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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