(CC-Prelims) Lesson 1: Basic Principles of Clinical Chemistry

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mole (mol)unit of quantity of substanceAmpere (A)unit of electric currentKelvin (K)SI unit for temperatureCandela (cd)SI unit for luminous intensitymoles of solute/volume of solution (mmol/L)It has been recommended that analytes be reported usingUnit per liter (U/L)Enzyme units are given as the international ________________ although katal unit has been adopted previously as SI unit, its use is limitedKataladopted oreviuous as enzyme's SI but its use is limitedpH scaleis retained for measurement of hydrogen ion concentrationsLiteris used as the reference volumeSI unitis used because compounds react on a molar basis, and expressions of amounts of substances in such terms allow for a better understanding of the relative proportion of compounds.conventional unitsMass concentration units (e.g. milligrams per deciliter); most labs in U.S. use conventional units; some medical journals use SI or bothImpuritiesmust be stated in the label to know if they will interfere with the specimen analysisPlasmathe fluid portion of unclotted bloodSerumfluid portion of clotted bloodAnticoagulanta drug that prevents clotting of the bloodReagentsMost instrument manufacturers make the reagents in a ready-to-use form or "kit" where all necessary reagents and respective storage containers are prepackaged as a unit requiring only the addition of water or buffer to the prepackaged components for reconstitution.Analytical Reagents Ultrapure Reagents Chemically Pure Reagents United States Pharmacopeia and National Formulary Technical/Commercial Grade6 Types of Chemicals/ Grades of Purity of Analytic Chemicals (AUCUNT)Analytical ReagentsA reagent that is suitable for most clinical laboratories; if this is contaminated the actual value cannot be determined; impurities must be stated to know if they can interfereUltrapure ReagentsChemicals that have been put through additional purification steps; are used in chromatography, molecular diagnosis, cellular acetateUltrapure reagentsUsed in procedures such as chromatography, atomic absorption, immunoassays, molecular diagnostics and other techniques that may require extremely pure chemicalsRNA & DNA (RT PCR)Molecular Diagnosis of Ultrapure ReagentsChemically Pure Reagentsimpurity limitations are not stated; not recommended for clinical laboratory unless further purification or reagent blank is included; preparation of these chemicals is not uniformUSP and NF gradeLowest grade of purity for an ingredient in a compoundUSP and NF gradeUsed to manufacture drugs; criteria of not being harmful to humans; chemicals are pure enough to be used in most chemical procedures; Based on the criterion of not being injurious to individualsUnited States PharmacopeiaUSP meaningNational FormularyNF meaningUnited States Pharmacology National FormulasUSPNF abbreviationTechnical/Commercial GradeUsed primarily in manufacturing and should never be used in the clinical laboratory but it still can be used when there is added purificationStandarda solution of known concentrationStandardIt is used in calibration of instruments/methodStandardA substance or solution in which the concentration is determinedStandardIt is used in the calibration of an instrument of methodAtomic Weight Standard Ultimate Standard Primary Standard Working Standard Secondary StandardDifferent Type of Standards (AUPWS)Atomic WeightIt is pure to the last moleculeGrade A IUPACGrade of Atomic Weight StandardInternational Union of Pure and Applied ChemistryWhat does IUPAC stand for?Primary Standarda highly purified solid compound used to check the concentration of the known solution in a titration; low concentration; used in the clinical laboratory99.98%What percentage of purity is Primary Standard?Grade CWhat grade is Primary Standard in IUPAC?Working Standard<0.05% particles, 99.95% pureGrade DWhat grade is Working Standard in IUPAC?Secondary Standarda substance whose purity has been established and verified by chemical analysis; low qualityWateruniversal solventWatera frequently used reagent in the reagentFiltration, Distillation, Ion Exchange, Reverse Osmosis, Ultraviolet OxidationMethods of Purification (FDIRU)Filtrationpassage of fluid through a permeable membrane from the area of higher to lower pressure; removed particulate matter; also used as a pre-treatmentFiltrationPassage through a filter or other material that prevents passage of certain molecules, particles or substancesFiltrationCan remove particulate matter from municipal water supplies before any additional treatmentsFiltrationUsed as a pre-treatmentActivated Carbon, Submicron FilterDifferent Types of Filtration (AS)Activated Carbon(filtration) chloride and organic matterSubmicron Filter(filtration) filters lower than micron.> 0.2 filtered < 0.2 fits the filterActivated carbonRemoves chloride and organic materialSubmicron filter0.2 microns Removes all particles that are larger than its sizeOnly particles that are larger than the filter size are not allowed to pass through; therefore, smaller particles can still contaminate the filtrate.Limitations of FiltrationDistillationThe process of vaporizing and condensing a liquid to purify or separate a volatile substance from a non volatile substrateVolatiles, Sodium, potassium, magnesium, carbonates and sulfatesLimitations in Distillation: Carryover of volatile impurities and entrapped water droplets may contain impurities such as:Ion ExchangeA process that removes ions to produce mineral-free waterinsoluble resin polymersAccomplished by passing feed water through columns containing ______________________ that exchange H+ and OH- ions for the impurities present in ionized from in the waterIon ExchangeAccomplished by passing feed water through columns containing insoluble resin polymers that exchange H+ and OH- ions for the impurities present in ionized from in the waterOnly ions are removed, therefore it is neither pure nor sterileLimitations in Ion ExchangeReverse OsmosisA process in which water is forced through a semipermeable membrane that acts as a molecular filter95-99 %, 90-97%Removes ____________ of organic compounds, bacteria and other particulate matter and ________ of all ionized and dissolved mineralsDoes not efficiently remove gassesLimitations of Reverse Osmosis254 nmUltraviolet Oxidation Uses ultraviolet radiation at the biocidal wavelength of __________Ultraviolet OxidationEliminates many bacteria and cleaves many ionizing organics that are then removed by deionizationRequires further purification methodsLimitation of Ultraviolet OxidationDistilled Deionized Reagent GradeTypes of Water (DDR)Distilled WaterPurified to remove almost all organic materialsRememberDistillation does not remove volatile organic impuritiesRememberWater may be distilled more than once to remove impuritiesDeionizedProduced from distilled water using either an anion or a cation exchange resin followed by replacement of the removed particles with hydroxyl or hydrogen ions respectivelyDeionizedGenerally, it is purified from previously treated water such as pre-filtered or distilled waterType 1[Reagent Grade] - Used for the procedures that require maximum water purity - Preparation of standard solutions - Tissue or cell culture - Final rinsing of glassware - Measurement of nanogram or sub nanogram concentrationWater is passed through 0.2 micron filter and Activated carbonHow to make water?≤ 10 cfu/mLType 1 Microbial content≤ 0.05mg/LType 1 Silicate contentenzyme analysis immunoassays fluorescence quantitation HPLCUltramicro chemical analysis of Type 1 Reagent Grade (EIFH)Type IIUsed for most laboratory determinations≤ 10^3 cfu/mLType 2 Microbial content≤ 0.1 mg/LType 2 Silicate contentType 3- For most qualitative measurement - For urinalysis, parasitology and histology - For washing glassware not requiring Type I or II reagent grade water (RGW)≤1.0 mg/LType 3 silicate contentCO2 free water- Obtained by boiling Type II RGW - Used (can remove) if CO2, ammonia, or O2 may affect analysisCarry Overleft behind previous substanceBorosilicate, Alumina-silicate, Low Actinic Glassware, Corning Boron Free GlassLaboratory Equipment-Glassware (BLGC)BorosilicateMost common type encountered in volume measurementBorosilicate- General purpose, for test tube and disposable glassware - With high thermal resistance and low alkali content - Should not be heated above strain point because rapid cooling cracks the glass easily when heated again512 degree celsiusstraining point of pyrexpyrex, kimaxexamples of borosilicateAlumina-silicate- 6x stronger than borosilicate - Strengthened chemically rather than thermally - Resist clouding and scratching better - Only half of the thermal resistance of borosilicate glasscorexAlumina-silicate exampleGraduated cylinder Thermometers Test tube for high speed centrifugationAlumina-silicate is used for (GTT)Low Actinic Glassware- High thermal resistanceLow Actinic GlasswareWith red color to permit adequate visibility of contents yet give maximum protection from lightLow Actinic GlasswareUsed for photosensitive materials like bilirubin standardsCorning Boron Free Glass- Highly resistant to alkali - Soft, poor heat resistancePolyethylene, Polypropylene, Polycarbonate, Teflon, Polyolefins, Polyvinyl ChloridePLASTICWARE (PPPTPP)Polyethylene- For inexpensive, disposable tubesPolyethyleneUsed for storage of alkaline solutionsPolypropylene- Withstands higher temperature - Becomes discolored by solventsPolycarbonate- Stronger, more heat resistant - Lower chemical resistance - For centrifugal tubes and graduated cylinders - AutoclavableTeflon- Almost chemically inert, very heat resistant - For stopcocks, stirring bars, cap liners and tubings - AutoclavablePolyolefins- Strong and resistant to elevated temperature - AutoclavablePolyvinyl Chloride- Soft and flexible - Used to construct tubingsPolyethylene, Polypropylene, Polycarbonate, Teflon, PolyolefinsPlasticware that are autoclavableMechanical, ElectronicAnalytical Balance (ME)Double pan analytical balance Single pan analytical balance Top loading balanceMechanical Balance Examples (DST)Double pan analytical balanceTwo pans of equal mass suspended from ends of a beam supported at its center by a knife-edge fulcrumDouble pan analytical balanceUseful for weighing to the nearest 0.1 gram Not to be used for measurement of standardsSingle pan analytical balance- Substitution balance; weights are removed from the side of the beam holding the unknown mass until equilibrium is restored - Unequal arms; single pan, provides greater precision and accuracySingle pan analytical balanceUsed for measuring primary standards, small amount of chemicals and for gravimetric calibration of pipets0.01 mgDigital Readout of Single Pan Analytical BalanceTop loading balance- Used for weighing larger amounts of chemicals for buffers, saline solution, etc. - Faster and easier but not preciseElectronic Balance- Have built-in provisions for tarring, so that the weight of the the container can be subtracted - They have a single pan, top loading or analytical, fast, and can be computer interfacedelectronic force compensationOn what principle does electronic balance operates?Transfer pipet Measuring/Graduated pipet MicropipettesTypes of pipettes (TMM)PipetteUsed in the transfer of a volume of liquid from one container to anotherTransfer PipetteDelivers fixed volume (more accurate and precise) and consist of cylindrical bulbs joined at both ends to a narrower glass tubingTransfer PipetteA calibration mark is etched around the upper section of the tube; The lower delivery tube is drawn out to a gradual taper.PipetteVOLUMETRIC SAMPLING AND DISPENSINGVolumetric PipetUsed for non-viscous fluidsStandard, reagents, dilution liquids, Plasma, serum, urineVolumetric Pipet is used for non-viscous fluids (SRDPSU)Volumetric PipetDrains by gravity by placing tip vertically against the side of accepting vessel but not touching the liquidVolumetric PipetDistinguished by bulb-like enlargement partway up the stemVolumetric PipetPipet that has the highest degree of accuracy and precisionTD PipetteDrains freely with the pipette touching the inner surface of the receiving vessel - Calibrated for the volume delivered - Calibrated to blow outTD Pipette calibration for the volume deliveredDo not attempt to wash out the film that adhere to the inside of the glass surfaceTD Pipette calibration to blow outRemaining liquid after free delivery must be blown out and added to the initial volumeTD Pipette calibration to blow outIndicated by opaque ring near mouthpiece of the pipetTo DeliverTD PipetteTC PipetteTo ContainTC Pipet- It is calibrated for the total volume of liquid held in the pipetteTC PipetteIt must be washed out completely for the delivery of complete volume0.5 mLMicropipettes that ranges up to this range __________ are calibrated to containOstwald-Folin PipetteIt is similar to volumetric pipet but have their bulb closer to the delivery tipwhole bloodOstwal-Folin pipette is used for viscous fluids like __________To Deliver, Blow outEtched rings are indicators that the glassware is __________Measuring/GRaduated Pipette- Delivers variable volume - Long cylindrical tubes drawn out a tip and calibrated in uniform fractional volume measurementMeasuring/GRaduated PipettePrinicipally used for measurment of reagents and not considered sufficiently accurate for measuring samples and calibratorsSerologic Pipette Mohr PipetteTypes of Measuring/Graduated Pipette (SM)Serologic Pipette- It has graduated marks up to the tip - has etched rings - calibrated to blow out - has alrger orifice than Mohr PipetteMohr Pipette- Calibrated between two marsk of the stem - Graduations do not go all the way to the tapered tip - Accuracy is greatest at full volumeMohr PipetteRequires controlled delivery of the solution between calibration marksMicropipette- Pipettes used for measurement of microfilters of volume - Piston operated, positive displacement - Uses disposable tips and detipers100 to 1000 uL or 1mLBlue Micropipette Tip2 to 200 uLYellow Micropipette Tip0.5 to 10 uLWhite Micropipette TipRoutine Blood Clots New Pipettes Grease Permanganate Stain Bacteriologic Glassware Heavy Metal Analysis Bromsulhalein DyeQuality Control and Preventive MaintenanceRoutine1. Rinse immediately after using, wash with low alkali, non-ion detergent 2. Rinse with tap water, 3-5x with Type 1 water5.5-5.7Final rinse must have a pH ofsoak in 10% NaOHblood clots quality control and preventive maintenanceSoak in 5% HCl / 5% nitric acidnew pipettes quality control and preventive maintenancesoak in any organic solvent 50% KOG or Contrad 70Grease quality control and preventive maintenancesoak in 50% HCl/ 1% FeSO4 in 25% H2SO4Permanganate Stainviolet staincolor of permanganate stain2-4$ cresol solution autoclave, thorough washingbacteriologic glasswarerinse in dilute acid to remove metal ion containment Dichromate - H2SOR Cleaning solution 1M HCl/ HNO3Heavy metal analysisBromsulhalein DyeIt is used to check random pieces of residual alakalin detergentPinkcolor of Bromsulhalein dye which means incomplete removal of detergent6 monthsCalibration is done everyClass A PipetteThis pioette does not require calibrationGravimetric MethodPRINCIPLE: weight of water delivered by the pipet is equal to the volume of liquid deliveredGravimetric MethodPRECISION: determined by pipetting 10 aliquots of water, weighing the aliquots and calculating the mean and the SDGravimetric MethodACCURACY: determined by correcting the mean for temperature and comparing the observed value to the expected value20 to 26 C; should not deviate more than 5% of the stated valueMean for TempSPECTROPHOTOMETRIC METHODPRINCIPLE: colored compound of known absorptivity is diluted into 10 test tubes using the pipet being checked ▪ ABSORBANCE OF EACH TUBE: obtained using measurements from a spectrophotometerSPECTROPHOTOMETRIC METHODPRECISION: determined by calculating the standard deviation of the aliquots; must be less than 5% margin of error ▪ ACCURACY: determined by comparison to a reference solutionCENTRIFUGATIONUses centrifugal force to separate the lighter portions from heavier portions of a solutionHorizontal Head Centrifuge Angle Head Centrifuge UltracentrifugeTypes of CentrifugeCENTRIFUGATIONRemove cellular elements from biological fluids to provide cell-free specimen for analysis o To concentrate cellular elements for microscopic examinationRPMrevolutions/rotations per minute;RPMspeed of the centrifugeRCFrelative centrifugal force;RCFforce required to separate substances in a solutionRCF = (1.118 x 10-5 )(r)(rpm)2formula for RCFHORIZONTAL HEAD CENTRIFUGE:swinging bucket typeANGLE HEAD CENTRIFUGE:less prone to heat build up due to air friction than HHC25 TO 52fixed angle of angle head centriULTRACENTRIFUGEheld at a fixed angle but generates tight sediments due to high speeds generated; highest centrifugal speedsULTRACENTRIFUGEfor lipoproteinm deparation