الرحــمـــــن الله الرحــمـــــن بـســـــم الله بـســـــمالرحيـــمالرحيـــم

Practical Clinical Practical Clinical PathologyPathology

IntroductionIntroduction Clinical hematologyClinical hematology Clinical Biochemistry UrinalysisUrinalysis Clinical ParasitologyClinical Parasitology

Clinical biochemistryClinical biochemistry

IntroductionIntroduction Total proteins AlbuminAlbumin Bilirubin (Total & Direct)Bilirubin (Total & Direct) GlucoseGlucose BUN = Blood Urea NitrogenBUN = Blood Urea Nitrogen CreatinineCreatinine PhosphorusPhosphorus Enzymes (ALT / AST)Enzymes (ALT / AST)

Measurement of total proteinsMeasurement of total proteins

Physical methodsPhysical methods RefractometryRefractometry

(Total Solids refractometer = T.S. (Total Solids refractometer = T.S. refractometer)refractometer)

Chemical methodsChemical methods Biuret’s method (colrimetric)Biuret’s method (colrimetric) Lowry assayLowry assay Dye binding methodDye binding method Precipitation by usingPrecipitation by using

TriChloro Acetic acidTriChloro Acetic acid Picric acidPicric acid

Physical methodsPhysical methodsRefractometryRefractometry

It depends on the change of It depends on the change of refractive index of the solution that refractive index of the solution that is proportional to the concentration is proportional to the concentration of proteinsof proteins

↑ ↑ Total proteins ↑ refractive indexTotal proteins ↑ refractive index

T.S. RefractometerT.S. Refractometer

T.S. RefractometerT.S. Refractometer

Chemical methodsChemical methods

Biuret’s methodBiuret’s method

(Colorimetric method)(Colorimetric method)

Total proteinsTotal proteins

99 PrinciplePrinciple SampleSample ReagentsReagents ProceduresProcedures WavelengthWavelength ReadingReading CalculationCalculation Normal valuesNormal values InterpretationInterpretation

Total proteinsTotal proteinsPrinciplePrinciple

Proteins in sample + Cu Proteins in sample + Cu ++++

Alkaline pH

(NaOH)

VioletViolet blueblue colour colour complexcomplex

• Measured colorimetrically at 546 nm wavelength• The intensity of the colour is directly proportional to the concentration of the proteins in the sample• ↑ Total proteins ↑ colour intensity

Total proteins Total proteins SampleSample

Serum or Plasma

Total proteinsTotal proteinsReagentsReagents

Total proteins reagent = Biuret Total proteins reagent = Biuret reagentreagent Copper Sulphate (CuSOCopper Sulphate (CuSO44)) Sodium hydroxide (NaOH)Sodium hydroxide (NaOH) Potassium iodidePotassium iodide Potassium sodium tartratePotassium sodium tartrate

Total protein Total protein standardstandard solution (10 g solution (10 g%)%)

Total proteinsTotal proteins (Procedures)(Procedures)

Blank Standard Test

Total protein reagent 1 ml 1 ml 1 ml

Standard - 0.01 ml (10 µl) -

Sample - - 0.01 ml (10 µl)

MixIncubate at room temperature for 5 minutes

Blank Standard TestBiuret reagent

0 -1 –

2 –3 –

4 –5 –

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Blank Standard TestStandard

0 -

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0 -

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Blank Standard Test

0 -

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Serum

Blank Standard Test

Total proteinsTotal proteinsWavelengthWavelength

546 nm546 nm

Total proteinsTotal proteinsReadingReading

Read OD of the standard and the Read OD of the standard and the test test

against the blank at 546 nmagainst the blank at 546 nm

OD standard = ……….? ……….OD standard = ……….? ……….

OD test = ……….? ……….OD test = ……….? ……….

Total proteinsTotal proteinsCalculationCalculation

According to Beer-According to Beer-Lambert lawLambert law

OD standard → Known conc.OD standard → Known conc. (10 g/dl)(10 g/dl)

OD test → T.P. conc. In the OD test → T.P. conc. In the sample sample

(Unknown)(Unknown)

OD test x Standard conc.T.P. conc. In sample = _____________________ OD standard

Total proteinsTotal proteinsNormal valuesNormal values

Total proteins normal valuesTotal proteins normal values

5.6 – 8 g/dl5.6 – 8 g/dl

Total proteinsTotal proteinsInterpretationInterpretation

↑ ↑ Increased values Increased values (Hyperproteinemia)(Hyperproteinemia)

Normal valueNormal value

↓ ↓ Decreased values Decreased values (Hypoproteinemia)(Hypoproteinemia)

Total proteinsTotal proteinsInterpretationInterpretation

↑ ↑ Increased values Increased values (Hyperproteinemia)(Hyperproteinemia)

Dehydration (relative increase)Dehydration (relative increase)

Inflammatory conditionsInflammatory conditions

(due to increase in acute phase (due to increase in acute phase proteins)proteins)

Total proteinsTotal proteinsInterpretationInterpretation

↓ ↓ Decreased values Decreased values (Hypoproteinemia) (Hypoproteinemia) MalnutritionMalnutrition MaldigestionMaldigestion MalabsorptionMalabsorption Liver diseasesLiver diseases Renal diseasesRenal diseases Loss of proteins (hemorrhage)Loss of proteins (hemorrhage)

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