Biochem Manual 2020-10-08 08 - 31 - 31 - 231030 - 153345

BIOCHEMISTRY
LABORATORY MANUAL
DEPARTMENT OF BIOCHEMISTRY
JAWAHARLAL INSTITUTE OF POSTGRADUATE
MEDICAL EDUCATION AND RESEARCH
An Institution of National Importance
(Ministry of Health and Family Welfare, Government of India)
Dhanvantari Nagar, PUDUCHERRY - 605 006
2019-2020
CONTENTS
TITLE PAGE NO.
I. GENERAL MODULE
1. Introduction To Clinical/Practical Biochemistry
i. Clinical Biochemistry- a corner stone of medicine; its 2

scope and future
ii. Safety measures and necessary precautions in lab 3
iii. A generalized concept of concentration and its 5

measurement
2. Colour Reactions Of Carbohydrates 7
3. Colour Reactions Of Proteins 13
4. Electrophoresis 17
5. Chromatography 19
II. HEMATOLOGY AND IMMUNOLOGY

1. Photometry 23
2. Immunoassays (RIA, ELISA & Chemiluminescence) 27
3. Hemoglobin And Its Derivatives 32
4. Case Reports 37
III. MUSCULOSKELETAL SYSTEM AND ANS

1. Estimation of Calcium in Serum 40
2. Estimate of Phosphate in Serum 36

IV. ENDOCRINE SYSTEM
1. Estimation of Glucose in Serum 47
2. Oral Glucose Tolerance Test 52
3. Case Reports 54
V. RESPIRATORY SYSTEM
1. Introduction to Acidimetry, pH Indicators, pH Meter, Buffers 58
2. Arterial Blood Gas Analysis and Its Interpretation 62
VI. CARDIOVASCULAR SYSTEM

1. Estimation of Serum Total Cholesterol 67
2. Point Of Care Testing (POCT) 71
3. Case Reports 72
VII. GASTROINTESTINAL SYSTEM, HEPATOBILIARY SYSTEM AND

NUTRITION
1. Estimation of Bilirubin in Serum 75
2. Case Reports 78
VIII. RENAL SYSTEM
1. Urinalysis :
Introduction 81
Normal Constituents of Urine 83
2. Abnormal Constituents of Urine 89
Dry Chemistry Methods (Dip Sticks) 99
3. Estimation of Urea in serum 100
4. Estimation of Creatinine in serum 103

5. Estimation of Uric acid in serum 107
6. Serum Electrolytes And Ion Selective Electrodes 110
7. Case Reports 113
IX. MOLECULAR BIOLOGY, CANCER BIOLOGY
1. Polymerase Chain Reaction (PCR) 121
X. APPENDICES
I. GENERAL MODULE
1
I.1. INTRODUCTION TO PRACTICAL BIOCHEMISTRY
CLINICAL BIOCHEMISTRY: A CORNERSTONE OF MEDICINE;
ITS SCOPE AND FUTURE
What is clinical biochemistry?
Clinical biochemistry (clinical chemistry, chemical pathology or pure blood chemistry) is
the area that is generally concerned with analysis of body fluids. Most of the current
laboratories are highly automated and use assays that are closely monitored and quality
controlled. These are performed on any kind of body fluid, but mostly on serum or
plasma. Serum is the yellow watery part of blood that is left after blood has been
allowed to clot and all blood cells have been removed. Serum is most easily separated
from the cells by centrifugation. Plasma is obtained by centrifuging the blood after
addition of anticoagulant.
Clinical chemistry reports provide critical information that saves lives, supports
health initiatives to improve population health.
Who is a clinical biochemist?

A clinical biochemist is a person who uses biochemistry to evaluate patient’s health.
S/he may be an assistant, technician, technologist, physician/surgeon (generally
referred as clinician), doctoral scientist or a developer of diagnostic products. Clinical
chemists have traditionally worked in clinical laboratories, but they also work in
research, manufacturing industries, pharmaceutical companies, commercial reference
laboratories, or academic institutes.
Clinical chemists do research and develop laboratory procedures that help
clinicians make earlier, precise diagnoses and tailor therapy for patients. Clinical
chemists educate patients and consult with clinicians on what the test results mean.
They implement newer diagnostic methods that are faster, accurate, precise, sensitive
and less expensive than the existing methods.
Automation in clinical biochemistry

Automation is the use of control systems, in concert with other applications of
information and communication technology (various softwares), to control machinery
and processes, reducing the need for human intervention.
Automation is changing laboratory and hospital operations. Sick or healthy,
people will always need medical information quickly, easily and at affordable means. A
new growth area, for example, is the use of "kits" in the home or at the hospital bedside,
such as those for pregnancy detection or glucose (diabetes) monitoring. The analytical
and operational capabilities of clinical chemistry analyzers will continue to evolve as
new technologies are developed. This will lead to greater advancement in automation
and enhanced ease of use. For example, robots may transport patient specimens to the
laboratory, or majority of the tests may be done at the patient's bedside.
2
SAFETY MEASURES AND NECESSARY PRECAUTIONS IN A
LABORATORY
There are standards designed to safeguard the health and lives of students and
personnel working in the laboratory. Following these procedures protects them from
accidents.
Safety measures in a laboratory

The control of potential biological hazards in the clinical laboratory is provided by the
use of standard work practices, commonly referred to as “Universal Precautions”.
Practicing universal precautions eliminates the need for using specific warning labels on
all specimens infected with hepatitis B virus (HBV) or human immunodeficiency virus
(HIV). Whether warning labels are used or not, all specimens must be treated as
capable of transmitting serious infection.
 Apron/over coats should be worn always while in the laboratory.
 Gloves should be worn when:
Performing routine laboratory work with any bio-fluids.
Touching mucous membranes and non-intact skin of patients.
Handling items (potentially) contaminated with blood or body fluids, including
specimen containers, laboratory instruments etc.
 Wash hands immediately after gloves are removed.
 Take extraordinary care to avoid accidental injuries (as caused by needles, lancets,
scalpel blades or laboratory instruments, etc.) while performing procedures, cleaning
instruments, handling sharp instruments, and disposing used needles.
Safety do’s and don’ts for students

Some basic do's are:
 Keep the work area neat, clean and free of any unnecessary objects.
 Laboratory coats/aprons must be worn at all times while in the laboratory.
 While heating a liquid, always keep the mouth of the test tube away from you and
your neighbors. Take care that it does not splash.
 Use a spatula to take a solid reagent from a container.
 Immediately report any spills, accidents, or injuries to a teacher.
 Be careful when handling hot glass wares in the laboratory. Use a test-tube holder
wherever necessary.
 Secure long hair and loose clothing (especially loose long dresses or scarves).
Synthetic wear can be avoided.
 Make sure no flammable solvents are in the surrounding area when lighting a flame.
 Treat every bio-fluid sample as potentially dangerous/hazardous.
Some basic don'ts are:

 Do not touch any corrosive chemicals with bare hands
 Do not heat flammable liquids directly. Use a hot water bath to heat the same.
 Do not contaminate the reagents. Use droppers while using reagents
 Do not leave lit Bunsen burners/spirit lamps unattended
 Do not eat, drink or smoke in laboratory
3
INSTRUCTIONS TO THE STUDENTS
1. All the students should be punctual in attending the practical classes. All should
wear neat white coat. Dress made from inflammable materials should be avoided.
2. Each student will be allotted a place in the working area of the laboratory. Glassware
items and other things will be distributed to each student and they should keep them
carefully on the table. Loss or breakage of any items should be reported to the
demonstrator then and there and might be liable for charges.
3. Each student should attend instruction or demonstration classes before starting the
practical.
4. A student can leave the laboratory only after completing all the experiments and
getting signature from the demonstrator in the observation note-book.
5. Each student should maintain a biochemistry practical record note book which
should be covered neatly. Instructions will be given for writing the record. The record
note book should be submitted within the stipulated time, as this will be counted for
internal assessment.
6. Pipetting should be done carefully. Concentrated acids, alkali or toxic substances
should not be pipetted by mouth.
7. Students should not discuss among themselves inside the laboratory. If they want
anything, they can approach demonstrators. Strict discipline is expected.
8. In the event of any accident such as spilling of acid over the body or eye, accidental
entry of acid/ alkali in mouth during pipetting, electric shock, gas leakage etc., it
should be reported to the staff immediately.
4
A GENERALIZED CONCEPT OF CONCENTRATION AND ITS
MEASUREMENT
Quantifying molecules
The molecular weight of a molecule is the sum of the mass numbers of all the atoms in
the molecule. One mole, or 6.023  1023 molecules, has a mass equal to the molecular
weight expressed in grams.
The SI unit of mass is the kilogram (kg), while the unit of length is the meter (m).
It follows that the SI unit of volume is the cubic meter (m 3). These units are far too large
for practical work in molecular biology, so one use smaller compatible units: the gram
(g) and the cubic decimeter (dm3). For all practical purposes, the liter is equivalent to
1dm3.
In the SI units the concentration is expressed in moles/L. It refers to the number
of molecules of a particular analyte in the given sample. However still in US, India and
many other countries, the concentration is expressed as mg/dL, although Australia, UK
and Europe use SI units.
Conversion of SI units to conventional units and vice-versa

If we divide the mg/dL figure by molecular weight, we will get mmol/L. Glucose for
example has a molecular weight of 180.
10 dL = 1 L
So 1mmol/L of glucose = 180 mg/L = 18 mg/dL
Conversion of units from one system to another is accomplished by use of a conversion
factor.
Ways of expressing concentrations

 Percentage by volume: 8% ethanol (8 volumes of ethanol in 100 volumes of
solution). Usually written (v/v).
 Percentage by mass: 10% sucrose (10 g sucrose in 100 ml of solution). Usually
written (w/v).
 Mass per volume: g/ml (mass of solute per unit volume of solvent).
 Mass per mass: g/100g (mass of solute per 100 g of solvent).
 Molality: Number of moles of solute per 1 kilogram of solvent.
 Molarity: Number of moles of solute per 1 liter of solution.
 Normality: Number of equivalent weights of solute per 1 liter of solution.
Molarity
The concentration of a substance in a solution is typically expressed as molarity (M),
which is the number of moles per liter. 1 M NaCl = 58.5 g of NaCl in 1 liter of solution
(molecular weight of NaCl is 58.5).
5
Normality
The definition of a normal solution is a solution that contains 1 gram equivalent weight
(EW) per liter solution. An equivalent weight is equal to the molecular weight divided by
the valence (replaceable H+ ions).
Normality refers to compounds that have multiple chemical functionalities, such as

sulfuric acid, H2SO4. 1 M solution of H2SO4 will contain only one mole of H2SO4 in 1
liter of solution, but if you titrate the solution with base, you will find that it contains two
moles of acid. This is because a single molecule of H2SO4 contains two acidic protons.
Thus, 1M solution of H2SO4 will be 2 N.
1 N HCL the MW= 36.5, the EW = 36.5 and 1 N would be 36.5 g/L
1 N H2SO4 the MW = 98, the EW = 49 and 1 N would be 49 g/L
1 N H3PO4 the MW = 98, the EW = 32.7 and 1 N would be 32.7 g/L
DATE :
NOTES :
6
I. 2. COLOUR REACTIONS OF CARBOHYDRATES
Introduction to carbohydrates
Carbohydrates are aldehyde or ketone derivatives of polyhydroxy alcohols. They are a
class of organic molecules with the general chemical formula
Cn (H2O)n, where n ≥ 3. They can be classified as follows:
CARBOHYDRATES
Monosaccharides Disaccharides Oligosaccharides Polysaccharides

e.g. Maltotriose
Raffinose
Aldoses Ketoses Reducing Non-reducing Homo Hetero

e.g. e.g. sugars sugars polysaccharides polysaccharides
Glucose Fructose e.g. e.g. e.g. Starch e.g. Heparin
Xylose Xylulose Lactose Sucrose Glycogen Chondroitin
Galactose Maltose Trehalose Inulin sulfate
Mannose
Ribose
1. MOLISCH’S TEST
This is the general test used to detect the presence of carbohydrates in solutions.
Principle
Conc. H2SO4 removes water molecules from carbohydrates forming furfural or

hydroxymethyl furfural, which reacts with α-naphthol to give violet colored complex.
Furfural is derived from the dehydration of pentoses and pentosans, while
hydroxymethylfurfural is produced from hexoses and hexosans.
Reagents
1. Molisch’s reagent – 1% alcoholic solution of α-naphthol

2. Conc. H2SO4
7
Test procedure Observation Inference
Take 2 ml of the given solution in a Violet ring at the Presence of
test tube. Add 6 drops of Molisch’s junction of two layers carbohydrate
reagent and slowly layer 2ml of
conc. H2SO4 along the sides of the
test tube, so the acid forms a layer Green ring Indicates excess of
at the bottom. Molisch’s reagent
Note: Glycoproteins and glycolipids will also answer this test.
2. IODINE TEST
This is the general test to indicate the presence of polysaccharides.
Principle
Polysaccharides that have six or more glucose units will interact with free iodine forming
characteristic colored adsorption complex, depending on the length and branching of
the chains. They form helical coils to which iodine gets attached.
On adding NaOH, iodine is trapped as sodium iodide and colour disappears. Colour
reappears on adding glacial acetic acid as iodine is again free.
Reagents
1. N/50 iodine
2. 5% sodium hydroxide (NaOH)
3. Glacial acetic acid

(a) To 2 ml of the given Characteristic color Presence of
solution in a test tube, add 2 e.g. Starch: blue color polysaccharides
drops of N/50 iodine. Note Dextrin: pink color
the color. Glycogen: Bordeaux red
Inulin: no color
(b) To the above solution,
add 1 ml of 5% NaOH . Note Disappearance of color on Presence of
the change in color. Then, adding NaOH and polysaccharides
add 0.5 ml of glacial acetic reappearance of characteristic
acid. Observe. color on adding acetic acid
3. BENEDICT’S TEST
This is the test for detecting the presence of reducing substances in solutions.
Principle
With mild alkali, sugars are converted into an enediol form, which has reducing
property. Enediol reduces Cu2+ to Cu+, forming CuOH, which on further heating gets
converted into Cu2O. Depending on the amount of reduction of CuSO4 taking place, the
color formed varies. It could be green, yellow, orange or red.
Non-reducing disaccharides answer this test, after acid hydrolysis.
8
Reagents
Benedict’s qualitative reagent is a mixture of copper sulfate, sodium citrate and sodium
carbonate. Copper sulfate provides cupric ions, sodium citrate keeps cupric ions in
solution and sodium carbonate provides an alkaline medium for the reaction

Take 5 ml of Benedict’s reagent in a test tube
and boil for 1 min. If there is no color change Green, yellow, Presence of
due to autoreduction, add 8 drops of the given orange or red reducing
sample and mix well. Boil for two minutes and color /precipitate substances
cool. (If there is color change, discard the
reagent and use fresh reagent)
Note:
(i)Substances like creatinine, uric acid, salicylic acid, homogentisic acid, vitamin C etc
having reducing property also give positive test, when present in appreciable amounts.
(ii)It is a semi quantitative test for detection of glucose. Approximate concentration of
glucose in urine may be ascertained for clinical purpose.
(iii)Benedict’s quantitative reagent has a slightly different composition. It contains
potassium thiocyanate and potassium ferrocyanide in addition to copper sulfate, sodium
citrate and sodium carbonate.
Color of the precipitate Approximate conc. of glucose (gm %)

Green (+) < 0.5
Yellow (++) 0.5 – 1.0
Orange (+++) 1.0 – 2.0
Red (++++) >2
4. MODIFIED BARFOED’S TEST

This test is used to differentiate monosaccharides from reducing disaccharides.
Principle
Monosaccharides reduce Cu2+ from cupric acetate into Cu+ in mild acidic medium,
which in turn reduces phosphomolybdic acid into molybdenum blue.
The milder condition allows oxidation of monosaccharides, but does not oxidize
disaccharides. Time of heating is carefully controlled so that disaccharides do not react
and only monosaccharides act.
Reagents
1. Barfoed’s reagent - copper acetate and glacial acetic acid (pH – 4.6)
2. Phosphomolybdic acid
9
To 2 ml of Barfoed’s reagent in a test
Presence of
tube, add 1 ml of the given solution Deep blue color
monosaccharides
and keep in boiling water bath for 2
minutes. Cool under running water. Presence of
Add phosphomolybdic acid drop by No color change reducing
drop, till the solution is clear. disaccharides
Note: Prolonged heating should be avoided, as it gives false positive result for
disaccharides which get hydrolyzed.
5. FOULGER’S TEST
This is a test to distinguish ketoses from aldoses.
Principle
Conc. H2SO4 removes water molecules from ketoses forming furfural or hydroxymethyl
furfural, which react with urea and stannous chloride to give blue colored complex.
Reagents
Foulger’s reagent which consists of urea and stannous chloride in 40% H2SO4

To 3 ml of Foulger’s reagent in a test
Blue color Presence of
tube, add 0.5 ml of given solution. Boil
ketose
vigorously for one minute. Cool.
Note: Glucose sometimes gives a green or amethyst colour.
6. SELIWANOFF’S TEST
This test is specific for ketoses.
Principle
Conc. HCl removes water molecules from ketoses forming hydroxymethyl furfural,
which reacts with resorcinol to give a cherry red complex.
Reagents
Seliwanoff’s reagent – .Resorcinol, Conc. HCl

Take 2 ml of Seliwanoff’s reagent in a
Presence of
test tube. Add 5 drops of given solution.
Red color ketose
Boil for 30 seconds. Cool.
Note: Glucose and Sucrose also give the color, if boiled for more than 30 seconds.
10
8. OSAZONE TEST
This test is based on the reducing property of sugars.
Principle
Reducing sugars give characteristic yellow crystalline osazones, when treated with
phenylhydrazine hydrochloride in the presence of sodium acetate and glacial acetic acid
(buffer pair pH – 5).
H-C=O H2N-NH-C6H5 H - C = N – NHC6H5 H - C = N – NHC6H5
H - C – OH + H - C – OH H2N-NH-C6H5 C=O
R H20 R R
Aldohexose Phenyl hydrazone Intermediate
Ammonia (NH3)
+ H2N-NH-C6H5
Aniline (C6H5NH2)
H20
H - C = N – NHC6H5
C = N – NHC6H5
Osazone
Observation Inference
Sheaves of corn/ hay stack Presence of glucose/ fructose/

appearance mannose
Powder puff appearance Presence of lactose
Sunflower appearance Presence of maltose
Fluffy ball appearance Presence of galactose
Broken needle appearance Presence of xylose
Hair thrown in water appearance Presence of arabinose
11
DATE :
NOTES :
12
I.3. COLOR REACTIONS OF PROTEINS
Color reactions of protein are due to the presence of amino acids. These reactions are
produced due to amino group, carboxyl group and the specific side chains of amino
acids.
1. BIURET TEST
This is the general test to detect the presence of proteins.
Principle
This test is answered by proteins with two or more peptide linkages.
CuSO4 reacts with peptide link forming co-ordination complex between cupric ions and
the nitrogen atoms of the peptide bonds forming a violet colored complex in alkaline
medium.
Reagents
- 5% sodium hydroxide
- 1% copper sulfate solution

Take 2 test tubes, in one add 3 Purple color in test tube
ml of given solution and in containing given sample Presence of two
another 3 ml of water(control). and blue color in the peptide bonds
In both the tubes add 10 drops control tube .
of 5% sodium hydroxide. Mix Pink color in test tube
well and add 3 drops of 1% containing given sample Presence of
copper sulfate solution. and blue color in the peptones
Observe both tubes. control tube
Note: Urea also gives this test positive (because of –CONH2).
2. NINHYDRIN TEST
This is a general test for α-aminoacids.
Principle
α-amino acids react with ninhydrin forming aldehyde, CO2, NH3 and hydrindantin
(reduced ninhydrin). Hydrindantin reacts with another molecule of ninhydrin and
released NH3 forming Ruhemann’s purple, a purple colored complex.
Reagents
- Ninhydrin reagent (oxidizing agent)

Take 1 ml of given solution Purple color Presence of α-amino
and add 3 drops of Ninhydrin acids
and boil for 2 min. Allow it to Presence of proline or
cool. Yellow color
hydroxyproline
13
3. XANTHOPROTEIC TEST
This is the test to detect the presence of benzene ring of aromatic amino acids.
Principle
Aromatic amino acids react with nitric acid on heating forming nitrated phenyl groups
which are yellow in color. Adding alkali leads to ionization of nitrated phenyl groups
which causes deepening of yellow color.
Reagents
- Conc. Nitric acid
- 40% Sodium hydroxide

Take 3 ml of protein solution in a test
tube. Add 1ml of concentrated Nitric acid Presence of
Deepening of
and boil for half a min. Cool and add aromatic amino
yellow color
conc. Ammonia or 40% sodium hydroxide acids
solution.
4. COLE’S MERCURIC NITRITE TEST (MODIFIED MILLON’S TEST)

This test is specific for phenolic group of tyrosine.
Principle:
Sulfuric acid reacts with sodium nitrite forming nitrous acid which reacts with phenolic
group of tyrosine forms nitrophenol derivative.
Reagents
- 10% Mercuric sulfate in 10% Sulfuric acid
- 1% Sodium nitrite solution.

To 1 ml of given solution add 1 ml of 10%
mercuric sulfate in 10% Sulfuric acid. Boil Red colored
Presence of
gently the test tube for 30 seconds. Cool solution or
tyrosine
and add 2-3 drops of 1% sodium nitrite precipitate
solution.
5. ALDEHYDE TEST
This is a test to detect indole group of tryptophan.
Principle
The indole ring of tryptophan reacts with formaldehyde in the presence of oxidizing
agents to form a violet colored complex in the form of a ring.
Reagents
- 1/500 Formalin
- 10% mercuric sulfate in 10% sulfuric acid (oxidizing agent)
- Conc.H2SO4
14
Take 1 ml of given solution in a test tube. Add
1 drop of 1/500 Formalin and 1 drop of 10% Purple colored
Presence of
mercuric sulfate in 10% sulfuric acid. Mix well. ring is formed at
tryptophan
Layer 3 ml of conc.H2SO4 along the sides of the junction
the test tube.
6. SAKAGHUCHI’S TEST
This is a test to detect guanidino group of arginine.
Principle
In alkaline medium α-naphthol combines with the guanidino group of arginine to form a
complex which is oxidized by bromine water or sodium hypochlorite to produce carbon
red color.
Reagents
- 5% sodium hydroxide
- Molisch’s reagent
- Bromine water/sodium hypochlorite

To 5 ml of given solution add 5 drops of 5%
sodium hydroxide and 4 drops of Molisch’s Red color is Presence of
reagent. Mix and add 10 drops of bromine formed arginine
water or sodium hypochlorite solution.
7. SULFUR TEST
This is a test to detect the presence of sulfur containing aminoacids.
Principle
Organic sulfur in the sulfur containing amino acids reacts with NaOH forming inorganic
sulfur, sodium sulfide. This sodium sulfide with lead acetate forms lead sulfide which is
black in color.
Reagents
- 40% NaOH
- Lead acetate

Take 2 ml of given solution in a test tube. Black or Presence of
Add 2 ml of 40% NaOH. Boil for 3 min. Add brownish cysteine/
1 ml of lead acetate solution. precipitate cystine
Note: Methionine does not answer this test as the sulfur group is not released by
alkaline digestion.
8. SODIUM NITROPRUSSIDE TEST

This is the test for free SH group.
15
Principle
Free SH group reacts with sodium nitroprusside in alkaline medium to give transient red
color.
Reagents
- Sodium nitroprusside
- 10% NaOH

To 2 ml of given solution, add 2drops of 40% Presence of
Transient red
NaOH. Heat for 1 min. Add 1 ml sodium free SH group
color
nitroprusside. (cysteine)
DATE :
NOTES :
16
I.4. ELECTROPHORESIS
Definition
Electrophoresis is a technique by which, charged particles or solutes in a liquid medium
are separated on the basis of their speed of migration in an applied electric field.
Principle
Separation of charged particles based on their migration in electric field.
Migration rate depends on :
1. Molecular weight- small molecular weight particle migrates rapidly.
2. Size and shape of molecule
3. Electrical field strength
4. Temperature of operation
5. Net electrical charge of the molecule – highly charged species migrate rapidly
affected by pH
affected by ionic strength of the buffer
Types of electrophoresis
Depending on the direction of run, it can be divided into two types. They are as follows:
1. Horizontal
2. Vertical
Horizontal electrophoresis: Agarose gel electrophoresis for serum proteins

Agarose is a linear polysaccharide made up of repeat unit – agarobiose (galactose+ 3, 6
anhydrogalactose). It is used to prepare supporting media.
Principle
In this procedure charged serum proteins are separated on the basis of their speed of
migration under electric field.
Reagents required
1. 0.05 M Barbitone buffer, pH 8.6
2. 1 gm% agarose gel
3. Cold acid ethanol(ethanol: water: acetic acid::70:25:5v/v): fixing agent
4. 90% Acetone: dehydrating agent
5. 1% amidoblack dye: staining
6. 0.1% bromophenol blue: tracking dye
The picture below shows the normal serum electrophoretogram
- + A- Albumin
α1- alpha-1 globulins
α2- alpha-2 globulins
β- beta globulins
γ- gamma globulins.
γ β α2 α1
A
17
Albumin, the largest band, lies closest to the anode. The 4 subsets of globulins are
separated in the order of alpha1, alpha2, beta and gamma, with the gamma band being
closest to cathode. Quantitative estimation of each fraction can be done by
densitometry.
Table below shows the different serum proteins that correspond to subsets of globulins.
Subsets of globulins Proteins
Alpha1-antitrypsin, alpha1-acid glycoprotein, thyroid
α1 globulins
binding globulin, transcortin.
α2 globulins Ceruloplasmin, α2-macroglobulin, haptoglobin
β globulins Transferrin, beta-lipoprotein
Immunoglobulins, C-reactive protein (CRP) is located in
γ globulins
the area between the beta and gamma components
Limitations
1. Plasma cannot be used for electrophoresis, as it contains fibrinogen, which
gives a band in gamma region. Hemolysed samples will also give a similar band
due to hemoglobin.
2. Lipemic samples cannot be used.
Clinical interpretation
The following table reveals the clinical conditions associated with changes in serum
electrophoretogram.
Increased Decreased
Nephrotic syndrome
Impaired liver function
Albumin Dehydration
Chronic infections
Malnutrition
α1
Pregnancy Alpha1-antitrypsin deficiency
globulins
Wilson's disease
Corticosteroid therapy
α2 Protein-losing enteropathies
Advanced diabetes mellitus
globulins Severe liver disease
Nephrotic syndrome
Malnutrition
Biliary cirrhosis
β globulins Cushing's disease Protein malnutrition
Iron deficiency anemia
Multiple myeloma
Amyloidosis
Waldenstrom's macroglobulinemia Agammaglobulinemia
γ globulins
Chronic infections (granulomatous Hypogammaglobulinemia
diseases)
Cirrhosis
18
I.5. CHROMATOGRAPHY
Definition
It is a biochemical technique used for the separation of components of mixtures. It
involves passing a mixture dissolved in a "mobile phase" through a stationary phase.
This separates the analyte to be measured from other molecules in the mixture and
allows it to be isolated.
Types and their principle

1. Partition chromatography: Differential distribution of solutes between a liquid
stationary phase on the "inert" solid supporting matrix and the mobile phase.
2. Normal phase chromatography or adsorption chromatography: The basis of
separation is the difference between adsorption and desorption of solutes at the
surface of stationary solid phase. It uses a polar stationary phase and a non-polar
aqueous mobile phase, and works effectively for separating analytes readily soluble
in non-polar solvents.
3. Reversed phase chromatography: It works with non-polar stationary phase and an
aqueous, relatively polar mobile phase.
4. Displacement Chromatography: A molecule with a high affinity for the
chromatography matrix (the displacer) will compete effectively for binding sites, and
thus displace all molecules with lesser affinities.
5. Size exclusion chromatography or gel permeation chromatography or gel
filtration chromatography: It separates particles based on their size.
6. Ion-exchange chromatography: This technique is based on exchange of ions
between charged stationary phase and ions of opposite charge in the mobile phase.
Ions of the same charge are excluded.
7. Bioaffinity chromatography: This chromatographic process relies on the property
of biologically active substances to form stable, specific, and reversible complexes.
SEPARATION OF AMINO ACIDS BY PAPER CHROMATOGRAPHY
Principle
This technique is based on partition chromatography. Aminoacids get partitioned
between polar stationary phase and the non-polar mobile phase, depending on their
polarity.
Reagents required :
Amino acid standards in 1 molar hydrochloric acid
Solvent: Butanol : Acetic acid : Water (4:1:1)
Stain: 1%Ninhydrin in butanol.
Rf value
Rf = Distance traveled by the compound
Distance traveled by the solvent
19
Aminoacids and their Rf value
Aminoacids Rf value Note: The symbol Rf stands for

Alanine 0.38 "retardation factor" or "ratio-to-
Arginine 0.20 front". It is expressed as a decimal
Asparagine 0.5 fraction. When the chromatography
Aspartic acid 0.24 conditions are duplicated, the same
Cysteine 0.4 average relative positions will turn
Glutamine 0.13 up for the solvent and solute; thus
Glutamic acid 0.30 the Rf value is a constant for a given
compound for a particular solvent
Glycine 0.26
system. The Rf value is a physical
Histidine 0.11 property for that compound. The Rf
Isoleucine 0.72 value is useful in identifying
Leucine 0.73 compounds.
Lysine 0.14
Methionine 0.55 However, Rf values vary with the
Phenylalanine 0.68 solvent systems. Since it is difficult
Proline (an imino acid, for different laboratories to exactly
0.43
stains yellow) duplicate conditions for a
Serine 0.27 chromatography experiment, Rf
Threonine 0.35 values are more useful for
Ttryptophan 0.66 comparisons within one lab than for
Tyrosine 0.45 comparisons of data from different
Valine 0.61 labs.
20
High performance liquid chromatography (HPLC)
High performance liquid chromatography is a highly improved form of column

chromatography. The solvent is forced through under high pressures down the column.
It uses an efficient column containing small particles of stationary phase, which gives a
much greater surface area for interactions between them and the molecules flowing
through it. This allows a much better separation of the components of the mixture. The
advantages of this method over column chromatography are that it is highly automated
and is an extremely sensitive detection method.
Applications of chromatography
1. Investigation of congenital metabolic disorders. Ion-exchange HPLC system is

more effective in separation and quantification of aminoacids, as compared to
paper or thin layer chromatography. This method is used for detection of maple
syrup urine disease and phenylketonuria.
2. Measurement of glycated hemoglobin (HbA1c) in diabetic patients. Cation
exchange resin coupled with HPLC is the reference method for HbA1c estimation.
3. Estimation of biogenic amines by Ion exchange chromatography in carcinoid
syndrome, pheochromocytoma and other conditions.
4. Antenatal screening for inborn errors of nucleotide metabolism by measuring
purine and pyrimidine levels in fetal RBC by ion-exchange chromatography for
the diagnosis of adenosine deaminase deficiency, xanthine oxidase deficiency
etc.
5. Identification and quantification of different urinary porphyrins and vitamins by
reverse phase HPLC system. For example – coproporphyrin, uroporphyrin,
Vitamin A, D, E and C.
6. Oxidative stress parameters like malondialdehyde can be measured by reverse
phase HPLC for research purpose.
21
II. HEMATOLOGY AND
IMMUNOLOGY
22
II.1. PHOTOMETRY
Definition
Photometry is defined as the measurement of light. Spectrophotometer and colorimeter
measure the intensity of light at selected wavelengths.
Principle
White light is made up of different colors or wavelengths of light. A colored
sample typically absorbs only one band of wavelength from white light. In a colorimeter,
a beam of white light is passed through a monochromator, which transmits only one
band of wavelength to the photodetector. The difference between the amount of light
transmitted by a sample (blank) and that transmitted by the given sample (test) is a
measurement of the absorbance of the given test sample. This is directly proportional to
the concentration of the substance producing the color and the path length.
In a colorimeter, the measurements are carried out only in the visible region of
the electromagnetic spectrum (400 – 700nm). Often filters are used for the preparation
of monochromatic light which results in poor spectral resolution. A spectrophotometer
uses prisms or gratings to separate light which results in better spectral resolution.
Further in a spectrophotometer measurements are carried out in the entire range of the
electromagnetic spectrum. Hence, spectrophotometer can measure biological
substances in near ultraviolet (UR), visible and near infrared (IR) range with more
precision, as compared to a colorimeter. Given below are the spectral characteristics of
UV, visible and IR rays.
Wavelength (nm) Region name Color observed

<220 Far UV Invisible
200-380 Near UV Invisible
380-440 Visible Violet
440-500 Visible Blue
500-580 Visible Green
580-600 Visible Yellow
600-620 Visible Orange
620-750 Visible Red
800-2500 Near IR Not visible
2500-15,000 Mid IR Not visible
15,000-1000000 Far IR Not visible
Beer-Lambert Law
Basic concepts:
Consider an incident light beam with intensity Io, passing through a cell containing a
solution of a compound, that absorbs light of certain wavelength. Given that the intensity
of the transmitted light is I, the transmittance (T) of light is defined as,
T = I/Io
A portion of the light might get reflected by the cuvette’s wall or absorbed by the solvent
used. To eliminate these factors, blank (reference cell) is used that is identical to the
sample cell, except that it lacks the compound of our interest, whose concentration
needs to be determined. In practice, the reference cell (blank) is inserted and instrument
23
is adjusted to 100% T , after which %T of the sample is measured. Transmittance varies
inversely and logarithmically with increase in the concentration of the compound in the
solution. Absorbance (A) is directly proportional to the concentration. Figures below
show the relationship between absorbance and %T.
%T A
Concentration Concentration
Thus, A = - log I/Io

A = - log T = log 1/T = log 100 - log %T = 2 – log %T
Optical density (OD)

Optical density is the term used for expressing the reading of absorbance. As it is
measured in logarithmic scale, values too low or too high are not acceptable for
accurate results (sensitive range is 0.1 to 0.6).
RELATIONSHIP BETWEEN TRANSMITTANCE, ABSORBANCE, AND

CONCENTRATION
Beer’s law
The intensity of the light absorbed is directly proportional to the concentration of the
colored compound in the given solution.
Lambert’s law
The amount of light absorbed by the colored solution is directly proportional to the path
length through which the light passes.
Combining these two principles, Beer-Lambert’s law can be expressed as:
A = ε cl
Where A is absorbance (no units, since A = log10Io/ I).
ε is proportionality constant defined as molar absorptivity or molar extinction coefficient.
l is the path length of the sample, that is, the path length of the cuvette in which the
sample is contained. It is expressed in centimetres.
c is the concentration of the compound in the solution, in moles/L.
24
Molar absorptivity
Molar absorptivity (ε) is the value of absorptivity (a) when the path length is 1 cm and
concentration is 1mol/L. The value of ε is constant for a given compound, at a given
wavelength under prescribed conditions of solvent, temperature, and pH. Value of ε is
useful in characterizing compounds and their purity.
Parts of a Colorimeter
1. Source of light (tungsten, mercury lamp)

2. Monochromator (filter)
3. Cuvette-sample holder (small glass tubes) – quartz (UV light), polystyrene(visible
light)
4. Photo cell-(Detector)
5. Read out device (Display-Galvanometer)
Light source Monochromator Cuvette Detector Galvanometer
Operation of colorimeter
1. Turn on the colorimeter and adjust the filter for the selected wavelength.
2. Insert a clean cuvette containing the blank into the holder. Be sure that the tube is
clean and free of finger prints. Adjust the meter to read 100% transmittance and
establish that your blank will give a reading of 100% transmittance (Zero
Absorbance).
3. To take a reading, insert a cuvette holding your test solution and read the
transmittance value directly on the scale.
4. Record the % transmittance of your solution
Experiment : Verification of Beer-Lambert Law using colorimeter
Reagents:
1. 1% potassium permanganate solution (KMnO4)
2. Distilled water
Procedure:
1. Pipette 1,2,3,4 and 5 ml of KMnO4 in 5 different test tubes.
2. Make up the volume to 10 ml in each test tube with distilled water.
25
3. Measure the absorbance of one of the solutions using different wavelengths.
Tabulate the readings. Choose the wavelength which allows the maximum
absorbance (minimum transmittance). This wavelength is denoted as λmax.
4. Measure the absorbance of all the 5 tubes (test and blank) at λmax. Tabulate the
readings.
5. Construct a graph with the absorbance on y-axis and concentration of standard
on x-axis. A straight line is obtained.
Advantages of spectrophotometer over colorimeter

1. Spectrophotometer is several times more accurate and sensitive than the
colorimeter since it uses a line of wavelength where as colorimeter uses a band
of wavelength.
2. Even minute quantities of the substance can be measured using
spectrophotometer.
3. Unlike colorimeter, spectrophotometer can be used in the infrared and ultraviolet
region also.
Applications of spectrophotometer
It can measure compounds with characteristic absorption maximum like
 Peptide bond (220 nm)
 Tryptophan (280 nm)
 Nucleic acids (260 nm)
 Bilirubin (450 nm)
 Deoxy-hemoglobin (565 nm)
 NADH/NADPH (340 nm)
 FAD+ (450 nm)
 FADH2 (570 nm)
 Porphyrin (400 nm)
 Potassium Permanganate (540 nm)
DATE :
NOTES :
26
II.2. IMMUNOASSAYS
(Radioimmunoassay, ELISA & Chemiluminescence)
Introduction
Immunoassays are analytical methods, which measure the concentration of a

substance in serum and other biological fluids, based on agglutination (antigen-antibody
binding) reactions. They can be used to quantify antigens and antibodies by labeling
them with different molecules.
Depending on the type of labels, immunoassay is classified as follows:
Types of labels Name of the method Examples for labels
Radioisotopes RIA(Radioimmunoassay) I125

ELISA(Enzyme Linked Alkaline phosphatase
Enzymes
ImmunoSorbent Assay) Horse radish peroxidase
Chemiluminescent
Chemiluminescence Acridinium ester
substances
Others include fluorescence excitation transfer immunoassay, bioluminescent

immunoassays, phosphorimmunoassays, quantum dot immunoassays, magnetic
immunoassay etc.
Radioimmunoassay (RIA)
Radioimmunoassay commonly uses I125, I131, H3, Co57 radioisotopes.
Types of RIA :
1. Competitive RIA
In competitive RIA, competition between radiolabeled and unlabeled antigen or antibody

in an antigen-antibody reaction is used to determine the concentration of unlabeled
antigen or antibody. This immunoassay takes advantage of specificity of antigen-
antibody interaction and its ability to measure very low quantities of radioactive
elements. Here a fixed concentration of labeled tracer antigen is incubated with a known
amount of antiserum.
When unlabeled antigen is added to this system, there is competition between labeled
tracer and unlabeled antigen for the limited and constant number of binding sites on the
antibody. Thus, the amount of tracer bound to antibody will decrease as the
concentration of unlabeled antigen increases. If the concentration of antigen in the
sample is more, we get lesser bound radioactive count per minute and vice versa.
Examples include the estimation of thyroid hormones, triiodothyronine (T 3) and
tetraiodothyronine (T4).
27
2. Non competitive RIA
Here, there is no competition for binding sites. A commonly used type of this method is
immunoradiometric assay (IRMA). It involves two high affinity monoclonal antibodies
against the antigen of interest. Among the two antibodies, one is used to capture the
antigen(capture antibody); the other called signal antibody is labeled with radioactive
substances for signaling. Both the antibodies bind the antigenic epitopes that are
different from each other. The two antibodies react simultaneously to the antigen in the
sample, which lead to the formation of a capture antibody- antigen- signal antibody
complex, or sandwich. Examples are estimation of TSH, insulin etc.
Applications of RIA
Measurement of different hormones, lipoproteins, oncoproteins, growth factors,
cytokines, bacterial antigens and other biological substances accurately.
Advantages of RIA
This technique has higher sensitivity and specificity and even minute amounts
(picograms) of substances can be analyzed.
Disadvantages of RIA
Since radioactive substances are used, stringent precautionary measures should
be followed and only approved laboratories can take up the assay.
The shelf life of reagent is short. For example I125 has half life of only 60 days.
Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA uses the catalytic properties of enzymes to detect and quantify immunological
reactions. Enzymes commonly used as labels are: Alkaline phosphatase, Horseradish
peroxidase, Glucose 6 phosphate dehydrogenase and β galactosidase.
In this type of assay, the antigen is immobilized and detection antibody is added, which
forms a complex with the antigen. The detection antibody is covalently linked to an
enzyme and adding a substrate to the detection antibody will produce a visible signal,
which measures the quantity of antigen in the sample. Newer assays employ
fluorogenic substrates enabling much higher sensitivity.
Types of ELISA
1. Dual antibody capture sandwich ELISA (DAS ELISA)

DAS ELISA requires two antibodies that recognize separate epitopes on the antigen to
be measured. The “capture” antibody, which is specific for the substance to be
measured, is first coated onto a microtiter plate. Samples and standards are then
incubated on the plate, and any antigen present subsequently binds to the capture
antibody. The bound antigen is detected using a secondary antibody thus creating the
“sandwich.” The detection antibody is conjugated with enzymes such as horse radish
peroxidase (HRP) and hence, addition of 3,3',5,5'-tetramethylbenzidine (TMB) substrate
28
lead to colorimetric reaction that can then be measured using a spectrophotometer. The
resulting color (optical density [OD]) is directly proportional to the amount of antigen
present in the sample.
Dual antibody capture sandwich ELISA
2. Indirect ELISA
In indirect ELISA, sample containing the antibodies bind to the specific antigens
coated onto the microtiter plate and detected using anti-antibody. It is used in
conditions where only one specific antibody is available to detect the antigen. An
example for this type is screening for HIV antibodies.
3. Competitive ELISA
In this type of ELISA, both the test antigen to be measured and the standard antigen
which is enzyme conjugated, are added to a microtiter plate, coated with antibodies
directed against the test antigen. The two forms of the antigen compete for binding
sites on the antibody-coated plate, which is proportional to their respective
concentration in the mixture. Measurement of the bound conjugated form of the
29
antigen is inversely proportional to the amount of test antigen, unlike in sandwich or
indirect ELISA. This is used for estimation of hormones like T 3 and T4.
Applications of ELISA
1. Identification of antibodies against pathogens. For example screening for anti-

hepatitis B and C antibodies, HIV antibodies etc in suspected individuals.
2. Measurement of autoantibodies in human serum in case of rheumatoid arthritis,
systemic lupus erythematosis etc.
3. Measurement of different hormones like thyroid hormones, growth hormones etc. In
infertility, luteinizing hormone levels aid in identification of the time of ovulation.
4. Estimating levels of hormones and cytokines present in minute quantities in
biological fluids.
5. Quantification of different tumor markers. Examples are CA-125 in ovarian cancer,
alpha fetoprotein in liver cancer, prostate specific antigen in prostate cancer etc.
6. Virulence in infectious diseases is determined by measuring the viral or bacterial
loads in human serum. For example, hepatitis B core antigen levels in infected
individuals.
7. Therapeutic drug monitoring.
Advantages of ELISA in comparison to RIA

1. Relatively safe as it does not involve radioactive materials.
2. Longer shelf life for reagents.
3. Lower time for standardization.
4. Relatively quick turnaround time.
CHEMILUMINESCENCE
Chemiluminescence is the generation of electromagnetic radiation as light, by the

release of energy from a chemical reaction. While the light can, in principle, be emitted
in the ultraviolet, visible or infrared region, those emitting visible light are the most
common. When this reaction involves enzymes, it is called bioluminescence. When a
chemiluminescent substance is activated, it is converted to an excited intermediate. The
decay of the intermediate from excited state to a lower energy level results in the
emission of light.
Types of chemiluminescence
1. Solid- phase reaction

This is used as a signaling method in ELISA, Western blot etc. Horseradish peroxidase
enzyme (HRP) is tethered to the molecule of interest (usually through labeling an
immunoglobulin that specifically recognizes the molecule). This enzyme complex
catalyzes the conversion of the excited chemiluminescent substrate, into a sensitized
reagent in the vicinity of the molecule of interest. This on further oxidation by hydrogen
peroxide, produces a triplet (excited) carbonyl; which emits light when it decays to the
30
singlet carbonyl. It allows detection of minute quantities of a biomolecule. Proteins can
be detected even in minute quantities (femtomoles)
2. Liquid-phase reactions
Luminol reaction is an example for this type. Luminol in an alkaline solution with
hydrogen peroxide, in the presence of an oxidant like iron or copper, produces
chemiluminescence.
The luminol reaction is:
luminol + H2O2 3-APA [¤] [3-aminophthalate] 3-APA + light.
3-APA [¤] is the excited state. It fluoresces as it decays to a lower energy level.
Other liquid phase chemilumiscence reagents are
1. Pyrogallol
2. Lucigenin
3. Aryl oxalates
Applications of chemiluminescence
1. Quantification of different hormones (thyroid hormones, cortisol, LH, FSH, HCG,

prolactin, testosterone, estrogen etc); tumor markers (CA-125, carcinoembryonic
antigen, alpha fetoprotein etc) and cardiac markers (troponins, homocysteine
etc),vitamins (folate, Vitamin B12)
2. Detection and quantitation of biomolecules in Western blots.
3. Analysis of metabolites, where the substrate isn't directly involved in
chemiluminescence reaction, but the product is. For example - homovanillic acid and 5-
hydroxyindole-3-acetic acid.
4. DNA sequencing using pyrosequencing.
31
II.3. HEMOGLOBIN AND ITS DERIVATIVES
Introduction
Hemoglobin (Hb) is a conjugated metalloprotein consisting of a proteinous globin part
and a non proteinous heme part, which has iron porphyrin compound as the prosthetic
group.
Derivatives of hemoglobin
1. Oxyhemoglobin: Hemoglobin + 4 molecules of oxygen
2. Carboxy hemoglobin: Hemoglobin + carbon monoxide
3. Methemoglobin: Hemoglobin in which the iron (Fe++) is oxidized to Fe+++
4. Hemochromogen: Denatured hemoglobin in which iron is in the Fe++ form
These derivatives can be detected by viewing the solution through a simple device
known as spectroscope.
Spectroscope
Hartridge reversion spectroscope is a simple device that resolves white light into its
seven component colors. It consists of a narrow slit through which light enters. A set of
prisms resolves the light that can be viewed through an eye piece. The wave length
region of light which the human eye can perceive ranges from 400 to 700 nm. When
day light is viewed through the spectroscope, a few dark lines are seen. The two
prominent lines are at 589 nm and 518 nm. They arise due to the absorption of light of a
particular wavelength by sodium and magnesium respectively, present in the solar
atmosphere. They are known as Fraunhofer’s lines. When a solution of hemoglobin is
viewed through a spectroscope, similar dark lines or bands are seen at definite
wavelengths. They arise due to absorption of light by hemoglobin. The absorption
maxima of these lines differ from one hemoglobin derivative to another, which is
successfully used in differential identification of these compounds.
Absorption characteristics of various derivatives of hemoglobin
Wavelength of the prominent

Name of the derivative Color
absorption (nm)
OxyHb Crimson red 540 (broad) & 576 (narrow)
Reduced Hb Purple 565 (broad & faint)
CarboxyHb Cherry red Nearly same as oxy Hb
MetHb Reddish brown 630
32
1. Study of various derivatives of hemoglobin by spectroscope
Test Observation Inference

a. OxyHb
2 bands will be seen in the
Prepare 1 in 200 dilution of Presence of
green portion of the
blood, take 5 ml in a clean test oxyHb is
spectrum at 576nm &
tube & examine under confirmed.
540nm respectively.
spectroscope.
b. DeoxyHb
To 5 ml of 1 in 200 dilution of Bright crimson red of Presence of
blood in a test tube, add stokes oxyHb is changed to deoxyHb is
reagent and mix. purple due to the formation confirmed.
of deoxyHb under
spectroscope. 2 bands of
oxyHb will be replaced by
single faint broad band
which occupies entire
space occupied by the 2
bands of oxyHb. Middle of
the faint band is at 565nm.
Color changes from purple Reoxygenation
Now shake the tube
to red. of deoxyHb.
vigorously.
c.CarboxyHb
a) Take 5 ml of given carboxy 2 bands will be seen in
Presence of
Hb in a test tube and observe almost the same region as
carboxyHb.
in spectroscope in oxyHb.
CO has more
b) Now add a pinch of sodium The carboxyHb does not affinity for Hb
hydrosulfite to carboxyHb in a get reduced. Unlike oxyHb, than oxygen &
tube. Mix gently. the 2 bands persist. carboxy Hb is
very stable.
d. MetHb
Take 5 ml of 1 in 50 dilution of
Red color will turn brown.
blood in a test tube. Add a Presence of
A prominent band in the
pinch of finely powdered metHb is
center of red region at 630
potassium ferricyanide and confirmed.
nm is seen.
shake well. Examine under
spectroscope.
33
1. Study of various derivatives of hemoglobin by spectroscope
(contd.)
Test Observation Inference

e. Preparation of hemin crystals
A drop of blood is spread on a glass slide to
form a thin film and it is dried over a low
Brown rhomboid
flame. 2 drops of Nippe’s fluid is added and
crystals of hemin/ Presence of
cover slip is placed in position. Then the slide
acid hematin hemin crystals
is heated gently until gas bubbles are formed.
chloride will be is noted.
Again 2 drops of Nippe’s reagent is run
seen.
underneath and the slide is heated gently
until gas bubbles are formed. The glass slide
is viewed under the microscope.
f. Hemochromogen Two bands are
seen in 555 nm
Presence of
(alpha) & 525 nm
hemochromog
(Beta). In very
en is
high dilution, only
confirmed
alpha band is
seen
Note:
Hemin is a reddish-brown crystalline chloride of heme, produced when hemoglobin
reacts with glacial acetic acid and potassium chloride.
Hemochromogen is an important derivative of Hb (denatured hemoglobin) in which
iron is in the Fe++ form. It absorbs the light in very great dilutions. This is important in
testing for presence of minute traces of blood in clinical medicine and in forensic
medicine to confirm suspected blood stains.
Composition of reagents
 Stokes reagent: 2 gms of ferrous sulfate + 3 gms of tartaric acid in 100 ml of

water. Take 5 ml of this and add ammonia till precipitate dissolves for use.
 Nippe’s fluid: Dissolve 0.1 gm each of potassium chloride, potassium bromide ,
potassium iodide, in 100 ml of glacial acetic acid.
Clinical significance
Oxyhemoglobin: Hb exhibits cooperative binding with oxygen. It gives up the oxygen

at low pressure and combines with it at high pressure. Hb can transport oxygen as long
as iron remains in bivalent state.
34
Carboxyhemoglobin: Hb has 200 times greater affinity for carbon monoxide (CO)
than for oxygen. Carboxyhemoglobin is a very stable compound. It can be dissociated
by oxygen under very high pressure.
Exposure to CO occurs in the following conditions:
 In mines.
 Heavy cigarette smoking.
 Exposure to automobile exhaust.
Clinical features include mild headache and dizziness in lower concentrations, to coma
& death in very high concentration.
Methemoglobin
A substitution of tyrosine for either proximal or distal histidine in alpha/beta chain of

hemoglobin locks the heme iron in its trivalent state (Fe +++) or when oxidizing agents
like potassium ferricyanide are added to Hb, Heme portion (Fe ++) is oxidized to hematin
(Fe+++) globulin part remain unaffected. Resulting derivative is called Methemoglobin
which is made up of globin conjugated with hematin, which cannot carry oxygen.
In the body the enzyme NADH-meth Hb reductase (Diaphorase 1) can reduce
small amount of meth Hb back to normal Hb. Congenital methemoglobinemia is due to
absence of this enzyme.
Note: The enzyme Diaphorase II is NADPH dependent meth Hb reductase, which is a

minor pathway for reducing meth Hb.
Methemoglobinemia
It is a condition where there is increased met Hb in blood. Clinical features when met Hb
concentration in blood increases to:
 Up to 15%: cyanosis
 >55%: hypoxia, coma
 > 70% : death
Acquired causes of methemoglobinemia

Amyl nitrate, dapsone, sulfonamides, chloroquine, aniline, nitrobenzene (shoe polish)
etc.
Note: Methylene blue which is a reducing agent can be used in treatment of this
condition.
35
DATE :
NOTES :
36
II.4.CASE REPORTS : ANAEMIA
QUESTION 1
A 50-year-old male is brought to the emergency department with complaints of malaise,

nausea and vomiting and fatigue with difficulty in maintaining balance. On physical
exam he appears malnourished. Neurologic examination reveals decreased vibration
and position sense. His blood count revealed a normal white blood cell count but does
show an anemia with large red blood cells.
a) What is the most likely cause of his anemia?
b) What is the molecular basis for the large erythrocytes?
QUESTION 2
An 18 year old female reported to the physician for consultation. She complained of
generalized weakness, lethargy and inability to do the routine work for the past four
months. On further questioning, she revealed that she was having excessive bleeding
during menstruation for the past six months. She also complained of breathlessness
and palpitations while climbing stairs . There was no history of any fever, drug intake or
abdominal discomfort.
On examination, her physician found that she had pallor and tachycardia.
Laboratory investigations:
Red Blood Cell Count -3.5 million/mm3
Hemoglobin (Hb) -7 g/dl
Haemtocrit (Hct)- 30%
Mean Corpuscular Volume (MCV) – low
Mean Corpuscular Hb Concentration (MCHC)- low
Serum Iron – 70 µg/dl (Ref Range:100-150 µg/dl)
Serum ferritin- 350 µg/L (Ref Range: 20-120 µg/L)
a) What is the probable diagnosis in this patient?
b) Mention the Recommended Dietary Allowance (RDA) for iron
c) What dietary advice should be given to this patient ?
37
QUESTION 3
A man aged 25 years presented in a hospital with complaints of abdominal pain and
passing red colored urine . History revealed that one week ago, he had very high fever
which was diagnosed as malaria. He was treated with chloroquine tablets after which
malarial fever subsided. After this he took primaquine tablets , as advised by the
physician as a prophylactic measure against recurrence of malaria. On examination, he
was pale , had tachycardia and mild splenomegaly
Laboratory features: Blood hemoglobin= 9 g/dl
RBC count = 3 million/cu mm
Serum total bilirubin = 4 mg/dL
Conjugated bilirubin = 0.4 mg/dL
Haptoglobin = 15 mg/dL ( Ref Range :100- 200 mg/dL )
Urine Blood = ++
Urine urobilinogen= present
a) What is the probable diagnosis ?
b) What is the biochemical cause for hemolysis ?
c) What is haptoglobin? What is the half life of haptoglobin ?
d) Name the drugs causing hemolysis in G6PD deficient individuals.
38
III. MUSCULOSKELETAL SYSTEM
& AUTONOMIC NERVOUS
SYSTEM
39
III.1. ESTIMATION OF CALCIUM IN SERUM
Calcium plays an important role in bone mineralization, blood coagulation, glucose
metabolism, hormone secretion, nerve conduction and muscle contraction. It is present
in three forms in blood; a protein bound fraction (40%), an ionized or free fraction (50%)
and the fraction which is complexed with small anions like phosphate, lactate and citrate
(10%). All the three forms can be quantitated though total calcium is most commonly
estimated.
Calcium can be estimated by :

1. Photometric methods
2. Titration methods
a. Iodometric titration
b. Baron and Bell method
BARON AND BELL METHOD
Principle
Calcium in the sample combines with calcein-thymophthalein indicator to form a bluish
green colored complex in a strong alkaline medium. On titration with EDTA solution,
calcium is released from this complex. The released complex gets chelated to EDTA.
The released indicator gives a mauve color indicating the end point of titration. The titre
value is compared with a standard solution treated same way and the serum level of
calcium is calculated.
Reagents
1. Alkaline buffer; pH 12.66 (contains glycine and NaOH)
2. Ethylene diamine tetraacetate disodium salt solution (EDTA)
3. Working standard calcium solution, 10 mg/100mL
4. Calcein-thymophthalein indicator
Procedure
Pipette 1 ml of serum into 5 ml of buffer solution taken in a small white dish and add a
pinch of indicator (about 1 mg). Titrate with EDTA solution from a micro burette
graduated to 0.01 ml. The end point is reached in aqueous solution when the color
changes from yellow green to mauve. Since the color is stable only for a few seconds,
titration should be carried out quickly. Titrate 1 ml of standard calcium solution and 1 ml
of water as blank and follow the same procedure.
Calculation
Serum calcium (mg/dL) = Titration of test- Titration of blank x 10

Titration of std- Titration of blank
40
SERUM CALCIUM ESTIMATION USING MODIFIED OCPC METHOD
SEMIAUTOANALYZER METHOD(demonstration)
Principle
In alkaline medium, the metal complexing dye CPC, forms a red chromophore with
calcium which is measured at 570 – 580 nm. 8-hydroxy quinoline prevents magnesium
ion from interference.
Reagents
1. Diethylamine
2. O-cresolphtalein complex (OCPC)
3. 8-hydroxy quinoline
4. Calcium standard (10 mg/dL)
Procedure
Take three test tubes and label as Blank ‘B’, Standard ‘S’ and Test ‘T’. Perform the
experiment as follows:
B S T
Working reagent 1000 μL 1000 μL 1000
μL
Standard - 10 μL -
Test - - 10 μL
Mix and incubate for 5 minutes at room temperature. Read the absorbance of standard
and sample against reagent blank.
Calculation
Calcium (mg/dL) = Absorbance of Test – Absorbance of Blank xconc. of standard
Absorbance of Std – Absorbance of Blank
Reference range of serum calcium
Total  9-11 mg/dL or 2.15-2.57 mmol/L
Free  4.5-5.5 mg/dL or 1.15-1.33 mmol/L
41
Hypocalcemia
Hypocalcemia is characterized by total calcium < 8.5 mg/dL

Hypocalcemia can be due to reduction in either bound or free calcium or both.
Hypoalbuminemia is the common cause of pseudohypocalcemia. 1gm albumin binds to
approximately 0.8 mg/dL of calcium. Low calcium levels are also seen in chronic renal
failure, hypomagnesemia, hypoparathyroidism etc.
Corrected total calcium in hypoalbuminemia

Serum proteins especially albumin and other organic and inorganic ions affect the
serum calcium levels.
Corrected total calcium (mg/dL) = Total calcium (mg/dL) +0.8(4- albumin (g/dL))
Hypercalcemia
Hypercalcemia is characterized by total calcium >11 mg/dL. It can be due to increased

intestinal absorption, increased renal retention or increased skeletal resorption. Primary
hyperparathyroidism is the most common cause. Hypercalcemia is also seen in
hyperthyroidism, multiple myeloma, leukemia, lymphoma, vitamin D intoxication. Free
calcium is the best indicator of calcium status.
DATE :
NOTES :
42
III.2. ESTIMATION OF PHOSPHATE IN SERUM
Phosphate is present in both organic and inorganic forms in blood. In labs inorganic
form is measured. In blood, about 10% of phosphate is protein bound, 35% complexed
with sodium, calcium and magnesium and rest 55% is free. Phosphate is necessary for
electrolyte transport, nerve function, muscle contractility and it is also an essential part
of ATP, NADP etc.
FISKE-SUBBAROW METHOD
Principle
The proteins are precipitated by trichloroacetic acid. The filtrate is treated with
ammonium molybdate to form phosphomolybdate and this is reduced by 1-amino 2-
naphthol 4-sulfonic acid (ANSA) to give a blue phosphomolybdate complex. The
intensity of color developed is directly proportional to amount of inorganic phosphate
present. The color is read at 680 nm.
Reagents
1. Trichloroacetic acid 10%
2. Molybdate reagent: 2.5% ammonium molybdate in 3 N sulfuric acid
3. ANSA 0.25%
4. Phosphate standard 0.04 mg/ml
Procedure
Preparations of protein free filtrate:

To 9 ml of 10% trichloroacetic acid (TCA) in a test tube, add 1 ml of serum drop by drop.
Mix well and allow it to stand for 10 minutes and filter.
Estimation of phosphorus in the protein free filtrate

Take four test tubes and label as ‘Blank (B)’, ‘Standard (S)’ and ‘Test (T 1 and T2).
Perform the experiment as follows:
Constituents B S T1 T2
Filtrate - - 5 ml 5ml
Distilled water 5 ml 4 ml - -
Std. - 1 ml - -
Molybdate 1 ml 1 ml 1 ml 1ml
reagent
Mix well
ANSA 0.4 ml 0.4 ml 0.4 ml 0.4ml
Distilled water 3.6 ml 3.6 ml 3.6 ml 3.6ml
Allow to stand for 10 minutes. Read absorbance at 680 nm filter.
43
Normally, both T1 and T2 would give same values if the procedure is followed
accurately. If the values of T1 and T2 are different, take average absorbance of T1 and
T2 for calculation
Calculation:
Serum inorganic phosphate (mg/dL) =
= Absorbance of Test x Amt. of std x 100

Absorbance of Std Vol of serum
= Absorbance of Test x 0.04 x 100

Absorbance of Std 0.5
= Absorbance of Test x 8
Absorbance of Std
SERUM PHOSPHATE ESTIMATION BY PHOSPHOMOLYBDATE METHOD
SEMIAUTOANALYZER METHOD (demonstration)
Principle
Ammonium molybdate + sulfuric acid + phosphorus phosphomolybdic
complex
The absorbance of phosphomolybdic complex formed is measured at 340 nm.
Reagents
1. Working Reagent containing

Ammonium molybdate
Sulfuric acid
2. Phosphorus standard (5 mg/dL)
Procedure
Take three test tubes and label as Blank ‘B’, Standard ‘S’ and Test ‘T’. Perform the
B S T
Working Reagent( 1000 1000 1000
μL)
Standard (μL) - 20 -
Test (μL) - - 20
Mix and incubate for 1 minute at 37oC. Read the absorbance of standard and sample
against reagent blank.
44
Calculation
Phosphorus = Absorbance of Test–Absorbance of Blank x conc. of standard

(mg/dL) Absorbance of Std – Absorbance of Blank
Reference range of serum inorganic phosphorus

Adults: 2.5-4.5 mg/dL or 0.81-1.45 mmol/L
Children: 4-7 mg/dL or 1.29-2.26 mmol/L
Hypophosphatemia
It is characterized by serum phosphate conc. <2.5 mg/dL. It may be caused by

intracellular shift, increased renal wasting, decreased intestinal absorption etc. Causes
include hyperparathyroidism, renal tubular defects, Vit D deficiency, malabsorption
syndrome.
Hyperphosphatemia
It is commonly due to inability of the kidney to excrete phosphate. Also due to

hypoparathyroidism, renal failure, acromegaly, acidosis.
DATE :
NOTES :
45
IV. ENDOCRINE SYSTEM
46
IV.1. ESTIMATION OF GLUCOSE IN SERUM
Glucose estimation is the most common investigation done in clinical biochemistry
laboratory.
Glucose can be estimated by

1. Non-enzymatic methods
a. Nelson-Somogyi method
b. Folin Wu method
c. Asatoor king method
d. O-toluidine method
2. Enzymatic methods
a. Hexokinase method
b. Glucose oxidase-peroxidase method
c. Glucose dehydrogenase method
Note: Non-enzymatic methods are not done now-a-days because these tests are mainly
based on the reducing property and hence substances with reducing property (NGRS)
like creatinine, uric acid and ascorbic acid interfere with the glucose estimation.
NON-ENZYMATIC METHOD
The commonly used manual method in clinical biochemistry laboratories is Nelson-

Somogyi method.
NELSON-SOMOGYI METHOD
Principle
Estimation of glucose by this method is based on its reducing property.
Proteins are precipitated using ZnSO4 and Ba(OH)2.
ZnSO4 + Ba(OH) 2  BaSO4
Non-glucose reducing substances (NGRS) are adsorbed on BaSO 4. Glucose in alkaline
medium is converted to enediol form, which reduces alkaline copper to cuprous oxide.
Cuprous oxide then reduces arsenomolybdic acid to molybdenum blue, which is bluish
green color. Color is read at 680 nm.
Reagents
1. Zinc sulfate
2. Barium hydroxide
3. Alkaline copper tartarate reagent
4. Arsenomolybdic acid
5. Glucose standard (working) – 2.5 mg/100ml
47
Procedure
Precipitation of proteins and preparation of protein free filtrate
In a test tube take 0.1 ml of serum, 3.5 ml of distilled water, 0.2 ml of ZnSO 4 and 0.2 ml
of Ba(OH) 2. Mix well and allow it to stand for 10 minutes and filter.
Estimation of glucose in the protein free filtrate
Constituents B S T1 T2
Filtrate (ml) - - 1 1
Distilled Water (ml) 1 - - -
Std. (ml) - 1 - -
Distilled Water (ml) 1 1 1 1
Alkaline Copper 2 2 2 2
tartarate (ml)
Mix well. Keep in boiling water bath for 10 min. Cool for 1 min.
Arsenomolybdic 1 1 1 1
acid (ml)
Distilled Water (ml) 5 5 5 5
Mix well till effervescence ceases. Read absorbance at 680 nm.
T2 for calculation.
Calculation
Glucose concentration (mg/dL) =
= Absorbance of Test – Absorbance of Blank x Amt. of standard x 100
Absorbance of Std – Absorbance of Blank Vol of sample in filtrate
= Absorbance of Test – Absorbance of Blank x 0.025 x 100

Absorbance of Std – Absorbance of Blank 0.025
= Absorbance of Test – Absorbance of Blank x 100

Absorbance of Std– Absorbance of Blank
PLASMA GLUCOSE ESTIMATION USING GLUCOSE OXIDASE METHOD
Semiautoanalyzer Method (demonstration)
Enzymatic method: Glucose oxidase-Peroxidase (GOD-POD) method

Principle
Enzymatic estimation of glucose is based on the following reaction.
GOD
Glucose + O2 + H2O H2O 2 + Gluconic acid
POD
2H2O 2 + Phenol + 4-Aminophenazone Quinoneimine + 4 H2O
48
Reagents required
1. Working reagent containing
- Tris-Buffer, (pH 7.40)
- Phenol
- Glucose oxidase
- 4-Aminophenazone
2. Standard (Conc. – 100 mg/dL)
Procedure
Take three test tubes and label as ‘Blank (B)’, ‘Standard (S)’ and ‘Test (T)’. Perform the
B S T
Working reagent (μl) 1000 1000 1000
Standard (μl) - 10 -
Test (μl) - - 10
Mix and incubate for 10 min at 37oC. Read the absorbance of standard and sample
against reagent blank at 670nm.
Calculation
Glucose (mg/dL)= Absorbance of Test – Absorbance of Blank xconc. of standard
Note:
This method can be used for serum, plasma or CSF.
Clinical Interpretation
Glucose concentration varies with the type of sample (serum, plasma, blood), as well as
site of blood collection (venous or capillary). Plasma glucose is preferred over serum
glucose. This is because glucose concentration decreases during coagulation process
and must be estimated as soon as possible after the sample has been withdrawn.
i. Fasting whole blood glucose conc. is ~10% to 12% lower than plasma glucose.
ii. Fasting capillary blood glucose conc. is 2-5 mg/dL higher than venous blood.
iii. Glycolysis decreases serum glucose by ~5% to 7% per hour. Separated serum
glucose conc. remains stable for upto 72 hrs at 4 oC. Glycolysis can be inhibited
by adding sodium fluoride (inhibits enolase).
Plasma glucose reference ranges
i. Fasting plasma glucose: < 110 mg/dL

ii. Postprandial plasma glucose: < 140 mg/dL (2 hrs)
iii. Random plasma glucose: <140 mg/Dl
49
Hypoglycemia (reduced blood glucose concentration )
 Plasma Glucose level of < 50 mg/dL is said to be hypoglycemia.
 Common causes include fasting, endocrinological disorders (insulinoma,
hypothyroidism, hypopituitarism, Addison’s disease), enzyme deficiencies etc.
Hyperglycemia (elevated blood glucose concentration)

Common causes include type-1 & type 2 diabetes mellitus, drugs (steroids, beta
blockers), endocrinological disorders (hyperthyroidism, hyperpituitarism, Cushing’s
disease, glucagonoma, and pheochromocytoma).
Diabetes mellitus (DM)

Diabetes mellitus is a metabolic disease due to absolute or relative insulin deficiency.
Types:
 Type 1 DM
 Type 2 DM
 Special cases: Gestational DM, Impaired glucose tolerance (IGT),
Impaired fasting glucose (IFG)
Biochemical investigations in Diabetes

Blood glucose level, lipid profile, kidney functions, glycated hemoglobin, fructosamine.
WHO criteria for the diagnosis of Diabetes Mellitus

 Symptoms of diabetes plus random blood glucose concentration ≥ 200 mg/dL or
 Fasting plasma glucose ≥ 126 mg/dL or
 Two-hour post-load plasma glucose ≥ 200 mg/dL during OGTT
Fasting plasma glucose between 110–125 mg/dL is impaired fasting glucose.

Plasma glucose between 140 and 199 mg/dL, 2 hr after oral glucose load is impaired
glucose tolerance.
(Reference:http://www.who.int/diabetes/publications/Definition%20and%20diagnosis%2
0of%20diabetes_new.pdf)
Oral glucose tolerance test (OGTT)

Indications:
1. Diagnosis of impaired glucose tolerance
2. To diagnose gestational diabetes
3. Evaluation of patient with unexplained nephropathy,neuropathy, retinopathy
with random glucose concentration less than 140mg/dL
4. Population studies for epidemiological data.
Procedure:
Patient should take normal carbohydrate diet for 3 days prior to test. After 10-14 hour
fast, fasting blood sample is collected. 75 g of glucose is given in 250-300 ml of water
(may be flavored).
After 2 hours sample is collected for estimation of blood glucose. (Classic GTT, samples
at 30, 60, and 90 minutes are no longer required).
50
Diagnosis of Gestational Diabetes Mellitus (GDM)
OGTT in pregnancy
 Procedure is same as described for regular GTT. Plasma glucose is measured
hourly for 3 hours.
 At least 2 values must exceed the following target value, to diagnose GDM
Fasting > 95 mg /dL
1 hour > 180 mg/dL
2 hour > 155 mg/dL
3 hour > 140 mg/dL
Glycated hemoglobin (HbA1c)

Hemoglobin becomes glycated by ketoamine reactions between glucose and other
sugars and the free amino groups on α and β chains (HbA 1). The major form of HbA1 is
hemoglobin A1c (HbA1c) where glucose is the carbohydrate moiety. HbA1c comprises 4–
6% of total hemoglobin A1. HbA1c is abnormally elevated in diabetic persons with
chronic hyperglycemia.
Since glycohemoglobins circulate within red blood cells whose life span lasts up
to 120 days, they generally reflect the state of glycemia over the preceding 8–12 weeks.
Any condition that shortens erythrocyte survival or decreases mean erythrocyte age (eg,
recovery from acute blood loss, hemolytic anemia) will falsely lower HbA 1c. Alternative
methods such as fructosamine (see below) should be considered for these patients.
Methods for measuring HbA1c include electrophoresis, cation-exchange
chromatography, boronate affinity chromatography and immunoassays.
Normal range of glycated hemoglobin is 4 – 6 % of total hemoglobin
Serum fructosamine
Fuctosamine is formed by nonenzymatic glycation of serum proteins (predominantly
albumin). Serum fructosamine generally reflects the state of glycemic control for only
the preceding 1–2 weeks. Reductions in serum albumin (eg, nephrotic state or hepatic
disease) will lower the serum fructosamine value.
Normal range of fructosamine in serum is 205 – 285 µmol/L
Note: Fructosamine is not used as a diagnostic test for diabetes.
DATE :
NOTES :
51
IV.2. ORAL GLUCOSE TOLERANCE TEST (OGTT)
In OGTT, a specific load of glucose is administered to the patient and blood samples
are subsequently checked to determine how quickly glucose is cleared from the
circulation.
Indications of OGTT
1. For confirming the diagnosis of diabetes mellitus in patients with impaired fasting
glucose (fasting blood glucose levels between 100-126mg/dL) or impaired
glucose tolerance (post prandial glucose levels between 140-200mg/dL).
2. For diagnosing gestational diabetes mellitus
Patient preparation
1. The patient is instructed not to restrict carbohydrate intake in the days or weeks
before the test.
2. The physical activity should not be restricted in the days before test.
3. The patient is instructed to fast (water is allowed) for 8–12 hours prior to the test.
4. The individual should not eat food, drink tea, coffee or alcohol, or smoke
cigarettes during the test, and should be seated.
Procedure
1. A zero time (baseline) blood sample is drawn.

2. The patient is then given a measured dose of glucose solution to drink within a 5
minute time frame. The dose of glucose administered vary in different clinical set
up. The glucose dose can be 50g in OGCT, 75 g or 100 g in adults, 1.75 g per
kg body weight in children (up to 75g).
3. Blood is drawn at specific intervals (usually at 1, 2 and 3 hours) for the
measurement of glucose. Concomitant urine samples can also be collected to
test for the presence of glucose.
Oral glucose challenge test (OGCT)
The oral glucose challenge test (OGCT) is a modification of OGTT, used to screen
pregnant women for gestational diabetes, at 28 weeks of pregnancy. It can be done at
any time of day, and overnight fasting is not a prerequisite. The test involves
administration of 50g of glucose, with a blood glucose reading after one hour. A blood
glucose value >140mg/dL is an indication for gestational diabetes, which needs to be
confirmed by 100g OGTT.
52
Interpretation of OGTT
1. For the diagnosis of gestational diabetes, two of the following criteria should be
met or exceed, according to Carpenter and Coustan
Fasting : 95mg/dL
1 hour : 180mg/dL
2 hour : 155mg/dL
3 hour : 140mg/dL
2. For the diagnosis of diabetes mellitus: A fasting blood glucose level of >126
mg/dL and a 2 hour post load level of >200mg/dL is indicative of diabetes
mellitus.
3. Renal glycosuria: Here, glucose is excreted in the urine despite normal or low
blood glucose levels. The normal renal threshold for excretion of glucose is
180mg/dL. In renal glycosuria, this threshold is lowered, leading to the
appearance of glucose in urine.
53
IV.3. CASE REPORTS : DIABETES MELLITUS PROFILE/ GLUCOSE
TOLERANCE TEST
QUESTION 1
The following are the plasma glucose report of three different subjects:
 Fasting plasma glucose level of Mr. X on two different occasions was 151 mg/dL
 Random plasma glucose level of Mr.Y on two different occasions was 130 mg/dL
a) Interpret the above values.
b) Who among Mr. X and Mr. Y is diabetic?
c) What would be the status of glycated hemoglobin in them? Justify.
QUESTION 2
A 50 year old male patient presented with fatigue, frequent urination, increased
appetite, recurrent infections and a non-healing ulcer in his right foot.
a) What is the probable diagnosis?
b) What are the relevant investigations?
c) Which is the standard test to assess long term glycemic control?
d) Name some conditions which will interfere with its estimation
QUESTION 3
A 14-year-old girl was admitted to a children's hospital in coma. Her mother stated that
the girl had been in good health until approximately 2 weeks previously, when she
developed a sore throat and moderate fever. She began to complain of undue thirst and
also started to get up several times during the night to urinate. The girl had started to
vomit, had become drowsy and difficult to arouse, and accordingly had been brought to
the emergency department. On examination she was dehydrated, her skin was cold,
she was breathing in a deep sighing manner and her breath had a fruity odour. Her
blood pressure was 90/60 and her pulse rate 115/min. She could not be aroused.
Laboratory Findings : Plasma or serum results:
 Glucose - 500 mg/dL (<200 mg/dL)
 Arterial blood pH - 7.07 (7.35–7.45)
 PCO2 - 27 (32–45 mm Hg)
 Bicarbonate - 6 mmol/L (22–30 mmol/L)
+
 Na - 136 mmol/L (136–146 mmol/L)
 Potassium - 5.5 mmol/L (3.5–5.0 mmol/L)
–
 Cl - 100 mmol/L (102–109 mmol/L)
54
 Anion gap - 35.5 (7–16 mmol/L)
 Osmolality - 325 mOsm/kg (275–295 mOsm/kg)
Urine results:
 Glucose - ++++ (Negative)
 Ketoacids - ++++ (Negative)
b) Why potassium levels are altered in this condition?
c) What are the factors which affect pH in this condition?
d) What is the cause for this condition?
e) What is osmolal gap?
f) Name any other clinical condition associated with ketosis
QUESTION 4
An apparently healthy man on a routine checkup was found to have blood glucose
80mg/dL & urine showed no abnormal constituents. After a heavy breakfast of one and
half hours his blood glucose was 140mg/dL & urine sample at that time was positive for
Benedict’s test.
b) What are normal fasting and post prandial blood glucose levels?
c) What is renal threshold for Glucose?
d) What is alimentary glycosuria?
e) Give some examples for Flat OGTT curve.
QUESTION 5
A pregnant women underwent an oral glucose tolerance test (OGTT) with 100 gram
glucose load at her 26th gestational week. Her Fasting Blood glucose level was 115
mg/dL , 1 hour after meals-180 mg/dL, 2 hour after meals-160 mg/dL and 3 hour after
meals-138 mg/dL.
a) Interpret the OGTT values & give your diagnosis
b) What are the precautions to be taken while performing OGTT ?
c) What are the complications that can be expected in this patient?
55
THYROID FUNCTION TESTS
QUESTION 1
A 27 year old female patient presented with history of lethargy and gain of weight. Her
thyroid profile is as under:
 Serum TSH : 20 mU/mL (Reference range : 0.6-5.0 mU/mL)
 Serum ‘Free T4’ : 0.4 ng/dL (Reference range : 0.89-1.76 ng/dL)
 Serum ‘Free T3’ : 1.5 pg/mL (Reference range : 2.3-4.2 pg/mL)
1. Comment on the thyroid function status. What is the diagnosis?
2. Why is free T4 a more reliable parameter for thyroid function than total T 4?
3. Name one technique used to estimate hormones and state the principle of
estimation
4. Explain the utility of thyroid antibodies in diagnosis.
QUESTION 2
A 25 yr old female presented with history of palpitation and loss of weight. Her thyroid
function report is given below:
 Serum TSH: 0.01 mU/mL (Reference range: 0.35-5.5 mU/mL)
 Serum ‘free T4’: 3.1 ng/dL(Reference range:0.89-1.76 ng/dL)
 Serum ‘free T3’: 6.2 pg/mL (Reference range:2.3-4.2pg/mL)
a) Comment on the thyroid function status. Mention the probable diagnosis.
b) Name any two techniques that can be used to assay thyroid hormone levels
c) Name the enzyme responsible for the conversion of T4 to T3. Which mineral is
required for its activity?
QUESTION 3
A 45 year old lady presented with fatigue, weight gain, menorrhagia, hoarseness of
voice and constipation. On examination, she had bradycardia.
a) What is the most probable diagnosis?
b) Name the investigation which helps in clinching the diagnosis. What is the
methodology used in this investigation?
c) Mention the principle of the above investigation.
d) Which other biochemical parameter will be increased in this condition? Explain

why.
56
V. RESPIRATORY SYSTEM
57
V.1. INTRODUCTION TO ACIDIMETRY, PH INDICATORS, PH
METER AND BUFFERS
Acid–Base reactions and pH
In acid–base reactions, a proton donor (acid) transfers a proton to a proton acceptor
(base). pH is defined as the negative logarithm of the hydrogen ion [H +] concentration in
a solution.
The pH scale is logarithmic: pH = −log [H+]
The pH of pure water is 7. pH indicates the concentration of protons in solution.
Water is subject to a self-ionization process as shown.
H2O H+ + OH−
The dissociation constant, KW, has a value of about 10-14, so in neutral solution of a salt
both the hydrogen ion concentration and hydroxide ion concentration are about 10-7 mol
dm-3. Thus,
pH + pOH = 14
pH = 14 – pOH
At pH = 7.0, [H ] = [OH-], and the aqueous solution is said to be neutral. Pure water is
+
said to be neutral. The pH for pure water at 25 °C (77 °F) is close to 7.0. pH values less
than 7.0 are acidic, while those with a pH value greater than 7.0 are alkaline or basic.
Biological systems operate in a narrow range of pH values. For most enzymes,
extremes of pH lead to irreversible denaturation and loss of biological activity. Thus in
laboratory, control of the pH of various solutions is critical, and the accurate
determination of pH is a routine laboratory operation.
Measurement of pH
pH indicators
An approximate measure of pH may be obtained by using a pH indicator. A pH indicator
is a substance that changes color, around a particular pH value. It is a weak acid or
weak base and the color change occurs at 1 pH unit on either side of its pKa value
(negative log of acid dissociation constant, Ka). For example, the naturally occurring
indicator litmus is red in acidic solution (pH<7) and blue in alkaline (pH>7) solution.
Universal indicator consists of a mixture of indicators such that there is a continuous
color change from about pH 2 to pH 10. Universal indicator paper is a paper that has
been impregnated with universal indicator.
Universal indicator components

Indicator Low pH color Transition pH range High pH color
Thymol blue (first transition) Red 1.2–2.8 Orange
Methyl red Red 4.4–6.2 Yellow
Bromothymol blue Yellow 6.0–7.6 Blue
Thymol blue (second transition) Yellow 8.0–9.6 Blue
Phenolphthalein Colorless 8.3–10.0 Purple
58
pH-meter
The device used to determine pH is called a pH-meter. A pH meter measures
essentially the electro-chemical potential between a liquid of known pH inside the glass
electrode (membrane) and a liquid of unknown pH outside. Because the thin glass bulb
allows mainly the small hydrogen ions to interact with the glass, the glass electrode
(indicator electrode) measures the electro-chemical potential of hydrogen ions or the
potential of hydrogen. To complete the electrical circuit, also a reference electrode is
needed. pH is measured using a setup with two electrodes: the indicator electrode and
the reference electrode. These two electrodes are often combined into one - a
combined electrode.
When the two electrodes are immersed in a solution, a small galvanic current is
established. The potential developed is dependent on both electrodes. Ideal measuring
conditions exist when only the potential of the indicator electrode changes in response
to varying pH, while the potential of the reference electrode remains constant.
The measured voltage can be derived by the Nernst equation as:

E = Ex + 2.303 RT log10 C
nF
E=Total potential developed between the indicator and the reference
electrodes(mV)
Ex = A constant that depends mainly on the reference electrode
R = Gas Constant
T = Absolute Temperature
F = Faraday's constant
n= number of charges on the ion
C= Concentration of the ion
The reference electrode, which traditionally used silver chloride (AgCl) has been
superseded by the calomel (mercurous chloride, HgCl2) electrode which uses mercuric
chloride (HgCl) in a potassium chloride (KCl) solution as a gel.
pH meter :
59
Buffer Solutions
Buffer solutions are solutions which resist changes in pH upon addition of relatively
small quantities of acids or bases. They therefore are used whenever a sensitive solute,
such as a protein, has to be protected from changes in pH during various extraction and
purification processes. Buffer solutions are prepared by mixing a weak acid (or weak
base) with its salts of strong base (or strong acid)
For a weak monoprotic acid HA dissociating in water,

HA + H2O H+ + A-
The acid dissociation constant Ka is given by: [H+] x [A-]
[HA]
Bearing in mind the definition of pH, and defining pK a = - log Ka gives the Henderson-
Hasselbalch equation:
The Henderson-Hasselbalch equation enables us to calculate the pH of a buffer

solution, if we know the relative concentrations of the conjugate acid/base partners and
the pKa of the conjugate acid. Note that when the two species are present at the same
concentration, that is [A-] = [HA], the log term vanishes and pH = pKa.
A buffer system is most effective when the pH of the system is near its pKa value. For
example a buffer that is most effective at a pH of about 4.5 is acetate buffer because
the pKa of acetate buffer falls in that range.
Buffer pKa
Phosphate 6.8
Bicarbonate 6.1
Acetate 4.76
Tris 8.3
For practical purposes, a buffer solution is effective in the range of pH = pKa ± 1.
EXPERIMENT : Measurement of pH
Preparation of buffers :
Acetate buffer (acidic range)
Mix 9.2 ml of 0.1 N acetic acid with 0.8 ml of 0.1 N sodium acetate.
Phosphate buffer (neutral range)

Mix 6 ml of 0.1 M disodium hydrogen phosphate (Na2HPO4) with 4 ml of 0.1 M
potassium dihydrogen phosphate (KH2PO4).
Carbonate buffer (alkaline range)

Mix 1.4 ml of 0.2 M sodium carbonate (Na2CO3) with 1.1 ml of 0.2 M sodium
bicarbonate (NaHCO3) and 7.5 ml water.
Check and record the pH of the above buffers using pH paper, indicator and pH meter.
60
DATE :
NOTES :
61
V.2. ARTERIAL BLOOD GAS ANALYSIS AND ITS
INTERPRETATION
Introduction
Metabolic processes continually produce acids. Hydrogen ion (H+) is especially reactive;
it can attach to negatively charged proteins and, in high concentrations, alter their
overall charge, configuration, and function. To maintain cellular function, the body has to
maintain blood H+ concentration within a narrow range (pH 7.35 to 7.45, where pH =
−log [H+]) and ideally 7.4. Disturbances of these mechanisms can have serious clinical
consequences.
Blood gas analysis

The Arterial Blood Gas (ABG) test measures the dissolved oxygen and carbon dioxide
in the arterial blood and also gives information about the acid-base status. The most
common site of blood collection is the radial artery at the wrist, but sometimes the
femoral artery is used. The syringe used for blood collection is pre-packed with a small
amount of heparin, to prevent coagulation. Care is taken to eliminate visible gas
bubbles, as they can dissolve in the sample and give inaccurate results. The syringe
should be sealed after the sample is drawn.
Blood Gas Analyzer (BGA)

It measures pH and partial pressures of oxygen and carbon dioxide in whole blood.
Standard blood tests can also be performed on arterial blood, such as measuring
glucose, lactate, hemoglobins, bilirubin and electrolytes.
Blood is collected from the patient and introduced into the analyzer. The analyzer
aspirates the blood into a measuring chamber which has ion selective electrodes (ISE
are sensitive only to the analyte of interest). The pH electrode compares a potential
developed at the electrode tip with a reference potential, the resulting voltage is
proportional to the concentration of hydrogen ions, [H+].
The pCO2 electrode is a pH electrode with a Teflon or silicone rubber CO2 semi
permeable membrane covering the tip. CO2 combines with water in the space between
the membrane and the electrode tip to produce free hydrogen ions in proportion to the
partial pressure of CO2. The voltmeter, although actually measuring[H+],is calibrated in
pCO2.
Similarly, in pO2 electrode, the O2 permeates through a polypropylene membrane
and reacts with water in the buffer, producing electric current which is measured by
voltmeter.
The pH is the measurement of free H+ ion concentration in circulating blood.
Blood gas analyzers directly measure pH and PCO2, and the HCO3– value is calculated
from the Henderson–Hasselbalch equation:
pH= 6.1 + log [HCO3-/0.3 x pCO2]
After analysis, the blood is automatically expelled into a waste container and the
sample path is cleaned. Results are displayed and may be printed as well.
62
Clinical interpretation
Normal blood gas analysis

pH 7.35–7.45
PaO2 80–100 mmHg
pCO2 35-45 mmHg
HCO3- 22–28 mEq/L
O2 saturation 96%–100%
Oxyhemoglobin dissociation curve No shift
Types of Acid–Base Disorders

There are two types of acid–base disorders: respiratory and metabolic. Primary
respiratory disorders affect blood acidity by causing changes in pCO 2, and primary
metabolic disorders are caused by disturbances in the HCO 3– concentration. pH is a
function of the ratio of HCO3– (regulated by the kidney) to pCO2 (regulated by the
lungs). The HCO3–/pCO2 relationship is useful in classifying disorders of acid-base
balance. Acidosis is due to gain of acid or loss of alkali; causes may be metabolic (fall in
serum HCO3–) or respiratory (rise in pCO2). Alkalosis is due to loss of acid or addition of
base and is either metabolic (rise in HCO3–) or respiratory (fall in pCO2).
Classification and characteristics of simple acid–base disorders
Acid – base Compensatory

Primary change
disorders response
Metabolic acidosis  HCO3–  pCO2
Metabolic alkalosis  HCO3–  pCO2
Respiratory acidosis  pCO2  HCO3–
Respiratory alkalosis  pCO2  HCO3–
The primary disturbances are usually accompanied by compensatory changes;

however, these changes do not fully compensate for the primary acid–base disorders
even if the disorders are chronic.
In mixed disorders, a combination of primary disturbances is present. Mixed disorders

should be suspected when the change in anion gap is significantly higher or lower than
the change in serum HCO3–.
63
Interpretation of ABG report
1. pH: The first step in an ABG interpretation is to look whether there is acidemia
(<7.35) or alkalemia (>7.45). Acidosis is due to gain of acid or loss of alkali; causes
may be metabolic (fall in serum HCO3–) or respiratory (rise in pCO2).
2. HCO3- levels: Bicarbonate levels <22 mEq/L in presence of acidosis imply metabolic
acidosis. Bicarbonate levels >28 mEq/L in presence of alkalosis imply metabolic
alkalosis.
3. pCO2 levels: pCO2 levels >45 mmHg in presence of acidosis imply respiratory
acidosis. pCO2 levels <35 mmHg in presence of alkalosis imply respiratory alkalosis.
Whenever there is a change in pH, compensation occurs. Respiratory Compensation

occurs in cases of metabolic acid-base disturbance and vice versa. Hence pCO2 &
HCO3- move in the same direction
 If HCO3- decreases (Metabolic acidosis) then pCO2 also decreases (Respiratory
compensation)
 If pCO2 increases (Respiratory acidosis), HCO3- also increases (Metabolic
compensation)
Standard bicarbonate (bicarbonate at normal temperature and pCO2)

Standard bicarbonate is an excellent measurement of the metabolic component. It is
defined as the bicarbonate concentration under standard conditions: pCO 2 = 40 mmHg,
and 37oC, and saturated with oxygen.
Base excess (range: -3 to +3)

The base excess is used for the assessment of the metabolic component of acid-base
disorders, and indicates whether the patient has metabolic acidosis or metabolic
alkalosis. A negative base excess indicates that the patient has metabolic acidosis
(primary or secondary to respiratory alkalosis). A positive base excess indicates that the
patient has metabolic alkalosis (primary or secondary to respiratory acidosis).
Metabolic acidosis
The low HCO3– results from the addition of acids (organic or inorganic) or loss of HCO3–.
Anion gap = Na+ - [Cl- + HCO3-], serum reference range = 7 – 12 mmol/L

This apparent gap is due to unmeasured anions (e.g. proteins, SO42-, HPO42-) that are
present in plasma.
Causes of metabolic acidosis

 High anion gap metabolic acidosis: Methanol or ethanol toxicity, Salicylate
toxicity, Uremia (kidney failure), Keto acidosis, Starvation, Lactic acidosis etc.
 Normal anion gap (hyperchloremic) metabolic acidosis: Renal tubular acidosis,
gastrointestinal fluid loss, severe diarrhea (e.g., cholera), Pancreatitis and
Intestinal fistula.
64
Metabolic alkalosis
It is characterized by a primary increase in serum [HCO3-]. Most cases originate with
volume concentration and loss of acid from the stomach or kidney.
Causes of metabolic alkalosis
 Chloride responsive metabolic alkalosis: Prolonged vomiting or nasogastric
suction (HCl loss), Pyloric and upper duodenal obstruction, Cystic fibrosis and
Diuretic therapy
 Chloride resistant metabolic alkalosis: Mineralocorticoid excess (primary &
secondary hyperaldosteronism), Glucocorticoid excess (Cushing syndrome) etc.
Respiratory alkalosis
Excessive ventilation causes a primary reduction in CO2 which in turn leads to
increased pH.
Causes of respiratory alkalosis
 Non Pulmonary stimulation of respiratory centre: Anxiety, Hysteria, Febrile
states, Meningitis, Encephalitis
 Pulmonary disorders: Pneumonia, Asthma, Pulmonary emboli, Interstitial lung
disease
 Ventilator induced hyperventilation
 Pregnancy
Respiratory acidosis
Respiratory acidosis is characterized by CO2 retention due to ventilatory failure.
Causes of respiratory acidosis
 Conditions that directly depress the respiratory center: Drugs (narcotics etc),
CNS disorders: trauma, tumors and comatose states.
 Conditions that affect the respiratory apparatus: Chronic obstructive pulmonary
diseases (COPD), Emphysema, Pulmonary fibrosis etc.
DATE :
NOTES :
65
VI. CARDIOVASCULAR SYSTEM
66
VI.1. ESTIMATION OF SERUM TOTAL CHOLESTEROL
Introduction
 Major constituents of plasma lipids are cholesterol and triacylglycerols (TAG)
 Cholesterol is synthesized in liver and transported in the form of LDL, HDL and
VLDL cholesterol in blood.
 Cholesterol is necessary for the formation of bile acids, steroid hormones and
vitamin-D.
Methods of estimation:
Non Enzymatic Methods
1. Zak’s method.
2. Liebermann-Burchard’s color reaction
3. Salkowski’s reaction
Enzymatic method
Cholesterol oxidase method (used in automated techniques)
ZAK’S METHOD
Principle
Proteins present in blood are precipitated by ferric chloride (FeCl3) in acetic acid.
Cholesterol present in protein free filtrate reacts with Conc.H2SO4 and undergoes
oxidation to form red-purple complex (Cholestapolyenesulfuricacid) which is read at
540nm with suitable blank and standard.
Reagents
1. FeCl3 reagent: 0.05% FeCl3.6H20 in glacial acetic acid (reagent should be aldehyde
free, tested by Hopkins Cole’s reaction).
2. Cholesterol working standard has 0.04 mg/ml in FeCl3 and acetic acid reagent.
3. Conc. H2SO4 reagent.
Procedure
Preparation of protein free filtrate :
In glass stopper centrifuge tube, add 0.1 ml of serum and 9.9 ml of FeCl3. Mix well and
keep aside for 10 to 15 minutes.
Filter or centrifuge. Transfer the supernatant to another test tube to get a clear protein
free filtrate.
Label 4 test tubes as Blank (B), Standard (S) and Test (T) and add reagents as shown
below.
Blank Standard Test 1 Test 2
FeCl3 reagent 3 ml ---- ---- ----
Cholesterol ---- 3 ml ---- ----
Standard
Protein free filtrate ---- ---- 3 ml 3 ml
Conc. H2SO4 2 ml 2 ml 2 ml 2 ml
67
We are not adding FeCl3 in standard because reagent preparation already has FeCl3 in
it. Mix well and allow it to stand for 20 -30 minutes for color development.
Take the absorbance of standard and test against blank at 540 nm in a colorimeter.
T2 for calculation
Calculation
Serum cholesterol (mg/dL) =
Absorbance of Std – Absorbance of Blank Vol. of sample in filtrate


ESTIMATION OF SERUM CHOLESTEROL BY CHOLESTEROL OXIDASE METHOD
Principle
Following reaction takes place by end point method
Cholesterol ester + H2O esterase cholesterol+ fatty acid

Cholesterol + O2 oxidase cholest-4 en -3 one +H2O2
2 H2O2 + 4aminoantipyrine + phenol peroxidase Quinoneimine dye + 4 H20
Absorbance of Quinoneimine is directly proportional to cholesterol concentration.
Procedure
Blank Standard Test
Working reagent 1000 1000 1000
(μL)
Standard (μL) --- 10 ---
Sample (μL) --- --- 10
Distilled water (μL) 10 --- ---
Mix and incubate for 5 min at 37°C. Read the absorbance at 505 nm.
Calculation
Total cholesterol(mg/dL) = Absorbance of Test x conc. of standard

Absorbance of Std
68
Interpretation of values
Normal Serum total Cholesterol = <200 mg/dL or 5.18 mmol/L
Interpretation of blood cholesterol
Desirable Borderline High

<200mg/dL 200-239mg/dL 240mg/dL
Causes of hypercholesterolemia and hypocholesterolemia

1. Hypercholesterolemia
 Nephrotic syndrome
 Diabetes mellitus
 Obstructive jaundice
 Myxoedema
 Congenital hypercholesterolemias
2. Hypocholesterolemia
 Thyrotoxicosis
 Malabsorption syndrome
 Abetalipoprotenemia
HDL- cholesterol - Normal range : > 40 mg/dL (35-80 mg/dL in males)

> 50 mg/dL (42-88 mg/dL in females)
Interpretation of values
1. Increased risk : < 40 mg/dL (in males)

< 50 mg/dL (in females)
2. Average risk : 40-50 mg/dL
3. Less than average risk : 60 mg/dL
Triacylglycerol (TAG) - Normal range : 60 - 165 mg/dL (in males)

40 - 140 mg/dL (in females)
Interpretation of serum triacylglycerol levels

 Desirable < 150 mg/dL
 Borderline 150-199 mg/dL
 High 200-400 mg/dL
 Very High > 500 mg/dL
69
LDL-cholesterol levels
1. Optimal <100 mg/dL
2. Near or above optimal 100-129 mg/dL
3. Border line 130-159 mg/dL
4. High 160-189 mg/dL
5. Very high >190 mg/dL
Friedwald’s formula can be used for estimation of LDL-cholesterol. There are however
few conditions where this formula is not sensitive. These include
 Excess chylomicrons
 Serum triglycerides >400 mg/dL
VLDL- Cholesterol = TAG/5 (virtually all plasma TAG is carried on VLDL in the fasting
condition)
Friedwald formula
LDL-C = Total cholesterol – (HDL-cholesterol +VLDL-cholesterol)
DATE :
NOTES :
70
VI.2. POINT OF CARE TESTING (POCT)
Point-of-care testing refers to the scope of laboratory tests that are performed where
patient care is delivered. This includes physician office testing as well as various
hospital locations outside the laboratory, such as the emergency department, operating
room, and intensive care unit. POCT brings laboratory testing to the site of the patient
encounter, rather than the traditional practice of obtaining a specimen and sending it to
the laboratory. Currently, more than 111 assays can be performed within this category.
EXAMPLES OF POCT
1. In Clinical Biochemistry-Glucose testing , hemoglobin A1c , creatinine , cardiac
markers, blood gas & electrolyte measurements, pregnancy tests, and intra-
operative immunoassay of parathyroid hormone (PTH) measurements.
2. In Hematology- Coagulation testing using POCT prothrombin time (PT) measure-
ment.
3. In Microbiology: POCT for human immunodeficiency virus (HIV), rapid group A
streptococcal antigen testing , respiratory syncytial viruses and influenza A and B
viruses.
GLUCOSE TESTING - GLUCOMETERS

Glucometers are the most frequently used POCT device. It can be used for quantitative
measurement of glucose in fresh capillary, venous, arterial whole blood sample. They
are not used for diagnosis of diabetes mellitus, but are used as an aid in monitoring the
effectiveness of blood glucose control regimens. Glucometers are biosensors as they
use enzymes as recognition agents- either glucokinase, glucose dehydrogenase, or
hexokinase with photometric or electrochemical detection. The sample is placed directly
on the test pad. Calibration may be performed automatically on some instruments or
performed with code test strips. Reportable range varies widely from instrument to
instrument.
The Reaction Principle

Glucose meters use the same chemical reactions that are used in glucose analysis in
the laboratory: glucose oxidase, hexokinase, and dehydrogenase. Most systems use
dehydrated reagents embedded in pads on plastic strips. The strip is inserted in the
meter, where the reaction is measured. The reaction may be a color change that is
measured by reflectance spectrophotometry, or the reaction may produce a change in
current that can be measured by electrochemistry.
Clinical utility of glucometer

Using glucometers, patients measure their own blood glucose concentration and modify
their insulin dose based on this glucose value (Self-monitoring of blood glucose). It
permits rapid and reasonably accurate measurements on a drop of whole blood.
71
VI.3.CASE REPORTS : CARDIAC PROFILE
QUESTION 1
A patient presented with severe chest pain which was radiating to his left arm. He was
diagnosed to have myocardial infarction.
Random blood glucose = 280 mg/dL (70-140 mg/dL)
Serum total cholesterol = 310 mg/dL ( <200 mg/dL)
Serum triglycerides = 180 mg/dL (<150 mg/dL)
CK- Total = 66 IU/L (20-170 IU/L)
CK-MB = 5 IU/L (< 6 IU/L)
Troponin I = 10 ng/ml (0-0.4 ng/ml)
Reference Ranges are given in the bracket.
a) Which is the first enzyme to be elevated in this condition?
b) Which enzyme can be measured for repeated Myocardial Infarction?
c) Name non enzymatic substances which can be measured in Myocardial

Infarction.
d) What is the reason for normal creatine kinase levels in this patient?
e) What is CK – MB index?
QUESTION 2
A50 year old obese man was brought to casualty with acute chest pain radiating to his
left arm of 3 hours duration. He is known diabetic patient and chronic smoker. The ECG
revealed ST segment elevation.
a) What are the biochemical tests to be done immediately to establish the
diagnosis?
b) What other parameters will you estimate in this patient?
c) Which marker will help you to diagnose reinfarction in this patient?
d) What is the dietary advice to be given to this patient?
e) What is ‘Flipped Pattern’?
72
LIPID PROFILE
QUESTION 1
A 48yr old male subject had following biochemical report:
 Serum cholesterol: 280 mg/dL
 Serum HDL-cholesterol: 28 mg/dL
 Serum Triacylglycerol: 200 mg/dL
a) Calculate his LDL-cholesterol level. What is the name of this formula?
b) Name two tests for detection of cholesterol.
c) Name any two drugs used therapeutically to lower the cholesterol levels.
QUESTION 2
A 5 year old boy presented with onset of jaundice since birth and multiple xanthomas
covering trunk and limbs. His total cholesterol level was 590 mg/dL and triglycerides
171 mg/dL. Echocardiography revealed mild aortic stenosis as a result of premature
atherosclerosis.
b) What are the consequences of increased cholesterol?
c) How can you manage this condition?
d) Which is the commonly recognized disorder of lipoprotein metabolism in

childhood?
QUESTION 3
A thirty year old male patient comes for a routine checkup. 5 ml venous blood was
collected for routine investigations. On separation, serum was opaque and milky in
appearance. Serum was kept at 4°C overnight and on the next day, white precipitates
were observed on the top of the tube. Lipid profile showed elevated triglyceride levels.
a) Name the enzyme defective in this case.
b) Which mucopolysaccharide acts as a anchor for this enzyme in capillaries of

organs ?
c) What is the effect of insulin on this enzyme ?
73
VII. GASTROINTESTINAL AND
HEPATOBILIARY SYSTEM
74
HEPATO-BILIARY FUNCTION TESTS
Introduction
Hepatobiliary function tests are biochemical investigations that are useful in detection
and diagnosis of liver and gall bladder dysfunction. They are also helpful in assessment
of disease severity prognosis and therapeutic monitoring of liver disease. The
commonly used biochemical parameters for this purpose are :
1. Bilirubins
2. Bilirubin fractionation
3. Total protein and Albumin
4. Alanine aminotransferase
5. Aspartate aminotransferase
6. Alkaline Phosphatase
7. Prothrombin time
VII.1. ESTIMATION OF BILIRUBIN IN SERUM
‘MALLOY AND EVELYN’ METHOD
Principle
The principle of this method is based on vandenberg reaction. In this reaction,
diazotized sulfanilic acid reacts with bilirubin to produce two Azodipyrroles.
They are as follows
 Direct bilirubin (conjugated) ( pH 7.4)

Serum + diazotized sulfanilic acid purple color
(azobilirubin)
 Total bilirubin ( pH 7.4 )

Serum + diazotized sulfanilic acid + accelerator purple color
(Incubate for 10 min)
 Indirect bilirubin (unconjugated) = Total – Direct bilirubin.
Reagents
1. Diazo reagent: Prepare freshly by mixing 0.3 ml of solution B with 10 ml of
solution A.
2. Solution A: Dissolve 1 gm of sulfanilic acid in 15 ml of conc.HCL and make upto
1 litre with water.
3. Solution B: Dissolve 0.5 gm of sodium nitrite in water and make up to 100 ml.
4. Diazo blank: 1.5% HCl
5. Bilirubin standard: 10 mg in 100 ml chloroform
75
Procedure
The following reagents were added to respective tubes as follows:

Reagents Std Standard Serum Total Total Direct Direct
blank Blank (Test 1) (Test 2) (Test 1) (Test 2)
Standard 0.2 0.2 ---- ---- ---- ---- ----
bilirubin (ml)
Serum (ml) ---- ---- 0.2 0.2 0.2 0.2 0.2
Methanol (ml) 4.3 4.3 ---- 2.5 2.5 ---- ----
Diazo reagent ---- 0.5 ---- 0.5 0.5 0.5 0.5
(ml)
Distilled water ---- ---- 4.3 1.8 1.8 4.3 4.3
(ml)
Diazo blank 0.5 ---- 0.5 ---- ---- ---- ----
(ml)
Mix and allow to stand for 30 minutes in dark. Compare the colors at 540nm.
Calculation
Total bilirubin/ Direct bilirubin (in mg/dL) =
= Absorbance of Test – Absorbance of Blank x Amt.of standard x 100
Absorbance of Std – Absorbance of Blank Vol of sample


BILIRUBIN ESTIMATION USING DIMETHYL SULFOXIDE (DMSO) ACCELERATOR
Principle
The azobilirubin produced by the reaction between bilirubin and the diazonium salt of
sulfanilic acid shows maximum absorption at 555nm in an acidic medium. The intensity
of the color produced is proportional to the quantity of bilirubin which has reacted. In the
absence of accelerator, only conjugated bilirubin reacts. In the presence of accelerator
(DMSO) non conjugated bilirubin also participates in the reaction.
Reagents
1. Bilirubin direct: Sulfanilic acid and hydrochloric acid
2. Bilirubin total: Sulfanilic acid, hydrochloric acid & dimethyl sulfoxide (DMSO)
3. Bilirubin serum blank: hydrochloric acid and sodium chloride
4. Sodium nitrite
5. Standard (0.04 mg/dL)
76
Procedure
The following reagents were added to respective tubes as follows:
Serum Blank Standard Test
Working reagent 1000 1000
(µl)
Bilirubin serum 1000 ----- -----
blank (µl)
Sample (µl) 50 ----- 50
Standard (µl) ------- 50 -------
Take absorbance at 546 nm. Take reading immediately for direct and after 10 min for
total.
Calculation
Bilirubin (mg/dL) = Absorbance of Test – Absorbance of Blank xconc. of standard
Reference range
Serum bilirubin: Total= <1 mg/dL
Direct =0.1–0.3 mg/dL
Indirect =0.2–0.7 mg/dL
Laboratory results in three different causes of jaundice

Condition Serum bilirubin Urine Urine bile salts & Fecal
urobilinogen bile pigments urobilinogen
(absent normally)
Hemolytic Increased indirect
Increased Absent Increased
jaundice bilirubin
Hepatocellular Increased direct & Decreased Present
Decreased
jaundice indirect bilirubin (if cholestasis) (if cholestasis)
Obstructive Increased direct Trace to
Absent Present
jaundice bilirubin absent
DATE :
NOTES :
77
VII.2. CASE REPORTS : HEPATOBILIARY FUNCTION TESTS
QUESTION 1
A 50 year old man was admitted in the hospital with complaints of loss of appetite and
itching. Following are the findings:
Blood examination
 Serum total bilirubin-------------------14 mg/dL (Reference range: 0.8 – 1.2 mg/dL)
 Serum conjugated bilirubin-----------13.2 mg/dL (Reference range: 0-0.2 mg/dL)
 Serum unconjugated bilirubin--------0.8 mg/dL (Reference range: 0.4-0.7 mg/dL)
 Serum AST-----------------------------40 IU/L (Reference range: 5-40 IU/L)
 Serum ALT---------------------------- 30 IU/L (Reference range: 5-45 IU/L)
 Serum alkaline phosphatase-------- 423 IU/L (Reference range: 80-120 IU/L)
Urine examination
 Bile salts-------------------------------+ +
 Bile pigments-----------------------+ +
 Urobilinogen-------------------------nil
a) Mention the type of jaundice that the patient is suffering from?
b) Why urobilinogen is absent in urine in this condition?
c) Which two other enzymes do you estimate in serum to confirm your diagnosis?
d) Name any two causes that can lead to this condition.
QUESTION 2
A newborn developed yellowish discoloration of the skin on Day 1. The blood chemistry
report of the newborn is as follows:
Blood examination
Serum total bilirubin------14 mg/dL (Reference range: 0.8 – 1.2 mg/dL)
Serum conjugated bilirubin------0.2 mg/dL (Reference range: 0 – 0.2 mg/dL)
Serum unconjugated bilirubin-----13.8 mg/dL (Reference range: 0.4 – 0.7 mg/dL)
a) Mention the type of jaundice that the newborn is suffering from.
b) Name two causes of this type of jaundice.
c) What would be the result of ‘Vandenberg Test’ in this case?
d) What is the role of methanol in Vandenberg test?
78
QUESTION 3
A teenager was brought to the clinic with complaints of nausea and vomiting, and
abdominal discomfort for the past one week. He had yellowish discoloration of skin,
sclera and mucous membrane and liver was palpable below the costal margin. He was
also passing dark urine. The serum and urine collected from the patient was deep
yellow. The biochemical report was as under :
 Total bilirubin : 15 mg/dL
 Direct bilirubin : 4 mg/dL
 Serum AST : 380 U/L
 Serum ALT : 800U/L
 Serum albumin : 2.8g/dl
 Urine bilirubin: 3+
a) What is the diagnosis? Justify .
b) Explain the reason for low albumin levels.
c) What is Van den berg test?
QUESTION 4
A 20 year old man came to a hospital with complaints of passing red colored urine and
pain abdomen. History of the patient revealed that he had undergone antimalarial
treatment one week before and had also taken primaquin the night before, as per the
advice of treating physician so as to prevent contracting malaria again. Blood reports
showed the following details :
 Total bilirubin -15mg/dL
 Direct bilirubin-1 mg/dL
 ALT-50 U/L
 Haptoglobulin – 10mg/dL (30-200 mg/dL)
a) What type of jaundice does this patient have ?
b) What is the most probable cause of these symptoms?
c) Explain the decreased level of haptoglobin in this patient.
79
VIII. RENAL SYSTEM
80
VIII.1. URINALYSIS
Introduction
Urine is a metabolic waste product, secreted by the kidneys. It is an amber colored
liquid. It consists of approximately 95% water, with the remaining being metabolic waste
products, which includes various inorganic, organic compounds.
Physical properties of urine

Following physical properties are discussed below:
 Volume
 Color
 Odor
 Specific gravity
 pH
1. Urine volume:
In normal adult urine volume is 1.2 to 1.5 L/day
Polyuria: Urine output > 3L/day. It is seen in diabetes mellitus, diabetes
Insipidus, end-stage renal disease etc.
Oliguria: Urine output < 500ml/day. It is seen in condition associated with
dehydration.
Anuria: Complete absence of urine formation usually or < 50ml /day. It is seen
in acute renal failure.
Nocturia: Excess excretion of urine at night. It is seen in patients with
prostatic enlargement, diabetes etc
2. Urine color:
Normal color of urine is amber, caused by urochrome pigment.
Dark colored urine is seen in condition associated with dehydration like
diarrhea , dysentery etc
Dark yellow colored urine is seen in obstructive jaundice
Red colored urine is seen in condition associated with hemoglobinuria,
hematuria etc
Black colored urine is seen in alkaptonuria
Reddish or brown colored urine is seen in porphyrias
3. Urine odor:
Faintly aromatic odor of urine is normal.
Long standing urine without any added preservative gives ammoniacal odor,
due to bacterial action which converts urea to ammonia.
Fruity odor – Diabetic ketoacidosis
Specific odor :
i. Mousy odor is associated with Phenylketonuria
ii. Burnt sugar odor is associated with Maple syrup urine disease etc.
81
4. Specific Gravity:
Specific Gravity of urine determines the concentrating ability of the kidneys. Normal
specific gravity of random urine sample ranges from 1.005 - 1.030. Its value varies in
random (spot) samples.
Minimum specific gravity after a standard water load should be > 1.003 in normal
individuals. Accepted range of urine specific gravity with adequate fluid intake is 1.016 –
1.022 over a 24 hour period.
The specific gravity of urine is:
Increased in diabetes mellitus, dehydration, and Syndrome of Inappropriate ADH
secretion (SIADH).
Decreased in diabetes insipidus, acute tubular necrosis, and psychogenic
polydipsia.
Fixed specific gravity of 1.010 seen in chronic renal failure.
Long’s coefficient refers to ‘total solids excreted in the urine’. The solid content of
1000ml of urine is calculated by multiplying the last 2 digits of specific gravity at 25oC by
2.6 and is expressed in gm/L.
Measuring Specific Gravity
Urinometer
Urinometer uses a glass float weighted with mercury and a stem with calibrations to
measure the buoyancy.
Fill up the 3/4th of the cylinder with the urine and gently float the urinometer in it. The
urinometer should not touch the sides or bottom of the cylinder. Note the reading which
gives the specific gravity of the urine at the temperature at which urinometer is
calibrated (15oC). Measure the room temperature. A value of 0.001 is added or
subtracted for every 3oC above or below15oC.
Limitations of urinometer relate to the fact that it requires large volume of urine. Also
the temperature corrections must be made on the readings. Corrections also are
recommended when glucose or higher amounts of proteins are present in the urine
sample.
5. pH of urine:
Normal pH range of urine is 4.8 – 8 with mean of 6.
High protein diet decreases pH of the urine.
Pure vegetarian diet increases alkalinity of urine.
Dip stick method for measuring pH of urine

Test pad is impregnated with the indicators, e.g. mixture of methyl red and bromothymol
blue. Methyl red is red at 4.2 and yellow above 6.2. Color change is appreciated exactly
at 60 sec and then it will be compared with color chart.
82
NORMAL CONSTITUENTS OF URINE
Normal constituents of urine
These include inorganic constituents e.g. calcium, phosphate, chloride and sulfur as
well as organic constituents e.g. urea, uric acid, ammonia, creatinine (commonly known
as non-protein nitrogenous substances) and urobilinogen.
1. TEST FOR CALCIUM AND PHOSPHATE

Principle
Calcium phosphate is precipitated in the alkaline medium. Calcium forms calcium
oxalate with potassium oxalate.
Phosphate in the urine reacts with ammonium molybdate to give yellow colored
ammonium phosphomolybdate.
Reagents
1. Conc. ammonia
2. Dilute acetic acid
3. Conc. nitric acid
4. Ammonium molybdate solution
5. Potassium oxalate solution

Take 5 ml of urine in a test
tube and add 1 ml of conc. Precipitate is Calcium phosphate
ammonia boil and then cool it. formed is precipitated.
Now Filter the above solution:
Wash the precipitate with
water and Divide the solution Presence of
into two. Canary yellow color phosphate
(a) To one portion, add 1 ml of
conc. nitric acid and 5 ml of
ammonium molybdate solution
and boil. White precipitate Presence of calcium
(b) To the other portion add 5
ml of 2% potassium oxalate
solution.
2. TEST FOR CHLORIDES
Principle
Chloride ion in urine reacts with silver nitrate to form silver chloride.
Conc. nitric acid is added to suppress the precipitation formed by phosphates and
urates.
83
Reagents
2. Silver nitrate solution
3. Urine sample

Take 5 ml of urine in a A white precipitate Presence of
test tube and add 1 ml of chlorides
conc. nitric acid and 2 ml
of 3% silver nitrate
solution.
3. TEST FOR SULFATES

Principle
(a) Inorganic sulfate reacts with barium chloride to form barium sulfate which is white
precipitate. Conc. HCl is added to suppress the precipitation formed by phosphates.
(b) Excess acid present in the solution hydrolyses ethereal sulfate to release sulfate
which forms barium sulfate with excess barium chloride.
Reagents
1. Conc. HCl
2. Barium chloride solution
3. Urine sample

(a) To 5 ml of urine, add A white precipitate Presence of
1ml of conc. HCl and 5 ml inorganic sulfates
of 10% barium chloride
solution.
(b) Filter. Boil the filtrate. Precipitate is formed Presence of ethereal
sulfate
4. TESTS FOR UREA

(a) HYPOBROMITE TEST
Principle
Sodium hypobromite splits urea into CO2 and N2. Carbon dioxide is converted into
bicarbonate and nitrogen is released as gas.
Reagents
1. Sodium hypobromite solution
2. Urine sample

Take 5 ml of urine in a test Effervescence Presence of urea
tube and add 1 ml of
alkaline sodium
hypobromite solution.
84
(b) SPECIFIC UREASE TEST:
Principle
Ammonia is liberated from urea present in urine by the action of urease, making the pH
alkaline.
The color of phenol red changes to red in alkaline pH.
Reagents
1. Phenol red
2. Horse gram powder

Take 5 ml of urine in a test tube and
add 2 drops of phenol red. In another
test tube take a spatula full of horse
gram powder (which contains the
enzyme urease) and add 5 ml of water Red color Presence of urea
and two drops of phenol red. Now,
adjust the pH to 7 (at which phenol red
is orange) by adding 2% acetic acid or
2% sodium carbonate. Mix the contents
of the both the tubes. Wait for 10
minutes. Note the color. Warm slightly if
necessary.
5. TEST FOR URIC ACID

(a) BENEDICT’S TEST
Principle
Uric acid acts as a reducing agent and reduces sodium tungstate to blue colored
tungsten blue in alkaline medium. Conc. HCl is added to suppress the precipitation of
phosphates and urates.
Reagents
1. Benedict’s uric acid reagent consists of a mixture of sodium tungstate, arsenic
acid, phosphoric acid and conc. HCl.
2. 20% sodium carbonate solution

Take 3 ml of urine in a test
tube and add 1 ml of Blue color Presence of uric acid
Benedict’s uric acid reagent
then add 1 ml of 20% sodium
carbonate solution.
Note: Don’t mouthpipette the benedict’s uric acid reagent as it has a virulent poison.
(b) SCHIFF’S TEST

Principle
In alkaline medium, uric acid reduces silver nitrate to silver which gives black stain.
85
Reagents
1. 2% sodium carbonate solution
2. Silver nitrate solution

Take 1 ml of urine in a test tube and add 2 Black stain Presence of uric
ml of 2% sodium carbonate solution. Pour on the filter acid
this mixture over the filter paper paper
moistened with silver nitrate solution.
6. TEST FOR AMMONIA

Principle:
Heating in alkaline medium converts ammonium ion present in urine to ammonia. The
ammonia so formed turns the phenolphthalein indicator pink.
Reagents:
1. Phenolphthalein
2. 2% sodium carbonate (Na2CO3)

To 5 ml of urine add a drop of
phenolphthalein and then add a few
drops of 2% Na2CO3 till the solution is Glass rod Presence of
faintly pink. Boil the test tube and hold turns pink ammonia
the glass rod dipped in phenolphthalein
at the mouth of the test-tube
7. JAFFE’S TEST
This is the test to detect creatinine in urine.
Principle:
Creatinine reacts with alkaline picrate to form creatinine picrate complex.
Reagents:
1. Saturated solution of picric acid
2. 10% sodium hydroxide

Take 5 ml of urine in a test tube and 5 ml Orange color Presence of
of water as control in another test tube. in test creatinine
Add 1 ml of saturated solution of picric
acid and 10 drops of 10% NaOH to both
the tubes.
8. EHRLICH’S TEST (MODIFIED WATSON & SCHWARTZ TEST)

This is the test for detection of urobilinogen in urine.
86
Principle:
Para-dimethyl amino benzaldehyde in conjunction with a color enhancer (saturated
sodium acetate) reacts with urobilinogen in a strongly acidic medium to produce a
pinkish-red color.
Reagents:
1. Ehrlich reagent (mixture of para-dimethyl amino benzaldehyde and conc. HCl)
2. Saturated sodium acetate
3. Chloroform

To 5 ml of urine add 5 ml of Chloroform layer becomes Presence of
Ehrlich Reagent. Allow it to pink/red (cherry red color may urobilinogen
stand for 10 minutes. Add 5 be seen in hemolytic jaundice)
ml saturated sodium acetate
and mix well. Later add 5 ml Aqueous layer is pink Presence of
of chloroform and mix. porphobilinogen
Reference ranges for normal constituents of urine

 Calcium: 0.1 - 0.3 g/day
 Chloride: 110 - 250 mEq/day
 Phosphate: 0.4 - 1.3 gm/day
 Sulfate (inorganic): 40 - 120 mEq/day
 Sodium: 20 - 220 mEq/day
 Potassium: 25 - 125 mEq/day
 Magnesium: 6 - 10 mEq/L
 Urea nitrogen : 10 - 20 gm/day
 Creatinine:
Male – 14 - 26 mg/kg/day
Female – 11 - 20 mg/kg/day
 Ammonia: 30 - 75 mEq/day
 Uric acid: 199 - 1094 mmol/mol creatinine
 Urobilinogen: Present normally and increased in hemolytic jaundice. It is absent
in obstructive jaundice.
Osmolality
Osmolality = 2 x Na+ + urea (mg/dL) + glucose (mg/dL)
6 18
+
Na is the major determinant of urine osmolality.
Urine osmolality: Reference range : 50 - 1200 mOsmol/kg water
Urine osmolality differentiates renal from non-renal water loss seen in hypernatremia.
 Urine osmolality >400 mOsm/kg implies normal renal water-conserving ability
 Urine osmolality <250 mOsm/kg is characteristic of central and nephrogenic
diabetes insipidus.
High urine osmolality in presence of hyponatremia suggests SIADH.
87
DATE :
NOTES :
88
VIII.2. ABNORMAL CONSTITUENTS OF URINE
Abnormal constituents in urine refer to the substances excreted in appreciable quantity
& can be detected by common laboratory tests. Urinalysis is non-invasive, inexpensive
and a quick procedure which can be used to screen many diseases. It gives an idea for
further management of the disease in clinical practice. It is also very easy and safe
method for self monitoring by the patients who are on long term treatment. The common
abnormal constituents in urine include glucose, protein, ketone bodies, blood, bile salts
and bile pigments.
TESTS FOR DETECTION OF PROTEINS IN URINE

Normal individuals excrete less than 150 mg/day of proteins which includes Tamm-
Horsfall protein, IgA; urokinase etc. Ideally 24 hour urine sample is preferred for
detecting Proteinuria.
Microalbuminuria (Paucialbuminuria)
This is a condition where there is loss of 30 to 300 mg of albumin per day. It is an early
marker of kidney disease.
QUALITATIVE TESTS FOR PROTEINURIA
1. HEAT COAGULATION TEST

It is regarded as bed side screening test. Hemoglobin, myoglobin, etc., are not
precipitated by heat coagulation.
Principle:
Heating causes denaturation of proteins which forms stable metaproteins.
Acetic acid will dissolve the precipitate formed by phosphates
Reagents:
1. 2% acetic acid

Take ¾th of a test tubeA cloudy white precipitate is Presence of
with urine. Heat only the
seen in the heated portion proteins/phosphates
upper 1/3rd portion of the
compared to lower portion
tube. which acts as the control
The precipitate remains Presence of
Now add a few drops of proteins
2% acetic acid
Note: The urine protein heat coagulation test remains the most commonly available
screening test for proteinuria in pregnancy in many parts of the developing world.
Results are reported as +1, +2, +3 and +4 based on the amount of turbidity present.
A proteinuria of ≥+1 on heat and coagulation test is very sensitive in detecting a
proteinuria of ≥500 mg/day (which refers to a proteinuria of ≥+2 on the dipstick test (dry
chemistry).
89
Interpretation of the heat coagulation test results
Negative No or mild turbidity observed. When the urine column in the tube is placed
/Trace in front of a typed sheet of paper, printed letters can be clearly read
through the tube.
+1 Definite turbidity observed. Printed letters can be clearly read through
tube.
+2 Definite turbidity observed. Printed letters cannot be read clearly through
the tube.
+3 Definite turbidity observed. Nothing can be observed through the tube.
+4 Protein clots are seen in the tube
2. HELLER’S TEST
Principle:
Acid denaturation of proteins.
Reagent:

To 3 ml conc. nitric acid in a
test tube add 2 ml of urine White ring will be seen at Presence of
along the sides of the test the junction of 2 fluids. proteins
tube.
Note: This is not a reliable test, although it is very sensitive. If urine has a high
concentration of urea, then urea nitrate complex will be formed at the junction of two
fluids giving a false positive test.
3. SULFOSALICYLIC ACID TEST

It is a very sensitive test to detect even small amount of protein in the urine, but false
positive results may be seen if the urine contains radiographic dyes, penicillin,
cephalosporins, sulfonamides, hemoglobin, myoglobin etc.
Principle:
Sulfosalicylic acid is an alkaloidal agent that neutralizes the charge on proteins and
produce precipitation
Reagent:
1. 20% sulfosalicylic acid

To 2 ml of urine, add few
Presence of
drops of 20% sulfosalicylic White precipitate
proteins
acid.
90
Quantitative measurement of urinary proteins include:
 Esbach’s albuminometer
 Lowry method
 Measurement of turbidity after mixing with trichloroacetic or sulfosalicylic acid
 Dye binding with Coomassie Brilliant blue and Pyrogallol red molybdate
Measurement of individual proteins

Immunoassay is the preferred method for the accurate & sensitive quantitation of
individual proteins
Clinical significance of proteinuria

 Screening for microalbuminuria should commence 5 yrs after the diagnosis of
Type 1 diabetes mellitus (DM)
 Screening for protenuria is adviced at the time of diagnosis of Type 2 DM.
 Proteinuria is a potent marker for severity and progression of diseases like
Preeclampsia, diabetes mellitus, hypertension and kidney diseases.
Note:
Strict control of diabetes should be achieved before investigating for
microalbuminuria & patients should not be screened during intercurrent illness.
Nephrotic syndrome is a clinical complex characterized by:

 Proteinuria >3.5 gms/1.73m2per 24 hrs
 Edema
 Hypoalbuminemia
 Hyperlipidemia
 Hypercoagulability
Common causes of Nephrotic syndrome include;
 Minimal change disease (most common cause in children)
 Diabetic nephropathy (most common cause in adults)
 Amyloidosis
 Systemic lupus erythematoses, & drugs like NSAIDs
Glomerulonephritis is characterized by:

 Hematuria
 Dysmorphic red cells, red cell casts
 Mild proteinuria (usually < 2 g/day)
 Dependent edema and hypertension.
 Acute renal insufficiency
Types of proteinuria
 Glomerular proteinuria
This type of proteinuria seen in any disease which causes damage to glomerular
basement membrane resulting in loss of high molecular weight proteins (IgG) along
91
with albumin. Generally proteinuria will be > 1gm/day. Eg: Diabetic nephropathy,
Immune complex diseases like systemic lupus erythematosis, etc.
 Tubular proteinuria
This type of proteinuria occurs as a result of decreased tubular reabsorptive capacity
and/or tubular damage, caused by nephrotoxic drugs, heavy metals or anoxia. In this
condition proteinuria usually will be less than 1gm/day, which includes lower
molecular weight proteins like retinol binding protein, beta-2 microglobulins,
enzymes like alkaline phosphatase, N –acetyl-glucosaminidase.
 Overflow proteinuria:
This type of proteinuria is due to increased blood concentration of freely filtered
proteins. Eg: Bence Jones protein (in Multiple Myeloma), Lysozyme, myoglobin.
 Functional Proteinuria:
This type of proteinuria occur in conditions like fever, post-exercise and postural
variation (orthostatic) and uncontrolled hypertension.
TESTS FOR DETECTION OF REDUCING SUBSTANCES IN URINE

4. BENEDICT’S TEST
For procedure refer to general reactions of carbohydrates
Reducing substances in urine
Carbohydrates Non-carbohydrates
Uric acid
Glucose Ascorbic acid
Fructose Homogentisic acid
Galactose Hippuric acid
Lactose Oxalic acid
Maltose Glucuronic acid
Arabinose Isoniazid
Xylose Salicylates
Ribose Creatinine
Cysteine
Clinical significance:
Merits & demerits of urine glucose testing
Normal excretion of glucose in urine varies from 1 to 15 mg/dL (0.1-0.8 mmol/L).
Examination of urine for glucose is rapid, non-invasive & inexpensive, which can only be
used for screening purpose.
It should not be used to diagnose diabetes mellitus and also to monitor the patients on
insulin therapy because of the following reasons:
1. Urine testing is not accurate.
2. Renal threshold varies widely among individuals.
92
3. Many factors like fluid intake, infection, ingestion of salicylates, ascorbic acid etc.,
influence the testing.
4. A negative result will not distinguish between hypoglycemia, euglycemia, or
hyperglycemia.
Renal glycosuria
Renal threshold for glucose can be defined as the “Blood glucose concentration above
which glucose appears in urine”. It varies from 160-180 mg/dL, lower in pregnancy and
childhood.
A decreased renal threshold for glucose is known as renal glycosuria.
TESTS FOR DETECTION OF KETONE BODIES IN URINE

Ketone bodies can be detected in urine (ketonuria) in conditions where there is
excessive formation and accumulation of ketone bodies in blood (ketonemia).
5. ROTHERA’S TEST (for acetone and acetoacetic acid)

Principle:
Acetone and acetoacetic acid react with sodium nitroprusside in the presence of alkali
to give a purple or orange color respectively.
Reagents:
Solid ammonium sulfate
Sodium nitroprusside
Ammonia solution

Saturate 5 ml of urine with Purple ring at the Presence of acetone
solid ammonium sulfate. Add junction of 2 liquids
3 drops of sodium
nitroprusside (2% solution) Orange ring at the Presence of acetoacetic
and mix well. Then add 2 ml junction of 2 liquids acid
of strong ammonia solution
slowly along the sides of the
tube.
Note: If purple ring is obtained, boil the urine and repeat the test. Absence of purple ring
in boiled sample of urine confirms the presence of acetone (acetone is volatile).
6. GERHARDT’S TEST (for acetoacetate)

Principle:
Acetoacetate reacts with ferric chloride to give a red color.
Reagents:
Ferric chloride
93
To 5 ml of urine, add 10 Purple/port-wine Presence of acetoacetate
drops of 10% ferric color is observed confirmed.
chloride to get maximum
precipitate of ferric
phosphate. Filter and
remove the precipitate. To
the filtrate, add excess of
ferric chloride. On boiling, acetoacetate
No color is present looses carbon dioxide and
Boil the urine thoroughly acetone is formed which will
and repeat the test not answer the test. But if
salicylates are present they
will answer the test even
after boiling.
Points to note:
Rothera’s test is much more sensitive than Gerhardt’s test.
Rothera’s test is not given by salicylates.
Gerhardt’s test can be used to differentiate between salicylates and acetoacetate in
urine.
Gerhardt’s ferric chloride test is for acetoacetate only.
Tests using nitroprusside are at least 10 times more sensitive to acetoacetate than
acetone and give no reaction with β-hydoxybutyrate.
Causes of ketoacidosis
Diabetes mellitus and alcohol consumption are the most common causes in adults.
Decreased availability of carbohydrates as in starvation or frequent vomiting.
Decreased use of carbohydrates as in diabetes mellitus, glycogen storage disease
(Von Gierke’s disease).
Inborn errors of aminoacid metabolism e.g. phenylketonuria, maple syrup urine
disease.
Other causes include isopropyl alcohol and salicylate poisoning.
Urine ketone testing is an important part of monitoring patients with diabetes mellitus
(particularly type I), pregnancy with pre-existing diabetes and gestational diabetes
mellitus.
DETECTION OF BILE SALTS & PIGMENTS IN URINE
Jaundice is a yellowish discoloration of the skin, the conjunctival membranes and other
mucous membranes caused by hyperbilirubinemia. Jaundice is seen when the serum
bilirubin level is >1.5 mg/dL. Choluric jaundice refers to presence of bile pigments in
urine. Only conjugated or water-soluble bilirubin seen in obstructive jaundice
94
(Regurgitation jaundice) can be excreted in urine. Acholuric jaundice is seen in
unconjugated hyperbilirubinemia (as in hemolytic jaundice). Urine will not test positive
for bile pigments.
7. HAY’S TEST FOR BILE SALTS

Principle:
This test is based on the principle that bile salts have the property of lowering surface
tension.
Reagents:
Sulfur powder

Take 5 ml of urine and 5 ml of distilled Sulfur powder sinks to Presence of
water (control) in two tubes labeled T the bottom of the tube bile salts.
and S. Sprinkle little quantity of sulfur containing urine, but it
powder over the surface of solution in floats in the control.
each tube.
NOTE:
Test tubes should be free of soap/detergent since they give false positive test.
8. PETTENKOFER’S TEST
To 5 ml of urine, 5 drops of 5% sucrose solution is added. The tube is kept in an
inclined position, and 2-3 ml of concentrated sulfuric acid is poured along the sides of
the tube. A red ring is produced if urine contains bile salts. Then contents are mixed
gently while keeping the tube under running water. The red color spreads throughout
the liquid.
This is a less sensitive test than Hay’s test, since protein and pigments in urine
may interfere.
Bile pigments
Bile pigments e.g. bilirubin and biliverdin are produced by the breakdown of heme in the
reticuloendothelial system. Bilirubin is in unconjugated form soon after it is produced
from heme and it gets conjugated with UDP glucuronic acid in liver. Bile contains
conjugated bilirubin which is excreted into intestines. In normal persons bile pigments
are not present in urine.
9. FOUCHET’S TEST FOR BILE PIGMENTS

Principle:
Barium sulfate is having the property of adsorbing the pigments Ferric chloride oxidizes
bilirubin to give biliverdin, which is greenish blue in color. This is a very sensitive test.
Reagents:
Fouchet’s reagent (mixture of trichloro acetic acid and 10% ferric chloride)
Solid ammonium sulfate
10% barium chloride
95
Take 3 ml of urine in a test tube. Add few A white
crystals of ammonium sulfate and dissolve by precipitate of
shaking. Add 2 ml of 10% barium chloride. barium sulfate is The given
Filter it by using filter paper. formed. urine sample
Unfold the filter paper and dry it. Add 1-2 contains bile
drops of Fouchet’s reagent to the precipitate Green color pigments.
in the filter paper.
10. GMELIN’S NITRIC ACID TEST

Principle: During stages of oxidation bile pigments undergo colour change
Take 5 ml of concentrated nitric acid in a test tube. Layer urine over the nitric acid
slowly. Ring produced will be green, blue or violet color at the junction of the liquids, if
urine contains bile pigments.
DETECTION OF UROBILINOGEN
Urobilinogen formed from bilirubin in the intestine by bacterial action. The greater part is
excreted in feces in which it exists as a mixture of stercobilinogen, stercobilin,
urobilinogen and urobilins, the latter being formed by oxidation of urobilinogen when
exposed to air. The remainder is absorbed from the intestine. But in a normal person,
this is mostly re-excreted by the liver. However little passes in to general circulation and
is excreted in urine. Urobilinogen is a colorless, water soluble compound which gives
red color with Ehrlich’s reagent.
11. EHRLICH’S TEST

For the procedure refer to test for normal constituents of urine.
Urine examination in a case of jaundice gives us an idea about the type of jaundice.
Traces of urobilinogen are found in normal persons. Urobilinogen is absent in urine in
obstructive jaundice. Due to absence of urobilinogen, stools are clay colored in
obstructive jaundice. Urobilinogen is increased in hemolytic jaundice.
Urinary findings in jaundice

Condition Urobilinogen Bilirubin
Healthy individuals Trace Nil
Hemolytic jaundice Increased Nil
Hepatic jaundice
Prodromal stage Increased Detectable
Icteric stage Undetectable Present
Recovery stage Detectable Falling
Obstructive jaundice Undetectable Present
96
DETECTION OF HEMATURIA (BLOOD IN URINE)
The glomeruli are not normally permeable to formed elements (RBC, WBC and
platelets) in blood. But if the glomeruli are damaged, the formed elements may appear
in the urine. Blood in urine may also be derived from the lower genitourinary tract .The
presence of blood in urine can be detected by microscopic, chemical, and spectroscopic
means.
12. ORTHOTOLIDINE TEST

Principle:
Heme has peroxidase like activity, which can split the hydrogen peroxide to release
nascent oxygen. O-tolidine will accept the oxygen to give a transient dark green color.
Reagents:
-O-tolidine
-Glacial acetic acid
-Hydrogen peroxide

Boil the urine sample to eliminate
pus cells which have peroxidase like
activity.
Take 6 drops of freshly prepared Transient dark
Presence of heme
O-tolidine in a test tube. Add 2 drops green color
of glacial acetic acid, 6 drops of
hydrogen peroxide and 4 drops of
boiled urine.
Benzidine test: It is less sensitive than O-tolidine. Moreover benzidine is carcinogenic.

Hence it is replaced by O-tolidine test.
Note:
Myoglobinuria can be present in conditions where there is injury to muscle tissue.
It also gives positive O-tolidine test. It can be differentiated from hemoglobinuria by
precipitating hemoglobin by saturating urine with 80% ammonium sulfate. Myoglobin in
urine can also be differentiated from hemoglobin by electrophoresis.
Hematuria is often visible to the naked eye on inspection of the urine. But, when RBC’s
are present in numbers less than 1,000/mm3 it can only be detected on microscopic
examination.
Hemoglobinuria can be differentiated from hematuria by microscopic examination.
Hematuria can be seen when there is damage to glomerulus as in acute
glomerulonephritis or in any injury of urinary tract (Eg: calculi).
97
DATE :
NOTES :
98
DRY CHEMISTRY METHODS (DIP STICKS)
Dry chemistry refers to the use of strips impregnated with dry reagents to which the
specimen is added. Urine Reagent Strips (URS) for Urinalysis are firm plastic strips to
which several different reagent areas are affixed. Applications of the dry chemistry that
were considered include well-patient screening, use in health clinics, mobile health
services, polyclinics and hospital in-patients including point-of-care testing in critical
care areas. With proper training, the dry chemistry systems can be operated by non-
laboratory technologists. However with respect to cost considerations addressing costs
of equipment, reagents, equipment maintenance and manpower, dry chemistry worked
out to be twice the cost of conventional laboratory testing.
Dry chemistry strips for urinary protein detection

Reagent: Tetra bromophenol blue with buffer
At a constant pH, the development of any green color is due to the presence of protein.
Colors range from yellow for a “Negative” reaction to yellow-green and green to blue-
green for a “Positive” reaction.
Dry chemistry strips for urinary glucose detection

This test is based on a double sequential enzyme reaction. One enzyme glucose
oxidase, catalyzes the formation of gluconic acid and hydrogen peroxide from the
oxidation of glucose. A second enzyme, peroxidase, catalyzes the reaction of hydrogen
peroxide with potassium iodide chromogen to oxidize the chromogen to colors ranging
from blue-green to greenish-brown through brown and dark brown.
Limitations: Moderate amounts of ketone may decrease color development. The
reactivity of the glucose test decreases as the ascorbic acid, uric acid etc., in urine
increases. Reactivity may also vary with temperature.
Dry chemistry strips for urinary ketone bodies detection

This test is based on the reaction of acetoacetic acid with sodium nitroprusside in a
strongly basic medium. The colors range from belgo or buff-pink color for a “Negative”
reading to pink and pink-purple for a “Positive” reading.
Limitations: Color reaction that could be interpreted as “positive” may be obtained with
urine specimens containing MESNA (2-mercaptoethane sulfonate sodium) or large
amounts of phenylketones or L-dopa metabolites.
Dry chemistry strips for urobilinogen detection

This test is based on a modified Ehrlich’s reaction in which p-diethylaminobenzaldehyde
reacts with urobilinogen in a strongly acid medium. Colors range from light pink to bright
magenta.
Reagent: 2.9% w/w p-diethylaminobenzaldehyde balanced with buffer.
Bile pigments
This is based on the coupling of bilirubin with a diazotized dichloroaniline in a strongly
acid medium. The colors range from light tan to reddish brown.
Reagent: 0.4% w/w 2, 4-dichloroaniline diazonium salt, balanced with buffer.
99
RENAL FUNCTION TESTS
Renal function tests are commonly performed to check the proper functioning of kidney.
Urea, creatinine and uric acid are some of the common waste products excreted by
kidneys. Increase in the levels of these waste products in blood indicates decline in
renal function. Hence, their estimation helps to monitor the renal function of an
individual. These tests include routine tests like urinalysis and estimation of urea,
creatinine, uric acid and electrolytes like sodium and potassium in both blood and urine.
To assess glomerular and tubular dysfunction in the kidney, following tests are done:
Glomerular function tests Tubular function tests

Clearance tests
1. Inulin 1. Urine: pH, specific gravity
2. Urea 2. Osmolality
3. Creatinine 3. Ammonium chloride load test
ESTIMATION OF NON-PROTEIN NITROGENOUS SUBSTANCES
VIII.3. ESTIMATION OF UREA IN SERUM

Urea is the end product of amino acid catabolism in our body. It helps in the
detoxification of ammonia in the body. It is synthesized in liver cells from ammonia and
CO2 and excreted by the kidneys. It is one of the non-protein nitrogenous substances
(NPN) in our body.
1. Non-enzymatic method
a. Diacetyl Monoxime method (DAM)
2. Enzymatic method (employs Urease enzyme)
a. Nessler’s method.
b. Berthelot reaction
c. Glutamate dehydrogenase method (GLDH)
DIACETYL MONOXIME (DAM) METHOD

Principle:
Proteins present in blood are precipitated by trichloroacetic acid (TCA). Urea present in
protein free filtrate reacts with Diacetyl Monoxime (DAM) in acid medium in the
presence of thiosemicarbazide to give a pink colored complex. The intensity of the color
is directly proportional to concentration of urea in the sample and is compared with a
standard urea solution similarly treated, in a colorimeter at 540nm.
Reagents
1. Trichloroacetic acid (10%)
2.Diacetyl Monoxime reagent
3.Thiosemicarbazide reagent
4.Acid Reagent (Phosphoric acid - Sulfuric acid)
5.Urea standard: 1 mg/dL
100
Procedure
Precipitation of proteins and preparation of protein free filtrate:
In a test tube take 0.1ml of serum, 3.4ml of distilled water and 1.5ml of 10% TCA in a
test tube. Mix well and keep aside for 10 minutes. Filter to get a clear protein free filtrate
Estimation of Urea in the protein free filtrate
Reagent B S T1 T2
Distilled water(ml) 1 - - -
Urea standard (ml) - 1 - -
Protein free filtrate (ml) - - 1 1
Diacetyl monoxime 1 1 1 1
reagent(ml)
Thiosemicarbazide (ml) 1 1 1 1
Acid reagent(ml) 3 3 3 3
Mix well and place the four tubes in boiling water-bath for exactly 15 minutes. Cool and
read the absorbance of standard and test against blank at 540 nm in a colorimeter.
T2 for calculation.
Calculation
Serum urea (mg/dL) =



UREA ESTIMATION BY GLUTAMATE DEHYROGENASE (GLDH) METHOD

Principle
Urea Urease NH3 + CO2
GLDH
NH4 + α-Ketoglutarate + NADH Glutamate + NAD+
Reagents
R1 – Buffer (pH = 7.55)
R2 – ADP, GLDH, Urease, NADH, α - Ketoglutarate
101
Procedure
Standard Test
Working reagent (µL) 1000 1000
Standard (µL) 10 -
Test (µL) - 10
Mix and read the absorbance (T1) 30 seconds after the
sample or standard addition. Exactly 60 seconds after the
first reading, take the second reading (T2) at 340nm.
Calculation
Urea (mg/dL) = T1 – T2 of sample x Conc. of the standard
T1 – T2 of standard
Blood Urea Nitrogen

Some laboratories estimate Blood Urea Nitrogen (BUN) instead of urea. Since the
molecular weight of urea and nitrogen are 60 and 28 respectively BUN estimates the
nitrogen content of urea. Urea concentration = BUN x (60/28), hence:
Blood Urea = Blood Urea Nitrogen (BUN) X 2.14 (in mg/dL)
Normal levels
Blood Urea = 15 - 40 mg/dL
BUN = 7 – 18 mg/dL
Clinical interpretation:
1. Blood urea increases in renal failure. Causes of renal failure can be classified as
follows
Pre renal: Hypovolemia like cardiac failure, trauma and burns
Renal: Acute glomerulonephritis, pyelonephritis and nephrotic syndrome
Post renal:Tumors and stones obstructing the flow of urine
2. Hemoconcentration, e.g. due to dehydration can also increase blood urea levels.
3. Condition in which blood urea level is decreased: Low protein intake, over hydration,
conditions causing protein loss (celiac disease, protein losing enteropathy).
DATE :
NOTES :
102
VIII.4. ESTIMATION OF CREATININE IN SERUM
Creatinine is the anhydride of creatine. Creatine is synthesized in kidney, liver and

skeletal muscles from the aminoacids glycine, arginine and methionine. Creatinine is
one of the non-protein nitrogenous substances (NPN) in our body.
JAFFE’S METHOD
Principle
Proteins present in blood are precipitated by tungstic acid. Creatinine in the protein free
filtrate reacts with alkaline picrate to form an orange-red colored complex of creatinine
picrate.
The intensity of this color is directly proportional to the concentration of creatinine in the
sample and is compared with a standard creatinine solution similarly treated, in a
colorimeter at 540 nm.
Reagents
1. 10% Sodium tungstate
2. 2/3 N sulfuric acid
3. 0.75 N Sodium hydroxide
4. 0.04 M Picric acid
5. Creatinine standard: 1 mg/dL
Procedure
Preparation of Protein free filtrate
In a test tube take 1ml of serum, 7ml of distilled water 1ml of 10% sodium
tungstate and 1ml of sulfuric acid. Mix well and keep aside for 10 minutes.
Filter to get a clear protein free filtrate.
Estimation of creatinine in the protein free filtrate

Reagents B S T1 T2
Distilled water (ml 3 - - -
Creatinine standard - 3 - -
Protein free filtrate - - 3 3
Picric acid (ml) 1 1 1 1
0.75 N NaOH (ml) 1 1 1 1
Mix well and allow it to stand for 15 minutes. Read the absorbance of standard and test
against blank at 540 nm (green filter) in a colorimeter. Normally, both T 1 and T2 would
give same values if the procedure is followed accurately. If the values of T1 and T2 are
different, take average absorbance of T1 and T2 for calculation.
103
Calculation
Serum Creatinine (mg/dL) =



CREATININE ESTIMATION BY JAFFE’S METHOD
Principle
Creatinine reacts with alkaline picrate to form an orange colored complex of creatinine
picrate.
Reagents
1. Creatinine dye reagent: Picric acid, surfactant
2. Creatinine base reagent: Sodium hydroxide, sodium phosphate
Procedure
Standard Test
Working reagent (µL) 1000 1000
Standard (µL) 100 -
Sample (µL) - 100
Mix and read the absorbance (T1) 60 seconds after the sample
or standard addition. Exactly 60 seconds after the first reading
take the second reading (T2) at 540 nm.
Calculation
Creatinine (mg/dL) = T1 – T2 of sample x Conc. of the standard
T1 – T2 of standard
Note: Jaffe’s reaction is not specific for creatinine and there are other substances in
blood like ketone bodies, pyruvate, glucose, cephalosporins, and amino acids (non-
creatinine chromogens) which also give orange color similar to creatinine.
True creatinine can be estimated by modification of Jaffe’s method by using Lloyd’s
reagent (hydrated Aluminium silicate) which adsorbs only creatinine.
Normal range for serum creatinine

Male: 0.7 – 1.4 mg/dL
Female: 0.6 – 1.2 mg/dL
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Creatinine levels are increased in renal failure (if more than 50% of kidney is damaged)
Prerenal – hypovolemia.
Renal-glomerulonephritis, nephrotic syndrome
Post renal- bladder outflow obstruction and musculoskeletal disorders like myotonic
dystrophy and myasthenia gravis.
CLEARANCE TESTS
The clearance is defined as the volume of blood/plasma completely cleared of a
substance per unit time and it is expressed as ml/min.
Clearance: mg of substance excreted per minute = UV

mg of substance per ml of plasma P
[U and P denote the concentration of substances in urine and plasma and V denote the
ml of urine excreted per minute]
The clearance of several substances like urea and creatinine has been used in the
study of renal function.
Urea clearance
It is the volume of plasma which contains the urea that is excreted in a minute by the
kidneys.
Urea clearance = U×V
P
U = Concentration of urea in urine (mg /100 ml)
P = Plasma urea concentration (mg/100ml)
V = Urine flow in one minute (ml/min)
The term maximum urea clearance is used when urine flow rate (v) is more than 2 ml
per minute and standard urea clearance is used when urine flow rate (v) is less than 2
ml per minute.
Maximum urea clearance = U×V

P
Normal range for maximum urea clearance is 60 – 95 ml/min
Standard urea clearance = U×√ V

P
Normal range for standard urea clearance is 40-65 ml/min
105
Creatinine clearance
It is the volume of plasma, which contains the creatinine that is excreted in a minute by
the kidneys.
Creatinine clearance = U×V
P
U = Concentration of creatinine in urine (mg /100 ml)
P = Plasma creatinine concentration (mg/100ml)
Normal levels: Males: 85 -125 ml/min, Females: 80-115 ml/min
Other method of estimating creatinine clearance is
Cockcroft-Gault formula:
Creatinine clearance = (140-age) x lean body mass in Kg

Plasma creatinine (mg/dL) x 72
1. Gold standard method for estimation of GFR is inulin continous infusion urinary
clearance method.
2. Silver standard method is inulin single bolus plasma clearance method.
3. Bronze standard method is creatinine and cystatin C clearance.
In the early stages of renal failure only blood urea is increased, where as serum
creatinine remains normal. So creatinine clearance is preferred over serum creatinine in
early stages of renal failure. Serum creatinine begins to rise only if more than 50% of
kidney is damaged. So serum creatinine estimation is preferred in the late stages of renal
failure.
Advantages of creatinine clearance over urea clearance

1. Not affected by age
2. Not affected by diet
3. Not affected by urine flow rate
DATE :
NOTES :
106
VIII.5. ESTIMATION OF URIC ACID IN SERUM
Uric acid is the end product of purine catabolism. It is one of the non protein nitrogenous
substance in our body.
1. Non enzymatic-Caraway’s method
2. Enzymatic-Uricase method
CARAWAY’S METHOD
Principle
Proteins present in blood are precipitated by tungstic acid. Uric acid in the protein free
filtrate reacts with phosphotungstic acid and sodium carbonate to give a blue color
complex. It is based on the reducing property of uric acid. The intensity of this color is
directly proportional to concentration of uric acid in the sample and is compared with a
standard uric acid solution similarly treated, in a colorimeter at 650 nm.
Reagents
1. 10% Sodium tungstate
2. 2/3 N Sulfuric acid
3. 20% sodium carbonate
4. Phosphotungstic acid
5. Standard uric acid – 1 mg/dL
Procedure
Preparation of Protein free filtrate
In a test tube take 1ml of serum, 7ml of distilled water 1ml of 10% sodium
tungstate and 1ml of sulfuric acid. Mix well and keep aside for 10 minutes. Filter to get a
clear protein free filtrate.
Estimation of Uric acid in the protein free filtrate

Reagents B S T1 T2
Distilled water (ml) 5 - - -
Uric acid standard (ml) - 5 - -
Protein free filtrate (ml) - - 5 5 Mix well
Sodium carbonate (ml) 1 1 1 1 and
Phosphotungstic acid(ml) 1 1 1 1 allow it
to stand for 5 minutes. Read the absorbance of standard and test against blank at 650
nm (red filter) in a colorimeter. Normally, both T1 and T2 would give same values if the
procedure is followed accurately. If the values of T1 and T2 are different, take average
absorbance of T1 and T2 for calculation.
107
Calculation
Serum Uric acid (mg/dL) =

Absorbance of Std – Absorbance of Blank Vol. of sample in filtrate


URIC ACID ESTIMATION BY URICASE METHOD
Semiautoanalyzer Method(demonstration)
Principle
Uricase
Uric acid + O2 + H2O Allantoin + CO2 +H2O2
Peroxidase
DHBS + 4- Aminoantipyrine + H2O2 Quinoneimine dye + H2O
(DHBS: 3,5-dichloro-2-hydroxybenzenesulfonic acid)
Reagents
- Pipes buffer (pH-7.5)
- 4 aminoantipyrine
- Uricase
- Peroxidase
- DHBS
Procedure
Take three test tubes and label as ‘Blank (B)’, ‘Standard (S)’ and ‘Test (T)’. Perform the
experiment as follows
B S T
Working reagent (µL) 1000 1000 1000
Distilled water (µL) 20 - -
Standard (µL) - 20 -
Test (µL) - - 20
Mix and incubate for 5 minutes at 370 C. Read the absorbance of
the sample and standard against blank at 670 nm.
108
Calculation
Uric acid (mg/dL) =Absorbance of Test – Absorbance of Blank xconc. of standard

Normal range: Serum: 2.5 – 6 mg/dL
Uric acid is the major product of catabolism of purine nucleosides: adenosine and
guanosine. It is increased in gout, renal failure, leukemia (as a result of enhanced
nucleic acid metabolism), severe PIH (pregnancy induced hypertension) etc. Hereditary
causes of hyperuricemia include: Von Gierke’s disease and Lesch Nyhan syndrome.
DATE :
NOTES :
109
VIII.6. SERUM ELECTROLYTES AND ION SELECTIVE
ELECTRODES
An ion selective electrode is a potentiometric electrode consisting of a membrane
selectively permeable to single ion species. The potential produced at the membrane-
sample interface is is proportional to the logarithm of ionic activity or concentration
according to the Nernst equation. The sensing part of the electrode is usually made as
an ion-specific membrane, along with a reference electrode. The most commonly used
ISE is the pH probe.
The types of ion-selective membrane used in ion-selective electrodes: glass, crystalline

membranes and ion-exchange resin membranes.
1. Glass membranes are made from an ion-exchange type of glass (silicate of

chalcogenide). This ISE has good selectivity for several single-charged cations;
mainly H+, Na+, and Ag+. An example for this type of electrode is the pH glass
electrode.
2. Crystalline membranes are made from mono- or poly-crystallites of a single
substance. They have good selectivity which can be for both cations and anions. An
example is the fluoride selective electrode based on LaF3 crystals.
3. Ion-exchange resins are based on special organic polymer membranes which
contain a specific ion-exchange substance (resin). These are the most commonly
used electrodes. These electrodes have low chemical and physical durability. An
example is the potassium selective electrode, based on valinomycin as an ion-
exchange agent.
Merits of ion exchange membranes

There is no background interference and relatively simple when compared to flame
photometry. Also less time consuming when compared to flame photometry.
Demerits of ion exchange membranes

In practice different electrodes from the same batch will differ in their properties. The
response of the electrode and galvanometer is temperature sensitive, and also 'drifts'
over time, requiring recalibration frequently during a series of measurements, ideally at
least one calibration sample before and after each test sample.
After immersion in the solution there is a transient 'settling time' which can be five
minutes or even longer, before the electrode and galvanometer equilibrate to a new
reading. The most serious problem limiting use of ion-selective electrodes is
interference from other, undesired ions.
They can measure the activity or concentration of analyte ions and metabolites. They
are useful in the analysis of biological fluids including blood, urine, plasma, saliva, spinal
fluid, and serum.
110
Therapeutic drug monitoring
Lithium is the mainstay of treatment in bipolar disorder. Lithium salts have a narrow
therapeutic window. Over-dosage may be fatal and toxic effects include tremor, ataxia,
nystagmus, renal toxicity, and convulsions. About 95% of the drug is excreted
unchanged through the kidneys within 24 hours. Due to its toxicity, close monitoring of
lithium concentration in biological fluids (e.g. serum, plasma, urine etc) is required
during the treatment. Lithium toxicity is exaggerated by sodium depletion. Hence during
treatment serum electrolytes (Na+ and K+) should also be monitored.
Electrolyte imbalances
The diagnosis and treatment of electrolyte disorders are based on serum and urine
electrolyte concentrations.
1. Hyponatremia
Hyponatremia is defined as a serum sodium concentration < 130 mEq/L. It is the
most common electrolyte abnormality observed in a general hospitalized population.
Hyponatremia usually reflects excessive water retention relative to sodium rather
than sodium deficiency. Assessment of ECF volume and serum osmolality is
essential to determine the etiology of hyponatremia.
Principal Causes of Hyponatremia
1. Adrenal gland insufficiency (Addison's disease)
2. Hypothyroidism
3. Medication e.g. thiazide diuretics, angiotensin II receptor blockers, angiotensin-
converting enzyme (ACE) inhibitors
4. Syndrome of inappropriate anti-diuretic hormone (SIADH)
5. Salt-wasting nephropathy
6. Primary polydipsia. In this condition, your thirst increases significantly, causing
increased fluid intake.
7. Chronic, severe vomiting or diarrhea and dehydration
8. Pseudohyponatremia: The aqueous phase is diluted by excessive proteins or
lipids. The body water and sodium are unchanged. This condition is seen with
hypertriglyceridemia and multiple myeloma.
2. Hypernatremia
Hypernatremia is defined as a serum sodium concentration > 145 mEq/L.
Hypernatremia occurs most commonly when water intake is inadequate, as in
patients with altered mental status. Rarely, excessive sodium intake may cause
hypernatremia.
Principal Causes of Hypernatremia
1. Primary hypodipsia (essential hypernatremia)
2. Renal loss e.g. intrinsic renal disease, loop diuretics and osmotic
diuresis(glucose, urea, mannitol)
3. Central and nephrogenic diabetes insipidus
4. Hypertonic fluid administration (hypertonic saline, NaHCO3 etc)
111
5. Mineralocorticoid excess e.g. adrenal tumors, Congenital adrenal
hyperplasia (11-hydroxylase deficiency)
3. Hypokalemia
Hypokalemia is defined as a serum potassium concentration < 3.5 mEq/L.
Principal Causes of Hypokalemia
 Shifting of potassium from extracellular space to intracellular space
o Rapid insulin treatment for diabetic ketoacidosis without potassium
supplementation
o Alkalosis
o Trauma (due to enhanced release of epinephrine)
 Extrarenal potassium loss (vomiting, diarrhea)
 Renal potassium loss (increased aldosterone or mineralocorticoid levels)
O Cushing's syndrome
O Primary hyperaldosteronism and congenital adrenal hyperplasia
4. Hyperkalemia
Hyperkalemia is defined as a serum potassium concentration > 5 mEq/L.
Principal Causes of Hyperkalemia
 Hyperkalemia usually develops in patients with advanced renal dysfunction.
 Medications: ACE inhibitors, angiotensin receptor blockers, potassium-sparing
diuretics, β-blockers, and NSAIDs.
 Thrombocytosis, leukocytosis, in vitro hemolysis may lead to
pseudohyperkalemia.
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VIII.7. CASE REPORTS : RENAL FUNCTION TESTS
QUESTION 1
A 60 year old diabetic man with uncontrolled diabetic status presents with generalized
edema.
His lab rept is as follows:
 Serum urea: 150 mg/dL (Reference range: 20-40 mg/dL)
 Serum creatinine: 4.0 mg/dL (Reference range: 0.8-1.2 mg/dL)
 Estimated GFR: 45 ml/min
a) What is the most probable diagnosis?
b) What is the normal range of GFR? Name any three substances used in
estimation of GFR in clinical practice.
c) Write any three characteristics of a substance that is ideal for measurement of

GFR.
QUESTION 2
A 20 year old female has a plasma creatinine concentration of 0.1 mg/dL and in 1 hour
excretes 60ml of urine with a creatinine concentration of 10.5 mg/dL.
a) Calculate the creatinine clearance and comment on the result.
b) What is the usefulness of estimating creatinine clearance in clinical practice?
c) Why is creatinine clearance considered superior to urea clearance?
QUESTION 3
A 6yr old boy presented with generalized edema. Following are the findings:
 Serum total protein: 4.9 g/dl
 Serum albumin: 1.5 g/dl
 Serum urea: 24mg/dL
 Serum creatinine: 0.6 mg/dL
 Serum cholesterol: 350 mg/dL
 Urinary protein: 4.8 gm/24hr
113
a) Which of the above parameters are showing abnormal result?
b) What is the most probable diagnosis?
c) Why is this condition associated with edema?
d) What will be the electrophoretic pattern in this condition?
QUESTION 4
A 64-year-old obese alcoholic male presented with severe pain in his right big toe. On
examination, his toe is found to be red and markedly swollen. His laboratory profile is as
follows:
 Fasting plasma glucose : 106 mg/dL

 Serum urea : 37 mg/dL
 Serum creatinine : 1.2 mg/dL
 Serum uric acid : 8.8 mg/dL
a) Mention the normal levels of all the above parameters.
b) What is the probable diagnosis?
c) What is the biochemical basis of this disorder ?
114
ACID-BASE / ELECTROLYTE DISORDERS
QUESTION 1:
A patient was operated for intestinal obstruction. He had continuous gastric aspiration
for past 3 days. The arterial blood gas results of the postoperative patient are
Reference range
pH : 7.54 ( 7.35 – 7.45)
pCO2 : 45 mm of Hg ( 35- 45 mm of Hg )
Plasma Bicarbonate : 36mmol/L ( 22 - 26 mmol/L)
Serum Sodium : 130 mmol/L ( 136 - 145 mmol/L)
Serum Potassium : 2.8 mmol/L ( 3.5 – 5 mmol/L)
Serum Chloride : 90mmol/L ( 96- 106 mmol/L)
a) What is the diagnosis?
b) What is the significance of serum potassium in relation to acid base status?
c) Calculate and comment on anion gap.
d) How is urine chloride level important in this patient?
QUESTION 2
A 52-year-old man is found at home hypotensive and confused. In the emergency

department, his blood pressure is 82/60 mmHg and his heart rate is 115 beats/ min. He
is confused and lethargic.
Laboratory data show:

Serum sodium :133 meq/L
Serum potassium : 2.4 meq/L
Serum chloride : 70 meq/L
Plasma HCO3 : 50 meq/L
BUN : 44 mg/dL
Serum creatinine : 1.7 mg/dL
An arterial blood gas shows PO2 of 62 mmHg, PCO2 of 49 mmHg and pH 7.66.
a) What acid-base disorder is present in this case?
115
b) What is meant by ‘anion gap’?
c) Calculate anion gap for this patient and comment.
d) Give some examples of high anion gap metabolic acidosis
QUESTION 3:
Following is the arterial blood gas report of a 17 year old female suffering from IDDM.
pH : 7.05
pO2 : 97 mm Hg
pCO2 : 20 mm Hg
HCO3 : 15 mEq/L
Anion gap : 30 mmol/L
a) Which acid-base imbalance is the patient suffering from? Justify.
b) What is anion gap? Why is it increased in this condition?
c) Give examples of few conditions where anion gap can be normal in this acid
base imbalance?
QUESTION 4:
Following is the arterial blood gas (ABG) report of a 24 yrs hyperventilating female who
presented to the hospital with hysteria
pH : 7.55
pCO2 : 27 mm Hg
pO2 : 105 mm Hg
HCO3 : 19 mEq/L
a) Which acid-base imbalance is the patient suffering from? Justify.
b) Which acid-base imbalance is characterized by CO2 retention due to ventilatory

failure? What would be the ABG report in this condition?
116
QUESTION 5 :
A 64 year old lady was brought to the Emergency Medical Services in unconscious
state. She is hypertensive and is on diet restriction. The laboratory findings were as
follows:
Reference range
Plasma glucose : 124 mg/dL (70 – 140 mg/dL)
Blood pH : 7.42 (7.35 – 7.45)
Serum sodium :121 mmol/L (135 – 145 mmol/L)
Serum potassium : 4.2 mmol/L (3.5 – 5.5 mmol/L)
Serum chloride : 105 mmol/L (96 – 106 mmol/L)
b) What may be the cause for this condition?
c) Name the machine used to measure pH.
d) What method is used to estimate the serum electrolytes?
QUESTION 6 :
The laboratory data of a patient in the Medical intensive care unit with septic shock are
given below
 Serum sodium : 127 mmol/L
 Serum potassium : 6.5 mmol/L
 Serum chloride : 92 mmol/L
 Serum bicarbonate: 5 mmol/L
 Plasma glucose : 50 mg/Dl
a) Write the normal values for the above parameters.
b) Interpret the given laboratory data.
c) Name the method used to estimate the serum electrolytes.
d) Calculate the anion gap for the above case and interpret the value.
117
INBORN ERRORS OF METABOLISM
QUESTION 1
A 1-year-old girl is brought to pediatrician’s office with concerns about her development.
She had an uncomplicated birth at term. The mother reports that the baby is not
achieving the normal milestones for a baby of her age. She also reports an unusual
odor to her urine and some areas of hypopigmentation on her skin and hair. The urine
collected is found to have a “mousy” odor.
a. What is the most likely diagnosis?
b. Which enzyme deficiency is responsible for this?
c. What is the biochemical basis of the hypopigmented skin and hair?
QUESTION 2
6 month old boy presented with mental retardation, microcephaly, hypopigmented skin
and hairs, eczema and “mousy” odour.
b) Name the screening test done for this condition.
c) Name the test performed to confirm the diagnosis.
d) Mention its principle.
QUESTION 3
A male baby of birth weight 3.7 kg developed jaundice from the 3 rd day of birth. On
examination it was seen that the infant had increased muscle tone and bilateral
cataract. On the 9th day, the child began vomiting and had convulsions. His liver was
found to be enlarged. Urinalysis showed positive result for reducing sugar. Milk feeding
was stopped and replaced by intra venous glucose. From 10th day onwards galactose
free formula milk was given. The child improved dramatically.
118
b) Explain the biochemical defect involved in this condition.
c) What is the reason for bilateral cataract in this baby?
d) What is the principle of Benedict’s test?
e) What are the other substances which will give positive result for Benedict’s Test?
QUESTION 4
A 40 year old patient presented with acute abdominal pain and neuropsychiatric
symptoms. The symptoms were alleviated by a carbohydrate rich diet. He gave history
of intake of barbiturates.
b) Name one test that can be done in urine to confirm the diagnosis.
c) Which analyte is increased in blood in this condition?
d) What is its absorption maximum?
QUESTION 5
A 5 year old boy was brought to the hospital with a complaint of abdominal swelling and
growth retardation. On examination, the liver was found to be enlarged. There was
increased serum uric acid and free fatty aid level associated with hypoglycemia. No
increase was found even after intravenous administration of glucagon
b) What is the enzyme defect involved?
c) What is the cause for increased uric acid level in this patient?
d) How can be specific diagnosis of this disorder made?
119
IX. MOLECULAR BIOLOGY AND
CANCER BIOLOGY
120
IX.1. POLYMERASE CHAIN REACTION (PCR)
Definition
It is a technique to amplify a single or few copies of a piece of DNA, generating millions

or more copies of a particular DNA sequence.
Common types of PCR
1. Quantitative PCR (Q-PCR): It is used to quantify a PCR product. Q-PCR is

commonly used to qualitative and quantitative determination of a DNA, cDNA or
RNA sequence is present in a sample. The Quantitative real-time PCR (QRT-PCR)
method uses fluorescent dyes, such as Sybr Green, or fluorophore-containing DNA
probes, such as TaqMan, to measure the amount of amplified product in real time
with highest level of accuracy.
2. Reverse Transcription PCR (RT-PCR): It is a method used to amplify, isolate or
identify a known sequence from a cellular or tissue RNA. The PCR is preceded by a
reaction using reverse transcriptase to convert RNA to cDNA. It is widely used in
gene expression studies.
Principle
The thermocycling steps consist of cycles of repeated heating and cooling of the
reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA
fragments) containing sequences complementary to the target region along with a DNA
polymerase are key components that enable selective and repeated amplification. As
PCR progresses, the DNA generated is itself used as a template for replication, setting
a chain reaction in which the DNA template is exponentially amplified.
Reagents required and their purpose
1. DNA template that contains the DNA region (target) to be amplified.

2. Two primers that are complementary to the 3' ends of each of the sense and
anti-sense strand of the DNA target. These are oligonucleotides with 18- 25 bp.
3. Taq polymerase or another DNA polymerase with a temperature optimum at
around 70°C. (Taq polymerase, an enzyme originally isolated from the bacterium
Thermus aquaticus. It is necessary for the polymerase to withstand higher
temperature during strand separation and lower temperature during DNA
synthesis). Taq polymerase is stable even at prolonged exposure at 95 0 C. This
eliminates the need of adding DNA polymerase enzyme after every cycle.
4. Deoxynucleoside triphosphates (dNTPs), the building blocks from which the
DNA polymerase synthesizes a new DNA strand.
5. Buffer solution, providing a suitable chemical environment for optimum activity
and stability of the DNA polymerase.
121
6. Divalent cations, magnesium or manganese ions; generally Mg2+ or Mn2+is
used.
Procedure of PCR
I. Pre PCR step:

DNA extraction is done by phenol chloroform method or commercially available kits.
II. Basic PCR step:
The above said reagents are added in appropriate amounts to polypropylene tubes
and kept in the thermocycler. The PCR usually consists of a series of 20 to 40
cycles; each cycle typically consists of following steps:
 Initialization step: This step consists of heating the reaction to a temperature of 94–
96°C (or 98°C if extremely thermostable polymerases are used), which is held for 1–
9 minutes. This is required for DNA polymerases which require heat activation as in
hot-start PCR.
 Denaturation step: This step is the first regular cycling event and consists of
heating up to 94–98°C for 20–30 seconds. It causes separation of DNA template
and primers by disrupting the hydrogen bonds between complementary bases of the
DNA strands, yielding single strands of DNA.
 Annealing step: The reaction temperature is lowered to 50–65°C for 20–40

seconds allowing annealing of the primers to the single-stranded DNA template.
Typically the annealing temperature is about 3-5oC below the Tm (transition
temperature) of the primers used. Stable DNA-DNA hydrogen bonds are only formed
when the primer sequence very closely matches the template sequence. The
polymerase binds to the primer-template hybrid and begins DNA synthesis.
 Extension/elongation step: The temperature at this step depends on the DNA

polymerase and commonly done at 72°C. The DNA polymerase synthesizes a new
DNA strand complementary to the DNA template strand by adding dNTPs that are
complementary to the template in 5' to 3' direction. The extension time depends both
on the DNA polymerase used and on the length of the DNA fragment to be
amplified.
 Final elongation: This step is occasionally performed at a temperature of 70–74°C

for 5–15 minutes after the last PCR cycle to ensure that any remaining single-
stranded DNA is fully extended.
III. Post PCR step: Agarose gel electrophoresis

This step is to separate DNA molecules amplified during PCR by size. This is
achieved by allowing negatively charged nucleic acid molecules to migrate through
an agarose matrix under an electric field. Generally 1-2% agarose gel is prepared
and this percentage varies with size of DNA to be separated. Higher the size, lower
122
will be the percentage of gel and vice versa. For staining the gel, DNA binding
agents like ethidium bromide (EtBr) is used. Since EtBr is carcinogenic, it is being
replaced by safe dyes like SYBR green etc. After staining the gel is visualized in UV
transilluminator for the presence of amplicons.
Applications of PCR
 Medical
1. Prenatal gene testing for genetic diseases
2. Tissue typing in transplantation
 Infectious disease
1. HIV
2. Tuberculosis
 Forensic
1. Genetic fingerprinting from a crime scene
2. Parental testing in case of sibling disparity
 Research
1. Southern or Northern blot hybridization
2. DNA cloning and sequencing
3. Sequence tagged sites in human genome project
4. Phylogenic analysis of DNA
5. Gene expression and mapping
DATE :
NOTES :
123
APPENDIX I
SAMPLE OF OSPE PERFORMANCE STATION

Question: Perform a suitable test to detect the presence of carbohydrate in the
given solution
NAME OF THE TEST: MOLISCH TEST
CODE SHEET
Procedure Marks
Takes 2 mL of the given solution 1
Adds 6 drops of Molisch’s reagent 1
Adds 2 mL of concentrated sulphuric acid

2
along the sides of test tube carefully
Obtains a violet ring at the junction of two

1
liquids
Clean work place 1
Principle / Explanation asked for 4
TOTAL MARKS 10
Principle :
Conc. H2SO4 by its dehydrating action removes water molecules from
carbohydrate forming furfural or hydroxyl methyl furfural which reacts with α-
naphthol to give violet colored complex.
124
APPENDIX II
OSPE Questions for performance tests
1. A child was brought to pediatrics clinic for suspected lactose intolerance.

Perform a suitable test in the given sample to confirm the excretion of
lactose in this condition. (Modified Barfoed’s test)
2. A 50 year old female presented with renal glycosuria. Perform a suitable

test in the given sample to confirm the excretion of glucose in this
condition (Molisch test)
3. A newborn was brought to pediatrics clinic with cataract. The pediatrician

suspected it to be a case of galactosemia. Perform a suitable test in the
given sample with the available reagents to confirm the reducing property
of the carbohydrate excreted in this condition (Benedict’s test)
4. A child was brought to pediatrics clinic for suspected hereditary fructose

intolerance. Perform a suitable test in the given sample to confirm the
excretion of fructose in this condition (Seliwanoff’s test)
5. Perform a suitable test in the given sample to confirm the presence of

aminoaciduria (Ninhydrin test)
6. A child was brought to pediatrics clinic for suspected Hartnup disease.

Perform a suitable test in the given sample to confirm the excretion of the
specific aminoacid in this condition. (Aldehyde test)
7. A newborn was brought to pediatrics clinic with complaints of passing dark

color urine. Perform a suitable test to detect the presence of the specific
aminoacid for the confirmation of alkaptonuria (Modified Millon test)
8. A newborn was brought to pediatrics clinic with suspected urea cycle

disorder defect. Perform a suitable test to detect the presence of the
specific aminoacid for the confirmation of hyperargininemia. (Sakaguchi
test)
9. A newborn was brought to pediatrics clinic with complaints of mental

retardation and mousy odor of the urine. Perform a suitable test in the
given sample to confirm the excretion of aromatic aminoacids in this
condition (Xanthoproteic test)
10. A 50 year old male was admitted with renal disorder in Medicine ward.
Perform a suitable test to confirm whether proteins are excreted in urine in
this condition. (Heat coagulation test)
125
11. The blood glucose level of a patient admitted in diabetic clinic was 300
mg/dL. Perform a suitable test with the available reagents to confirm
whether glucose is excreted in urine in this condition. (Benedict’s test)
12. A 30 year old diabetic patient was admitted the in Medicine ICU with
disorientation and confusion. Perform a suitable test to confirm whether
ketone bodies are excreted in urine in this condition. (Rothera’s test)
13. Perform a suitable test in the given urine sample with the available
reagents to confirm the presence of obstructive jaundice in a patient with
chronic alcoholism. (Hay’s test)
14. Perform a suitable test in the given urine sample with the available
reagents to confirm the presence of hematuria in a patient with renal
stones. (Orthotolidine test)
* * * * *
126
APPENDIX III
LABORATORY REFERENCE RANGES

CLINICAL BIOCHEMISTRY
ROUTINE INVESTIGATIONS
BLOOD GLUCOSE
Fasting 70 – 100 mg/dL
Random 70 – 140 mg/dL
2 hour postprandial <140 mg/dL
OGTT( In Pregnancy)
Fasting <95 mg/dL
1 hour <180 mg/dL
2 hour <155 mg/dL
3 hour <140 mg/dL
RENAL PROFILE
 Blood urea 15 – 40 mg/dL
 Serum creatinine 0.7 – 1.2 mg/dL
 Uric acid 2.5 – 7 mg/dL (Males)
1.5 – 6 mg/dL (Females)
SERUM ELECTROLYTES
 135 – 145 mmol/L
+
Na
 3.5 – 5 mmol/L
+
K
 96 – 106 mmol/L
-
Cl
 0.6 – 1.2 mmol/L
+
Li (Therapeutic)
 4.5 – 5.6 mg/dL
2+
Ca ( Ionic)
 9 – 11 mg/dL
2+
Ca (Total)
 Inorganic phosphorus 2.5 -4.5 mg/dL
 Magnesium 1.8 – 3 mg/dL
LIPID PROFILE
 Total Cholesterol <200 mg/dL
 Triglycerides <150 mg/dL
 HDL Cholesterol >40 mg/dL
 LDL Cholesterol <100 mg/dL
 VLDL Cholesterol <30 mg/dL
HEPATOBILIARY PROFILE
 Total Bilirubin 0.4 – 1.2 mg/dL
 Direct Bilirubin 0.1 – 0.4 mg/dL
 Indirect Bilirubin 0.3 – 0.8 mg/dL
 Total Protein 6.3 – 8.3 g/dL
 Albumin 3.5 – 5.5 g/dL
 Globulin 2.5 – 3.5 g/dL
 AST 0 – 40 IU/L
 ALT 0 – 45 IU/L
 GGT 1 – 50 IU/L
 Alkaline phosphatise 30 – 125 IU/L
127
MYOCARDIAL PROFILE
 LDH 60 – 200 IU/L
 CK total 20 – 170 IU/L
 CK – 2 < 6 IU/L
CSF
 Protein 15 – 40 mg/dL
 Glucose 40 – 70 mg/dL
 Chloride 116 – 130 mmol/L
OTHERS
 Serum Amylase 28 – 100 IU/L
 Serum Acid Phosphatase 1.5 – 4.5 IU/L
 Plasma Fibrinogen 200 – 400 mg/dL
 Prothrombin time 12 – 18 Sec ( INR: 1 – 1.2)
SPECIAL INVESTIGATIONS
THYROID PROFILE
 Total T4 Adults:4.5 – 10.9 μg/dL
Children:
1 – 3 days:11.8 – 22.6 μg/dL
1 – 2 weeks : 9.9 -16.6 μg/dL
1 – 4 months: 7.2 - 14.4 μg/dL
4 -12 months:7.8 -16.5 μg/dL
1 -5 years: 7.3 – 15 μg/dL
5 – 10 years:6.4 – 13.3 μg/dL
11 – 15 years:5.6 – 11.7 μg/dL
 Free T4 Adults:0.89 – 1.76 ng/dL

Children:
1 – 4 days:2.2 – 5.3 ng/dL
2 weeks – 20 years :0.8 – 2 ng/dL
 Total T3 Adults:0.6 – 1.81 ng/mL
Children :
1 – 3 days :1 – 7 ng/mL
1 – 11 months :1.05 – 2.45 ng/mL
1 – 5 years:1.05 – 2.69 ng/mL
6 – 10 years:0.94 – 2.41 ng/mL
11 – 15 years : 0.82 – 2.13 ng/mL
 Free T3 Adults:2.3 – 4.2 pg/mL
 TSH Adults <55 years:0.35 – 5.5 μIU/mL
Adults >55 years:0.5- 8.95 μIU/mL
Children :
1 – 4 days:1 – 39 μIU/mL
2 – 20 weeks :1.7 – 9.1 μIU/mL
21 weeks – 20 years: 0.7 – 6.4 μIU/mL
128
ASSAYS RELATED TO PREGNANCY, PRENATAL SCREEHING & INFERTILITY
 Testosterone 241 – 827 ng/dL (Males)
14 – 76 ng/dL (Females )
 LH Males:
23 -70 years :1.5 – 9.3 mIU/mL
>70 years :3.1 – 34.6 mIU/mL
Females:
Follicular phase: 1.9 – 12.5 mIU/mL
Mid cycle Peak :8.7 – 76.3 mIU/mL
Luteal phase : 0.5 – 16.9 mIU/mL
Pregnant : <0.1 – 1.5 mIU/mL
Post menopausal : 15.9 – 54 mIU/mL
Contraceptives: 0.7- 5.6 mIU/mL
 FSH Males:
23 – 70 years: 1.4 – 18.1 mIU/mL
Females:
Follicular phase: 2.5 -10.2 mIU/mL
Mid cycle peak :3.4 -33.4 mIU/mL
Luteal phase : 1.5 – 9.1 mIU/mL
Pregnant : <0.3 mIU/mL
Post menopausal : 23 – 116.3 mIU/mL
 Estradiol Pre pubertal child: 3 – 10 pg/mL
Males : 10 - 50 pg/mL
Females:
Follicular Phase:20 – 250 pg/mL
Midcycle:35 – 570 pg/mL
Luteal phase:23 – 256 pg/mL
Post menopausal: <21 pg/mL
 Progesterone Prepubertal child : 7 - 52 ng/dL
Males : 13 - 97 ng/dL
Females :
Follicular phase : 15 – 70 ng/dL
Luteal phase : 200 – 2500 ng/dL
Pregnant Female
First trimester : 725 – 4400 ng/dL
Second trimester : 1950 – 8250 ng/dL
Third trimester : 6500 -22,900 ng/dL
 Prolactin Males:2.1 – 17.7 ng/mL
Females:
Non pregnant:2.8 – 29.2 ng/mL
Pregnant: 9.7 -208.5 ng/mL
Post menopausal : 1.8 – 20.3 ng/mL
 Total human chorionic gonadotropin Males : <5 IU/L
Females:
Non pregnant : <5 IU/L
Pregnant :
4 weeks : 5 – 100 IU/L
5 weeks : 200 – 3000 IU/L
6 weeks : 10,000 – 80,000 IU/L
7-14 weeks : 90,000 – 5,00,000 IU/L
15-26 weeks : 5000 – 80,000 IU/L
27-40 weeks : 3000 – 15,000 IU/L
Trophoblastic disease : >1,00,000 IU/L
129
TUMOR MARKERS
 Alpha feto protein Gestational age:
15 weeks : 31.3 ng/mL
16 weeks: 36.3 ng/mL
17 weeks: 42 ng/mL
18 weeks :48.7 ng/mL
19 weeks:56.5 ng/mL
20 weeks:65.4 ng/mL
(-For screening of Down syndrome, values less
than above are abnormal.
-For screening of neural tube defects, values
more than twice the above are abnormal)
 Prostate specific antigen < 4 ng/mL
 CA 125 < 35 U/mL
 Carcinoembryonic antigen < 3 ng/mL
OTHERS
 Serum Cortisol 4.3 – 22.4 μg/dL
 Serum PTH 10 – 65 pg/mL
 Serum Iron 60 - 160 μg/dL (Males)
35 – 145 μg/dL (Females)
 Serum Ferritin 20 – 250 ng/mL ( Males)
10 – 120 ng/mL (Females)
 Plasma Homocysteine 3.7 – 14 μmol/l
 Serum Vitamin B12 >201 pg/mL
 Serum Folic acid >5.38 ng/mL
 ADA CSF:< 10 U/L
Other fluids : < 30 U/L
 Glycated haemoglobin (Whole Blood) 4 -6 %
 Serum ceruloplasmin 20- 60 mg/dL
 Troponin T 0 – 0.1 ng/mL
 Troponin I 0 – 0.4 ng/mL
 Sweat chloride 5 – 35 mmol/l
URINE INVESTIGATIONS
 24 Hour Protein <150 mg/dL
 Spot Protein creatinine ratio <0.2 mg/mg
 pH 4.6 – 8
 Specific gravity 1.016 – 1.022
 Sodium 40 – 220 mmol/day (Males)
27 – 287 mmol/day (Females)
 Potassium 25 – 125 mmol/day
 Chloride 110 – 250 mmol/day
 Osmolality 60 – 1200 mOsm/kg
 Magnesium 6 – 10 mmol/L
 Phosphorus 400 – 1300 mg/day
 Calcium 100 – 300 mg/day
 Uric Acid 250 – 750 mg/day
 Creatinine 1 – 2 g/day
 Creatinine clearance 85 – 125 mL/min ( Males)
80 – 115 mL/min (Females)
 Microalbuminuria 30 – 300 mg/day
130

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BIOCHEMISTRY

TITLE PAGE NO.

i. Clinical Biochemistry- a corner stone of medicine; its 2

ii. Safety measures and necessary precautions in lab 3

iii. A generalized concept of concentration and its 5

2. Colour Reactions Of Carbohydrates 7

3. Colour Reactions Of Proteins 13

II. HEMATOLOGY AND IMMUNOLOGY

2. Immunoassays (RIA, ELISA & Chemiluminescence) 27

3. Hemoglobin And Its Derivatives 32

III. MUSCULOSKELETAL SYSTEM AND ANS

2. Estimate of Phosphate in Serum 36

2. Oral Glucose Tolerance Test 52

1. Introduction to Acidimetry, pH Indicators, pH Meter, Buffers 58

2. Arterial Blood Gas Analysis and Its Interpretation 62

VI. CARDIOVASCULAR SYSTEM

2. Point Of Care Testing (POCT) 71

VII. GASTROINTESTINAL SYSTEM, HEPATOBILIARY SYSTEM AND

VIII. RENAL SYSTEM

Normal Constituents of Urine 83

2. Abnormal Constituents of Urine 89

Dry Chemistry Methods (Dip Sticks) 99

3. Estimation of Urea in serum 100

4. Estimation of Creatinine in serum 103

6. Serum Electrolytes And Ion Selective Electrodes 110

7. Case Reports 113

IX. MOLECULAR BIOLOGY, CANCER BIOLOGY

1. Polymerase Chain Reaction (PCR) 121

Who is a clinical biochemist?

Automation in clinical biochemistry

Safety measures in a laboratory

Safety do’s and don’ts for students

Some basic don'ts are:

Conversion of SI units to conventional units and vice-versa

Ways of expressing concentrations

Normality refers to compounds that have multiple chemical functionalities, such as

Monosaccharides Disaccharides Oligosaccharides Polysaccharides

Aldoses Ketoses Reducing Non-reducing Homo Hetero

Conc. H2SO4 removes water molecules from carbohydrates forming furfural or

1. Molisch’s reagent – 1% alcoholic solution of α-naphthol

Test procedure Observation Inference

Test procedure Observation Inference

Color of the precipitate Approximate conc. of glucose (gm %)

4. MODIFIED BARFOED’S TEST

Test procedure Observation Inference

Test procedure Observation Inference

H-C=O H2N-NH-C6H5 H - C = N – NHC6H5 H - C = N – NHC6H5

Sheaves of corn/ hay stack Presence of glucose/ fructose/

Powder puff appearance Presence of lactose

Sunflower appearance Presence of maltose

Fluffy ball appearance Presence of galactose

Broken needle appearance Presence of xylose

Hair thrown in water appearance Presence of arabinose

Test procedure Observation Inference

Test procedure Observation Inference

Test procedure Observation Inference

4. COLE’S MERCURIC NITRITE TEST (MODIFIED MILLON’S TEST)

Test procedure Observation Inference

Test procedure Observation Inference