Dr. S. H. Burungale

1. Clinical Analysis
Dr. S. H. Burungale

11. Estimation of cholesterol

18. Estimation of urea

22. Components of Blood
Blood can be divided into
1. Plasma
2. cellular Elements
Blood
Plasma Cellular Elements
Serum Fibrinogen erythrocytes Leukocytes platelets
R.B.C. W.B.C. Thrombocytes

23. Blood Components
1. Blood Plasma : Blood plasma contains 91% to 92
% Water and 8 to 9% Solids.
solid portion consists of following substances:
a. Organic substances :7 to 8 % 1. Proteins 2.
Harmones 3. Antibodies 4. enzymes 5. Natural fats 6.
Non proteins.
b. Respiratory gases: R O2 and CO2
c. Inorganic Substances : sodium chloride and ca, K,
Bicarbonates, Iodine and Iron.

24. Cellular Elements
This portion is about 45% of total volume of blood in
which free cells are suspended in plasma . These
cells are known as blood cells or Corpuscles.
Erythrocytes R.B.C. : bone marrow hemoglobin
Leukocytes W.B.C. : body defence , production of
antibodies, Neutrophil Basophil ,Eosinophil,Lyphocytes,
Monocytes
Platelets Thrombocytes: large cells, thermocytes
clotting cells, 250000 to 500000 /cc of blood.

25. Collection and Preservation of
Samples
Collection of blood of HIV Infected Pateint: use hand gloves.
2. Collection of Blood for Serum analysis :
collection of blood in dry test tube and hemolysis destruction of blood for
librating hemoglobin and other cells. Serum is separated by centrifuge .
3. Collection of blood for Co2 analysis
4. Collection of Blood for Glucose analysis

26. Determination of the blood glucose concentration :
• Glucose is the main sugar that the body makes from the food in the diet.
It is carried through the bloodstream to provide energy to all cells in the
body.
• Glucose is a simple sugar (a monosaccharide). The body produces it from
protein, fat and, in largest part, carbohydrate.
• Ingested glucose is absorbed directly into the blood from the intestine and
results in a rapid increase in blood glucose.

27. Two Methods of Measuring Blood Glucose Level :
1. Reduction method, which is based on the ability of glucose to reduce
Cu++ to Cu+ less sensitive, substances that could reduce Cu++ :
fructose, galactose, vitamin C, uric acid, etc.
2. Enzymatic method (more specific and precise result) : Glucose is
oxidized by glucose oxidase gluconic acid + H2O2  red dye.

28. Principle of the test :
• Glucose present in the plasma is oxidized by the enzyme
(Glucose oxidase( to gluconic acid with the liberation of hydrogen
peroxide, which is converted to water and oxygen by the enzyme
peroxidase .
• 4-aminophenazone ,an oxygen acceptor, takes up the oxygen and together
with phenol forms Red colored complex which can be measured at 500nm.
Glucose oxidase (GOD) catalyzes the oxidation of glucose according to the following
equation :
GOD
Glucose + O2 Gluconic acid + H2O2
The hydrogen peroxide (H2O2) which is formed, in the presence of peroxidase (POD) reacts
with 4-aminophenazone and phenol, and gives rise to 4-benzoquinon monoiminophenazon
(a red dye)
POD
H2O2 + 4-aminophenazone + phenol 4 H2O + 4 p-benzoquinon
monoiminophenazone
(red color)

29. • Procedure :
• Centrifuge 3 ml EDTA blood, 2000 rpm for 10 min.
The plasma will be separated from the blood cells. Use plasma for sample.
• Pipette into each of the three reaction tubes according to the following table

30. • Mix the content of each tube well, then incubate them at 37oC for 10
minutes .
• Using cuvette tube, read the sample and standard absorbance against the
blank at 500 nm.
Calculation:
Calculate the concentration of BL in the provided fasting blood samples
using the absorbance reading of standard glucose and applying the following
equation:
Glucose (mg/dl) = (Abs. sample/Abs. standard)* standard conc. (100mgdl) .
Normal Range :
Normal (fasting BSL = 70 – 110 mg/dl)

31. Notes :
1. By this method the blood glucose level can be measured linearly up to 500
mg/dl.
2. If the blood glucose level is higher than 500 mg/dl, dilute the plasma three
times by adding 2 volumes of normal saline, then repeat the procedure.
Multiply the result *3.
3. The color produced will be stable for 2 hours.

41. Introduction
• The key roles which plasma proteins play in bodily
function, together with the relative ease of assaying
them, makes their determination a valuable diagnostic
tool as well as a way to monitor clinical progress.
• In very general terms, variations in plasma protein
concentrations can be due to any of three changes:
– rate of protein synthesis,
– rate of removal,
– the volume of distribution.
Mohammed Laqqan

42. Proteins: Common properties
• In spite of functional differences between the various
serum proteins, they have certain common
biophysical and biochemical properties. These
include:
– a basic composition of carbon, hydrogen, nitrogen
and oxygen;
– a backbone of covalent peptide bonds which join
the amino acid units together; and
– absorption maxima in the ultraviolet region.
• Based on these properties, laboratory methods have
been developed to determine the concentration of
proteins in serum,
Mohammed Laqqan

43. Serum Total Protein
• Serum total protein, also called plasma total protein or total
protein, is a biochemical test for measuring the total amount of
protein in blood plasma or serum.
• Protein in the plasma is made up of albumin and globulins.
• Note: the globulin in turn is made up of α1, α2, β, and γ
globulins.
• These fractions can be quantitated using protein
electrophoresis, but the total protein test is a faster and cheaper
test that estimates the total of all fractions together.
• The traditional method for measuring total protein uses the
biuret reagent, but other chemical methods are also available.
Mohammed Laqqan

44. Methods of Total Protein Analysis
• Method 1: Kjeldahl; quantitative, protein nitrogen
determination
• Method 2: Biuret; quantitative, increased absorption
at 540 nm;
Mohammed Laqqan

45. Specimen
• Serum and plasma may be used, and all usually
yield comparable results, though, because of the
presence of fibrinogen, plasma levels for total
protein are 2 to 4 g/L higher than serum levels.
• A fasting specimen is not required but may be
desirable to decrease lipemia.
• Total protein is stable in serum and plasma for
– 1 week at room temperature,
– and for at least 2 months at –20° C
Mohammed Laqqan

46.  Hypoproteinemia
– Malnutrition and/or malabsorption
– Excessive loss as in renal disease, GI leakage,
– excessive bleeding, severe burns
– Excessive catabolism
– Liver disease
 Hyperproteinemia
– Dehydration
– Monoclonal increases
– Polyclonal increase
• Only disorders affecting the concentration of albumin and/or the
immunoglobulins will give rise to abnormal total protein levels.
• Other serum proteins are never present in high enough concentrations for
changes to have a significant overall effect.
Mohammed Laqqan

47. The Biuret Method
• The Biuret reagent is made of (NaOH) and copper (II) sulfate
(CuSO4), together with potassium sodium tartrate
(KNaC4H4O6).
– A blue reagent which turns violet in the presence of
proteins.
• The Sodium hydroxide does not participate in the reaction at
all, but is merely there to provide an alkaline medium so that
the reaction can take place.
Mohammed Laqqan

48. Principle: Biuret Method
• Peptide bonds of proteins react with tartrate-
complexed cupric ions in alkaline solutions to form a
colored product.
• In a positive test, a copper(II) ion is reduced to
copper(I), which forms a complex with the nitrogens
and carbons of the peptide bonds in an alkaline
solution.
• A violet color indicates the presence of proteins.
• The intensity of the color, and hence the absorption at
540 nm, is directly proportional to the protein
concentration, and can be determined
spectrophotometrically at 540 nm.
Mohammed Laqqan

49. Reference range
• Reference range for total proteins is 66.6 to 81.4 g/L
• Results for males are approximately 1 g/L higher than results
for females; this difference is probably not of clinical
significance.
• In newborns, the mean serum protein concentration is 57 g/L,
increasing to 60 g/L by 6 months and to adult levels by about 3
years of age.
• Serum protein levels of premature infants can be much lower
than that of full term infants, ranging from 36 to 60 g/L.
Mohammed Laqqan

50. Albumin
• Albumin is the most abundant circulating plasma
protein (40–60 % of the total)
• Playing important roles in the maintenance of the
colloid osmotic pressure of the blood, in transport of
various ions, acids, and hormones.
• It is a globular protein with a molecular weight of
approximately 66,000 D and is unique among major
plasma proteins in containing no carbohydrate.
• It has a relatively low content of tryptophan and is an
anion at pH 7.4.
Mohammed Laqqan

51. Analysis Methods
• Method 1: Precipitation; quantitative
– Salt fractionation, Acid fractionation
– Principle of analysis: Changes of net charge of protein
result in precipitation
• Method 2: Tryptophan content; quantitative
– Principle of analysis:
– Glyoxylic acid + tryptophan in globulin Purple chromogen
(Amax, 540 nm); Total protein – globulin = albumin.
• Method 3: Electrophoresis; quantitative
– Principle of analysis: Albumin is separated from other
proteins in electrical field; percent staining of albumin
fraction multiplied by total protein value
Mohammed Laqqan

52. • Method 4: Dye binding, quantitative
– Methyl orange; BCG (bromcresol green); BCP (bromcresol
purple);
• Method 5: Dye binding; semiquantitative
– Bromphenol blue in test strip changes color from yellow to
blue in presence of albumin most commonly used test for
urine protein
Mohammed Laqqan

53. • Specimen: Serum is the specimen of choice, but heparinized
plasma can also be used if precautions are taken to prevent
heparin interferences.
• Interfering Factors
– Albumin is decreased in:
• Pregnancy (last trimester, owing to increased plasma
volume)
• Oral birth control (estrogens) and other drugs.
• Prolonged bed rest.
• IV fluids, rapid hydration, overhydration.
Albumin Reference Interval for Serum
Age Men (g/L) Women (g/L)
21–44 33.3–61.2 27.8–56.5

54. Clinical Significance
• Plasma albumin levels, although important for management
and follow-up, have very little value in clinical diagnosis.
• Hyperalbuminemia is usually attributable to
• dehydration or hemoconcentration.
• Hypoalbuminemia is usually the result of
• hemodilution,
• a rate of synthesis less than the albumin loss,
• diseases that cause a large albumin loss from urine,
skin, or intestine,
• increased catabolism observed in fevers, untreated
diabetes mellitus, and hyperthyroidism.
Mohammed Laqqan

55. Dye-binding Techniques
• Serum albumin is most often assayed using dye-binding
techniques.
• Albumin preferentially binds to anionic dyes that do not attract
globulins
• Bromcresol purple (BCP) and bromcresol green (BCG) are
most commonly used
• The amount of light absorbed by the albumin –dye complex is
proportional to the amount of albumin present
Mohammed Laqqan

56. Estimation of Serum albumin

60. Estimation of Serum Creatinine
Principle
Sample
Reagents
Procedures
Wavelength
Reading
Calculation
Normal values
Interpretation

61. 1Jaffe method(Manual method) Disadvantage: non
creatinine chromagen interfere with the reaction e.g.
protein , glucose, Ketones, Pyruvate, Bilirubin…….
2. Kinetic analyses modes(Automated method)

64. Read the OD test & Stand. against the blank at 510 nm
Read the sample against its blank at 510 nm O.D. =…………..

66. Definition of bilirubin
 Bilirubin is the water insoluble breakdown product of normal
heme catabolism
 It’s a yellow pigment present in bile ( a fluid made by the liver) ,
urine and feces .
 Heme is found in hemoglobin, a principal component of RBCs
[Heme: iron + organic compound “porphyrin”].
 Heme source in body:
 80% from hemoglobin
 20% other hemo-protein: cytochrome, myoglobin)

67. Heme and bilirubin
 Heme four pyrrols rings connected together to form
(porphyrin).
 Bilirubin consists of open chain of four pyrrols-like rings

68.  Direct bilirubin: is conjugated (water soluble bilirubin) in
aqueous solution it reacts rapidly with reagent (direct reacting).
 Indirect bilirubin: is unconjugated (water insoluble bilirubin)
because it is less soluble in it reacts more slowly with reagent
(reaction carried out in methanol).
- in this case both conjugated and unconjugated bilirubin are
measured given total bilirubin. Unconjugated will calculated by
subtracting direct from total and so called indirect.
 Total bilirubin = D+ ID
• Knowing the level of each type of bilirubin has diagnostic
important.

69. Procedure
Measuring serum bilirubin level
Principle:
 Kit components
 Sulfanalic acid reagent
 Sodium nitrate reagent
 Methanol reagent
 Bilirubinequavalent standard (5mg/dl T.bil; 2.5 mg/dl D.bil)
Sulphanalic acid + NaNO3 diazotized sulphanalic acid (DSA)
DSA + Bilirubin “D” Azobilirubin “purple”
Bilirubin “ID”+ DSA + accelerator Total bil.
(methanol )

70. Procedure:
TestTest blankEquivalent
Standard
1.4 ml1.4 ml1.4mlSulfanilic
acid
25ml-25mlNaNO3
-25mldis. H2O
Mix, stand for 1 min
100 µl
(serum)
100 µl
(serum)
100 µl
(Standard)
Sample
- Read the Abs after 1 min at 540nm, (Blank dis. H2O)
Use this to calculate D.bil
1.5 ml1.5 ml1.5 mlMethanol
- Mix by inversion, stand 5 min or more
- Read Abs. at 540nm (Blank dis. H2O)
Use this to calculate T.bil
Pour Bilirubinequavelant standard in clean cuvette read Abs. at
540nm, (blank dis. H O)

71. Calculations
• Conc. of Bilirubin equavelant is 5mg/dl for T.bil, and 2.5 mg/dl for D.bil
D.Bil:
Abs (test) - Abs (test blank) X 2.5 mg/dl
Abs of bilirubin equivalent
T.Bil:
Abs (test) - Abs (test blank) X 5 mg/dl
• To convert to mmol/L multiply by 17.1
Abs of Bilirubin equivalent

72.  Normal Results
 It is normal to have some bilirubin in your blood.
 Normal levels are:
 Direct (also called conjugated) bilirubin: 0 to 0.5 mg/dL
 Total bilirubin: 0.3 to 1.9 mg/dL
 Note: mg/dL = milligrams per deciliter
 Normal value ranges may vary slightly among different
laboratories.

73. Thank You