Liver function test is done to examine the healthy status of
the liver, the test is mostly requested for those suspects of liver disease, or
those infected with Hepatitis virus, mostly Hepatitis B and C.
PARAMETER FOR THE ANALYSIS
1.
Total Bilirubin (TB)
2.
Conjugated Blirubin (CB)
3.
Total protein (TP)
4.
Albumin (AL)
5.
Globulin (GL)
ESTIMATION OF BILIRUBIN USING VAN DEN BERGH DIAZO REAGENT
REAGENT
1.
SULPHANILIC ACID REAGENT
COMPOSITION
Sulphanilic acid 5.0g
Hydrochloric acid concentration
15ml
Distilled
water 1litre
PREPARATION
Dissolved 5.0g
of Sulphanilic acid in 600ml warm distilled water, when dissolved, add 15ml hydrochloric
acid concentration and make up the solution to 1litre with distilled water.
2.
SODIUM NITRATE SOLUTION
COMPOSITION
Sodium nitrate 5.0g
Distilled water 100ml
PREPARATION
Dissolved 5.0g
of Sodium nitrate in 80ml warm distilled water, and make up the solution to
100ml with distilled water.
When store at
4ºC the reagent is active for 2 weeks.
3.
DIAZO REAGENT SOLUTION
COMPOSITION
Reagent 1 10.0ml
Reagent 2 0.25ml
PREPARATION
Mix 10ml of reagent 1 with 0.25ml of
reagent 2. The reagent is stable for 3 hour so it must be prepared for every
use.
4.
CAFFEINE REAGENT SOLUTION
COMPOSITION
Sodium benzoate 75g
Caffeine
50g
Sodium acetate anhydrous 75g
EDTA
1g
Distilled water 1litre
PREPARATION
Dissolved 75g of
sodium benzoate, 50g caffeine, 57g sodium acetate anhydrous, and 1g EDTA in
600ml of distilled water, and make up the solution to 100ml with distilled
water
5.
ALKALINE TARTARATE
COMPOSITION
Sodium hydroxide 100g
Sodium potassium tartarate 350g
Distilled water 1litre
PREPARATION
Dissolved 100g
of sodium hydroxide, and 350g of sodium potassium tartarate, in 750ml of
distilled water, and make up the solution to 1litre with distilled water
6.
ASCOBIC ACID SOLUTION
COMPOSITION
Ascorbic acid 0.02g
Distilled water 5ml
PREPARATION
Dissolved 0.20g
of ascorbic acid, in 5ml of distilled water
Make freshly for
use every day
7.
STANDARD BILIRUBIN SOLUTION (200µmol/l)
COMPOSITION
Hepatits antigen free non icteric
human serum
Pure bilirubin
11.7mg
Dimethylsulphoxide
4ml
PREPARATION
Dissolved 11.7mg
of pure bilirubins, and in 4ml of Dimethylsulphoxide by warming and 35ºC, shake
the solution at interval until total dissolved.
Make up the
volume to 100ml with serum.
Distribute 1ml
amounts in small vials or containers and store in deep freeze until required.
NB: There are commercially available of this
reagent in the store selling laboratory reagent.
METHOD
Set up four
tubes as shown bellow for total bilirubin (TB);
|
REAGENT
|
TOTAL
BILIRUBIN
|
BLANK
|
STANDARD
|
STANDARD
BLANK
|
|
Serum
|
0.2ml
|
|
|
|
|
Standard
200µmol/l
|
|
|
0.2ml
|
|
|
Distilled
water
|
0.8ml
|
0.8ml
|
0.8ml
|
0.8ml
|
|
Diazo reagent
|
0.5ml
|
|
0.5ml
|
|
|
caffeine
|
2.0ml
|
|
2.0ml
|
|
Leave at room temperature for 10 minute and
continue as shown bellow
|
Ascorbic acid
|
0.1ml
|
0.1ml
|
0.1ml
|
0.1ml
|
|
Diazo reagent
|
|
0.5ml
|
|
0.5ml
|
|
serum
|
|
0.2ml
|
|
|
|
Standard
200µmol/l
|
|
|
|
0.2ml
|
|
Caffeine
|
|
2.0ml
|
|
2.0ml
|
|
Alkaline
tartarate
|
1.5ml
|
1.5ml
|
1.5ml
|
1.5ml
|
Set the
instrument to zero reading with the blank using absorbance of 600nm, and read
the tubes, record your reading.
Set only only
single tube for conjugated bilirubin as shown bellow;
|
REAGENT
|
TEST TUBE FOR
CB
|
|
serum
|
0.2ml
|
|
Distilled
water
|
0.8ml
|
|
Diazo reagent
|
0.5ml
|
Leave at room
temperature for 10 minute and continue as shown bellow
|
Ascorbic acid
|
0.1ml
|
|
Caffeine
|
2.0ml
|
|
Alkaline
tartarate
|
1.5ml
|
Read as of total bilirubin illustrated above.
CALCULATION
Abs TB
(or CB) –Abs B
µmol/l of Bilirubin =
x 200
Abs S
– Abs SB
To convert to
mg/dl = µmol/l of Bilirubin / 17
Normal CB 3.5µmol/l
of Bilirubin
Normal TB 17µmol/l
of Bilirubin
ESTIMATION OF
TOTAL PLASMA PROTEIN
Protein is
produce in the cytoplasm of cell by RNA (ribose nucleic acid). The protein is a
sequential arrangement of amino acid linked together by peptide bond. Protein
performs many function in the body system, bellow are the function perform in
the body;
1.
Protein offer protection to body system by defending
the body from foreign inversion.
2.
Protein help maintain body PH level
3.
The metabolism of protein help to release amino acid
which can be used to produce another protein in the body
4.
Protein helps transporting lipid molecule within the
body system.
5.
Protein help
maintain the osmotic pressure of the blood in the body system
There is two
methods adopted for examination of protein in the plasma, they are;
1.
Kjeldahl method
2.
Biuret method
But we will use
biuret method for our analysis.
BIURET METHOD
FOR ESTIMATION OF PLASMA TOTAL PROTEIN
REAGENT
1.
BIURET REAGENT (STOCK SOLUTION)
COMPOSITION
Sodium potassium tartarate 45g
[0.2N (0.8%) NaOH] sodium
hydroxide 1litre
Copper sulphate
15g
Potassium iodide
5.0g
PREPARATION
Dissolved 45g of
Sodium potassium tartarate, in 400ml of [0.2N (0.8%) NaOH] sodium hydroxide,
add 15g Copper sulphate follow by 5.0g of Potassium iodide, and make up the
solution to 1litre with [0.2N (0.8%) NaOH] sodium hydroxide
2.
BIURET WORKING SOLUTION
COMPOSITION
Potassium iodide
5g/l
[0.2N (0.8%) NaOH] sodium
hydroxide 1lite
PREPARATION
Dissolved 5g/l
of Potassium iodide, with 400ml of [0.2N (0.8%) NaOH] sodium hydroxide, and
make up the solution to 1litre with [0.2N (0.8%) NaOH] sodium hydroxide
TO BE COUNTINUE ON THE NEXT POST
Comments
Post a Comment