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1006
Reagents
Rapid Troponin I Test device (test strips) provided in a kit by the manufacturer.
Carefully follow instructions given in the insert.
Supplies not provided in the kit
• Serum collection containers
• Timer or clock
Storage: Store the test device at 2–30°C. Do not freeze.
Precautions
Do not use commercial kits with expired date.
Quality control
• The control band is an internal reagent and procedural control. It will give colour
reaction if the test has been performed correctly and the reagents are reactive.
• Good laboratory practice recommends the daily use of control materials to validate the
reliability of the device.
Medical Laboratory Technology: Volume 3
Blood urea nItrogen (Bun)
Urea is the nal degradation product of protein and amino acid metabolism. In protein catabolism the proteins are broken down to amino acids and deaminated. The ammonia formed
in this process is synthesized to urea in the liver. The urea passes into the blood and ltered
out of the kidneys and excreted in the urine. This is the most important catabolic pathway
for eliminating excess nitrogen in the human body. If the kidneys do not remove urea, the
concentration in the blood is increased which is the sign of renal dysfunction.
Clinical significance
The determination of serum blood urea nitrogen currently is the most widely used screening
test for the evaluation of kidney function. The test is frequently requested along with the
serum creatinine test since simultaneous determination of these two compounds appears
to aid in the dierential diagnosis of pre-renal, renal and post-renal hyperuremia. There
could be pre-renal clinical problems that results in an increase protein breakdown like
glomerulonephritis, chronic nephritis, nephrotic syndrome, etc. Hence the results must be
carefully evaluated. Acute upper gastrointestinal bleeding can cause increase in protein into
the gut which aer absorption may cause high level of BUN. Looking to the other side of the
kidney, post-renal problems like obstruction of urinary tract may also cause increase in urea.
The value must, however, be compared with creatinine level before concluding the diagnosis.
Post-renal problems lead to increase of both urea and creatinine proportionately. In other
words, the ratio does not change signicantly.
Normal range
Urea: 15.0–38.5 mg/dL (values vary with diet)
Specimen
Serum specimen is commonly submied for reporting blood urea concentration. Since urea
may be lost through bacterial action, the specimen should be analyzed within 2 h aer blood
collection or should be preserved by refrigeration.
Interfering substances: Avoid using haemolyzed and icteric serum to prevent loss of urea to
bacterial contamination. Moderate haemolysis (0.15 g dL Hgb), bilirubin levels up to 20 mg/dL,
and moderate lipaemia do not cause signicant interference with this method.
Routine Biochemical Test Procedures
1007
Diacetyl monoxime method (colorimetric)
The diacetyl monoxime methodology for BUN determination is direct and measures
a chromogen formed from the condensation of urea with diacetyl. This condensation
methodology does not suer from ammonia interference and utilizes less caustic reagents
than other methods. Diacetyl monoxime is hydrolyzed under acidic conditions to produce
diacetyl which then condenses with urea to form a pink chromogen that is measured at
520 nm. Thiosemicarbazide and ferric ions are employed to enhance the colour development.
Reagents
• BUN colour reagent—16.6 mmol/L diacetyl monoxime (Reagent #1): BUN colour reagent
should be a clear to pale yellow solution. Darkening or formation of a precipitate in the
reagent would indicate contamination and the reagent should be discarded.
• BUN acid reagent—0.21 mmol/L ferric chloride, 0.01 mol/L phosphoric acid, and
1.9 mol/L sulphuric acid (Reagent #2): The BUN acid reagent should be a clear colourless solution. Failure to achieve assay values on freshly prepared control sera may indicate reagent deterioration.
• Urea nitrogen standard solution—Concentration is provided by the manufacturer;
follow direction for reconstitute. Store the solution in refrigerator at 0–5°C. The nal
concentration of the standard is usually 100 mg/dL which widely varies.
Warning: All chemicals should be considered poisonous and harmful. Avoid contact to
all chemicals with skin, eyes, or clothing. Flush aected area with water and seek medical
aention. All specimens are potential biohazard.
Materials required but not provided in the kit
• Spectrophotometer or suitable instrument to read well, calibrated to read absorbance
at 520 nm.
• Test tubes and cuvees.
• Automatic pipeor capable of dispensing 0.02 mL (20 µL), 1.5 mL, and 3 mL.
• Timer or stopwatch.
• Boiling water bath (100°C) or use a heat block capable of maintaining that temperature.
Procedure
1. Label three or more test tubes or cuvees as blank, standard, Test 1, Test 2, etc.
2. To each, add 1.5 mL BUN colour reagent.
3. To blank (B), add 20 µL water. To standard (S), add 20 µL of urea nitrogen standard
solution, and to tube marked T add 20 µL serum or plasma.
4. Mix contents by gentle swirling.
5. Add 3.0 mL BUN acid reagent into all tubes and mix well.
6. Incubate for 10 min at 100°C in a heat block or 8 min in a boiling water bath.
7. Remove all tubes and cool to room temperature using cold tap water.
8. Mix all tubes with hand or Vortex.
9. Set the wavelength of the spectrophotometer at 520 nm (or use appropriate lter in case
of colorimeter). Set the “Zero” with the reagent blank (use water in place of serum).
10. Read the absorbance of standard (or control) and that of test specimen (patient’s serum).
Note Final colour is stable for 6 h at room temperature. Calculate the concentration of
urea in serum specimen.
Calculations
Conc. of urea in serum (mg/dL) = AT/AS × Conc. of standard × Serum dilution factor
Where,
A
= Absorbance reading of the test specimen against blank
T
A
= Absorbance reading of the standard against blank
S
1008
Medical Laboratory Technology: Volume 3
Note If the concentration of urea in the specimen is more than 150 mg/dL, the specimen
must be diluted with saline and test repeated. Multiply results by the dilution factor (total
volume/amount of specimen) when calculating the unknown.
Example 1 mL of serum mixed with 2 mL of saline, the dilution factor = 3/1 = 3 ×
Quality Control
Linearity extends to 150 mg/dL. Check the linearity for every batch of chemicals used. The
two levels of assayed control, i.e., Monitrol I and Monitrol II, can be diluted to get various
concentrations of urea to make the standard curve.
Enzymatic method
There are many kits available in the market for the assay of urea by enzymatic method.
Of these, the microplate method is more popular. This allows smaller size of the sample and
large number of assays. The depressions on the plate are used as cuvees. Hence, in the
long-run, microplates may be cost eective as it requires smaller amount of reagents. We
have chosen one of the microplate-based kit for the purpose of illustration. Instruction may
vary with manufacturers. Hence, always follow the instructions given in the kit. In absence of
microplate, cuvees may be used aer making a few changes.
Principle
Enzyme-based assay procedure utilizes urease to specically detect urea in serum. In presence
of urea, urease produces ammonia as follows:
CO(NH2)2 + H2O
Urea Ammonia
Urease
CO2 + 2NH
3
The ammonia produced from the urea is then directly detected by a colorimetric chemical
reaction.
The method is rapid, accurate, and proven to be reliable. The linearity range of urea
concentration in this assay is 1–20 mg/dL. If the concentration is beyond this range, the
specimen is diluted with saline and the dilution factor is used during calculation. The kit
contains urea standards to construct a linear calibration curve and verify assay performance.
The kit is designed to be used with a microplate reader.
Specimen and its preparation
Always try to get fresh specimen and run the test immediately. Standing specimen may lose
urea.
1. Allow blood sample to coagulate in a microfuge tube for 20 min at room temperature.
2. Centrifuge for 5 min at 9,000 rpm.
3. Transfer the supernatant (serum) to a clean tube.
4. If the samples cannot be tested within 6 h of collection, store them at 4°C and test no
later than 3 days aer collection.
Note Immediately before testing, dilute the serum 1 : 4 (dilution factor 5×) with normal
saline or PBS (phosphate buered saline).
Kit Contents
• Store the kit in a refrigerator. The shelf life is 6 months when the kit is properly stored.
• Microtitre Plate (1 × 96-well plate that contains 8 wells in 12 strips).
• Urea Standard: 0–20 mg/dL (higher standards may be included).
• Urease Mix: 1 vial (keep refrigerated)
• Alkaline hypochlorite solution: 20 mL
Routine Biochemical Test Procedures
Storage and Shelf Life
• Do not intermix reagents from dierent kits or dierent lots.
• Try to maintain a laboratory temperature of 20–25°C (68°–77°F).
• Use only distilled-deionized water since water quality is very important.
Reconstituting the reagents
• Urease mix: It comes in lyophilized form in the kit and need to be reconstituted. Follow
manufacturer’s instruction. Here is a general outline: Add exactly 20 mL of deionizeddistilled water to the urease mix powder (in kit). Mix by swirling or inverting several
times until the powder is dissolved. Wait for 10 min at room temperature. The urease mix
is stable for 4 months in the refrigerator aer reconstitution with water. The reconstituted
urease mix, however, can be le at room temperature for short periods prior to use.
Between uses, the reconstituted urease mix should be stored in the refrigerator. Discard
the urease mix 4 months aer reconstitution.
• Urease standard: Reconstitute by direction provided by the manufacturer. The nal
concentration of the standard is 20 mg/dL.
Materials/Equipment required but not provided with the kit
• Microtitre plate with 96-wells
• Microtitre plate reader (with 620 nm absorbance lter)
• Microcentrifuge
• Microcentrifuge tubes
• Automatic pipeors
• Normal saline or PBS (Phosphate-buered saline, pH 7.4)
Note You can use narrow cuvees aer making necessary alteration in the procedure.
1009
Warnings and Precautions
• Read the instructions thoroughly as provided by the manufacturer.
• Standard curve should be made with each kit and check the linearity.
• Do not use the kit past the expiration date.
• When pipeing samples or reagents into an empty microtitre plate, place the pipee
tips in the lower corner of the well, making contact with the plastic.
• A standard curve must be constructed for each new kit. Here the absorbance of the
standard (blank corrected) is ploed (y-axis) against the concentration (x-axis) and
check the linearity. The curve should be linear up to 100 mg/dL.
Procedure
1. Set-up
• Warm up kit reagents to room temperature and turn on the plate reader. This will
allow the light source to warm up.
• Set absorbance wavelength to 620 nm (other wavelengths in the range of 590–630 nm
can also be used for the assay, but the sensitivity will be slightly lower).
• Before testing, dilute serum samples 1 : 4 in either normal saline or PBS. Here the
dilution factor = 5 × (1 mL + 4 mL = 5 mL, so the dilution is ve times; 1 made to 5).
2. Add 5 µL of diluted serum (see Step #1) to the microplate wells. Duplicate sample is
recommended.
3. Then add 150 µL of reconstituted urease mix solution to the wells.
4. Tap the plate gently several times to mix the sample and enzyme.
5. Allow to incubate for 15 min at room temperature.
1010
Medical Laboratory Technology: Volume 3
6. Add 150 µL of alkaline hypochlorite to each well. This stops the reaction in progress.
7. Incubate for another 10 min at room temperature.
8. Read the absorbance in duplicate at 620 nm.
Calculation
Calculation of urea concentration is made by standard formula:
BUN concentration (mg/dL) = AT/AS × 5 (Dilution factor)
Where,
A
= Absorbance of Test (specimen)
A
T
= Absorbance of Standard
S
5 = Dilution factor
urIc acId
Uric acid is a heterocyclic compound of carbon, nitrogen, oxygen and hydrogen with formula
C5H4N4O3. It forms ions and salts known as urates and acid urates such as ammonium acid
urate. Uric acid is the end product of nucleoprotein metabolism with low threshold excretory
product.
Clinical significance
High blood concentrations of uric acid can lead to gout. The chemical is associated with
other medical conditions including diabetes and formation of ammonium acid urate kidney
stones. The determination of serum uric acid has diagnostic value in dierentiating gout
from other causes of arthritis. Uric acid levels are also increased in renal failure, uraemia
and leukaemia.
Normal range
Serum uric acid (Adult) – Male: 3–7 mg/dL; Female: 2-6 mg/dL
A serum uric acid >12 mg/dL calls for further investigation.
Principle
Determination of serum uric acid by enzymatic method is a two-step reaction. In the rst
step, the enzyme uricase (present in working reagent) acts on the uric acid present in the
specimen to catalyse the spliing of uric acid. This reaction produces hydrogen peroxide
(H2O2). In the second step, another enzyme, peroxidase (present in the working reagent)
oxidizes the H2O2 releasing nascent oxygen that oxidizes the phenolic chromogen (present in
the reaction mixture). The oxidized form of the chromogen is a coloured compound, whose
intensity can be measured at 500 nm (500–530, green lter).The intensity (absorbance) of red
colour is proportional to the amount of uric acid present in the serum sample.
Here are the steps of chemical reactions:
2O2
Uricase
Peroxidase
Allantoin + H2O2 + CO
H2O + [O]
2
Uric acid + 2H2O + O2
H
Phenolic chromogen + [O] Coloured chromogen
Specimen
Serum, blood collected by routine procedure.
Routine Biochemical Test Procedures
Reagents
A. Stock reagents: This comes in lyophilized form in the kit. Reconstitute according to
manufacturer’s direction.
•Buer (pH 7.5) 100 mmol/L
•Uricase 100 IU/L
•Peroxidase 140 IU/L
•Chromogen 2.5 µmol/L
•Surfactants/stabilizer
Stability of reagents: Lyophilized reagents are stable at 2–4°C. Reconstituted reagents
must be stored in the refrigerator.
B. Uric acid standard: This comes as standard solution (5.0 mg/dL).
C. Working reagent solution or enzyme reaction mixture is made from the lyophilized
stock reagent. The stock is diluted with distilled water according to manufacturer’
direction. Working reagent is stable at 2–4°C for 60 days. 1 mL of working reagent is
used in testing.
Procedure
1. Label three test tubes as blank (B), standard (S), and test (T). If there are more specimens,
name the test tubes T1, T2, T3, etc., according to the number of tests to be done.
2. Pipee 1.0 mL of working reagent solution in all test tubes.
3. Add:
0.02 mL of water in the blank tube (B).
0.02 mL of standard (5 mg/dL) in the tube marked S (standard).
0.02 mL of serum specimen in tube marked as T (Test).
Continue if more serum specimens are to be tested (T1, T2, T3, etc.).
4. Keep the test tubes at room temperature 25 ± 5°C for 10 min.
5. Transfer the contents of the tubes to cuvees to take absorbance readings at 500 nm
(use green lter for colorimeters).
6. Set the zero of colorimeter (spectrophotometer) with the blank. Read the absorbance of
test specimen (AT) and that of the standard (AS) against the blank which is used to set
the zero.
7. Calculate the concentration of uric acid in test specimens.
1011
Calculation
Serum uric acid (mg/dL) = AT/AS × 5
Where,
A
= Absorbance of test (specimen)
T
A
= Absorbance of standard
S
5 = Concentration of standard in mg/dL
AT/AS ratio remains constant for the same kit.
Note
• Method is linear up to 25 mg/dL.
• Glucose, bilirubin, ascorbic acid, urea, proteins, and haemoglobin do not interfere with
this test, if present in moderate concentrations.
Determination of Uric Acid in Urine
1. Dilute urine 1 : 10 in distilled water and perform the test as described above.
2. Modify Calculation by multiplying with 10 (dilution) to report the concentration of uric
acid in urine.
Urine uric acid (mg/dL) = AT/AS × 5 × 10
1012
Medical Laboratory Technology: Volume 3
creatInIne
Creatinine is a breakdown product of creatinine phosphate in muscle, and is usually produced
at a fairly constant rate by the body (depending on muscle mass). It is a substance stored
in muscles and used for energy. Creatinine is removed from plasma through glomerular
ltration and is then excreted in the urine without being reabsorbed by the tubules to any
signicant extent. When renal function is impaired, blood creatinine levels rise, but more
than 50% of kidney function is lost before this happens. So, elevation usually indicates
signicant insuciency. Other reasons for the increase of serum creatinine level are shock,
water imbalance, dehydration and ureter blockage. Simultaneous determination of urea and
creatinine is desirable in order to trace the aetiology as both increases with decreased kidney
function. Creatinine determinations have one advantage over urea determinations as they are
not aected by a high protein diet as is the case for urea levels. Determination of creatinine
clearance is a highly sensitive test for measuring the glomerular ltration rate.
Clinical significance
Creatinine is produced from creatine which provides energy to the function of muscles. Thus,
it is a waste product of the body that comes from muscle metabolism. Creatinine leaves the
body through the urine. Thus, creatinine levels in the blood are related to excretion rate by
the kidneys. Increased concentrations of creatinine in serum indicate that the kidneys are
not functioning properly (diseased or damaged). The test is simple but it is unsuitable for
detecting early-stage of kidney disease. A beer estimation of kidney function is given by
calculating the estimated glomerular ltration rate (eGFR). eGFR can be accurately Calculated
using serum creatinine concentration. Many laboratories will automatically Calculate eGFR
when a creatinine test is requested. Urine creatinine concentration is also checked during
standard drug tests. But one should keep in mind that the trend of serum creatinine levels
over time is more important than absolute creatinine level.
Normal range
Creatinine is typically reported in mg/dL but also expressed as umol/L. For the conversion
of creatinine concentration in mg/dL unit to the International unit of µmol/L, apply the
following formula:
mg/dL × 88.4 = µmol/L
Serum creatinine range:
Males: 0.7 to 1.3 mg/dL (60–110 µmol/L)
For women: 0.6 to 1.q mg/dL (45–90 µmol/L)
Urine: 90–150 mg/dL (100 times higher than serum)
Urinary discharge per day: 1.0–2.0 g/day
Laboratory assay
Two methods are available for the laboratory assay of creatinine—colorimetric method and
enzymatic method. Only the colorimetric method will be described here as the enzymatic
method is more expensive with no additional advantage.
Specimen and its preparation
Blood is collected in plain tube through venepuncture. Prepare the serum specimen by
routine procedure:
• Clot the blood at room temperature
• Centrifuge
• Separate the serum
Routine Biochemical Test Procedures
• Alternatively, use the serum separator tubes (not cost eective in developing countries).
• At least 20 µL of serum will be needed for each test.
Caution Avoid hemolysis as it may release erythrocyte creatinine into the serum. Creatinine
in serum is reported stable for 2 days at 4°C (refrigerator) and 3 months when frozen and
properly protected against evaporation.
1013
Alkaline picrate method (colorimetric)
Colorimetric assay kits are available in the market which makes the test simple, direct, and
automation compatible. Because of increasing popularity, we have described here the procedure for microplate instead of cuvee. With minor adjust of volume, the kit can be used for
cuvee as well.
Principle
At high pH (alkaline), creatinine reacts with picrate reagent forming a red-coloured complex.
This is called Jaee reaction.
Creatinine + Alkaline Picrate Red Coloured Complex
The intensity of red colour, as measured at 510 nm, is proportional to the concentration of
creatinine in the sample.
Reagents
Most kits provide sucient prepared reagents necessary for 40 duplicate specimens. Kits can
be stored at room temperature, except the standard which is stored in the refrigerator. The
shelf life is 6 months if properly stored.
• Microtitre plate
• Microplate cover sheet
• Creatinine colour reagent (Reagent #1) 20 mL (picric acid)
• Creatinine buer reagent (Reagent #2) 20 mL
• Creatinine standard (20 mg/dL) 0.75 mL (reconstitute)
Note Reagent mix refers to mixing of Reagent 1 and 2 and keep it ready for the test.
Special instructions
• A new standard curve must be established for each new kit in order to check the linearity.
The creatinine standard provided in the kit should be used to calibrate the assay.
• Strictly follow manufacturer’s instructions.
• While preparing standard curve using microplate, always remember: Add standards to plate
only in the order from low concentration to high concentration, as this will minimize the
risk of compromising the standard curve.
• Treat all specimens as potential biohazard.
• Wipe up any spillage since dried picrate (picric acid) is explosive.
• Handle all reagents cautiously.
Additional materials required but not provided in the kit
• Microtitre plate reader (510 nm) that reads absorbance
• Water bath or incubator to maintain 37°C temperature
• Centrifuge (for preparing serum)
• Deionized-distilled water (superpure)
• PBS (phosphate buer saline, pH 7.3)
• 1.5 mL microfuge tubes
• Multichannel pipee or repeating pipeor (recommended but not required)
1014
Medical Laboratory Technology: Volume 3
Working reagent preparation
Mix equal amount of Reagent #1 (colour reagent) and Reagent #2 (buer reagent) as needed
for the number of tests to be performed. This will be referred as “Reagent Mix”.
For example: You will need a total of 300 µL per testing. Hence if you have to perform 10 tests,
combine 1.6 mL of Reagent # 1 with 1.6 mL of Reagent #2 in order to yield a total of 3.2 mL of
reagent mix. This will allow you to perform 10 tests (each requiring 300 uL of total mixture),
implying that you will need 3.0 mL of mixture leaving behind 0.2 mL (20 µL) excess reagent
to avoid entry of any air bubble into the pipee.
Procedure
The general summary of the procedure is given below. Aer preparing the sera, the assay is
performed by adding reagent mix into microplate wells containing 10 µL sera. Aer a brief
incubation, the absorbance of each well at 510 nm is then measured using a plate reader. The
concentration of creatinine in each sample is then directly determined from the 510 nm absorbance. The step-by-step-account of these steps is given below:
1. Use three microplate-wells as blank (B), standard (S) and test specimen (T). If more than
one specimen is there, mark them as T1 T2, etc.
2. Add 10 µL of water in well-marked B, 10 µL of test specimen in well-marked T, and
10 µL of standard (20 mg/dL) in the well-marked S.
3. Add 300 µL of reagent mix in all the wells. (Note Reagent mix is the mixture of Reagent
1 and 2 in equal amounts. This has been explained earlier).
4. Carefully cover the wells with adhesive cover sheet, mix in the shaker and incubate at
37°C for 15 min.
5. Aer incubation, carefully remove the adhesive cover sheet and zero the absorbance
reading of the instrument with blank.
6. Treat the test (T) and standard (S) in the same way and measure the absorbance of each
specimen (AS) and that of the standard (AT) at 510 nm. As the instrument is zeroed with
blank, all absorbance readings are against the blank (0 mg/dL).
7. Samples with values above 20 mg/dL should be diluted with PBS or normal saline and
re-tested. (Multiply results by dilution factor).
Calculation
Concentration of creatinine in specimen (mg/dL) = AT/A
× 20 × DF
S
Where,
A
= Absorbance of test specimen
A
T
= Absorbance of standard
S
20 = Concentration of standard (20 mg/dL)
DF = Dilution factor (in case the specimen is diluted)
Preparation of standard curve
Within limits there is a linear relationship between the concentration of creatinine in the
specimen and the absorbance at 510 nm. Therefore, a standard curve provides a reference
for the linear range of the assay. Once the linearity is established, the concentration can be
calculated mathematically, as long the concentration falls within the linearity range: AT/AS,
where AT is the absorbance of the Specimen (Test) and As is the absorbance of the Standard.
How to make dilutions of the standard to be used in preparing the standard curve?
Label four centrifuge tubes to prepare various dilutions of the standard.
The four dilutions of the standard (plus one blank, #5) are made as follows:
Routine Biochemical Test Procedures
1015
Table showing the dilution process that gives various standards to plot the standard curve
Tube # 1 2 3 4 5
Creatinine standard (20 mg/dL)
Water (deionized/distilled)
Creatinine concentration
100 µL 50 µL 25 µL 5 µL 0 µL
µL 50 µL 75 µL 95 µL 100 µL
0
20 mg/dL 10 mg/dL 5 mg/dL 1 mg/dL 0 mg/dL (blank)
10 µL of each diluted standard is used for reaction.
Determination of creatinine clearance
The group of tests, generally referred to as renal clearance tests, are useful in assessing the
capacity of kidneys to eliminate (or clear) certain substances present in plasma. Two of these
substances are routinely considered—creatinine and urea. Clearance study of creatinine has
proved to be more reliable and reproducible because the serum level of urea varies considerably depending on the type of food taken by the patient. The request for the clearance study
is made by the physician when the patient shows an increase of non-protein nitrogenous
constituents of blood—mainly urea, creatinine and uric acid. The correct assessment of the
functional capacity of the kidney comes from the clearance study (Chapter 34 of this volume).
BIlIruBIn
Bilirubin is one of the degradation products of haemoglobin formed when red blood cells
die in the spleen, liver, and bone marrow. Bilirubin is conjugated with glucuronic acid in
the liver to form a soluble compound. This conjugated bilirubin passes down the bile duct
and is excreted into the gastrointestinal tract. An unconjugated, albumin bound form is also
present in the circulation. It is insoluble and does not normally pass through the kidneys
into the urine. Thus, in serum, bilirubin exists as insoluble unconjugated form (also indirect
bilirubin), or soluble glucuronide conjugated form (also called direct bilirubin). Conjugated
bilirubin moves into the bile duct of the liver and then to the gall bladder. When stimulated
by eating, bile (including the conjugated bilirubin) is excreted into the small intestine, where
bilirubin is converted into urobilinogen.
Clinical significance
Jaundice is caused by the elevation of bilirubin in blood. It gives a yellowish discolouration
of skin, mucous membranes, and whiteness in eyes. Bilirubin is a brownish yellow substance
found in bile. It is produced in liver from old red blood cells aer they cease functioning.
Bilirubin is normally taken out of the body through stool and urine. This is why they have the
normal yellowish colour. If the liver does not function properly, bilirubin is back-red into the
blood stream. This clinical condition is jaundice.
Bilirubin is a key diagnostic indicator. High levels of bilirubin result when too much
haemoglobin is broken down or the removal of bilirubin does not function properly. The
accumulation of bilirubin in the body causes jaundice. Jaundice occurs in case of toxic infectious diseases of the liver, e.g.,viral hepatitisor obstruction of the bile duct and also in case of
incompatible blood transfusion. In newborn babies, excess bilirubin can lead to retardation or
physical abnormalities; early detection is therefore extremely important.
Useful information may be obtained by determining which form of bilirubin is elevated.
Unconjugated or indirect bilirubin can be determined by subtracting the direct bilirubin level
from the total bilirubin result. High levels of conjugated or direct bilirubin indicate that bile
is not being properly excreted; therefore an obstruction may be present in the bile duct or gall
bladder. High levels of unconjugated bilirubin indicate that too much haemoglobin is being
destroyed or that the liver is not actively treating the haemoglobin it is receiving.