الرحــمـــــن الله الرحــمـــــن بـســـــم الله بـســـــمالرحيـــمالرحيـــم

Practical Clinical Practical Clinical PathologyPathology

IntroductionIntroduction Clinical hematologyClinical hematology Clinical Biochemistry UrinalysisUrinalysis Clinical ParasitologyClinical Parasitology

Clinical BiochemistryClinical Biochemistry

Introduction Total proteinsTotal proteins AlbuminAlbumin GlucoseGlucose Bilirubin (Total & Direct)Bilirubin (Total & Direct) PhosphorusPhosphorus BUN = Blood Urea NitrogenBUN = Blood Urea Nitrogen CreatinineCreatinine EnzymesEnzymes

Clinical BiochemistryClinical Biochemistry DefinitionDefinition SampleSample

Arterial and venous bloodArterial and venous blood Serum and PlasmaSerum and Plasma PrecautionsPrecautions

ReagentsReagents KitsKits

Measurment of solutesMeasurment of solutes ColorimetryColorimetry Beer Lambert lawBeer Lambert law Calculation of concentrationCalculation of concentration BlanksBlanks EnzymesEnzymes

Quality controlQuality control

Definition of Clinical Definition of Clinical biochemistrybiochemistry

The part of clinical pathology that deals The part of clinical pathology that deals

with chemical analysis of body fluids, with chemical analysis of body fluids,

excretions and tissues for the purpose excretions and tissues for the purpose

of diagnosis of disease conditions.of diagnosis of disease conditions.

Arterial and venous Arterial and venous bloodblood

ArterialArterialBright redBright redDifficult to sample Difficult to sample from because it’s from because it’s deepdeepSuitable for blood Suitable for blood gas analysisgas analysisHealing is slowHealing is slow

VenousVenousDark redDark redEasy to sample Easy to sample from because it’s from because it’s superficiealsuperficiealUsed for most lab. Used for most lab. TestsTests

Healing is rapidHealing is rapid

Serum and PlasmaSerum and PlasmaSerumSerum

Blood collected Blood collected WITHOUT WITHOUT anticoagulantanticoagulant

Contains CalciumContains CalciumTotal proteins in Total proteins in serum < plasmaserum < plasmaMore preferrable in More preferrable in biochemical and biochemical and enzymatic assaysenzymatic assays

PlasmaPlasmaBlood collected Blood collected WITHWITH anticoagulantanticoagulant

Not contain Calcium Not contain Calcium except heparinized except heparinized samplesampleRetains coagulation Retains coagulation factors as fibrinogen factors as fibrinogen and prothrombinand prothrombin

Followed by centrifugation for separation

Precautions of samplesPrecautions of samples

GenerallyGenerally Avoid HemolysisAvoid Hemolysis Avoid lipemiaAvoid lipemia

Special precautionsSpecial precautions Glucose:Glucose: use Sodium fluoride as use Sodium fluoride as

anticoagulantanticoagulant Urea:Urea: Don’t use sodium fluoride Don’t use sodium fluoride Bilirubin:Bilirubin: Avoid exposure to direct sunlight Avoid exposure to direct sunlight

Reagents (Kits)Reagents (Kits)

Commercial ready made kitsCommercial ready made kitsAvailable for analysis of biochemical Available for analysis of biochemical constituents in different body fluidsconstituents in different body fluids

(Instead of preparing reagents in your (Instead of preparing reagents in your lab)lab)

Colorimetry

ColorimetryColorimetry

I0: Original light

I: Light absorbed

= OD = Absorbance

I1: Light transmitted

↑ Colour of the solution → ↑ the Optical Denisty (Absorbance)

Colorimeter vs Spectrophotometer

InfraRed

UltraViolet

Visible LightColorimeter

Spectrophotometer

Beer Lambert lawBeer Lambert law

Under suitable conditions,Under suitable conditions,

The amount of The amount of light absorbedlight absorbed by a by a coloured coloured

solution, when illuminated with solution, when illuminated with light of a light of a

suitable wavelengthsuitable wavelength, is directly , is directly proportional to proportional to

the the conc. of colored solutionconc. of colored solution..

Beer Lambert lawBeer Lambert law

ODStanda

rdMeasur

ed

Conc.Standa

rdKnown

ODTest

(Sample)

Measured

Conc.Test

(Sample)

Unknown ?

Beer Lambert lawBeer Lambert law

Conc.Test

(Sample)

Unknown ?

=

ODTest

(Sample)

Measured

Conc.Standa

rdKnown

ODStanda

rdMeasur

ed

Ways to obtain the Ways to obtain the concentration of certain concentration of certain biochemical constituentbiochemical constituent

Beer Lambert lawBeer Lambert law

Standard calibration curveStandard calibration curve

Standard tableStandard table

Standard factorStandard factor

BlanksBlanks

A solution used for zero adjustment A solution used for zero adjustment of the colorimeter or of the colorimeter or spectrophotometer before spectrophotometer before measuring the OD (absorbance) of measuring the OD (absorbance) of standard or teststandard or test

It can be eitherIt can be either Reagent blankReagent blank Sample blankSample blank

Reagent blankReagent blank

Contains all reagent added to the Contains all reagent added to the samplesample

WITHOUTWITHOUT

The sampleThe sample

Used for most tests in biochemistryUsed for most tests in biochemistry

Sample blankSample blank

Contains the sample + reagentsContains the sample + reagents

WITHOUTWITHOUT

One reagent essential to complete the One reagent essential to complete the reactionreaction

Used when the sample has pronounced Used when the sample has pronounced colour as in case of jaundice (so used for colour as in case of jaundice (so used for BilirubinBilirubin))

EnzymesEnzymes

Because of their small concentration Because of their small concentration in the blood, ACTIVITY of the enzymes in the blood, ACTIVITY of the enzymes is measured instead of concentrationis measured instead of concentration

Enzyme activity can be obtained by Enzyme activity can be obtained by measuring the amount of:measuring the amount of:

a- substrates consumed or a- substrates consumed or

b- the product obtainedb- the product obtained

EnzymesEnzymes

Methods for enzymes determination Methods for enzymes determination are:are: End point End point KineticKinetic

Enzyme activity is expressed as ……. Enzyme activity is expressed as ……. U/LU/L

Quality controlQuality control

Control sample Control sample

(sample with known conc. Of certain solute (sample with known conc. Of certain solute e.g: urea)e.g: urea)

is used every now and thenis used every now and then

Control sample should be assayed in the Control sample should be assayed in the same way the samples are analyzedsame way the samples are analyzed

i.e.: same reagents, instruments and i.e.: same reagents, instruments and calculations.calculations.

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