CLINICAL CLINICAL CHEMISTRYCHEMISTRY

Julius P. Mario, RMT, MS Chem.Julius P. Mario, RMT, MS Chem.

The purpose & function of laboratorians The purpose & function of laboratorians through clinical pathology & laboratory through 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 prognosis Establishing a prognosis

4.4. Detecting disease through case finding or screeningDetecting disease through case finding or screening

5.5. Monitoring follow-up therapyMonitoring follow-up therapy

Clinical ChemistryClinical Chemistry

The The systematic studysystematic study of of

biochemical processesbiochemical processes associated associated with with health & diseasehealth & disease & &

the the measurement of constituentsmeasurement of constituents in in body fluids or tissuesbody fluids or tissues to to

facilitate facilitate diagnosis of disease.diagnosis of disease.

SCOPE OF CLINICAL CHEMISTRYSCOPE OF CLINICAL CHEMISTRY

Biochemistry

InstrumentationComputers

Pharmacology Endocrinology

Analytical ChemistryToxicologyImmunology

CLINICAL CHEMISTRY

It is much easier to It 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 of Malfunction 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. Malignancy Malignancy

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

Point-of-care TestingPoint-of-care Testing

(POCT)(POCT) Also known as Also known as near-patient testingnear-patient testing, , alternate-site alternate-site

testingtesting or or 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

Addresses Addresses acute patient needsacute patient needs

Instrumentation includes Instrumentation includes portable chemistry portable chemistry analyzers, glucometers, BG Analyzers, analyzers, glucometers, BG Analyzers, hemoglobin meters hemoglobin meters && coagulation testing coagulation testing

Specimens or samples Specimens or samples areare analyzedanalyzed

while while substances in them aresubstances in them are measured measured or or quantitated.quantitated.

Substances measured in serum fall generally Substances measured in serum fall generally into the following categories:into the following categories:

1.1. Substances Substances normally presentnormally present with a function in the with 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

3.3. Substances Substances released from cells as a result of released from cells as a result of cell damage & abnormal permeability or cell 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

Quick QuizQuick Quiz Tissue injury is greatly associated Tissue injury is greatly associated

with elevated levels of bloodwith elevated levels of blood

a. a. electrolyteselectrolytes b. b. hormoneshormones

c. c. enzymesenzymes d. d. albuminalbumin

Electrochemical methods are ideal for Electrochemical methods are ideal for the measurement of serum/plasmathe measurement of serum/plasma

a. a. glucoseglucose b. b. enzymesenzymes

c. c. proteins & amino acids proteins & amino acids d. d. metal metal ionsions

Laboratory TestsLaboratory Tests Chemical testsChemical tests

Direct or IndirectDirect or IndirectColorimetric Colorimetric RedoxRedoxCondensationCondensation

Degradative testsDegradative tests ( (EnzymaticEnzymatic))One-step or Multi-stepOne-step or Multi-stepNADH ConsumptionNADH Consumption

Measurement of ActivityMeasurement of Activity (not (not concentration)concentration)

Multi-methodMulti-method TitrimetricTitrimetric ElectrochemicalElectrochemical

Quick QuizQuick Quiz Identify the Identify the analyte measuredanalyte measured using the using the

following tests and the following tests and the typetype of each of each test:test:

1. 1. Jaffe reactionJaffe reaction2. 2. Hantzsch testHantzsch test3. 3. GOD-PAPGOD-PAP4. 4. Clark-CollipClark-Collip5. 5. Schales and SchalesSchales and Schales6. 6. Evelyn-MalloyEvelyn-Malloy

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 = important for clinical decisionsdecisions

= less than 2 hour TAT= less than 2 hour TAT

ROUTINEROUTINE = non urgent; standard or = non urgent; standard or special special teststests

= generally 4-6 hours= generally 4-6 hours

BreakableBreakable

ReusableReusable

Ideal for acidic solutionsIdeal for acidic solutions

High thermal & corrosion High 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

GLASS: 5 GENERAL TYPESGLASS: 5 GENERAL TYPES1.1. 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 are Most common are PyrexPyrex, , KimaxKimax and and ExaxExax

Corex Corex - 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, graduated higher temperature thermometers, graduated

cylinders and centrifuge tubescylinders and centrifuge tubes..

VycorVycor- - recommended for use in recommended for use in application involving high application involving high temperature and drastic heat shock and extreme temperature and drastic heat shock and extreme chemical chemical treatment with acids and dilute alkaliestreatment with acids and dilute alkalies- heatable to 900- heatable to 900ooC and withstand downshock from 900C and withstand downshock from 900ooC to C to ice waterice water- ideal for - ideal for ashing and ignition techniquesashing and ignition techniques

2. High silica glass2. High silica glass- - 96% silica content 96% silica content comparable to comparable to fused quartzfused quartz in in its its thermal endurance, chemical stability and thermal endurance, chemical stability and electrical characteristicselectrical characteristics

- - radiation resistant, good optical radiation resistant, good optical qualities and qualities and temperature temperature capabilitiescapabilities

- ideal for - ideal for high precision analytical workhigh precision analytical work and and can can also be used for also be used for optical reflectors and optical reflectors and mirrorsmirrors

3. Glass with high resistance to alkalies3. Glass with high resistance to alkalies- - boron-freeboron-free glassware for strong alkali solutions glassware 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

4. Low actinic glass4. Low actinic glass- - amber or red-coloredamber or red-colored to reduce the amount of light to reduce the amount of light passing 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 labile substances in the 300-500nm

range range (bilirubin, carotene and (bilirubin, carotene and vitamin A)vitamin A)

5. Standard flint glass5. Standard flint glass- - soda-lime glasssoda-lime glass which is composed of a mixture of which 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 reagent bottles andand

disposable laboratory disposable laboratory glasswaresglasswares- release alkali into the pipetted liquid - release alkali into the pipetted liquid

Quick QuizQuick Quiz Which of the following is an aluminum-Which of the following is an aluminum-

silicate glass that is at least six times silicate glass that is at least six times stronger than borosilicate and is resistant stronger than borosilicate and is resistant to alkaline etching and scratching?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 The tinted glass which is ideal for delayed testing of light-sensitive substances istesting of light-sensitive substances is

a. a. standard flintstandard flint b. b. soda limesoda lime

c. c. low actiniclow actinic d. d. boron-freeboron-free

1.1. Polyolefins Polyolefins (polyethylenes, (polyethylenes, polypropylenes)polypropylenes)

Unique group of Unique group of resins resins with relatively inert with relatively inert chemical propertieschemical properties

Unaffected by acids (however, conc. sulfuric acid Unaffected by acids (however, conc. sulfuric acid slowly attacks polyethylene at RT), slowly attacks polyethylene at RT),

alkalies, alkalies, salt solutions and most aqueous salt solutions and most aqueous solutionssolutions

Aromatic, aliphatic and chlorinated hydrocarbons Aromatic, aliphatic and chlorinated hydrocarbons cause moderate swelling at RTcause moderate swelling at RT

Organic acids, essential oils and halogens slowly Organic acids, essential oils and halogens slowly penetrate these plastics penetrate these plastics

PLASTICWARES

Polyolefins (polyethylenes, polypropylenes)Polyolefins (polyethylenes, polypropylenes)

Strong oxidizing agents attack this group at Strong oxidizing agents attack this group at elevated temperatures onlyelevated temperatures only

Polypropylene is more expensivePolypropylene is more expensive than than polyethylene therefore the latter is polyethylene therefore the latter is used in used in most disposable plastic waresmost disposable plastic wares

Polypropylene is sterilizablePolypropylene is sterilizable however it however it absorbs pigment and tends to absorbs pigment and tends to become become discoloreddiscolored

PLASTICWARES

2. Polycarbonate resin2. Polycarbonate resin

twice as strong as polypropylene (from –100twice as strong as polypropylene (from –100ooC to C to +160+160ooC)C)

Chemical resistance is not as wide as the Chemical 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 for Ideal for centrifuge tubes and centrifuge tubes and graduated cylindersgraduated cylinders

3. Tygon3. Tygon

Nontoxic, clear plastic of modified PVC Nontoxic, clear plastic of modified PVC (polyvinylchloride)(polyvinylchloride)

Used extensively for the Used extensively for the manufacture of ACA manufacture of ACA tubingstubings

Flexible and used to handle most Flexible and used to handle most chemicalschemicals

Can be Can be steam-autoclaved or chemically steam-autoclaved or chemically sterilizedsterilized

Tubing is soft and flexible and quickly slips over Tubing is soft and flexible and quickly slips over tubulatures but tubulatures but gripping tightly on glass or gripping tightly on glass or metalsmetals

4. Teflon fluorocarbon resins4. Teflon fluorocarbon resins

Almost chemically inert with Almost chemically inert with high corrosion high corrosion resistance at extreme temperaturesresistance at extreme temperatures

For For cryogenic experiments and work at cryogenic experiments and work at high temperatures over extended high temperatures over extended periods (-270periods (-270ooC to +255C to +255ooC)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 but it can be scratched and misshapedscratched and misshaped

Quick QuizQuick Quiz The internal tubings of clinical analyzers The internal tubings of clinical analyzers

should be made of should be made of

a. a. Teflon fluorocarbonTeflon fluorocarbon b. b. polycarbonatepolycarbonate

c. c. TygonTygon d. d. polyvinylchloridepolyvinylchloride

The advantages of this plastic is that it can The advantages of this plastic is that it can be made into very thin sheets and can be made into very thin sheets and can withstand extreme temperatures.withstand extreme temperatures.

a. a. PolyolefinsPolyolefins b. b. PolycarbonatePolycarbonate

c. c. TygonTygon d. d. TeflonTeflon

PIPETSPIPETSTwo General TypesTwo General Types

1.1. Volumetric or Transfer pipetVolumetric or Transfer pipet

Designed Designed to deliver (TD) a fixed volume of to deliver (TD) a fixed volume of liquidliquid

Consists of a Consists of a cylindrical bulbcylindrical bulb joined at both ends to joined at both ends to narrower glass tubingnarrower glass tubing

Calibration mark is etched around the upper suction tube Calibration 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 for used for accurate measurements of aliquots accurate measurements of aliquots of nonviscous samples, filtrates, controls, of nonviscous samples, filtrates, controls, and standard solutionsand standard solutions

OSTWALD-FOLIN PIPETOSTWALD-FOLIN PIPET

Similar to volumetric pipet but have their bulb Similar to volumetric pipet but have their bulb closer to the delivery tipcloser to the delivery tip

Used for Used for measuring viscous fluids as measuring viscous fluids as blood or serum blood or serum

Has an Has an etched ring near the etched ring near the mouthpiece (blowout pipet)mouthpiece (blowout pipet)

Blown out only when the fluid has drained to Blown out only when the fluid has drained to the last drop in the delivery tipthe last drop in the delivery tip

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 Graduated uniformly along its

lengthlength Two types which are calibrated for delivery are Two types which are calibrated for delivery are

availableavailable

Mohr pipetMohr pipet

- - calibrated between two markscalibrated between two marks on the stemon the stem

Serological pipetSerological pipet

- has - has graduation marks down on graduation marks down on the tipthe tip and blown out to deliver the entire and blown out to deliver the entire volume of the pipet if etched ring or double volume of the pipet if etched ring or double rings are evident near the mouth of the pipetrings are evident near the mouth of the pipet

Serological pipetSerological pipet

Intended for the Intended for the delivery of delivery of predetermined predetermined volumesvolumes

Principally used for the Principally used for the measurements of measurements of reagents reagents and and are not are not

generally generally considered considered accurate enough for accurate enough for measuring viscous samples and measuring viscous samples and

standardsstandards

Serological pipets have larger orifice than Serological pipets have larger orifice than the Mohr pipet and thus drain fasterthe Mohr pipet and thus drain faster

MICROPIPETSMICROPIPETS

SahliSahli (TC) (TC) Kirk Kirk (TC)(TC)

Lang-LevyLang-Levy (TD/ TC) (TD/ TC) OverflowOverflow (TC) (TC) Capillary Capillary (TC)(TC)

Sanz PipetSanz Pipet (TD=TC) (TD=TC) UnopetteUnopette (TC) (TC)

Semiautomatic pipetSemiautomatic pipet Eppendorf Eppendorf (TD/TC)(TD/TC)

Quick QuizQuick Quiz The College of American Pathologists The College of American Pathologists

(CAP) requires that volumetric pipets (CAP) requires that volumetric pipets and flasks be certified asand flasks be certified asa. a. Class AClass A b. b. Class BClass Bc. c. Class CClass C d. d. Class DClass D

““To deliver”(TD) pipettes are identified To deliver”(TD) pipettes are identified bybya. a. two etched bands near the two etched bands near the mouthpiecemouthpieceb. b. self-draining capacityself-draining capacityc. c. dual purpose pipet labelsdual purpose pipet labelsd. d. blue graduation levelsblue graduation levels

CHEMICALSCHEMICALSThe The 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 labeled They are labeled Analytical ReagentAnalytical Reagent (AR) or ACS which state (AR) or ACS which state the actual purity of each lot. These include chemicals such as: the actual purity of each lot. These include chemicals such as:

spectrograde, spectrograde, nanograde, and nanograde, 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 for b. making up reagents for spectrophotometry, chromatography, spectrophotometry, chromatography, trace metal analysis, atomic absorption spectrophotometry, trace metal analysis, atomic absorption spectrophotometry, fluorometry and standardizationfluorometry and standardization procedures. procedures.

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 produced injurious to humans and are used to produced drugs.drugs. They are not pure enough for use in They are not pure enough for use in most chemical procedures.most chemical procedures.

Less pure grade chemicals are referred to as Less pure grade chemicals are referred to as purified and technical. The purified and technical. The chemically purechemically pure (CP) (CP) or or pure gradepure grade chemicals do not recommend for chemicals do not recommend for reagent preparations.reagent preparations.

TechnicalTechnical or or commercialcommercial grade reagents are grade reagents are primarily used in manufacturing and should not primarily used in manufacturing and should not be used in the clinical laboratory. The technical be used in the clinical laboratory. The technical grade is the lowest quality and should not be grade is the lowest quality and should not be used for analytical work.used for analytical work.

STANDARDSSTANDARDSA A primary standardprimary standard (PS) is a chemical that is the (PS) is a chemical that is the

highest purity and can be measured directly to highest purity and can be measured directly to produce a substance of exact known concentration.produce a substance of exact known concentration.

The The 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 and that

thethe working standards be 99.95% working standards be 99.95% purepure..

Grade AGrade A - atomic weight standards - atomic weight standardsGrade BGrade B – ultimate standards – ultimate standardsGrade CGrade C* – primary standards with >0.002% impurity* – primary standards with >0.002% impurityGrade DGrade D* – working standards with >0.5% impurity* – working standards with >0.5% impurityGrade EGrade E – secondary standards derived from using – secondary standards derived from using

Grade C standardsGrade C standards**commercially availablecommercially available

The The 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 Standard Standard Reference MaterialsReference Materials (SRM) are used as (SRM) are used as primary standard materials in the clinical primary standard materials in the clinical laboratory. They are relatively expensive and laboratory. They are relatively expensive and should be used as a should be used as a reference for the reference for the comparison of commercially obtained comparison of commercially obtained standards and reagents.standards and reagents.

A A secondary standardsecondary standard is a substance of is a substance of lower purity whose concentration is determined lower purity whose concentration is determined by comparison to a primary standard.by comparison to a primary standard.

Quick QuizQuick Quiz Regarded as the ultimate standard by IUPAC Regarded as the ultimate standard by IUPAC

isisa. a. Grade AGrade A b. b. Grade BGrade Bc.c. Grade C Grade C d. d. Grade DGrade D

What is the most common use of a What is the most common use of a standard in the clinical laboratory?standard in the clinical laboratory?

a. a. to verify accuracy of resultsto verify accuracy of resultsb. b. to standardize the testingto standardize the testingc. c. to compare its value with the results to compare its value with the results

obtainedobtained d. d. to obtain the concentration of the to obtain the concentration of the

substance testedsubstance tested

WATERWATERReagent grade waterReagent grade water (RGW) is water (RGW) is water

suitable for reagent and standard preparation. Most suitable for 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 all is purified to remove almost all organic materials. Water may be distilled more than organic materials. Water may be distilled more than once and each distillation cycle will remove once and each distillation cycle will remove impurities.impurities.

Deionized waterDeionized water is produced from distilled water is produced from distilled water using either an anion or cation exchange resin using either an anion or cation exchange resin followed by replacement of the removed particles followed by replacement of the removed particles with hydroxyl or hydrogen ions respectively.with hydroxyl or hydrogen ions respectively.

The The College of American PathologistsCollege of American Pathologists (CAP) and the (CAP) and the National National Committee for Clinical LaboratorCommittee for Clinical Laboratoryy Standards Standards (NCCLS) issued (NCCLS) issued minimum specifications for RGW. The NCCLS requirements for each minimum specifications for RGW. The NCCLS requirements for each type are tabulated below:type are tabulated below:

SpecificationsSpecifications Type IType I Type II Type III Type II Type III

Bacterial content (maximumBacterial content (maximum <10<10 10 1022 NANA colony forming unit/mL)colony forming unit/mL)

pHpH NANA NA NA NANA

Resistivity (megaohms/cmResistivity (megaohms/cm 1.0 (in-line) 1.01.0 (in-line) 1.0 0.10.1 at 25at 25ooC)C)

Silicates, mg/L SiOSilicates, mg/L SiO22 maximum maximum 0.050.05 0.1 0.1 1.01.0

Particulate matter (filter in system)Particulate matter (filter in system) 0.22 0.22 µmµm NA NA NANA

Organic contaminantsOrganic contaminants activated carbonactivated carbon NA NA NANA filter in systemfilter in system

Type I RGWType I RGW is used for procedures that require maximum is 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 subnanogram measurement of nanogram or subnanogram

concentration concentration 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 in is used in most laboratory determination in chemistry, hematology, microbiology, immunology and other chemistry, 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 and examinations, most procedures in urinalysis, parasitology and histology, washing glasswares and procedures not requiring histology, 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 may This is used when carbon dioxide, ammonia and oxygen may affect analysis.affect analysis.

CLEANING GLASSWARES AND PLASTIC CLEANING GLASSWARES AND PLASTIC WARESWARES Routine washing may be done by soaking in dilute bleach followed Routine washing may be done by soaking in dilute bleach followed

by drying in an oven, soaking in 20% nitric acid solution for 12-24 by 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 solution For 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 or For iron determination, soak in 1:2 dilution of conc. HCI solution or 1:3 dilution of conc. HNO1:3 dilution of conc. HNO33

Quick QuizQuick Quiz What is the most appropriate term to What is the most appropriate term to

describe water that is used as a solvent describe water that is used as a solvent in the clinical laboratory?in the clinical laboratory?a. a. distilled waterdistilled water b. b. deionized deionized waterwaterc.c. reagent grade water reagent grade water d.d. tap water tap water

The type of water desired for use in The type of water desired for use in test methods requiring maximum test methods requiring maximum accuracy and precision isaccuracy and precision isa. a. distilleddistilled b. b. pure gradepure gradec. c. type Itype I d. d. type IItype II

What is effectively removed by activated What is effectively removed by activated charcoal adsorption in water purification?charcoal adsorption in water purification?

a. a. charged chemicalscharged chemicals b. b. microbes and microbes and pyrogenspyrogens

c. c. toxic gasestoxic gases d. d. organic compoundsorganic compounds

WhatWhat purification method is ideal for the purification method is ideal for the removal of pyrogens and microorganisms?removal of 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 distillation and deionization combinedcombined

Chemical HazardsChemical Hazards

Caustic Caustic – can cause burns– can cause burns FlammableFlammable – also combustible, that is, – also combustible, that is,

can can easily catch fireeasily catch fire CorrosiveCorrosive – harmful to mucous – harmful to mucous

membranes, 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 – cause changes in RNA or

DNADNA TeratogenTeratogen – cause birth defects – cause birth defects

Quick QuizQuick Quiz Xylene, ethanol, methanol, and acetone Xylene, ethanol, methanol, and acetone

would be in which hazard class?would be in which hazard class?a. a. corrosivecorrosive b. b. flammableflammablec. c. oxidizeroxidizer d. d. all of the aboveall of the above

Precautions such asPrecautions such as using a fume hood, using a fume hood, wearing rubber gloves, donning a wearing rubber gloves, donning a respirator, and cleaning glassware with a respirator, and cleaning glassware with a strong acid or organic solvent are strong acid or organic solvent are consistent with working withconsistent with working witha. a. corrosivescorrosives b. b. carcinogenscarcinogensc. c. azidesazides d. d. all reagentsall reagents

CENTRIFUGE CENTRIFUGE

The The 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.

All laboratories are required to have All laboratories are required to have safety safety showers, eyewash stations and fire showers, eyewash stations and fire extinguishersextinguishers and to periodically test and and to periodically test and inspect the equipment of proper operation. inspect the equipment of proper operation. Other items that must be available for Other items that must be available for personnel include fire blankets, spill kits and personnel include fire blankets, spill kits and first aid supplies.first aid supplies.

Mechanical pipetting devices must be used Mechanical pipetting devices must be used for manipulating all types of liquids in the for manipulating all types of liquids in the

laboratory, including water. laboratory, including water. Mouth Mouth pipetting is strictly pipetting is strictly prohibited.prohibited.

Quick QuizQuick Quiz Which of the following may be safely Which of the following may be safely

pipetted by mouth?pipetted by mouth?a. a. serumserum b. b. urineurinec. c. saline solutionsaline solution d. d. none of the none of the aboveabove

The speed of the centrifuge should be The speed of the centrifuge should be checked at least once every three (3) checked at least once every three (3) months with a /anmonths with a /ana. a. tachometertachometer b. b. wiperwiperc. c. potentiometerpotentiometer d. d. ergometerergometer

BIOLOGIC SAFETYBIOLOGIC SAFETY

All samples and other body fluids should be All samples and other body fluids should be collected, transported, handled and collected, transported, handled and

processed using processed using strict strict precautions.precautions.

Gloves, gowns and face protection must be Gloves, gowns and face protection must be used if used if splash or splatteringsplash or splattering is likely to is likely to

occur.occur.

Specimen should remain Specimen should remain cappedcapped during during centrifugation because biologic centrifugation because biologic

specimens could produce finely specimens could produce finely dispersed aerosolsdispersed aerosols that that

are a are a high-risk high-risk source of infection.source of infection.

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 disinfected must 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 or Use 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% bleach the 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 biohazard Dispose all materials in appropriate biohazard containers.containers.

Occupational Safety and Health Organization AdministrationOccupational Safety and Health Organization Administration (OSHA) “Blood –Borne Pathogens” standard requires (OSHA) “Blood –Borne Pathogens” standard requires written 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 to blood and body fluidsfluids

Category II- Category II- regular exposureregular exposure to blood and to blood and body fluidsbody fluids

Category III- Category III- no exposureno exposure to blood and body to blood and body fluidfluid

Employers must offer Hepatitis B vaccine at no costEmployers must offer Hepatitis B vaccine at no cost 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 strategic should be installed in strategic places to facilitate manipulations of infectious material. It places to facilitate manipulations of infectious material. It reduces risk of exposurereduces risk of exposure of laboratory personnel and of laboratory personnel and patients.patients.

SAFETY AGAINST EXPOSURE TO TOXIC SAFETY AGAINST EXPOSURE TO TOXIC CHEMICALSCHEMICALS

OSHA published its OSHA published its Hazard Communication StandardHazard Communication Standard in in 1983 to minimize the incidence of chemically related 1983 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 should a. Manufacturers of chemicals should evaluate the hazards of evaluate the hazards of the chemicalsthe chemicals they produce and they produce and develop hazard develop hazard

communication programscommunication programs for employees for employees exposed to exposed to hazardous chemicals.hazardous chemicals.

b. Clinical laboratories should b. Clinical laboratories should develop and institute a develop and institute a chemical hygiene plan.chemical hygiene plan.

c. Hospitals and laboratories are obliged to c. Hospitals and laboratories are obliged to maintain an maintain an inventory of all hazardous substancesinventory of all hazardous substances used in the used in the

workplace.workplace.

OSHA requires that OSHA requires that material safety data sheetsmaterial safety data sheets ( (MSDSMSDS) be ) be provide for each chemical by manufacturers and provide for each chemical by manufacturers and suppliers and must be made available on site for suppliers and must be made available on site for laboratory personnel. laboratory personnel.

MSDS will specifically include MSDS 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; and spill 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.

The The National Fire Protection AssociationNational Fire Protection Association (NFPA) has (NFPA) has developed the Hazards Identification system to developed the Hazards Identification system to provide common, recognizable warning signs for provide 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 a coded, 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 a Contained within each color-coded diamond is a number ranging from 0 to 4, indicating the severity of number 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)

Special ConsiderationsSpecial Considerations

Water Reactive Water Reactive

Oxidizing Agent Oxidizing Agent

Radioactive Radioactive

Poison Poison

Ox

ELECTRICAL SAFETYELECTRICAL SAFETY

Lockout/tag outLockout/tag out malfunctioning electrical or malfunctioning electrical or mechanical equipment until services. mechanical equipment until services. ReportReport any small shocks, unplug and any small shocks, unplug and

tag equipment until serviced.tag equipment until serviced.

If a severely shocked person can not let go of If a severely shocked person can not let go of instrument, instrument, unplug it without touching unplug it without touching the person or knock person loose the person or knock person loose with nonconductive materialwith nonconductive material such as such as wood.wood.

RADIATION SAFETYRADIATION SAFETY A radiation-safety policy should include environmental and A radiation-safety policy should include environmental and

personnel protection.personnel protection.

All areas where radioactive materials are used or stored All areas where radioactive materials are used or stored must be posted with must be posted with caution signscaution signs and and traffic in these traffic 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 of Records must be maintained as to the quantity of radioactive material on hand as well as the quantity that is radioactive material on hand as well as the quantity that is disposed. disposed. Records must be maintained for the length of Records 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 dose The exposure limit (maximum permissible dose equivalents is equivalents is 5000 mrem/year whole body5000 mrem/year whole body))

The The Wipe test (Leak test)Wipe test (Leak test) involves wiping laboratory involves wiping laboratory surfaces with moistened absorbent material and the surfaces with moistened absorbent material and the radiation contained in each wipe is counted. radiation contained in each wipe is counted.

Quick QuizQuick Quiz A corrosive material was spilled onto the hand A corrosive material was spilled onto the hand

of a laboratorian. After diluting the material of a laboratorian. After diluting the material under running cold water, what should be done under running cold water, what should be done next?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 dilute spills and remove it in a biohazard bagbiohazard bag

d. d. go to the emergency roomgo to the emergency room

Which of the following information is found in Which of the following information is found in the MSDS?the MSDS?

a.a. Health hazard data Health hazard data b. b. Fire & explosion Fire & explosion hazard datahazard data

c. c. First Aid measures First Aid measures d.d. All of the aboveAll of the above

FIRE SAFETYFIRE SAFETY

Fires have been divided into four classes on Fires have been divided into four classes on the nature of the combustible material and the nature of the combustible material and requirements for extinguishments.requirements for extinguishments.Class A-Class A- ordinary combustible solid ordinary combustible solid

materialsmaterials such as paper, such as paper, wood, wood, plastic and rubberplastic and rubber

Class B-Class B- flammable liquids/ gases and flammable liquids/ gases and combustible petroleum productscombustible petroleum products

Class C-Class C- energized electrical equipmentenergized electrical equipmentClass D-Class D- combustible/reactive metalscombustible/reactive metals such such

as Mg, Na and Kas Mg, Na and K Class EClass E – – nuclear reactionnuclear reaction Class KClass K – – cooking mediacooking media

(Type A) Pressurized water extinguishers(Type A) Pressurized water extinguishers (foam and (foam and multipurpose dry-chemical types) are for multipurpose 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 for extinguishers are used for Class B and C firesClass B and C fires. .

(Type C) Halogenated hydrocarbon(Type C) Halogenated hydrocarbon extinguishers are extinguishers are recommended particularly for use with recommended particularly for use with computer computer equipment. equipment.

(Type D) Class D(Type D) Class D fires present special problems and fires present special problems and extinguishments is left to trained fire fighters using extinguishments is left to trained fire fighters using special dry-chemical extinguishersspecial dry-chemical extinguishers..

Personnel should know the location and type of portable Personnel should know the location and type of portable fire extinguisher near the work area and know how to use fire extinguisher near the work area and know how to use an extinguisher before a fire occurs. an extinguisher before a fire occurs.

Quick QuizQuick Quiz A fire extinguisher used in the event A fire extinguisher used in the event

of an electrical fire should include of an electrical fire should include which of the following classifications ?which of the following classifications ?a. a. type Atype A b. b. type Btype Bc. c. type Ctype C d. d. type Dtype D

In the Hazards Identification System, In the Hazards Identification System, what hazard does the blue diamond what hazard does the blue diamond positioned to the left identify ?positioned to the left identify ?a. a. flammableflammable b. b. healthhealthc. c. reactivityreactivity d. d. contactcontact

SAFETY AWARENESS FOR CLINICAL SAFETY AWARENESS FOR CLINICAL LABORATORY PERSONNELLABORATORY PERSONNEL

Health Care Organizations focus their responsibility in Health Care Organizations focus their responsibility in protecting their employees from infection especially protecting their employees from infection especially against against hepatitis B virus (HBV), hepatitis B virus (HBV), human immunodeficiency virus human immunodeficiency virus (HIV) & TB bacilli.(HIV) & TB bacilli.

The The Centers for Disease Control (CDC)Centers for Disease Control (CDC) updated updated the 1983 Guidelines for Isolation Precautions in the 1983 Guidelines for Isolation Precautions in hospitals with the release of its Universal Precautions hospitals 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.

The The CDC recommends that blood and body fluid CDC recommends that blood and body fluid precautionsprecautions should be consistently used for all should be consistently used for all patients regardless of their blood-borne infection patients regardless of their blood-borne infection status.status.

Potentially infectious materials include:Potentially infectious materials include:a. a. body fluids (semen, vaginal body 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 by Infective agents may be inactivated by = heat sterilization (250= heat sterilization (250OOC for 15 minutes),C for 15 minutes), = ethylene oxide (450-500 mg/L at 55-60= ethylene oxide (450-500 mg/L at 55-60OOC)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 makes a very effective and economical economical disinfectant inactivating disinfectant inactivating HBV in 10 HBV in 10 minutes minutes

andand HIV in 2 minutes. HIV in 2 minutes.

DISPOSAL OF HAZARDOUS MATERIALSDISPOSAL OF HAZARDOUS MATERIALS

There are four basic waste disposal techniques. There are four basic waste disposal techniques. These include:These include:

flushing down the drain to flushing down the drain to the sewer the sewer systemsystem

IncinerationIncineration

landfill buriallandfill burial

recyclingrecycling

It is permissible toIt is permissible to flush water-soluble substances down the flush water-soluble substances down the drain with large quantities of water.drain with large quantities of water.

Strong acids and bases Strong acids and bases should beshould be neutralized before neutralized before disposaldisposal

Foul smelling chemicals Foul smelling chemicals should should never be disposed of down never be disposed of down the drainthe drain..

Other liquid wastes, including flammable solvents, Other liquid wastes, including flammable solvents, must be must be collected in approved containers and segregated into collected in approved containers and segregated into compatible classescompatible classes..

flammable material can be burned in specially designed flammable material can be burned in specially designed incinerators with after burners and scrubbers to remove incinerators 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.

Biohazardous WastesBiohazardous Wastes

Medical waste is defined as a Medical waste is defined as a special special waste waste from from health care facilitieshealth care facilities and is further defined and is further defined as as solid wastesolid waste that, if improperly treated or that, if improperly treated or handled handled may transmit infectious diseasesmay transmit infectious diseases..

it comprises of it comprises of animal waste, bilk blood and blood animal waste, bilk blood and blood products, microbiologic wastes, pathologic products, microbiologic wastes, pathologic wastes and sharps.wastes and sharps.

The approved methods for treatment and The approved methods for treatment and disposition of medical wastes are disposition of medical wastes are incineration, incineration, steam sterilization, burial, thermal steam sterilization, burial, thermal inactivation, chemical disinfection, or inactivation, chemical disinfection, or encapsulation in a solid matrix.encapsulation in a solid matrix.

Quick QuizQuick Quiz The major job-related hazard in clinical The major job-related hazard in clinical

laboratories produces symptoms of malaise, laboratories produces symptoms of malaise, anorexia, nausea, vomiting, fatigue, diarrhea, anorexia, nausea, vomiting, fatigue, diarrhea, and abdominal tenderness isand abdominal tenderness isa. a. AIDSAIDS b. b. salmonellosissalmonellosisc. c. tuberculosistuberculosis d. d. hepatitishepatitis

A laboratory professional was askedA laboratory professional was asked to send a to send a blood specimen from an HIV-positive patient blood specimen from an HIV-positive patient to a laboratory for special tests. The specimen to a laboratory for special tests. The specimen must be properly labeled and shipped as must be properly labeled and shipped as a. a. infectious substanceinfectious substance b. b. diagnostic diagnostic specimenspecimenc. c. biological productbiological product d. d. clinical clinical specimenspecimen

UNITS OF MEASUREMENTUNITS OF MEASUREMENT The The Systeme InternationaleSysteme Internationale (SI) defines seven (SI) defines seven base unitsbase units as shown. as shown.

QuantityQuantity Basic Unit Basic Unit SymbolSymbol

Length Length metermeter mmMassMass kilogramkilogram kgkgTimeTime secondsecond ssElectric currentElectric current ampereampere AATemperatureTemperature KelvinKelvin KKLuminous intensityLuminous intensity CandelaCandela cdcdAmount of substanceAmount of substance molemole molmolCatalytic amountCatalytic amount katalkatal

SI-Derived UnitsSI-Derived Units are obtained by combining two are 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

Activity (radionuclide)Activity (radionuclide) Becquerel Becquerel BqBq

Volume Volume cubic metercubic meter mm33

The National Institute of Standards and The National Institute of Standards and Technology (NIST)Technology (NIST) recognizes the use of recognizes the use of five specific types of calibrating weights. five specific types of calibrating weights.

Class S weightsClass S weights – for monthly calibration – for monthly calibration of of analytical balances analytical balances

Class M weightsClass M weights - to calibrate the weights - to calibrate the weights themselves since their quality is that of themselves since their quality is that of

primary primary standards. standards.

Class S-1Class S-1 - used for routine analytical - used for routine analytical work. work.

Class P weightsClass P weights - are allowed greater - are allowed greater tolerance tolerance levels than Class S-1levels than Class S-1

Class J weightsClass J weights - used for microanalytical - used for microanalytical work.work.

The The accuracy of the thermometer used to monitor the accuracy of the thermometer used to monitor the incubation temperature of an instrumentincubation temperature of an instrument should be should be verified every 6 to 12 months. verified every 6 to 12 months.

Reference thermometersReference thermometers must be must be certified by the NISTcertified by the NIST. .

For the For the monitoring of enzymatic reactionsmonitoring of enzymatic reactions, , thermometers should thermometers should agree within 0.1agree within 0.1ooC with the NIST C with the NIST reference thermometerreference thermometer..

Thermometers used to Thermometers used to check refrigerator and freezer check refrigerator and freezer temperaturestemperatures should should agree within 1.0agree within 1.0ooC with the C with the reference thermometerreference thermometer. Thermometers should be . Thermometers should be discarded if they differ by more than 1discarded if they differ by more than 1ooC from the C from the referencereference. .

On a daily basis, the temperature of the incubation area On a daily basis, the temperature of the incubation area should be checked, and the thermometer itself should be should be checked, and the thermometer itself should be observed for splits in the mercury column. observed for splits in the mercury column.

It is imperative that It is imperative that preventive preventive maintenance proceduresmaintenance procedures be performed be performed and the results recorded for all laboratory and the results recorded for all laboratory instrumentation. instrumentation.

This includes This includes maintenance of analytical maintenance of analytical balances, refrigerators, freezers, balances, refrigerators, freezers, centrifuges, ovens, water baths, centrifuges, ovens, water baths, heating blocks, thermometers, heating blocks, thermometers, pipettors, dilutors, automated pipettors, dilutors, automated analyzers, and all other laboratory analyzers, and all other laboratory equipment used for analyzing equipment used for analyzing specimens. specimens.

Preventive maintenance is performed at Preventive maintenance is performed at scheduled times such as scheduled times such as per shift, daily, per shift, daily, weekly, monthly, or yearly.weekly, monthly, or yearly.

Quick QuizQuick Quiz SI units are designated units employed by SI units are designated units employed by

the International System of Units. The unit the International System of Units. The unit class that encompasses the seven class that encompasses the seven fundamental quantities of measurement isfundamental quantities of measurement isa. a. basebase b. b. primaryprimaryc. c. derivedderived d. d. elementalelemental

Which of the folllowing is most commonly Which 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 electric currentcurrentc. c. massmass d. d. substance substance

concentrationconcentration

FACTORS THAT INFLUENCE FACTORS THAT INFLUENCE LABORATORY TESTING LABORATORY TESTING

Increased Increased DecreasedDecreasedExercise Exercise Immediate effects:Immediate effects:

Alanine and lactate Alanine 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 Glucose Glucose

metabolites and metabolites and

12-14 hrs for lipid profile12-14 hrs for lipid profile))

EFFECT ON LABORATORY RESULTS

Increased Increased 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

Diet Diet 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 Serum Serum to saturated fatto saturated fat cholesterol cholesterol

Purine-richPurine-rich UratesUrates

High caffeineHigh caffeine FFA, catecholamine releaseFFA, catecholamine release

Bananas, pineapple,Bananas, pineapple, Serotonin in bloodSerotonin in blood tomatoes and tomatoes and 5’HIAA in urine5’HIAA in urine avocadosavocados

Alcohol ingestionAlcohol ingestionImmediateImmediate Lactate, urate, ketone bodies,Lactate, urate, ketone bodies,

and FFAand FFA

Chronic abuseChronic abuse HDL-cholesterol, GGT, and MCVHDL-cholesterol, GGT, and MCV

Increased Increased DecreasedDecreased

Tobacco smokingTobacco smoking Acute effectsAcute effects Catecholamines, cortisolCatecholamines, cortisol EosinophilEosinophil

neutrophils, monocytesneutrophils, monocytesand FFAand FFA

Chronic effectsChronic effects CO-Hb, MCV and leukocyteCO-Hb, MCV and leukocytecountcount

Prolonged tourniquet applicationProlonged tourniquet application Serum enzymes, protein,Serum enzymes, protein,protein-bound substances,protein-bound substances,cholesterol, TAG, Ca, bloodcholesterol, TAG, Ca, bloodcell concentration cell concentration (hemoconcentration(hemoconcentration)**)**

Postural changesPostural changes same as ** due to hydrostaticsame as ** due to hydrostatic efflux of waterefflux of water

Stress Stress lactate, FFA and alterationlactate, FFA and alteration iin electrolyte levelsn electrolyte levels

Quick QuizQuick Quiz

Which of the following substances does Which of the following substances does not require the patient a fasting blood not require the patient a fasting blood specimen?specimen?a. a. uric aciduric acid b. b. triglyceridestriglyceridesc. c. cholesterolcholesterol d. d. phosphatesphosphates

Expected to be increasedExpected to be increased in blood in in blood in situationssituations characterized by characterized by hemoconcentration ishemoconcentration isa. a. calciumcalcium b. b. potassiumpotassiumc. c. ferritinferritin d. d. creatininecreatinine

BLOOD COLLECTION BLOOD COLLECTION Venipuncture (Phlebotomy)Venipuncture (Phlebotomy)

Venous blood is Venous blood is deoxygenated blooddeoxygenated blood that that

contains contains substances that come from substances that come from metabolic activities of different organsmetabolic activities of different organs..

Venous blood is mostly used in analysis of Venous blood is mostly used in analysis of blood blood chemistry and immunologic studies.chemistry and immunologic studies.

Venous blood is more easily collected than arterial Venous blood is more easily collected than arterial blood.blood.

Maybe obtained via Maybe obtained via one-movement one-movement or or 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

Complications of venipuncture-classified as Complications of venipuncture-classified as immediate or delayedimmediate or delayed and and local or systemiclocal or systemic

HematomaHematoma due to missed veindue to missed vein Collapsed small veinsCollapsed small veins due to excessive pull of due 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 and like hepatitis B and

AIDSAIDS

ConsiderationsConsiderations

Clean the venipuncture site with 70% alcohol or Clean the venipuncture site with 70% alcohol or 1% iodine.1% iodine.

Apply the tourniquet several inches above the Apply the tourniquet several inches above the puncture site.puncture site.

Never leave the tourniquet longer than one Never leave the tourniquet longer than one minute.minute.

The needle, as it enters the skin, is positioned at The needle, as it enters the skin, is positioned at approximately 15-degree angle to the site with the approximately 15-degree angle to the site with the bevel upbevel up..

Ensure prompt and adequate mixture of blood Ensure prompt and adequate mixture of blood and anticoagulant to prevent coagulation and to and 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 with Blood should not be drawn in an area with intravenous line. intravenous line.

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 (2) Wash hands in warm, running water with the chlorhexidine gluconate hand washing product chlorhexidine gluconate hand washing product (approved by the Infection Control Committee), or if (approved by the Infection Control Committee), or if not visibly contaminated with a commercial foaming not visibly contaminated with a commercial foaming hand wash product before and after each patient hand wash product before and after each patient collection.collection.

(3) Gloves are to be worn during all phlebotomies, and (3) Gloves are to be worn during all phlebotomies, and changed between patient collections. Palpation of changed between patient collections. Palpation of phlebotomy site may be performed without gloves phlebotomy site may be performed without gloves providing the skin is not broken.providing the skin is not broken.

(4) A lab coat or gown must be worn during blood (4) A lab coat or gown must be worn during blood collection procedures.collection procedures.

(5) Needles and hubs are single use and are (5) Needles and hubs are single use and are disposed of in an appropriate 'sharps' disposed of in an appropriate 'sharps' container as one unit. NOTE: container as one unit. NOTE: Needles are Needles are never recapped, removed, broken, or bent after never recapped, removed, broken, or bent after phlebotomy procedure.phlebotomy procedure.

(6) Gloves are to be discarded in the appropriate (6) Gloves are to be discarded in the appropriate container immediately after the phlebotomy container immediately after the phlebotomy procedure. All other items used for the procedure. All other items used for the procedure must be disposed of according to procedure must be disposed of according to proper biohazardous waste disposal policy.proper biohazardous waste disposal policy.

(7) Contaminated surfaces must be cleaned with (7) Contaminated surfaces must be cleaned with freshly prepared 10% bleach solution.  All freshly prepared 10% bleach solution.  All surfaces are cleaned daily with bleach.surfaces are cleaned daily with bleach.

(8) In the case of an accidental needlestick, (8) In the case of an accidental needlestick, immediately wash the area with an immediately wash the area with an antibacterial soap, express blood from the antibacterial soap, express blood from the wound.wound.

Factors to consider in site Factors to consider in site selectionselection

* Extensive scarring or healed burn areas should be * Extensive scarring or healed burn areas should be avoidedavoided

* Specimens should not be obtained from the arm * Specimens should not be obtained from the arm on the same side as a mastectomy.on the same side as a mastectomy.

* Avoid areas of hematoma.* Avoid areas of hematoma.

* If an IV is in place, samples may be obtained * If an IV is in place, samples may be obtained below but NEVER above the IV site.  below but NEVER above the IV site. 

* Do not obtain specimens from an arm having a * Do not obtain specimens from an arm having a cannula, fistula, or vascular graft.cannula, fistula, or vascular graft.                    

* Allow 10-15 minutes after a transfusion is * Allow 10-15 minutes after a transfusion is completed before obtaining a blood sample.completed before obtaining a blood sample.

Which of the following are Which 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 ___Recapping or removal of needles after

withdrawing the withdrawing the needle.needle.______Reuse of gloves for as long as they were Reuse of gloves for as long as they were

autoclaved autoclaved beforehand.beforehand.___Weekly cleaning of all lab surfaces with ___Weekly cleaning of all lab surfaces with

10% bleach 10% bleach solution.solution.______Outpatients are identified by making them Outpatients are identified by making them

say their name 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.

______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 Mixing of anticoagulated blood by shaking the

tube gently tube gently 5-8 times.5-8 times.

___Obtaining a blood sample from a patient who ___Obtaining a blood sample from a patient who underwent underwent blood transfusion just 30 minutes blood transfusion just 30 minutes ago.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 Avoiding to touch again a thoroughly cleaned

puncture 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 Collecting PST before a dark green heparinized tube in tube in one-movement technicone-movement technic..

Quick QuizQuick Quiz Which of the following is an unnecessary step Which of the following is an unnecessary step

in the proper phlebotomy procedure?in the proper phlebotomy procedure?

a. a. recapping the needle after userecapping the needle after use

b. b. placing bandage over the punctured placing bandage over the punctured sitesite

c. c. washing the hands prior to washing the hands prior to venipuncturevenipuncture

d. d. sterilizing the puncture sitesterilizing the puncture site

Which vein cannot be used in obtaining Which vein cannot be used in obtaining venous blood in an adult?venous blood in an adult?

a. a. radial veinradial vein b. b. jugular veinjugular vein

c. c. ankle veinankle vein d. d. dorsal hand dorsal hand veinvein

Arterial punctureArterial puncture

Arterial blood is Arterial blood is oxygenated bloodoxygenated blood. It is . It is uniform in composition throughout the body.uniform in composition throughout the body.

Arterial blood is used to measure Arterial blood is used to measure oxygen oxygen tension, carbon dioxide tensiontension, carbon dioxide tension andand blood pH.blood pH.

Blood gas analysesBlood gas analyses (BGA) are critical to patients (BGA) are critical to patients with with pulmonary problems, oxygen pulmonary problems, oxygen therapy, cardiovascular problems and therapy, cardiovascular problems and those undergoing major operationsthose undergoing major operations. .

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 are the preferred sites.sites.

Each site of arterial puncture uses different gauge of Each 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 at In 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 is used as anticoagulant for arterial puncture. puncture.

Complications of arterial punctureComplications of arterial puncture HematomaHematoma due to increased pressure in due to increased pressure in

the arterythe artery

Restriction of blood flowRestriction of blood flow due to reflex due to reflex constriction constriction ((arterial spasmarterial spasm))

Temporary discomfortTemporary discomfort ((aching, aching, throbbing, tenderness, sharp throbbing, tenderness, sharp sensation, and cramping)sensation, and cramping)

Thrombosis, hemorrhage and Thrombosis, hemorrhage and infectioninfection

ConsiderationsConsiderations Intense care should be administered to patients undergoing Intense care should be administered to patients undergoing

arterial puncture.arterial puncture.

Sites that are irritated, edematous, near a wound or in an Sites that are irritated, edematous, near a wound or in an area of arteriovenous (AV) shunt or fistula should not be area 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 coolant Samples are placed in ice water or other coolant (temperature of (temperature of 1-51-5ººCC)) to to minimize leukocyte minimize leukocyte consumption of oxygen.consumption of oxygen.

Capillary blood may be a Capillary blood may be a suitable substitute for suitable substitute for arterial blood determination of pH and arterial blood determination of pH and pCO2pCO2 provided that the site must be warmed prior to provided that the site must be warmed prior to collection. Warning increases blood flow through capillaries collection. Warning increases blood flow through capillaries and arterioles and result in arterial-rich blood.and arterioles and result in arterial-rich blood.

Quick QuizQuick Quiz Which test does not require an arterial blood Which test does not require an arterial blood

sample?sample?a. a. oxygen tensionoxygen tension b. b. blood pHblood pH

c. c. pCOpCO22 determination determination 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 Arterial blood collection always requires the

use of anticoagulant.use of anticoagulant.b. b. Arterial blood is a must in blood gas Arterial blood is a must in blood gas

analyses.analyses.c. c. Arterial blood collection requires a glass Arterial blood collection requires a glass

syringe to minimize leukocyte consumption syringe to minimize leukocyte consumption of gases.of gases.

d. d. Arterial blood has lower pCOArterial blood has lower pCO22, therefore, is , therefore, is less acidic than venous blood.less acidic than venous blood.

Skin puncture/Capillary punctureSkin puncture/Capillary punctureCapillary blood is an Capillary blood is an admixture of venous, admixture of venous,

arterial and capillary blood and may arterial and capillary blood and may contain tissue fluidscontain tissue fluids..

Skin punctures are suitable in Skin punctures are suitable in pediatric patients, pediatric patients, obese patients with thrombotic obese patients with thrombotic tendencies and severe burnstendencies and severe burns.. It isIt is often often preferred to geriatric patientspreferred to geriatric patients due to due 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 4 or 4thth

fingers may be used in older infantsfingers may 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

Capillary blood can be collected for analysis of blood Capillary 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 in The best method of arterial blood collection in newborn newborn is the is the umbilical artery catheterumbilical artery catheter..

Skin puncture site must be warmed before collection Skin puncture site must be warmed before collection to increase the blood flow (to increase the blood flow (arterializationarterialization). ). Thermal Thermal using dry heat or paper towel with using dry heat or paper towel with

warm water (39-42warm water (39-42OOC)C)

Mechanical Mechanical byby fflicking with index finger until licking with index finger until flushing is observedflushing is observed

Chemical Chemical using using Trafuril paste or Histamine creamsTrafuril paste or Histamine creams

Arterial blood collected from capillary Arterial blood collected from capillary puncture may yield puncture may yield unreliableunreliable results if: results if: Systolic pressures is less than 95 Systolic 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 2.00 Puncture 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.

Quick QuizQuick Quiz Which test requires finger skin puncture?Which test requires finger skin puncture?

a. a. Rumpel-LeedeRumpel-Leede b. b. Schryver and Schryver and WaughWaugh

c. c. Ivy Bleeding TimeIvy Bleeding Time d. d. Lee-WhiteLee-White

Which blood collection technic requires an Which blood collection technic requires an arm maneuver known as Allen test to check arm maneuver known as Allen test to check for collateral circulation?for collateral circulation?

a. a. phlebotomyphlebotomy b. b. arterial puncturearterial puncture

c. c. skin punctureskin puncture d. d. peripheral peripheral puncturepuncture

VACUTAINER/EVACUATED TUBESVACUTAINER/EVACUATED TUBES

These are tubes for blood collection which These are tubes for blood collection which are are 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 culture Blood culture tubestubes, , red topred top, , blue topblue top, , green topgreen top, , lavender lavender toptop and and gray topgray top

COLORCOLOR ADDITIVEADDITIVE ACTIONACTION USEUSE

LavenderLavender EthylenediaminetetraaceticEthylenediaminetetraacetic chelates calcium hematologic assayschelates calcium hematologic assays Acid (EDTA)Acid (EDTA) lead assay, CEA lead assay, CEA

** Versene Versene (disodium salt) (disodium salt) Determination and Determination and * * Sequestrene Sequestrene (dipotassium salt)(dipotassium salt) cell counts cell 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 or or None but containsNone but contains Allows blood to clot Most chemistry tests Allows blood to clot Most chemistry tests

Red BlackRed Black separator material separator material and serves as aand serves as a

barrier between cellsbarrier between cellsand serumand serum

YellowYellow Citrate dextrose Citrate dextrose preserves RBCs Blood culture preserves RBCs Blood culture

GreenGreen Heparin (Na+, Li+, Heparin (Na+, Li+, inhibits thrombin ammonia CO-Hb inhibits thrombin ammonia CO-Hb

or, NHor, NH44++)) activation and activation and

methemoglobinmethemoglobin

COLORCOLOR ADDITIVEADDITIVE ACTION ACTION USEUSE

OrangeOrange ThrombinThrombin accelarated clotaccelarated clot STAT serum STAT serum teststests

Blue Blue Buffered citrateBuffered citrate binds calcium binds calcium Coagulation assays Coagulation assays like PT & APTTlike PT & APTT

BlackBlack Buffered sodium Buffered sodium binds calciumbinds calcium Westergren ESR Westergren ESR citratecitrate

GrayGray NaF/KNaF/K22CC22OO44 Inhibits glycolytic Inhibits glycolytic GlucoseGlucoseenzyme enzyme enolaseenolase and determination and determinationact as anticoagulantact as anticoagulant

IodoacetateIodoacetate inhibits glycolytic inhibits glycolytic Glucose Glucose enzyme enzyme glyceraldehyde glyceraldehyde determination determination3-phosphate 3-phosphate dehydrogenasedehydrogenase

Anticoagulant InterferenceAnticoagulant Interference Dilution errorsDilution errors especially oxalates which are highly especially oxalates which are highly

osmoticosmotic

Inhibition of plasma enzyme activitiesInhibition of plasma enzyme activities activities especially activities especially with with fluoridefluoride which is an enzyme poison, which is an enzyme poison, EDTAEDTA which which chelates metallic enzymes activators. chelates metallic enzymes activators. OxalateOxalate inhibitsinhibits AMS, LD and ACP, and AMS, LD and ACP, and citratecitrate which which inhibitsinhibits AMS AMS

Oxalates, citrate and EDTA Oxalates, citrate and EDTA lower plasma calcium levelslower plasma calcium levels

False increase in electrolyte analysesFalse increase in electrolyte analyses due to the due to the anticoagulants in the salt formanticoagulants in the salt form

  If multiple tubes are needed, the If multiple tubes are needed, the proper order of drawproper order of draw to to avoid avoid cross contamination and erroneous resultscross contamination and erroneous results is as follows:  is as follows: 

1st - 1st -  Blood culture vials or bottles, sterile Blood culture vials or bottles, sterile ( (yellowyellow or or yellowyellow--blackblack top) top)

2nd - 2nd - Coagulation tubeCoagulation tube ( (light bluelight blue top) NOTE: top) NOTE: If just a routine If just a routine coagulation assay is the only test ordered, then a single light coagulation assay is the only test ordered, then a single light blue top tube may be drawn. If there is a concern regarding blue top tube may be drawn. If there is a concern regarding contamination by tissue fluids or thromboplastins, then one may contamination by tissue fluids or thromboplastins, then one may draw a non-additive tube first, and then the light blue top tube.draw a non-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 or or gold gold top). Contains a gel separator and clot top). Contains a gel separator and clot activator.activator.2. Sodium heparin (2. Sodium heparin (dark greendark green top) top)3. PST (3. PST (light greenlight green top). Contains lithium heparin top). Contains lithium heparin anticoagulant and a gel anticoagulant and a gel separator.separator.4. EDTA (4. EDTA (lavender lavender top)top)5. ACDA or ACDB (5. ACDA or ACDB (pale yellowpale yellow top). Contains acid citrate top). Contains acid citrate dextrose.dextrose.6. Oxalate/fluoride (6. Oxalate/fluoride (light graylight gray top) top)

Quick QuizQuick Quiz The light-sensitive substance that The light-sensitive substance that

provides the normal straw color of serum provides the normal straw color of serum and plasma isand plasma is

a. a. urochromeurochrome b. b. bilirubinbilirubin

c. c. flavin compoundsflavin compounds d. d. beta-carotenebeta-carotene

TheThe vacutainer required in the vacutainer required in the measurement of serum ammonia ismeasurement of serum ammonia is

a. a. green topgreen top b. b. red topred top

c. c. blue topblue top d. d. lavender toplavender top

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-Interfering substances are co-precipitated during clotting such as precipitated during clotting such as LPLLPL

2.2. Optically clearerOptically clearer3.3. Free from anticoagulant interferenceFree from anticoagulant interference

Must ideally reach the laboratory within 45 minutesMust ideally reach the laboratory within 45 minutes Agitation must be avoided during transportAgitation must be avoided during transport Use amber containers for photolabile substancesUse amber containers for photolabile substances Transport in ice (4 deg C) those specimens for Transport in ice (4 deg C) those specimens for BGA, BGA,

renin, enzymes and catecholaminesrenin, enzymes and catecholamines

Specimen InterferenceSpecimen InterferenceLysis 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 called Lysis of RBCs is called laking or hemolysislaking or hemolysis which may which may occur occur in vivo or in vitroin vivo or in vitro

In vitro hemolysisIn vitro hemolysis is more common which may be due is more common which may be due to:to:

1.1. Use of vacuum tubesUse of vacuum tubes2.2. Vigorous mixingVigorous mixing3.3. Use of too narrow or too wide needle boresUse of too narrow or too wide needle bores4.4. Effect of alcoholEffect of alcohol5.5. Centrifugation and separation stepsCentrifugation and separation steps

Hemolysis is visible only not until a Hemolysis is visible only not until a 200 mg/L of 200 mg/L of hemoglobin level in presenthemoglobin level in present

Icteresia (Icteric serum)Icteresia (Icteric serum) Intensely yellow serum sampleIntensely yellow serum sample due to due to

elevated bilirubinelevated bilirubin value value

Jaundice in a patient is caused by a Jaundice in a patient is caused by a bilirubin bilirubin level of greater than 430 level of greater than 430 µM (25 mg/L)µM (25 mg/L)

Bilirubin interferes with tests using dyes and Bilirubin interferes with tests using dyes and turbidity teststurbidity tests

Interference due to bilirubin may be Interference due to bilirubin may be minimized by sample blankingminimized by sample blanking or dual or dual wavelength method known as the wavelength method known as the Allen Allen correctioncorrection method method

Lactescence (Lipemic serum)Lactescence (Lipemic serum) Obtained normally after a meal due to Obtained normally after a meal due to elevated elevated

chylomicronschylomicrons

Characterized by Characterized by milky or highly turbid serummilky or highly turbid serum

Lactescence appears when the Lactescence appears when the TAG level reaches 4.6 mM (4g/L)TAG level reaches 4.6 mM (4g/L)

Errors due to lipemia may be Errors due to lipemia may be corrected by ultracentrifugation of corrected by ultracentrifugation of the serum samplethe serum sample

Grounds for rejecting a specimenGrounds for rejecting a specimen Inadequate sample identificationInadequate sample identification

Insufficient volume of specimen Insufficient volume of specimen collectioncollection

Inappropriate collection tubeInappropriate collection tube

HemolysisHemolysis

Improper transportationImproper transportation

InterferencesInterferences

Last Quick QuizLast Quick Quiz Which sample below will likely require Which sample below will likely require

ultracentrifugation to minimize sample ultracentrifugation to minimize sample interference?interference?a. a. icteresiaicteresia b. b. lipemialipemiac. c. lakinglaking d. d. all of the aboveall of the above

Prompt separation of the clot from serum Prompt separation of the clot from serum should always be observed because this should always be observed because this substance substantially decrease by 5% per substance substantially decrease by 5% per hour if serum is left in contact with clot. This hour if serum is left in contact with clot. This substance issubstance isa. a. sodiumsodium b. b. albuminalbuminc. c. glucoseglucose d. d. phosphatephosphate

QUALITY MANAGEMENTQUALITY MANAGEMENTQuality Assurance (QA)Quality Assurance (QA)

includes maneuvers encountered in the includes maneuvers encountered in the analyticanalytic, , pre-analytic and post-pre-analytic and post-analytic phasesanalytic phases of laboratory testing of 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

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

Analytical phaseAnalytical phase includes includes 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 includes includes reporting out results of analysis (manual or reporting out results of analysis (manual or

computerized)computerized) physician contactphysician contact

Post-analytical phase also includesPost-analytical phase also includes

monitors quality performance starting from monitors quality performance starting from the ordering of a laboratory determination the 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 requires involves total quality control which requires constant attention of all involved in the constant attention of all involved in the laboratory testinglaboratory testing

Quality Control (QC)Quality Control (QC) is concerned with the is concerned with the analytic phaseanalytic phase of QA of QA

monitors the monitors the over-all reliability of over-all reliability of laboratory resultslaboratory results in terms of in terms of accuracy accuracy and precisionand precision

relies on the diligent and persistent execution of the relies 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

has two major typeshas two major types

External QC (Interlaboratory QC)External QC (Interlaboratory QC) was was established byestablished by BelkBelk which monitors primarily thewhich monitors primarily the accuracyaccuracy of laboratory tests; the use ofof laboratory tests; the use of Youden Youden plotsplots

Internal QC (Intralaboratory QC)Internal QC (Intralaboratory QC) was was established byestablished by Levey Levey which primarily monitors the which primarily monitors the day-to-dayday-to-day performance of laboratory tests -performance of laboratory tests - precisionprecision assessment. Intralaboratory QC can assessment. Intralaboratory QC can be based either from the results of control be based either from the results of control specimens or on the results of patient specimens; specimens or on the results of patient specimens; the use ofthe use of Levey-Jennings chartLevey-Jennings chart

Quick Quick QuizQuiz Which QC chart is a graphic Which QC chart is a graphic

representation of the acceptable limits of representation of the acceptable limits of variation in the results of an analytical variation in the results of an analytical method?method?a. a. GaussianGaussian b.b. Youden Youdenc.c. Levey-Jennings Levey-Jennings d.d. Cusum Cusum

Which of the following has as its purpose Which of the following has as its purpose to promote the incorporation of principles to promote the incorporation of principles of quality management and QA into daily of quality management and QA into daily work routines?work routines?a. a. ISO 9000ISO 9000 b.b. NCCLS NCCLSc.c. CAP CAP d.d. NIST NIST

ErrorsErrorsAnalytical errorsAnalytical errors are usually are usually systematic systematic

errors or determinate errorserrors or determinate errors that are that are caused by some factors in the analytical caused by some factors in the analytical system such as system such as erroneously calibrated erroneously calibrated pipettor, deteriorating reagent pipettor, deteriorating reagent and and fluctuating electrical current.fluctuating electrical current.

Personnel or operator errorsPersonnel or operator errors are usually are usually called called random errors or indeterminate random errors or indeterminate errorserrors that usually affect several analyses. that usually affect several analyses. Examples are Examples are mislabeling the specimen, mislabeling the specimen, wrong number entry wrong number entry and and instability of instability of needle due to electronic component of needle due to electronic component of instrumentinstrument..

Interpretation of the Levey-Interpretation of the Levey-Jennings ChartJennings 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

Trend Trend suggests a suggests a systematic driftsystematic drift or error. or error. Values move continuously away from the Values move continuously away from the mean in just one direction. Among its causes mean in just one direction. Among its causes include deteriorating reagents, changes in include deteriorating reagents, changes in standard concentration or failing instrument.standard concentration or failing instrument.

Shift Shift also suggests also suggests systematic errorsystematic error. Seen as . Seen as an abrupt change from the established mean an abrupt change from the established mean and continue in a linear fashion parallel to the and continue in a linear fashion parallel to the mean. mean.

Increased dispersionIncreased dispersion suggests suggests random random errorerror. High or low outliers are frequently . High or low outliers are frequently observed. Causes of this are variations in observed. Causes of this are variations in operation of instrument, interfering operation of instrument, interfering substances, electronic fluctuations, and substances, electronic fluctuations, and clerical errors.clerical errors.

Quick QuizQuick Quiz Which of the following terms refers to Which of the following terms refers to

deviation from the true value caused by deviation from the true value caused by indeterminate errors inherent in every indeterminate errors inherent in every laboratory management?laboratory management?

a.a. random error random error b.b. standard error of the mean standard error of the meanc.c. parametric analysis parametric analysis d.d. nonparametric analysis nonparametric analysis

When comparing a potential new test with a When comparing a potential new test with a comparative method in order to bring a new comparative method in order to bring a new method into the laboratory, one observes error method into the laboratory, one observes error that is consistently affecting results in one that is consistently affecting results in one direction. This type of error isdirection. This type of error is

a.a. systematic error systematic error b.b. random error random errorc.c. constant systematic error constant systematic error d.d. proportional proportional

random random errorerror

Westgard Multi-Rule Westgard Multi-Rule TechnicTechnicRule Rule MeaningMeaning

1:2s1:2s = one control observation exceeds the = one control observation exceeds the control limit set at control limit set at M±2SD; warning ruleM±2SD; warning rule

1:3s1:3s = reject when one observation exceeds = reject when one observation exceeds M±3SD; suggests M±3SD; suggests random errorrandom error

2:2s2:2s = reject when two consecutive observations = reject when two consecutive observations exceed the same exceed the same M+2SD or the same M-M+2SD or the same M-2SD; suggests systematic 2SD; suggests systematic errorerror

R:4sR:4s = reject when one control observation in the = reject when one control observation in the run exceeds its run exceeds its M+2SD and another exceeds M+2SD and another exceeds M-2SD; suggests M-2SD; suggests random errorsrandom errors

4:1s4:1s = reject when four consecutive control = reject when four consecutive control observations exceed observations exceed the same M+1SD limit or the same M+1SD limit or the same M-1SD limit; suggests the same M-1SD limit; suggests systematic systematic errorerror

10:Mean10:Mean= reject when 10 consecutive control = reject when 10 consecutive control observations observations fall on one side of the mean; fall on one side of the mean; suggests systematic errorsuggests systematic error

Quick Quick QuizQuiz What does the preparation of a Levey-Jennings What does the preparation of a Levey-Jennings

chart for any single constituent of serum require?chart for any single constituent of serum require?

a. a. analysis of control serum over a period of 20 analysis of control serum over a period of 20 consecutive daysconsecutive days

b.b. 20-30 analyses of the control serum, on 1 day, 20-30 analyses of the control serum, on 1 day, in one in one batchbatch

c.c. analyses consistently performed by one person analyses consistently performed by one person

d.d. weekly analyses of the control serum for 1 weekly analyses of the control serum for 1 monthmonth

In addition to utilizing Levey-Jennings charts, what In addition to utilizing Levey-Jennings charts, what other criteria should be applied to interpret other criteria should be applied to interpret internal QC data?internal QC data?

a. a. Westgard multiruleWestgard multirule b.b. Cusum Cusum

c.c. linear regression linear regression d.d. Youden Youden

Accuracy and PrecisionAccuracy and PrecisionAccuracyAccuracy is the extent to which the is the extent to which the mean mean

measurement is close to the true value.measurement is close to the true value. The accuracy of the method is reflected by its The accuracy of the method is reflected by its

ability to obtain the same values of the ability to obtain the same values of the reference samples of known reference samples of known concentration. concentration. Expressed asExpressed as % absolute % absolute error.error.

% absolute error = true value – actual value x 100%% absolute error = true value – actual value x 100% true valuetrue value

PrecisionPrecision is the is the reproducibility reproducibility of a of a laboratory determination when it is run repeatedly laboratory determination when it is run repeatedly under identical conditionsunder identical conditions

precision is commonly expressed in terms of precision is commonly expressed in terms of standard deviation (SD), variance standard deviation (SD), variance or or coefficient of variation (CV)coefficient of variation (CV)

Standard deviation =Standard deviation = ΣΣ (M – x) (M – x)22

n-1n-1

Variance = (SD)Variance = (SD)22

Coefficient of variation = SD x Coefficient of variation = SD x 100%100%

MM

Reliability Reliability refers to the ability of a refers to the ability of a test to test to maintain its accuracy and maintain its accuracy and precision for an extended period precision for an extended period of timeof time..

Quick QuizQuick Quiz Which of the following terms refers to Which of the following terms refers to

the closeness with which the measured the closeness with which the measured value agrees with the true value?value agrees with the true value?

a. a. random errorrandom error b.b. precision precision

c.c. accuracy accuracy d.d. variance variance

Given that a method mean is 25 mg/dl Given that a method mean is 25 mg/dl and the SD is 1.2 mg/dl, what would be and the SD is 1.2 mg/dl, what would be the CV?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%

SDSD is a measure of dispersion of the values around is a measure of dispersion of the values around the the 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 +/-3 SD around the meanmean

most laboratories choose the most laboratories choose the 95% confidence limit95% confidence limit in in expressing precisionexpressing precision

the the medianmedian is the value that is middlemost in an array of is the value that is middlemost in an array of numbers while the numbers while the modemode is the value that occurs most is the value that occurs most frequently. frequently.

In a In a Gaussian distributionGaussian distribution, the mean, median and , the mean, median and mode are very close in value as shown by its bell-shaped mode are very close in value as shown by its bell-shaped curve.curve.

Quick QuizQuick Quiz In a QC program the confidence interval In a QC program the confidence interval

has been set at 95%. How many test has been set at 95%. How many test results are expected to fall beyond the results are expected to fall beyond the established limits?established limits?

a. a. 1 in 51 in 5 b.b. 1 in 10 1 in 10

c.c. 1 in 20 1 in 20 d.d. 1 in 95 1 in 95

What percentage of values will fall What percentage of values will fall between ±2SD in a Gaussian between ±2SD in a Gaussian distribution?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%

SensitivitySensitivity is the ability of the test to is the ability of the test to detect the detect the smallest amount of the analyte in a smallest amount of the analyte in a solution or samplesolution or sample. It expresses the ability of . It expresses the ability of the test to be positive in the presence of the analyte the test to be positive in the presence of the analyte or the disease. A highly sensitive test is or the disease. A highly sensitive test is characterized by a characterized by a decreased probability of decreased probability of obtaining false negative resultsobtaining false negative results..

SpecificitySpecificity refers to the ability of the test to refers to the ability of the test to detect analyte without detecting other detect analyte without detecting other analytes that are also present in the analytes that are also present in the samplesample. It expresses the ability of the test to be . It expresses the ability of the test to be negative in the absence of the analyte or the negative in the absence of the analyte or the disease. A highly specific test leads to a disease. A highly specific test leads to a decreased probability of obtaining false decreased probability of obtaining false positive results.positive results.

Control Control is a solution (usually is a solution (usually pooled pooled serum samplesserum samples) whose constituents are ) whose constituents are diversediverse but are known ( but are known (a range of a range of values per analytevalues per analyte). This can be run ). This can be run simultaneously with the simultaneously with the TestTest to to check, check, verify or validate the accuracy of the verify or validate the accuracy of the results.results.

StandardStandard is a solution of is a solution of a particular a particular analyteanalyte of known characteristics and of known characteristics and known value (known value (exact concentrationexact concentration). It ). It is used as is used as reference for the calculation reference for the calculation of the value of the Unknownof the value of the Unknown..

Quick QuizQuick Quiz To determine the predictive value of a positive To determine the predictive value of a positive

test, all of the following parameters must be test, all of the following parameters must be known known exceptexcept

a. a. sensitivitysensitivity b.b. specificity specificity

c.c. disease prevalence disease prevalence d.d. precision precision

Which activity will not check on the accuracy Which activity will not check on the accuracy of the results obtained?of the results obtained?

a. a. make a Youden plot make a Youden plot b.b. use of abnormal use of abnormal controlcontrol

c. use of normal control c. use of normal control d.d. use of a use of a calibrator calibrator (standard)(standard)

Figures of Merit of Figures of Merit of Analytical MethodsAnalytical Methods

MajorMajor1. 1. AccuracyAccuracy

2. Precision2. Precision

3. Limit of detection3. Limit of detection

4. Applicable concentration 4. Applicable concentration range orrange or

linear rangelinear range

5. Sensitivity5. Sensitivity

6. Selectivity6. Selectivity

MinorMinor

1. 1. SpeedSpeed

2. Ease and 2. Ease and convenienceconvenience

3. Skill required of the 3. Skill required of the operatoroperator

4. Cost of analysis and 4. Cost of analysis and availability of availability of

equipmentequipment

5. Per sample cost5. Per sample cost

EndEnd