Biochemistry Lab Manual

AMRITA SCHOOL OF MEDICINE
FARIDABAD, HARYANA
DEPARTMENT OF BIOCHEMISTRY
I MBBS - JOURNAL
FARIDABAD
LABORATORY MANUAL
OF
PRACTICAL BIOCHEMISTRY
FIRST YEAR M.B.B.S.
NAME OF STUDENT:
ROLL NO: YEAR:

EDITORIAL BOARD
Convener Dr (Maj Gen) T K Saha

Professor & Head
Co-ordinator Dr A K Harith
Professor
Dr AK Singhal
Associate Professor
Members Dr Diravyaseelan M
Assistant Professor
Dr Nidhi Thakur
Assistant Professor
Dr Vivek Sharma
Tutor
Dr Alka Yadav
Tutor
Priya Koundal
Tutor
Pratibha
Tutor
EDITORIAL BOARD
In keeping with dramatic recent advances in the biological sciences, we have endeavoured to
create a comprehensive, yet updated instruction model for the students of Amrita School of
Medicine, Faridabad.
Despite our efforts, it is more than likely that some errors-typographic, grammatical, spelling
or technical may have inadvertently crept in. Readers are requested to bring such errors to
our notice so that the necessary corrections could be carried out. Kindly send your comments
to:
Professor & Head

Department of Biochemistry
Amrita School of Medicine
Faridabad, Haryana-121002
FARIDABAD
DEPARTMENT OF BIOCHEMISTRY
CERTIFICATE
This is to certify that
Roll No. has satisfactorily carried out the practical work in Biochemistry as
prescribed by Amrita Vishwa Vidyapeetham, Faridabad for I M.B.B.S. Examination.
1st Term:
2nd Term:
Teacher in Charge
Professor & Head
Department of Biochemistry
CONTENTS
S. No. Competency No. & Competency Competency Page Date Signature

No.
Introduction BI11.1 1
1. Vacuum-assisted Blood Collection BI11.1 5
tubes and anticoagulants
2. Biomedical Waste Management BI11.1 9
3. Commonly used Laboratory apparatus BI11.1, 13
and equipment BI11.19
4. Principles of BI11.6, 19
Colorimetry/Spectrophotometry BI11.18
5. Determination of the absorption BI11.6, 25
Maximum (Λ Max) of a solution BI11.18
6. Effect of change of concentration on BI.11.6, 27
absorbance: Constructing a standard BI11.18
curve
7. pH and Buffers BI11.2 31
8. Estimation of amylase activity BI11.17 35
9. Effects of pH and EDTA on amylase BI11.17 39
activity
10. Estimation of Plasma Glucose BI11.17, 41
BI11.21
11. Glucose Tolerance Test BI11.17 47
12. Estimation of Triglycerides BI11.10 51
13. Estimation of Serum Cholesterol and BI11.9 55
HDL Cholesterol
14. Urine analysis-I BI11.3, BI11.4 61
15. Urine analysis-II BI11.4, 67
BI11.20
16. Estimation of Blood urea BI11.17, 75
BI11.21
17. Estimation of Serum Creatinine BI11.7, 79
BI11.21
18. Estimation of Urine Creatinine BI11.7 83
19. Estimation of Serum Calcium BI11.11 87
20. Estimation of Serum Inorganic BI11.11 91
phosphorus
21. Estimation of Serum uric acid BI11.17 93
22. Renal Function Test BI11.17, 97
Bi11.22
23. Estimation of Serum Bilirubin BI11.12 101
24. Estimation of Total Protein, Albumin BI11.8, 107
i
S. No. Competency No. & Competency Competency Page Date Signature
No.
and Determination of A:G ratio BI11.22
25. Estimation of Transaminase (AST/ALT) BI11.13 113

26. Estimation of Serum Alkaline BI11.14 119
Phosphatase
27. Evaluation of Liver Function Test BI11.17 123
28. Analysis of CSF BI11.15 127
28A Estimation of proteins in CSF 131
28B Estimation of glucose in CSF 133
29. Chromatography BI11.5, 137
BI11.16
30. Electrophoresis BI11.16 141
31. Blood Gas Analyzer BI11.16 145
32. Thyroid Function Tests BI 6.13 153
BI 6.14
33. Screening for Inborn Errors of BI11.5 157
Metabolism
34. DNA Isolation BI11.16 167
35. Polymerase Chain Reaction BI11.16 169
36. Glycated Hemoglobin BI4.7 173
37. Polyacrylamide Gel Electrophoresis BI11.16 175
38. Immunochmistry Techniques BI11.19 177
39. Radio Immuno Assay BI11.16 181
40. ELISA BI11.16 183
41. Quality Control in Clinical Laboratory BI11.16 187
42. Normal Ranges
Remarks:
Date: Signature:
ii
INTRODUCTION
The marked increase in the number and availability of laboratory diagnostic procedures has helped
rapidly diagnose and manage many clinical conditions. Usually, combinations of lab tests are done;
individual lab tests are rarely ordered. The Physician, however, should be judicious in selecting the
tests. A trend is also emerging to conduct certain biochemical investigations, which could reveal a
predisposition to specific disease processes in healthy individuals. The physician can then suggest
preventive measures to the person, e.g., elevated plasma cholesterol levels persisting for a long time
contribute to the development of cardiovascular diseases. These individuals should be advised for
appropriate dietary and lifestyle modifications to prevent or delay the development of a serious
disease.
Metabolic changes associated with specific disorders may give rise to a change in the biochemical
profile of a body fluid, e.g., blood Glucose in Diabetes Mellitus, serum Creatinine in renal failure,
etc. Hence, specific investigations are performed in body fluids when a particular disease is
suspected.
From a clinical point of view, one purpose of performing a test could be to corroborate a diagnosis or
rule it out. Other tests may be done to assess the severity of a disease process or monitor its progress.
Still, others may evaluate or monitor a therapeutic regimen's effectiveness or potential side effects;
specific tests can explain the prognosis of the disease and its probable outcome.
The final interpretation of the results of investigations, whether biochemical or of any other category
should be aligned with the overall context of the disease process and the patient’s clinical profile.
Biological samples
Some of the body fluids that are used for biochemical investigations are given below.
Body fluid Method of collection Examples of Investigations performed

Whole blood Obtained by arterial or Blood gases
venipuncture; collected with
anticoagulants like heparin.
Plasma Blood is collected with Enzymes, metabolites, and electrolytes
anticoagulants and
centrifuged; the supernatant
is plasma.
Serum Blood is collected without Enzymes, metabolites, and electrolytes
1
Body fluid Method of collection Examples of Investigations performed
anticoagulant, centrifuged
after clotting; the
supernatant is serum.
Urine Directly passed into a Glucose, proteins, bile salts, bile pigments, blood,
container or through a and steroids
catheter introduced into the
bladder
Cerebrospinal Lumbar puncture from Glucose and protein
fluid subarachnoid space
Gastric juice Aspiration by Ryle’s tube HCl, blood
Serous fluids Needle puncture to the Proteins
serous space (e.g. Pleural
and peritoneal)
Sweat Soaked into a filter paper Chloride
Table 1.1 List of biological samples used for biochemical investigations.
Presentation of results
1. Metabolites (glucose, urea, etc.) are expressed as mg/dL or mmol/L.
2. Electrolytes (Na+, K+) as mmol/L or mEq/L.
3. Enzyme activity is expressed as International Unit per liter (IU/L).
Enzyme activity is sometimes expressed in conventional units (e.g., amylase in Somogyi units and
phosphatase in King-Armstrong units)
Chemical units
 Molar solutions: Contain 1g molecular weight of the solute/L of solution. 1 Molar solution
of H2SO4 contains 98.08 g H2SO4/L (MW of H2SO4 = 98.08).
 Normal solution: Contain 1g equivalent weight of the solute/liter. 1 mole HCl, 0.5 mole
H2SO4, and 0.333 mole H3PO4 in 1000 mL of solution in water have a normality of 1.
No. of moles x valency = No. of equivalents

Molarity x valency = Normality
The following equations define the expression of concentrations:
Molarity of a solution = No. of moles of solute / No. of liters of solution
Normality of a solution = No. of g equivalents of solute / No. of liters of solution
2
The unit of measure commonly used to express the concentrations of electrolytes in plasma is
milliequivalents (mEq/L) or millimoles (mmol/L).
No. of mEq = [Mass (g) x 1000] / Equivalent Weight (g)

= [Mass (g) x 1000 x valency] / MW
No. of mmol = [Mass (g) x 1000] / MW
mg/100 mL can be converted to mEq/L or mmol/L as follows:

mEq/L = [(mg/100 mL) x 10 x valency] / atomic or molecular mass
mmol/L = [(mg/100 mL) x 10] / atomic or molecular mass
Example: If serum sodium is 322 mg/100 mL [3220 mg/L], the atomic weight of Na = 23, valency =
1 mEq/L = (322 x 10 x 1) / 23 = 140
Enzyme activity When enzymes are quantitated relative to their activity rather than directly
measuring their concentration, activity units report enzyme levels. International unit (IU) is defined
as the amount of enzyme that will catalyze the reaction of 1 μmol of substrate per minute under
specified conditions of temperature, pH, substrates, and activators. The enzyme concentration is
usually expressed in international units per liter (IU/L). The unit of enzyme recognized by the
International System of Units is the katal (mol/s).
Interpretation of results
Values obtained with a particular parameter are interpreted as increased, decreased, or within the
normal (reference) range.
Reference values
Values obtained from individuals in good health as judged by other clinical and laboratory parameters
after suitable standardization and statistical analysis under definite laboratory conditions.
Normal (reference) range

The normal range of a parameter is that value within which more than 95% of the normal health
population lies.
Quality control
Quality control (QC) is all the processes involved in assuring the accuracy and precision of the
analytical processes and detection of immediate error. As most of the clinical decisions are made on
the laboratory results, it is essential that the results generated by the laboratory meet the minimum
specified quality standards. Quality Control process are activities done in the laboratory to ensure that
the quality of the reports generated meet the specified standards.
Accuracy: Accuracy is defined as how close the observed value is to the true value of the analyte.
3
Precision: Refers to the reproducibility of the results. It is expressed in terms of Standard
Deviation(SD). The smaller the SD observed, the better the precision is. However, it is important to
note that Precision alone does not guarantee the Accuracy of the result.
Laboratory hazards
 Biological hazards: Every patient’s specimen must be treated as potentially infectious. Blood
samples from high-risk patients (like AIDS, Hepatitis B, etc.) should be collected, transported,
handled & processed using strict precautions. Gloves, masks, and gowns should be worn.
Specimens should remain “capped” during centrifugation to prevent the formation of infective
aerosols.
 Physical & chemical hazards: Careless handling of apparatus and reagents is a common
cause of laboratory accidents resulting in burns or fires that must be reported and treated
promptly. Hence, it is necessary to ensure the safety of self, personnel, and equipment.
Safety rules to be followed in the laboratory
1. Good personal behavior/habits

a. Students must wear laboratory overalls, whenever they are in the laboratory for prac-
tical classes.
b. Long hair should not be left loose.
c. One should not eat or drink in the work area.
d. No mouth pipetting in the lab. Auto pipettes and dispensers should be used.
2. Good housekeeping
a. Work area should be kept free of chemicals, dirty and broken glassware.
b. Chemicals should be stored in the proper place.
c. All reagents and solutions should be neatly labeled.
3. Good laboratory technique
a. New or unfamiliar equipment should only be operated once you have been instructed
about it.
b. All instructions & labels should be carefully read.
c. For safe handling, use, and disposal of chemicals, one must know their properties and
hazards.
d. One should be aware of emergency procedures and become familiar with the location
of fire exits and fire extinguishers.
4
1. VACUUM-ASSISTED BLOOD COLLECTION TUBES AND ANTICOAGULANTS
A vacuum-assisted blood collection tube is a sterile glass or plastic tube with a closure that is
evacuated to create a vacuum inside the tube, facilitating the draw of a predetermined volume of
sample. These tubes are widely utilized in healthcare settings for drawing blood samples for various
laboratory tests. They are available in different sizes with specific color-coded tops indicating the
tube content.
Most blood collection tubes contain an additive that either accelerates the clotting of the blood (clot
activator) or prevents the blood from clotting (anticoagulant).
Figure 1.1 Different types of sample collection tubes
Blood collection tube top colours
1. Red
2. Yellow
3. Light Blue
4. Green
5. Lavender
6. Grey
5
Tube additives
Most sample collection tubes contain additives. If the additive is an anticoagulant, the blood will not
clot, and the specimen will be whole blood that can be centrifuged to obtain plasma. All other
additives and additive-free tubes (e.g., glass red top) produce serum specimens.
An additive functions optimally when the tube is filled to its stated volume and gently inverted
immediately after collection to mix the additive with the blood. Specimen quality can be
compromised if a tube is partially filled. Shaking or vigorous mixing can haemolyse the blood,
making it unsuitable for testing. Additive reliability is guaranteed until expiration if the tube is
handled and stored correctly. Expiration dates should be checked, and expired tubes should be
discarded.
The most common additives are the following:

Anticoagulants: They prevent blood from clotting and include Ethylene Diamine Tetra Acetic acid
(EDTA), Citrate, Heparin, and Oxalates. Each is designed for use in certain types of testing, and it is
essential to use the correct one.
Anti glycolytic agents: They prevent glycolysis, which can decrease glucose concentration by up to
10 mg/dL per hour. The most commonly used antiglycolytic agent is Sodium Fluoride (NaF) which
preserves glucose for up to 3 days and inhibits bacterial growth. NaF inhibits the enzyme Enolase in
the glycolytic pathway. Inhibitors of the Glycolytic pathway is used because RBCs do not have
Mitochondria. NaF is often combined with Potassium Oxalate as an anticoagulant for the collection
of plasma specimens for glucose estimation.
Clot activators: Pro-coagulation factors (thrombin) and substances like silica enhance clotting by
providing more surface area for platelet activation. The clot activators in gel separator tubes and
plastic red-top tubes are typically silica.
Thixotropic gel separators (Yellow Tube) are inert substances in or near specific tubes' bottoms.
During centrifugation, the gel lodges between the cells and the fluid, forming a physical barrier
preventing cells from metabolizing substances in the serum or plasma.
Trace element–free Tubes

Trace element–free tubes are as contamination free as possible. They are used to collect specimens
for trace elements, toxicology, nutrient, and other tests. As the analytes being tested is in very minute
quantity, these special tubes are required to prevent false positive results.
6
Order of draw
The order of draw is a particular sequence of tube collection that reduces the risk of specimen
contamination either by microorganisms (e.g., blood cultures) or additive used in the sample
collection tube. For example Potassium salt of EDTA is used in the Lavender tube. If the Red Tube
(for serum) is collected after the Lavender tube then there is chances that the Potassium of the
anticoagulant contaminates the sample in the serum tube giving falsely high results of Potassium in
the patient. To prevent such occurrence, we have to follow the correct order of draw.
The sequence of collection of evacuated tubes in a multi-draw should be in the order as follows:
1. Sterile tube (e.g., blood culture)

2. Coagulation tube (e.g., blue top)
3. Serum tube with or without clot activator, with or without gel (e.g., red top/yellow top)
4. Heparin tube with or without gel separator (e.g., green top)
5. EDTA tube with or without gel separator (e.g., lavender top)
6. Glycolytic inhibitor tube (e.g., gray top)
7
Different types of blood collection tubes
Red Top Yellow Top Light blue Top
• Additive: Clot activator and gel • Additive: Sodium citrate

 Additive: None or contains silica
• What additive does: On centrifugation,
particles that act as clot activators. • What additive does: Binds
the serum comes above the gel and the and removes calcium to pre-
 What additive does: Clot activator
clot (RBC) stay below the gel. Im- vent blood from clotting
promotes blood clotting with glass
portance of this is that the sample probe
or silica particles. • Uses: Coagulation tests in he-
of the analyser does not get blocked by
 Uses: In Biochemistry, blood clots. matology
Immunology and Serology
• Uses: Same as the red top tubed.
Green Top Lavender Top Gray Top
• Additive: Heparin (Sodium/Lith- • Additive: EDTA

ium/Ammonium) • Additive: Sodium Fluoride
• What additive does: Removes cal-
• What additive does: Inactivates and potassium oxalate
cium, preventing the clotting of blood
thrombin to prevent clotting. • What additive does: So-
• Uses: Hematology (CBC) and blood
• Uses: Chemistry Testing (Plasma dium Fluoride-Antiglyco-
bank
determinations in chemistry): am- lytic agent Potassium oxalate
monia, carboxyhemoglobin & -anticoagulant
STAT electrolytes, chromosome • Uses: Glucose
screening, insulin, renin, and aldos-
terone
Table 1.1 Different types of blood collection tubes
8
2. BIOMEDICAL WASTE MANAGEMENT
Bio-medical waste is any waste generated during human beings' diagnosis, treatment, immunization,
or research activity. The waste produced during healthcare activities carries a higher potential for
infection and injury than any other type of waste. Hence, proper disposal of biomedical waste is of
utmost importance.
Bio-medical waste management rules recommend using appropriate color-coded containers in which
the various types of biomedical waste must be disposed of. Figure 2.1 depicts the current biomedical
waste management protocol at Amrita Hospital.
Figure 2.1 Biomedical waste management protocol

9
Category Type of waste Treatment and disposal Type of
bag/container
Yellow 1. Human and animal anatomi- Incineration Yellow non-
cal waste. chlorinated
plastic
2. Items contaminated with Incineration
bag/container
body fluids, like dressings,
with a biohazard
plaster casts, cotton swabs,
sign
and bags containing residual
or discarded blood or blood
components.
3. Expired or discarded medi- Incineration/return to the
cines/cytotoxic waste, in- manufacturer
cluding all items contami-
nated with cytotoxic drugs.
4. Chemical waste: disinfect-
ants or chemicals used to
produce disinfectants. Incineration/encapsulation
in hazardous waste
5. Discarded linen or mattress
storage facility
contaminated with blood or
body fluids. Masks and
gowns. Incineration
6. Microbiology, biotechnol-
ogy, and other clinical labor-
atory waste. Blood bags, lab
cultures, stocks or speci- Autoclaving followed by
mens of microbes, live or at- incineration
tenuated vaccines, human
and animal cell cultures
used in research, dishes, and
devices used for cultures.
Red 1. Contaminated waste (recy- Autoclaving followed by Red colored non-
clable): waste generated shredding, then sent to chlorinated
from disposable items such authorized recyclers. plastic bag/
as tubing, bottles, IV sets, container with a
catheters, urine bags, sy- biohazard sign
ringes (without needles),
vacutainers, and gloves.
White 1. Sharps including metals: Autoclaving followed by Puncture-proof,
Needles, syringes with fixed shredding/ mutilation leak-proof, and
needles, needles from needle tamper-proof
tip cutter or burner, scalpels, container.
10
Category Type of waste Treatment and disposal Type of
bag/container
blades, or any other contam-
inated sharp objects that
may cause punctures and
cuts.
Blue 1. Glassware and metallic Cleaning followed by Puncture-proof,
body implants: broken or disinfection or leak-proof, and
discarded and contaminated autoclaving, then send for tamper-proof
glass, including medicine vi- recycling container.
als and ampules, except
those contaminated with cy-
totoxic wastes.
Black 1. General solid waste: office Disposal on secured Black-colored
waste like paper, plastic co- landfill plastic bags/
vers, and disposable con- containers.
tainers.
Table 2.1 Biomedical waste management protocol.
11
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12
3. COMMONLY USED LABORATORY APPARATUS AND EQUIPMENT
Glassware
Glassware is made up of complex silicates containing boron dioxide. They resist acid, alkali,
temperature, and radiation corrosion and have a low expansion coefficient. Some of the most used
glassware in the biochemistry laboratory are:
1. Flasks
a. Conical flasks – for heating/boiling fluids
b. Volumetric flasks – it is graduated to measure the exact volumes of liquids. Standard
solutions are prepared by using the volumetric flask. It is available in different vol-
umes from 10 mL to 5 L.
c. Round and flat-bottomed flasks - for preparing solutions.
2. Beaker: a cylindrical container for storing, mixing, and heating liquids. It can also be used to
estimate volume.
3. Bottles: different kinds of bottles include:
a. Specimen bottles with top screws.
b. Reagent bottles.
13
4. Funnel: It holds filter papers to transfer liquids or fine-grained substances into containers with
small openings.
5. Measuring jar/measuring cylinder: commonly used for measuring liquids and preparation
of reagents. There are available in different volumes. While measuring liquids, the upper me-
niscus is considered for colored solutions, and the lower meniscus is considered for colorless
solutions.
6. Test tubes: small glass tubes used to handle chemicals, especially for qualitative experiments
and assays.
7. Pipettes: Pipettes are used to transfer the measured volume of liquid. Types of pipettes in-
clude:
a. Serological/blowout pipettes: marking is right up to the tip of the lower end. Contents
must be blown out till the last drop.
b. Mohr pipette/non-blowout pipette: no graduation till the last, and the contents will not
be delivered until the last marking.
14
c. Volumetric pipette/bulb pipette: It has a large bulb with an extended narrow portion
above it with a single graduation mark.
d. Micro pipettes/auto pipettes: variable and fixed micropipettes are available to transfer
small quantities of samples.
e. Micro syringes: used for precision sampling in chromatography (like high-perfor-
mance liquid chromatography and gas chromatography)
f. Pasteur pipettes: plastic or glass pipettes used to transfer small amounts of liquids.
Pasteur pipettes are not graduated.
g. Transfer pipettes/Beral pipettes: They are like Pasteur pipettes but are made from a
single piece of plastic, and their bulb can serve as liquid-holding chambers.
h. Ostwald Folin pipette: A special pipette used in measuring viscous fluids such as
whole blood.
15
Commonly used laboratory equipment
1. Centrifuge
Centrifugation is a technique used to separate particles using a centrifugal field. The particles are
suspended in a liquid medium and placed in a centrifuge. The tube is then placed in a rotor and
rotated at a certain speed. Rotation of the rotor about a central axis generates a centrifugal force
upon the particles in the suspension. Particles that differ in density, size, or shape sediment at
different rates. The rate of sedimentation depends upon:
1. The applied centrifugal field
2. Density and radius of the particle
3. Density and viscosity of the suspending medium
The tubes/centrifuge cups in the rotor head must be balanced before centrifugation. Tubes should be
capped appropriately, and the centrifuge lid should be closed during centrifugation. This will prevent the
release of infectious material inside the centrifuge by aerosol formation.
Uses: Separation of serum or plasma in clinical biochemistry laboratories.
2. Water bath
It is a device that maintains water at a constant temperature. It can provide temperatures ranging
from room temperature to 100°C.
Uses: To incubate samples in water at a constant temperature.
3. Weighing balance
Weighing balances are used for measuring large quantities of materials, while analytical balances
are used for accurately weighing small quantities. Chemicals should be weighed in a container/wax
paper, not directly on the pan.
16
4. Hot air oven
It is a double-walled steel chamber controlled thermostatically and electrically heated to dry and
sterilize the lab glassware. Sterilizing time varies through – 3hrs at 140°C, 1hr at 160°C, 30min at
180°C. Hot air ovens are unsuitable for sterilizing culture plates, masks, etc.
5. Autoclave
It utilizes steam under high pressure for sterilizing.
Uses: For sterilization purposes - surgical instruments, culture media, rubber material, gowns, and
dressings, and pre-disposal treatment and sterilization of biomedical waste material.
6. Magnetic stirrer
A magnetic stirrer employs a rotating magnetic field to cause a stir bar immersed in a liquid to spin
quickly, thus stirring it.
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18
4. PRINCIPLES OF COLORIMETRY/SPECTROPHOTOMETRY
When light passes through a colored solution, specific wavelengths are selectively absorbed. The plot
of absorbance (A) also called Optical Density (OD) against wavelength (λ) is called the absorption
spectrum of the compound in solution. The wavelength at which maximum absorption occurs is called
that compound's absorption maximum (λ max). The light that is not absorbed is transmitted through
the solution and gives the solution its color. This is called transmittance (T).
If the concentration of the substance in solution is increased linearly, absorbance rises linearly, and
transmittance decreases exponentially. Hence absorbance is directly proportional to the concentration
of the substance and inversely proportional to transmittance. The term absorbance can be defined as
1
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒(𝐴) = 𝑙𝑜𝑔
𝑇
Absorbance has no units. Photometric instruments electronically convert the measured transmittance
to absorbance values.
Photometric instruments measure transmittance, which is defined as follows:
𝐼𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦𝑜𝑓𝑡ℎ𝑒𝑒𝑚𝑒𝑟𝑔𝑒𝑛𝑡(𝑡𝑟𝑎𝑛𝑠𝑚𝑖𝑡𝑡𝑒𝑑)𝑙𝑖𝑔ℎ𝑡
𝑇𝑟𝑎𝑛𝑠𝑚𝑖𝑡𝑡𝑎𝑛𝑐𝑒𝑇 =
𝐼𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦𝑜𝑓𝑡ℎ𝑒𝑖𝑛𝑐𝑖𝑑𝑒𝑛𝑡𝑙𝑖𝑔ℎ𝑡
Transmittance is usually expressed in a range of 0 to 100%.
Thus photometry is chiefly governed by two laws: When a parallel beam of monochromatic light
passes through a solution, the absorbance (A) of the solution is directly proportional to the
concentration (c) of the compound in the solution. This is Beer's law.
Each successive solution layer absorbs a constant proportion of the light entering the solution,
although the absolute amount entering each layer diminishes progressively. Therefore, absorbance is
directly proportional to the thickness or length of the light path (l) through the solution. This is
Lambert's law.
Absorbance (a)  Concentration (c) (Beer's law) --------------(1)
Absorbance  Path Length (Lambert's law) -----------(2)
By combining (1) and (2), we get
A = ε c l
ε, the proportionality constant, is termed the molar absorption coefficient.
19
It is specific for a given substance at a given wavelength. It is the absorbance of one molar solution
of a substance with a light path of one centimeter. Beer's law applies only to dilute solutions. In
colorimetry, the absorption coefficient is not usually used. The concentration of an unknown solution
can be determined by using equation 1, which is derived as follows:
The absorbance of test sample (At) = ε x concentration of test (Ct) x l
The absorbance of the standard sample (As) = ε x concentration of the standard (Cs) x l
Hence,
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒𝑜𝑓𝑡𝑒𝑠𝑡(𝐴𝑡)
𝐶𝑜𝑛𝑐. 𝑜𝑓𝑡𝑒𝑠𝑡(𝐶𝑡) = × 𝐶𝑜𝑛𝑐. 𝑜𝑓𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑(𝐶𝑠)
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒𝑜𝑓𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑(𝐴𝑠)
The light path, l, is usually kept constant in photometric measurements at 1 cm. This is the diameter
of the tube (called the cuvette) containing the solution.
A standard (or calibrator) represents the substance whose concentration is sought to be determined.
The concentration of the compound in the test sample is obtained by comparing its absorbance with
that of a known concentration of a standard solution. Ideally, a series of standards of known
concentration are prepared to get a standard (calibration) curve. This helps to determine the range of
concentrations over which Beer's law is obeyed.
Appropriate blanks to exclude the absorbance contributed by the solvents and reagents used- i.e., by
anything other than the compound of interest- are also essential for any photometric measurement.
Colorimetry
Colorimetry uses the basic principles of photometry, but the solutions must be colored, i.e., absorb
light in the visible range. To utilize this principle in clinical biochemistry tests, colorless compounds
are converted into colored compounds using chemical reactions. Under defined reaction conditions,
the color formed is proportional to the amount of the original colorless compound.
Spectrophotometry
Spectrophotometry works on the same principle as colorimetry, but it covers a broad range of
wavelengths (ultraviolet, visible, and infrared)
Photometric instruments
Colorimeter
A Colorimeter measures the intensity of light transmitted through a colored solution. It uses light in
the visible range. Light from a tungsten lamp is passed through a suitable filter to obtain light of a
desired wavelength, then passed through the solution in a cuvette. Transmitted light falls on a
20
photocell, generating a current proportional to the light intensity. The photocell is connected to a
galvanometer, which is used to measure percentage transmission or absorbance (Figure 4.1).
Figure 4.1 Diagrammatic representation of a colorimeter
Use of blank: Three types of blanks are used to eliminate errors.

1. Water blank: Used to adjust the Absorbance or Optical Density (OD) to zero and % T to 100.
2. Reagent blank: The preparation of a blank is essential in colorimetric analysis because the
colored reagents themselves absorb some light. Therefore, the blank is prepared by adding all
reagents except the substance to be estimated.
3. Serum blank: In case the serum has high colour (as in cases of sever jaundice where the serum
colour becomes deep yellow) then the serum also needs to be included in the blank. In such a
case we use serum blank (read note in serum bilirubin estimation)
Use of the standard solution: It is the solution of the known concentration of the substance in pure
form to be estimated. The concentration and OD of the standard solution are known; therefore, the
unknown concentration can be calculated.
Operation steps
1. Proper filter is selected.
2. Instrument is set to zero O.D or 100% T with distilled water blank.
3. O.D. of reagent blank is read.
4. O.D. of standard and unknown solutions are read.
21
Spectrophotometer
In contrast to a colorimeter, a spectrophotometer is more sensitive and covers a broad range of
wavelengths. Spectrophotometer uses light sources that emit light in the spectrum's ultraviolet,
visible, and infrared regions. The desired wavelength is selected using a prism or diffraction grating,
and narrower bandwidths than what is possible in a colorimeter can be chosen. Since light in the
ultraviolet and infrared ranges is also emitted, the compound to be estimated does not necessarily
have to be colored. It can be measured directly if they significantly absorb, even at these wavelengths.
This offers a significant advantage over the colorimeter, which is restricted to the visible range. Using
a narrower bandwidth than is available with ordinary filters, the absorbance is often higher, and the
relation between absorbance and concentration remains linear over a wide range in a
spectrophotometer compared to a colorimeter. A spectrophotometer is usually required if very dilute
solutions are to be measured.
Analytical methods depending on ultraviolet absorption are commonly used in clinical chemistry and
research. Examples include serum enzyme assays of glucose, urea, uric acid, etc., which take
advantage of the UV absorption of NADH and NADPH coenzymes at 340 nm. For such methods, a
spectrophotometer is essential.
Microplate reader
A microplate reader is a modification of the spectrophotometer, which enables the quantitation of up
to 96 samples per microplate. Microplates usually have a 12 x 8 well format. (Figure 4.2).
Figure 4.2 Microplate reader
Automated and Semi-automated clinical chemistry analyzers

Automated and semi-automated clinical chemistry analyzers have spectrophotometers as one of their
integral components, along with components that carry out repetitive tasks like pipetting, dispensing,
22
and mixing. They help in the processing, transportation, and testing of many clinical specimens in an
efficient manner.
Figure 4.3 Semi-automatic (A) and Automated (B) biochemistry analyzer
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24
5. DETERMINATION OF THE ABSORPTION MAXIMUM (Λ MAX) OF A SOLUTION
When light passes through a colored solution, specific wavelengths are selectively absorbed. The plot
of absorbance (A) against wavelength (λ) is called the absorption spectrum of the compound in
solution. The wavelength at which maximum absorption occurs is called that compound's absorption
maximum (λmax).
Procedure
1. Set the filter at the lowest available wavelength. (e.g., 400nm)
2. Take DW as blank and adjust the absorbance of the instrument to zero.
3. Record the absorbance of the standard solution (KMNO4 solution of 12mg/dL)
4. Repeat the same procedure for subsequent wavelengths in the colorimeter (e.g., 450nm,
500nm, etc.).
5. Plot the Wavelength (X-axis) vs Absorbance (Y-axis) in a graph sheet.
6. The wavelength that gives maximum absorbance is the λmax of the test solution.
Table for identifying the λmax of the KMNO4 solution.
Wavelength (nm) Absorbance (OD)
25
26
6. EFFECT OF CHANGE OF CONCENTRATION ON ABSORBANCE:
CONSTRUCTING A STANDARD CURVE
A standard curve, also known as a calibration curve, is a graphical representation of the relationship
between the concentration of a substance (analyte) and a measurable response. It is a fundamental
tool in biochemistry and is used to quantify the concentration of unknown samples based on their
measured response.
To create a standard curve, a series of samples with known analyte concentrations are prepared and
analyzed using an analytical method, such as Colorimetry or Spectrophotometry. The instrument's
response (e.g., absorbance) to each known concentration is plotted on a graph, usually with the
concentrations on the x-axis and the instrument's response on the y-axis.
The resulting curve is typically linear, although it can be other shapes depending on the nature of the
analyte and the detection method. This curve serves as a reference, allowing us to determine the
concentration of an unknown sample by measuring its response and then using the standard curve to
interpolate or extrapolate the corresponding concentration.
Figure 6.1: Example of a standard curve
Procedure
The standard solution of KMnO4 given to you has a concentration of 12 mg/dL. Plot a standard curve
using different concentrations of KMnO4 by making dilutions of 1.2, 2.4, 4.8, 9.6, and 12mg/dL (10
mL each).
1. Set the filter at the predetermined λ max of the test solution (ref to the results of chapter 6)
2. Set the instrument using the DW as BLANK
3. Record the absorbance of the test solution at different dilutions.
27
4. Start measuring the most dilute solution continuing through the most concentrated.
Table for making dilutions for KMNO4 solution
Required dilution Volume of KMNO4 Volume of DW Total volume

1 1.2 mg/dL 10 mL
2 2.4 mg/dL 10 mL
3 4.8 mg/dL 10 mL
4 9.6 mg/dL 10 mL
5 12 mg/dL 10 mL 0 mL 10 mL
Table for making the standard curve of KMNO4
Concentration Absorbance
1 1.2 mg/dL
2 2.4 mg/dL
3 4.8 mg/dL
4 9.6 mg/dL
5 12 mg/dL
Plot the absorbance (Y-axis) vs. concentration (X-axis) graph and obtain the standard curve.
Uses of the standard curve

Some of the uses of standard curves in biochemistry include:
1. Quantitative analysis: Standard curves are used to determine the concentration of an unknown
sample based on its measurable response. For instance, in glucose estimation assays, a
standard curve is generated using known concentrations of glucose and the absorbance values,
and the amount of glucose in the patient sample is quantified by measuring the absorbance
and comparing it to the standard curve.
2. Validation of assay linearity and sensitivity: Standard curves assess an assay's linear range and
sensitivity. A well-constructed standard curve should demonstrate a linear relationship
between concentration and response within a specific range.
3. Assay calibration: Standard curves are essential to calibrate instruments and validate assay
performance to ensure consistent and accurate results over time.
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29
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30
7. pH AND BUFFERS
Measurement of pH
All the biochemical reactions in the body take place in an aqueous environment, and enzymes catalyze
most of these reactions. Enzymes are optimally active at a particular H+ ion concentration. The H+
ion concentration varies very little in any biochemical fluid or environment. Depending on the
situation, this variation is arrested by some bases or acids which absorb or donate H+ ions. This
phenomenon is called buffering.
H+ ions can be measured and expressed as mol/L. However, there is a more convenient way to express
H+ ion concentration, i.e., pH. pH is defined as the negative logarithm of H+ ion concentration.
pH = - log [H+]
In aqueous solutions, the pH ranges from 0 to 14. The H+ or OH- ion molar concentration in pure
water is 1 x I0-7mol/L. Pure water is considered neutral, with a pH close to 7.0 at 25 °C. With water,
at a neutral point, [H+] = [OH-] = 10-7 mol/L. pH at a neutral point is -log [10-7mol/L], So neutral pH=
7. pH above 7 indicates that the solution is alkaline, and pH less than 7 is acidic. 1M HCl would give
a pH of 0. 1M NaOH would give a pH of 14.
The pH of blood is tightly regulated between 7.35 and 7.45 (slightly alkaline), while gastric juice has
a pH of ~1.0 (highly acidic).
pH meter
The most accurate method for routine pH measurement is the pH meter, in which a change in [H+] is
measured as a change in electrical potential. If a metal rod is placed in a solution of its salt, it acquires
some electric potential. If two dissimilar metals are dipped into their salts' solutions, the potential
difference can be measured or calculated from the two separate potentials generated. A standard
electrode is thus required, against which the potential of all other electrodes can be compared. This
is the standard hydrogen electrode, consisting of a platinum rod dipped in an aqueous solution with a
given H+ activity in which hydrogen gas is bubbled continuously at 1-atmosphere pressure. But, as
this is too cumbersome to be used as a reference electrode for routine use, other secondary reference
electrodes of known potential in relation to the standard hydrogen electrodes are used.
The most commonly used secondary reference electrode is the calomel electrode, consisting of
mercury-mercurous chloride in contact with a saturated solution of KCl. Its potential is pH-
independent. The other secondary reference electrode is silver-silver chloride in contact with a
saturated KCl solution.
31
The glass electrode is the most commonly used pH-dependent unit in the pH meter. Certain types of
borosilicate glass are permeable to H+ but not to other cations and anions. Therefore, if a thin glass
membrane separates two solutions of different pH, a potential difference is generated across the
membrane, the magnitude of which is given by the equation
𝑅𝑇
𝐸 = 2.303 𝑙𝑜𝑔
𝐹
Where E = potential, R = gas constant, T = absolute temperature
F = Faraday constant, [H+]i = concentration on the inside which is fixed (0.1N HCl), and [H+]o =
concentration on the outside (test sample).
The voltage measured by such a system is primarily the difference between the glass and the reference
electrodes. It is linearly related to the pH of the test solution, i.e., [H+]o. The system, therefore, consists
of the glass electrode in contact with the solution to be measured, the calomel reference electrode, a
KCl bridge (the KCl should flow slowly into the sample), and the measuring device (meter) (Figure
1). These are designed so that pH 7 gives a zero potential. High resistances are used, so that little
current is drawn from the circuit (a sizeable current flow could change the ionic concentration).
Certain precautions have to be observed while using a pH meter. The glass electrode is fragile and
must be handled with care. It must not be left to dry. It is usually kept dipped in 3M KCl.
The temperature compensation dial must be set before calibrating, as the potential produced depends
on temperature. The meter must be calibrated first with a standard buffer of pH 7, then with a standard
of pH 4 (if the test sample is acidic), or with a standard of pH 9 or 10 ( if the test sample is basic).
Figure 7.1 pH meter (A) and the schematic diagram of a pH meter (B)
32
Ion-Selective Electrodes
An ion-selective electrode (ISE), also known as a specific ion electrode, is a transducer that converts
the concentration of a particular ion dissolved in a solution into an electrical potential. ISEs can
measure the concentrations of various ions, such as hydrogen, sodium, potassium, calcium, and
chloride.
An ion-selective electrode consists of an ion-selective membrane, an inner reference electrode, and a
reference electrode in contact with the sample solution. The ion-selective membrane is a barrier
between the sample solution and the two electrodes, allowing only the specific ion of interest to
penetrate. The reference electrode allows a reliable measure of the ion activity in the solution,
regardless of any changes in the sample’s pH or temperature. The inner reference electrode provides
a fixed electrical reference point and compares the ion activity in the sample solution against a
predetermined ion concentration.
The critical component of the ion-selective electrode is the ion-selective membrane, which consists
of an insoluble material permeable to a specific ion. This permeability facilitates an ion exchange
between the sample solution and the membrane, creating an equilibrium between the two components.
Precautions while using a pH meter

1. While a pH meter is not used, it should always be dipped in a storage solution so the glass
electrode doesn’t dry out. Drying the glass electrode will affect the pH measurement. The
most used storage solution is 3M KCl.
2. Do not scrub the surface of the glass electrode. Scrubbing will damage the semipermeable
membrane and affect the pH measurement. Remove any water or solution from the glass elec-
trode by gently dabbing the electrode with absorbent paper.
3. Glass electrodes should be cleaned using deionized water between every measurement.
4. Ensure the calibration and measurements are carried out at room temperature. The temperature
will alter the pH. Some pH meters come with a thermometer and adjust the measured pH
based on the temperature.
Steps involved in operating a pH meter

1. Before using the pH meter, remove the glass electrode from the storage buffer and wash it
with deionized water.
2. Calibration: Turn on the pH meter. Dip the electrode in the pH 7 buffer solution. Wait for
the reading to stabilize, then adjust the pH meter to read 7 if necessary. Repeat the same pro-
cess with the pH 4 (and/or pH 10) buffer solution. Adjust the meter as needed. Some pH meters
have automatic calibration; follow the manufacturer's instructions.
33
3. Sample preparation: Ensure your sample is at room temperature. Stir or mix the sample
gently to homogenize it.
4. Measuring pH: Immerse the clean electrode into the sample solution. Avoid hitting the sides
or bottom of the container. Allow the reading to stabilize. pH values typically stabilize within
a few seconds to a minute. Record the pH reading from the meter's display.
5. Cleaning and storage: Rinse the electrode with distilled water after each measurement. If the
electrode is not used immediately, store it in a storage solution. Properly clean the electrode
at the end of the day or when switching to a different sample type.
34
8. ESTIMATION OF AMYLASE ACTIVITY
Amylase (EC 3.2.1.1)

Amylases are a group of hydrolases that split complex carbohydrates of D-glucose residues linked by
alpha-1,4-glycosidic bonds. Hence it is known as 1,4 alpha-D Glucan Glucanohydrolase. The enzyme
does not hydrolyze the alpha-1,6-linkages at branch points. For straight-chain polyglucans (e.g.,
amylose), maltose and some residual glucose are the end products. In contrast, the end products of
branched-chain polyglucans (e.g., amylopectin and glycogen) are maltose, glucose, and limit dextrin.
Estimation of serum amylase

To estimate amylase activity, serum or heparinized plasma should be used since all other
anticoagulants bind calcium.
Amyloclastic method
These assays monitor the decrease in substrate concentration, i.e., starch. This can be done by
Iodimetry. In Iodimetry, large starch chains in helical form react with molecular iodine to form a deep
blue starch-iodine complex. The hydrolysis of starch is accompanied by a gradual loss of ability to
bind iodine and hence a decrease in color.
Saccharogenic method
Amylase hydrolyses starch to Maltose. The amount of Maltose produced is estimated by measuring
the reduction. In the following procedure, reduction of 3,5- Dinitro Salicylic Acid (DNSA) is used
to assess the Maltose (a reducing sugar) formed in the reaction.
Chromogenic method
These assays use artificially synthesized dye-labeled amylase substrates. These substrates are
synthesized by linking amylose or amylopectin to indicator groups like 4-Nitrophenol. The amount
of dye released into the solution is directly proportional to the amylase activity. This is the currently
recommended method by the IFCC (International Federation of Clinical Chemistry and Laboratory
Medicine) for estimating serum amylase activity.
The International Unit of amylase activity

One unit of amylase activity is defined as the activity of enzyme that liberates reducing substances
with a reducing value equivalent to 1 µmol of glucose, in a 1-minute reaction, at 40°C and pH 7.
35
The activity of enzymes is generally expressed in International Units per Litre of serum (IU/L)
Precautions
1. Avoid pipetting by mouth to prevent contamination by salivary amylase.
2. Avoid anticoagulants that chelate calcium, such as oxalate, citrate, and EDTA.
Reagents: Buffer, starch solution of pH 7, chromogen solution containing DNSA, and a stop solution
consisting of NaOH
Procedure
B (mL) S (mL) T (mL) C (mL)
Substrate 1.5 1.5 1.5 1.5
Buffer (pH 7.0) 1 1 1 1
DDW 0.5 - - -
Std glucose (1.5 mg/mL) - 0.5 - -
Pre-incubate at 40°C for 3 minutes.
Serum - - 0.5 -
Incubate at 40oC for 15 minutes.
NaOH (to stop further 0.5 0.5 0.5 0.5

reaction)
Serum - - - 0.5
DNSA (chromogen reagent) 1 1 1 1
Keep in a boiling water bath for 5 minutes. Measure the absorbance at 540 nm.
OD
Calculation
The activity of the enzyme =
𝑂𝐷𝑜𝑓𝑇 − 𝑂𝐷𝑜𝑓𝐶
× 𝑎𝑚𝑜𝑢𝑛𝑡𝑜𝑓𝑠𝑡𝑑.× 𝑡𝑖𝑚𝑒𝑓𝑎𝑐𝑡𝑜𝑟 × 𝑣𝑜𝑙𝑢𝑚𝑒𝑓𝑎𝑐𝑡𝑜𝑟
𝑂𝐷𝑜𝑓𝑆 − 𝑂𝐷𝑜𝑓𝐵
36
Normal Range
Serum Amylase activity
Amylase activity in serum: 31 - 107 IU/L (by IFCC method)
Significance of control test tube in the enzyme activity estimation experiments

Enzyme activity estimation usually estimates the amount of product formed. Sometimes, the
measured product will already be present in some small amount in the serum sample. For example,
while estimating the amylase activity in serum, the glucose or maltose present in the serum will give
color along with the glucose and maltose produced through the amylase activity. We use the control
test tube to account for the product already in the sample. In the control tube, the serum is added after
the enzymatic reaction is stopped. The color produced in the control tube represents the product
already present in the serum sample. Subtracting the absorbance of the control tube from the “test”
tube gives the product formed and, in turn, the enzyme activity.
Clinical significance
Serum amylase measurement is used as a screening test for acute pancreatitis in patients with acute
abdominal pain or back pain and other features suggestive of acute pancreatitis. The rise of serum
amylase levels in acute pancreatitis is rapid and transient, reaching a peak within the first 12 – 72 hrs
after onset and returning to normal, usually in 3-4 days. The serum amylase activity rises 4 to 6-fold
over the reference range. Hence, it is a sensitive marker of acute pancreatitis. However, serum
amylase levels also rise in several other clinical conditions (mentioned below), some of which mimic
acute pancreatitis clinically. Thus, amylase is not a specific marker for acute pancreatitis. That is why
serum lipase is used to confirm the diagnosis of acute pancreatitis. Serum lipase is both highly
sensitive and highly specific for acute pancreatitis.
If other causes of hyperamylasaemia are ruled out, serum amylase activity more than the reference
range usually leads to the diagnosis of acute pancreatitis. However, diagnosing any clinical condition
depends not entirely on biochemical investigations alone. The physician must consider all the
available information (i.e., clinical history of the patient, clinical examination findings, radiology
findings, biochemical investigations, etc.) to arrive at a diagnosis.
37
Causes of hyperamylasaemia and hyperamylasuria are:
1. Pancreatic disease
a. Acute pancreatitis
b. Chronic ductal obstruction
c. Pancreatic trauma
d. Pancreatic carcinoma
2. Non-pancreatic disorders
a. Salivary gland lesions
i. Mumps
ii. Calculus
b. Renal insufficiency
c. Tumor hyperamylasemia
i. Carcinoma of the lung
ii. Carcinoma of the esophagus
iii. Breast carcinoma
iv. Ovarian carcinoma
d. Peritonitis
38
9. EFFECTS OF pH AND EDTA ON AMYLASE ACTIVITY
Effect of pH on enzyme activity

Enzymes typically are most active in a narrow pH range. There are a variety of reasons for this
phenomenon. A change in the pH of the medium will cause a change in the 3D structure of the proteins
(tertiary and quaternary), which would impact the catalytic site and hence the rate of enzyme activity.
Other factors affecting enzyme activity
Apart from pH, several other factors, like temperature, ionic strength, etc., influence enzyme activity
for similar reasons as pH. Some enzymes like amylase need co-factors like Ca2+ for their optimal
activity. If chelators like EDTA are present, the activity of these enzymes would be sub-optimal.
Other factors influencing enzyme activity include enzyme concentration, substrate concentration, and
the presence or absence of inhibitors/activators.
Factors that affect the frequency of Factors that affect the energy barrier of
collisions the reaction
Substrate concentration Temperature
Enzyme concentration pH
Co-enzyme concentration Modifier (activator or inhibitor)
Reagents: Buffers of pH 7 and 4, starch solution, EDTA, chromogen solution containing DNSA, and
stop solution consisting of NaOH.
Procedure
B (mL) S (mL) T1 (mL) T2 (mL) T3 (mL) C (mL)
Substrate 1.5 1.5 1.5 1.5 1.5 1.5

Buffer (pH 7.0) 1 1 1 1
Buffer (pH 4.0) 1
EDTA (pH 7.0) 1
DDW 0.5
Std glucose 0.5
(1.5 mg/mL)
39
Pre-incubate at 40°C for 3 minutes.
B (mL) S (mL) T1 (mL) T2 (mL) T3 (mL) C (mL)
Serum 0.5 0.5 0.5
Incubate at 40oC for 15 minutes.

NaOH 0.5 0.5 0.5 0.5 0.5 0.5
Serum 0.5
DNSA 1 1 1 1 1 1
Keep in a boiling water bath for 5 minutes. Measure the absorbance at 540 nm.
OD
Calculations
The activity of the enzyme =
𝑂𝐷𝑜𝑓𝑇 − 𝑂𝐷𝑜𝑓𝐶
× 𝑎𝑚𝑜𝑢𝑛𝑡𝑜𝑓𝑠𝑡𝑑.× 𝑡𝑖𝑚𝑒𝑓𝑎𝑐𝑡𝑜𝑟 × 𝑣𝑜𝑙𝑢𝑚𝑒𝑓𝑎𝑐𝑡𝑜𝑟
𝑂𝐷𝑜𝑓𝑆 − 𝑂𝐷𝑜𝑓𝐵
40
10. ESTIMATION OF PLASMA GLUCOSE
Introduction
Estimating glucose in the blood has become a routine investigation in clinical practice for screening
and following up on metabolic disorders like diabetes mellitus. It is usually measured in the plasma.
In some point-of-care testing devices like glucometers, capillary blood (whole blood) can also be
used to measure glucose. However, being arterial blood, the values are slightly higher than that of
plasma venous blood.
Collection of blood sample

The sample is collected in a vial containing Sodium Fluoride (NaF) and Potassium Oxalate (1:3).
Both act as anticoagulants, and NaF additionally prevents glycolysis in RBCs by inhibiting the
enzyme “Enolase.” To avoid glycolysis, the sample can be centrifuged and the plasma can be
separated from the cells. Collecting serum may delay the analysis, thereby exposing the glucose to
glycolysis by the blood cells. Hence, the estimation of glucose in plasma is preferred.
The following samples are commonly tested for glucose:

1) Fasting blood glucose (FPG): A blood sample is collected after overnight fasting (fasting is
defined as no calorie intake for at least 8 hrs).
2) Post-prandial blood glucose: Here, blood is collected 2 hrs after ingestion of a meal.
3) Random blood glucose: Blood is collected anytime without considering the last meal.
4) 2-hour post glucose (2HPG): Blood is collected in a fasting individual 2hrs after 75gm an-
hydrous glucose load, dissolved in 240-300 mL of water.
Methods of estimation
1. Enzymatic method (preferred method)
a. Glucose oxidase and Peroxidase (GOD-POD) method
b. Hexokinase method
2. Non-enzymatic method
a. Folin Wu method
b. King and Asatoor (modification of Folin Wu)
c. Ortho-toluidine method
41
Glucose oxidase method
Principle
Glucose is oxidized by glucose oxidase to gluconic acid and H2O2. The peroxidase enzyme reduces
H2O2 to produce water and nascent oxygen [O]. [O] reacts with 4 amino antipyrine (4 AAP) to give
pink colored compound, which is measured colorimetrically at 530 nm.
Glucose oxidase
Glucose Gluconic acid + H2O2
Peroxidase
H2O2 H2O + [O]
Peroxidase
H2O2 + 4AAP + 3,5-DHBS Quinoneimine dye + 2H2O
4-AAP - 4 Aminoantipyrine
DHBS - 3, 5-Dicholoro-2-hydroxybenzene sulfonate
Reagents
1. Color reagent: glucose oxidase, peroxidase & 4-aminophenazone
2. Standard glucose: 100 mg/dL
Procedure
Standardization of a semi-auto biochemistry analyser:
Standardizing a semi-auto analyzer is critical in ensuring laboratory test results' accuracy, precision,
and reliability. It involves a calibration procedure to establish a consistent relationship between the
instrument's measurements and the actual concentrations of analytes. After washing the flowcell of
the semi autoanalyser we run the reagent blank. We instruct the instrument to assume the OD of the
reagent blank as zero value. Then we run a standard of known concentration and instruct the
equipment to assign the OD of the standard to the concentration of the standard. A factor is created
by dividing the value of the concentration of the Standard with that of the OD of the standard. This
factor is then multiplied to the OD of all the test to determine the concentration of the analyte in the
test sample.
Steps involved in operating a semi-auto biochemistry analyser

1. Rinse the flow cell with a minimum of 3-4 mL of distilled water.
2. Perform the calibration – run reagent blank and set OD to Zero. Then run the standard and
calibrate the equipment to obtain the factor.
3. Prepare the sample/reagent mixture as per instructions.
4. Aspirate the test/control samples.
5. Record the absorbance of each sample. Most machines will provide the calculated levels of
the analyte using the calibration.
42
Instrument care
1. Use the instrument within 10-35°C; maximum humidity: 85% at 30°C.
2. Turn the instrument OFF & cover it with a dust cover when unused.
3. Never use highly acidic/alkali reagents or solutions to be aspirated in the flow cell.
4. Don’t allow the reactive reagent to stay in the flow cell; always wash the flow cell with dis-
tilled water after use.
Follow the given protocol for the development of color and estimation of optical densities.
STEP 1. Blank (B) Standard (S) Test (T)

Distilled Water 20 μL - -
Glucose standard - 20 μL -
Test - - 20 μL
Enzyme Reagent 1000 μL 1000 μL 1000 μL
STEP 2. Mix well & incubate for 25 minutes at 15-25°C or 10 minutes at 37°C.
STEP 3 Add 2mL of distilled water in all test tubes.
STEP 4 Read colorimetrically at 540 nm.
OD
Calculation
Blood Glucose (mg/dL)
𝑂𝐷𝑇 −𝑂𝐷𝐵
= 𝑂𝐷𝑆 −𝑂𝐷𝐵
× 𝐶𝑜𝑛𝑐. 𝑜𝑓𝑆𝑡𝑑. (𝑚𝑔⁄𝑑𝐿)
= mg/dL
Normal Range
 Fasting blood glucose: 70-100 mg/dL (plasma)
 Post prandial blood glucose: 70 - 140 mg/dL
43
Interpretation
Hyperglycaemia: when the plasma glucose level is equal to or exceeds 100 mg/dL during fasting
conditions or 2-hr post-prandial glucose level equivalent to or exceeds 140 mg/dL on more than one
occasion.
It is found in the following conditions:
1. Physiological
a. Alimentary: After a high carbohydrate diet
b. Emotional: stress, anger, anxiety
c. Pregnancy
2. Pathological
a. Diabetes mellitus
b. Hyperthyroidism
c. Hyperadrenalism
d. Hyperpituitarism
e. Diseases of the pancreas, like pancreatitis and carcinoma
f. Cerebrovascular accidents
Hypoglycaemia: when the plasma glucose level falls below 70 mg/dL. It is found in the following
conditions:
1. Physiological
a. During starvation
b. After severe exercise
2. Pathological
a. Due to excess insulin e.g.
i. Excessive dose of insulin
ii. No food intake after insulin administration
iii. Tumours of the pancreas (insulinoma)
b. Inborn Errors of Metabolism: Glycogen Storage Disease 1, Galactosemia.
44
American Diabetes Association (ADA) criteria for diagnosis of Diabetes Mellitus:
Fasting plasma glucose ≥ 126 mg/dL

2 hr plasma glucose during an OGTT ≥ 200 mg/dL
HbA1c ≥ 6.5%
Random plasma glucose in patients with typical symptoms of ≥ 200 mg/dL
hyperglycaemia or hyperglycaemic crisis.
In the absence of symptoms of hyperglycaemia, the first three criteria listed above should be
confirmed by repeat testing on a different day.
Glycated haemoglobin (HbA1c)

It is a form of haemoglobin measured primarily to identify the average plasma glucose concentration
over prolonged periods. It is formed in a non-enzymatic glycation pathway by haemoglobin’s
exposure to plasma glucose. Normal levels of glucose produce a normal amount of glycated
haemoglobin. As the average amount of plasma glucose increases, the fraction of glycated
haemoglobin increases predictably. Haemoglobin A1c was first separated from other forms of
haemoglobin using a chromatographic column. The glucose molecule is attached to N-terminal
valines of the β-polypeptide chains of normal adult haemoglobin. The HbA1c formation is
proportional to the blood glucose concentrations because the average red blood cell life is 120 days,
and the glycated haemoglobin level reflects the average blood glucose level during the previous 2 to
3 months.
The potential benefits of using HbA1c include:
1. Doesn’t require fasting.

2. Faster and easier diagnostic test
3. Less interference of test results because of stress and illness.
HbA1c level Interpretation

< 5.7% Normal
5.7% - 6.4% Pre-diabetes
≥ 6.5% Diabetes Mellitus
POCT (point of care testing) in diabetes mellitus

Testing that occurs close to the patient rather than the central lab. It is performed by non-lab-trained
individuals - nurses, physicians, paramedics, or the patient (self-testing). Glucometer is an example
of POCT testing.
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11. GLUCOSE TOLERANCE TEST
Glucose tolerance is defined as the capacity of the body to tolerate an extra load of glucose. Usually,
the blood glucose level remains relatively constant, the fasting being <100 mg/dL. After a meal, there
is a temporary rise to about 160 mg/dL, which returns to normal within 2 hrs. The initial test of choice
for diagnosis of Diabetes is Glucose fasting and 2HPG. However, in cases of impaired glucose
tolerance in borderline cases of Diabetes Mellitus or in the diagnosis of Gestational Diabetes, Oral
Glucose Tolerance Test (OGTT) is done.
Two opposing forces are responsible for the time course of the glucose concentration in OGTT:
a) Gastric emptying rates and intestinal absorption determine the initial rate of increase.
b) Increase in glucose concentration stimulates insulin release from the pancreas. The rate of
fall in blood glucose is determined by insulin response.
Indication of GTT
1. Patients with transient or sustained glycosuria, with no symptoms of diabetes mellitus, with
normal fasting or PP blood glucose.
2. Patients with symptoms of DM but no glycosuria and normal fasting blood glucose.
3. Patients with a strong family history of diabetes mellitus.
Precautions
1. The patient should be on an unrestricted diet (150 g of carbohydrates/day) for at least three
days.
2. Fasting should not be less than 8 hrs. Only water is permitted after dinner.
3. The patient should not be taking drugs that affect carbohydrate metabolism.
4. During the test, the patient’s activity should be mild to moderate, and abstain from smoking.
The patient should be at rest mentally.
Factors affecting GTT

1. Starvation/ ingestion of a high-fat diet
2. Exercise
3. Pregnancy – tolerance is decreased.
4. Illness – stress causes decreased tolerance, so patients recovering from surgery, burns, or
childbirth should be allowed two weeks before the test.
5. Physiologically decreased tolerance with age
6. Drugs- drugs must be withdrawn before the test, e.g., oral contraceptives, thiazide diuretics,
insulin, oral hypoglycemic agents, salicylates
7. Liver diseases
47
Oral GTT: Procedure
Glucose load: Glucose (anhydrous) at a rate of 1.75 g/kg body weight (usually 75 g of anhydrous
glucose or 82 g of commercial glucose) is given to the patient with water over 5 minutes. After the
glucose load, no calories in any form are given during the entire duration of the test. Water, however,
can be taken. If the patient vomits during the procedure (glucose is a gastric irritant), the test results
are invalid and must be repeated.
Sample collection: The following venous samples using fluoride as an anticoagulant are collected for
estimating venous plasma glucose:
1. A fasting sample: A fasting state is between 8 to 10 hrs of the last feed. The period is chosen
when the liver's glycogen stores are depleted, and gluconeogenesis has not started.
2. Samples at half an hour intervals for a total period of 2 hrs.
Graph: The values of the venous plasma glucose are plotted against time.
The following types of responses are commonly seen:
• Normal response: Fasting blood sugar is normal. At 1 hour reaches a peak but remains below
the renal threshold of 180 mg/dL. It returns to normal fasting level within 2 hrs and 30
minutes.
• Diabetic curve: Fasting levels are 126 mg/dL or more. The highest value is usually reached
after 1 to 1.5 hrs. Glycosuria is generally seen. The blood glucose level does not return to the
fasting level at any time within 2 hrs and 30 minutes.
• Renal glycosuria: Due to the lowering of renal threshold, one or more urine samples contain
glucose. It is usually an accidental discovery & is often asymptomatic. Causes are late stages
of pregnancy, acute kidney disease, etc.
• Lag/alimentary curve: Fasting blood glucose is normal. Due to rapid absorption, the
maximum level is reached within 30 minutes, which crosses the renal threshold, so the
corresponding urine samples show glycosuria. Hypoglycemic levels may be reached at the
end of 2 hrs. It is assumed that a greater rise in plasma glucose is due to a delay in the insulin
mechanism coming into action or an increased rate of glucose absorption from the intestine
following the rapid emptying of the stomach.
• The flat curve of enhanced glucose tolerance: The fasting plasma glucose is normal.
Throughout the test, glucose level does not vary ± 20 mg/dL. Causes are impaired
carbohydrate absorption, hypothyroidism, hypoadrenocorticism, hypopituitarism, etc.
48
49
DIPSI (Diabetes in Pregnancy Study Group India) guidelines
Methodology:
Test for diagnosis: “A Single Test Procedure” (Recommended by Ministry of Health
Government of India)
Target population: All pregnant women need to be screened for GDM twice:
 1st test is done during first ANC visit.
 If first test is negative and was done before 24 weeks, 2nd test is done between 24-28th weeks.
It is important to ensure second test as most of the pregnant women develop blood sugar
intolerance during this period.
Step 1: 75 g glucose is to be given orally after dissolving in approximately 300 ml water irrespective
of the fasting state. The intake of the solution has to be completed within 5-10 minutes.
Step 2: Plasma glucose level should be evaluated 2 hours after the oral glucose load.
If vomiting occurs within 30 minutes of oral glucose intake, the test has to be repeated the next day.
If vomiting occurs after 30 minutes, the test continues.
Interpretation: The threshold plasma glucose level of ≥140 mg/dL is taken as cut off for diagnosis
of GDM
50
12. ESTIMATION OF TRIGLYCERIDES
Introduction
Triglycerides (TG) or triacylglycerols are esters of glycerol with fatty acids. They comprise three
molecules of fatty acids attached to a single molecule of glycerol by ester bonds.
They are the storage form of lipids in the adipose tissue and constitute 95% of tissue storage fat; they
also form the core lipid component of lipoproteins and are the predominant form of glyceryl ester
found in plasma. Their primary function is to provide energy in conditions like starvation.
Triglycerides form the major proportion of dietary fat. After ingestion, triglycerides are digested
(hydrolysed) in the duodenum and the proximal ileum. Through the action of pancreatic and intestinal
lipases and in the presence of bile acids, which activate lipases, they are hydrolysed to glycerol,
monoglycerides and fatty acids. After absorption, triglycerides are resembled from glycerol,
monoglycerides and fatty acids in the intestinal epithelial cells and are combined with cholesterol and
Apo B-48 to form chylomicrons.
Glycerol phosphate oxidase (GPO) method

Principle
The triglycerides are estimated after enzymatic hydrolysis with lipase, followed by the estimation of
released glycerol through a series of reactions. The indicator is a quinoneimine formed from
hydrogen-peroxide, 4-aminophenazone, and 4-chlorophenol under the catalytic influence of
peroxidase as described below:
POD: Peroxidase, GK: Glycerol kinase, GPO: Glycerol phosphate oxidase.
The intensity of chromogen (Quinoeimine) formed is proportional to the triglyceride concentration

in the sample when measured at 505nm (500-540 nm).
Type of sample: Use a non-haemolytic serum or plasma (heparin or EDTA)
Reagents
The reagent mix consists of lipoprotein lipase, glycerol kinase, glycerol phosphate oxidase,
peroxidase, chromogenic substrate (4-amino antipyrine), and buffer (pH 7.0)
51
Materials provided
1. Enzyme reagent
2. Triglyceride standard (200 mg/dl)
Procedure
STEP 1. Blank (B) Standard (S) Test (T)

Working Reagent 1000µL 1000µL 1000µL
Distilled water 10µL - -
Standard - 10µL -
Test - - 10µL
STEP 2. Mix & incubate for 10 min at 37°C.
STEP 3. Add 2 mL Distilled water
STEP 4. Read absorbance at 505 nm (500-540 nm) against reagent blank.
Step 5. OD
Calculations
Triglycerides (mg/dL) = Abs of Test X Conc. of standard (mg/dL)
Abs of Standard
= mg/dL
As per the National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III)
guidelines, the desirable level of Triglycerides should be less than 150 mg/dL.
Reference Intervals
Category Serum TG levels
Desired level < 150mg/dL
Borderline 150–200 mg/dL
High 200–500 mg/dL
Very high > 500 mg/dL
52
Clinical significance
Patient preparation: Triglyceride levels must be measured only after 12-14 hrs of strict fasting.
The patient must sit comfortably in a well-ventilated & lightened room for 5 min.
Hypertriglyceridemia: Hypertriglyceridemia can be a consequence of genetic abnormalities, called
familial hypertriglyceridemia, or the result of secondary causes, such as hormonal abnormalities
associated with the pancreas, adrenal glands, and pituitary
Primary hypertriglyceridemia: Endocrine disorders such as

1. Diabetes mellitus
2. Hypothyroidism
3. Cushing’s syndrome
Genetic Disorders
1. Familial Hypertriglyceridemia
2. Familial Combined Hyperlipoproteinemia.
A moderate increase is seen in the following:

2. Von Gierke’s disease (Type 1 glycogen storage disease)
3. Alcoholism
4. Hypothyroidism
5. Nephrotic syndrome
6. During estrogen administration, including oral contraceptive preparations.
7. Ethanol ingestion
8. Drugs that may cause hypertriglyceridemia include ß- blockers, thiazides & synthetic retin-
oids.
9. Congenital Abetalipoproteinemia
10. Malnutrition
Serum triglyceride values are decreased with the following:

1. Large doses of ascorbic acid
2. During administration of heparin & oral hypoglycemic drugs
3. Therapeutic agents that reduce high triglyceride levels include clofibrate & bezafibrate.
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13. ESTIMATION OF SERUM CHOLESTEROL AND
HIGH-DENSITY LIPOPROTEIN (HDL) CHOLESTEROL
Introduction
Estimating Serum Cholesterol is important since increased serum cholesterol is one of the main
causative factors for atherosclerotic heart diseases like myocardial infarction, cerebral haemorrhage
& thrombosis. Cholesterol is the most abundant sterol in human tissues & body fluids. It is present in
blood in two forms: free (30%) and esterified (70%). It is transported bound to lipoproteins, mainly
HDL & LDL. It has many important functions in the body.
High-density lipoprotein (HDL) transports cholesterol from peripheral tissues to the liver for
excretion. The measurement of HDL cholesterol provides valuable information for assessing
coronary heart diseases.
Methods of estimation of total cholesterol

• Enzymatic method
Cholesterol oxidase-peroxidase method
• Non-enzymatic method
Zak’s method
Total cholesterol by cholesterol oxidase - peroxidase method

Principle
Cholesterol esterase is used to free the cholesterol from cholesterol ester. Free cholesterol is oxidized
by cholesterol oxidase & produces hydrogen peroxide, which gives pink color on reacting with phenol
& 4-amino antipyrine.
Esterase
Cholesterol ester + H2O Cholesterol + Free fatty acid
Oxidase
Cholesterol + O2 Cholesterol-4 ene-3 one + H2O2
Peroxidase
H2O2 + Phenol + 4-amino antipyrine Quinoneimine + H2O
Reagents
The reagent mix contains Cholesterol Esterase, Cholesterol Oxidase, Peroxidase, Chromogenic
Substrate (4 – Amino Antipyrine), And Buffer.
Cholesterol standard: 200 mg/dL
55
Procedure
STEP 1. Blank (B) Standard (S) Unknown (U)

Working Reagent 1000 μL 1000 μL 1000 μL
Standard - 20 μL -
Test - - 20 μL
STEP 2. Mix well and incubate at 37°C for 10 minutes.
STEP 3. Add 2mL Distilled water in each tube.
STEP 4. Read colorimetrically at 540nm.
Step 5. OD
Calculations
Serum cholesterol (mg/dL)
𝑂𝐷𝑈 −𝑂𝐷𝐵
× 𝐶𝑜𝑛𝑐. 𝑜𝑓𝑆𝑡𝑑(𝑚𝑔⁄𝑑𝐿)
= mg/dL
Normal range
1. Total serum cholesterol: < 200 mg/dL

2. LDL cholesterol: < 100 mg/dL
3. HDL cholesterol:
a. Males: 40 – 60 mg/dL
b. Females: 50-60 mg/dL
56
Summary of the Third Report of the National Cholesterol Education Programme (NCEP)
Expert Panel on Detecting and Evaluating Blood Cholesterol in Adults
Parameter Value in mg/dL Inference

Total Cholesterol < 200 Desirable
200 – 239 Borderline high
≥ 240 High
HDL Cholesterol ˂ 40 Low
≥ 60 High
LDL Cholesterol < 70 Therapeutic option for high-risk patients
< 100 Optimal
100 – 129 Near optimal/ above optimal
130 – 159 Borderline high
160 – 189 High
≥ 190 Very high
Interpretation
Hypercholesterolemia: Abnormally high levels of serum cholesterol lead to the deposition of
cholesterol in the arterial walls (atherosclerosis). It may be observed in:
2. Nephrotic syndrome
3. Obstructive jaundice
4. Hypothyroidism:
Hypocholesterolaemia: may be observed in

1. Hyperthyroidism
2. Pernicious anemia
3. Haemolytic anemia
4. Malabsorption syndrome
57
HDL cholesterol by PEG/CHOD - PAP method
Principle
When the serum is reacted with polyethylene glycol (PEG) in precipitating reagent, LDL and VLDL
cholesterol precipitates. HDL cholesterol in the supernatant reacts with cholesterol oxidase (CHOD)
and cholesterol esterase (CHER) to produce H2O2. H2O2, and quinine dye, react with Peroxidase
producing a color that is measured colorimetrically.
HDL Fatty Acid Cholesterol-4 ene-3 one + H2O2+ Free fatty acid
2H2O2 +4-AAP +Phenol Quinoneimine + 5 H2O
Reagents
Reagent 1 contains Polyethylene-Glycol-Methyl Ester and Buffer. Reagent 2 contains Cholesterol
Esterase, Cholesterol Oxidase, Peroxidase, Chromogenic Substrate (4-Amino Antipyrine), Detergent,
and Buffer.
Procedure
1. Dilution of serum and standard
Take 0.2 mL of serum and standards in two separate test tubes. Add 1.8 mL normal saline to
each and mix.
2. Colour development
Reagent R1 375 μL 375 μL 375 μL
Standard - 5 μL -
Serum - - 5 μL
STEP 3. Reagent R2 125 μL 125 μL 125 μL
STEP 5. Add 2.5 mL of Distilled Water
STEP 6 Read final absorbance at 600/700 nm filter against reagent blank.
STEP 7. OD
58
Calculations
𝑂𝐷𝑈 −𝑂𝐷𝐵
× 𝐶𝑜𝑛𝑐. 𝑜𝑓𝑆𝑡𝑑(𝑚𝑔⁄𝑑𝐿)
= mg/dL
Interpretation
High-density lipoproteins (HDL) are one of the significant classes of plasma lipoproteins. They are
synthesized in the liver as complexes of apolipoprotein and phospholipid. They can pick up
cholesterol from arteries to the liver, which is converted to bile acids and excreted in the intestine.
HDL cholesterol is a marker for risk assessment for atherogenic plaque formation. An inverse
relationship exists between HDL Cholesterol (HDL C) levels in serum and the incidence/prevalence
of coronary heart disease (CHD).
HDL-C levels are higher with

1. Regular physical activity
2. Regular exercise
HDL-C levels are lower in

1. Malnutrition
2. Frequent fasting/starvation
3. Lack of physical activity/exercise
5. Hypothyroidism
6. Liver disease
7. Chronic smoking
8. Usage of anabolic steroids
9. Certain rare dyslipidemia (e.g., Tangier disease)
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14. URINE ANALYSIS - I
Urine: A clear, pale yellow, slightly acidic, aromatic fluid with a specific gravity (1.010-1.022) of
miscellaneous composition. This includes dissolved compounds, fragments of cells, intact cells,
proteinaceous casts and crystals.
Water, inorganic salts (sulphates, phosphates and chlorides of Na+/K+, ammonia, etc.), nitrogenous
organic compounds - urea, uric acid, Creatinine, hippuric acid, etc.), organic sulphur containing
compounds - Ethereal sulphates, conjugated bile acids and salts, etc.), ketone bodies, proteins, bile
pigments are the usual constituents.
The biochemical tests done in this part is for substances detectable in amounts not usually
associated with a disease state with the assays used.
Physical characteristics:
(a) Volume: Normal volume of urine excreted is 800-1800mL/24 hr.
(b) Colour: Normal urine is pale yellow coloured due to the pigment – urochrome.
(c) Appearance: Freshly voided urine is clear, but becomes turbid on standing due to
precipitation of phosphates and urates.
(d) Odour: Normally aromatic, but turns ammoniacal on standing due to urea
splitting organisms liberating ammonia.
(e) Specific gravity: The specific gravity of urine is measured using Urinometer. The
instrument is calibrated at 150C and hence, temperature correction is applied for room
temperature, which is as follows:
(i) For every 30C rise in room temperature, add 0.001 to the observed specific gravity.
(ii) For every 30C fall in room temperature, subtract 0.001 from the observed specific
gravity. The normal specific gravity is 1.010 to 1.022.
(f) Total solids: Last 2 digits of specific gravity are multiplied by 2.66 (Long’s
coefficient). The units for total solids are G/L.
(g) pH: The pH of freshly voided urine is slightly acidic (approximately 5-6), but becomes
alkaline on standing. Very often urine excreted after ingestion of vegetarian diet may be
alkaline and that excreted after intake of non- vegetarian diet may be acidic.
61
CHEMICAL CONSTITUENTS
I. Inorganic constituents:
Cations like Na+, K+, Ca2+, Mg2+ are excreted as salts in association with anions like Cl-, SO42-,
PO42- etc.
(a) Chlorides: are excreted mainly in the form of sodium chloride. The amount of sodium
chloride excreted varies between 5 to 25 g per day depending upon the dietary intake.
(b) Sulphates: is ingested from sulphur containing amino acids cystine, cysteine, methionine
and also from chondroitin sulphates. The sulphur is metabolized and excreted in two
forms such as:
i. Ethereal sulphates
ii. Inorganic sulphates
(c) Phosphates: are derived chiefly from the metabolism of foods containing the element and
tissue components such as phosphoproteins, nucleoproteins, nucleotides and
phospholipids. The quantity excreted is extremely variable as it depends on the nature of
the diet. Phosphorus is excreted in urine largely as inorganic phosphate and to a small
extent in organic form (< 4% of total).
(d) Calcium: Under normal dietary intake, urinary excretion accounts for about 5–40% of total
calcium, the remainder being excreted in the faeces. An adult excretes an average of 200–
300 mg of calcium daily.
(e) Ammonia: Under normal dietary conditions, an adult excretes about 0.3 to 1.2 g (average
0.7 g) of ammonia nitrogen daily which is derived from dietary amino acids.
II. Organic Constituents:
(a) Ethereal sulphates: Ethereal sulphates consist of sodium and potassium salts of sulphuric
acid esters of phenols such as indoxyl, skatoxyl, phenol and cresol. These are
detoxication compounds of phenols and are formed in liver. Indoxyl and skatoxyl
sulphates are formed by putrefactive decomposition of tryptophan in the intestine.
(b) Urea: Urea is the principal end product of protein (amino acid) metabolism. About 30g
urea is excreted per day. The amount excreted depends on the protein intake.
(c) Uric acid: Uric acid is the chief end product of purine catabolism. The quantity of uric
acid excreted in urine generally varies between 0.5 to 1.0 g/24 hrs. On the purine free
diet, the uric acid excretion may fall to 0.1g/day, while on a high purine diet, the daily
excretion may rise to 2.0 g.
62
(d) Creatinine: is an excretory product formed during muscle metabolism from creatine
phosphate. It is an internal anhydride of creatine. The amount of Creatinine excreted in
urine is 1.2-1.7 g/24 hrs. It is purely endogenous and does not depend on the dietary
intake of proteins. The amount of Creatinine excreted in urine is dependent on muscle
mass.
(e) Hippuric acid: represents a detoxification product of benzoic acid with glycine. The
benzoic acid is present in many fruits, vegetables and also as food preservative. The
amount of hippuric acid excreted per day in urine varies between 0.1 to 1.0 g/24 hrs with
an average of about 0.7 g/24 hrs.
Note: Several other substances are not detected in appreciable amounts.
PHYSICAL CHARACTERISTICS
Tests Observations
a) Volume
b) Colour
c) Appearance
d) Odour
e) pH
f) Specific gravity
Room temperature
Calibration temperature
Temperature difference
Observed specific gravity
Therefore, corrected specific gravity
= Observed Sp. Gr. ± 0.001 x Temp difference

3
g) Total solids
= Last two digits of Sp. Gr. x 2.66
63
CHEMICAL TESTS
Protocol
Test Observation Inference

Calcium
Take 5ml of urine in a test Turbidity appears. Salts of calcium oxalate are
tube and add 5 drops of 1% precipitated.
acetic acid followed by 5ml
of 2% potassium oxalate.
Phosphates
Phosphates present react with
Acidify 3ml of urine with 2- Canary yellow turbidity
ammonium molybdate to form
3 drops of concentrated
ammonium phosphomolybdate.
HNO3. Add 3ml of
ammonium molybdate
solution and heat.
INORGANIC CONSTITUENTS

Sulphates
To 2ml of urine, add 2-3 Curdy white ppt. Inorganic sulphates are precipitated as
drops of concentrated HCl BaSO4.
and then 1ml of BaCl2
solution (10%).
Chlorides are precipitated as AgCl.
Chlorides Curdy white ppt.
To 2ml of urine, add a few
drops of concentrated HNO3
and then 2ml of silver nitrate
solution.
Salts of ammonia are unstable and on
Nitrogenous constituents Pink spot appears on
heating in alkaline medium,
(NH3) the filter paper
decompose to form vapours of NH3
Take 2 mL solution ‘A’ in a which disappears which being alkaline turns
test tube and heat. When phenolphthalein pink.
quickly
fumes start appearing, hold
the filter paper pre-
moistened with 3–4 drops of
phenolphthalein close to the
mouth of the test tube.
64
ORGANIC CONSTITUENTS:

Urea
1 mL urine + 1 mL of urea Solution turns pink. Pink colored complex of urea with
color reagent. Place it in a diacetylmonoxime semicarbazide
boiling water bath for 10
minutes.
Creatinine
Test: To 5ml of urine Orange colored Creatinine present in sample (as
sample, add 2ml of saturated solution seen. Creatinine picrate)
picric acid solution and a
few drops of 10% NaOH.
Negative control: A blank
may be put up by taking 5ml No change in color.
of water and adding picric
acid and NaOH to it.
Uric acid
Take 2 ml of urine sample,
add a few drops of
Well-developed blue Uric acid present in sample (as
phosphotungstic acid and a complex with tungstan blue).
color seen.
few drops of 20% sodium
carbonate.
Hippuric acid
2 mL urine + dilute FeCl3
drop by drop. Cream colored Precipitate of ferric hippurate.
turbidity.
Ethereal sulphate
2mL urine + 2mL Baryta White colored turbidity Ethereal sulphates present in sample.
mixture. Add 2mL conc. (dissociation on boiling with conc.
HCl. and boil for 2 minutes HCl. liberates inorganic sulphates,
in beaker and cool at room which form white precipitate of barium
temperature sulphate).
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15. URINE ANALYSIS - II
Under pathological conditions, urine excreted by patient shows the presence of constituents present
in amounts indicative of disease.
The biochemical tests done in this part is for substances detectable in amounts usually
associated with a disease state with the assays used.
These include tests for proteins, sugar, blood, bile salts, bile pigments and ketone bodies.
Physical Characteristics:
1. Volume:
(a) Polyuria: Volume more than 3000mL/24 hrs

It is observed in Diabetes mellitus, Diabetes insipidus, excess water intake, intake of
diuretics like caffeine, alcohol etc.
(b) Oliguria: Volume less than 400mL/24 hrs
It is observed in fluid deprivation, excess fluid loss as in haemorrhage and neurogenic shock,
dehydration, acute glomerulonephritis, obstruction in the urinary tract.
(c) Anuria: Urine output less than 100mL per day.
It is observed in shock and renal failure.
2. Appearance:
(a) Clear when fresh

(b) Turbidity in urine implies the presence of pus or bacteria
(c) Turbidity on standing is due to precipitation of phosphates
(d) Smokiness is due to RBCs
(e) Milky appearance of urine is due to the presence of fat droplets (Chyluria)
3. Colour:
(a) Dark yellow in high fever (due to concentrated urine) and jaundice
(b) Smoky red due to the presence of RBCs
(c) Yellowish brown or greenish due to the presence of bile pigments
(d) Dark brown/black in Alkaptonuria
4. Odour:
(a) Ammonical on standing due to urea splitting organisms

(b) Fruity odour is due to the presence of acetone in ketoacidosis
67
(c) Mousy odour in Phenylketonuria
(d) Smell of maple syrup in congenital Maple syrup urine disease
5. pH:
(a) Alkaline: Metabolic Alkalosis in severe vomiting

Respiratory Alkalosis in hyperventilation
(b) Acidic: Metabolic Acidosis in starvation and diabetic ketosis

Respiratory Acidosis in depression of respiratory centre
6. Specific gravity: It is a tubular function test.
(a) Increase in specific gravity is observed when concentrated urine is excreted
(b) Presence of abnormal constituents like glucose or proteins increases specific gravity
(c) In Diabetes insipidus, low specific gravity is observed due to polyuria
CHEMICAL TESTS
1. Tests for Protein:

(a) Sulphosalicylic acid test: (Bedside White precipitate Protein present
test. In emergency, it can be performed
when few drops of urine is available)
3mL urine + sulphosalicylic acid drop
wise.
(b) Acid precipitation test: White ring at the Protein present in given
In a tube, take 2mL urine and add 0.5 mL junction of two sample
conc. H2SO4 slowly along the sides of the layers
tube.
(c) Heat coagulation test: Coagulum formed. Albumin is present in given

sample. At pH of 5.4,
Take 2/3rd of the test tube with urine
albumin is near to its PI and
sample (approximately 5mL) and acidify
can easily be denatured by
it by adding 2-3 drops of acetic acid. Heat
heating to form &
the upper 1/3rd portion of the urine
Coagulum.
sample.
If a white precipitate If the precipitate disappears
is formed then add 1 then it was due to
ml of 1% acetic acid phosphates present in the
urine. If the precipitate
persists then it is due to
proteins.
68
*Note: On addition of chlorophenol red
a) If the colour of urine becomes yellow (pH is acidic), add 2% Na2CO3 till colour turns
pink.
b) If the colour of urine becomes red (pH is alkaline), add 1% acetic acid till colour turns
pink
Interpretation:
Presence of proteins in urine is generally called as Proteinuria.
(a) Glomerulonephritis
(b) Nephrotic Syndrome
(c) Acute and chronic kidney disease
(d) Urinary tract infection.
(e) Bence Jones proteinuria seen in multiple myeloma.
(f) Proteinuria in pregnancy suggests bad prognosis.
Note: As long as the glomerulus is intact, proteinuria is unlikely.
Bence Jones Proteinuria occurs due to excretion of light chain of immunoglobulin (kappa / lambda)
seen in Immunoglobulin producing tumors of plasma cells called Multiple Myeloma. On heating
urine from room temperature to 100oC, these proteins precipitate at around 60oC and then re-solubilise
once again at around 80oC. When the urine is cooled, they precipitate once again at around 60oC and
disappear at around 40oC. Now a days direct immune-turbidometric tests are available for proper
quantification of individual light chains in the urine.
Tamm-Horsfall protein: This protein is normally found in urine but not in serum. It is a mucoprotein
that is produced in the renal tubules. It is the major protein constituent of urinary casts. Its
concentration in urine is around 40 mg/ day.
2. Test for Glucose:

Benedict’s qualitative test: Green or yellowish Glucose is present.
green or orange or red
Heat 5mL of Benedict’s reagent False positive: Creatinine, Vit C,
precipitate.
to check for the presence of any Glucoronates, Salicylates, PAS
reducing substances already and Isoniazid
present in the reagent. Then add
0.5 mL (8 drops) of urine. Boil
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Control: To check for the presence of any
reducing substances already
5ml Benedict’s reagent and boil No change in colour
present in the reagent. If the
colour of the control changes then
the Benedicts reagent is
contaminated.
Interpretation
Presence of glucose in urine is called as glycosuria. Colour of the precipitate indicates severity of
glycosuria as follows:
Colour of the precipitate Degree of Glycosuria Approx. Glucose Conc. (g%)
Green colour Trace Up to 0.5

Green precipitate + 0.5-1.0
Yellow precipitate ++ 1.0-1.5
Orange precipitate +++ 1.5-2.0
Brick Red precipitate ++++ 2.0 or >
Presence of glucose in urine is found in Diabetes mellitus & Renal glycosuria
3. Test blood and blood pigments- Benzidine test:

Principle: This test detects the presence of peroxidase activity in the given sample. H2O2 is
decomposed by the peroxidase activity in the test sample to liberate nascent oxygen. This then
oxidizes benzidine to form a bluish-green colored complex.
H2O2 H2O + (O) Benzidine + (O) Blue-green Complex

To 3mL of Urine add 3mL of Bluish green colour Presence of Haemoglobin/
Benzidine reagent and 1mL of seen Myoglobin
H2O2
Control: To 3mL of urine add Colour remain The colour of this is the colour of the
3mL of Benzidine reagent. Add greyish brown urine with the reagent. Absence of
1mL of water in place of H2O2. H2O2 does no result in production of
any additional colour.
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Interpretation
Presence of blood and intact RBCs in urine is called haematuria, while presence of haemoglobin in
urine is called is haemoglobinuria.
Haematuria is seen in:
(a) Acute glomerulonephritis
(b) Urinary Tract infection.
(c) Trauma to urinary tract
(d) Cancer of the urinary bladder (painless hematuria)
Haemoglobinuria is due to intravascular haemolysis of RBCs, which occurs in:
a) Malaria
b) Severe burns
c) Haemolytic jaundice
Differentiating Myoglobinuria from Haemoglobinuria:
a) Appearance – Reddish brown to black.
b) Demonstrate that colour produced is due to protein. Treat urine with TCA/SSA and centrifuge.
If the colour disappears then it is confirmed that the colour was due to protein.
c) Perform Benzidine test. If the result is protein, then it confirms that the protein is either
Haemoglobinuria / Myoglobinuria.
d) Salting out by fully saturated Ammonium SO4: Saturate the urine with Ammonium Sulfate.
Shake and then centrifuge. Ammonium Sulfate salts out Hemoglobin but not Myoglobin as
Hemoglobin is a large molecule with 4 side chains. If after centrifuging the colour disappears
than it is due to Hemoglobin. If the colour persists then it is due to Myoglobinuria.
4. Test for bile salts and bile pigments:
(a) Tests for bile salts:

Hays Sulphur powder test:
Fill 3/4th test tube with urine and Sulphur powder sinks to the Surface tension reducing
sprinkle a pinch of Sulphur bottom substance present in given sample.
powder. Indicates probable presence of bile
Control: salts
Fill 3/4th test tube with water Sulphur powder floats
Surface tension reducing
and sprinkle a pinch of Sulphur
substance is absent in control
powder.
sample.
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Interpretation:
Bile salts undergo enterohepatic circulation. In normal persons, there is only a trace of bile salts in
urine. Cholestasis leads to an increased concentration of bile salts in serum & results in its excretion
in considerable amounts e.g. in obstruction of common bile duct & in drug induced cholestasis.
(b) Test for bile pigments:

Fouchet’s test:
5mL urine + 1mL magnesium Colour changes from Bile pigments are adsorbed by
sulphate + 2mL 10% BaCl2. yellow to pista green Barium Sulphate (formed by the
Filter and add few drops of reaction of MgSO4 and BaCl2). This
Fouchet’s reagent to the is then oxidised to Biliverdin (green)
precipitate on the filter paper. and Bilicyanin (blue) by FeCl3 of the
Fouchet's reagent.
Interpretation
Bilirubin is found in the urine in extrahepatic obstructive jaundice and in those types of hepatic
jaundice in which there is cholestasis. It is thus an indicator of regurgitation of conjugated bilirubin
which, being water soluble, passes into the urine. It is not present in the urine in prehepatic jaundice.
Urinary Findings in Jaundice
Haemolytic Hepatocellular/Hepatic Obstructive

Bilirubin Absent + ++
Bile salts Absent + ++
Urobilinogen ++ Normal Absent
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5. Tests for ketone bodies
a) Rothera’s test: 5mL urine + A deep purple ring is Acetone and acetoacetic acid
solid (NH4)2SO4 until observed at the junction present. Na-nitroprusside
saturation + a drop of of two layers reacts with both acetone
freshly prepared sodium and acetoacetic acid to
nitroprusside. Mix and add produce a purple coloured
0.5mL strong ammonia compound.
carefully by the side of the
tube.
b) Gerhardt’s test: 3mL Portwine red colour Acetoacetic acid present.
urine + Ferric chloride drop Acetoacetic acid reacts with
wise ferric chloride to form ferric
acetoacetate
Interpretation:
Ketone bodies excreted are acetone, acetoacetic acid and β-hydroxybutyric acid. Excessive excretion
of ketone bodies occurs in a state of ketosis, characterized by a fruity odour of breath due to acetone
exhalation. The unifying thing between starvation and severe Diabetes is intracellular structure with
arrest of the TCA cycle which is seen in:
(a) Uncontrolled diabetes mellitus
(b) Starvation
(c) Severe vomiting
Note:
1) All tests for abnormal constituents of urine may not always be positive in a given urine sample.
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16. ESTIMATION OF BLOOD UREA BY DIACETYL MONOXIME METHOD (DAM)
Principle
Urea reacts with DAM in strongly acidic condition at nearly 1000C to give a pink coloured product
which is measured colorimetrically at 540nm. Thiosemicarbazide and ferric chloride are added to
catalyse the reaction and stabilize the intensity of colour. Advantages of this method are that it
measures urea directly, therefore ammonia present in reagents and in atmosphere does not interfere.
This method requires deproteinization and boiling. This method is linear upto 300mg% urea. For
higher values, the blood sample should be diluted.
Reagents
(a) Sodium tungstate, 10%

(b) Sulphuric acid, 2/3 N
(c) Urea acid reagent: Sulphuric acid, ortho phosphoric acid, ferric chloride
(d) Urea colour reagent: Diacetyl monoxime, thiosemicarbazide
(e) Standard urea solutions: 25, 50,100 mg/dL
Protocol
1. Preparation of protein free filtrate:
a. Distilled water 7mL

b. Blood or plasma 1mL
c. 10% Sodium tungstate 1mL
d. 2/3 N H2SO4 1mL
Mix. Keep at room temperature for 5 minutes and filter. Treat standards in the same way as blood.
2. Color development:
Follow the given protocol for development of color and estimation of optical densities
Distilled water 1.0 mL - -
Protein free filtrate - - 1.0 mL
Urea standard diluted - 1.0 mL -
Urea acid reagent 2.5 mL 2.5 mL 2.5 mL
Urea colour reagent 2.5 mL 2.5 mL 2.5 mL
STEP 2. Keep in boiling water bath for 20 minutes. Cool slowly to room temperature.
STEP 3. Read colorimetrically at 540 nm (green filter).
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Calculations:
Blood urea (mg/dL)
= ODU-ODB x Conc. of Std (mg/dL) x Vol. of Std.
ODS-ODB Vol. of Plasma
= mg/dL
Conversion of Blood Urea to BUN:
Urea contains two atoms of nitrogen and its molecular weight is 60, of which 28 is due to nitrogen.
Hence, the relationship of total blood urea to Blood urea nitrogen (BUN) is given by the ratio
60
= 2.14
28
Therefore,
Blood urea nitrogen (mg/dL) = Blood urea (mg/dL)
2.14
i.e. Blood urea (mg/dL) = Blood urea nitrogen (mg/dL) x 2.14
Results
Blood urea nitrogen = mg /dL
Blood urea = mg/dL
Normal Range
On ordinary diet, the normal range for Blood urea nitrogen is 7 – 20 mg /dL and that of Blood urea
is 15 – 40 mg/dL.
Interpretation:
Urea is the main end product of protein catabolism. Its blood level depends on dietary intake of
protein. It is synthesized in liver and constitutes a major non-protein nitrogenous (NPN) substance.
Causes of raised urea levels:
a) Dehydration: Low blood volume will result in a low GFR. The rate of urine flow will be
low. Substantial amount of urea gets reabsorbed in the loop of Henle causing a raised Urea.
b) Shock: Reasons same as above.
c) Renal disorders / Pathology -
 Acute Kidney Injury.
 Glomerulonephritis
 Obstructive disorders of the Urinary tract – e.g. stones / tumors
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Physical variations:-
1) Higher in males than females.

2) Gradual rise with age.
3) Varies with the amount of protein in the diet.
4) Lower level in early pregnancy probably due to hemodilution.
Pathological causes:-
Pre renal: - Severe dehydration, vomiting, diarrhoea, hypovolemic shock.
Renal: - Renal failure, Nephritis, Glomerulonephritis.
Post renal: - Obstruction to urine flow e.g. stones, cancer of urinary tract, hypertrophy of prostate.
Low levels of Urea:
Rarely seen. May be on the lower side of normal. True low urea are very rare. In very severe liver
diseases, where the Urea cycle stops, Urea may be low (the serum ammonia levels are high in this
case).
Use of blood urea estimation in the assessment of renal function:
Since route of urea excretion is through kidneys, blood urea level is estimated to assess renal function.
As blood urea level is affected by dietary protein and other pre-renal factors, serum Creatinine
estimation should also be done simultaneously.
Other Methods of Urea estimation
Enzymatic methods-
Urea is decomposed to ammonia and carbon dioxide in the presence of enzyme urease.
NH2.CO.NH2 + H2O 2 NH3 + CO2
1. Nessler’s method- gives low results at higher values of urea, so not preferred.
2. Berthelot’s reaction- the ammonia formed from urease action reacts with phenol in the presence of
hypochlorite to form an indophenol, which with alkali gives a blue coloured compound.
3. Kinetic Assay using glutamate dehydrogenase- Ammonia, formed from urease action, in the
presence of glutamate dehydrogenase combines with alpha keto glutarate to form glutamate.
Alpha ketoglutarate + NH3 + NADH+ Glutamate + H2O + NAD+
In this reaction utilization of NADH+ results in a decrease in absorbance at 340 nm, which is
measured to calculate urea concentration.
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17. ESTIMATION OF SERUM CREATININE BY JAFFE’S METHOD
Principle
Proteins are precipitated from serum using Sodium Tungstate. Creatinine present in protein free
filtrate reacts with Alkaline Picrate solution to form a reddish orange coloured complex of Creatinine
picrate, the intensity of which is measured colorimetrically at 540 nm. A standard Creatinine solution
is treated similarly and the colour intensities are compared.
Reagents
(a) Sodium tungstate, 10 %

(b) Sulphuric acid, 2/3 N
(c) Saturated picric acid
(d) NaOH, 10 %
(e) Standard Creatinine solutions (0.6, 1.2, 2.4 mg/dL)
Protocol
1. Preparation of protein free filtrate:

(a) Distilled water 7mL
(b) Blood or plasma 1mL
(c) 10% Sodium tungstate 1mL
(d) 2/3 N H2SO4 1mL
Mix. Keep at room temperature for 5 minutes and filter. Treat standards in same way as the blood.
Follow the given protocol for development of color and estimation of optical densities
Diluted standard - 5.0 mL -
Alkaline picrate** 2.5 mL 2.5 mL 2.5 mL
STEP 2. Mix well. Keep at room temperature for 15 minutes.
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**Alkaline picrate is prepared by mixing 10mL of saturated picric acid with 2mL of 10% NaOH.
Calculations
Serum Creatinine (mg/dL)
ODU-ODB ODS-
= x Conc. of Std (mg/dL) x Vol. of Std
ODB
Vol. of serum
The two chief disadvantages with this method are:-
1. Lack of specificity: - Only 80% of the colour is due to creatinine in serum. The remaining colour
is due to the presence of non-specific chromogens that react with picric acid e.g. ketone bodies, uric
acid, urea, protein, pyruvate, ascorbate and glucose, hence false values may be obtained.
2. Sensitivity: - Sensitivity to certain variables like pH, temperature, etc.
Result
Serum Creatinine = mg/dL
Normal Range
The normal range for serum Creatinine is 0.6 – 1.2 mg/dL (for males)
0.5 – 0.9 mg/dL (for females)
Other Methods of Creatinine estimation:
Jaffe’s method is a non-specific method. Non Creatinine Chromogens found in serum and urine
samples can falsely react with Picric acid and give colours. To overcome this problem there are two
strategies:
a) Kinetic Jaffe’s method: Two readings are taken after 20 and 80 sec of mixing the reagents and
sample and then the concentration of Serum Creatinine is estimated.
b) Specific Method: Using a specific enzyme – Creatininase converts creatinine to Creatine.

Creatinase then converts Creatine into sarcosine & urea. The Sarcosine gets oxidized to H2O2 which
subsequently gets reduced to form a coloured chromogen. Thus this method can specifically estimate
the concentration of Serum/Urine Creatinine without any interference.
Interpretation:
Creatinine is a metabolite of Creatine, a normal constituent of muscles. Serum level of Creatinine

does not depend upon diet. Kidneys excrete it and hence its estimation is done to assess kidney
function.
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Serum Creatinine is increased in
a) Urinary tract obstruction

b) Chronic nephritis
c) Renal failure
d) Diabetic Nephropathy
e) Acute Glomerulonephritis
While it is very intuitive to think that Serum Creatinine levels would increase in muscular dystrophy,
in practicality it does not happen as the kidney has tremendous reserves for excreting the excess of
Creatine released from the damaged muscles.
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18. ESTIMATION OF URINE CREATININE BY JAFFE’S METHOD
Principle:
Diluted urine is treated with alkaline picrate solution to form a reddish orange coloured complex of
Creatinine picrate, the intensity of which is measured colorimetrically at 540nm. A standard
Creatinine solution is treated similarly and the colour intensities are compared. The colour intensity
is directly proportional to the concentration of Creatinine. For creatinine clearance, a 24 hour urine
specimen is collected and one blood sample is taken during the day.
Reagents:
(a) Alkaline picrate

(b) Standard Creatinine solution (10 mg/L)
Protocol:
I. Dilution of urine:
Dilute urine 1:100 with distilled water. Treat the standards in the same way as the blood.
II. Colour development:
Blank (B) Standard (S) Unknown (U)
Distilled water 3.0mL - -
Creatinine standard diluted - 3.0mL -
Dilute urine - - 3.0mL
Alkaline picrate 1.5mL 1.5mL 1.5mL
Mix and keep at room temperature for 10 minutes.

Read colorimetrically at 540 nm / green filter.
Calculations:
Urine Creatinine (g/L)
= ODU-ODB x Conc. of Std.(mg/L) x Vol. of Std.
ODS-ODB Volume of Urine x 1000
= g/L
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Urinary excretion of Creatinine per 24 hrs can be calculated as follows:
Urine Creatinine (g/24 hrs) = Urine Creatinine (g/L) x Volume of urine excreted/24 hrs (L)
Normal Range
The normal range for urine Creatinine is 0.8 – 1.5 g/24 hrs
Interpretation
High urine Creatinine levels are observed in Acromegaly, gigantism & Diabetes mellitus
Low urine Creatinine levels are observed in advanced renal diseases
Urine creatinine and serum creatinine share an inverse relationship. Conditions which increase the
serum creatinine levels will invariably result in decreased urine creatinine levels.
Result
Urine Creatinine = g/L
Note
Creatinine is filtered at glomerulus and is neither reabsorbed nor secreted by the renal tubules except
when the plasma Creatinine levels are very high. Hence, the Creatinine clearance can be estimated
to measure glomerular filtration rate (GFR).
Creatinine clearance is defined as the quantity of blood or plasma completely cleared of Creatinine
per unit time.
Creatinine clearance (mL/ minute) = U x R or U x V

P P
Where U= Urine Creatinine (mg/dL)

P= Plasma Creatinine (mg/dL)
R= Rate of urine flow (mL/minute)
V= Volume of urine flow (mL/minute)
Normal Range
Creatinine Clearance: Males: 115 ± 15 mL/minute.
Females: 110 ± 10 mL/minute.
Note: Currently Creatinine Clearance is calculated by formulas based on the serum Creatinine and
the body surface area, weight and age of the patient. This Creatinine Clearance gives an estimate of
eGFR i.e estimated GFR.
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19. ESTIMATION OF SERUM CALCIUM USING O- CRESOLPHTHALEIN
COMPLEXONE (OCPC) METHOD
Principle
The metal complexing dye OCPC binds tightly with calcium in an alkaline medium pH-10.0 to form
a pink-colored complex absorbance of which is measured at 578 nm. 8 hydroxyquinolene prevents
interference by Mg2+.
Calcium + OCPC Calcium-Cresolphthalein complexone complex
Reagents
1. Calcium standard 10 mg/dL
2. Colouring reagent: Contains OCPC along with hydroxyquinoline, urea, ethanol, and acetic
acid diethanolamine buffer (2M), pH 11.7.
Protocol
Reagents Blank Standard Unknown
Serum _ _ 0.01mL
Calcium Standard (10 mg/ dL) _ 0.01mL _
Distilled water 0.01 mL _ _
Coloring reagent 1.0 mL 1.0 mL 1.0 mL
Mix and read absorbance at 578 nm.
Optical density: (B): (S): (U):
Calculations
(a) Serum Calcium (mg/dL)
(ODU - ODB)
= x Conc. of Std (mg/dL)
(ODs- ODB)
= mg/dL
87
mg/dL of calcium x 10 x valency
(b) Serum Calcium (mEq/L) =
Atomic weight of calcium
mg/dL of calcium x 10 x 2
=
40
mg/dL of calcium
=
2
= mEq/L
Results
Serum calcium = mg/dL
= mEq/L
Normal range
Normal range for serum - Total Calcium is 8.7 – 10.2 mg/dL
- Ionized Calcium is 4.5 – 5.3 mg/dL
Serum calcium below 7 mg/dL causes tetany, which results in severe convulsions and could be fatal.
Discussion
Calcium in blood exists in three forms:
1. 50% is free ionized calcium, the physiologically active form.
2. 45% is bound to plasma proteins (80% to Albumin; 20% to Globulin)
3. Remainder (5%) forms a complex with small organic molecules.
NOTE: The ionic calcium can bind to / disassociate from the plasma proteins based on the pH of the
blood. In alkalosis, the proteins are more negatively charged and hence a larger fraction of the ionic
calcium bind to the plasma proteins creating a decrease in the ionic calcium levels. This is the basis
of tetany seen in hysterical patients who hyperventilate (respiratory alkalosis). It is important to note
88
that the total calcium levels in these patients even in the presence of frank tetany would be normal as
there is a reduction in the ionic calcium concentration due to binding with plasma protein.
In acidosis, the exact opposite occurs.
Serum calcium concentration is affected by:
1. Absorption of calcium from the gastrointestinal tract
2. Parathyroid hormone secretion and
3. Changes in serum phosphorous concentration
Interpretation
A. Hypercalcemia is observed in
1. Multiple Myeloma – because of an increase in the gamma globulin fraction
2. Hyperparathyroidism
3. Hypervitaminosis D
4. Neoplastic disease of the bone
5. Raised serum protein (because calcium is bound to serum proteins)
B. Hypocalcemia is observed in
1. Hypoparathyroidism
2. Hypovitaminosis D (Rickets, Osteomalacia)
3. Chronic renal failure
4. Malabsorption
Other methods of Calcium estimation: -
1) Indirect titration: - Calcium is precipitated as oxalate and then estimated by titration with
permanganate.
2) Fluorescent method: - Based on titration of calcium fluorescent complexes.
3) Atomic absorption spectrometry: - Sensitive and specific method.
4) Ion selective electrode: - Measures ionized calcium.
Note: Clinical symptoms of hypercalcemia or hypocalcaemia occur mainly due to the free ionic
calcium in the blood. Serum calcium is not a good indicator of the ionic calcium in the blood. The
hormonal regulation of calcium is mainly sensitive to ionic calcium and not the total serum calcium.
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20. ESTIMATION OF SERUM INORGANIC PHOSPHORUS
BY MODIFIED FISKE SUBBAROW METHOD
Principle
Phosphate reacts with ammonium molybdate to form Ammonium phosphomolybdate which is
reduced by reducer ANSA (Amino Naphthol Sulphonic acid) to molybdenum blue. The blue colour
is measured colorimetrically at 670nm.
Reagents
1. Trichloracetic acid (TCA), 10 %
2. Molybdate l (Higher acidity)
3. Molybdate II (Lower acidity)
4. 1,2,4-amino naphthol sulphonic acid (ANSA)
5. Working phosphorus standard (2.5, 5.0, 10.0 mg/dL)
Protocol
1. Preparation of protein-free filtrate (PFF):
a. Serum 1.0 mL
b. 10% Trichloroacetic acid 9.0 mL
Mix. Incubate at room temperature for 10 minutes. Filter and use. Treat the standards in the same way as
the blood.

Molybdate I 1.0 mL 1.0 mL -
Molybdate II - - 1.0 mL
Distilled water 8.6 mL 3.6 mL 3.6 mL
ANSA 0.4 mL 0.4 mL 0.4 mL
STEP 2. Mix and keep at room temperature for 4 minutes only.
STEP 3. Read colorimetrically at 670 nm (red filter)
STEP 4. Optical density
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Calculations
Serum inorganic phosphorus (mg/dL)
ODU-ODB
ODS-ODB
Vol. of Serum
Result
Serum inorganic phosphorus = mg/dL
Normal range
The normal range for serum inorganic phosphorus is 2.5 – 4.5 mg/dL. Children have a slightly higher
normal range, i.e., 4 – 6 mg/dL.
Interpretation
Inorganic phosphorus in blood exists as the phosphates of the alkali and alkaline earth metals like
Na3PO4, Na2HPO4, Ca3PO4, Ca (OH)2, and Ca2HPO4.
The product of Ca x P usually is 40.
A. Hyperphosphatemia is seen in:
1. Chronic nephritis: With increasing renal failure, the serum inorganic phosphorus rises
2. Hypoparathyroidism: An increase in serum inorganic phosphorus level is associated with low
serum calcium level.
3. Hypervitaminosis D
B. Hypophosphatemia is seen in:
1. Hyperparathyroidism
2. Fanconi’s syndrome: decreased renal tubular reabsorption.
3. Hypovitaminosis D (rickets, osteomalacia)
4. Renal rickets
5. Physiological fall occurs whenever there is increased carbohydrate utilization, e.g., in the
treatment of diabetic coma by injection of insulin.
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21. ESTIMATION OF SERUM URIC ACID BY PHOSPHOTUNGSTIC ACID METHOD
(CARAWAY’S METHOD)
Principle
Uric acid in the protein-free serum filtrate reduces phosphotungstic acid reagent in the presence of an
alkaline medium (sodium carbonate) to form a blue-colored phosphotungstic complex (tungsten
blue). The color intensity of the complex is measured colorimetrically at 670 nm and compared with
a uric acid standard treated similarly. The color intensity is directly proportional to the concentration
of the uric acid.
Reagents
1. Sodium tungstate, 10%
2. Sulphuric acid, 2/3 N
3. Sodium carbonate, 14 %
4. Phosphotungstic acid
5. Standard uric acid solution (3.0, 6.0, 12.0 mg/dL)
Protocol
Preparation of protein-free filtrate:
1. Distilled water 8.0 mL
2. Serum 1.0 mL
3. 10% Sodium tungstate 0.5 mL
4. 2/3 N H2SO4 0.5 mL
Mix. Keep at room temperature for 5 minutes and filter. Treat the standards in the same way as the
serum.
Color development:
Follow the given protocol for the development of color and estimation of absorbance.
14% sodium carbonate 1.0 mL 1.0 mL 1.0 mL
Phosphotungstic acid 1.0 mL 1.0 mL 1.0 mL
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STEP 3. Read colorimetrically at 670 nm (red filter).
STEP 4. Optical density
Calculations
Serum uric acid (mg/dL)
ODU-ODB
ODS-ODB
Vol. of Serum
Result
Serum uric acid = mg/dL
Normal Range
The normal range for serum uric acid is:

Males: 3.1 – 7.0 mg/ dL
Females: 2.5 – 5.6 mg/ dL
Interpretation
A. Hyperuricemia is seen in:

1. Gout (primary and secondary gout)
2. Lactic acidosis
3. Diabetic Ketoacidosis
4. Leukaemia
5. Lesch-Nyhan’s syndrome
6. Von-Gierke’s disease
7. Polycythaemia
8. Multiple myeloma
9. Glomerulonephritis
B. Hypouricemia is seen in:

1. Wilson’s disease
2. Fanconi’s syndrome
3. Xanthinuria (congenital xanthine oxidase deficiency)
4. Severe liver disease
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22. RENAL FUNCTION TEST
Kidney is a versatile organ involved in:

a) Excretion of waste products of the body
b) Homeostasis of water and electrolyte
c) Acid Base Homeostasis
d) Production of Erythropoietin and Vit D.
Disorders of the renal function can be evaluated by the following tests:
Screening procedure:
Urine examination: Presence of Proteins and urinary casts highly suggestive of renal disease.
Serum Urea and Creatinine: Derangement of these parameters suggests of renal disease.
Note: these parameters can be normal even in cases of renal diseases as the kidney has a high
functional reserve.
Evaluation of the Kidney function can be done in the following headings
A) Evaluation of the Glomerular Function: Clearance test and eGFR

B) Evaluation of the Tubular function: Specific gravity
C) Evaluation of the ability of the kidney to regulate Acid Base homeostasis
A) Evaluation of Glomerual Function:

Clearance tests is defined as the volume of plasma completely cleared of a substance in a unit time.
For a substance to be use for clearance study, it should be neither be reabsorbed or secreted by the
tubules. Its unit is mL/min.
Inulin, a homopolymer of fructose is the best substance for clearance study.
Creatinine, too is used for clearance study as it is:

 Endogenously produced by the body.
 Levels are constant throughout the day
 Not much affected by diet.
 Only a small amount of it is secreted by the distal convoluted tubule.
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Clearance is calculated by the formula
UxR
B
Where, U is the concentration of Creatinine in the urine
R is the rate of urine formation (in mL/min)
B is the concentration of Creatinine is the blood
eGFR : Large number of studies done to evaluate renal function has come up with a formula where
the GFR can be estimated (eGFR) from the serum Creatinine levels.
MDRD equation is eGFR = 186 x (Creat / 88.4)-1.154 x (Age)-0.203 x (0.742 if female)
Cockcroft-Gault formula = (140-age) * (Wt in kg) * (0.85 if female) / (72 * Cr)
This test offers an advantage in that a single serum Creatinine estimation can give a rough
estimation of the eGFR.
B) Evaluation of Tubular Function:

Specific Gravity of the urine: defined as the ratio of the weight of urine to that of equal weight of
water, is a good test to evaluate the Tubular function.
a) Water Deprivation test: In case a person is deprived of water for a long period of time (say
6 to 8 hrs), the body tries to conserve the water by increasing tubular absorption of water. This
in turn reflects as a raise in the urine specific gravity. Classically, the early morning urine
sample specific gravity is tests and a high Specific gravity indicates that the kidneys ability to
concentrate urine is good.
b) Water loading test: In case the body is loaded with water, say 1 to 2 litres of fluid, the body
will try to excrete the excess of water in the urine. This will bring down the specific gravity
of the urine showing the tubules ability to loose water.
C) Ability to handle Acid Base Homeostasis:

Loading the body with a fixed amount of Ammonium Chloride and seeing how low the urine pH
becomes as well as how much of Ammonium salts are excreted indicate the ability of the kidney to
handle Acid Base Homeostasis.
Complete details of the above tests can be obtained from standard text books.
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Questions for which answers need to be found out:
a) Why is urea high in cases of dehydration?
b) Other substances which have been used for clearance studies
c) Role of Cystatin C in evaluating renal function.
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23. ESTIMATION OF SERUM BILIRUBIN BY MALLOY AND EVELYN METHOD
Principle
Serum bilirubin can be present in two forms: conjugated (with glucuronic acid) or Direct and
Unconjugated or Indirect bilirubin. Total Bilirubin is sum of the Conjugated and Unconjugated
bilirubin.
The test is based on the Van den Bergh reaction. Bilirubin in serum couples with diazotized
sulphanilic acid to form a pink-colored compound, azobilirubin. The intensity of color produced is
measured colorimetrically at 530 nm. The color intensity is directly proportional to the concentration
of the bilirubin.
Bilirubin glucuronide (direct or conjugated bilirubin) reacts with the diazo reagent in an aqueous
solution, whereas unconjugated bilirubin requires solubilization (by methanol, caffeine, etc.) to react.
Adding alcohol solubilizes unconjugated bilirubin, which then combines with the diazo reagent to
form azobilirubin.
The following optical readings will be measured to estimate Direct and Total bilirubin levels.
1. Sc: Serum Control (Serum Blank)
2. S: Bilirubin standard
3. Cc: Conjugated Bilirubin control
4. Cu: Conjugated Bilirubin unknown
5. Tc: Total Bilirubin control
6. Tu: Total Bilirubin unknown
Reagents:
1. Diazo reagent: Sulphanilic acid and sodium nitrite in hydrochloric acid.
2. Hydrochloric acid, 0.15 N (used as Diazo blank)
3. Methanol (ice-cold)
4. Working standard, Methyl red solution: O.D. of given methyl red standard corresponds to
O.D. of azobilirubin formed in serum sample containing 2.0, 4.0, 8.0 mg/dL bilirubin.
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Protocol:
Color development: Follow the given protocol for the development of color and estimation of optical
densities.
STEP 1.
Standard Conjugated Bilirubin Total Bilirubin
Sc S Cc Cu Tc Tu
Distilled water 8.0 mL - 6.2 mL 6.2 mL 2.2 mL 2.2 mL
Bilirubin standard - 8.0 mL - - - -
Serum - - 1.0 mL 1.0 mL 1.0 mL 1.0 mL
0.15 N HCl - - 0.8 mL - 0.8 mL -
Diazo Reagent (D) - - - 0.8 mL - -
Diazo Reagent (T) - - - - - 0.8 mL
Methyl alcohol - - - - 4.0 mL 4.0 mL

(ice cold)
STEP 2. Mix the contents of individual tubes after every addition.
STEP 3. Incubate the tests in the dark (Direct – 5 min, Total – 15 min)
STEP 5. Sc: S: Cc: Cu: Tc: Tu:
Calculations
O.D. of methyl red solution used as standard corresponds to O.D. of azobilirubin formed in serum
containing 7 mg/dL of bilirubin treated with diazo reagent. Hence,
(a) Conjugated bilirubin (mg/dL)
ODCU – ODCC
= ODS-ODSC x Conc. of Std (mg/dL)
= mg/dL
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(b) Total bilirubin (mg/dL)
ODTU - ODTC
= ODS-ODSC
x Conc. of Std (mg/dL)
= mg/dL
Unconjugated bilirubin (mg/dL) = Total bilirubin (mg/dL) - Conjugated bilirubin (mg/dL)
= mg/dL
Results
Total bilirubin = mg/dL
Conjugated bilirubin = mg/dL
Unconjugated bilirubin = mg/dL
Normal range
The normal range for total bilirubin is 0.1 – 1.0 mg/dL.
Conjugated Bilirubin: 0.1 – 0.4 mg/dL (bilirubin maybe up to 30% of the total)
Unconjugated Bilirubin: 0.2 – 0.9 mg/dL.
Interpretation
Two unique features of this test:
a) Use of serum control: When the bilirubin levels increase, the colour of the serum also
increases and this would impact the final readings of the absorbance. Hence to know how
much additional colour was produced by the reaction, we cannot use a plain reagent blank –
which does not have the colour of the serum. So a serum blank is made which contains serum,
and the reagent (less a colorless vital ingredient) which will have the same characteristics of
the reagent and the serum but the reaction will not occur due to the lack of the vital ingredient.
In this case the diazo reagent has HCL and Sulphanalic acid. For the control we use the same
Diazo reagent minus the Sulphanalic acid i.e. HCL only.
b) Use of Artificial Standard: Bilirubin is an unstable compound and liable to photo degradation.
Methyl red dye has the same absorbance characteristics of Azobilirubin, and is used as an
artificial standard.
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Normal serum bilirubin is less than 1.0 mg/dL. Hyperbilirubinemia of more than 2 mg/dl results in
clinical jaundice. Subclinical jaundice is between 1-2 mg/dl. Hyperbilirubinemia may be due to:
1. Unconjugated hyperbilirubinemia (retention jaundice) - due to retention in the circulation

of the increased amount of unconjugated bilirubin.
a. Haemolytic (pre-hepatic jaundice) due to –
i. Thalassemia
ii. Spherocytosis
iii. G6PD deficiency
iv. Mismatch transfusion
b. Non-haemolytic jaundice due to –
i. Neonatal jaundice – due to hepatic immaturity
ii. Defect in Bilirubin conjugation: Crigler Najjar syndrome I and II
2. Conjugated hyperbilirubinemia (regurgitation jaundice): Due to regurgitation into circula-
tion of conjugated bilirubin, which would normally pass along the biliary system.
a. Impaired transport to biliary canaliculi – e.g., in
i. Dubin- Johnson syndrome.
ii. Rotor syndrome.
iii. Cholestasis: Bile stones
iv. Tumors of the biliary radicles
v. Carcinoma head of the pancreas – obstruction of the opening of the bile duct
into the duodenum
vi. Intra hepatic cholestasis.
3. Mixed hyperbilirubinemia - Both unconjugated and conjugated bilirubin increase in liver
cell necrosis. Unconjugated bilirubin is retained due to impaired hepatic uptake and conjuga-
tion. Conjugated bilirubin is regurgitated due to impaired transport to bile capillaries and in-
trahepatic cholestasis. Hepatocellular necrosis may result from-
a. Virus, e.g., Hepatitis A, B, C, D, E, etc.
b. Drugs and poisons, e.g., cytotoxic drugs, isoniazid, alcohol, etc.
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24. ESTIMATION OF TOTAL PROTEINS, ALBUMIN AND DETERMINATION OF
A/G RATIO
Estimation of serum total protein by Biuret method

Principle
This is a very specific method for protein estimation. In an alkaline medium, copper reacts with the
peptide bonds in the proteins to give a purple colour (minimum 2 peptide bonds are required for
colour formation). The intensity of the colour developed is proportional to the concentration of the
protein in the solution. The intensity of the colour obtained is read in a colorimeter at 540nm and
compared to a standard treated similarly.
Reagents
(a) Biuret reagent: Sodium-potassium tartarate, copper sulphate, potassium iodide and
sodium hydroxide
(b) Normal saline
(c) Protein Standard
Protocol
1. Dilution of serum and standard:

Take 0.2mL of serum and standards in two separate test tubes. Add 1.8mL normal saline to
each and mix.
2. Colour development:
Follow the given protocol for development of color and estimation of optical densities.
STEP 1. Blank (B) Standard Test
Normal saline 2.0 mL - -

Diluted serum - - 2.0 mL
Biuret reagent 5.0 mL 5.0 mL 5.0 mL
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Calculations
Serum total proteins (g/dL)
Vol. of Std.
ODU-ODB ODS-
=
ODB x Conc. of Std (g/dL) x
Vol. of Serum
= g/dL
II. Estimation of serum albumin by Bromocresol green (BCG) binding method

Principle
Albumin in serum has an affinity for anionic forms of many indicators at pH below 5. Bromocresol
green (BCG), an anionic dye binds tightly to albumin when added to serum. The increase in
absorption of light is directly proportional to the albumin concentration.
Reagents
(a) Bromocresol Green dye
(b) Normal saline
(c) Albumin standard solution (0.5 g/dL)
Procedure
3. Dilution of serum and standard:
Take 0.2mL of serum and standard in one test tube and standards in two separate test tubes.
Add 1.8mL normal saline to each and mix.
4. Colour development:
Follow the given protocol for development of color and estimation of optical densities.
Normal saline 0.2 mL - -

Diluted serum - - 0.2 mL
Working BCG dye 5.0 mL 5.0 mL 5.0 mL
STEP 3. Read colorimetrically at 620 nm (red filter).
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Calculations
Serum Albumin (g/dL)
= ODU-ODB ODS-x Conc. of Std (g/dL) x Vol. of Std.
ODB
Vol. of Serum
= g/dL
III. Determination of A/G ratio

Total protein (g/dL) = Albumin (g/dL) + Globulin (g/dL)
Serum Globulin (g/dL) = Serum Total Proteins (g/dL) – Serum Albumin (g/dL)
= g/dL
A/G ratio = Serum Albumin
Serum Globulin
Results
Serum total protein = g/dL
Serum albumin = g/dL
Serum globulin = g/dL
A/G ratio =
Normal Range
Serum total Protein: 6 g/dL – 8 g/dL
Serum Albumin : 3.5 g/dL – 5.5 g/dL
Serum Globulin : 2.0 g/dL - 3.5 g/dL
A/G ratio : 1.2 – 2.5
Interpretation
Hyperproteinaemia:
Elevated serum total protein concentration is seen in following conditions:
(a) Haemoconcentration:
i) In dehydration due to inadequate water intake
ii) Excessive water loss as in severe vomiting, diarrhoea
iii) Prolonged use of tourniquet while collecting the sample.
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(b) Infections: Due to increase in the Gamma globulin fraction from the immune competent cells
– the plasma cells and lymphocytes.
(c) Kala Azar is classical disease where there is a substantial rise in the plasma proteins.
(d) Multiple Myeloma: Tumor of the plasma cells. Results in unregulated and gross increase of
monoclonal antibodies.
Hypoproteinaemia:
Hypoproteinaemia is often due to decreased serum albumin concentration. It occurs in following
conditions:
(a) Chronic Liver Disease: Liver is the main organ that produces most of the plasma
proteins (apart from the immunoglobulins)
(b) Haemodilution due to water intoxication or salt retention.
(c) Loss of proteins from blood:
(i) In urine, due to nephritic syndrome, chronic glomerulonephritis and diabetes
(ii) From skin in severe burns
(d) Malnutrition disorders when protein intake is diminished - Kwashiokar.
A/G ratio:
A/G ratio can be altered when
a) There is a decrease in albumin relative to Globulin
(ii) Severe liver disease – where the synthesis of Albumin is less
(iii) Nephrotic syndrome and other renal disease where the loss of albumin by the kidney
is more.
b) There is an increase in immunoglobulin content :
(i) Severe infection (Kala Azar)
(ii) Multiple Myeloma
Note: In the olden times the AG ratio was very important in differentiating between acute and chronic
liver disease. However, now a days, the AG ratio does not have much significance. A value of serum
albumin of less than 3.2 g/dL indicate that the liver disease is chronic.
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Other Methods of Serum Protein Estimation:
Bradford method: Coomassie Brilliant Blue complexes with protein (Absorption maxima - 595 nm).
Free dye binds with basic amino acids (arginine and lysine) in the protein. This method is simple and
very sensitive. Colour development is rapid but not very stable. Sensitivity is 20 µg/ ml.
Lowry’s method: This is also a sensitive method. It involves the formation of a copper-protein
complex in alkaline solution as in the Biuret reaction and the reduction of phosphotungstic and
phosphomolybdic acids into tungsten blue and molybdenum blue respectively, by the copper
catalysed oxidation of aromatic amino acids (tyrosine and tryptophan). λmax is 660 nm. Sensitivity
is 10 µg/ ml.
BCA method: Proteins reduce alkaline Cu (II) to Cu (I) in a concentration dependent manner.
Bicinchoninic Acid (BCA) is a highly specific chromogenic reagent for Cu (I), forming a complex
with an absorbance maximum at 562 nm. Because of this property, the resultant absorbance at 562
nm, is directly proportional to the protein concentration. Sensitivity is 0.5 µg/ ml.
Other Method for Serum Albumin Estimation

Bromocresol Purple: Bromocresol Purple is a dye that binds more specifically to Albumin and
hence measures it better than BCG. It is especially important in measuring albumin less than 3gm/dL.
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25. ESTIMATION OF SERUM TRANSAMINASES: ASPARTATE TRANSAMINASE
(AST/SGOT EC 2.6.1.1) ALANINE TRANSAMINASE (ALT/SGPT EC 2.6.1.2)
Modified Method of Reitman & Frankel

Principle
Transaminase is an enzyme catalyzing transfer of amino groups from an alpha-amino acid to an alpha-
keto acid. ALT in serum catalyzes the transamination reaction between its substrates, L-alanine and
α-ketoglutarate, yielding L-glutamate and pyruvate. Similarly, AST in serum catalyzes the
transamination reaction between its substrates, L-aspartate and α-ketoglutarate, yielding L-glutamate
and oxaloacetate. Oxaloacetate is spontaneously decarboxylated to pyruvate.
L-Alanine transaminase
α -Ketoglutarate + Alanine Glutamate + Pyruvate
(ALT/SGPT)
Aspartate transaminase
α Ketoglutarate + L-Aspartate Glutamate + Oxaloacetate
(AST/SGOT)
Spontaneous decarboxylation
Oxaloacetate Pyruvate
CO2
The pyruvate produced by transamination reacts with 2, 4 dinitrophenylhydrazine (DNPH) in an
alkaline medium to form reddish-brown colored hydrazone, which is measured colorimetrically at
530 nm and compared with a pyruvate standard treated similarly.
Reagents
1. Phosphate buffer (pH 7.4)
2. SGPT substrate containing L-alanine and α-ketoglutarate
3. SGOT substrate containing L-aspartate and α-ketoglutarate
4. 2,4-dinitrophenyl hydrazine (DNPH)
5. 0.4 N NaOH
6. Standard sodium pyruvate solution (22 mg/dL, i.e. 2000 μmol/L)
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Protocol
Label four test tubes as ‘Blank,’ ‘Standard,’ ‘Control,’ and ‘Unknown’ and make the following
additions:
Blank (B) Standard Control(C) Unknown (U)

(S)
Step 1 Buffered substrate* 1.2mL 1.0mL 1.0mL 1.0mL
Step 2 Pyruvate standard - 0.2mL - -
Step 3 Incubate at 370 C for 5 minutes.
Step 4 Serum - - - 0.2mL
Step 5 Incubate at 370 C for 30 min for SGPT & 60 min for SGOT.
Step 6 DNPH 1.0mL 1.0mL 1.0mL 1.0mL
Step 7 Serum - - 0.2mL -
Step 8 Allow to stand for 20 minutes at room temperature.
Step 9 0.4 N NaOH 10 mL 10mL 10mL 10mL
Step 10 Mix and keep room temperature for 10 minutes and read at 530 nm / green filter.
Step 11 Optical Density:
* Use respective substrates for estimations of SGPT and SGOT.
Calculations
The activity of transaminases is expressed in terms of International Units.
Definition of International Units/Litre (IU/L):
The international unit is defined as micromoles of pyruvate formed at 370C in one minute by enzyme
activity in 1000mL of serum.
Serum transaminase activity in IU/L
Vol. of Std 1
= ODU-ODc ODS-x Conc. of Std (μmol/L) x x
ODB Vol. of Serum Incubation Period (min)
1 1
ODU-ODc ODS-
= x 2000 x 0.2 x x
ODB 0.2 30 (SGPT) or 60 (SGOT)
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Therefore,
SGPT activity in IU/L

ODU-ODc
= x 66.66
ODS-ODB
= IU/L
SGOT activity in IU/L

ODU-ODc
= x 33.33
ODS-ODB
= IU/L
Normal range
Activity of SGPT = 5 – 30 IU/L
Activity of SGOT = 5 – 35 IU/L
Interpretation
Both transaminases (SGPT and SGOT) are widely distributed in the heart, liver, skeletal muscles, and
kidneys. However, the liver and heart are particularly rich in AST/SGOT. Any damage to these
organs that involves necrosis of the cells or increased cell permeability can be expected to raise the
serum enzyme activity. AST/SGOT is also expressed in the RBCs. A slight increase in AST relative
to ALT is noted whenever there is a destruction of RBCs, as in haemolytic anaemia.
SGOT activity
In Viral Hepatitis and other forms of liver disease associated with hepatic necrosis, serum SGOT and
SGPT levels are elevated even before the clinical signs and symptoms of the disease, such as jaundice,
appear. SGPT activity is often higher than SGOT activity. Raised SGOT activity is also observed in
hepatocellular damage due to hepatotoxic drugs, infective hepatitis, and primary or secondary liver
cancer. Still, a rise in SGOT activity in these conditions is much less than SGPT activity. After acute
myocardial infarction, increased SGOT activity appears in serum because of high SGOT
concentration in the heart muscle. SGOT may also be high in case of Muscular disorder and
haemolytic anaemia (because SGOT is present even in the RBCs). In alcohol induced liver disease
the SGOT levels are higher than the SGPT levels as alcohol is a mitochondrial poison and the SGOT
from the mitochondrial fraction is also released increasing the total SGOT level.
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SGPT activity
The chief application of determination of SGPT activity is in the diagnosis of hepatobiliary diseases.
A marked rise in SGPT activity is seen in infective hepatitis and other inflammatory conditions of the
liver. A moderate increase in SGPT activity is observed in obstructive jaundice. Moderately raised
SGPT levels are also seen in biliary cirrhosis, primary and secondary liver cancer. Rise is also seen
in toxic hepatitis due to drugs and heavy metals. SGPT has a longer half-life than SGOT, and the
levels remain higher than SGOT. SGPT is not expressed significantly in other tissues. Hence it is a
good maker for liver disease.
Liver enzymes in fulminant hepatic failure: Whenever there is a massive necrosis of the
hepatocytes, as in Fulminant Hepatitis, initially, the SGOT and SGPT levels will be very high, as they
are released from the massive hepatocyte necrosis. Over a while, when most of the viable hepatocytes
are destroyed, no hepatocytes will be available to release the enzymes, and one can observe
paradoxically low AST and ALT levels on the background of increasing serum bilirubin and
deteriorating patient condition. Hence the serum transaminase levels (and, for that matter, any clinical
enzymology results) should be evaluated based on the patient's clinical condition.
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26. ESTIMATION OF SERUM ALKALINE PHOSPHATASE (ALP, EC 3.1.3.1)
ACTIVITY
Estimation of Serum Alkaline Phosphate by King & Armstrong Method

Principle
Phosphatases are enzymes that hydrolyze monophosphoric esters to liberate phosphoric acid.
Enzyme alkaline phosphatase hydrolyses the organic phosphates like disodium phenyl phosphate
under defined conditions of time, temperature, and pH.
Alkaline phosphatase
Disodium phenyl phosphate Phenol + Phosphoric acid
(pH = 10, temp = 370C, time = 15 minutes)
The liberated phenol reacts with 4-amino antipyrine and potassium ferricyanide, to give reddish-
brown color. The color intensity is read colorimetrically at 530 nm and compared with a standard
phenol solution treated similarly.
Reagents
1. Substrate: Disodium phenyl phosphate

2. Buffer: Sodium carbonate – sodium bicarbonate (pH = 10)
3. NaOH, 0.5 N
4. NaHCO3, 5%
5. 4-aminoantipyrine (AAP)
6. Potassium ferricyanide
7. Standard Phenol solution: Concentration = 1 mg/dL
Protocol
Blank (B) Standard (S) Control (C) Unknown (U)
Step 1 Substrate 1.1mL 1.1mL 1.0mL 1.0mL
Buffer - - 1.0mL 1.0mL
Distilled water 1.0mL - - -
Phenol standard - 1.0mL
Step 2 Incubate at 370C for 5 minutes.
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Blank (B) Standard (S) Control (C) Unknown (U)
Step 3 Serum - - - 0.1mL
Step 4 Incubate at 370C for 15 minutes.
Step 5 0.5 N NaOH 0.8mL 0.8mL 0.8mL 0.8mL
5% NaHCO3 1.2mL 1.2mL 1.2mL 1.2mL
Serum - - 0.1mL -
AAP 1.0mL 1.0mL 1.0mL 1.0mL
Potassium 1.0mL 1.0mL 1.0mL 1.0mL

ferricyanide
Step 6 Mix well and read immediately at 530 nm / green filter.
Step 7 Optical density
Calculations
Serum alkaline phosphatase activity is expressed in King Armstrong (KA) units and International
Units (IU/L).
King Armstrong units (KA units)

KA units are defined as milligrams of phenol liberated by enzyme activity in 100 mL of serum at
370C in 15 minutes.
Serum Alkaline Phosphatase Activity (KA units)
ODU- ODc
= x Conc. of Std (mg/dL) x Vol. of Std.
ODS- ODB
Vol. of Serum
ODU-ODc 1
= x 1x
ODS-ODB 0.1
= KA units/dL (1 KA unit/dL =7.1 IU/L)
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International Units/L (IU/L)
IU/L is defined as micromoles of phenol liberated by enzyme activity in 1000mL of serum at 37 0C
in 1 minute.
Normal range
The normal range for serum alkaline phosphatase activity is 50 - 130 IU/L in adults & is up to about
two & a half times greater in children particularly during periods of active growth.
Interpretation
Alkaline phosphatase are expressed in liver, bone, placenta, intestinal epithelial cells and kidney in
significant quantity. They exist as Isoenzymes and also as Isoforms.
Difference between Isoenzymes and Isoform of Enzymes:

Isoenzymes are produced from different genes by bring about the catalysis of same reaction. There
are different gene products.
Isoforms are products from the same gene, catalyzing the same reaction but differing due to post
translational modification (especially based on the carbohydrates attached to them post translation.
Alkaline Phosphatase has two isoenzymes (Liver, Bone and Kidney are a set of isoforms on one
isoenzyme, and the Intestinal and Placental ones are isoforms of another isoenzyme)
Use or enzyme control: In this test an enzyme control is also used. In the Enzyme Control we do the
test in the similar way that the other test tubes are treated, except that the serum is added to this test
tube after the incubation step. In this way, in the control no additional products will be formed, and
this test tube will serve as a serum blank.
Increased serum alkaline phosphatase activity is seen in various physiological and pathological
conditions.
Physiological conditions:
1. Slightly higher levels are found in late pregnancy & after parturition (due to placental fraction)
2. High values are found in children, particularly during the growth period (due to the bone
fraction involved in bone growth and remodeling)
3. Post prandial samples would also have a high Alkaline Phosphatase level owing the intestinal
fraction being released into the serum.
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Pathological conditions:
Bone diseases with high osteoblastic activity:
1. Paget’s disease
2. Hyperparathyroidism with bone involvement
3. Rickets
4. Osteomalacia
5. Bone tumors
Hepatobiliary diseases:
1. Obstructive jaundice
2. Infective hepatitis
3. Biliary cirrhosis
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27. EVALUATION OF LIVER FUNCTION TEST
The liver is the pivotal organ of metabolism in the body. It has many functions and equally large
reserves, so liver function abnormalities indicate substantial derangement of liver function.
Acute vs chronic liver disease:
While most liver diseases present with loss of appetite, yellowish discoloration of the eye, and dark
urine, this clinical presentation can represent:
Acute liver injury: The liver has been damaged recently.
Chronic liver disease: Seen in cases of Hepatitis B or C virus. In this, the virus enters the body and
starts destroying the liver. The initial presentation may not be clinically visible. The destruction
process is gradual and continuous; if the liver reserves are not destroyed, the patient does not present
with any abnormalities. Over 10 – 20 years, when substantial damage to the liver has been done, the
patient presents with jaundice. While the presentation history is only a few days old, the liver has
been progressively destroyed for 10 – 20 years, indicative of chronic disease.
Distribution of enzymes used to evaluate liver disease:
Significance of liver function test:

When a clinician encounters a case of jaundice, the following questions come to their mind:
1. Are we dealing with liver disease?
2. If yes, how bad is it?
3. What kind of jaundice is it? Pre-hepatic, hepatocellular, or post-hepatic?
4. What is the current state of the disease?
5. Is it an acute or chronic liver disease?
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6. Is the patient responding to my treatment?
Liver function tests are designed to answer all these questions.
1. Are we dealing with liver disease?

Ans: a raised serum bilirubin indicates a liver disease. The only exception is pre-hepatic jaun-
dice and jaundice of the newborn.
2. If yes, how bad is it?

Ans: higher the serum bilirubin levels, the more severe the liver disease.
3. What kind of jaundice is it – pre-hepatic, hepatocellular, or post-hepatic?

Ans: unconjugated hyperbilirubinemia is seen in hemolytic jaundice and physiological
jaundice of the newborn.
Predominantly conjugated hyperbilirubinemia is seen in hepatocellular or obstructive
Jaundice.
Serum enzymes help differentiate between the last two conditions:
Serum transaminase (ALT and AST) are increased in hepatocellular jaundice.
Serum alkaline phosphatase (ALP) is increased in obstructive jaundice.
4. What is the current state of the disease?

Ans: the levels of the serum enzymes give a rough estimate of how severe the liver disease is.
In the case of very severe liver disease, the serum enzymes may be low due to a lack of viable
cells from which the enzymes are released.
5. Is it an acute or chronic liver disease?

Ans: serum albumin is a single parameter helpful in differentiating between acute and chronic
liver disease. The liver solely produces albumin. Albumin has a long half-life in the serum.
Also, the liver has tremendous secretory reserves for the production of albumin. Hence if the
serum albumin levels are less than 3.2gm/dL, it indicates that there has been a substantial loss
of liver function suggestive of chronic liver disease.
6. Is the patient responding to my treatment?

Ans: progressive improvement of the enzymes and a fall in the serum bilirubin levels indicate
improvement of the liver function.
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APPROACH TO ELEVATED SERUM BILIRUBIN LEVELS
*Isolated GGT may be due to intake of alcohol or enzyme induction by drugs (barbiturates & Dilantin)
*5’NT is a marker of cholesteric liver disease

Questions for which answers need to be known:
1. Role of GGT as an LFT

2. Causes of hepatic encephalopathy
3. Role of prothrombin time as a liver function test
4. Urinary findings in various cases of hyperbilirubinemia
5. Named diseases of hyperbilirubinemia and metabolic defects
6. Cause of neonatal jaundice and why phototherapy helps such children
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28.ANALYSIS OF CEREBROSPINAL FLUID (CSF)
The CSF is a clear, colorless fluid formed as an ultrafiltrate of plasma by the choroid plexus. The
CSF acts as a cushion that protects the brain from shocks. It also has a vital role in providing nutrition
to the brain.
Composition of CSF
The composition of CSF is essentially the same as that of brain ECF and is primarily determined at
the cell surfaces on which it is produced (choroid plexus) and where it is absorbed (arachnoid villi).
Its ionic composition is the same as that of plasma for some components and different for others. In
general, CSF glucose concentration is 60% of serum. Sodium, chloride, and magnesium are the same
or greater than serum, but potassium, calcium, and glucose are lower than serum. Active transport in
and out of the CSF space is probably responsible for maintaining this difference.
1. Total volume = 150 ml
2. Specific gravity = 1.006-1.008
3. pH = 7.31 -7.40
4. Normal pressure = 60-250 mm of water, or 7-20 mm of Hg
5. Color = colorless
6. Transparency = clear, free of clots ; Osmolarity = 292-297mOsm/l
7. Cellularity = nil or less than 5 lymphocytes or monocyte / mm3
8. Glucose = 40-70 mg/dl
9. Protein = 15-50 mg/dl
10. Na+ = 138-150 mEq/L
11. CI - = 116-122 mEq/L
12. HCO3 = 20-24 mmol/L
13. CSF proteins' normal A: G ratio is 3: 1.
14. Ratio of serum: CSF protein is 200: 1.
The total volume of CSF is about 150ml (30ml within cerebral ventricles and 120 ml in subarachnoid
space. In the subarachnoid space, 85ml of the total is in the spinal part, and 35 ml is in the cranial part).
Functions
1. Mechanical support (cushion effect)

2. Removal of metabolic waste products
3. Transport of biologically active compounds
4. Maintenance of the chemical environment of the brain
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CSF collection
Quinke first developed the lumbar puncture (LP) technique or spinal tap in 1891. CSF is collected by
lumbar puncture in which a fine bore needle (22 or 24 L.P needle) is passed between the 3rd and 4th
lumbar vertebrae into the subarachnoid space with the patient lying in the lateral decubitus position
and the fluid allowed to flow automatically. The whole procedure is done under strict asepsis. The
first few drops of the fluid are discarded, and the rest is collected in sterile containers. The specimen
is divided into 3 aliquots:
1. Chemistry and Serology

2. Microscopy
3. Bacteriology
In neonates, before the closure of the Anterior Fontanelle, CSF can be collected from the lateral
ventricles by passing the needle through the Anterior Fontanelle. Important to note that there is some
difference in the composition of the CSF in the lumbar region and the lateral ventricle and this has to
be kept in mind while interpreting the result.
Physical characteristics of CSF

Appearance: Normal CSF is clear.
1. Turbidity is found in bacterial meningitis.

2. The highly purulent sample is found in streptococcal meningitis.
3. Presence of RBCs gives a reddish appearance.
Color: Normal CSF is colorless.

1. Reddish appearance of CSF is due to a traumatic tap
2. Yellow color is observed in jaundice and long standing subarachnoid haemorrhage.
3. Greenish color is seen in pneumococcal meningitis.
Difference between a normal and a traumatic tap
Normal CSF Tap:

The procedure is typically well-tolerated and causes minimal discomfort. After the procedure,
patients might experience mild headaches or pain at the needle insertion site, which usually resolves
within a day or two.
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Traumatic CSF Tap:
A traumatic CSF tap, also called a "bloody tap," occurs when the needle punctures a blood vessel in
the area surrounding the spinal canal, mixing blood with the collected cerebrospinal fluid sample.
This can happen for various reasons, such as improper technique, difficult anatomy, or using larger
needles. A traumatic tap can lead to a falsely elevated white blood cell count, red blood cell count,
and protein levels in the collected CSF sample. This could complicate the interpretation of the test
results and lead to unnecessary further investigations. In a traumatic tap, the initial few samples may
show reddish colour with gradually decreases to a clear colour.
Indication of CSF examination

A wide range of disorders can produce changes in CSF composition. The most typical indication for
a lumbar puncture to remove CSF is meningitis or infection.
1. Infections:
a. Meningitis (Bacterial commonest)
b. Encephalitis
c. Viral Meningits
d. TB meningitis
2. Cerebrovascular
a. Sub-arachnoid hemorrhage
3. Dementia and degenerative
a. Alzheimer’s disease
4. Neoplastic
a. Meningial carcinomatosis
b. Secondary deposits
5. Demyelinating disorders
a. Multiple sclerosis
6. Autoimmune
a. Sarcoidosis
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28(A). ESTIMATION OF PROTEINS IN CSF BY TURBIDIMETRIC METHOD
Principle
The estimation of CSF proteins is based on the principle of turbidimetry. Turbidity causes the
decrease in intensity of the incident beam of light as it passes through a solution of particles, which
do not show a tendency to settle. The measurement of this decrease in intensity of the incident light
beam caused by scattering, reflectance, and absorption of the light is called turbidimetry. Proteins are
precipitated from CSF using trichloroacetic acid. The turbidity is then read at 620 nm and compared
with the turbidity produced in a standard protein solution treated similarly.
Reagents
(a) Trichloroacetic acid, 3%
(b) Standard protein solution (50 mg/dL)
Protocol
Label three test tubes as ‘Blank,’ ‘Standard,’ and ‘Unknown’ and make the following additions.
Blank (B) Standard (S) Unknown (U)
Distilled water 1.0mL - -
Standard protein solution - 1.0mL -
CSF sample - - 1.0mL
Trichloroacetic acid 4.0mL 4.0mL 4.0mL
Mix and read colorimetrically at 620 nm / red filter.
Optical density:
Mix and read colorimetrically at 620 nm / red filter.
131
Calculations
CSF proteins (mg/dL)
ODU-ODB
ODS-ODB
Vol. of Serum
= mg/dL
Result
CSF protein = mg/dL
Normal range
The normal range for CSF proteins is 15 – 45 mg/dL.
Interpretation
The concentration of CSF proteins increases due to the increased blood-brain barrier permeability to
plasma proteins. The increase in CSF proteins is observed in the following conditions:
1. Inflammation due to
a. Bacterial Meningitis
b. Viral meningitis
c. Encephalitis
d. Poliomyelitis
2. Obstruction to the flow of CSF
3. GB Syndrome (associated with albumino-cytological disassociation)
4. Hemorrhage
5. High intracranial pressure due to brain tumors
6. Traumatic injury
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28 (B) ESTIMATION OF CSF GLUCOSE BY GLUCOSE OXIDASE METHOD
Principle
Glucose oxidase catalyzes the oxidation of glucose to glucuronic acid & hydrogen peroxide. This
hydrogen peroxide is broken down to water & nascent oxygen by peroxidase in the presence of an
oxygen acceptor, which itself is converted to a colored compound, the amount of which can be
measured colorimetrically at 530 nm. This method is similar to the one used in estimating plasma
glucose.
Glucose oxidase
Glucose Gluconic acid + H2O2
Peroxidase
H2O2 + Chromogenic O2 acceptor Chromogen (measured) + H2O
Reagents
1. Color Reagent: Glucose oxidase, peroxidase & 4-aminophenazone
2. Standard Glucose Reagent: 100 mg/dL
Protocol
STEP 1.
Blank (B) Standard (S) Test (T)
Glucose standard - 20 μL -
Test - - 20 μL
Enzyme Reagent 1000 μL 1000 μL 1000 μL
STEP 2. Mix well & incubate for 25 minutes at 15-25°C or 10 minutes at 37°C.
STEP 3. Read colorimetrically at 530 nm.
STEP 4: Optical density:
Calculations
CSF Glucose (mg/dl)
= mg/dL
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Normal range
The normal range for CSF glucose level is approximately 2/3rd of the plasma glucose when the patient
has been fasting for over 4 hrs. In a non-diabetic patient, the CSF glucose is usually 50-80 mg/dL.
Interpretation
 An increase in CSF glucose is observed in the following conditions:
(a) Diabetes mellitus
(b) CNS tumors
(c) CNS syphilis
 A decrease in CSF glucose is observed in the following conditions:
a) Bacterial meningitis: Very low values are observed as the inflammatory cells consume
all the glucose.
b) T.B. Meningitis: Lower levels are observed.
c) Viral meningitis: Occasionally, lower levels are observed.
For clinical diagnosis of different kinds of meningitis, the following parameters are assessed in the
CSF:
Normal Viral Bacterial Tubercular
Appearance Clear Clear Turbid Turbid
(cobweb coagulum)
White blood < 5/mm3 ↑ ↑↑ ↑

cells
Predominant Lymphocytes Lymphocytes Neutrophils Lymphocytes
Cells
Glucose 2/3rd Blood No change ↓↓↓ ↓

glucose
Proteins 15-45 mg/dL No change/ mild ↑↑↑ ↑↑

elevation
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29.CHROMATOGRAPHY
Definition
Chromatography is the process of separation of components of a mixture based on differential
distribution between two immiscible phases:
1. Stationary phase
2. Mobile phase
The porous medium through which the mobile phase migrates is called the support.
1. Stationary phase: In some chromatographic techniques, solid support forms the stationary
phase, while in others, it adsorbs the liquid phase, which acts as a stationary phase. Thus, the
stationary phase is either solid or liquid.
2. Mobile phase: Mobile phase is the one that flows over or through the stationary phase and
carries the sample along with it when the separation of its components results. It is liquid or
gas.
Physicochemical principles of chromatography
1. Adsorption equilibrium: Solubility of a component in the mobile phase and adsorption in

the stationary phase decides separation. The component, which is less soluble and strongly
adsorbed, will be retained on the support, while more soluble and less adsorbed will flow out
faster.
2. Partition equilibrium: Stationary and mobile phases are immiscible with each other. Inert
support may be needed for the stationary phase. The stationary phase is liquid, and the mobile
phase may be liquid or gaseous. Separation occurs due to the differential solubility of
components in two phases.
3. Gel filtration: As the solute mixture passes through a column of porous beads, the solute
separates based on molecular size. The smaller particles pass into the pores of the beads and
are retained in the column and come out later, whereas bigger particles pass between the beads
and come out earlier. The elution time for each solute is a function of the molecular size.
Therefore, gel filtration is also called molecular sieving. It is used for the determination of
molecular weight. Examples of gels used are Sephadex, agarose, and polyacrylamide.
4. Ion exchange equilibrium: In this, charged solutes are separated. The solute mixture to be
separated is allowed to pass through a column of ion exchange resins (i.e., insoluble polymers
containing ionic groups). There are two types of ion exchange resins
137
a. Cation exchange resins contain negatively charged ions and retain positively charged
ions, e.g., Dowax 50 b
b. Anion exchange resins contain positively charged ions and retain negatively charged
ions, e.g., DEAE and cellulose.
Techniques of chromatography
1. Paper chromatography
Types:
a. Ascending: Mobile phase moves up.
b. Descending: Mobile phase moves down
c. Radial: Circular sheet of paper is kept horizontally, and the mobile phase moves radi-
ally.
d. Two-dimensional – see discussion below.
Support medium: A sheet of Whatman filter paper no. 3 (square or circular) is soaked in an
aqueous solvent.
The solvent mixture contains non-polar and polar solvents (Butanol: Acetic acid: Water). The
paper absorbs a polar solvent and acts as a stationary phase, whereas the non-polar solvent acts as
the mobile phase.
Procedure
Paper is soaked in the solvent mixture in a tray, which absorbs the aqueous part of the solvent
mixture. The sample, the components of which are to be separated, is spotted near the lower edge
of the filter paper along with known standards. The samples are applied repeatedly (allowing
drying in between) with the help of fine capillaries. The size of the spot should be tiny. The paper
is then mounted on a stand and kept in a solvent tray inside an air-tight chromatography chamber.
The solvent mixture is allowed to run almost to the upper edge of the paper. After the run, the
paper is removed, dried, and sprayed with the appropriate staining solution. The colored spots are
identified based on Rf values specific to different substances.
Distance travelled by solute.

Rf (Ratio of fronts) =
Distance travelled by solvent.
For quantitation, the spots may be cut out and eluted, i.e., dissolved in a suitable solvent, and the
intensity of the color is measured on a photoelectric colorimeter.
138
Two-dimensional paper chromatography:
This technique is used for the separation of substances with similar Rf values. Separation is first
done in ascending direction with one solvent mixture. The paper is then removed, turned through
the right angle, and the run is repeated with a different solvent mixture.
Uses of paper chromatography:

Paper chromatography is used for the separation of amino acids and sugars.
2. Thin layer chromatography (TLC)
Thin-layer chromatography and paper chromatography are related techniques concerning sample
application, development, detection, and quantitation. In TLC, a thin layer of adsorbent, such as
silica gel, usually 0.2 mm thick, is applied uniformly to a glass or plastic plate.
Advantages of TLC over paper chromatography:

1. Greater Speed
2. Greater sensitivity
3. Small sample volume
4. Good resolution
Uses of TLC:
 Separation of neutral lipids and phospholipids
 For determination of lecithin/sphingomyelin ratio in cases of respiratory distress syndrome
 Rapid identification of compounds in cases of poisoning.
3. Gas chromatography
It is used for separating volatile mixtures or after forming volatile derivatives. It is divided into
two types:
1. Gas-solid chromatography: The stationary phase is solid with a large surface area.
2. Gas-liquid chromatography: The stationary phase is a non-volatile liquid coated on an inert
solid.
In both, the mobile phase is an inert gas, which carries the solute molecules to be separated, e.g.,
Argon.
4. High-pressure liquid chromatography (HPLC)

This highly sensitive and versatile technique can be applied to the resolution of different types of
biomolecules. In this technique, a solvent mixture is pumped at high pressure through a tightly
packed column of an adsorbent to give a constant flow rate. Using powered material in the column
139
increases the surface area for better separation/resolution. Using high pressure increases the speed
of separation and reduced the run time. Separation of components occurs in the column, coming
in the effluent, and is detected by a suitable detector.
Applications of chromatography
1. Adsorption chromatography separates compounds like carotenoids, lipids, and fat-soluble
vitamins.
2. Partition chromatography: Used to separate amino acids and sugars.
3. Gel-filtration chromatography: Used to separate and purify proteins and enzymes, polysac-
charides, and nucleic acids.
4. lon-exchange chromatography: Used to separate amino acids, peptides, enzymes, isoen-

zymes, and hormones.
140
30. ELECTROPHORESIS
Definition
Electrophoresis is a technique used to separate a mixture of charged particles by migration under the
influence of an electric field. Many important biological molecules like proteins, nucleotides,
nucleoproteins, etc., possess ionizable groups, which can act as cations or anions at a given pH and
migrate towards oppositely charged electrodes.
Electrophoretic migration depends on the following:
1. Net electrical charge on the molecule

2. Size and shape of the molecule
3. Ionic strength and pH of buffers
4. Strength of the electric field
5. Temperature
6. Nature of supporting medium
Types of electrophoresis:
1. Moving boundary electrophoresis:

In moving boundary electrophoresis, described by Tiselius, the macromolecules are present
freely in the solution, and the separation occurs only at the boundaries. In contrast, the bulk
of the solution remains homogeneous. This method is rarely used.
2. Zone electrophoresis:
This is the most common type of electrophoresis. In zone electrophoresis, the sample is
applied as a spot or band on a chemically inert and homogenous supporting medium on which
the separation of the components takes place in the form of zones or bands.
Depending on the support medium used for separation, zone electrophoresis is further
classified as:
a) Paper electrophoresis
b) Cellulose acetate electrophoresis
c) Gel Electrophoresis
141
Apparatus:
Electrophoresis is carried out in a tank suitable for supporting medium, e.g., paper or gel. The
significant parts of the tank include:
1. Buffer chambers
2. Electrodes
3. Electrical connections to the power supply
4. Space for keeping support medium, e.g., paper and slides of agar gel
A power pack is used to supply either constant current or constant voltage.
Technique for serum protein electrophoresis

The serum sample to be separated is applied to a supporting medium (paper, cellulose acetate, agar
gel, polyacrylamide gel, etc.), and electrophoresis is carried out at desired constant voltage or constant
current in the presence of a suitable buffer of pH 8.6. After completion of electrophoresis, the
supporting medium is placed in a fixative (7% acetic acid) to prevent diffusion of separated fractions.
The separated fractions are then visualized using appropriate stains, e.g., Bromophenol blue and
Amino Schwartz for plasma proteins and Sudan black for lipoproteins.
Quantification of each fraction is done by the following methods:
1. Densitometry
2. Elution followed by colorimetry or spectrophotometry of the eluted fractions.
The normal electrophoresis shows the following bands:
1. Albumin – Wide dense band present on the +ve electrode side.

2. α1 Band – α1 antitrypsin, trans globulin (TGB) , α1 acid glycoprotein & α1 lipoprotein
3. β Region – Transferrin, β – lipoproteins, complements (C-3 & C-4 in fresh sample).
4. ϒ Region – Immunoglobulins
M Spike
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Applications
Types of Electrophoresis Support medium Applications

Paper electrophoresis Whatman paper No. 1 Separation of peptides, serum proteins,
nucleic acids, nucleotides, and charged
carbohydrate derivatives.
Cellulose acetate Cellulose acetate strips Separation of glycoproteins,

electrophoresis in which hydroxyl lipoproteins, and haemoglobin
groups of cellulose are variants.
acetylated with acetic
anhydride
Gel electrophoresis:
Separation is based on the
following:
1. Charge
2. Molecular size
Agarose gel Composed of agarose Separation of:

electrophoresis and agaropectin
1. Plasma proteins
2. Lipoproteins
3. Lactate dehydrogenase isoen-
zymes
4. Haemoglobins
Immunodiffusion and
immunoelectrophoresis
PCR amplification products
Starch gel electrophoresis Partially hydrolysed Separation of isoenzymes of lactate

starch dehydrogenase and alkaline
phosphatase
Polyacrylamide gel Polyacrylamide gel Separation of plasma proteins,

electrophoresis layers of varying pore enzymes, isoenzymes, and lipoproteins
sizes – provides better resolution.
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31.BLOOD GAS ANALYZER
The acid-base and electrolyte balance governs the homeostatic mechanism of the body. Measuring
blood pH, pCO2, and pO2 aids in the assessment of an individual’s acid-base status. Blood gases and
pH are determined electrochemically by the blood gas analyser.
The blood gas analyzer is designed to quantitatively determine 125μl of a blood sample for pH, pCO2,
pO2, and Hb by the electrode and a potentiometer. Based on these, it can calculate additional
parameters like plasma HCO3-, actual base excess, total CO2, oxygen saturation and oxygen content
in the blood. In some instruments, a single blood sample is simultaneously in contact with pH, pCO2,
and pO2 electrodes. In others the electrodes are in contact with separate sub-samples.
Capillary samples, in addition to arterial blood samples can also be used. The sample required is 50μl.
Most accurate results are obtained using arterial blood collected with small amount of heparin
(1mg/mL) as an anticoagulant and read as soon as possible.
Analysis of pH
Modern pH electrode measuring systems comprise a glass electrode, a reference electrode, and a
liquid junction between the two electrodes. Reference electrodes are usually Ag/AgCl electrodes and
calomel electrodes.
The potential of the glass electrodes varies with changes in the pH. H+ in the blood sample will
exchange with metallic ions in the glass membrane of the glass electrode. The glass layer of the
electrode must be hydrated for the electrode to function correctly.
Analysis of pCO2
One measures pCO2 by isolating a glass electrode in a weak bicarbonate buffer and separating this
buffer from the blood sample by a membrane permeable to CO2 in the specimen. The CO2 diffuses
into a bicarbonate buffer solution inside the electrode, and the following reaction occurs:
CO2 + H2O H2CO3 H+ + HCO3-
The change in H+ concentration in the buffer is measured inside the pCO2 electrode by same type of
components found in the pH electrode. The gas-permeable membrane of the pCO2 electrode excludes
H+ in the solution so that the pH of the specimen will not affect the observed pH change inside the
pO2 electrode induced by diffusion of CO2 from the specimen into the electrode.
Analysis of pO2
Measurement of pO2 relies on the electrochemical measurement of O2 that diffuses across a gas-
permeable membrane into an electrolyte within a Clark electrode, comprising a silver anode and
platinum cathode immersed in an electrolyte buffer. The buffer solution consists of KCl and
145
phosphate buffer or buffered KOH in water. The electrical potential of the cathode is held constant
with respect to that of the anode. O2 present in the blood sample diffuses across the gas-permeable
membrane into the electrolyte buffer, where it is reduced at platinum cathode as follows:
O2 + 2H+ + 4e- H2O2 + 2e- 2OH-
The reduction of oxygen at the cathode results in the current flow between the cathode and the anode.
The amount of current flow is measured and related to the amount of O2 in the sample.
A pO2 and pCO2 electrode can be calibrated with a flowing gas or a liquid sample that has been
equilibrated with gas. The gases used for instrument calibration are usually humidified at 37 0C to
saturate the gases with water vapor. Saturating the gases with water is necessary to eliminate the
problem of maintaining dry gas for instrument calibration.
Measured values
1. Air pressure (PL): The current air pressure is measured continuously by an integrated air
pressure sensor. The value of pO2 and pCO2 are dependent on the current air pressure. This
is taken into account when the electrodes are calibrated. The air pressure measured at the time
of the calibration is considered for the measurements.
2. Partial pressure of oxygen (pO2): Measured in mmHg / Kpa.
3. Partial pressure of carbon dioxide (pCO2): Parameter for the respiratory component of
the acid-base balance. It is measured in mmHg / Kpa.
4. pH
5. Electrolytes like K+, Na+ Ca++, Li+, Cl -
6. Haemoglobin
Calculated parameters
1. Actual bicarbonate (HCO3- ): Parameter for the non-respiratory component of the acid-base
balance, influenced by the lung function and calculated from pH & pCO2 using Henderson-
Hasselbalch equation.
2. Standard bicarbonate (HCO3-S): Parameter for a non-respiratory component of acid-base
balance. It measures bicarbonate concentration in plasma at a pCO2 of 40mmHg, a
temperature of 370C & complete O2 saturation of Hb.
3. Base excess (BE): BE is defined as a titrable base on titration to normal pH (7.4) at normal
pCO2 (40mmHg) and normal temperature (370C) and is expressed in mmol/L. It is positive
if there is actual excess of base or deficit of acid.
4. Total carbon dioxide (TCO2): Total CO2 is that liberated from dissolved CO2 and HCO3-
present in plasma when blood is drawn anaerobically and not further treated. This method
146
relies on the colour change of an indicator in a carbonate-bicarbonate buffer following a
release of CO2 on acidification of the sample.
5. Buffer bases (BB): BB shows the sum of all buffer’s anion concentrations in blood (Hb,
HCO3-, proteins, phosphate).
6. Oxygen saturation of hemoglobin (O2 SAT): O2 saturation shows the percentage of possible
bonding points of the Hb occupied by O2. O2 saturation is independent of Hb concentration.
7. Oxygen concentration (O2 CT): O2 CT is the sum of physically dissolved & chemically
bonded O2 & is mainly determined by pO2 and Hb.
8. P50 (Semi saturation pressure): The semi-saturation pressure P50 shows the oxygen partial
pressure at which the Hb is 50% loaded with O2.
9. Alveolar–arterial oxygen pressure difference is defined as the difference in O2 content
between alveolar air and the arterial blood (measured pO2).
10. Hematocrit is defined as the percentage of red blood cells to the total blood volume.
Reference values
Base excess of blood -2.3 to 2.3 mmol/L
Actual bicarbonate 24.0 to 33.0 mmol/L
Std. bicarbonate 22.4 to 25.8 mmol/L
Arterial pCO2 34 to 45 mmHg or 1.02 to 1.35 mmol/L
pH 7.36 to 7.45
Procedure
When the ‘Sample Light’ turns on, open the flap and inject the sample by pressing the aspirator
button. The ‘Ready Light’ turns on, and the flap is closed. Sample details are fed with the help of a
keyboard, i.e., body temperature in centigrade, arterial/venous blood, and patient identification. Wait
for the machine to give results on the screen.
Precautions
1. Arterial blood is to be used. Capillary blood is similar to arterial. However, it can only be
assumed to be so if circulation is good. If hands are cold, warm them to 45 – 500C for at least
4 minutes before taking the sample. On pricking the skin, the blood should be freely flowing.
2. Blood must not be exposed to air. This can be avoided when collecting directly into capillary
tubes.
147
3. Fear and painful arterial puncture may induce hyperventilation. If patient is hyperventilating
then don’t attempt ABG immediately. Reassure the patient.
4. A correction can be made for low body temperature in hypothermia. The measurements are
made at 370C. Then if ‘T’ is the actual body temp pH at T0C = pH at 370C + 0.014(37 – T)
Types of possible acid-base disturbances:
1. Metabolic acidosis: Primary decrease in HCO3- with compensatory increase arise either from
increased production of acids or decreased removal of acid, which depletes HCO3-.
2. Respiratory acidosis: Increased pCO2 arises when there is impaired excretion of CO2 from
breathing and rebreathing air containing CO2.
3. Metabolic alkalosis: Primary increase in plasma HCO3- is due to accumulation of base or
loss of acid other than H2CO3. This can occur if an excessive dose of NaHCO3 is given in
treating metabolic acidosis or with massive blood transfusion with blood containing sodium
citrate.
4. Respiratory alkalosis: The primary decrease in pCO2 is due to excessive ventilation. This
occurs physiologically at high altitudes. The stimulus is oxygen lack, but it can be produced
by excessive rapid and deep respiration in normal persons. It can be clinically seen in patients
with
a. Encephalitis.
b. Hysterical attacks.
148
Interpretation of acid-base disorder
Condition pH HCO3-- levels PCO2 Interpretation
Metabolic Acidosis Low Low Normal Respiratory compensation

has not yet started.
Metabolic Acidosis Low Low Low Body starts

with respiratory hyperventilation (reducing
compensation PCO2) in an attempt to
lose acids
Metabolic High High Normal Respiratory compensation

Alkalosis has not started
Metabolic High High High Breathing reduces in order

Alkalosis with to accumulate PCO2 (a
Respiratory form of retainable acid)
compensation
Respiratory Low Normal High Renal Compensation has

Acidosis not started
Respiratory Low High High The kidney retains

Acidosis with bicarbonates to balance
Renal the acid retained by
Compensation decreased respiration.
Respiratory High Normal Low Hyperventilation results in

Alkalosis loss of CO2, causing
alkalosis.
Respiratory High Low Low The kidney also tries to

Alkalosis with lose bicarbonates to
Renal compensate for the
compensation alkalosis.
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32.THYROID FUNCTION TESTS
Abnormalities of thyroid function: Among the endocrine glands, thyroid is the most susceptible
for hypo- or hyperfunction.
Three abnormalities associated with thyroid functions are known.
1. Goitre: Any abnormal increase in the size of the thyroid gland is known as Goitre. Enlargement
of thyroid gland is mostly to compensate the decreased synthesis of thyroid hormones and is
associated with elevated TSH. Goitre is primarily due to a failure in the autoregulation of T3 and
T4 synthesis. This may be caused by deficiency or excess of iodide
2. Hyperthyroidism: This is also known as thyrotoxicosis and is associated with overproduction

of thyroid hormones. Hyperthyroidism is characterized by increased metabolic rate (higher
BMR), nervousness, anxiety, and often protrusion of eyeballs (exophthalmos). Hyperthyroidism
is caused by Grave's disease, which is an autoimmune disease occurring due to the presence of
antibodies which bind & stimulate the TSH receptors in the thyroid gland. These auto antibodies,
known as thyroid stimulating IgG or long acting thyroid stimulator (LATS), cause excess
production of T3 & T4. Thyrotoxicosis is diagnosed by estimation of T4 (elevated) and TSH
(decreased) in plasma.
3. Hypothyroidism: This is due to impairment in the function of thyroid gland that often causes
decreased circulatory levels of T3 and T4. Hypothyroidism is characterized by reduced BMR,
sensitivity to cold, dry skin etc. Hypothyroidism in children is known as cretinism while in adult
myxoedema. Serum cholesterol level is increased in hypothyroidism (increased synthesis by
stimulating the HMG CoA reductase enzyme & decreased catabolism). It diagnosed by
estimation of T4 (decreased) and TSH (elevated) in plasma. Thus, Cholesterol estimation may
be utilized for monitoring thyroid therapy.
Once clinical or subclinical hypothyroidism is confirmed, the etiology is usually easily

established by demonstrating the presence of TPO (thyroperoxidase) and Tg (thyroglobulin)
antibodies, which are present in >95% of patients with autoimmune hypothyroidism.
Hashimoto’s Thyroiditis is the most common cause of autoimmune hypothyroidism.
153
Secretion, Metabolism & Regulation of Thyroid Hormone
Interpretation of Thyroid Function Tests
Name of disorder TSH FT4

Primary Hypothyroidism High Low
Subclinical Hypothyroidism High Normal
Secondary Hypothyroidism Low Low
Primary Hyperthyroidism Low High

Subclinical
Hyperthyroidism Low Normal
Secondary Hyperthyroidism High High
154
Flow chart to evaluate Thyroid Function
Measure
TSH
Low High
Measure FT4
High Normal Low Normal

Hyperthyroidism Subclinical Hypothyroidism Subclinical
Hyperthyroidism Hypothyroidism
Check
Anti-TPO
Antibodies
Positive Negative
Autoimmune Non- Autoimmune
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33. SCREENING FOR INBORN ERRORS OF METABOLISM
Inborn Errors of Metabolism (IEM) is not a single disease entity, but a collection of different clinical
conditions having a similar cause – deficiency of a key enzyme in a metabolic pathway. Because of
the deficiency of a key enzyme the patient has the following:
(a) Decreased synthesis of an essential metabolite produced in that pathway

(b) Accumulation of substrate before the enzyme block and
(c) Conversion of substrate into other, often undesirable, compounds
Individually these IEM diseases is rare. But if the whole spectrum of IEM is taken as one single entity,
the disease is very common. They are detected most frequently in infancy, often fatal at an early age,
and if untreated, they cause irreversible damage to the organs. Also, the disease can be inherited by
the next offspring.
Manifestations of these disorders differ widely, and these can be prevented or mitigated if diagnosis
is achieved early and appropriate treatment is instituted promptly. Several screening tests are
available to detect a suspected case. Still, the final diagnosis is only possible by measuring the enzyme
levels/activity in the patients WBCs, cultured fibroblast cells or cultured cells from the amniotic fluid.
The latter gives the physician a prenatal diagnosis. Molecular genetic techniques are increasingly
being adopted to diagnose inheritable metabolic disease at the level of DNA or RNA, with the help
of chorionic villus sampling or amniocentesis. Currently extensive research is being done in trying to
detect genetic diseases of the developing foetus by examining cell free DNA of the foetus circulating
in the maternal serum.
A list of some of the common IEMS are:
Phenylketonuria
Phenylketonuria is the most common metabolic error (1:10,000 live births). It shows an autosomal
recessive pattern of inheritance. It is caused by phenylalanine hydroxylase deficiency or absence,
leading to phenylalanine accumulation. Excess phenylalanine is converted to phenylpyruvate,
phenylacetate, and phenyl-lactate, giving rise to neurotoxic symptoms. Accumulated phenylalanine
is a competitive inhibitor of tyrosinase, the key enzyme in melanin synthesis, which leads to
hypopigmentation.
157
As shown above, a tetrahydrobiopterin (BH4) deficiency in the presence of normal phenylalanine
hydroxylase can lead to PKU. This condition is known as malignant PKU.
Clinical features: Mental retardation, seizures, psychotic behaviour, eczema, the mousy odour of
urine, hypopigmentation.
Alkaptonuria
This was the first disease identified as an inborn error of metabolism. Alkaptonuria is an autosomal
recessive amino acid storage disorder due to the deficiency of homogentisic acid oxidase.
Accumulation of alkapton bodies in the connective tissues leads to blackish discolouration of
cartilages and skin known as ‘Ochronosis’.
Clinical Features: Generally, the disease manifests in middle age, as degenerative arthritis, renal
stones, or nephrosis, due to the accumulation of homogentisic acid in respective tissues. Deposits
may also be seen in the sclera, ears, heart, tympanic membrane, and larynx.
One of the key features of this disease is the fact that the urine turns black on prolonged standing.
Patients have only a minimal increase in the concentration of homogentisic acid in their blood because
the kidneys rapidly filter it.
Maple syrup urine disease

This is an autosomal recessive disorder in which deficiency of the branched chain α-keto acid
dehydrogenase enzyme leads to a block in the metabolism of branched-chain amino acids (valine,
leucine, isoleucine).
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Presenting symptoms are commonly of the CNS. Babies may be normal at birth but subsequently
present with poor feeding, lethargy, vomiting, ataxia, convulsions, and spasticity. These are due to
the accumulation of toxic metabolites in the brain. The characteristic feature is the smell of maple
syrup (burnt sugar) in the urine due to keto acids.
Glucose -6- Phosphate Dehydrogenase (G6PD) deficiency

Glucose -6- phosphate dehydrogenase is an enzyme of the hexose-monophosphate pathway. This is
a pathway in which no ATP is generated but reducing equivalents such as NADPH are generated, and
pentose sugars are formed.
The protective action of NADPH:

G6PD helps in the conversion of glucose -6- phosphate to 6-phosphogluconate, in the presence of
which NADP is converted to NADPH. NADPH helps to keep glutathione in the reduced form and
provides reducing equivalents for various synthetic pathways in the cytoplasm. The reduced
glutathione, which helps reduce hydrogen peroxide, formed because of oxidative injury. In the RBC,
hydrogen peroxide results in the conversion of haemoglobin to methaemoglobin.
G6PD deficiency is an X-linked recessive disorder. In this enzyme deficiency, the life span of the
RBC is decreased due to oxidative damage leading to haemolytic anaemia. During oxidant stress, the
RBC cannot generate NADPH in large amounts, leading to haemolysis. Drugs causing haemolysis in
G6PD deficient individuals include:
(a) Sulfonamides
(b) Chloroquine, primaquine
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(c) Phenacetin
(d) Para-amino salicylic acid
(e) Nalidixic acid
(f) Quinine
(g) Probenecid
Clinical features may vary with the type of deficiency.
Screening test
Methylene blue reduction test: Sodium nitrite and methylene blue are added to a citrated blood
sample. The sodium nitrite changes Hb2+ to Hb3+ which makes the sample brown. Methylene blue
induces the enzyme G6PD to produce NADPH which converts the Hb3+ back to Hb2+making the
sample red once again. Deficiency leads to the persistence of the brown color.
Confirmatory test: The spectrophotometric method for detecting NADPH levels in the sample is
confirmatory.
Galactosemia
This refers to any of the three inborn enzyme deficiencies of the galactose utilization pathways.
Classical Galactosemia: This is due to the enzyme galactose-1 phosphate uridyl transferase, which
is typically associated with cataracts, mental retardation, and cirrhosis. It is an autosomal recessive
disease.
The second disorder, galactokinase deficiency, primarily leads to cataract formation. The third and
rarest is UDP-galactose 4-epimerase deficiency. The galactose is converted to galactitol in the lens
by Aldol reductase, leading to cataract formation.
The clinical features present within days of birth. The infant shows reluctance to feed, vomiting, poor
nutrition, and failure to thrive. Jaundice and hepatomegaly may develop. Cataracts are usually absent
at birth, and mental retardation, which develops, is irreversible even after omitting milk feeds. The
cause of death is generally sepsis.
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Screening tests - Beutler enzyme spot test is done to detect reducing sugars in the urine other than
glucose.
Galactose -1- phosphate uridyl transferase activity is monitored on blood dried on a filter disc with
the aid of Galactose-1-PO4, UDP-glucose, NADP+, phosphoglucomutase, glucose-6-phosphate
dehydrogenase followed by measurement of the absorbance of reduced NADP+ under UV light.
Confirmatory test: By enzyme assays on filter discs on which blood is dried
Inherited disorders of mucopolysaccharides

Mucopolysaccharidoses are a broad spectrum of disorders due to the deficiency of a group of enzymes
that degrade three classes of mucopolysaccharides, dermatan, keratan, and heparan sulfate.
Depending on the enzyme deficiency, this disorder has at least eight variants.
Clinical features
The general phenotype includes coarse facies, corneal clouding, hepatosplenomegaly, joint stiffness,
hernias, dysostosis multiplex, and mucopolysaccharide excretion in the urine. Acidic
mucopolysaccharides yield acetyl hexosamine and a hexose.
Diagnosis: This may be done by identification of metachromatic staining of Reilly bodies from
leucocytes and bone marrow. It may also be done by measurement of 24 hrs of uronic acid in urine.
Alcian blue spot test

In this method, 10 mL of urine is dried on filter paper, and then Alcian Blue is added. After rinsing it
in water, it is washed with glacial acetic acid followed by water. The urine appears as a blue spot on
a white background due to increased mucopolysaccharides in the urine.
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Screening of Inborn errors of metabolism by tandem mass spectrometry
Tandem mass spectrometry (MS/MS) is an essential tool for neonatal screening and diagnosis of
inborn errors of metabolism. It permits detecting and quantifying many IEMs in a single blood spot.
Furthermore, in a single analytical run, multiple metabolites can be measured.
Mass spectrometry identifies and quantifies analytes based on their molecular mass and charge. For
analysis by a mass spectrometer, the analyte of interest must be converted into ions. An
electromagnetic field separates these ions, and the separated ions are detected by a quantitative
method and processed into mass spectra according to their mass-to-charge ratio (m/z).
Figure 1 MS/MS amino acid profiles from neonatal specimens tested negative (normal, left panel)
and confirmed PKU-positive (right panel).
URINARY SCREENING TESTS FOR INBORN ERRORS OF METABOLISM

URINE TESTS FOR PHENYLKETONURIA
Experiment Observation Inference
Ferric Chloride Test:

Take 1 mL of urine and add Dark green color develops, Phenylpyruvate is present
2 drops of ferric chloride which persists for 2-4min
solution. Add H2SO4
dropwise to dissolve any
precipitate.
Dinitrophenyl hydrazine
test (DNPH):
To 2 mL urine, add an equal
The appearance of a yellow Presence of Ketoacids
volume of DNPH & mix it.
precipitate (phenylpyruvate)
Examine after 10 min.
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URINE TESTS FOR ALKAPTONURIA
Ferric chloride test:

Take 1 mL of urine and add Dark blue/violet color Homogentisic acid is
2 drops of ferric chloride develops. present.
solution. Add H2SO4
precipitate.
Benedict’s test:
Heat 5mL of Benedict’s The appearance of a brown Homogentisic acid may be
reagent to check for the color present.
presence of any reducing
substances already present
in the reagent. Then add 0.5
mL (8 drops) of urine. Boil
URINE TESTS FOR MAPLE SYRUP URINE DISEASE
Ferric chloride test:

Take 1 mL of urine and add Greenish grey color MSUD may be present
2 drops of ferric chloride develops, which persists for
solution. Add H2SO4 2-4mins
precipitate.
Dinitrophenyl hydrazine
test (DNPH):
To 2 mL urine, add an equal
The appearance of a yellow Presence of ketoacids
volume of DNPH & mix it.
precipitate
Examine after 10 mins.
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URINE TEST FOR HOMOCYSTINURIA
Cyanide-Nitroprusside
test:
Take 5 drops of urine in a
watch glass. Add a drop of
ammonium hydroxide & 2
drops of sodium cyanide.
Deep red color Cystine & Homocysteine
Mix & wait for 10 mins.
present
Then add a drop of 1%
sodium nitroprusside.
URINE TEST FOR GLUCOSE-6-PHOSPHATE DEHYDROGENASE

DEFICIENCY
Bernstein method: The time required for the

dye to change to reduced
Fresh blood is haemolysed
form (colorless) is noted,
and to it, add freshly
which may be more than 2
prepared indophenol dye,
hrs in G6PD deficient
NADP & G6P & phenazine G6PD deficiency
individuals.
methosulphate are added.
Add sodium nitrite & The sodium nitrite changes
methylene blue. Hb (Fe2+) to Hb (Fe3+) and
gets converted back to Hb
(Fe2+) using methylene
blue. Deficiency leads to the
persistence of the brown
color.
URINE TEST FOR PENTOSURIA
Bials test:
Take 1 mL of urine and add Blue-green color Pentose is present
2 mL of Bials reagent. Heat
on a flame till boiling
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URINE TEST FOR GALACTOSEMIA/GALACTOSURIA
Benedict’s test:
Heat 5mL of Benedict’s A reddish-brown color is Reducing sugars may be
reagent to check for the obtained. present.
presence of any reducing
substances already present
in the reagent. Then add 0.5
mL (8 drops) of urine. Boil
Methylamine test:
Add 5 mL of urine to 1 mL Appearance of red color Presence of Galactose
of methylamine
hydrochloride solution and 2
mL of 20% NaOH. Heat at
56 C for 30 min. cool
URINE TEST FOR MUCOPOLYSACCHARIDES
Alcian test:
10 mL urine is dried on filter Blue spot appears Mucopolysaccharides
paper, and Alcian blue is present (Conc.: >100 µ/dL)
added. After rinsing it in
distilled water, wash it in
glacial acetic acid followed
by distilled water.
Cetyl trimethyl
ammonium bromide
(CTAB) test:
Turbidity appears within Presence of
Place 5 mL of urine in a
5mins mucopolysaccharides
tube. Add 1 mL 5% CTAB
in citrate buffer.
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34. DNA ISOLATION
DNA, or deoxyribonucleic acid, is the double helical hereditary material in humans and almost all
other organisms. Nearly every cell in a person’s body has the same DNA. Most of the DNA is located
in the cell nucleus (nuclear DNA), but a small amount can also be found in the mitochondria
(mitochondrial DNA or mt DNA).
DNA can be isolated from any nucleated cell. The commonly used samples are leucocytes from
peripheral blood, bone marrow, amniotic fluid cells, chorionic villi cells, muscles biopsy & saliva.
The process involves the following steps:
(a) Cell lysis: Disruption of the cell membrane

(b) Removal of cellular proteins
(c) Precipitating the DNA with ethanol
(d) Quantitation and purity measurement of DNA
Methods of DNA extraction:
(a) Liquid phase

(a) Organic (phenol-chloroform) extraction
(b) Non-organic (Proteinase K and salting out) extraction
(b) Solid phase
(a) Silica-based method.
(b) Magnetic bead method
(c) Chelex (ion exchange resin) extraction
Principle of DNA extraction
Phenol-chloroform extraction method
This method relies on phase separation by centrifuging a mix of the aqueous sample and a solution
containing water-saturated phenol chloroform.
First, the cells are lysed using a lysis buffer containing detergent at a high temperature. The lysis
buffer contains Proteinase K, which denatures all the intracellular proteins released during the process
of cell lysis. This is important as many proteins (including haemoglobin) inhibit PCR reactions.
After lysis of the cells and denaturation of the proteins, the DNA is extracted in the aqueous phase by
treating the sample with Phenol, Chloroform Isoamyl alcohol mixture [Note:
Phenol/Chloroform/Isoamyl alcohol mixture is made in a ratio of 25:24:1]. The DNA remains in the
aqueous phase, while the cell debris and proteins are trapped in the organic phase.
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The DNA in the upper aqueous phase is recovered by precipitation with ice-cold ethanol or
isopropanol. After drying the DNA sample, it is reconstituted in tris buffer. The DNA extracted is
checked for quality and quantity using a nanodrop/picodrop analyzer or electrophoresis.
Figure 1 Phenol-chloroform extraction method
Advantages:
(a) Most effective at extracting large amounts of DNA.

(b) Can be used on a wide range of samples.
Disadvantages:
(a) Slow, labor-intensive, and involves toxic chemicals (phenol and chloroform).
Uses of DNA extraction:
1. Diagnostic purposes: e.g., Mutation analysis in sickle cell anemia.

2. Forensic applications
3. Constructing genomic libraries
4. Detect genomic polymorphisms.
5. Genetic engineering
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35.POLYMERASE CHAIN REACTION (PCR)
PCR is a molecular technique by which a specific sequence of a DNA molecule can be amplified.
This can be a single gene, a part of a gene, or a non-coding sequence. Most PCR methods amplify
DNA fragments of up to 10 kilobase pairs (kb). The basic principle behind the polymerase chain
reaction is in vitro enzymatic replication. The DNA generated in this replication is used as a template
as PCR progresses, setting in motion a chain reaction in which the DNA target is exponentially
amplified. With PCR, it is thus possible to amplify a single or few copies of a piece of DNA across
several orders of magnitude, generating millions or more copies of the DNA piece of interest.
Materials required
Primer pair: A primer is a nucleic acid strand or a related molecule that is a starting point for DNA
replication. A primer is required because most DNA polymerases, enzymes that catalyze the
replication of DNA, cannot begin synthesizing a new DNA strand but can only add to an existing
strand of nucleotides.
These are the oligonucleotides, 18-25 bp in length, each complementary to a stretch of DNA (in the
flanking region) to the 3' side of the region to be amplified.
Buffer: Tris-Cl buffer adjusts this reaction's optimum pH (8.3 to 8.8 at room temperature).
Taq polymerase: The DNA polymerase used in PCR is derived from Thermus aquaticus, which is
stable even at prolonged exposures at 95°C. This removes the requirement of adding the enzyme after
every cycle.
dNTPs: Usually, equimolar concentrations (200-250 µM each) of dATP, dCTP, dGTP, and dTTP
are used. Taq adds these dNTPs to the growing DNA strands according to the complementarity of the
template DNA strand.
Template DNA: 50-100 ng of pure DNA is the starting material for any PCR reaction. The DNA
should be free of any contaminating proteins which can inhibit the reaction.
PCR machines or thermal cyclers vary the temperatures cyclically, thus helping to automate the
process. After the ‘n’ number of cycles, theoretically, 2n number of copies of the target DNA will be
formed from each initial template.
PCR – steps and principle
1. Genomic DNA denaturation – 94oC for 10 min

2. The PCR cycle comprises 30-40 cycles of
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a. Denaturing
b. Annealing
c. Chain elongation
3. Final Elongation – 72oC for 5 minutes
4. Visualization of PCR products
Genomic DNA denaturation step

Genomic DNA extracted is usually very large and needs a long time to denature to give the primers
access. This is generally done at 94oC for 10 minutes. At this time, the genomic DNA denatures into
a single strand, making it possible for the primers to access the target DNA site.
The PCR cycle:

This is done cyclically and includes three steps:
1. Denaturation is done at 94oC – to separate the formed PCR products into single strands. In
contrast to the genomic denaturation step, the duration of denaturation is approximately 30
seconds. This is because the PCR products formed are rarely over 1000 base pairs long, and
these can be satisfactorily denatured quickly.
2. Annealing: This temperature is specific for each PCR amplification we plan to do. At this
temperature, the primer binds to the DNA. A high annealing temperature will not allow the
primer to attach to the DNA, and we will get false negative results. A low annealing temper-
ature will result in non-specific binding of the primer to the DNA resulting in false positive
results. We need to standardize the annealing temperature to get the desired product.
3. Chain Elongation: Done usually at 72oC for 30 sec or more, depending upon the expected size
of the PCR fragment.
The entire cycle is repeated nearly 30 – 40 times, with each cycle producing 2n copies at the end of
‘n’ cycles.
Final chain elongation:
During the PCR cycle, there are chances that a few strands would not have been wholly synthesized.
This will result in PCR fragments of varying sizes. A 10-minute final chain elongation step is given
at 70oC for 10 minutes to create uniformity. All the incomplete strands would be completed at this
stage resulting in the uniform size of the PCR fragments.
Visualization of the PCR products
At the end of the PCR cycle, even if the target DNA is amplified, it is not visible. To detect the PCR
products, we need to do gel electrophoresis of the PCR mixture and visualize it under UV light using
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suitable dyes. As UV light harms the eyes, visualization is done in a gel documentation system, and
digital images are taken and evaluated.
Application of PCR
The PCR product formed can be used for multiple applications:
1. RFLP – Restriction Fragment Length Polymorphism

2. Cloning
3. DNA sequencing
Applications
a) For diagnosis of genetic disorders. (e.g., sickle cell anemia)

b) For rapid diagnosis of infectious diseases [e.g., viruses (like HIV), bacteria (like mycobacte-
rium tuberculosis)].
c) Monitoring disease progression (e.g., viral copy number in HIV, hepatitis B).
d) Detection of drug resistance. (e.g., in diseases like TB).
e) In forensic science (e.g., for paternity determination).
f) Cloning of PCR products for further applications like insertion into expression vectors.
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36.GLYCATED HEMOGLOBIN (HBAIC)
Glycation is the irreversible, non-enzymatic addition (covalent linkage) of a sugar (e.g.

glucose) residue to terminal amino groups of proteins. The amount of H𝑏1𝑐 represents
integrated values of glucose over the preceding 6-8 weeks. It has become a reliable indicator
of efficacy of therapy. It should be routinely monitored very 3-4 months in diabetics. It is a
useful indicator in evaluating glucose metabolism during pregnancy.
Human adult hemoglobin consists of Hb𝐴1 (97%), Hb𝐴2 (2.5%), and HbF (0.5%). On
chromatographic analysis, three fractions Hb𝐴1𝛼 , Hb𝐴1𝑏 , Hb𝐴1𝑐 , are collectively referred to
as Hb𝐴1 or fast hemoglobins or glycohemoglobins or Glycated hemoglobins. Hb 𝐴1𝑐 is the
major fraction constituting approximately 80% of Hb𝐴1 . Hb𝐴1𝑐 is formed by condensation of
glucose with N-terminal valine of each ẞ chain of Hb𝐴1 to form an unstable Schiff base
(aldimine or pre𝐴1𝑐 ) which then undergoes an Amadori rearrangement to form a stable
ketoamine Hb𝐴1𝑐
METHOD FOR DETERMINATION:

1) By separation based on charge differences, like ion exchange chromatography, HPLC,
electrophoresis & isoelectric focusing.
2) Chemical analysis (Colorimetry, spectrophotometry)
3) Structural differences (affinity chromatography, immunoassay).
PRINCIPLE OF ION EXCHANGE CHROMATOGRAPHY:

Whole blood is mixed with lysing reagent to prepare a hemolysate. This is then mixed with
a weakly binding cation exchange resin. The non-glycated hemoglobin binds to the resin
leaving Glycated hemoglobin free in the supernatant. The Glycated hemoglobin is
determined by measuring the absorbance of the Glycated hemoglobin fraction and the total
hemoglobin.
REFERENCE INTERVAL
Values for Glycated hemoglobin are usually expressed as percent of total blood hemoglobin.
Non-diabetic: <5.7%
Impaired glucose tolerance: 5.7% - 6.4%
Diabetic: > or = 6.5%
Goal of Treatment for glycaemic control in Diabetics: < 7%
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37.POLY ACRYLAMIDE GEL ELECTROPHORESIS (PAGE)
PRINCIPAL:
When placed in an electric field, molecules with a net charge, such as proteins, move towards
one electrode or the other, a phenomenon known as electrophoresis.
In polyacrylamide gel electrophoresis the molecular separation is based on the size as well
as net charge. Gels are commonly made up of polyacrylamide, which is chemically inert and
polymerizes rapidly. The pore size is controlled by appropriate concentration of acrylamide
and the cross-linking reagent, methylene bisacrylamide. Higher the concentration of
acrylamide, smaller the pore size in the gel. In simplest native PAGE, the buffer has a pH of
approximately 9 therefore; proteins have net negative charges and migrate towards anode.
SDS-PAGE:
It is the most widely used method for analysing protein mixtures qualitatively. SDS (sodium
dodecyl sulphate) is an anionic detergent which denatures proteins and confers a negative
charge to the polypeptide chains. So the protein or polypeptides are separated on the basis
of their molecular mass.
ISOELECTRIC FOCUSSING:
In this proteins are separated by electrophoresis in a pH gradient in a gel. Each protein
migrates through the gel until it reaches the point where it has no net charge i.e. its
isoelectric pH.
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38.IMUNOCHEMICAL TECHNIQUES
Immunological reactions between antigen and antibody from the basis of a diverse range of
sensitive & specific clinical assays. Antigens or immunogens are defined as substances that
induce an immune response. The proteins that combine specifically with antigens are
termed antibodies. Antibodies are produced by plasma cell.
In vitro antigen antibody reactions provide methods for the diagnosis of disease and for the
identification and quantitation of antigens or antibodies. Antigen antibody reaction is
affected by antigen antibody binding, the antibody affinity & avidity and cross reactivity.
Antigen antibody binding forces are like Van der Wall forces, hydrogen bonds and ionic
bonds. These in turn are affected by environmental factors like pH, temperature and salt
concentration.
Antigen antibody reaction can be detected by

Precipitation reaction: When antigen is soluble.
Agglutination reaction: for cellular antigen/particular antigen.
Antibody Affinity- It is sum total of all non-covalent interactions between Ag and Ab.
Stronger the bonds, higher the affinity.
Antibody Avidity- Interaction of one epitope with Ab enhances the chances of interaction of
another epitope of an immunogen. Multivalent Ab IgM has higher avidity than IgG though
IgM has low affinity than IgG.
Cross Reactivity- Sometimes Ab raised against one antigen (x) reacts with unrelated Ag (y),
if this Ag (y) has similar epitope as of ‘x’. This is called cross creativity. Affinity of cross
reactivity is generally low.
PRECIPITATION REACTIONS
When a soluble Ag having atleast two Ab binding epitopes react with corresponding
antibodies, it leads to formation of large Ag- Ab complexes visible to naked eye as
precipitates. This happens because of cross linking of Ag through Ab. Such antibodies are
known as Precipitins. For precipitation reaction to occur, both Ag and Ab have to be in
optimal concentration. If either Ag or Ab is in excess, it leads to small complexes which fail
to precipitate.
Zone of Equivalence- If increasing amount of Ag is added to different test tubes containing
fixed amount of Ab the tubes showing maximum precipitation indicates optimal
concentration of Ag and Ab. In graphical representation, this zone of optimal ratio is called
as Zone of Equivalence (ZOE).
Immunological Techniques Based on Precipitation

Various qualitative and quantitative techniques are performed in semisolid gel medium
using agar or agarose. They help in diagnosing various infections like HIV, HBV etc.
QUALITATIVE METHODS:
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a) Double immunodiffusion (Ouchterlony method) – Antigen and serum containing antibodies are
placed on a separate wells that are cut into gel. Ag and Ab (if present) diffuse towards each other
through the agar and form band of precipitates.
b) Immunoelectrophoresis (IEP) – Here the Ag mixture is first separated by electrophoresis on agarose
gel e.g. HIV antigens. Then a trough is cut parallel to line of electrophoresis. Patient’s serum is put
in this trough. If antibody against any Ag is present in serum, it will be seen as precipitation band.
QUANTITATIVE METHODS:
a) Radial immunodiffusion (Mancini Method) – Ag is placed in a well and allowed to diffuse into agar
containing Ab to produce radial precipitation zone in form of a circle. Its area is proportional to the
concentration of Ag. Concentration of unknown Ag can be known by comparing it with standard
curve which is obtained using known concentration of Ag.
b) Rocket immunoelectrophoresis – Wells are cut in gel containing Ab. Then Ag (serum) is put in this
well and electrophoresis is done. Precipitation appears in the form of ‘Rocket’ and height being
proportional to concentration. This can be compared with standard curve obtained by using
increasing concentration of Ag.
AGGLUTINATION REACTIONS
When the Ag is of a particulate nature its reaction with specific Ab leads to formation of
visible clumping due to cross linking. This reaction is called agglutination. The Ab producing
these reactons are called agglutinins.
Excess of Ab does not allow agglutination as it leads to univalent binding with antigen
without crosslinking of antigen. This phenomenon is called as prozone effect.
These reactions are used in blood grouping, diagnosing infections like typhoid, syphilis and
autoimmune diseases like rheumatoid arthritis.
Different types of agglutination reactions are:

A) DIRECT AGGLUTINATION TEST-
1. HAEMAGGLUTINATION: It is used for blood grouping. RBCs are mixed with antibodies to blood
group antigens A and B on a side. If antigen is present on RBCs surface they clump together and
agglutinate.
2. BACTERIAL AGGLUTINATION: This is used to detect bacterial infections. Infection leads to antibody
synthesis in body, so serum samples of patient is placed in increasing dilution in different test tubes.
The bacteria ex. S. Typhi which causes typhoid is added to each test tube. If sample has antibody
against S. Typhi, agglutination can be seen. This test is known as WIDAL test. The test tube with
maximum dilution showing agglutination indicates the titre of antibody. If tube with 1: 200 dilution
shows agglutination and next tube of 1:400 dilution does not show, then their titre is 200. So
agglutination titre is defined as the reciprocal of greatest serum dilution showing agglutination.
B) INDIRECT AGGLUTINATION TEST- In this method, soluble antigen which normally do not cause
agglutination are used. They are coated on synthetic beads like latex particles. These are then used
to detect antibodies in serum e.g. VDRL for Syphilis and Rheumatoid factor for Rheumatoid arthritis.
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39.RADIOIMMUNOASSAY
Radioimmunoassay (RIA) was first developed by Yalow & Berson in 1959 as a sensitive and
specific technique for measuring insulin levels, for which Yalow received Nobel Prize in 1977.
PRINCIPLE:
In this technique specific Ab against the hormone is attached to the plate. Fixed amount of
Labelled Hormone (labelled Ag) and patient’s sample containing Unlabelled Hormone
(unlabelled Ag) is added. After incubation excess is washed. Competition between the
unlabelled hormones in the patient’s sample and the added labelled hormone for limited
number of Ag binding sites forms the basis of assay. Using various concentrations of
unlabelled Hormone (Ag) a standard curve can be drawn which will allow hormone
quantitation of any unknown sample. When radioactivity is measured in the bound Ag*Ab
fraction, it will be inversely proportional to (Ag).
A hypothetical example of tubes in RIA can be:

From the amount of labelled antigen bound at various concentration of unlabelled Ag
concentration. A curve can be conducted which will allow computation of any unlabelled
unknown Ag conc.
Radioactive labels:
ϒ-Emitters-most commonly used is 𝐼125 with half-life of 60 days. Others are cobalt, selenium.
Β-Emitters- not commonly used examples are tritium (𝐻 3 ) or 𝐶 14 very long half-life.
ADVANTAGES:
1) Highly sensitive technique.
2) Same equipment and operating system can be used for different substances and if necessary the
technique can be automated to handle larger workloads.
3) Can be used to assay many non-protein compounds also for which Ab can be produced, e.g.
hormones & drugs.
4) It is most commonly used to estimate the levels of insulin and other hormones.
DISADVANTAGES:
1) High cost of equipment.
2) Requirement of trained personnel.
3) Misleading results if the antigen has a well-defined biological activity.
4) Hazards of handling radioactive material.
APPLICATIONS:-
- Hormone assay.
- Tumor Marker assay etc.
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40.ENZYME LINKED IMMUNOASSAY (ELISA)
Enzyme immunoassay (EIA) or Enzyme linked Immunoassay (ELISA) is used to detect or

quantitate the substance in nano/micro amount that are present in body fluid. Enzyme
Labelled antibodies (Ab’s) or antigens (Ag’s) that is conjugates are allowed to react with
ligand and subsequently enzyme substrate is added. The decrease in substrate concentration
or increase in product concentration is measured and used to either detect or quantitate the
ligand. It is similar in principle to RIA but depends on an enzyme rather than radioactive label.
ELISA- Mainly 3 types

1. Indirect ELISA
2. Sandwich ELISA
3. Competitive ELISA
INDIRECT ELISA –
Serum or sample containing primary Ab1 is added to an Ag coated microtitre well and
allowed to react with the bound Ag. After any free Ab is washed away the presence of Ab
bound to Ag is detected by adding an enzyme conjugate Ab (anti-isotype Ab, secondary)
which binds to the primary Ab. Any free Ab is washed away & a substrate for the enzyme is
added. The coloured reaction product that forms is measured by specialized
Spectrophotometeric plate readers.
The colour developed is proportionate to Ab concentration in Patient’s Serum. Serum Ab to
HIV can be detected within 6 wks of infection.
SANDWICH ELISA –
Ag can be detected or quantitated by this ELISA. In this technique the Ab (rather than Ag) is
immobilized on a microtitre well. Sample containing Ag is added & allowed to react with
bound Ab and washed. Then 2nd Ab to which enzyme is linked is added. Excess washed off
and substrate is added and color developed is measured.
COMPETITIVE ELISA –
Ab is first incubated in solution with a sample containing Ag. The Ag-Ab mixture is then added
to an Ag coated microtitre well. The more the Ag present in sample. The less free Ab will be
available to bind the Ag coated well. Addition of an enzyme conjugated secondary Ab
specifically for the isotype of the primary Ab can be used to quantitate the amount of
primary Ab bound to the well as in the indirect ELISA. In the competitive assay however the
higher the concentration of Ag in the original sample, the lower the absorbance.
COMMONLY USED ENZYME LABELS:

Alkaline phosphate, horseradish peroxidase, glucose 6 phosphate dehydrogenase & β-
galactosidase. Enzymes can be used as labels because of their amplifying effect. A single
molecule of enzyme typically converts 103 - 104 molecules of substrate into products per
minute.
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DETECTION SYSTEM
1) Photometry: Used for compounds, which can be measured photometrically. This is
popular because of availability of compact, high performance photometers which are
versatile, reliable, simple to operate and relatively inexpensive.
2) Fluorescent & chemiluminescent measurements.
APPLICATIONS:
- Diagnosis of infections
- Hormone assays
- Tumor marker assay.
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186
41.QUALITY CONTROL IN CLINICAL LABORATORY
The main objective the lab is to have accurate values of all biochemical
parameters. Lab results have to be reliable and accurate to fulfil their role in prevention,
screening diagnosis and treatment. Strict quality control needs to be maintained in the lab.
Total quality management is the foundation by which clinical laboratories are operated and
quality improvement.
TERMINOLOGY IN QUALITY CONTROL

1. Accuracy- Closeness of the estimated value with its True value.
2. Precision- The reproducibility of the analysis. It is expressed as variation of results
obtained by repeated determinations of a same sample for the same analyte.
3. Reliability- If the result is both accurate and precise then it is a reliable test.
4. Sensitivity- It is the percentage of the positive results in-patients with the disease, e.g. test
for PKU is highly (100%) sensitive.
5. Specificity- It is the percentage of negative results among people who do not have the
disease, e.g. test for PKU is highly (99%) specific.
Approach to Quality Assurance:
1. CONTROL MATERIALS-
The performance of analytical method can be monitored by analyzing
samples whose concentration are known and then by comparing the observed values with
the known values. These controls or standards (Known value) are run on the daily basis
before analyzing patient’s samples to know the performance of the test.
Controls or standards should be-
 Stable materials
 Same matrix as the serum sample
Bovine materials are used because of safety and ready availability. Concentration of
analyte should be in normal and abnormal ranges to monitor the performance of the test.
Control Sera are supplied as lyophilized or freeze dried materials that are reconstituted by
adding distilled water or specific diluents.
INTERNAL QC- Pooled sera or material intended for quality control can be used as IQC.
It is required for the daily monitoring of the precision and the accuracy of the analytical
test method.
EXTERNAL QC- Quality control materials supplied by external agency and the results
provided by the lab are compared with other lab using the same method and instrument
for that analyte. It is important for maintaining the long-term accuracy. It is sent by an
external agency like CMC vellore to the labs.
2. PRECISION: Monitoring of QC over a period of time.
3. CONTROL CHARTS:
Control charts are graphically displays showing the observed results versus the
time when the observations were made. To monitor the results on daily and periodic basis,
the values of IQC are represented on chart by line of upper and lower control limits. When
the plotted points fall within the control limit it indicates some problem.
187
LEVEY JENNINGS CONTROL CHARTS- Mostly labs follow these charts. The
control materials are analysed for at least 20 different days to calculate the mean and
standard deviation. When the value exceeds the control limits, the test is out of control
and should not be reported.
4. SOURCES OF ERRORS IN THE LABORATORY

1) Pre Analytical Errors-
a) Test ordering:
 Tests ordered are inappropriate.
 Handwriting is not clear.
 Tests are done on wrong patient.
b) Sample collection:
 Incorrect tube or container chosen.
 Improper volume.
 Improper processing (transport, handing).
 Incorrect patient identification.
 Invalid specimen (hemolysed, lipemic, contaminated or too diluted).
 Collected at wrong time.
 Taken from same side of IV drip.
2) Analytical errors-
 Instrument not calibrated properly.
 Quality control not proper.
 Specimen mix up.
 Insufficient volume of specimen.
 Interfering substance present.
Types of errors:
1. SYSTEMATIC ERROR- Due to calibration problem and is reflected as a shift in mean
value of test.
2. RANDOM ERROR- Lack of reproducibility may be caused because of errors in:
 Pipetting
 Dissolving reagent
188
 Mixing
 Lack of stability of temperature in water bath
 Timing regulation
 Photometric and other sensors
Percentage Error = Actual value – Obtained value X 100

Actual value
MAINTENANCE OF QUALITY ASSURANCE:
1. Adequate Infrastructure
2. Trained Lab staff
3. Monitoring of Lab errors
4. Quality control maintenance
5. Problem solving and feedback mechanism.
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42.NORMAL RANGES
Parameters Normal Range
Protein (serum), total 6.7- 8.6 g/dL
Albumin 3.5 – 5.5 g/dL
Aspartate Transaminase 5 – 35 lU/L
Alanine Transaminase 5 – 30 lU/L
Alkaline Phosphatase 33 – 96 lU/L
Bilirubin -Total 0.1 -1.0 mg/dL
- Conjugated 0.1 – 0.4 mg/dL
- Unconjugated 0.2-0.9 mg/dL
Calcium – Total 8.7 – 10.2 mg/dL
- Ionized 4.5 – 5.3 mg/dL
Cholesterol < 200 mg/dL
Triglycerides < 150 mg/dL
Creatinine, Serum
Males 0.6 – 1.2 mg/dL
Females 0.5 - 0.9 mg/dL
Glucose, Blood
Fasting 70 - 100 mg/dL
Post Prandial < 140 mg/dL
Glucose, CSF 50 – 80 mg/dL
Inorganic Phosphorus
Adults 2.5 – 4.5 mg/dL
Children 4.0 – 6.0 mg/dL
Potassium 3.5 – 5.5 mEq/L
191
Parameters Normal Range
Protein, CSF 15 – 45 mg/dL
Sodium 136-145 mEq/L
Blood Urea nitrogen 7.0 – 20.0 mg/dL
Blood Urea 15 - 40 mg/dL
Uric acid
Males 3.1 – 7.0 mg/dL
Females 2.5 – 5.6 mg/dL
Amylase 31- 107 IU/L
192

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AMRITA SCHOOL OF MEDICINE

FIRST YEAR M.B.B.S.

ROLL NO: YEAR:

Convener Dr (Maj Gen) T K Saha

Professor & Head

This is to certify that

Professor & Head

S. No. Competency No. & Competency Competency Page Date Signature

25. Estimation of Transaminase (AST/ALT) BI11.13 113

Body fluid Method of collection Examples of Investigations performed

No. of moles x valency = No. of equivalents

No. of mEq = [Mass (g) x 1000] / Equivalent Weight (g)

mg/100 mL can be converted to mEq/L or mmol/L as follows:

Normal (reference) range

Safety rules to be followed in the laboratory

1. Good personal behavior/habits

Figure 1.1 Different types of sample collection tubes

Blood collection tube top colours

The most common additives are the following:

Trace element–free Tubes

1. Sterile tube (e.g., blood culture)

Red Top Yellow Top Light blue Top

• Additive: Clot activator and gel • Additive: Sodium citrate

Green Top Lavender Top Gray Top

• Additive: Heparin (Sodium/Lith- • Additive: EDTA

Table 1.1 Different types of blood collection tubes

Figure 2.1 Biomedical waste management protocol

Photometric instruments measure transmittance, which is defined as follows:

Transmittance is usually expressed in a range of 0 to 100%.

Absorbance (a)  Concentration (c) (Beer's law) --------------(1)

Absorbance  Path Length (Lambert's law) -----------(2)

By combining (1) and (2), we get

ε, the proportionality constant, is termed the molar absorption coefficient.

The absorbance of test sample (At) = ε x concentration of test (Ct) x l

Figure 4.1 Diagrammatic representation of a colorimeter

Use of blank: Three types of blanks are used to eliminate errors.

Figure 4.2 Microplate reader

Automated and Semi-automated clinical chemistry analyzers

Figure 4.3 Semi-automatic (A) and Automated (B) biochemistry analyzer

Table for identifying the λmax of the KMNO4 solution.

Wavelength (nm) Absorbance (OD)

Figure 6.1: Example of a standard curve

Required dilution Volume of KMNO4 Volume of DW Total volume

Table for making the standard curve of KMNO4

Uses of the standard curve

Where E = potential, R = gas constant, T = absolute temperature

Precautions while using a pH meter

Steps involved in operating a pH meter

Amylase (EC 3.2.1.1)

Estimation of serum amylase

The International Unit of amylase activity

Buffer (pH 7.0) 1 1 1 1

Std glucose (1.5 mg/mL) - 0.5 - -

Pre-incubate at 40°C for 3 minutes.

Incubate at 40oC for 15 minutes.

NaOH (to stop further 0.5 0.5 0.5 0.5

DNSA (chromogen reagent) 1 1 1 1

Significance of control test tube in the enzyme activity estimation experiments

Effect of pH on enzyme activity

Substrate 1.5 1.5 1.5 1.5 1.5 1.5