Basics of Clinical Chemistry PDF

Basics of Clinical Chemistry Basics of Clinical Chemistry Units of Measure Units of Measure SI system seven basic units: Based on the metric system Prefix used with SI units:...

Basics of Clinical Chemistry Basics of Clinical Chemistry Units of Measure Units of Measure SI system seven basic units: Based on the metric system Prefix used with SI units: Prefix used with SI units: Base Quantity Name Symbol Factor Prefix Symbol Factor Prefix Symbol Length Meter m 10 exa E 10 liter, meter, gram basic unit Mass Kilogram kg 10 peta P 10 deci d Time Seconds s 10 tera T 10 centi c Electric current Ampere A 10 giga G 10 milli m Thermodynamic Kelvin K 10 mega M 10 micro u 10 kilo k 10 nano n Amount of Substance Mole Mol 10 hecto h 10 pico p Luminous intensity Candela cd 10 deka da 10 femto F 10 liter, meter, gram basic unit 10 atto a Note: (-) to the left; (+) to the right Basics of Clinical Chemistry Basics of Clinical Chemistry Common Chemistry Analytes Reagents Chemicals Labels for reagents 1. State of actual impurities 2. List of maximum allowable impurities 3. Clearly printed with percentage of impurities 4. Initials of its grade of purity OSHA requirement 1. Indicate the lot number 2. Physical hazard 3. Biologic health hazard 4. Precautions for safe use 5. Storage Safety Data Sheet Formerly known as ________________. A required document provided by the manufacturer which contains technical data sheets for each chemical manufactured are only required for hazardous chemicals not primarily intended for use by the general consumer UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) Basics of Clinical Chemistry Basics of Clinical Chemistry Reagents Reagents Grades of Purity Reference Material ACS Standard Grade Reference AKA as ________________. Primary Standard High purified chemical Material For Laboratory Use measured directly to produce a substance of exact known concentration Suitable for use in most analytic procedures Ultrapure Chemical Have undergone additional purification steps Standard Reference Materials Used instead of ACS primary standard material Used in specific procedures as: chromatography, atomic absorption, Certified reference materials immunoassays, molecular diagnostics, and standardizations used in clinical chemistry laboratories US Pharmacopeia (UPS) or used to manufacture drugs used to verify calibration of accuracy/bias assessments ________________. pure enough to be used in most chemical procedures but may or may not meet all assay requirements. Chemically Pure/Pure Grade preparations of these chemicals is not uniform (impurity limitations Secondary Standard substance of lower purity are not stated) its concentration is determined by comparison with a primary standard not recommended for reagent preparation in clinical laboratories depends not only n its composition but also on the analytic reference unless further purification or a reagent blank is included method Technical or Commercial Grade Primarily in manufacturing NEVER used in Clinical Laboratory Basics of Clinical Chemistry Basics of Clinical Chemistry Reagents Reagents Water Specifications Water Specification ________________ most frequently used reagent in the laboratory Clinical Laboratory Reagent Processes required in preparation of reagent grade water (CLRW): Substantially Purified Water For lab procedures including reagent and standard preparation Water Distilled Water Water purified by ________________. routine testing 1. Pre-filters are glass or cotton microfibers that remove 98% of the Deionized Water Water purified by _______________________________. meet the CLSI requirements particulate matter Reverse Osmosis Water (RO Purified water by ________________: pumps water across a can replace type I and II 2. Activated carbon removes organic matter and chlorine water) semipermeable membrane water 3. A submicron filter removes all particles or microorganisms larger than Other Purification Methods Ultrafiltration; UV light; Sterilization; Ozone treatment the membrane pore size. 4. Reverse osmosis is process that removes 89-95% of bacteria and Reagent Grade Water Laboratory required water QC and Impurity Testing of Water organic and other particulate matter. Categories of reagent grade 1. Clinical laboratory reagent water 1. Microbial Monitoring 5. Ion exchange is a system of resin cartridges or tanks connected in series water 2. Special regent water 2. Resistivity that remove cations and anions to make deionized water. 3. Instrument feed water 3. pH 4. Water supplied by methods 4. Pyrogens manufacturer 5. Silica 5. Autoclave and wash water 6. Organic contaminants 6. Commercially bottled purified water Basics of Clinical Chemistry Basics of Clinical Chemistry Reagents Reagents Water Specification CLSI-CLRW Guidelines Special Reagent Water May require different preparation than CLRW according to intended use, Type I Most stringent requirement and generally suitable for routine laboratory such as sterility specification for tissue or organ culture, nucleic acid most pure use content for DNA testing, metal content for trace metal analysis, etc. requires maximum purity Required for critical lab applications (HPLC), mobile phase preparation, used for preparation of blanks and sample dilution for analytic techniques Instrument Feed Water Used for internal instrument rinsing, making dilutions, etc., and need to standard solutions, buffers preparation of buffers and media for mammalian cell culture, and IVF, meet manufacturer’s specifications. and controls reagents for molecular biology applications, preparation of solutions for for quantitative analysis electrophoresis and blotting Water Supplied by Method Label states intended use: Do not substitute for CLRW or SRW unless Manufacturer label indicates it is of such quality. Type II used in buffers, pH solutions and microbiological culture media preparation Autoclave and Wash Water Purified to contain only low levels of organics, inorganics and particulate preparation of reagents for chemical analysis (TYPE III) matter so it does not leave residue on glassware or contaminate for clinical analyzers cell culture incubators solutions and media in autoclaves. feed water to type I systems for qualitative analysis in chemistry, hematology and microbiology Commercially Bottled Purified Exercise care because some plastic containers permit microorganism Type III glassware rinsing Water growth due to air permeability. filling autoclaves, heating baths and humidifiers feed water to type I systems Basics of Clinical Chemistry Basics of Clinical Chemistry Solution Properties Solution Properties CONCENTRATION Percent Solution Equal parts per hundred or the amount of solute per 100 total units w/w, v/v, w/v (most common) Solute substance that is dissolved in a liquid number of moles per 1L of solution; M = moles/Liter 1 moles of solute per 1kg of solvent; molality = moles/kg solvent number of gram equivalent weights per 1L of solution; N = gEw/L Solvent liquid in which the solute is dissolved SOLUTION SATURATION Dilute relatively little solute Concentrated large quantity of solute in solution Solution biologic solute + biologic fluid Saturated excess undissolved solute particles Supersaturated greater concentration of undissolved solute COLLIGATIVE PROPERTIES Basic properties of Concentration, saturation, colligative properties, Vapor Pressure pressure at which the liquid solvent is in equilibrium with the water vapor solution redox potential, conductivity, density, pH and Freezing point temperature at which the vapor pressure of the solid and liquid phases are the same ionic strength most commonly used and preferred and uses wheatstone bridge Boiling point temperature at which vapor pressure of the solvent reaches one atmosphere Osmotic Pressure pressure that opposes osmosis when a solvent flows through a semipermeable membrane to establish equilibrium between compartments of differing concentration Basics of Clinical Chemistry Basics of Clinical Chemistry Clinical Laboratory Supplies Clinical Laboratory Supplies Basics of Clinical Chemistry Basics of Clinical Chemistry Clinical Laboratory Supplies Clinical Laboratory Supplies Basics of Clinical Chemistry Basics of Clinical Chemistry Clinical Laboratory Supplies Pipettes Cylindrical glass tube used in measuring fluids, calibrated to deliver, or transfer, a specified volume from one vessel to another Most basic pipette: glass Routinely used: automatic Classification of Pipette According to design To contain (TC) To deliver (TD) Do not deliver the same volume Delivers or dispenses stated when the liquid is transferred into volume (exact amount) a container Designed to drain by gravity, referred to as rinse-out pipettes should not be blown out Must be completely transferred for Designed to meet requirements of accurate measurement class A type Basics of Clinical Chemistry Basics of Clinical Chemistry Pipettes Pipettes According to drainage characteristics TRANSFER PIPETTES BLOW OUT SELF-DRAINING Dispenses one volume without further subdivisions has a continuous etched ring No etched ring or markings last drop of liquid should be expelled by blowing Contents drain by gravity Volumetric pipette bulb closer to the center Self-draining pipette out Ex: ________________, ________________, single calibration mark Highest accuracy and precision Ex:______________, ________________ ________________ for non-viscous or aqueous Should only be used once prior to solutions cleaning According to type used when diluting standards, TRANSFER GRADUATED/MEASURING calibrators, or QC materials Dispense one volume without further subdivisions Dispenses several different volumes Ex: Volumetric, Ostwald-Folin, Pasteur, Automatic Ex: Serologic, Mohr, Bacteriologic, Ball, Kolmer, or Ostwald-Folin With bulb closer to the delivery tip for viscous fluids macro or micro pipettes Kahn, Micropipet blow out pipette Note: Blowout = OS Pasteur No calibration marks Used to transfer solutions without Self-draining = MVP Not for quantitative analytic consideration for specific volume Graduated = Serologic, Mohr, Micropipette, Bacteriologic, Ball, Kolmer, or Khan techniques w/ Bulb: Ostwald-Folin, Volumetric Basics of Clinical Chemistry Basics of Clinical Chemistry Pipettes Pipettes MEASURING PIPETTES/GRADUATED AUTOMATIC PIPETTES capable of dispensing several different volumes most routinely used in clinical chemistry laboratory Serologic with graduation marks down to tip Air displacement relies on a piston for creating suction to draw the sample into a disposable not accurate enough for measuring samples or standards tip (tips are only used once) can be used for serial dilutions and measuring reagents piston does not come in contact with the liquid blow out pipette Mohr no graduation marks to tip Positive displacement tips are reusable self-draining pipette piston is moved in the pipette tip or barrel much like a hypodermic syringe tip should be allowed to touch the vessel while pipette is draining Micropipette For volumes ranging from 1 to 1000 uL Dispensors and dilutor Obtain the liquid from a common reservoir and dispense it repeatedly Single calibration mark Dilutor often combines sampling and dispensing function Filled by capillary action must be rinsed out with diluent to deliver exact amount Basics of Clinical Chemistry Basics of Clinical Chemistry Pipettes Basic Separation Techniques Calibration of Pipettes A. Centrifugation: process in which centrifugal force is used to separated solid matter from a liquid suspension Gravimetric Most accurate method; delivering and weighing a solution of known Centrifugal force depends on three variables; mass, speed, and radius specific gravity, such as water Speed of centrifuge is checked using a tachometer or strobe light. This This method verifies the amount of liquid dispense by a pipette should be done ________________. The weight of water is proportional to the volume of water pipetted All equipment and water must be at room temperature before beginning Disinfected: ________________ Filtration: can utilize filter papers to separate solids form liquids Liquid that passes through the filter paper is called filtrate Spectrophotometric Secondary method B. Dialysis: another method for separating macromolecules from a solvent or Absorbance of potassium dichromate and p-nitrophenol delivered: this small substances method uses a dye of known concentration and water. A specific amount of A solution is out into a bag or is contained on one side of a semipermeable dye is pipetted into a specific volume of water. membrane Large molecules are retained within the sack or on one side of the membrane, while smaller molecules and solvents diffuse out. Basics of Clinical Chemistry Basics of Clinical Chemistry Basic Separation Techniques Point of Care Testing TYPES OF CENTRIFUGE Horizontal-head or cups holding the tubes of material to be centrifuged occupy a vertical position Defined as those analytical patient-testing activities provided within the ________________ when the centrifuge is at rest but assume a horizontal position when the centrifuge institution, but performed outside the physical facilities of the clinical revolves laboratories ________________ or cups are held in a rigid position at a fixed angle ( ____ to ____ degrees) Usually performed by ________________ Angle Head this position makes the process of centrifuging more rapid than the horizontal Rapid Centrifuge centrifuge Also known as: near patient testing, extra-laboratory analyses, ancillary testing, there is also less chance that the sediment will be disturbed when the centrifuge bedside testing, physician’s office testing, and alternative site testing stops POCT bring the laboratory to the patients used when rapid centrifugation of solutions containing small particles is needed Ex: microhematocrit centrifuge Major advantage: faster delivery of results Ultracentrifuge high-speed centrifuge used to separate layers of different specific gravities Smaller volume of sample allows POCT use in neonatal and pediatric x 10000 rpm commonly used to separate ________________ population and is ideal for those patients requiring frequent testing. usually refrigerated to counter heat produced through friction POCT is more expensive than the cost of central laboratory testing as a result of Cytocentrifuge uses a very high torque and low inertia motor to spread monolayers of cells rapidly the higher disposable and reagent costs of the POCT analyzers. However, across a special slide for critical morphologic studies uses blood, urine, body fluid, or any other liquid specimen that can be spread on a overall costs of patient care is arguably smaller due to improved patient work- slide flows. Basics of Clinical Chemistry Basics of Clinical Chemistry Automation Automation Automation Parameters/Terminologies Automation Parameters/Terminologies Able to perform individual tests or panels, and allow for STAT samples to be Amount of time to generate on result added to the run ahead of other specimens, any test on any sample in any Dead volume Amount of serum that cannot be aspirated sequence Carry over The contamination of a sample by a previously aspirated sample All samples are loaded at the same time and a single test is performed on each sample. Reflex testing Use of preliminary test results to determine if additional tests should be multi-sample, single test ordered or cancelled on a particular specimen; performed automated or manually Parallel Testing More than one test is analyzed concurrently on a given specimen single sample, multi-test Dry reagent can be packaged as lyophilized or tablet form that must be reconstituted with a buffer or reagent grade water and manually fed into the machine Sequential Analysis Multiple tests analyzed one after another on a given specimen can be spread over a support material and assembled into a single-use slide Open reagent system system wherein reagents form other manufacturers can be used Liquid regent directly pipetted with the instrument and mixed with the sample Closed reagent system Only the manufacturer’s reagent may be used Reaction temp 37C and 30C Throughput Maximum number of tests generated per hour Kinetic Determination of sample concentration is based on change in absorbance over time End point Incubated for a specific time, absorbance determined, related to calibrators for calculation of sample concentration Basics of Clinical Chemistry Laboratory Safety Automation THREE BASIC APPROACHES Safety Equipment The employer is required by law to have designated safety equipment available, but it is also the CONTINUOS FLOW CENTRIFUGAL DISCRETE responsibility if the employee to comply with all safety rules and use to use safety equipment. All laboratories are required to have safety showers, eyewash stations, and fire extinguishers and to periodically inspect the equipment for proper operation reagents, diluents and samples uses acceleration and each sample is It is recommended that safety showers deliver 30 to 50 gallons of water per minute at 20 to 50 pounds per are pumped sequentially deceleration of a centrifuge compartmentalized; multiple square inch and be located in areas where corrosive liquids are stored or used though a system of continuous rotor to transfer the reagents tests, one sample at a time Eye wash stations must be accessible (within 100 ft. or 10s travel) in laboratory areas presenting chemical or tubing and sample from one chamber handled separately in its own biological exposure hazards to another vessels Fire blankets, spill kits first-aid supplies Biosafety Cabinets remove particles that may be harmful to the employee who is working with potentially infectious biologic Parallel testing – multi-test, one Batch analysis – multi-sample, Random access capability specimens sample single test Batch analysis BSCs are designed to offer various levels of protection, depending on the biosafety level of the specific Significant carry-over effect and laboratory wasteful use of reagent CDC and the NIH have described four levels of biosafety Laboratory Safety Laboratory Safety Comparison of Biosafety Cabinet Characteristics Fume Hoods and PPE Chemical Fume Hoods Fume hoods are required to contain and expel noxious and hazardous chemical reagents The hood should never be operated with the sash fully opened, and a maximum operating sash height should be established and conspicuously marked Periodically evaluate ventilation by measuring face velocity with a calibrated velocity meter Velocity at the face of the hood should be 100 to 120 feet per minute and fairly uniform across the entire opening Personal Protective Equipment safety glasses rubberized sleeves goggles laboratory coats visors proper footwear work shields respirators (with HEPA) gloves (polyvinyl for latex allergies) hand washing Proper Donning and Doffing Donning _____________ > _____________ > _____________ > _____________ Doffing _____________ > _____________ > _____________ > _____________ > Handwash  Laboratory Safety Laboratory Safety Biologic Safety: Biologic Safety: All blood sample and other body fluids should be collected, transported, and handled, and processed using standard precaution. Finely dispersed aerosols from centrifugation of biologic specimens are high-risk source of infection. Bloodborne Pathogens Ideally, specimens should remain capped during centrifugation, or several minutes should be allowed ____________________ – to minimize bloodborne pathogen exposure. The plan must be available to all to elapse after centrifugation is complete before opening the lid. employees whose duties may result in reasonably anticipated occupational exposure to blood or other As a preferred option, the use of sealed-cup centrifuge is recommended. potentially infectious materials Airborne Pathogens SPILLS A TB exposure control program must be established, and risks to laboratory workers must be assessed. 1. Alert others in area of the spill The CDC guidelines require the government of a tuberculosis infection control program by any facility 2. Wear appropriate protective equipment involved in the diagnosis or treatment of cases of confirmed infectious TB. 3. Use mechanical devices to pick up broken glass or other sharp objects TB isolation areas with specific ventilation controls must be established in health-care facilities. 4. Absorb the spill with paper towel, gauze pads or tissue Those workers on high-risk areas may be required to wear respirator for protection 5. Clean the spill site using a common aqueous detergent All health-care workers considered to be at risk must be screened for TB infection. 6. Clean the spill site using approved disinfectant or 10% bleach, using appropriate contact time 7. Rinse the spill site with water 8. Dispose of all material in appropriate biohazard containers Laboratory Safety Laboratory Safety Fire Safety: Fire Safety: CLASS OF COMBUSTIBLE TYPE OF COMMENTS Notes: FIRE MATERIAL FIRE EXTIGUISHER MULTIPURPOSE DRY CHEMICAL FIRE EXTINGUISHER: CLASS ABC A Ordinary combustible; wood, Pressurized water, Dry chemicals CLASS ABC CO2 FIRE EXTINGUISHER: CLASS BC paper, fabric/cloth, plastic and leaded stream dry chemicals Halogenated hydrocarbon extinguishers: recommended for use with computer equipment Inspect fire extinguishers __________ to ensure they are mounted, visible, accessible, and charged. B Flammable liquid and gases; Dry chemicals Do not use water to extinguish petroleum products, gasoline, CO2 thinners, greases Halon RACE PASS C Live electrical equipment; Dry chemicals Never use water. Dry chemicals may computers, analyzers, CO2 damage electrical equipment RESCUE – anyone in immediate PULL the pin electrical wirings Halon CO2 leaves no residue and is a good danger AIM nozzle at the base of the fire choice for computers and analyzers ALARM – activate the institutional fire SQUEEZE the trigger D Combustible metals; Mg, Na, Sand or Ceramic barrier Leave to professional firefighters; only alarm system SWEEP the nozzle side to side K, Al Metal X try to isolate burning metal from CONTAIN – close all doors to combustible surfaces with sand and or potentially affected areas ceramic barrier EXTINGUISH/EXIT – if possible, E Arsenal None Allowed to burn out and nearby attempt to extinguish the fire; exit the materials are protected area K Cooking media; oil, Liquid designed to prevent vegetable, animal oil, fats splashing and cool the fire. Laboratory Safety Laboratory Safety NFPA Hazard Diamond: Chemical Safety: Developed a standard hazard identification system. Hazard Color 0 1 2 3 4 SDS/MSDS – Safety Data Sheet Health Blue No hazard can cause can cause temporary can cause can be lethal a major source of safety information for employees who may use hazardous materials in their work significant incapacitation or serious or employers are responsible for obtaining the SDS from the manufacturer irritation residual injury permanent injury Chemical Hygiene Plan Flammability Red Will not burn must be must be heated or in can be ignited will vaporize and preheated for high ambient temp to under almost all burn at normal A plan that provides procedures and work practices for regulating and reducing exposure of lab personnel ignition to occur burn ambient temps temp to hazardous chemicals Reactivity Yellow Stable high temp makes violent chemical may explode may explode at Chemical Hygiene Officer unstable change at high temp or from high temp normal temp pressures or shock and pressure must be designated for any lab using hazardous chemicals Special White OXY – oxidizer; ACID – acid; ALK – alkaline; COR – corrosive; W – use no water; - radioactive Storage and Handling Chemicals Notes: Develop respect for all chemicals and have complete knowledge of their properties Store according to their chemical properties; arrangement depends on quantities of chemical needed and 0 no hazard the nature or type of chemicals. It must not be based solely on alphabetical order 1 slight hazard The storeroom should be organized so that each class of chemicals is isolated in an area that is not used for 2 moderate hazard routine work. 3 serious hazard (extreme danger) 4 extreme hazard (death) Laboratory Safety Laboratory Safety Chemical Safety: Chemical Safety: STORAGE REQUIREMENTS Flammable/ among the most hazardous materials in the clinical chemistry laboratory SUBTANCE STORED SEPARATELY Combustible classified according to flash point; the temp at which sufficient vapor is given off to form an Chemicals ignitable mixture with air Flammable liquids Flammable solids Flammable liquid – has a flash point below 37.8C (100F) Mineral acids Organic acids Combustible liquids – has a flash point at or above 37.8C (100F) Caustics Oxidizers Corrosive injurious to the skin or eyes by direct contact or to the tissues of the respiratory and Perchloric acid Water-reactive substances Chemicals gastrointestinal tract if inhaled or ingested external exposure to concentrated corrosives can cause severe burns and require Air-reactive substances Others immediate flushing with copious amount of clean water Heat reactive substances requiring refrigeration Reactive substances that under certain conditions can spontaneously explode or ignite or that Unstable substances (shock sensitive explosives) Chemicals evolve heat or flammable or explosive gases Carcinogenic substances that have been determined to be cancer-causing agents Note: Chemicals If chemical spill occurs: 1. assist/evaluate personnel 2. confinement and clean up of the spill Laboratory Safety Specimen Collection and Handling Waste Disposal: Blood collection equipment: Nonsterile, disposable latex, nitrile, neoprene, polyethylene, and vinyl examination gloves are acceptable for phlebotomy procedures BLACK non - infectious DRY waste Antiseptics Tourniquet GREEN non – infectious WET waste 70% Alcohol most commonly used antiseptic for routine purpose restrict venous flow blood collection YELLOW infectious and pathological time should not be longer than _______________ Benzalkonium used for skin cleansing for ethanol testing YELLOW W/BLACK BAND chemical waste or with heavy metals Chloride (for blood alcohol testing) prolonged Hemoconcentration: increased enzymes, (Zephiran) application proteins, protein-bound substances, ORANGE radioactive waste 1:750 potassium RED sharps 70% Alcohol + used for ____________ Note: incorrect application and fist exercise can Iodine “double antisepsis” result in erroneous test results use of tourniquet in lactate determination Chlorhexidine recommended by CLSI for blood culture in may result in falsely elevated results Gluconate infants, 2 months and older, and patients BP cuff as tourniquet: 40 to 60 mmHg with iodine sensitivity. Standard: ______ mmHg Obese: _______ mmHg __ to __ inches above the site Specimen Collection and Handling Specimen Collection and Handling Blood collection equipment: Blood collection equipment: Needles Most Common Anticoagulant Used Notes: higher gauge = smaller bore (vice versa) EDTA for hematology cell counts and cell morphology STANDARD GAUGE: ______ MOA: chelates calcium LENGTH: 1 and 1.5 inches long Lavender-top: K2-EDTA;plastic, spray-dried, recommended by ICSH, K3-EDTA; liquid form in BEVEL UP during extraction glass ETS: most common and efficient system preferred by the CLSI for collecting blood samples Pink tubes used in immunohematology for ABO grouping, Rh typing, antibody screening White top tubes contain EDTA and gel for molecular diagnostics Parts: Multi-sample needles; has 2 pointed parts Tube holder Sodium Light Blue Black top Evacuated tubes Citrate for coagulation testing as it preserve labile factors (5 for Westergren ESR and 8) Blood to AC ratio = 4:1 Factors: Shelf life is defined by the stability of the additive and vacuum retention most evacuated tubes have at least 12-month shelf-life Blood to AC ratio = 9:1 Ambient temp low temp = increase blood volume draw (vice versa) MOA: chelates calcium Heparin MOA: accelerates the action of antithrombin III, neutralizing thrombin and prevents formation Altitude high altitude = low blood volume draw of fibrin Humidity different humidity conditions can affect only plastic evacuated tubes due to greater permeability Green Top Tube to water vapor relative to glass Lithium Heparin Sodium Heparin Light citrate theophylline adenosine dipyridamole (CTAD) is a special additive mixture for coagulation used for most chemistry test except not used for sodium assays, but testing that is sensitive to light and found only in glass evacuated tubes lithium and folate recommended for trace elements, leads and toxicology (injectables) Specimen Collection and Handling Specimen Collection and Handling Blood collection equipment: General Method of Blood Collection: Arterial Puncture Skin Puncture Venipuncture arterial blood for pediatric px (for routine assays venous blood obtained in px’s artery requiring small amounts of blood) obtained in px’s vein oxygenated In neonate: __________ deoxygenated Most Common Anticoagulant Used bright red In older children: __________ dark red color for blood gas analysis and pH useful also in adults with extreme sites: antecubital fossa, veins on wrist Sodium Gray top: used for glucose measurement because of its antiglycolytic properties preventing measurement obesity, severe burns, and thrombotic and dorsal aspect of the hands, veins Fluoride glycolysis for ______ days preferred AC: ______________ tendencies, with POCT or with on the ankle (needs consent) In bacterial septicemia, fluoride inhibition of glycolysis is neither adequate nor effective in Most preferred site: _______ > brachial patients performing test at home best site: __________ preserving glucose > femoral > scalp > umbilical Often preferred in geriatric px, second choice: cephalic the anticoagulant is potassium oxalate or Na2EDTA Best site for ABG: indwelling umbilical because their skin is thinner and less third choice: basilic artery elastic Clot Red top: used for most chemistry, blood bank, and immunology assay Angle: ____ to ____; 90 if femoral for NBS tubes Red/gray and gold-top: contains clot activator and separation gel: most often used for ___________________ chemistry test done before blood is collected thixotropic gel can absorb certain drugs causing false lowered results from radial artery to determine Ex: Phenytoin, phenobarbital, lidocaine, quinidine and carbamazepine whether the ulnar artery can Tubes containing gels are not used for BB or IS provide collateral circulation Specimen Collection and Handling Specimen Collection and Handling General Method of Blood Collection: Order of Draw Sample Variations ETS and Syringe Skin Puncture 1. Blood culture 1. Capillary blood gas 2. Coagulation tubes 2. Slide/Blood smear 3. Serum tubes with clot 3. EDTA tubes activator or gel separator 4. Other anticoagulated tubes 4. Heparin tubes 5. Serum tubes 5. EDTA tubes 6. Glycolytic inhibitor Topics of Discussion Topics of Discussion Analytical Methods: Analytical Methods: SPECTROPHOTOMETRY Principle: substance absorbs light at unique wavelength; based on __________, where the amount of light is BLANK CORRECTION/BLANKING: proportional to the amount of substance that is present ensures the accuracy of the test results by eliminating interfering substances inherent with the sample or PARTS OF A SPECTROPHOTOMETER reagent. provides polychromatic light  Reagent Blank corrects for the absorbance interference caused by the color of the reagent mixture of any reagents that would be presented to the detector for analysis of a minimizes unwanted or stray light and prevents scattered light test sample and isolates specific or individual wavelength of light is analyzed to determine if it contributes to the measurement signal controls the width of light beam (bandpass) used to zero the instrument BEFORE measuring test samples and other blanks holds the solution whose concentration is to be measured  Sample Blank used to correct non-specific absorbance of substances in the sample refers to using the sample for zeroing an instrument DURING a test procedure detects and converts transmitted light into photoelectric energy  Solvent Blank up from the solvents contained in the solution presented to the instrument displays output of the detection system may be used during validation to assess any interferences which may be present Wavelength Range Common light source Cuvette in the solvent < 400 near ultraviolet deuterium or mercury arc Quartz (silica) 400 – 700 visible light incandescent tungsten or tungsten iodide Borosilicate > 700 near infrared incandescent tungsten or tungsten iodide Quartz (silica) Topics of Discussion Topics of Discussion Other Methods: Quality Assurance and Quality Control:  FEP measures light emitted by ___________________ such as Sodium, Potassium and Lithium QUALITY ASSURANCE TERMINOLOGIES  AAS measures light absorbed by ___________________; useful for analytes not easily excited by flame Quality Assessment A program that monitors the total testing process with the aim of such as Magnesium and Calcium providing the highest quality patient care; formerly: Quality Assurance  Fluorometry atoms absorb light at specific wavelength and emit light at longer wavelength (lower energy); for drugs and hormones; uses 2 monochromators; susceptible to quenching Quality control A system that verifies the reliability of analytical test results through the use of standards, controls and statistical analysis  Chemiluminescence involves a chemical reaction to produce light via oxidation of luminol, acridinium esters or dioxetanes; usually for immunoassays Quality System In an institution, a comprehensive program in which all areas of  measures the amount of light blocked (reduction in light) by the particles in suspension; for operation are monitored to ensure quality with the aim of providing the proteins in urine and CSF highest quality patient care  measures light at an angle from the light source (light scattering); used for antigen-antibody LEAN SYSTEM reaction TEAM ROLES BELT COLORS DEDICATE % OF TIME  Potentiometry measurement of potential (voltage) between two electrodes in a solution Project coaches/ leaders Black Belt 100  Coulometry measures quantity of electricity (in coulombs) needed to convert an analyte to a different oxidation state Project Team members Green Belt & Yellow Belt Green: 20 Yellow: full day  Voltammetry method in which potential is applied to an electrochemical cell and the resulting current is Project Sponsors Blue Belt measured  Amperometry measurement of __________________ produced by an oxidation-reduction reaction Topics of Discussion Topics of Discussion Quality Assurance and Quality Control: Quality Assurance and Quality Control: PHASES OF TESTING /TOTAL TESTING PROCESS PARAMETERS OF QUALITY CONTROL Pre-analytical Analytical Post-analytical Parameter Description Factors that affect the laboratory Verification of instrument linearity, Timely and accurate laboratory results Ability of an analytical method to measure the smallest concentration of the analyte of results due to handling of the precision, accuracy and overall reporting and other aspects that occur interest specimen sample prior to analysis reliability through the use of standard after the analysis phase 32% to 75% of all testing errors occur materials, QC samples, procedures, Delta check Ability of an analytical method to measure only the analyte of interest in the pre-analytic phase and QC rules The nearness or closeness of the assayed value to the true or target value QUALITY MANAGEMENT ERRORS Estimated using 3 types of studies: recovery, interference and patient sample comparison Pre-analytical Analytical Post-analytical Ability of an analytical method to give repeated results on the same sample that agree 1. Patient misidentification 1. Sample misidentification 1. Patient misidentification with one another 2. Wrong test ordered 2. Erroneous instrument calibration 2. Poor handwriting 3. Incorrect urine specimen type 3. Reagent deterioration 3. Transcription error Ability of an analytical method to maintain accuracy and precision over an extended collected 4. Poor testing technique 4. Poor quality of printer period of time during which equipment, reagent and personnel may change 4. Insufficient urine volume (QNS) 5. Instrument malfunction 5. Failure to send report Degree by which a method is easily repeated 5. Delayed transport of urine to lab 6. Interfering substances present 6. Failure to call critical values 6. Incorrect preservation 7. Misinterpretation of QC Data 7. Failure to identify interfering Ability of analytical method to detect the proportion of individuals with the disease 7. Specimen collection technique 8. Laboratory Staff competence substance 8. Monitoring of specimen condition 8. Turnaround time, cost analysis Ability of the analytical method to detect the proportion of individuals without the disease Topics of Discussion Topics of Discussion Quality Assurance and Quality Control: Quality Assurance and Quality Control: RANDOM ERROR VS SYSTEMATIC ERROR QUALITY CONTROL CHART RANDOM ERROR SYSTEMATIC ERROR Cumulative Sum Graph Youdin/Twin Plot Levey Jennings  Detects: Imprecision  Detects: Inaccuracy earliest detection of compare results obtained on a Most widely used chart that  Parameters: SD and CV  Parameters: Mean systematic errors; needs to high and low control serum allows laboratorians to apply  Remedy: Re-assay using same reagent  Remedy: Stepwise Evaluation  Test used for determination: Replication Experiment  Test used for determination: Sample Comparison be computerized from different laboratories multirules without the use of Method aka CUSUM Graph computers Preferred QC Graph 1. Pipetting error/ reagent dispensing 1. Improper calibration 2. Sample evaporation 2. Deterioration of reagents & control materials Test value: 20 runs 3. Mislabeling of samples 3. Improperly made standard solutions Needs: Mean and SD 4. Improper mixing of sample & reagent 4. Contaminated solutions 5. Temperature fluctuation 5. Wear and tear of instruments 6. Voltage fluctuations not compensated for by the 6. Sample instability instrument circuitry 7. Unstable and inadequate reagent blanks 7. Electro-optical mechanism 8. Leaky ion selective electrode (ISE) 8. Environmental conditions 9. Failing instrumentation/ Instrument drift & 9. Variation in handling techniques: pipetting, mixing, malfunction timing, 10. Poorly written procedures 10. Variation in operators Topics of Discussion Topics of Discussion Quality Assurance and Quality Control: Quality Assurance and Quality Control: WESTGARD RULES WESTGARD RULES Rule Illustration Explanation Type of Error Comments Rule Illustration Explanation Type of Error Comments 12S 1 control above or below 2 SD Random Warning rule initiates testing 13S 1 control >±3 SD from mean Random Rejection rule from mean. of other rules. If Warning flag of no violation of possible change other rules, run in accuracy or is considered in precision. control Topics of Discussion Topics of Discussion Quality Assurance and Quality Control: Quality Assurance and Quality Control: WESTGARD RULES WESTGARD RULES Rule Illustration Explanation Type of Error Comments Rule Illustration Explanation Type of Error Comments 22S 2 consecutive controls >±2 Systematic Rejection rule R4S 2 consecutive controls differ Random Rejection rule SD by >4 SD from mean on same side Topics of Discussion Topics of Discussion Quality Assurance and Quality Control: Quality Assurance and Quality Control: WESTGARD RULES WESTGARD RULES Rule Illustration Explanation Type of Error Comments Rule Illustration Explanation Type of Error Comments 41S 4 consecutive controls >1 SD Systematic Rejection rule 10x 10 consecutive controls on Systematic Rejection rule from mean on same side of same side mean Topics of Discussion Topics of Discussion Quality Assurance and Quality Control: Quality Assurance and Quality Control: SUMMARY OF WESTGARD CONTROL RULES DIAGNOSTIC EFFICIENCY RANDOM 12S 1 control value exceeds the mean ±2SD PARAMETERS OF DIAGNOSTIC EFFICIENCY 13S 1 control value exceeds the mean ±3SD Diagnostic Sensitivity Proportion with the disease who have a positive test result R4S 1 control value exceeds +2SD and another exceeds -2SD Diagnostic Specificity Proportion without the disease who have a negative test result SYSTEMATIC 22s 2 consecutive control values exceed the same mean +2SD or -2SD Positive Predictive value is the probability that people with a positive test result indeed do have the condition of interest 41s 4 consecutive control values exceed ±1SD Negative Predictive value is the probability that people with a negative screening test result indeed do not 10x 10 consecutive control values fall on 1 side or other side of the mean have the condition of interest. Abrupt change in the mean Has the Condition Does not have the Diagnostic sensitivity : ______ % 6 or more consecutive control values distribute themselves on one side or either condition Diagnostic specificity : ______ % side of the mean but maintain a constant level Positive 85 12 PPV : ______ % Main cause: Improper calibration of instrument Negative 5 76 NPV : ______ % TREND/______ 6 consecutive control values gradually increase or decrease REFERENCE INTERVAL STUDIES Main cause: Deterioration of reagents Establishing a reference interval 120 healthy individuals OUTLIER Control values that are far from the main set of values Verifying a reference interval (Transference) 20 healthy individuals Topics of Discussion Topics of Discussion Quality Assurance and Quality Control: Quality Assurance and Quality Control: MEASURE OF CENTRAL TENDENCY QUALITY CONTROL MEAN average INTERNAL (INTRALABORATORY) EXTERNAL (INTERLABORATORY) National Reference Laboratories: MEDIAN midpoint of distribution; the middle of the data after the data have been rank ordered; often used with skewed data Quality Control Quality Control CC: _________ MODE seldom used; refers to the most frequent observation performed by the lab personnel Proficiency testing programs Hema: _________ used to describe data that seem to have two centers (bimodal) using control materials of that periodically provide Infectious: _________ MEASURE OF DISPERSION