clinical chemistry
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thanks to collage of medtech southwestern university

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clinical chemistry Presentation Transcript

  • 1. CLINICALCLINICAL CHEMISTRYCHEMISTRY Julius P. Mario, RMT, MS Chem.Julius P. Mario, RMT, MS Chem.
  • 2. The purpose & function of laboratoriansThe purpose & function of laboratorians through clinical pathology & laboratorythrough clinical pathology & laboratory medicine are to assist clinicians in:medicine are to assist clinicians in: 1.1. Confirming or rejecting a diagnosisConfirming or rejecting a diagnosis 2.2. Providing guidelines in patient managementProviding guidelines in patient management 3.3. Establishing a prognosisEstablishing a prognosis 4.4. Detecting disease through case finding orDetecting disease through case finding or screeningscreening 5.5. Monitoring follow-up therapyMonitoring follow-up therapy
  • 3. Clinical ChemistryClinical Chemistry TheThe systematic studysystematic study ofof biochemical processesbiochemical processes associatedassociated withwith health & diseasehealth & disease && thethe measurement of constituentsmeasurement of constituents inin body fluids or tissuesbody fluids or tissues toto facilitatefacilitate diagnosis of disease.diagnosis of disease.
  • 4. SCOPE OF CLINICAL CHEMISTRYSCOPE OF CLINICAL CHEMISTRY Biochemistry InstrumentationComputers Pharmacology Endocrinology Analytical ChemistryToxicologyImmunology CLINICAL CHEMISTRY
  • 5. It is much easier toIt is much easier to detect a MALFUNCTIONdetect a MALFUNCTION than to elucidate or document the cause.than to elucidate or document the cause. Malfunction of a cell maybe caused by a variety ofMalfunction of a cell maybe caused by a variety of factorsfactors 1.1. Destruction by trauma or by invasive agentsDestruction by trauma or by invasive agents 2.2. Genetic deficiency of a vital enzymeGenetic deficiency of a vital enzyme 3.3. Insufficient supply of one or more essential nutrientsInsufficient supply of one or more essential nutrients 4.4. Insufficient blood supplyInsufficient blood supply 5.5. Insufficient oxygen supplyInsufficient oxygen supply 6.6. MalignancyMalignancy 7.7. Accumulation of waste productsAccumulation of waste products 8.8. Failure of a control systemFailure of a control system 9.9. Defect in the cellular recognition of certain signalsDefect in the cellular recognition of certain signals
  • 6. Point-of-care TestingPoint-of-care Testing (POCT)(POCT)  Also known asAlso known as near-patient testingnear-patient testing,, alternate-sitealternate-site testingtesting oror patient-focused testingpatient-focused testing  Used in emergency dept., operating suites, clinics,Used in emergency dept., operating suites, clinics, health maintenance organization (HMO),health maintenance organization (HMO), physicians, offices & nursing homesphysicians, offices & nursing homes  AddressesAddresses acute patient needsacute patient needs  Instrumentation includesInstrumentation includes portable chemistryportable chemistry analyzers, glucometers, BG Analyzers,analyzers, glucometers, BG Analyzers, hemoglobin metershemoglobin meters && coagulation testingcoagulation testing
  • 7. Specimens or samples areSpecimens or samples are analyzedanalyzed whilewhile substances in them aresubstances in them are measuredmeasured oror quantitated.quantitated.
  • 8. Substances measured in serum fall generallySubstances measured in serum fall generally into the following categories:into the following categories: 1.1. SubstancesSubstances normally presentnormally present with a function in thewith a function in the circulationcirculation  electrolytes  TAG, cholesterol  hormones  vitamins  glucose  TP  albumin  individual proteins 2. Metabolites- nonfunctioning waste products in the process of being cleared  urea  creatinine  uric acid  ammonia  bilirubin
  • 9. 3.3. SubstancesSubstances released from cells as a result ofreleased from cells as a result of cell damage & abnormal permeability orcell damage & abnormal permeability or abnormal cellular proliferationabnormal cellular proliferation enzymes such as LD, ALT, AST, CK, AMS,enzymes such as LD, ALT, AST, CK, AMS, GGT, ALP & ACPGGT, ALP & ACP ferritinferritin 4. Drugs & toxic substances  antibodies  substances of abuse  therapeutic drugs  poisons
  • 10. Quick QuizQuick Quiz  Tissue injury is greatly associated with elevatedTissue injury is greatly associated with elevated levels of bloodlevels of blood a.a. electrolyteselectrolytes b.b. hormoneshormones c.c. enzymesenzymes d.d. albuminalbumin  Electrochemical methods are ideal for theElectrochemical methods are ideal for the measurement of serum/plasmameasurement of serum/plasma a.a. glucoseglucose b.b. enzymesenzymes c.c. proteins & amino acidsproteins & amino acids d.d. metal ionsmetal ions
  • 11. Laboratory TestsLaboratory Tests  Chemical testsChemical tests Direct or IndirectDirect or Indirect ColorimetricColorimetric RedoxRedox CondensationCondensation  Degradative testsDegradative tests ((EnzymaticEnzymatic)) One-step or Multi-stepOne-step or Multi-step NADH ConsumptionNADH Consumption  Measurement of ActivityMeasurement of Activity (not concentration)(not concentration)  Multi-methodMulti-method  TitrimetricTitrimetric  ElectrochemicalElectrochemical
  • 12. Quick QuizQuick Quiz  Identify theIdentify the analyte measuredanalyte measured using theusing the following tests and thefollowing tests and the typetype of each test:of each test: 1.1. Jaffe reactionJaffe reaction 2.2. Hantzsch testHantzsch test 3.3. GOD-PAPGOD-PAP 4.4. Clark-CollipClark-Collip 5.5. Schales and SchalesSchales and Schales 6.6. Evelyn-MalloyEvelyn-Malloy
  • 13. LAB REQUESTSLAB REQUESTS  ““STAT”STAT” = immediate; urgent requests;= immediate; urgent requests; emergencyemergency = usually less than 1 hour TAT= usually less than 1 hour TAT  PRIORITYPRIORITY = important for clinical decisions= important for clinical decisions = less than 2 hour TAT= less than 2 hour TAT  ROUTINEROUTINE = non urgent; standard or special= non urgent; standard or special teststests = generally 4-6 hours= generally 4-6 hours
  • 14.  BreakableBreakable  ReusableReusable  Ideal for acidic solutionsIdeal for acidic solutions  High thermal & corrosionHigh thermal & corrosion resistanceresistance  Good optical propertiesGood optical properties  Low costLow cost Glasswares Plasticwares Highly shock proof; shatterproof Disposable Ideal for alkaline solutions Relatively inert Flexible For cryogenic experiments
  • 15. GLASS: 5 GENERAL TYPESGLASS: 5 GENERAL TYPES 1.1. High thermal resistant glassHigh thermal resistant glass  Borosilicate glass with low alkali contentBorosilicate glass with low alkali content  Resists heat, corrosion and thermal shockResists heat, corrosion and thermal shock  Most common areMost common are PyrexPyrex,, KimaxKimax andand ExaxExax CorexCorex - 6x stronger than borosilicate glass- 6x stronger than borosilicate glass -- Alumina-silicate glassAlumina-silicate glass - Better able to resist clouding due to to alkali or scratching- Better able to resist clouding due to to alkali or scratching - Ideal for- Ideal for higher temperature thermometers, graduatedhigher temperature thermometers, graduated cylinders and centrifuge tubescylinders and centrifuge tubes.. VycorVycor -- recommended for use inrecommended for use in application involving highapplication involving high temperature and drastic heat shock and extremetemperature and drastic heat shock and extreme chemical treatment with acids and dilute alkalieschemical treatment with acids and dilute alkalies - heatable to 900- heatable to 900oo C and withstand downshock from 900C and withstand downshock from 900oo C toC to ice waterice water - ideal for- ideal for ashing and ignition techniquesashing and ignition techniques
  • 16. 2. High silica glass2. High silica glass -- 96% silica content96% silica content comparable tocomparable to fused quartzfused quartz inin itsits thermal endurance, chemical stability andthermal endurance, chemical stability and electrical characteristicselectrical characteristics -- radiation resistant, good optical qualities andradiation resistant, good optical qualities and temperature capabilitiestemperature capabilities - ideal for- ideal for high precision analytical workhigh precision analytical work and canand can also be used foralso be used for optical reflectors and mirrorsoptical reflectors and mirrors 3. Glass with high resistance to alkalies3. Glass with high resistance to alkalies -- boron-freeboron-free glassware for strong alkali solutionsglassware for strong alkali solutions and digestion with strong alkaliesand digestion with strong alkalies - less thermal resistant than borosilicate glass- less thermal resistant than borosilicate glass - known as- known as soft glasssoft glass
  • 17. 4. Low actinic glass4. Low actinic glass -- amber or red-coloredamber or red-colored to reduce the amount of light passingto reduce the amount of light passing through the substance within the glasswarethrough the substance within the glassware - highly protective laboratory glassware for handling- highly protective laboratory glassware for handling heat-heat- labile substances in the 300-500nm rangelabile substances in the 300-500nm range (bilirubin, carotene and vitamin A)(bilirubin, carotene and vitamin A) 5. Standard flint glass5. Standard flint glass -- soda-lime glasssoda-lime glass which is composed of a mixture ofwhich is composed of a mixture of oxides of Si, Ca and Naoxides of Si, Ca and Na - Lowest in cost and readily fabricated- Lowest in cost and readily fabricated - poorly resistant to high temperature and sudden changes in- poorly resistant to high temperature and sudden changes in temperaturetemperature - easy to melt and shape thus used as- easy to melt and shape thus used as reagent bottles andreagent bottles and disposable laboratory glasswaresdisposable laboratory glasswares - release alkali into the pipetted liquid- release alkali into the pipetted liquid
  • 18. Quick QuizQuick Quiz  Which of the following is an aluminum-silicate glassWhich of the following is an aluminum-silicate glass that is at least six times stronger than borosilicatethat is at least six times stronger than borosilicate and is resistant to alkaline etching and scratching?and is resistant to alkaline etching and scratching? a.a. KimaxKimax b.b. PyrexPyrex c.c. Corning boron freeCorning boron free d.d. CorexCorex  The tinted glass which is ideal for delayed testing ofThe tinted glass which is ideal for delayed testing of light-sensitive substances islight-sensitive substances is a.a. standard flintstandard flint b.b. soda limesoda lime c.c. low actiniclow actinic d.d. boron-freeboron-free
  • 19. 1.1. PolyolefinsPolyolefins (polyethylenes, polypropylenes)(polyethylenes, polypropylenes) Unique group ofUnique group of resinsresins with relatively inertwith relatively inert chemical propertieschemical properties Unaffected by acids (however, conc. sulfuric acidUnaffected by acids (however, conc. sulfuric acid slowly attacks polyethylene at RT), alkalies,slowly attacks polyethylene at RT), alkalies, salt solutions and most aqueous solutionssalt solutions and most aqueous solutions Aromatic, aliphatic and chlorinated hydrocarbonsAromatic, aliphatic and chlorinated hydrocarbons cause moderate swelling at RTcause moderate swelling at RT Organic acids, essential oils and halogens slowlyOrganic acids, essential oils and halogens slowly penetrate these plasticspenetrate these plastics PLASTICWARES
  • 20. Polyolefins (polyethylenes, polypropylenes)Polyolefins (polyethylenes, polypropylenes) Strong oxidizing agents attack this group atStrong oxidizing agents attack this group at elevated temperatures onlyelevated temperatures only Polypropylene is more expensivePolypropylene is more expensive thanthan polyethylene therefore the latter is used inpolyethylene therefore the latter is used in most disposable plastic waresmost disposable plastic wares Polypropylene is sterilizablePolypropylene is sterilizable however ithowever it absorbs pigment and tends to becomeabsorbs pigment and tends to become discoloreddiscolored
  • 21. PLASTICWARES 2. Polycarbonate resin2. Polycarbonate resin  twice as strong as polypropylene (from –100twice as strong as polypropylene (from –100oo C toC to +160+160oo C)C)  Chemical resistance is not as wide as theChemical resistance is not as wide as the polyolefinspolyolefins  Unsuitable for use with bases as amines,Unsuitable for use with bases as amines, ammonia and alkalies as well as oxidizing agentsammonia and alkalies as well as oxidizing agents  Glass-clear and shatterproofGlass-clear and shatterproof  Ideal forIdeal for centrifuge tubes and graduatedcentrifuge tubes and graduated cylinderscylinders
  • 22. 3. Tygon3. Tygon  Nontoxic, clear plastic of modified PVCNontoxic, clear plastic of modified PVC (polyvinylchloride)(polyvinylchloride)  Used extensively for theUsed extensively for the manufacture of ACAmanufacture of ACA tubingstubings  Flexible and used to handle most chemicalsFlexible and used to handle most chemicals  Can beCan be steam-autoclaved or chemically sterilizedsteam-autoclaved or chemically sterilized  Tubing is soft and flexible and quickly slips overTubing is soft and flexible and quickly slips over tubulatures buttubulatures but gripping tightly on glass or metalsgripping tightly on glass or metals
  • 23. 4. Teflon fluorocarbon resins4. Teflon fluorocarbon resins  Almost chemically inert withAlmost chemically inert with high corrosion resistancehigh corrosion resistance at extreme temperaturesat extreme temperatures  ForFor cryogenic experiments and work at highcryogenic experiments and work at high temperatures over extended periods (-270temperatures over extended periods (-270oo C toC to +255+255oo C)C)  Pure translucent white and inertPure translucent white and inert  Easy to clean and fast dryingEasy to clean and fast drying but it can be scratchedbut it can be scratched and misshapedand misshaped
  • 24. Quick QuizQuick Quiz  The internal tubings of clinical analyzers should beThe internal tubings of clinical analyzers should be made ofmade of a.a. Teflon fluorocarbonTeflon fluorocarbon b.b. polycarbonatepolycarbonate c.c. TygonTygon d.d. polyvinylchloridepolyvinylchloride  The advantages of this plastic is that it can be madeThe advantages of this plastic is that it can be made into very thin sheets and can withstand extremeinto very thin sheets and can withstand extreme temperatures.temperatures. a.a. PolyolefinsPolyolefins b.b. PolycarbonatePolycarbonate c.c. TygonTygon d.d. TeflonTeflon
  • 25. PIPETSPIPETS Two General TypesTwo General Types 1.1. Volumetric or Transfer pipetVolumetric or Transfer pipet DesignedDesigned to deliver (TD) a fixed volume of liquidto deliver (TD) a fixed volume of liquid Consists of aConsists of a cylindrical bulbcylindrical bulb joined at both ends tojoined at both ends to narrower glass tubingnarrower glass tubing Calibration mark is etched around the upper suction tubeCalibration mark is etched around the upper suction tube Lower delivery tube is drawn out to a fine tipLower delivery tube is drawn out to a fine tip used forused for accurate measurements of aliquots ofaccurate measurements of aliquots of nonviscous samples, filtrates, controls, andnonviscous samples, filtrates, controls, and standard solutionsstandard solutions
  • 26. OSTWALD-FOLIN PIPETOSTWALD-FOLIN PIPET  Similar to volumetric pipet but have their bulbSimilar to volumetric pipet but have their bulb closer to the delivery tipcloser to the delivery tip  Used forUsed for measuring viscous fluids as blood ormeasuring viscous fluids as blood or serumserum  Has anHas an etched ring near the mouthpieceetched ring near the mouthpiece (blowout pipet)(blowout pipet)  Blown out only when the fluid has drained toBlown out only when the fluid has drained to the last drop in the delivery tipthe last drop in the delivery tip
  • 27. 2. Graduated or Measuring Pipets2. Graduated or Measuring Pipets  Plain, narrow tube drawn out to a tipPlain, narrow tube drawn out to a tip  Graduated uniformly along its lengthGraduated uniformly along its length  Two types which are calibrated for delivery areTwo types which are calibrated for delivery are availableavailable Mohr pipetMohr pipet -- calibrated between two markscalibrated between two marks on theon the stemstem Serological pipetSerological pipet - has- has graduation marks down on the tipgraduation marks down on the tip and blown out to deliver the entire volume ofand blown out to deliver the entire volume of the pipet if etched ring or double rings arethe pipet if etched ring or double rings are evident near the mouth of the pipetevident near the mouth of the pipet
  • 28. Serological pipetSerological pipet  Intended for theIntended for the delivery of predetermineddelivery of predetermined volumesvolumes  Principally used for thePrincipally used for the measurements ofmeasurements of reagentsreagents andand are not generallyare not generally considered accurate enough forconsidered accurate enough for measuring viscous samples andmeasuring viscous samples and standardsstandards  Serological pipets have larger orifice thanSerological pipets have larger orifice than the Mohr pipet and thus drain fasterthe Mohr pipet and thus drain faster
  • 29. MICROPIPETSMICROPIPETS  SahliSahli (TC)(TC)  KirkKirk (TC)(TC)  Lang-LevyLang-Levy (TD/ TC)(TD/ TC)  OverflowOverflow (TC)(TC)  CapillaryCapillary (TC)(TC)  Sanz PipetSanz Pipet (TD=TC)(TD=TC)  UnopetteUnopette (TC)(TC)  Semiautomatic pipetSemiautomatic pipet EppendorfEppendorf (TD/TC)(TD/TC)
  • 30. Quick QuizQuick Quiz  The College of American Pathologists (CAP)The College of American Pathologists (CAP) requires that volumetric pipets and flasks berequires that volumetric pipets and flasks be certified ascertified as a.a. Class AClass A b.b. Class BClass B c.c. Class CClass C d.d. Class DClass D  ““To deliver”(TD) pipettes are identified byTo deliver”(TD) pipettes are identified by a.a. two etched bands near the mouthpiecetwo etched bands near the mouthpiece b.b. self-draining capacityself-draining capacity c.c. dual purpose pipet labelsdual purpose pipet labels d.d. blue graduation levelsblue graduation levels
  • 31. CHEMICALSCHEMICALS TheThe American Chemical SocietyAmerican Chemical Society (ACS) established the(ACS) established the specifications of chemicals in the analytical grade category.specifications of chemicals in the analytical grade category. The analytical reagent grade chemicals are of very high purity.The analytical reagent grade chemicals are of very high purity. They are labeledThey are labeled Analytical ReagentAnalytical Reagent (AR) or ACS which state(AR) or ACS which state the actual purity of each lot. These include chemicals suchthe actual purity of each lot. These include chemicals such as:as:  spectrograde,spectrograde,  nanograde, andnanograde, and  High Performance Liquid Chromatography (HPLC)High Performance Liquid Chromatography (HPLC) grade.grade. The AR grade chemical are used in:The AR grade chemical are used in: a.a. most analytical laboratory proceduresmost analytical laboratory procedures b. making up reagents forb. making up reagents for spectrophotometry,spectrophotometry, chromatography, trace metal analysis, atomic absorptionchromatography, trace metal analysis, atomic absorption spectrophotometry, fluorometry and standardizationspectrophotometry, fluorometry and standardization procedures.procedures.
  • 32. United States PharmacopoeiaUnited States Pharmacopoeia (USP) and the(USP) and the National FormularyNational Formulary (NF) grade chemicals are not(NF) grade chemicals are not injurious to humans and are used to producedinjurious to humans and are used to produced drugs.drugs. They are not pure enough for use in mostThey are not pure enough for use in most chemical procedures.chemical procedures. Less pure grade chemicals are referred to asLess pure grade chemicals are referred to as purified and technical. Thepurified and technical. The chemically purechemically pure (CP)(CP) oror pure gradepure grade chemicals do not recommend forchemicals do not recommend for reagent preparations.reagent preparations. TechnicalTechnical oror commercialcommercial grade reagents aregrade reagents are primarily used in manufacturing and should notprimarily used in manufacturing and should not be used in the clinical laboratory. The technicalbe used in the clinical laboratory. The technical grade is the lowest quality and should not begrade is the lowest quality and should not be used for analytical work.used for analytical work.
  • 33. STANDARDSSTANDARDS AA primary standardprimary standard (PS) is a chemical that is the(PS) is a chemical that is the highest purity and can be measured directly tohighest purity and can be measured directly to produce a substance of exact known concentration.produce a substance of exact known concentration. TheThe International Union of Pure and Applied ChemistryInternational Union of Pure and Applied Chemistry (IUPAC) requires that(IUPAC) requires that PS be at least 99.98% purePS be at least 99.98% pure and that theand that the working standards be 99.95% pureworking standards be 99.95% pure.. Grade AGrade A - atomic weight standards- atomic weight standards Grade BGrade B – ultimate standards– ultimate standards Grade CGrade C* – primary standards with >0.002% impurity* – primary standards with >0.002% impurity Grade DGrade D* – working standards with >0.5% impurity* – working standards with >0.5% impurity Grade EGrade E – secondary standards derived from using– secondary standards derived from using Grade C standardsGrade C standards **commercially availablecommercially available
  • 34. TheThe National Bureau of StandardsNational Bureau of Standards (NBS) now(NBS) now National Institute of Science and TechnologyNational Institute of Science and Technology (NIST) purified materials known as(NIST) purified materials known as StandardStandard Reference MaterialsReference Materials (SRM) are used as(SRM) are used as primary standard materials in the clinicalprimary standard materials in the clinical laboratory. They are relatively expensive andlaboratory. They are relatively expensive and should be used as ashould be used as a reference for thereference for the comparison of commercially obtainedcomparison of commercially obtained standards and reagents.standards and reagents. AA secondary standardsecondary standard is a substance of loweris a substance of lower purity whose concentration is determined bypurity whose concentration is determined by comparison to a primary standard.comparison to a primary standard.
  • 35. Quick QuizQuick Quiz  Regarded as the ultimate standard by IUPAC isRegarded as the ultimate standard by IUPAC is a.a. Grade AGrade A b.b. Grade BGrade B c.c. Grade CGrade C d.d. Grade DGrade D  What is the most common use of a standard inWhat is the most common use of a standard in the clinical laboratory?the clinical laboratory? a.a. to verify accuracy of resultsto verify accuracy of results b.b. to standardize the testingto standardize the testing c.c. to compare its value with the results obtainedto compare its value with the results obtained d.d. to obtain the concentration of the substanceto obtain the concentration of the substance testedtested
  • 36. WATERWATER Reagent grade waterReagent grade water (RGW) is water suitable(RGW) is water suitable for reagent and standard preparation. Mostfor reagent and standard preparation. Most procedures use distilled water or deionized water.procedures use distilled water or deionized water. Distilled waterDistilled water is purified to remove almost allis purified to remove almost all organic materials. Water may be distilled moreorganic materials. Water may be distilled more than once and each distillation cycle will removethan once and each distillation cycle will remove impurities.impurities. Deionized waterDeionized water is produced from distilled wateris produced from distilled water using either an anion or cation exchange resinusing either an anion or cation exchange resin followed by replacement of the removed particlesfollowed by replacement of the removed particles with hydroxyl or hydrogen ions respectively.with hydroxyl or hydrogen ions respectively.
  • 37. TheThe College of American PathologistsCollege of American Pathologists (CAP) and the(CAP) and the NationalNational Committee for Clinical LaboratorCommittee for Clinical Laboratoryy StandardsStandards (NCCLS) issued(NCCLS) issued minimum specifications for RGW. The NCCLS requirements for eachminimum specifications for RGW. The NCCLS requirements for each type are tabulated below:type are tabulated below: SpecificationsSpecifications Type IType I Type II Type IIIType II Type III Bacterial content (maximumBacterial content (maximum <10<10 101022 NANA colony forming unit/mL)colony forming unit/mL) pHpH NANA NANA NANA Resistivity (megaohms/cmResistivity (megaohms/cm 1.0 (in-line) 1.01.0 (in-line) 1.0 0.10.1 at 25at 25oo C)C) Silicates, mg/L SiOSilicates, mg/L SiO22 maximummaximum 0.050.05 0.10.1 1.01.0 Particulate matter (filter in system)Particulate matter (filter in system) 0.220.22 µmµm NANA NANA Organic contaminantsOrganic contaminants activated carbonactivated carbon NANA NANA
  • 38. Type I RGWType I RGW is used for procedures that require maximumis used for procedures that require maximum water purity such as :water purity such as : preparations of standard solutions,preparations of standard solutions, ultramicro chemical analysis,ultramicro chemical analysis, measurement of nanogram or subnanogrammeasurement of nanogram or subnanogram concentrationconcentration and tissue or cell culture (or both).and tissue or cell culture (or both). Type II RGWType II RGW is used in most laboratory determination inis used in most laboratory determination in chemistry, hematology, microbiology, immunology and otherchemistry, hematology, microbiology, immunology and other chemical laboratory areas.chemical laboratory areas. Type III RGWType III RGW is used in most qualitative measurement /is used in most qualitative measurement / examinations, most procedures in urinalysis, parasitology andexaminations, most procedures in urinalysis, parasitology and histology, washing glasswares and procedures not requiringhistology, washing glasswares and procedures not requiring Type I or Type II water.Type I or Type II water. Carbon dioxide-free water is obtained by boiling Type II water.Carbon dioxide-free water is obtained by boiling Type II water. This is used when carbon dioxide, ammonia and oxygen mayThis is used when carbon dioxide, ammonia and oxygen may affect analysis.affect analysis.
  • 39. CLEANING GLASSWARES AND PLASTIC WARESCLEANING GLASSWARES AND PLASTIC WARES  Routine washing may be done by soaking in dilute bleach followedRoutine washing may be done by soaking in dilute bleach followed by drying in an oven, soaking in 20% nitric acid solution for 12-24by drying in an oven, soaking in 20% nitric acid solution for 12-24 hours and soaking in acid-dichromate solution.hours and soaking in acid-dichromate solution.  For blood clots, soak in 10% NaOHFor blood clots, soak in 10% NaOH  For new pipets, soak in 5% HCI or 5% HNOFor new pipets, soak in 5% HCI or 5% HNO33  For metal ion determination, soak in 20% nitric acidFor metal ion determination, soak in 20% nitric acid  For grease, soak in any organic solvent or 50% KOHFor grease, soak in any organic solvent or 50% KOH..  For permanganate stains, soak in 50% HCI or a mixture of 1%For permanganate stains, soak in 50% HCI or a mixture of 1% ferrous sulfate in 25% sulfuric acidferrous sulfate in 25% sulfuric acid  For bacteriologic glassware, soak in 2% to 4% cresol solutionFor bacteriologic glassware, soak in 2% to 4% cresol solution followed by autoclaving and thorough washingfollowed by autoclaving and thorough washing  For iron determination, soak in 1:2 dilution of conc. HCI solution orFor iron determination, soak in 1:2 dilution of conc. HCI solution or 1:3 dilution of conc. HNO1:3 dilution of conc. HNO33
  • 40. Quick QuizQuick Quiz  What is the most appropriate term to describeWhat is the most appropriate term to describe water that is used as a solvent in the clinicalwater that is used as a solvent in the clinical laboratory?laboratory? a.a. distilled waterdistilled water b.b. deionized waterdeionized water c.c. reagent grade waterreagent grade water d.d. tap watertap water  The type of water desired for use in testThe type of water desired for use in test methods requiring maximum accuracy andmethods requiring maximum accuracy and precision isprecision is a.a. distilleddistilled b.b. pure gradepure grade c.c. type Itype I d.d. type IItype II
  • 41.  What is effectively removed by activated charcoalWhat is effectively removed by activated charcoal adsorption in water purification?adsorption in water purification? a.a. charged chemicalscharged chemicals b.b. microbes and pyrogensmicrobes and pyrogens c.c. toxic gasestoxic gases d.d. organic compoundsorganic compounds  WhatWhat purification method is ideal for the removal ofpurification method is ideal for the removal of pyrogens and microorganisms?pyrogens and microorganisms? a.a. activated charcoal adsorptionactivated charcoal adsorption b.b. semipermeable membrane filtrationsemipermeable membrane filtration c.c. reverse osmosisreverse osmosis d.d. distillation and deionization combineddistillation and deionization combined
  • 42. Chemical HazardsChemical Hazards  CausticCaustic – can cause burns– can cause burns  FlammableFlammable – also combustible, that is, can– also combustible, that is, can easily catch fireeasily catch fire  CorrosiveCorrosive – harmful to mucous membranes,– harmful to mucous membranes, skin, eyes or tissuesskin, eyes or tissues CarcinogenCarcinogen – can produce or incite cancer– can produce or incite cancer  MutagenMutagen – cause changes in RNA or DNA– cause changes in RNA or DNA  TeratogenTeratogen – cause birth defects– cause birth defects
  • 43. Quick QuizQuick Quiz  Xylene, ethanol, methanol, and acetone would be inXylene, ethanol, methanol, and acetone would be in which hazard class?which hazard class? a.a. corrosivecorrosive b.b. flammableflammable c.c. oxidizeroxidizer d.d. all of the aboveall of the above  Precautions such asPrecautions such as using a fume hood, wearingusing a fume hood, wearing rubber gloves, donning a respirator, and cleaningrubber gloves, donning a respirator, and cleaning glassware with a strong acid or organic solvent areglassware with a strong acid or organic solvent are consistent with working withconsistent with working with a.a. corrosivescorrosives b.b. carcinogenscarcinogens c.c. azidesazides d.d. all reagentsall reagents
  • 44. CENTRIFUGECENTRIFUGE TheThe relative centrifugal forcerelative centrifugal force (RCF) is obtained by(RCF) is obtained by using a nomogram or by using this formula:using a nomogram or by using this formula: RCF = 1.118 x 10-5 x r x rpm2 Where: r = radius in cm. between the axis of rotation and the center of the centrifuge tube rpm = speed in revolutions per minute The CAP recommends cleaning of any spills or debris such as blood, glass and others daily; checking of the timer, brushes and speed every three months. The speed of the centrifuge is checked using the tachometer or strobe light.
  • 45.  All laboratories are required to haveAll laboratories are required to have safetysafety showers, eyewash stations and fireshowers, eyewash stations and fire extinguishersextinguishers and to periodically test andand to periodically test and inspect the equipment of proper operation.inspect the equipment of proper operation. Other items that must be available forOther items that must be available for personnel include fire blankets, spill kits andpersonnel include fire blankets, spill kits and first aid supplies.first aid supplies.  Mechanical pipetting devices must be usedMechanical pipetting devices must be used for manipulating all types of liquids in thefor manipulating all types of liquids in the laboratory, including water.laboratory, including water. MouthMouth pipetting is strictly prohibited.pipetting is strictly prohibited.
  • 46. Quick QuizQuick Quiz  Which of the following may be safely pipettedWhich of the following may be safely pipetted by mouth?by mouth? a.a. serumserum b.b. urineurine c.c. saline solutionsaline solution d.d. none of the abovenone of the above  The speed of the centrifuge should be checkedThe speed of the centrifuge should be checked at least once every three (3) months with a /anat least once every three (3) months with a /an a.a. tachometertachometer b.b. wiperwiper c.c. potentiometerpotentiometer d.d. ergometerergometer
  • 47. BIOLOGIC SAFETYBIOLOGIC SAFETY  All samples and other body fluids should beAll samples and other body fluids should be collected, transported, handled andcollected, transported, handled and processed usingprocessed using strict precautions.strict precautions.  Gloves, gowns and face protection must beGloves, gowns and face protection must be used ifused if splash or splatteringsplash or splattering is likely tois likely to occur.occur.  Specimen should remainSpecimen should remain cappedcapped duringduring centrifugation because biologiccentrifugation because biologic specimens could produce finelyspecimens could produce finely dispersed aerosolsdispersed aerosols that are athat are a high-riskhigh-risk source of infection.source of infection.
  • 48. Any blood, body fluid or other potentially infectious material spillAny blood, body fluid or other potentially infectious material spill must be cleaned up and the area of equipment disinfectedmust be cleaned up and the area of equipment disinfected immediately.immediately. Recommended clean upRecommended clean up includes the following:includes the following: A.A. Wear appropriate protective equipmentWear appropriate protective equipment B.B. Use mechanical devices to pick up broken glass orUse mechanical devices to pick up broken glass or other sharp objects.other sharp objects. C.C. Absorb the spill with paper towels, gauze pads or tissue,Absorb the spill with paper towels, gauze pads or tissue, etc.etc. D.D. Clean the spill site using common aqueous detergentClean the spill site using common aqueous detergent.. E.E. the spill site using approved disinfectant or 10% bleachthe spill site using approved disinfectant or 10% bleach using appropriate contact time.using appropriate contact time. F.F. Rinse the spill site with water.Rinse the spill site with water. G.G. Dispose all materials in appropriate biohazardDispose all materials in appropriate biohazard containers.containers.
  • 49. Occupational Safety and Health OrganizationOccupational Safety and Health Organization AdministrationAdministration (OSHA) “Blood –Borne Pathogens”(OSHA) “Blood –Borne Pathogens” standard requires writtenstandard requires written “Exposure Control Plan”“Exposure Control Plan” Categories of exposure are the following:Categories of exposure are the following: Category I-Category I- daily exposuredaily exposure to blood and body fluidsto blood and body fluids Category II-Category II- regular exposureregular exposure to blood and bodyto blood and body fluidsfluids Category III-Category III- no exposureno exposure to blood and body fluidto blood and body fluid Employers must offer Hepatitis B vaccine at noEmployers must offer Hepatitis B vaccine at no costcost to all personnel in Category I and II.to all personnel in Category I and II. Biological safety cabinetsBiological safety cabinets should be installed in strategicshould be installed in strategic places to facilitate manipulations of infectious material.places to facilitate manipulations of infectious material. ItIt reduces risk of exposurereduces risk of exposure of laboratory personnel andof laboratory personnel and patients.patients.
  • 50. SAFETY AGAINST EXPOSURE TO TOXIC CHEMICALSSAFETY AGAINST EXPOSURE TO TOXIC CHEMICALS OSHA published itsOSHA published its Hazard Communication StandardHazard Communication Standard inin 1983 to minimize the incidence of chemically related1983 to minimize the incidence of chemically related occupational illnesses and injuries in the workplaces.occupational illnesses and injuries in the workplaces. OSHA requires the following:OSHA requires the following: a. Manufacturers of chemicals shoulda. Manufacturers of chemicals should evaluate the hazards ofevaluate the hazards of the chemicalsthe chemicals they produce andthey produce and develop hazarddevelop hazard communication programscommunication programs for employees exposed tofor employees exposed to hazardous chemicals.hazardous chemicals. b. Clinical laboratories shouldb. Clinical laboratories should develop and institute adevelop and institute a chemical hygiene plan.chemical hygiene plan. c. Hospitals and laboratories are obliged toc. Hospitals and laboratories are obliged to maintain anmaintain an inventory of all hazardous substancesinventory of all hazardous substances used in theused in the workplace.workplace.
  • 51. OSHA requires thatOSHA requires that material safety data sheetsmaterial safety data sheets ((MSDSMSDS) be) be provide for each chemical by manufacturers andprovide for each chemical by manufacturers and suppliers and must be made available on site forsuppliers and must be made available on site for laboratory personnel.laboratory personnel. MSDS will specifically includeMSDS will specifically include  the chemical identity;the chemical identity;  chemical and common name;chemical and common name;  physical and chemical characteristics;physical and chemical characteristics;  signs and symptoms of exposure;signs and symptoms of exposure;  routes of entry;routes of entry;  exposure limits;exposure limits;  carcinogenic potential;carcinogenic potential;  safe handling procedures;safe handling procedures;  spill cleanup procedures; andspill cleanup procedures; and  emergency first-aid.emergency first-aid. MSDSs contain information on the nature of the chemical,MSDSs contain information on the nature of the chemical, precautions if spilled and disposal recommendations.precautions if spilled and disposal recommendations.
  • 52.  TheThe National Fire Protection AssociationNational Fire Protection Association (NFPA) has(NFPA) has developed the Hazards Identification system todeveloped the Hazards Identification system to provide common, recognizable warning signs forprovide common, recognizable warning signs for chemical hazards. The system consists of four color-chemical hazards. The system consists of four color- coded, diamond-shaped symbols arranged to form acoded, diamond-shaped symbols arranged to form a larger diamond shape.larger diamond shape. (top) designates(top) designates flammabilityflammability (left) indicates(left) indicates health hazardshealth hazards (right) indicates(right) indicates reactivity-stabilityreactivity-stability (bottom) indicates(bottom) indicates special considerationspecial consideration  Contained within each color-coded diamond is aContained within each color-coded diamond is a number ranging from 0 to 4, indicating the severity ofnumber ranging from 0 to 4, indicating the severity of the respective hazard (0 = none and 4 = extreme)the respective hazard (0 = none and 4 = extreme)
  • 53. Special ConsiderationsSpecial Considerations  Water ReactiveWater Reactive  Oxidizing AgentOxidizing Agent  RadioactiveRadioactive  PoisonPoison Ox
  • 54. ELECTRICAL SAFETYELECTRICAL SAFETY  Lockout/tag outLockout/tag out malfunctioning electrical ormalfunctioning electrical or mechanical equipment until services.mechanical equipment until services. ReportReport any small shocks, unplug andany small shocks, unplug and tag equipment until serviced.tag equipment until serviced.  If a severely shocked person can not let go ofIf a severely shocked person can not let go of instrument,instrument, unplug it without touchingunplug it without touching the person or knock person loosethe person or knock person loose with nonconductive materialwith nonconductive material such assuch as wood.wood.
  • 55. RADIATION SAFETYRADIATION SAFETY  A radiation-safety policy should include environmental andA radiation-safety policy should include environmental and personnel protection.personnel protection.  All areas where radioactive materials are used or storedAll areas where radioactive materials are used or stored must be posted withmust be posted with caution signscaution signs andand traffic in thesetraffic in these areas should be restricted to essential personnel onlyareas should be restricted to essential personnel only..  Records must be maintained as to the quantity ofRecords must be maintained as to the quantity of radioactive material on hand as well as the quantity that isradioactive material on hand as well as the quantity that is disposed.disposed. Records must be maintained for the length ofRecords must be maintained for the length of employment plus 30 yearsemployment plus 30 years..  Radiation monitoring utilizes film badge or survey meter.Radiation monitoring utilizes film badge or survey meter. The exposure limit (maximum permissible doseThe exposure limit (maximum permissible dose equivalents isequivalents is 5000 mrem/year whole body5000 mrem/year whole body))  TheThe Wipe test (Leak test)Wipe test (Leak test) involves wiping laboratoryinvolves wiping laboratory surfaces with moistened absorbent material and thesurfaces with moistened absorbent material and the radiation contained in each wipe is counted.radiation contained in each wipe is counted.
  • 56. Quick QuizQuick Quiz  A corrosive material was spilled onto the hand of aA corrosive material was spilled onto the hand of a laboratorian. After diluting the material under running coldlaboratorian. After diluting the material under running cold water, what should be done next?water, what should be done next? a.a. consult the MSDSconsult the MSDS b.b. wipe up spills with paper towelswipe up spills with paper towels c.c. dilute spills and remove it in a biohazard bagdilute spills and remove it in a biohazard bag d.d. go to the emergency roomgo to the emergency room  Which of the following information is found in the MSDS?Which of the following information is found in the MSDS? a.a. Health hazard dataHealth hazard data b.b. Fire & explosion hazard dataFire & explosion hazard data c.c. First Aid measuresFirst Aid measures d.d. All of the aboveAll of the above
  • 57. FIRE SAFETYFIRE SAFETY Fires have been divided into four classes onFires have been divided into four classes on the nature of the combustible material andthe nature of the combustible material and requirements for extinguishments.requirements for extinguishments. Class A-Class A- ordinary combustible solidordinary combustible solid materialsmaterials such as paper,such as paper, wood,wood, plastic and rubberplastic and rubber Class B-Class B- flammable liquids/ gases andflammable liquids/ gases and combustible petroleum productscombustible petroleum products Class C-Class C- energized electrical equipmentenergized electrical equipment Class D-Class D- combustible/reactive metalscombustible/reactive metals suchsuch as Mg, Na and Kas Mg, Na and K Class EClass E –– nuclear reactionnuclear reaction Class KClass K –– cooking mediacooking media
  • 58.  (Type A) Pressurized water extinguishers(Type A) Pressurized water extinguishers (foam and(foam and multipurpose dry-chemical types) are formultipurpose dry-chemical types) are for Class AClass A fires.fires.  (Type ABC) Multi-purpose dry chemical and carbon(Type ABC) Multi-purpose dry chemical and carbon dioxidedioxide extinguishers are used forextinguishers are used for Class B and C firesClass B and C fires..  (Type C) Halogenated hydrocarbon(Type C) Halogenated hydrocarbon extinguishers areextinguishers are recommended particularly for use withrecommended particularly for use with computercomputer equipment.equipment.  (Type D) Class D(Type D) Class D fires present special problems andfires present special problems and extinguishments is left to trained fire fighters usingextinguishments is left to trained fire fighters using special dry-chemical extinguishersspecial dry-chemical extinguishers..  Personnel should know the location and type of portablePersonnel should know the location and type of portable fire extinguisher near the work area and know how to usefire extinguisher near the work area and know how to use an extinguisher before a fire occurs.an extinguisher before a fire occurs.
  • 59. Quick QuizQuick Quiz  A fire extinguisher used in the event of anA fire extinguisher used in the event of an electrical fire should include which of theelectrical fire should include which of the following classifications ?following classifications ? a.a. type Atype A b.b. type Btype B c.c. type Ctype C d.d. type Dtype D  In the Hazards Identification System, whatIn the Hazards Identification System, what hazard does the blue diamond positioned to thehazard does the blue diamond positioned to the left identify ?left identify ? a.a. flammableflammable b.b. healthhealth c.c. reactivityreactivity d.d. contactcontact
  • 60. SAFETY AWARENESS FOR CLINICALSAFETY AWARENESS FOR CLINICAL LABORATORY PERSONNELLABORATORY PERSONNEL  Health Care Organizations focus their responsibility inHealth Care Organizations focus their responsibility in protecting their employees from infection especiallyprotecting their employees from infection especially againstagainst hepatitis B virus (HBV), humanhepatitis B virus (HBV), human immunodeficiency virus (HIV) & TB bacilli.immunodeficiency virus (HIV) & TB bacilli.  TheThe Centers for Disease Control (CDC)Centers for Disease Control (CDC) updatedupdated the 1983 Guidelines for Isolation Precautions inthe 1983 Guidelines for Isolation Precautions in hospitals with the release of its Universal Precautionshospitals with the release of its Universal Precautions in 1987 because of the growing concern about HIV.in 1987 because of the growing concern about HIV.  TheThe CDC recommends that blood and body fluidCDC recommends that blood and body fluid precautionsprecautions should be consistently used for allshould be consistently used for all patients regardless of their blood-borne infectionpatients regardless of their blood-borne infection status.status.
  • 61.  Potentially infectious materials include:Potentially infectious materials include: a.a. body fluids (semen, vaginalbody fluids (semen, vaginal secretions, pericardial fluid, peritoneal fluid,secretions, pericardial fluid, peritoneal fluid, synovial fluid, pleural fluid, amniotic fluid,synovial fluid, pleural fluid, amniotic fluid, saliva, tears, CSF, urine and breast milk)saliva, tears, CSF, urine and breast milk) b.b. unfixed tissues, organs or blood slidesunfixed tissues, organs or blood slides  Infective agents may be inactivated byInfective agents may be inactivated by = heat sterilization (250= heat sterilization (250OO C for 15 minutes),C for 15 minutes), = ethylene oxide (450-500 mg/L at 55-60= ethylene oxide (450-500 mg/L at 55-60OO C)C),, = 2% glutaraldehyde,= 2% glutaraldehyde, = 10% hydrogen peroxide= 10% hydrogen peroxide,, = 10% formaldehyde= 10% formaldehyde,, = 5-25% hypochlorite= 5-25% hypochlorite,, = 10% v/v with tap water of a common household= 10% v/v with tap water of a common household bleachbleach makes a very effective and economicalmakes a very effective and economical disinfectant inactivatingdisinfectant inactivating HBV in 10 minutesHBV in 10 minutes andand HIV in 2 minutes.HIV in 2 minutes.
  • 62. DISPOSAL OF HAZARDOUS MATERIALSDISPOSAL OF HAZARDOUS MATERIALS There are four basic waste disposalThere are four basic waste disposal techniques. These include:techniques. These include: flushing down the drain toflushing down the drain to the sewer systemthe sewer system IncinerationIncineration landfill buriallandfill burial recyclingrecycling
  • 63.  It is permissible toIt is permissible to flush water-soluble substances down theflush water-soluble substances down the drain with large quantities of water.drain with large quantities of water.  Strong acids and basesStrong acids and bases should beshould be neutralized beforeneutralized before disposaldisposal  Foul smelling chemicalsFoul smelling chemicals shouldshould never be disposed of downnever be disposed of down the drainthe drain..  Other liquid wastes, including flammable solvents,Other liquid wastes, including flammable solvents, must bemust be collected in approved containers and segregated intocollected in approved containers and segregated into compatible classescompatible classes..  flammable material can be burned in specially designedflammable material can be burned in specially designed incinerators with after burners and scrubbers to removeincinerators with after burners and scrubbers to remove toxic products of combustion.toxic products of combustion.  solid chemicals wastes that are unsuitable for incinerationsolid chemicals wastes that are unsuitable for incineration must be buried in a landfill.must be buried in a landfill.
  • 64. Biohazardous WastesBiohazardous Wastes  Medical waste is defined as aMedical waste is defined as a specialspecial wastewaste fromfrom health care facilitieshealth care facilities and is further definedand is further defined asas solid wastesolid waste that, if improperly treated orthat, if improperly treated or handledhandled may transmit infectious diseasesmay transmit infectious diseases..  it comprises ofit comprises of animal waste, bilk blood and bloodanimal waste, bilk blood and blood products, microbiologic wastes, pathologicproducts, microbiologic wastes, pathologic wastes and sharps.wastes and sharps.  The approved methods for treatment andThe approved methods for treatment and disposition of medical wastes aredisposition of medical wastes are incineration,incineration, steam sterilization, burial, thermalsteam sterilization, burial, thermal inactivation, chemical disinfection, orinactivation, chemical disinfection, or encapsulation in a solid matrix.encapsulation in a solid matrix.
  • 65. Quick QuizQuick Quiz  The major job-related hazard in clinical laboratoriesThe major job-related hazard in clinical laboratories produces symptoms of malaise, anorexia, nausea,produces symptoms of malaise, anorexia, nausea, vomiting, fatigue, diarrhea, and abdominal tendernessvomiting, fatigue, diarrhea, and abdominal tenderness isis a.a. AIDSAIDS b.b. salmonellosissalmonellosis c.c. tuberculosistuberculosis d.d. hepatitishepatitis  A laboratory professional was askedA laboratory professional was asked to send a bloodto send a blood specimen from an HIV-positive patient to a laboratoryspecimen from an HIV-positive patient to a laboratory for special tests. The specimen must be properlyfor special tests. The specimen must be properly labeled and shipped aslabeled and shipped as a.a. infectious substanceinfectious substance b.b. diagnostic specimendiagnostic specimen c.c. biological productbiological product d.d. clinical specimenclinical specimen
  • 66. UNITS OF MEASUREMENTUNITS OF MEASUREMENT  TheThe Systeme InternationaleSysteme Internationale (SI) defines seven(SI) defines seven base unitsbase units asas shown.shown. QuantityQuantity Basic UnitBasic Unit SymbolSymbol LengthLength metermeter mm MassMass kilogramkilogram kgkg TimeTime secondsecond ss Electric currentElectric current ampereampere AA TemperatureTemperature KelvinKelvin KK Luminous intensityLuminous intensity CandelaCandela cdcd Amount of substanceAmount of substance molemole molmol Catalytic amountCatalytic amount katalkatal
  • 67. SI-Derived UnitsSI-Derived Units are obtained by combining twoare obtained by combining two or more basic units by multiplication or division.or more basic units by multiplication or division. Derived QuantityDerived Quantity Derived UnitDerived Unit SymbolSymbol Substance concentrationSubstance concentration moles per cubic metermoles per cubic meter mol/mmol/m33 or mol m-or mol m-33 ConductanceConductance SiemensSiemens SS ResistanceResistance ΩΩ Activity (radionuclide)Activity (radionuclide) BecquerelBecquerel BqBq VolumeVolume cubic metercubic meter mm33
  • 68. The National Institute of Standards andThe National Institute of Standards and Technology (NIST)Technology (NIST) recognizes the use of fiverecognizes the use of five specific types of calibrating weights.specific types of calibrating weights. Class S weightsClass S weights – for monthly calibration of– for monthly calibration of analytical balancesanalytical balances Class M weightsClass M weights - to calibrate the weights- to calibrate the weights themselves since their quality is that of primarythemselves since their quality is that of primary standards.standards. Class S-1Class S-1 - used for routine analytical work.- used for routine analytical work. Class P weightsClass P weights - are allowed greater tolerance- are allowed greater tolerance levels than Class S-1levels than Class S-1 Class J weightsClass J weights - used for microanalytical work.- used for microanalytical work.
  • 69. TheThe accuracy of the thermometer used to monitor the incubationaccuracy of the thermometer used to monitor the incubation temperature of an instrumenttemperature of an instrument should beshould be verified every 6 to 12verified every 6 to 12 months.months. Reference thermometersReference thermometers must bemust be certified by the NISTcertified by the NIST.. For theFor the monitoring of enzymatic reactionsmonitoring of enzymatic reactions, thermometers should, thermometers should agree within 0.1agree within 0.1oo C with the NIST reference thermometerC with the NIST reference thermometer.. Thermometers used toThermometers used to check refrigerator and freezercheck refrigerator and freezer temperaturestemperatures shouldshould agree within 1.0agree within 1.0oo C with the referenceC with the reference thermometerthermometer. Thermometers should be. Thermometers should be discarded if they differ bydiscarded if they differ by more than 1more than 1oo C from the referenceC from the reference.. On a daily basis, the temperature of the incubation area should beOn a daily basis, the temperature of the incubation area should be checked, and the thermometer itself should be observed for splits inchecked, and the thermometer itself should be observed for splits in the mercury column.the mercury column.
  • 70. It is imperative thatIt is imperative that preventive maintenancepreventive maintenance proceduresprocedures be performed and the results recordedbe performed and the results recorded for all laboratory instrumentation.for all laboratory instrumentation. This includesThis includes maintenance of analyticalmaintenance of analytical balances, refrigerators, freezers, centrifuges,balances, refrigerators, freezers, centrifuges, ovens, water baths, heating blocks,ovens, water baths, heating blocks, thermometers, pipettors, dilutors, automatedthermometers, pipettors, dilutors, automated analyzers, and all other laboratory equipmentanalyzers, and all other laboratory equipment used for analyzing specimens.used for analyzing specimens. Preventive maintenance is performed at scheduledPreventive maintenance is performed at scheduled times such astimes such as per shift, daily, weekly, monthly,per shift, daily, weekly, monthly, or yearly.or yearly.
  • 71. Quick QuizQuick Quiz  SI units are designated units employed by theSI units are designated units employed by the International System of Units. The unit class thatInternational System of Units. The unit class that encompasses the seven fundamental quantities ofencompasses the seven fundamental quantities of measurement ismeasurement is a.a. basebase b.b. primaryprimary c.c. derivedderived d.d. elementalelemental  Which of the folllowing is most commonlyWhich of the folllowing is most commonly represented by a derived unit ?represented by a derived unit ? a.a. amount of substanceamount of substance b.b. electric currentelectric current c.c. massmass d.d. substancesubstance concentrationconcentration
  • 72. FACTORS THAT INFLUENCEFACTORS THAT INFLUENCE LABORATORY TESTINGLABORATORY TESTING IncreasedIncreased DecreasedDecreased ExerciseExercise Immediate effects:Immediate effects: Alanine and lactateAlanine and lactate FFAFFA Long-term effectsLong-term effects CK, LD, AST, platelets,CK, LD, AST, platelets, testosterone,testosterone, androstenedione and LHandrostenedione and LH FastingFasting Most analytesMost analytes ((8-12 hrs for common8-12 hrs for common bilirubin, FFA, TAGbilirubin, FFA, TAG GlucoseGlucose metabolites andmetabolites and 12-14 hrs for lipid profile12-14 hrs for lipid profile)) EFFECT ON LABORATORY RESULTS
  • 73. IncreasedIncreased DecreasedDecreased After eatingAfter eating K, TAG (chylomicrons),K, TAG (chylomicrons), ALP intestinal isoenzyme,ALP intestinal isoenzyme, turbidity of serum and plasmaturbidity of serum and plasma DietDiet High meatHigh meat NPNs (esp. urea, ammonia,NPNs (esp. urea, ammonia, and urate except creatinine)and urate except creatinine) High ratio of unsaturatedHigh ratio of unsaturated SerumSerum to saturated fatto saturated fat cholesterolcholesterol Purine-richPurine-rich UratesUrates High caffeineHigh caffeine FFA, catecholamine releaseFFA, catecholamine release Bananas, pineapple,Bananas, pineapple, Serotonin in bloodSerotonin in blood tomatoes andtomatoes and 5’HIAA in urine5’HIAA in urine avocadosavocados Alcohol ingestionAlcohol ingestion ImmediateImmediate Lactate, urate, ketone bodies,Lactate, urate, ketone bodies, and FFAand FFA Chronic abuseChronic abuse HDL-cholesterol, GGT, and MCVHDL-cholesterol, GGT, and MCV
  • 74. IncreasedIncreased DecreasedDecreased Tobacco smokingTobacco smoking Acute effectsAcute effects Catecholamines, cortisolCatecholamines, cortisol EosinophilEosinophil neutrophils, monocytesneutrophils, monocytes and FFAand FFA Chronic effectsChronic effects CO-Hb, MCV and leukocyteCO-Hb, MCV and leukocyte countcount Prolonged tourniquet applicationProlonged tourniquet application Serum enzymes, protein,Serum enzymes, protein, protein-bound substances,protein-bound substances, cholesterol, TAG, Ca, bloodcholesterol, TAG, Ca, blood cell concentrationcell concentration (hemoconcentration(hemoconcentration)**)** Postural changesPostural changes same as ** due to hydrostaticsame as ** due to hydrostatic efflux of waterefflux of water StressStress lactate, FFA and alterationlactate, FFA and alteration iin electrolyte levelsn electrolyte levels
  • 75. Quick QuizQuick Quiz  Which of the following substances does not requireWhich of the following substances does not require the patient a fasting blood specimen?the patient a fasting blood specimen? a.a. uric aciduric acid b.b. triglyceridestriglycerides c.c. cholesterolcholesterol d.d. phosphatesphosphates  Expected to be increasedExpected to be increased in blood in situationsin blood in situations characterized by hemoconcentration ischaracterized by hemoconcentration is a.a. calciumcalcium b.b. potassiumpotassium c.c. ferritinferritin d.d. creatininecreatinine
  • 76. BLOOD COLLECTIONBLOOD COLLECTION  Venipuncture (Phlebotomy)Venipuncture (Phlebotomy)  Venous blood isVenous blood is deoxygenated blooddeoxygenated blood that containsthat contains substances that come from metabolic activities ofsubstances that come from metabolic activities of different organsdifferent organs..  Venous blood is mostly used in analysis ofVenous blood is mostly used in analysis of bloodblood chemistry and immunologic studies.chemistry and immunologic studies.  Venous blood is more easily collected than arterialVenous blood is more easily collected than arterial blood.blood.  Maybe obtained viaMaybe obtained via one-movementone-movement oror two-two- movement techniques.movement techniques. Sites of venipuncture includes:Sites of venipuncture includes:  Antecubital fossa veins (most preferred site) –Antecubital fossa veins (most preferred site) – median cubital veins and cephalic veinsmedian cubital veins and cephalic veins  Wrist veins, ankle veins, hand veinsWrist veins, ankle veins, hand veins
  • 77.  Complications of venipuncture-classified asComplications of venipuncture-classified as immediate or delayedimmediate or delayed andand local or systemiclocal or systemic  HematomaHematoma due to missed veindue to missed vein  Collapsed small veinsCollapsed small veins due to excessive pull ofdue to excessive pull of plungerplunger  SyncopeSyncope  Excessive bleedingExcessive bleeding  Thrombosis of veinThrombosis of vein  Infection is situInfection is situ  Blood-borne infectionBlood-borne infection like hepatitis B andlike hepatitis B and AIDSAIDS
  • 78. ConsiderationsConsiderations  Clean the venipuncture site with 70% alcohol orClean the venipuncture site with 70% alcohol or 1% iodine.1% iodine.  Apply the tourniquet several inches above theApply the tourniquet several inches above the puncture site.puncture site.  Never leave the tourniquet longer than oneNever leave the tourniquet longer than one minute.minute.  The needle, as it enters the skin, is positioned atThe needle, as it enters the skin, is positioned at approximately 15-degree angle to the site with theapproximately 15-degree angle to the site with the bevel upbevel up..  Ensure prompt and adequate mixture of bloodEnsure prompt and adequate mixture of blood and anticoagulant to prevent coagulation and toand anticoagulant to prevent coagulation and to prevent formation of “unwanted” blood clot.prevent formation of “unwanted” blood clot.  Blood should not be drawn in an area withBlood should not be drawn in an area with intravenous line.intravenous line.
  • 79. Safety Measures in PhlebotomySafety Measures in Phlebotomy (1) Observe universal (standard) safety precautions. (1) Observe universal (standard) safety precautions.  (2) Wash hands in warm, running water with the chlorhexidine(2) Wash hands in warm, running water with the chlorhexidine gluconate hand washing product (approved by the Infectiongluconate hand washing product (approved by the Infection Control Committee), or if not visibly contaminated with aControl Committee), or if not visibly contaminated with a commercial foaming hand wash product before and after eachcommercial foaming hand wash product before and after each patient collection.patient collection. (3) Gloves are to be worn during all phlebotomies, and changed(3) Gloves are to be worn during all phlebotomies, and changed between patient collections. Palpation of phlebotomy site may bebetween patient collections. Palpation of phlebotomy site may be performed without gloves providing the skin is not broken.performed without gloves providing the skin is not broken. (4) A lab coat or gown must be worn during blood collection(4) A lab coat or gown must be worn during blood collection procedures.procedures.
  • 80. (5) Needles and hubs are single use and are disposed of in an(5) Needles and hubs are single use and are disposed of in an appropriate 'sharps' container as one unit. NOTE:appropriate 'sharps' container as one unit. NOTE: NeedlesNeedles are never recapped, removed, broken, or bent afterare never recapped, removed, broken, or bent after phlebotomy procedure.phlebotomy procedure. (6) Gloves are to be discarded in the appropriate container(6) Gloves are to be discarded in the appropriate container immediately after the phlebotomy procedure. All other itemsimmediately after the phlebotomy procedure. All other items used for the procedure must be disposed of according toused for the procedure must be disposed of according to proper biohazardous waste disposal policy.proper biohazardous waste disposal policy. (7) Contaminated surfaces must be cleaned with freshly(7) Contaminated surfaces must be cleaned with freshly prepared 10% bleach solution.  All surfaces are cleaned dailyprepared 10% bleach solution.  All surfaces are cleaned daily with bleach.with bleach. (8) In the case of an accidental needlestick, immediately wash(8) In the case of an accidental needlestick, immediately wash the area with an antibacterial soap, express blood from thethe area with an antibacterial soap, express blood from the wound.wound.
  • 81. Factors to consider in site selectionFactors to consider in site selection * Extensive scarring or healed burn areas should be avoided* Extensive scarring or healed burn areas should be avoided * Specimens should not be obtained from the arm on the same side* Specimens should not be obtained from the arm on the same side as a mastectomy.as a mastectomy. * Avoid areas of hematoma.* Avoid areas of hematoma. * If an IV is in place, samples may be obtained below but NEVER* If an IV is in place, samples may be obtained below but NEVER above the IV site. above the IV site.  * Do not obtain specimens from an arm having a cannula, fistula, or* Do not obtain specimens from an arm having a cannula, fistula, or vascular graft.vascular graft.                      * Allow 10-15 minutes after a transfusion is completed before* Allow 10-15 minutes after a transfusion is completed before obtaining a blood sample.obtaining a blood sample.
  • 82. Which of the following areWhich of the following are allowed in phlebotomy?allowed in phlebotomy? ________Observe universal safety precautions.Observe universal safety precautions. ___Handwashing before and after phlebotomy.___Handwashing before and after phlebotomy. ______Collecting blood without a lab gown.Collecting blood without a lab gown. ___Recapping or removal of needles after withdrawing the___Recapping or removal of needles after withdrawing the needle.needle. ______Reuse of gloves for as long as they were autoclavedReuse of gloves for as long as they were autoclaved beforehand.beforehand. ___Weekly cleaning of all lab surfaces with 10% bleach___Weekly cleaning of all lab surfaces with 10% bleach solution.solution. ______Outpatients are identified by making them say their nameOutpatients are identified by making them say their name and date of birth.and date of birth. ___Doing the venipuncture twice on a patient.___Doing the venipuncture twice on a patient.
  • 83. ______Obtaining blood below the IV line of a patient.Obtaining blood below the IV line of a patient. ______Mixing of anticoagulated blood by shaking the tube gentlyMixing of anticoagulated blood by shaking the tube gently 5-8 times.5-8 times. ___Obtaining a blood sample from a patient who underwent___Obtaining a blood sample from a patient who underwent blood transfusion just 30 minutes ago.blood transfusion just 30 minutes ago. ______Drawing blood from the feet.Drawing blood from the feet. ___Applying tourniquet for two minutes.___Applying tourniquet for two minutes. ______Avoiding to touch again a thoroughly cleaned punctureAvoiding to touch again a thoroughly cleaned puncture site.site. ___Collecting SST before a yellow vacutainer in one-___Collecting SST before a yellow vacutainer in one- movement technic.movement technic. ______Collecting PST before a dark green heparinized tube inCollecting PST before a dark green heparinized tube in one-movement technicone-movement technic..
  • 84. Quick QuizQuick Quiz  Which of the following is an unnecessary step in theWhich of the following is an unnecessary step in the proper phlebotomy procedure?proper phlebotomy procedure? a.a. recapping the needle after userecapping the needle after use b.b. placing bandage over the punctured siteplacing bandage over the punctured site c.c. washing the hands prior to venipuncturewashing the hands prior to venipuncture d.d. sterilizing the puncture sitesterilizing the puncture site  Which vein cannot be used in obtaining venousWhich vein cannot be used in obtaining venous blood in an adult?blood in an adult? a.a. radial veinradial vein b.b. jugular veinjugular vein c.c. ankle veinankle vein d.d. dorsal hand veindorsal hand vein
  • 85. Arterial punctureArterial puncture  Arterial blood isArterial blood is oxygenated bloodoxygenated blood. It is uniform in. It is uniform in composition throughout the body.composition throughout the body.  Arterial blood is used to measureArterial blood is used to measure oxygen tension,oxygen tension, carbon dioxide tensioncarbon dioxide tension andand blood pH.blood pH.  Blood gas analysesBlood gas analyses (BGA) are critical to patients(BGA) are critical to patients withwith pulmonary problems, oxygen therapy,pulmonary problems, oxygen therapy, cardiovascular problems and those undergoingcardiovascular problems and those undergoing major operationsmajor operations..
  • 86.  Sites of arterial punctureSites of arterial puncture  Radial arteriesRadial arteries  Brachial arteriesBrachial arteries  Femoral arteriesFemoral arteries  Radial and brachial arteriesRadial and brachial arteries are the preferred sites.are the preferred sites.  Each site of arterial puncture uses different gauge ofEach site of arterial puncture uses different gauge of needles:needles:  Brachial artery- 18-20 gaugeBrachial artery- 18-20 gauge  Radial artery- 23-25 gaugeRadial artery- 23-25 gauge  In collecting arterial blood, needle is positioned atIn collecting arterial blood, needle is positioned at different angles in each site:different angles in each site:  Brachial artery- 45-60 degreesBrachial artery- 45-60 degrees  Femoral artery- 90 degreesFemoral artery- 90 degrees  HeparinHeparin is used as anticoagulant for arterial puncture.is used as anticoagulant for arterial puncture.
  • 87.  Complications of arterial punctureComplications of arterial puncture  HematomaHematoma due to increased pressure indue to increased pressure in the arterythe artery  Restriction of blood flowRestriction of blood flow due to reflexdue to reflex constrictionconstriction ((arterial spasmarterial spasm))  Temporary discomfortTemporary discomfort ((aching,aching, throbbing, tenderness, sharp sensation, andthrobbing, tenderness, sharp sensation, and cramping)cramping)  Thrombosis, hemorrhage and infectionThrombosis, hemorrhage and infection
  • 88. ConsiderationsConsiderations  Intense care should be administered to patients undergoingIntense care should be administered to patients undergoing arterial puncture.arterial puncture.  Sites that are irritated, edematous, near a wound or in anSites that are irritated, edematous, near a wound or in an area of arteriovenous (AV) shunt or fistula should not bearea of arteriovenous (AV) shunt or fistula should not be selected for arterial puncture.selected for arterial puncture.  Samples are placed in ice water or other coolantSamples are placed in ice water or other coolant (temperature of(temperature of 1-51-5ººCC)) toto minimize leukocyteminimize leukocyte consumption of oxygen.consumption of oxygen.  Capillary blood may be aCapillary blood may be a suitable substitute forsuitable substitute for arterial blood determination of pH and pCO2arterial blood determination of pH and pCO2 provided that the site must be warmed prior to collection.provided that the site must be warmed prior to collection. Warning increases blood flow through capillaries andWarning increases blood flow through capillaries and arterioles and result in arterial-rich blood.arterioles and result in arterial-rich blood.
  • 89. Quick QuizQuick Quiz  Which test does not require an arterial blood sample?Which test does not require an arterial blood sample? a.a. oxygen tensionoxygen tension b.b. blood pHblood pH c.c. pCOpCO22 determinationdetermination d.d. plasma hemoglobinplasma hemoglobin  Which statement is false regarding arterial blood?Which statement is false regarding arterial blood? a.a. Arterial blood collection always requires the useArterial blood collection always requires the use of anticoagulant.of anticoagulant. b.b. Arterial blood is a must in blood gas analyses.Arterial blood is a must in blood gas analyses. c.c. Arterial blood collection requires a glass syringeArterial blood collection requires a glass syringe to minimize leukocyte consumption of gases.to minimize leukocyte consumption of gases. d.d. Arterial blood has lower pCOArterial blood has lower pCO22, therefore, is less, therefore, is less acidic than venous blood.acidic than venous blood.
  • 90. Skin puncture/Capillary punctureSkin puncture/Capillary puncture Capillary blood is anCapillary blood is an admixture of venous, arterial andadmixture of venous, arterial and capillary blood and may contain tissue fluidscapillary blood and may contain tissue fluids.. Skin punctures are suitable inSkin punctures are suitable in pediatric patients, obesepediatric patients, obese patients with thrombotic tendencies and severe burnspatients with thrombotic tendencies and severe burns It isIt is often preferred to geriatric patientsoften preferred to geriatric patients due todue to thinness and less elasticity of skin.thinness and less elasticity of skin. Sites of skin punctureSites of skin puncture  InfantsInfants  Lateral plantar heel surfaceLateral plantar heel surface  Median plantar heel surfaceMedian plantar heel surface  ChildrenChildren  Plantar surface of the big toePlantar surface of the big toe  Plantar surface of the last digit of the 2Plantar surface of the last digit of the 2ndnd , 3, 3rdrd or 4or 4thth fingersfingers may be used in older infantsmay be used in older infants  Lateral side of the finger adjacent to the nailLateral side of the finger adjacent to the nail  Ear lobeEar lobe
  • 91.  Capillary blood can be collected for analysis of bloodCapillary blood can be collected for analysis of blood gas (site must be pre-warmed before collection).gas (site must be pre-warmed before collection).  The best method of arterial blood collection inThe best method of arterial blood collection in newbornnewborn is theis the umbilical artery catheterumbilical artery catheter..  Skin puncture site must be warmed before collectionSkin puncture site must be warmed before collection to increase the blood flow (to increase the blood flow (arterializationarterialization).). ThermalThermal using dry heat or paper towel withusing dry heat or paper towel with warm water (39-42warm water (39-42OO C)C) MechanicalMechanical byby fflicking with index finger untillicking with index finger until flushing is observedflushing is observed ChemicalChemical usingusing Trafuril paste or Histamine creamsTrafuril paste or Histamine creams
  • 92.  Arterial blood collected from capillaryArterial blood collected from capillary puncture may yieldpuncture may yield unreliableunreliable results if:results if:  Systolic pressures is less than 95Systolic pressures is less than 95 mmhgmmhg  Cardiac output is severely restrictedCardiac output is severely restricted  Vasoconstriction is presentVasoconstriction is present  Puncture depth ranges from 0.85 to 2.00Puncture depth ranges from 0.85 to 2.00 mm deep and 1.75-3.00 mm in length.mm deep and 1.75-3.00 mm in length.
  • 93. Quick QuizQuick Quiz  Which test requires finger skin puncture?Which test requires finger skin puncture? a.a. Rumpel-LeedeRumpel-Leede b.b. Schryver and WaughSchryver and Waugh c.c. Ivy Bleeding TimeIvy Bleeding Time d.d. Lee-WhiteLee-White  Which blood collection technic requires an armWhich blood collection technic requires an arm maneuver known as Allen test to check for collateralmaneuver known as Allen test to check for collateral circulation?circulation? a.a. phlebotomyphlebotomy b.b. arterial puncturearterial puncture c.c. skin punctureskin puncture d.d. peripheral punctureperipheral puncture
  • 94. VACUTAINER/EVACUATED TUBESVACUTAINER/EVACUATED TUBES  These are tubes for blood collection whichThese are tubes for blood collection which areare color-coded based on the anticoagulantcolor-coded based on the anticoagulant presentpresent. They come in various sizes; 2, 5, 7,. They come in various sizes; 2, 5, 7, and 10 ml.and 10 ml.  Blood is drawn in this order:Blood is drawn in this order: Blood cultureBlood culture tubestubes,, red topred top,, blue topblue top,, green topgreen top,, lavenderlavender toptop andand gray topgray top
  • 95. COLORCOLOR ADDITIVEADDITIVE ACTIONACTION USEUSE LavenderLavender EthylenediaminetetraaceticEthylenediaminetetraacetic chelates calcium hematologic assayschelates calcium hematologic assays Acid (EDTA)Acid (EDTA) lead assay, CEAlead assay, CEA ** VerseneVersene (disodium salt)(disodium salt) Determination andDetermination and ** SequestreneSequestrene (dipotassium salt)(dipotassium salt) cell countscell counts RedRed NoneNone Allows blood to clot Most chemistry,Allows blood to clot Most chemistry, immunologic andimmunologic and blood bank testsblood bank tests RedRed GrayGray oror None but containsNone but contains Allows blood to clot Most chemistry testsAllows blood to clot Most chemistry tests Red BlackRed Black separator materialseparator material and serves as aand serves as a barrier between cellsbarrier between cells and serumand serum YellowYellow Citrate dextroseCitrate dextrose preserves RBCs Blood culturepreserves RBCs Blood culture GreenGreen Heparin (Na+, Li+,Heparin (Na+, Li+, inhibits thrombin ammonia CO-Hbinhibits thrombin ammonia CO-Hb or, NHor, NH44 ++ )) activation and methemoglobinactivation and methemoglobin
  • 96. COLORCOLOR ADDITIVEADDITIVE ACTIONACTION USEUSE OrangeOrange ThrombinThrombin accelarated clotaccelarated clot STAT serumSTAT serum teststests BlueBlue Buffered citrateBuffered citrate binds calciumbinds calcium Coagulation assaysCoagulation assays like PT & APTTlike PT & APTT BlackBlack Buffered sodiumBuffered sodium binds calciumbinds calcium Westergren ESRWestergren ESR citratecitrate GrayGray NaF/KNaF/K22CC22OO44 Inhibits glycolyticInhibits glycolytic GlucoseGlucose enzymeenzyme enolaseenolase and determinationand determination act as anticoagulantact as anticoagulant IodoacetateIodoacetate inhibits glycolyticinhibits glycolytic GlucoseGlucose enzymeenzyme glyceraldehydeglyceraldehyde determinationdetermination 3-phosphate3-phosphate dehydrogenasedehydrogenase
  • 97. Anticoagulant InterferenceAnticoagulant Interference  Dilution errorsDilution errors especially oxalates which areespecially oxalates which are highly osmotichighly osmotic  Inhibition of plasma enzyme activitiesInhibition of plasma enzyme activities activitiesactivities especially withespecially with fluoridefluoride which is an enzymewhich is an enzyme poison,poison, EDTAEDTA which chelates metallic enzymeswhich chelates metallic enzymes activators.activators. OxalateOxalate inhibitsinhibits AMS, LD and ACP,AMS, LD and ACP, andand citratecitrate whichwhich inhibitsinhibits AMSAMS  Oxalates, citrate and EDTAOxalates, citrate and EDTA lower plasma calciumlower plasma calcium levelslevels  False increase in electrolyte analysesFalse increase in electrolyte analyses due to thedue to the anticoagulants in the salt formanticoagulants in the salt form
  • 98.   If multiple tubes are needed, theIf multiple tubes are needed, the proper order of drawproper order of draw toto avoid crossavoid cross contamination and erroneous resultscontamination and erroneous results is as follows: is as follows:  1st - 1st -  Blood culture vials or bottles, sterile Blood culture vials or bottles, sterile ((yellowyellow oror yellowyellow--blackblack top)top) 2nd -2nd - Coagulation tubeCoagulation tube ((light bluelight blue top) NOTE:top) NOTE: If just a routine coagulation assayIf just a routine coagulation assay is the only test ordered, then a single light blue top tube may be drawn. If there is a concernis the only test ordered, then a single light blue top tube may be drawn. If there is a concern regarding contamination by tissue fluids or thromboplastins, then one may draw a non-regarding contamination by tissue fluids or thromboplastins, then one may draw a non- additive tube first, and then the light blue top tube.additive tube first, and then the light blue top tube. 3rd -3rd - Non-additive tube or Serum tubeNon-additive tube or Serum tube Last draw - additive tubes in this order:Last draw - additive tubes in this order: 1. SST (1. SST (redred--graygray oror goldgold top). Contains a gel separator and clot activator.top). Contains a gel separator and clot activator. 2. Sodium heparin (2. Sodium heparin (dark greendark green top)top) 3. PST (3. PST (light greenlight green top). Contains lithium heparin anticoagulant and a geltop). Contains lithium heparin anticoagulant and a gel separator.separator. 4. EDTA (4. EDTA (lavenderlavender top)top) 5. ACDA or ACDB (5. ACDA or ACDB (pale yellowpale yellow top). Contains acid citratetop). Contains acid citrate dextrose.dextrose. 6. Oxalate/fluoride (6. Oxalate/fluoride (light graylight gray top)top)
  • 99. Quick QuizQuick Quiz  The light-sensitive substance that provides the normalThe light-sensitive substance that provides the normal straw color of serum and plasma isstraw color of serum and plasma is a.a. urochromeurochrome b.b. bilirubinbilirubin c.c. flavin compoundsflavin compounds d.d. beta-carotenebeta-carotene  TheThe vacutainer required in the measurement of serumvacutainer required in the measurement of serum ammonia isammonia is a.a. green topgreen top b.b. red topred top c.c. blue topblue top d.d. lavender toplavender top
  • 100. Specimen Handling and ProcessingSpecimen Handling and Processing SerumSerum  20-30 minutes is the ideal clotting time20-30 minutes is the ideal clotting time  Generally more preferred than plasmaGenerally more preferred than plasma 1.1. Interfering substances are co-precipitatedInterfering substances are co-precipitated during clotting such as LPLduring clotting such as LPL 2.2. Optically clearerOptically clearer 3.3. Free from anticoagulant interferenceFree from anticoagulant interference  Must ideally reach the laboratory within 45Must ideally reach the laboratory within 45 minutesminutes  Agitation must be avoided during transportAgitation must be avoided during transport  Use amber containers for photolabileUse amber containers for photolabile substancessubstances  Transport in ice (4 deg C) those specimens forTransport in ice (4 deg C) those specimens for BGA, renin, enzymes and catecholaminesBGA, renin, enzymes and catecholamines
  • 101. Specimen InterferenceSpecimen Interference Lysis of cells or Laking (Hemolyzed serum)Lysis of cells or Laking (Hemolyzed serum)  Results in leakage of intracellular substancesResults in leakage of intracellular substances  Lysis of RBCs is calledLysis of RBCs is called laking or hemolysislaking or hemolysis which may occurwhich may occur in vivo or in vitroin vivo or in vitro  In vitro hemolysisIn vitro hemolysis is more common which mayis more common which may be due to:be due to: 1.1. Use of vacuum tubesUse of vacuum tubes 2.2. Vigorous mixingVigorous mixing 3.3. Use of too narrow or too wide needle boresUse of too narrow or too wide needle bores 4.4. Effect of alcoholEffect of alcohol 5.5. Centrifugation and separation stepsCentrifugation and separation steps  Hemolysis is visible only not until aHemolysis is visible only not until a 200 mg/L200 mg/L of hemoglobin level in presentof hemoglobin level in present
  • 102. Icteresia (Icteric serum)Icteresia (Icteric serum)  Intensely yellow serum sampleIntensely yellow serum sample due todue to elevated bilirubinelevated bilirubin valuevalue  Jaundice in a patient is caused by aJaundice in a patient is caused by a bilirubin level of greater than 430bilirubin level of greater than 430 µM (25µM (25 mg/L)mg/L)  Bilirubin interferes with tests using dyesBilirubin interferes with tests using dyes and turbidity testsand turbidity tests  Interference due to bilirubin may beInterference due to bilirubin may be minimized by sample blankingminimized by sample blanking or dualor dual wavelength method known as thewavelength method known as the AllenAllen correctioncorrection methodmethod
  • 103. Lactescence (Lipemic serum)Lactescence (Lipemic serum)  Obtained normally after a meal due toObtained normally after a meal due to elevated chylomicronselevated chylomicrons  Characterized byCharacterized by milky or highly turbidmilky or highly turbid serumserum  Lactescence appears when theLactescence appears when the TAG levelTAG level reaches 4.6 mM (4g/L)reaches 4.6 mM (4g/L)  Errors due to lipemia may beErrors due to lipemia may be correctedcorrected by ultracentrifugation of the serumby ultracentrifugation of the serum samplesample
  • 104. Grounds for rejecting a specimenGrounds for rejecting a specimen  Inadequate sample identificationInadequate sample identification  Insufficient volume of specimen collectionInsufficient volume of specimen collection  Inappropriate collection tubeInappropriate collection tube  HemolysisHemolysis  Improper transportationImproper transportation  InterferencesInterferences
  • 105. Last Quick QuizLast Quick Quiz  Which sample below will likely requireWhich sample below will likely require ultracentrifugation to minimize sample interference?ultracentrifugation to minimize sample interference? a.a. icteresiaicteresia b.b. lipemialipemia c.c. lakinglaking d.d. all of the aboveall of the above  Prompt separation of the clot from serum shouldPrompt separation of the clot from serum should always be observed because this substancealways be observed because this substance substantially decrease by 5% per hour if serum is leftsubstantially decrease by 5% per hour if serum is left in contact with clot. This substance isin contact with clot. This substance is a.a. sodiumsodium b.b. albuminalbumin c.c. glucoseglucose d.d. phosphatephosphate
  • 106. QUALITY MANAGEMENTQUALITY MANAGEMENT Quality Assurance (QA)Quality Assurance (QA)  includes maneuvers encountered in theincludes maneuvers encountered in the analyticanalytic,, pre-analytic and post-analyticpre-analytic and post-analytic phasesphases of laboratory testingof laboratory testing Pre-analytical phasePre-analytical phase includes:includes:  test orderingtest ordering  specimen collectionspecimen collection  transport of the specimen in the laboratorytransport of the specimen in the laboratory
  • 107. Pre-analytical phase includes:Pre-analytical phase includes:  processing of specimenprocessing of specimen  entering patient informationentering patient information  centrifugingcentrifuging  separating aliquoting specimenseparating aliquoting specimen  delivery to proper laboratory locationdelivery to proper laboratory location
  • 108. Analytical phaseAnalytical phase includesincludes  specimen analysis (manual or automated)specimen analysis (manual or automated)  use of commercial controlsuse of commercial controls  record keepingrecord keeping Post-analytical phasePost-analytical phase includesincludes  reporting out results of analysis (manual orreporting out results of analysis (manual or computerized)computerized)  physician contactphysician contact
  • 109. Post-analytical phase also includesPost-analytical phase also includes  monitors quality performance starting frommonitors quality performance starting from the ordering of a laboratory determinationthe ordering of a laboratory determination to its reporting, interpretation of results,to its reporting, interpretation of results, and then application to patient careand then application to patient care  involves total quality control which requiresinvolves total quality control which requires constant attention of all involved in theconstant attention of all involved in the laboratory testinglaboratory testing
  • 110. Quality Control (QC)Quality Control (QC)  is concerned with theis concerned with the analytic phaseanalytic phase of QAof QA  monitors themonitors the over-all reliability of laboratoryover-all reliability of laboratory resultsresults in terms ofin terms of accuracy and precisionaccuracy and precision  relies on the diligent and persistent execution of therelies on the diligent and persistent execution of the following QC associated activities:following QC associated activities:  assay of control samplesassay of control samples  instrument maintenanceinstrument maintenance  statistical data analysesstatistical data analyses  proficiency testing surveyproficiency testing survey
  • 111.  has two major typeshas two major types External QC (Interlaboratory QC)External QC (Interlaboratory QC) was establishedwas established byby BelkBelk which monitors primarily thewhich monitors primarily the accuracyaccuracy ofof laboratory tests; the use oflaboratory tests; the use of Youden plotsYouden plots Internal QC (Intralaboratory QC)Internal QC (Intralaboratory QC) was establishedwas established byby LeveyLevey which primarily monitors the day-to-daywhich primarily monitors the day-to-day performance of laboratory tests -performance of laboratory tests - precisionprecision assessment. Intralaboratory QC can be basedassessment. Intralaboratory QC can be based either from the results of control specimens or oneither from the results of control specimens or on the results of patient specimens; the use ofthe results of patient specimens; the use of Levey-Levey- Jennings chartJennings chart
  • 112. Quick QuizQuick Quiz  Which QC chart is a graphic representation of theWhich QC chart is a graphic representation of the acceptable limits of variation in the results of anacceptable limits of variation in the results of an analytical method?analytical method? a.a. GaussianGaussian b.b. YoudenYouden c.c. Levey-JenningsLevey-Jennings d.d. CusumCusum  Which of the following has as its purpose to promoteWhich of the following has as its purpose to promote the incorporation of principles of qualitythe incorporation of principles of quality management and QA into daily work routines?management and QA into daily work routines? a.a. ISO 9000ISO 9000 b.b. NCCLSNCCLS c.c. CAPCAP d.d. NISTNIST
  • 113. ErrorsErrors Analytical errorsAnalytical errors are usuallyare usually systematic errors orsystematic errors or determinate errorsdeterminate errors that are caused by some factors inthat are caused by some factors in the analytical system such asthe analytical system such as erroneously calibratederroneously calibrated pipettor, deteriorating reagentpipettor, deteriorating reagent andand fluctuatingfluctuating electrical current.electrical current. Personnel or operator errorsPersonnel or operator errors are usually calledare usually called random errors or indeterminate errorsrandom errors or indeterminate errors that usuallythat usually affect several analyses. Examples areaffect several analyses. Examples are mislabeling themislabeling the specimen, wrong number entryspecimen, wrong number entry andand instability ofinstability of needle due to electronic component ofneedle due to electronic component of instrumentinstrument..
  • 114. Interpretation of the Levey-Jennings ChartInterpretation of the Levey-Jennings Chart SubstanceSubstance +3SD+3SD +2SD+2SD +1SD+1SD MeanMean -1SD-1SD -2SD-2SD -3SD-3SD Days of a particular monthDays of a particular month Shift Dispersion Trend
  • 115. TrendTrend suggests asuggests a systematic driftsystematic drift or error. Valuesor error. Values move continuously away from the mean in just onemove continuously away from the mean in just one direction. Among its causes include deterioratingdirection. Among its causes include deteriorating reagents, changes in standard concentration or failingreagents, changes in standard concentration or failing instrument.instrument. ShiftShift also suggestsalso suggests systematic errorsystematic error. Seen as an abrupt. Seen as an abrupt change from the established mean and continue in achange from the established mean and continue in a linear fashion parallel to the mean.linear fashion parallel to the mean. Increased dispersionIncreased dispersion suggestssuggests random errorrandom error. High or. High or low outliers are frequently observed. Causes of this arelow outliers are frequently observed. Causes of this are variations in operation of instrument, interferingvariations in operation of instrument, interfering substances, electronic fluctuations, and clerical errors.substances, electronic fluctuations, and clerical errors.
  • 116. Quick QuizQuick Quiz  Which of the following terms refers to deviationWhich of the following terms refers to deviation from the true value caused by indeterminate errorsfrom the true value caused by indeterminate errors inherent in every laboratory management?inherent in every laboratory management? a.a. random errorrandom error b.b. standard error of the meanstandard error of the mean c.c. parametric analysisparametric analysis d.d. nonparametric analysisnonparametric analysis  When comparing a potential new test with a comparativeWhen comparing a potential new test with a comparative method in order to bring a new method into themethod in order to bring a new method into the laboratory, one observes error that is consistentlylaboratory, one observes error that is consistently affecting results in one direction. This type of error isaffecting results in one direction. This type of error is a.a. systematic errorsystematic error b.b. random errorrandom error c.c. constant systematic errorconstant systematic error d.d. proportional randomproportional random errorerror
  • 117. Westgard Multi-Rule TechnicWestgard Multi-Rule Technic RuleRule MeaningMeaning 1:2s1:2s = one control observation exceeds the control limit set at= one control observation exceeds the control limit set at M±2SD; warning ruleM±2SD; warning rule 1:3s1:3s = reject when one observation exceeds M±3SD; suggests= reject when one observation exceeds M±3SD; suggests random errorrandom error 2:2s2:2s = reject when two consecutive observations exceed the same= reject when two consecutive observations exceed the same M+2SD or the same M-2SD; suggests systematicM+2SD or the same M-2SD; suggests systematic errorerror R:4sR:4s = reject when one control observation in the run exceeds its= reject when one control observation in the run exceeds its M+2SD and another exceeds M-2SD; suggestsM+2SD and another exceeds M-2SD; suggests random errorsrandom errors 4:1s4:1s = reject when four consecutive control observations exceed= reject when four consecutive control observations exceed the same M+1SD limit or the same M-1SD limit; suggeststhe same M+1SD limit or the same M-1SD limit; suggests systematic errorsystematic error 10:Mean10:Mean= reject when 10 consecutive control observations= reject when 10 consecutive control observations fall on one side of the mean; suggests systematic errorfall on one side of the mean; suggests systematic error
  • 118. Quick QuizQuick Quiz  What does the preparation of a Levey-Jennings chart forWhat does the preparation of a Levey-Jennings chart for any single constituent of serum require?any single constituent of serum require? a.a. analysis of control serum over a period of 20analysis of control serum over a period of 20 consecutive daysconsecutive days b.b. 20-30 analyses of the control serum, on 1 day, in one20-30 analyses of the control serum, on 1 day, in one batchbatch c.c. analyses consistently performed by one personanalyses consistently performed by one person d.d. weekly analyses of the control serum for 1 monthweekly analyses of the control serum for 1 month  In addition to utilizing Levey-Jennings charts, what otherIn addition to utilizing Levey-Jennings charts, what other criteria should be applied to interpret internal QC data?criteria should be applied to interpret internal QC data? a.a. Westgard multiruleWestgard multirule b.b. CusumCusum c.c. linear regressionlinear regression d.d. YoudenYouden
  • 119. Accuracy and PrecisionAccuracy and Precision AccuracyAccuracy is the extent to which theis the extent to which the meanmean measurement is close to the true value.measurement is close to the true value.  The accuracy of the method is reflected by itsThe accuracy of the method is reflected by its ability to obtain the same values of the referenceability to obtain the same values of the reference samples of known concentration.samples of known concentration. Expressed asExpressed as %% absolute error.absolute error. % absolute error = true value – actual value x 100%% absolute error = true value – actual value x 100% true valuetrue value PrecisionPrecision is theis the reproducibilityreproducibility of a laboratoryof a laboratory determination when it is run repeatedly underdetermination when it is run repeatedly under identical conditionsidentical conditions  precision is commonly expressed in terms ofprecision is commonly expressed in terms of standard deviation (SD), variancestandard deviation (SD), variance oror coefficient ofcoefficient of variation (CV)variation (CV)
  • 120. Standard deviation =Standard deviation = ΣΣ (M – x)(M – x)22 n-1n-1 Variance = (SD)Variance = (SD)22 Coefficient of variation = SD x 100%Coefficient of variation = SD x 100% MM ReliabilityReliability refers to the ability of arefers to the ability of a test totest to maintain its accuracy and precision for anmaintain its accuracy and precision for an extended period of timeextended period of time..
  • 121. Quick QuizQuick Quiz  Which of the following terms refers to theWhich of the following terms refers to the closeness with which the measured value agreescloseness with which the measured value agrees with the true value?with the true value? a.a. random errorrandom error b.b. precisionprecision c.c. accuracyaccuracy d.d. variancevariance  Given that a method mean is 25 mg/dl and theGiven that a method mean is 25 mg/dl and the SD is 1.2 mg/dl, what would be the CV?SD is 1.2 mg/dl, what would be the CV? a.a. 2.1%2.1% b.b. 2.4%2.4% c.c. 4.8%4.8% d.d. 9.6%9.6%
  • 122.  SDSD is a measure of dispersion of the values aroundis a measure of dispersion of the values around thethe mean and in normal or Gaussian distribution,mean and in normal or Gaussian distribution, 68%68% of the values fall withinof the values fall within +/-1 SD around the mean+/-1 SD around the mean 95%95% of the values fall withinof the values fall within +/-2 SD around the mean+/-2 SD around the mean 99.7%99.7% of the values fall withinof the values fall within +/-3 SD around the mean+/-3 SD around the mean  most laboratories choose themost laboratories choose the 95% confidence limit95% confidence limit inin expressing precisionexpressing precision  thethe medianmedian is the value that is middlemost in an array ofis the value that is middlemost in an array of numbers while thenumbers while the modemode is the value that occurs mostis the value that occurs most frequently.frequently.  In aIn a Gaussian distributionGaussian distribution, the mean, median and mode, the mean, median and mode are very close in value as shown by its bell-shaped curve.are very close in value as shown by its bell-shaped curve.
  • 123. Quick QuizQuick Quiz  In a QC program the confidence interval hasIn a QC program the confidence interval has been set at 95%. How many test results arebeen set at 95%. How many test results are expected to fall beyond the established limits?expected to fall beyond the established limits? a.a. 1 in 51 in 5 b.b. 1 in 101 in 10 c.c. 1 in 201 in 20 d.d. 1 in 951 in 95  What percentage of values will fall betweenWhat percentage of values will fall between ±2SD in a Gaussian distribution?±2SD in a Gaussian distribution? a.a. 34.13%34.13% b.b. 68.26%68.26% c.c. 95.45%95.45% d.d. 99.74%99.74%
  • 124. SensitivitySensitivity is the ability of the test tois the ability of the test to detect thedetect the smallest amount of the analyte in a solution orsmallest amount of the analyte in a solution or samplesample. It expresses the ability of the test to be. It expresses the ability of the test to be positive in the presence of the analyte or thepositive in the presence of the analyte or the disease. A highly sensitive test is characterized by adisease. A highly sensitive test is characterized by a decreased probability of obtaining false negativedecreased probability of obtaining false negative resultsresults.. SpecificitySpecificity refers to the ability of the test torefers to the ability of the test to detectdetect analyte without detecting other analytes that areanalyte without detecting other analytes that are also present in the samplealso present in the sample. It expresses the ability of. It expresses the ability of the test to be negative in the absence of the analytethe test to be negative in the absence of the analyte or the disease. A highly specific test leads to aor the disease. A highly specific test leads to a decreased probability of obtaining false positivedecreased probability of obtaining false positive results.results.
  • 125. ControlControl is a solution (usuallyis a solution (usually pooled serumpooled serum samplessamples) whose constituents are) whose constituents are diversediverse but arebut are known (known (a range of values per analytea range of values per analyte). This). This can be run simultaneously with thecan be run simultaneously with the TestTest toto check, verify or validate the accuracy of thecheck, verify or validate the accuracy of the results.results. StandardStandard is a solution ofis a solution of a particular analytea particular analyte ofof known characteristics and known value (known characteristics and known value (exactexact concentrationconcentration). It is used as). It is used as reference for thereference for the calculation of the value of the Unknowncalculation of the value of the Unknown..
  • 126. Quick QuizQuick Quiz  To determine the predictive value of a positive test, allTo determine the predictive value of a positive test, all of the following parameters must be knownof the following parameters must be known exceptexcept a.a. sensitivitysensitivity b.b. specificityspecificity c.c. disease prevalencedisease prevalence d.d. precisionprecision  Which activity will not check on the accuracy of theWhich activity will not check on the accuracy of the results obtained?results obtained? a.a. make a Youden plotmake a Youden plot b.b. use of abnormal controluse of abnormal control c. use of normal controlc. use of normal control d.d. use of a calibratoruse of a calibrator (standard)(standard)
  • 127. Figures of Merit of AnalyticalFigures of Merit of Analytical MethodsMethods  MajorMajor 1.1. AccuracyAccuracy 2. Precision2. Precision 3. Limit of detection3. Limit of detection 4. Applicable concentration range or4. Applicable concentration range or linear rangelinear range 5. Sensitivity5. Sensitivity 6. Selectivity6. Selectivity
  • 128.  MinorMinor 1.1. SpeedSpeed 2. Ease and convenience2. Ease and convenience 3. Skill required of the3. Skill required of the operatoroperator 4. Cost of analysis and4. Cost of analysis and availability of equipmentavailability of equipment 5. Per sample cost5. Per sample cost
  • 129. EndEnd