Basic Principles and Practice PDF

Basic Principles and Practice  Reage ntsclinical labs use reagents Most prepared by manufacturers. Knowledge of chemicals, standards, solutions, buffers, and water requirements is still useful, though. Chemicals – Varying grades of purity – Analytic reagent grade (AR) or...

Basic Principles and Practice  Reage ntsclinical labs use reagents Most prepared by manufacturers. Knowledge of chemicals, standards, solutions, buffers, and water requirements is still useful, though. Chemicals – Varying grades of purity – Analytic reagent grade (AR) or reagent grade are best for lab use. – Specifications for AR chemicals established by American Committee Society (ACS) – Labels on reagents indicate grade and percentage of impurities.  Reage nts Materials Reference – Clinical chemistry involves analysis of biologic fluids. – Standards of traditional analytic chemistry don’t readily apply. – Calibrators and standards for lab are certified by NIST(National Institute of Standards and Technology) instead of “primary standards.” Water Specifications – Water is most-used reagent in lab. – Labs require reagent grade water. – CLSI provides three specifications of water Type I reagent grade water, Type II reagent grade water, and Type III reagent grade water – Minimum testing requires resistivity and microbiology culture – Resistivity measure of the ion content ( ions interfere with assays) – Bacteria may inactivate reagents or alter optical properties of test solution  Reage ntsSpecifications Water – Methods of water purification: Distillation – removes bacteria, particulates, and dissolved ionized solids Deionization – removes dissolved ionized gases and solids Reverse osmosis Ultrafiltration and nanofiltration – Combination of purification methods is needed to produce reagent grade water.  Reage nts Properties Solution – Terms and definitions Solute: a substance dissolved in a liquid Analytes: biologic solutes Solvent: liquid in which solute is dissolved Solution: solute plus solvent – Concentration percent solution parts per hundred or the amount of solute per 100 total units of solution Weight per weight (w /w), volume per volume (v/ v), weight per volume (w /v) Molarity – number of moles per 1 L of solution (mol/L) Molality – number of moles per 1 kg of solvent (mol/kg)  Reage ntsProperties ⚫ Solution ⚫ Colligative properties: osmotic pressure, vapor pressure, freezing point, boiling point ⚫ Redox potential: measure of ability of solution to accept or donate electrons ⚫ Oxidizing agents – accept electrons ⚫ Reducing agents – donate electrons ⚫ Conductivity: measure of how well electricity passes through a solution ⚫ pH: inverse log of hydrogen ion concentration ⚫ pH = -log[H+] ⚫ Buffers: weak acids or bases and their related salts that minimize changes in hydrogen ion concentration ⚫ Most important acid base balance system in the body is bicarbonate buffer ⚫ CO2+H2O = H2CO3 = HCO3- + H+ ⚫ Henderson hasselbalch equation describes the dissociation characteristics of weak acids and bases ⚫ pH= pKa + log [A-] [HA] Laboratory Plasticware and Glassware ⚫ Class A - lab supplies meeting the NIST (National Institute of Standards and Technology) tolerance of accuracy ⚫ Glassware and Plasticware ⚫ Laboratory vessels: flasks, beakers, graduated cylinders ⚫ Pipets: glass or plastic utensils used to transfer liquids ⚫ Burets: look like wide, long, graduated pipets with stopcock at one end ⚫ Syringes: sometimes used for transfer of small volumes in blood gas analysis or separation techniques Laboratory Plasticware and Glassware ⚫ Containers and Receivers ⚫ Beakers ⚫ Erlenmeyer Flasks ⚫ Test Tubes ⚫ Reagent Bottles Laboratory Pipettes ⚫ PipettingTerms ⚫ Calibrated To-Deliver (TD) Pipettes – after the liquid is expelled, the liquid on the walls of the pipette is allowed to remain- no need to rise ⚫ Calibrated To-Contain (TC) Pipettes - after the liquid drains freely the pipette is rinsed out in the diluent so the entire volume contained is delivered- you need to rinse ⚫ Frosted Bands – indicate the pipette should drain freely and the last drop is blown out- it has nothing to with delivery or containing. We need to push more to push out whats left in the tip ⚫ No Frosted Bands – the pipette is allowed to drain and the last drop should remain in the tip Laboratory Pipettes ⚫ Pipettes ⚫ Graduated or Mohr Pipettes – last calibration ending above the delivery tip, marked TD and no frosted bands not for calibrators or controls ⚫ Serologic Pipettes – graduated all the way to the tip, marked TD and has frosted bands making it a “blow out pipette”, more inaccurate than other pipettes, never use for calibrators or controls- for serial dilutions ⚫ Volumetric Pipettes – marked with single calibration line, marked TD with no frosted bands, it is not suitable for variable measurements, always use for standards (calibrators) and controls- do not use for serial dilutions but can be used for calibrations ⚫ Ostwald-folin – single calibration, marked TD, has frosted bands, used for viscous fluid like blood or fluids that don’t drain like water Laboratory Pipettes Laboratory ⚫ Select the smallest pipette that can be used for Pipettes measurement, never use a pipette that is ten times the measurement ⚫ Immerse tip in liquid to a level that will remain in solution after volume has been withdrawn without touching the vessel walls(to avoid bubbles,etc) ⚫ Hold pipette upright, not at an angle ⚫ Suction is applied to the mouth of the pipette until fluid is above the desired graduation line, then remove pipette from liquid ⚫ Wipe the pipette tip and drain until the meniscus touches the desired calibration line ⚫ Hold the pipette in a vertical position and with the exception of the Mohr pipette, hold the tip against the side of the receiving vessel, the contents is allowed to drain Laboratory Pipettes Laboratory Pipettes ⚫ Glass Pipette Errors Volumes ⚫Dirty pipette ⚫Not draining vertically ⚫Drawing fluid too far above the above calibration ⚫Failure to wipe off pipette ⚫Wiping pipette after it has been adjusted to desired volume Laboratory Pipettes ⚫ Pipetting Technique Using Micropipettors (To-Contain Pipettes) ⚫ Prewet pipette tip ⚫ Work at temperature equilibrium ⚫ Examine the tip before dispensing sample ⚫ Use standard mode pipetting ⚫ Pause consistently after aspiration ⚫ Pull the pipette tip straight out ⚫ Minimize handling of the pipette and tip ⚫ Immerse the tip to the proper depth ⚫ Use the correct pipette tip ⚫ Use consistent plunger pressure and speed Ten TipsVideo Laboratory Pipettes Laboratory Centrifuges ⚫ Centrifugation is used in the separation of a solid material from a liquid by applying rapid rotation or spinning. ⚫ Precipitate = sediment packed at the bottom of the centrifuge tube ⚫ Supernatant = liquid or top portion of fluid at the top of the centrifuge tube Laboratory Centrifuges ⚫ Centrifugation ⚫Common applications ⚫Separating serum or plasma from blood cells ⚫Separating supernatant from a precipitate in analytic reaction ⚫Separating two immiscible liquids or expelling air ⚫Centrifuges classified according to the following: ⚫Rotor head type (fixed, hematocrit, horizontal, angled) ⚫Maximum speed attainable Centrifuge ⚫ Microfuges – used to spin special microtubes at high speeds types 14,000RPM ⚫ Microhematocrit centrifuge – spins capillary tubes at high speeds for hematocrit measurement ⚫ Fixed-Angle Rotor – A rotor that supports tubes at a fixed angle; also called fixed head, spins faster and gives a slanted faster separation, used for blood ⚫ Horizontal Rotor – A rotor that allows tubes to swing from a vertical to a horizontal position; also called the horizontal head, gives horizontal separation, better for liquids, used for blood and urine ⚫ Ultracentrifuge – A very high-speed centrifuge capable of obtaining extremely high speeds 40,000 RPM, used for gram stains and differentials of body fluids- it will produce heat so it needs to be stored in the fridge : DNA/RNA/CSF Balanced Centrifuge ⚫ Why balance a centrifuge? To protect the mechanical life of the centrifuge; reduce vibration and avoid breakage of glass and spillage ⚫ Properly balanced centrifuge: one of even placement and one of opposition Laboratory Centrifuges Specimen Considerations ⚫ Types of Samples ⚫ Whole blood: both liquid portion (plasma) and cellular components (red and white blood cells, platelets) ⚫ Plasma: clear yellow supernate ⚫ Serum: remaining liquid after clotting ⚫ Arterial blood: to measure blood gases and pH ⚫ Urine ⚫ Cerebrospinal fluid ⚫ Paracentesis fluids: pleural, pericardial, peritoneal ⚫ Amniotic fluids Specimen Considerations ⚫ Blood Sample Specimen Considerations ⚫ Tubes ⚫ Blue top – contains sodium citrate anticoagulant, spun and used for coagulation studies ⚫ Red, Gold, and Speckled top – contain no anticoagulants, with or without clot activator, with or without gel separator, used in chemistry, must allow to clot before spinning ⚫ Green top – anticoagulant heparin, with or without gel, used in chemistry, don’t have to wait for clot formation before spinning ⚫ Lavender top – anticoagulant EDTA, whole blood unspun, used in hematology ⚫ Unacceptable Specimens (evaluate plasma and serum) Uri ne ⚫ Types of Urine Specimens ⚫ Random Specimen - UA ⚫ First Morning Specimen - UA ⚫ Midstream Clean-Catch Specimen – UA or culture ⚫ 24-Hour or Timed Specimen – Creatinine Clearance ⚫ Catheter Collection Specimen – Better for Culture ⚫ Suprapubic Aspiration Specimen – Best for Culture ⚫ Pediatric Specimen Uri ne ⚫ Preservation of Urine Specimens ⚫ Labeling and Processing of Urine Specimens ⚫ Labels ⚫ Collection Date and Time ⚫ Collection Method ⚫ Proper Preservation ⚫ Light Protection Body Fluids Specimens For Culture ⚫ Throat Culture Collection ⚫ Wound Culture ⚫ Sputum Culture ⚫ Stool Culture ⚫ Urine Culture ⚫ Blood Culture