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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 –
0 -
1 –
2 –
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4 –
5 –
0 -
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2 –
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5 –
0 -
1 –
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0 -
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0 -
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Blank Standard TestStandard
0 -
0.01 –
0.02 –
0.03 –
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0.06 –
0.07 –
0.08 –
0.09 -
0 -
0.01 –
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0.04 –
0.05 –
0.06 –
0.07 –
0.08 –
0.09 -
0 -
0.01 –
0.02 –
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0.05 –
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0.08 –
0.09 -
Blank Standard Test
0 -
0.01 –
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0 -
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0.07 –
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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)
THANKS FOR ATTENTION
ALL THE BEST
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