coat, and, if necessary, a mask or face shield. ○ N95 Respirator (removes at least 95% of particulates) 3. Contain the Spill: ○ For liquids: Cover with absorbent material (e.g., paper towels or spill pads). ○ For solids: Avoid scattering the material. 4. Decontaminate: ○ For biological spills: Apply a disinfectant like 10% bleach and allow it to sit for 10–15 minutes. ○ For chemical spills: Follow the specific Material Safety Data Sheet (MSDS) instructions for neutralization or cleaning. 5. Clean Up: ○ Use a disposable scoop or tongs to pick up debris. ○ Place all waste in a biohazard or chemical waste bag. 6. Dispose of Waste Properly: ○ Label and seal the waste container. ○ Dispose of it in accordance with your institution's hazardous waste protocols. 7. Decontaminate Tools and Hands: ○ Wipe down equipment used during cleanup. ○ Wash hands thoroughly after removing gloves. B. Large Spill Cleanup (e.g., over 100 mL or high-risk agents) 1. Evacuate and Notify: ○ Evacuate the area immediately. ○ Alert your supervisor and contact your lab’s safety officer or emergency response team. 2. Secure the Area: ○ Place warning signs or barriers to keep others out. 3. Follow Institutional Protocols: ○ Specialized teams may handle high-risk spills (e.g., hazardous chemicals, infectious agents). 2. PPE Donning and Doffing Steps Properly putting on and removing Personal Protective Equipment (PPE) is crucial to prevent contamination. A. PPE Donning (Putting On) 1. Inspect Equipment: ○ Check for tears, punctures, or damage in gloves, gowns, and other PPE. 2. Put on PPE in the Following Order: ○ Gown: Fully cover the torso, arms, and legs. Fasten ties or Velcro securely at the neck and back. ○ Mask or Respirator: Fit the mask snugly over the nose and mouth. For respirators (e.g., N95), conduct a fit check by exhaling sharply to ensure no air escapes. ○ Goggles or Face Shield: Place over eyes or face to protect against splashes or aerosols. ○ Gloves: Extend gloves to cover the cuffs of the gown. 3. Ensure Fit and Coverage: ○ Adjust as needed for full protection without gaps. B. PPE Doffing (Removing) 1. Remove PPE in the Following Order (to minimize self-contamination): ○ Gloves: Pinch the outside of one glove near the wrist and pull it off, turning it inside out. Hold the removed glove in the opposite hand. Slide fingers under the wrist of the second glove and peel it off over the first glove. ○ Gown: Untie or break the ties at the back. Pull the gown away from the body, turning it inside out as you remove it. Dispose of it in the appropriate container. ○ Goggles or Face Shield: Remove by handling the headband or ear loops only. Avoid touching the front surface.  ○Mask or Respirator: Remove by untying or lifting the straps from behind the head. Avoid touching the front of the mask. 2. Dispose of PPE: ○ Place all used PPE in designated waste containers (e.g., biohazard or chemical waste bins). 3. Perform Hand Hygiene: ○ Wash hands thoroughly with soap and water or use an alcohol-based hand sanitizer. General Tips for Spill Cleanup and PPE Use Preparation: Always know the location of spill kits, eyewash stations, and emergency showers. Practice: Review and practice donning/doffing procedures to build muscle memory. Use Proper Disinfectants: For biological spills, a 10% bleach solution or an EPA-approved disinfectant is most effective. Avoid Cross-Contamination: Do not touch your face or personal items (e.g., phone, glasses) while wearing PPE. By following these procedures, you can ensure safety and reduce risks in the laboratory environment. 3. Laboratory Math (6%) Overview: Essential for solution preparation, calculations, and interpreting lab results. Key Topics: Dilutions: ○ Formula: C1V1=C2V2 ○ Example: Preparing 10 mL of a 1:10 dilution from a stock solution. Unit Conversions: ○ 1 mL = 1000 µL, 1 mg = 0.001 g. ○ 1 lb = 454 grams ○ C=5/9(F-32) Molarity (M): ○ Concentration of a solution (mol/L).  ○ Example: To make 1 M NaCl, dissolve 58.44 g in 1 L of water. Normality (N): ○ Number of equivalents per liter of solution. Study Tips: Practice dilution and molarity problems. Familiarize yourself with common lab equipment for measuring volumes. 4. Hematology & Hemostasis (14%) Overview and General Vocabulary: Covers blood components and clotting mechanisms. SKILL V-A/B: PREPARING/STAINING A BLOOD SMEAR, pg. 234-239 Tips: NEED a feathered edge PRACTICE PRACTICE PRACTICE THIS!!! How you hold the slide is subjective, some people hold it in their hand, but I find it easiest and most traditional to have it flat on the table Use quick and smooth movements, not hesitating to avoid a choppy smear Hold the slide in place with one hand BY THE EDGES, smear with the other Be very careful when you’re inserting the cannula into the blood tube, as it is a needle My perfect stains after about 40 trashed ones :) Wright’s stain: polychromatic stain, combination of methylene blue (BASE blue stain), eosin (ACID red/orange stain), and methanol (fixative)  ○ Quick stain: smear is dipped sequentially for a set amount of time and is rinsed and air dries - 2-5 min, good for beginners but not amazing results ○ Two-step method: smear is placed on the rack and flooded with Wright’s stain ○ Automatic stainers also exist (and are used by most hospitals) ○ A PROPERLY stained smear should appear pinkish-blue to the naked eye Under microscope, RBCs should be pink/tan, nucleus should be purple Blood Morphology Feathered edge should be LOCATED with 10X and should be EXAMINED with oil-immersion objective (usually 100X) for differential count Granulocytic (myeloid) cells develop in bone marrow ○ Neutrophil: WBC with neutral-staining cytoplasmic granules Baby Neutrophil=Band cell ○ Eosinophil: WBC with granules that tend to stain red-orange  ○ Basophil: WBC with granules that have an affinity for basic stain (light purple) Lymphocyte: Produced in bone marrow, produce antibodies, smallest WBC, usually smooth and stains purple, cytoplasm is blue Monocyte: Arise from stem cells, influenced by CSFs to become monocytes instead of N, E, or B, largest WBC, nucleus is oval or horseshoe, kind of looks like a brain, irregular outline ○ Phagocytosis + antigen processing In a normal count, N + B = 85-95%, M + E + B = 5-15%, use serpentine pattern to count Reticulocyte Count: method of estimating immature RBCs in circulating blood ○ Commonly used to diagnose/monitor anemia Anisocytosis: different sizes of RBCs in a patient’s blood Poikilocytosis: significant variation in RBC shape in a patient’s blood Leukocytosis: too many WBCs ○ Neutrophilia, Eosinophilia, Basophilia Leukopenia: too few WBCs Condition Effect on WBCs Bacterial Infections Increased total, N, bands Viral Infections Decreased total, L Mononucleosis Increased total, L  Parasitic/allergic reactions Increased E Leukemia Increased total, L Cell Morphology Conditions Size Normocytic Normal, 6-8 micro(m) Microcytic Iron deficiency anemia Macrocytic Vitamin B12/folate deficiency Shape Round, biconcave disk Normal Sickle (drepanocyte) Sickle cell disease Spherical Hereditary spherocytosis Hemoglobin Content Normochromic Normal Hypochromic Iron deficiency anemia Hyperchromic Appearance caused by thickness of cells in spherocytosis Mean Cell Volume (MCV of RBC)= (Hematocrit (percent) / RBC) x 10 Mean Cell Hemoglobin (MCH in RBC)= (Hemoglobin (grams) / RBC) x 10 Mean Cell Hemoglobin Concentration (MCHC)= (Hemoglobin (grams) / Hematocrit (percent) x 100 Study of the cellular components of blood Arteries: strongest type of blood vessel, from the heart to the body [Aorta] Veins: carry deoxygenated blood from capillaries to the heart [superior/inferior vena cava] Capillaries: smallest blood vessels, have thin walls allowing fluid, nutrients, and waste products to easily pass through [BOTH oxygenated and deoxygenated blood here] Plasma: complex solution in which blood cells are suspended [90% water], 55% of total blood volume ○ Erythrocyte Sedimentation Rate (ESR): sed rate, measures the rate in which erythrocytes sediment in blood under standard conditions Normal = slow sedimentation rate  Inflammation/Anemia/Pregnancy = fast sedimentation rate ○ IN plasma, erythrocytes usually aggregate/clump, abnormal = rouleaux Quantitative [counts of blood components - cell counter/hematology analyzer] vs Qualitative [blood components observed for size, shape, and maturity] Hematocrit and Hemoglobin are tests commonly performed to help diagnose anemia ○ Based on the principle of separating the cellular elements of blood from plasma ○ (Top to bottom) Capillary tube - plasma - buffy coat - red blood cells - plug ○ Normal value: 42-52% M, 36-48% F adults ○ Heme (iron), globin (4 protein chains), determines oxygen-carrying capacity of blood Can be measured using specific gravity technique (estimate), Drabkin’s reagent, cyanmethemoglobin, hemiglobincyanide (using spectrophotometer) 3 main types of Hemoglobin (Hgb): Hgb A1 (95-98%), Hgb A2 (2-3%), Hgb F (2% in babies) ○ Electrical-Impedance Cell Counting BCs diluted in an electrolyte solution that conducts electricity, cells kind of interrupt the electricity that flows through and so instrument records quantity and size of cells in the sample ○ Light-Scatter Cell Counting Laser beam forces cells into single-file line, cells scatter light depending on their shape and SIZE, detected by sensors ○ Differential Cell Counting: Provides a breakdown of the cell population rather than just the quantity Cells must be subject to a specific reagent that shrinks cytoplasm to a different degree which allows them to be sorted ○ Hemacytometer: heavy glass slide with 2 counting areas, must be used with coverglass, filled with micropipet [NO OVERFLOW/AIR BUBBLES] Used for manual blood counts, fertility testing WBC: area of count is subjective RBC: large square in the middle used for counting Platelets: entire large center square used for counting Use microscope with 10X, then 40X to view Begin in upper left corner, proceed in serpentine manner for count Calculating the cell count: 2 𝐶𝑒𝑙𝑙𝑠/𝑚𝑖𝑐𝑟𝑜𝐿 = (𝑎𝑣𝑔 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 (0. 1))/𝐴(𝑚𝑚) 𝑥 𝐷𝑒𝑝𝑡ℎ(0. 1 𝑚𝑚) Microscope: light microscope vs electron microscope Light: specimen is illuminated using a lamp or light source ○ Binocular bright-field [viewing stained specimens] ○ Phase-contrast [urine sediments/platelet counts, transparent cells] ○ Epi-fluorescence [UV light used, mycobacteria, antibodies]  Electron: greater magnification and resolving power ○ Transmission electron [minute details, nuclear structure, electrons pass through] ○ Scanning electron [3-D image, surface examination, electrons bounce off] !!! With oil immersion, never use COARSE ADJUSTMENT Key Topics: Blood Components: ○ Red Blood Cells (RBCs): Transport oxygen using hemoglobin. ○ White Blood Cells (WBCs): Fight infections; types include neutrophils, lymphocytes, and monocytes. ○ Platelets (Thrombocytes): Aid in blood clotting. Fragments of the cytoplasm of megakaryocytes (large cells in bone marrow), important for hemostasis  Hematological Tests: ○ CBC (Complete Blood Count): Measures RBCs, WBCs, hemoglobin, and platelets. ○ Peripheral Blood Smear: Examines blood cell morphology. Hemostasis: ○ Intrinsic Pathway: Activated by trauma within blood vessels. ○ Extrinsic Pathway: Activated by external injury. ○ Tests: Prothrombin Time (PT), Activated Partial Thromboplastin Time (aPTT). Capillary Tubes and Collection ○ Capillary tubes are slender tubes about 7 cm long and primarily used for microhematocrit measurements Red ring on one end = heparin Blue ring = nonheparinized, used with blood already with an anticoagulant ○ Capillary punctures should be on fingertips, first drop must be wiped away as it contains tissue fluid which can dilute blood and activate clotting Vacuum Tubes ○ Venipuncture: most common methods of obtaining blood for laboratory exams ○ Tubes can be sterile/non-sterile, glass/plastic ○ Tubes with anticoagulants contain exact amount required for the amount of blood the tube will draw ○ Tubes MUST be filled to their stated capacities to not risk incorrect ratio  ○ Hemolysis: rupture of RBCs, can cause erroneous hematology and blood chemistry results: happens when you poke the vein too close to the surface, or the collection tube is shaken harshly Stopper Color Anticoagulant Examples of use Red None Tests that require serum: blood chemistries/serology Red/Gray None Serum-separator tube, tests that require serum Lavender, pink EDTA Most hematological tests, blood-typing Green Heparin Special chemistry tests, certain lymphocyte studies, lupus erythematosus test Light/Royal (3.2-3.8%) Sodium Most coagulation studies Blue Citrate or nothing Yellow ACD Solution Blood banking studies Gray Glycolic inhibitor + Certain glucose tests, legal alcohol anticoagulant (Potassium Oxalate) Black Buffered Sodium Citrate Westergren ESR (speed of RBC settling) ABO Blood Grouping: The ABO blood group system categorizes blood into four types: A, B, AB, and O, based on the presence of A and/or B antigens on red blood cells and antibodies in plasma. Blood type compatibility for donations is as follows: Type O: Universal donor for red cells (no antigens). Can only receive from O. Type A: Can donate to A and AB. Receives from A and O. Type B: Can donate to B and AB. Receives from B and O. Type AB: Universal recipient for red cells (both antigens present). Can only donate to AB. Hemostasis: process of stopping the loss of blood from blood vessels  Coagulation Factors: plasma proteins (except for Ca++), ultimately form a fibrin clot 1. Conversion of prothrombin to thrombin in the presence of thrombokinase and calcium 2. Conversion of fibrinogen to fibrin due to actions of thrombin Fibrin clot is temporary, gets eaten by enzymes (fibrinolysis) [plasminogen & plasmin involved] ○ Rapid test for D-dimer comes from here, because they want to detect the fragments of fibrin and fibrinogen that can interfere with polymerization and aggregation TF also = Thromboplastin Coagulation Cascade  ○ (1) Common, (2) Intrinsic, (3) Extrinsic pathways https://www.khanacademy.org/science/health-and-medicine/advanced-hematologic-syste m/bleeding-and-impaired-hemostasis/v/coagulation-cascade The coagulation cascade is divided into three major phases: A. Initiation Begins with vascular injury and exposure of tissue factor. Activates the extrinsic pathway. B. Amplification The intrinsic pathway is activated to amplify the clotting signal. Platelets and clotting factors are recruited. C. Propagation The common pathway generates large amounts of thrombin, leading to fibrin clot formation. 2. Steps of the Clotting Pathway A. Intrinsic Pathway Triggered by damage to the endothelium (inner lining of blood vessels). It involves factors present in the blood. 1. Activation of Factor XII (Hageman Factor): ○ Damaged endothelium exposes collagen. ○ Factor XII is activated to XIIa. 2. Activation of Factor XI: ○ XIIa converts Factor XI into XIa. 3. Activation of Factor IX: ○ XIa activates Factor IX to IXa, in the presence of calcium (Ca²⁺). 4. Formation of Tenase Complex: ○ IXa combines with cofactor Factor VIIIa on platelet surfaces, activating Factor X. B. Extrinsic Pathway Triggered by external trauma that exposes tissue factor (TF). It is a quicker pathway than the intrinsic pathway. 1. Release of Tissue Factor (TF): ○ Damaged tissues expose TF, a membrane protein. 2. Activation of Factor VII:  ○ TF binds to circulating Factor VII, converting it to VIIa. 3. Activation of Factor X: ○ The TF-VIIa complex activates Factor X to Xa, in the presence of Ca²⁺. C. Common Pathway Where the intrinsic and extrinsic pathways converge. 1. Activation of Factor X: ○ Both pathways generate activated Factor X (Xa). 2. Formation of Prothrombinase Complex: ○ Xa combines with cofactor Factor Va on platelet surfaces. ○ Converts prothrombin (Factor II) into thrombin (Factor IIa). 3. Thrombin's Key Roles: ○ Converts fibrinogen (Factor I) into fibrin monomers. ○ Activates Factor XIII to stabilize the fibrin clot. ○ Amplifies clotting by activating Factors V, VIII, and XI. 4. Fibrin Clot Formation: ○ Fibrin monomers polymerize to form insoluble fibrin strands. ○ Factor XIIIa cross-links fibrin to stabilize the clot. 3. Regulation of the Clotting Pathway The body tightly regulates clot formation to prevent excessive bleeding or clotting. Natural Anticoagulants: Antithrombin III: Inhibits thrombin and Factors IXa, Xa, XIa, and XIIa. Protein C and Protein S: Degrade Factors Va and VIIIa. Tissue Factor Pathway Inhibitor (TFPI): Inhibits the TF-VIIa complex. Fibrinolysis: Once healing begins, plasminogen is converted to plasmin, which breaks down fibrin clots. Hemostasis Disorders: ○ Hemophilia A & B, inherited on X chromosome A: Classic, caused by deficiency in VIII B: Christmas, caused by deficiency in IX ○ *any disease named after a person is usually inherited Bleeding Time: ○ Measured to evaluate function of platelets and small blood vessels, incision in capillaries: Ivy (more common) / Duke (rarely used) method  Accepted time reference: Ivy - 2-9 min, Duke - 1-3 min Prothrombin Time: ○ Most frequently performed tests, evaluates function of extrinsic and common pathways of hemostasis, used pre-surgery and to monitor anticoagulant therapy Accepted time reference: 10-13 sec Kind of weird because it’s very sensitive and you can have different results at different labs, so use ISI (international sensitivity index) / INR (international normalized ratio) 1:9 coag to blood, 3.2% soln of sodium citrate, 4.5 mL Many POC (point-of-care) instruments used to measure coagulation testing Activated Partial Thromboplastin Time: ○ Used to monitor heparin therapy and screen for function of intrinsic and common pathways, try to form a fibrin clot Accepted time reference: 31-39 sec D-dimers are created by XDPs, which are like the fibrin segments (FDPs), but they are cross-linked and usually signify a more serious condition ○ Tests are good for diagnosing DIC (disseminated intravascular coagulation), DVT (deep vein thrombosis), and pulmonary embolism Less than 0.20 microgram/mL is negative Greater than 0.20 microgram/ML is positive  2. White Blood Cell (WBC) Count 3. Platelets 4. Reticulocyte Count 5. Immunology (16%) SKILL IV: ABO Blood Grouping pg. 386-393 Blood is observed for agglutination - agglutination is a positive reaction, absence is a negative reaction ○ Recorded with a (+) or (0) Reactions of ABO Blood groups with anti-A and anti-B sera Blood Group Anti-A Anti-B A + 0 B 0 +  AB + + O 0 0 ABO forward and reverse grouping results ABO Group Forward Reverse Cells Plasma Anti-A Anti-B A Cells B Cells O Cells O 0 0 + + 0 A + 0 0 + 0 B 0 + + 0 0 AB + + 0 0 0 Forward grouping: identifies antigens present on RBCs by creating a suspension of cells with anti-A/B serum and observing for agglutination after centrifuging/sitting for 15-30 min ○ Agg. graded by 0, w+, 1+, 2+, 3+, 4+ Reverse grouping: identifies the antibodies present in a patient’s serum/plasma by reacting the plasma with a commercial 2-5% suspension of group A/B cells and observing for agglutination ○ 2 drops of plasma added to 3 tubes (a, b, control), one drop of group A added to tube a, one drop of group B added to tube b, one drop of suspension added to control ○ + indicates antibody present corresponds to antigen Control tube should ALWAYS be negative since it only contains patient’s plasma and cells Rh Blood group: composed of many antigens, products of inherited genes ○ Major antigen: Rh D, then come C, c, E, and e ○ People must be tested before transfusions to ensure compatibility of blood Also identifies females at risk of giving birth to infants with hemolytic disease of the newborn (HDN) where antibody from mom destroys fetal RBCs ○ Testing can be performed by slide, tube, or gel methods D-neg type must be confirmed by test for weak D If transfused with the wrong type, your body can produce antibodies that destroy donor blood cells, causing a transfusion reaction ALTHOUGH BOTH IMPORTANT, blood type is usually more important than Rh factor in transfusions because it can cause more severe reactions if its mismatched Key Topics: Innate Immunity: ○ First-line defense (e.g., skin, mucous membranes). ○ Includes phagocytes like macrophages. Adaptive Immunity: ○ Specific response involving T-cells and B-cells. ○ Produces memory cells for long-term immunity. Antibodies: ○ Proteins that target specific antigens. ○ Types: IgG, IgM, IgA, IgD, IgE. (from most to least abundant) ○ Monoclonal (1 class & specificity) vs Polyclonal (produced by more than one cell line & specificity)) Antigens: ○ Foreign substances in the body ○ Molecules, viruses, blood cells, tumor cells, bacteria, fungi Serological Tests: ○ ELISA (Enzyme-Linked Immunosorbent Assay): Detects antibodies/antigens. ○ Western Blot: Confirms presence of specific proteins. Lymphocytes: ○ Important cells that help bring about the specific immune response T lymphocytes: (cell-mediated immunity) protection against viruses, fungi, tumor cells, and intracellular orgs. Secrete lymphokines/cytokines which are like little messenger molecules that communicate and help regulate response B lymphocytes: (humoral immunity) primary protection against bacteria, toxins, and circulating agents, produce antibodies Plasma cells are differentiated B lymphocytes Vaccines stimulate humoral immunity Immunoglobulins: ○ Antibodies, circulate in blood and make up about 10-15% of serum Antibody for A blood group antigen = anti-A Antibody for B blood group antigen = anti-B Immune Diseases ○ Autoimmune Rheumatoid Arthritis 75-85% of people have rheumatoid factors (RFs) in their serum, leads to RF test ○ A diagnosis of RA cannot be ruled out solely on the basis of a negative test because not everyone has RFs Lupus erythematosus Type 1 diabetes  Myasthenia gravis ○ Hypersensitivities Rhinitis Asthma Dermatitis ○ Acquired immunodeficiencies Infections Systemic disease Malignancies Reaction to drugs Irradiation ○ Malignancies Lymphomas Leukemias Multiple myeloma ○ Congenital immunodeficiencies DiGeorge syndrome Agammaglobulinemia SCID (severe combined immune deficiency) Precipitation: the formation of an insoluble complex when a specific antibody is reacted with a soluble antigen ○ Measured with Radial immunodeficiency, Agar precipitation, rocket electrophoresis, immunoelectrophoresis Nephelometry: allows precipitation tests to be automated ○ Principle: suspension of small particles will scatter light when a beam is passed through it Complement fixation: sensitive method for detecting an antigen-antibody reaction and detect specific antibodies in patient serum Labeled antibody techniques: ○ Enzyme immunoassays, radioimmunoassays, flow cytometry, etc. Immunohematology (Blood bank!!!): study of human blood groups ○ Includes: evaluation of blood donors, collection and processing of donor blood, testing patient blood for blood group antigens, matching patient with compatible blood, tissue typing, forensic studies, paternity tests, and genetic studies ○ Standards for blood banking issued by the AABB (American Association of Blood Banks) Approx. 15 million units of blood are donated each year Apheresis: process of removing one blood component and returning the remaining components to the donor ○ ABO blood grouping is based on the presence or absence of two blood group antigens (anti-A and anti-B) ABO Blood Group Antigen on RBC Antibody in Serum  A A Anti-B B B Anti-A AB A&B Neither O Neither Both ○ Testing patient blood for the presence of blood group antibodies is called reverse grouping Performed by reacting serum/plasma with RBCs whose A and B antigens are known Steps for Serological Testing Serological testing is used to detect and measure antibodies or antigens in a patient’s blood to diagnose infections, autoimmune disorders, or determine immunity. It involves various techniques like enzyme-linked immunosorbent assay (ELISA), agglutination, and immunofluorescence. Below are the typical steps for performing serological tests. 1. Preparation a. Collect the Sample: ○ Obtain the patient’s blood sample via venipuncture (for serum) or fingerstick (for plasma). ○ Centrifuge the sample to separate serum or plasma from the blood cells. b. Label Samples: ○ Label the tubes or vials with patient details and sample identification to avoid mix-ups. 2. Choose the Appropriate Serological Test Select the test based on the suspected condition or pathogen. Common tests include: ○ ELISA (Enzyme-Linked Immunosorbent Assay): For detecting antibodies or antigens. ○ Agglutination tests: For detecting antigens or antibodies (e.g., blood typing). ○ Western Blot: For detecting specific antibodies (e.g., HIV testing). ○ Immunofluorescence: To detect antibodies or antigens by fluorescence under a microscope. ○ Complement fixation: For detecting specific antibodies to a pathogen. 3. Prepare Reagents and Controls a. Prepare Antigens/Antibodies: ○ Obtain purified antigens or antibodies that are specific to the pathogen of interest. ○ In some cases, conjugated (linked) antibodies (e.g., enzyme or fluorochrome-conjugated) are used. b. Prepare Positive and Negative Controls: ○ Controls are used to verify the accuracy of the test. A positive control contains known antigens/antibodies, while a negative control does not. 4. Perform the Test a. For ELISA: 1. Coating: Place antigens or antibodies onto the test wells of a microplate. 2. Blocking: Add a blocking agent (e.g., bovine serum albumin) to prevent non-specific binding. 3. Sample Addition: Add the patient’s serum or plasma to the wells. 4. Incubation: Allow time for the antigen-antibody interaction to occur, usually for 30 minutes to 1 hour. 5. Washing: Remove excess unbound material by washing the wells. 6. Detection: Add enzyme-conjugated secondary antibody (if detecting antibodies), followed by a substrate that produces a color change if binding occurs. 7. Reading the Results: Measure the absorbance (color intensity) using a spectrophotometer or colorimeter. b. For Agglutination: 1. Place antigen or antibody solution on a slide or microtiter plate. 2. Add the patient’s serum to the test surface. 3. Stir and look for visible clumping or agglutination, which indicates a positive result. c. For Immunofluorescence: 1. Apply patient sample (e.g., serum) to a slide with immobilized antigen or antibody. 2. Incubate for the required time. 3. Wash off excess sample and add fluorescently labeled secondary antibodies. 4. Examine under a fluorescence microscope for a positive signal (fluorescent glow). 5. Interpretation of Results  a. Positive Result: ○ The presence of specific antibodies or antigens is detected. ○ In ELISA, the color change indicates a reaction between the antigen and antibody. ○ In agglutination tests, visible clumping indicates a positive result. b. Negative Result: ○ No specific antibodies or antigens were detected. ○ In ELISA, no color change means no binding of antigen and antibody. ○ In agglutination, no clumping indicates a negative result. c. Control Comparison: ○ Ensure that controls function correctly to validate the test results. 6. Reporting Results Record the test results, including any relevant patient details and the interpretation of the findings. For certain tests, a titer may be reported, which indicates the concentration of antibodies present in the serum. In cases of doubt or ambiguous results, repeat testing or confirmation tests (e.g., Western Blot for HIV) may be recommended. 7. Clean-Up and Waste Disposal Properly dispose of any used reagents, patient samples, and materials (e.g., gloves, pipette tips, test tubes) according to biohazard and chemical waste protocols. Clean the workspace to avoid cross-contamination for future tests. 8. Quality Control and Validation Perform routine quality control (QC) checks using control sera to verify that the test systems and reagents are functioning as expected. Document and maintain QC logs for compliance with laboratory standards. Additional Considerations Sensitivity and Specificity: The effectiveness of the test depends on its sensitivity (ability to detect true positives) and specificity (ability to detect true negatives).  Timing: Serological tests may need to be repeated over time to detect changes in antibody levels, such as in the case of diagnosing infections with late seroconversion. Clinical Correlation: Always interpret serological results in conjunction with clinical findings and other diagnostic tests. 6. Urinalysis (8%) Overview: Analyzes physical, chemical, and microscopic properties of urine. Key Topics: Physical Properties: ○ Color (normal: pale yellow). ○ Clarity (normal: clear). ○ Smell (normal: no odor/neutral) Urine can also smell like ammonia (dehydration), sweet (diabetes), fishy (genital infection), rotten eggy (tumor), or bad (food/medications) Chemical Analysis: ○ Test strips detect glucose, ketones, proteins, and pH. Microscopic Examination: ○ Detects crystals (e.g., calcium oxalate), casts, and cells. Major function of the kidneys: ○ Elimination of metabolic and toxic waste products from the body ○ Regulation of acid-base balance (pH) ○ Regulation of the composition and volume of body fluids ○ Production of hormones necessary for proper function of body tissues and organs ○ ○ Each kidney has about 1 million nephrons, composed of a glomerulus and its associated renal tubule Glomerulus: filtering unit Renal Tubule: (1) proximal convoluted tubule, (2) loop of Henle, (3) distal convoluted tubule concentrates glomerular filtrate, forms urine ○ Urine: 95% water, 5% is solutes Urea is highest in concentration (breakdown product of amino acids) ○ ○ Hormones that influence or are produced by the kidneys Hormone Function Influence Aldosterone Regulates electrolytes, especially K Antidiuretic hormone (ADH) Regulates water reabsorption Atrial natriuretic peptide Influences sodium excretion Parathyroid hormone (PTH) Regulates calcium reabsorption Calcitonin Inhibits calcium reabsorption Produced Erythropoietin Stimulates red blood cell synthesis Active Vitamin D3 Influences bone calcium levels  Renin Influences blood pressure ○ Healthy adults excrete 1-2 L of urine per day ○ Abnormal urinalysis results can be seen in (1) disorders or diseases of the urinary tract, (2) disease in other parts of the body affects kidney function/urine composition Can cause changes in urine volume, color, transparency/clarity, odor, cells present, and chemical constituents Urine Testing: ○ Urine specimens must be clearly labeled with patient’s name, date, and time of collection ○ Urine specimens must be examined within 1 hour of collection Deterioration of urine can be prevented by storing in the dark in a lidded container at 4-6 degrees celsius for up to 4 hours Bacteria can grow if the urine sits, making the pH level rise and it will skew test results ○ A clean-catch urine specimen is required if the urine is to be cultured for bacteria Physical Characteristics of Urine: ○ Color: ranges from pale to yellow to amber, variations can be caused by diet, medications, physical activity, and disease Yellow: produced by urochrome, regular Red: cloudy=hematuria, clear=hemo/myoglobin, porphyrins can cause urine to be red or wine-red Brown or Black: acidic urine causes hemoglobin to darken, also melanin or melanoma Yellow-Brown or Green-Brown: caused by bilirubin or bile pigments (hepatitis) ○ Transparency (Clarity): reported as clear (normal), hazy (slightly cloudy), cloudy (turbid), or milky Hazy: mucus, talcum powder, squamous epithelial cells Cloudy: calcium oxalate, uric acid crystals, amorphous phosphates/urates ABNORMAL: Cloudy-red: RBCs Turbid/cloudy: WBCs, bacteria, yeasts, renal epithelial cells, lipids Opalescent, milky: Fats, lipids ○ Odor: normal urine has a characteristic aromatic and not unpleasant odor Uncontrolled diabetes = fruity urine due to ketones PKU = mousy/musty odor Maple syrup urine disease… If urine is allowed to break down it will smell like ammonia UTI = foul, pungent odor Garlic/asparagus can also produce an abnormal odor  ○ Specific Gravity: ratio of the weight of solution compared to the weight of an equal volume of distilled water at the same temperature Normal range: 1.005-1.030, most samples fall between 1.010-1.025 Highest in the first morning specimen Indicator of real tubular function, assesses the ability of the kidneys to reabsorb essential chemicals and water from the glomerular filtrate Darker = more concentrated = higher specific gravity Can be measured with urinometer or refractometer Chemical Examination of Urine: Substance Tested Description Reference Value Glucose Glycosuria detects if blood glucose Negative level has exceeded renal threshold Bilirubin Primary bile pigment, can indicate Negative liver disease, bile duct obstruction, or hepatitis Ketones Produced when the body burns fat Negative rather than sugar for energy, occurs in uncontrolled diabetes Blood Hematuria can occur in infection, Negative trauma, bleeding in kidneys, menstruation, or tumor pH Measure of acidity, can change with 4.5-8.0 diet, medications, or kidney and metabolic diseases Protein Proteinuria is an indicator of renal Negative to trace disease but can also be a result of diet and albumin Urobilinogen Bilirubin degradation product 0.1-1.0 mg/dL formed by intestinal bacteria, can be increased in hepatic or hemolytic disease Bacteria (nitrite) Gram-negative bacteria convert Negative nitrate to nitrite, indicates bacterial UTI but can also be negative in an infection Leukocyte Esterase Enzyme contained by neutrophils Negative that indicates infection or inflammation bc of presence of leukocytes in urine  Specific Gravity Reflects kidney’s ability to 1.005-1.030 concentrate urine ○ WBCs and RBCs at a microscopic level should be 0-4/HPF ○ Epithelial and Casts should be occasional Urine pregnancy tests are based on the detection of the human chorionic gonadotopic (hCG) hormone in urine ○ Rapid tests and hCG tests must be interpreted carefully because the treatment has the potential to cause damage to a fetus 7. Clinical Chemistry (14%) Overview: Focuses on biochemical analysis of body fluids. Usually use serum to test. Key Topics: Pleural Fluid: the fluid in the space between the pleural membrane of the lung and the inner chest wall Synovial Fluid: a viscous fluid secreted by membranes lining the joints Instrumentation: ○ Spectrophotometry: Measures light absorbance to determine concentration. ○ Centrifuges: spin samples at high speeds, forcing the heavier particles to the bottom of the container Clinical centrifuge: model that can be used for urinalysis or serum separation [0-3,000 rpm, 5-50 mL] Serological centrifuge: small model used in blood banking to spin small tubes [2-3 mL] Microcentrifuge/Microfuge: spin special microtubes for measuring microhematocrits [0.5-1.5 mL, up to 14,000 rpm] A chemistry profile commonly tests for substances like electrolytes (sodium, potassium, chloride), blood glucose, liver function markers (ALT, AST, bilirubin), kidney function markers (BUN, creatinine), calcium, total protein, albumin, and lipids (cholesterol, triglycerides) depending on the specific panel, which can include a Basic Metabolic Panel (BMP) or a Comprehensive Metabolic Panel (CMP) Electrolytes: Measures essential minerals like sodium, potassium, chloride, and sometimes magnesium, which are crucial for fluid balance and nerve function. ○ Sodium (Na⁺): affect water retention, 135–148 mEq/L  ○ Potassium (K⁺): affect muscle function, can be decreased by vomiting or diarrhea 3.5–5.4 mEq/L ○ Chloride (Cl⁻): affect dehydration, 98–108 mEq/L ○ Bicarbonate (HCO₃⁻): affect blood pH [buffer] 22–28 mEq/L Liver function tests: Includes enzymes like alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactase dehydrogenase (LDH),gamma glutamyl transferase (GGT) and alkaline phosphatase (ALP) to assess liver health. ○ Source of cholesterol ○ Bilirubin measured to screen for liver or gallbladder dysfunction ○ Rise in serum enzymes usually reflects injury to tissue ○ Alanine Aminotransferase (ALT): 7–56 U/L ○ Aspartate Aminotransferase (AST): 10–40 U/L ○ Alkaline Phosphatase (ALP): 44–147 U/L ○ Gamma-Glutamyl Transferase (GGT): 9–48 U/L Kidney function tests: Measures blood urea nitrogen (BUN) and creatinine to evaluate kidney function. ○ BUN: influenced by diet, hormones, and kidney function ○ Creatinine: waste product of creatine which is stored in muscle and used for energy - impairment of urine formation/excretion ○ Uric acid: used to diagnose gout, can also rise after radiation therapy Proteins: Measures total protein (6-8 g/dL) and albumin (3.8-5 g/dL) levels, which can indicate nutritional status and liver function. ○ Serum and albumin protein are usually measured simultaneously, in the A/G (albumin to globulin) ratio Globulin = total protein - albumin Calcium: Checks for calcium levels, important for bone health. 8.7-10.5 mg/dL ○ Phosphorus: influenced by calcium and certain hormones, higher in children because of higher GH, reference is 3.0-4.5 Cardiac Function: Creatine Kinase (CK) is an enzyme measured to help diagnose myocardial infarction Iron: 65-165 microg/dL, deficiency can lead to anemia Basic Metabolic Panel (BMP): Includes core electrolytes, kidney function markers, glucose, and calcium. Comprehensive Metabolic Panel (CMP): Expands on the BMP by adding additional liver function tests and other metabolic markers. Lipid Panel: Specifically measures cholesterol, HDL, LDL, and triglycerides to assess cardiovascular risk. Over 107 million Americans have over 200 mg/dL cholesterol ○ HDL cholesterol: good cholesterol, is transported to liver to be broken down ○ LDL cholesterol: bad cholesterol, transports to be stored as fat HDL/LDL = heart attack risk factor ○ Triglycerides should be less than 150 mg/dL Good indicator for the presence or development of metabolic syndrome that causes coronary heart disease Blood glucose: Checks for blood sugar levels, important for diabetes monitoring.  ○ Disorders: (1) Diabetes mellitus [increased glucose], and (2) hypoglycemia [decreased glucose] ○ Oral Glucose Tolerance Test (OGTT) is reliable and intricate but not commonly used because it takes around 3-5 hours to administer ○ Fasting Blood Glucose (FBG): 60–100 mg/dL ○ Postprandial (After Meal) Glucose: