(An Exclusive Biotechnology Institute)

Training report in:

Clinical Biochemistry and Haematology
Session 2011-12




Submitted By:- Submitted To:
Mahendra Patidar Mr. Gokulendra Singh Bhati
Bsc.Biotechnology 2
nd
yr Teacher incharge





I express my sincere thanks toMr. Gokulendra Singh Bhati, teacher incharge for Clinical
Biochemistry and Hematology training module, Dr. B. Lal Institute of Biotechnology for the
guidance and encouragement during the course of this work.

I am also grateful to Dr. B. Lal Sir, Director, Dr. B. Lal Institute of Biotechnology for
providing all required research facilities.

I wish to express my gratitude & thanks to my parents and my friends for their help and
co operation.
















Clinical Biochemistry (a.k.a. chemical pathology, medical biochemistry or pure blood
chemistry) is the area of pathology that is generally concerned with analysis of bodily fluids.
All biochemical tests come under chemical pathology. These are performed on any kind of
body fluid such as blood, serum or plasma.
y Body fluids such as blood contain many thousands of different substances. These
include materials such as :
Glucose ingested in food
Ions such as sodium
Hormones controlling biological processes
Drugs and toxins
An enormous range of different proteins and other substances, often reflecting cell
turnover or response to illness.
y Serum is the yellow watery part of blood that is left after blood has been allowed to
clot and blood cells have been removed.
y Plasma is essentially the same as serum, but is obtained by centrifuging the blood
without clotting. Plasma therefore contains all of the clotting factors including
fibrinogen.
y Clinical biochemistry applies basic biochemistry and analytical chemistry to medical
diagnosis, treatment and management.
It provides a sound, objective basis on which to gauge the :
Extent of a clinical disorder
Biochemical consequences of a particular disease process
Response to therapy


General Instruments to be Used in Laboratory
Various types of instruments are used in the clinical laboratory and their working is based on
varied sophisticated techniques.
Following are the various instruments and techniques used in the clinical laboratory:
1. Balances a common balance is used to find out the mass of a substance by comparing it
with known masses. These are useful for the preparation of qualitative,
quantitative and standard solutions and reagents.
2. Hot plate and Magnetic stirrer the instrument is based on the principle of rotating
magnetic fields produced in metal plate. This is used for the mixing of the
solutes in the solution. Magnetic stirrer performs this function and hot plate
provides the necessary heat.
3. Centrifuge - they are designed to accelerate the sedimentation process by using centrifugal
force. The centrifuge is capable of producing speeds upto 100,000 Rpm with
relative centrifugal force (RCF) values to 600,000g are called
Ultracentrifuges.
RCF = R(RPM)
2
11810
-7

where, R = radius of Rotor
It is based on the principle of centrifugal force, which acts on a substance in circular motion
towards periphery.
Used for: a) Separation of serum or plasma from RBC
b) Separation of sediment in urine.
c) Separation of protein free filtrate.
4. Hot air oven when electricity is passed through the heating coils, electrical energy is
converted to heat energy. The temperature is controlled by a thermostat.
Uses: a) Dry sterilization.
b) Preparation of anticoagulant bulbs.
c) Drying glassware.
d) Heating of chemicals used for the preparation of primary standards.
5. Incubators these are mainly used for:
Determination of enzymes in the specimen by end point reaction methods.
Determination of Glucose, urea, uric acid, cholesterol etc by enzymatic
methods.
Growing micro-organisms on culture media.
6. Constant temperature bath this is used to carry out various chemical reactions at specific
temperatures depending upon the requirement of an experiment. The
temperature of the water bath is controlled by thermostatic arrangement.
7. Colorimeters and Spectrophotometers - the body fluids such as blood (serum and plasma)
CS and urine etc contain several organic and inorganic substances. In
colorimetric determinations, a specific reagent is used which reacts with the
specific component to form a coloured complex. The concentration of the
coloured complex is directly proportional to the concentration of the
component in the specimen. The depth of the coloured complex is measured
on a photometer or spectrophotometer. The readings are compared with the
known primary standards.
The principle behind the working of spectrophotometers is Beer-Lamberts law.
11. pH Meters used to determine the pH of the given solutions, buffers etc.


Preparation of Reagents and Buffers
Various chemicals and reagents are used in the laboratory.
Solution It is the combination of substance-a solute and a solvent. Dissolved substance is
called solute and substance present in relatively greater quantity in solution is solvent.
Reagents - Any chemical compound or mixture of compounds usually employed in chemical
analysis.
Preparation of reagents Reagents are required for qualitative and quantitative tests
involving photometric, titrimatric, chromatographic etc.
Types of Solutions and Reagents
1. Normal Solutions
2. Molar Solutions
3. Percent Solutions
4. Buffer Solutions
5. Buffered Substances
6. Indicators
7. Primary Standards
8. Complex Reagents
1. Normal Solutions these are used as reference standard solutions for the preparation of
fresh normal solution.
Normality is defined as the number of gram equivalent weight of a solute per litre of its
solution.
N =

2. Molar Solutions in this we have to find out the molecular weight of the chemical and
dissolve that much amount in distilled water.
It may be defined as the measure of concentration of a solute in a solution or units of moles
of solute per litre of the solution.
M =



3. Percent Solutions concentrations of solution is expressed as:
type the percent is calculated from the weight of solute in gram
divided by the volume(in ml).
type the percent is calculated from the volume(in ml) of solute per 100ml
of solution.
4. Buffer Solution it is the one that resists pH change on addition of acid or alkali. Buffer
solution can be prepared by combining :
a) Weak acid and its salt.
b) Weak base and its salt.
c) Acidic salt and basic salt.



Qualitative Estimation
(A) For Protien
AIM: To Perfom Hellers test for proteins.
PRINCIPLE:
Alkaloid reagents precipitate the protein Hellers test mostly used in pathology laboratories
for the determination the albumin in urine. In this test proteins get denatured when acid is
added & this form a white coagulum.
REQUIRMENTS:
Conc.HNO3,5% Egg white solution ,test tubes,test tube holders etc.
PROCEDURE:
1. Take 3ml of conc.HNO3 in a test tube.
2. Add 5% egg white solution with the help of pipette in such a manner that it form
upper layer
3. Mix gradually by rotator b/w palms.
4. Observe the test tube.

OBSERVATION:

A white ring of form at the junction of two solutions.

RESULT:
It shows the precipitation of protein in egg white by alkaloid reagent HNO3


Qualitative Assessment of Lipids
(A) Sudan dye Test
AIM:
To perform sudan dye test for lipids.
PRINCIPLE:
Sudan dye has specific property of dissolving in salts and their derivatives. These impart a
clear red colour to the solution. Sudan staining is the use of sudan dyes to stain sudanophillic
substances, usually lipids. Sudan dyes have high affinity for fats, therefore they are used to
demonstrate triglycerides, lipids and lipoproteins.
Requirements
Sample
Sudan dye
Spirit lamp
Test tube with stand and holder
Procedure
1. Take 2.5 ml of the sample and add a pinch of sudan dye.
2. Heat on burner.
Observation
A red colour is produced due to dissolution of the dye in the fat.
Result
Lipid is present in given sample.

Quantitative Assessment of Carbohydrates
AIM:
Quantitative determination of urine glucose by Benedicts method.
PRINCIPLE:
The Benedicts quantitative reagent contains potassium thiocynite and potassium ferrocynide
in addition ti sodium citrate,sodium carbonate and copper sulphate. Glucose reduces cupric
ions in the solution to cuprous ions, which reacts with potassium thiocynate to form white
coloured cuprous thiocyanate.
10 mg of glucose completely reduces 5 ml of benedicts quantitative reagent.
REAGENTS:
1. Sodium citrate
2. Anhydrous sodium carbonate
3. Potassium thiocynate
4. Distilled water
5. Copper sulphate: 18 gm
6. 5% w/v potassium ferrocynide : 5 ml
Make the final volume to 1l using distilled water.
Requirements:
Porcelain dish
5 ml graduated pipette
Glass rod
Burner
Anhydrous sodium carbonate
Procedure
1. Take 5ml of the reagents in a porcelain dish.
2. Add 2-3gm anhydrous sodium carbonate, mix well.
3. Heat the mixture to the boiling point.
4. Add urine drop wise by using a 5 ml graduated pipette, with constant stirring by glass
rod,till the blue colour of the reagents disappears and white ppt is formed.
5. Note titration reading.




Calculation:
Urinary glucose- mg/dl:
10 x 10
Titration reading(ml)

10 x 10
1.8

Result55.55










(D) For Enzyme

Aim:- Check the catalytic Action of Ptyalin enzyme
principle: - Enzymes act as catalyst for the chemical reactions. They are organic
protenaceous substances and act only on a particular substrate. In animals enzymes are
most active between r 35 C to r 44 C.
In this test we check the catalytic action of ptyalin enzyme. Ptyalin is a salivary
enzymes called as amylase and it act on starch and glycogen converting them in to maltose.

Requirements:- 0.1N iodine solution, 1% starch solution, test tube, holder etc .

Procedure:-
1. In order to obtain your own saliva, rinse your mouth with water first. Again keep 10 -
15 ml of warm water ( r r 40 30 ) in to mouth and rotate it by tongue for 2 minutes
then collect the water in a beaker.
2. Prepare 0.1N iodine solution and 1% starch solution.
3. Take 5ml of saliva solution
4. Add 5 ml of 1% starch solution
5. Add 2-3 drops of iodine solution
6. Observe the color.

Observation:- Blue colour is observed.

Result:- Salivary digestion of starch has been demonstrated.


Test Principle
Glucose reacts with ortho-toluidine in hot acidic medium to form a green colored complex.
The intensity of the final color produced is measured by using a photometer at 620 nm to
660 nm. The measured color intensity is directly proportional to the concentration of
glucose in the specimen.
Normal values
Serum/plasma (fasting): 70&110 mg/dl.
Serum/plasma: Post prandial (PP) (2 Hrs after
lunch): up to 130 mg/dl.
Specimen collection
Patient should fast for 12-16 hrs.
Fasting sample: (F) Collect 2 to 2 ml of bl ood in a fluoride bulb.
Post-glucose sample (PG): After collecting the fasting blood sample, give the patient
75 gm glucose (1.75gm glucose /kg) Collect 2 to 2 ml of blood exactly after 2 hrs.
Post prandial sample (PP) patient should re[ort to the laboratory 2 hrs after lunch 2 to
3 ml of blood is collected in a fluoride bulb.

Sample material: Plasma


Aim- Quantitative Determination of Plasma (or Serum) Glucose by Ortho -
toluidine method.
Clinical significance
Increased glucose levels may be found in different conditions such as diabetes
mellitus, hyperthyroidism, hyperpituitarism, adrenocortical hyperthyroidism, and
occasionally in hepatic disorders . In diabetic treatment overdose of insulin may
cause hypoglyccemia, Low fasting blood glucose values are associated with
hypothyroidism, hypopituitarism & hypoadrenalism.
Requirements
1) Burette or a dispenser.
2) Test-tubes : (15 x25mm)
3) Push burton pipette
4). Gucose standard : 100 mg/ dl
5.) Bunsen burner or hotplate
6.) Water bath
7.) Stows
8.) Centrig
Preparation and stability of the reagents.

1) Orthotoluidine reagents: It contains 940 ml of glacial acetic acid, 60 ml of
orthotoluidine and 1.5 gm of thiourea. The reagent is corrosive and should not be
pipette by mouth. It is stable for at least 6 months when stored at 2 -8
o
C in an amber
colored bottle. It may solidify if stored cold. Allow it to thaw at room temperature
before use. If stored col d. Allow it to thaw at room temperature before use. If stored
at room temperature, it may give low O.D. readings after a month. However, the
Usefulness of the reagent is not affected.

2) Glucose standard: 100 mg/dl, in saturated benzoic acid. It is stable f or one year at
2-8
o
C.

Additional Reagent
3) 5G/DL trichloroacetic acid. This reagent is stable at room temperature (25
o
C+5
o
C) for
one year.

Procedure


Test Standard Blank
1) Glucose reagent, ml 5.0 5.0 5.0
2) Serum/plasma,ml 0.05 - -
3) Glucose std: 100
mg/dl.ml)
- 0.05 -
4) Distilled water,ml - - 0.05

Mix thoroughly, and places the tubes in the boiling water -bath for exactly 10min. By
using tap water cool the tubes to room temperature. Measure the optical densities
of test & standard against blank at 640 nm. (red filter, 620-660 nm)

Calculations
Serum or plasma glucose, mg/dl = O.D. Test ------------------- X 100
OD Standard

If the sample is grossly hemolysed, lipemic or icteri c, an alterative method is used.

Quantitative Assessment of Proteins
(A) Quantitative determination of total serum proteins by Biuret Method
ReagentsNaOH, 6.0M - Dissolve 60gm of NaOH in distilled water and dilute to 250ml.
Store in a tightly closed polyethylene bottle at room temperature.
Biuret Reagent Dissolve 1.5gm of copper(II) sulphate and 2.5g of potassium
iodide. After solids have dissolved add 50ml of 6.0M NaOH and dilute to a total
volume of 50ml with distilled water. Store in a tightly capped polyethylene bottle
at room temperature. The reagent is stable for six months.
Biuret Reagent Blank Prepare exactly as same in step 2 but omit the copper(II)
sulphate.
Sodium Azide solution, 1.5mM Add 0.05g of sodium azide to250ml of
distilled water. Dilute to a total volume of 500ml.
Protein Standard Dissolve 1.0g of 7ml of sodium azide solution. Dilute to
10ml total volume with sodium azide solution.
Standard Solutions Dilute the protein standard to 20, 40, 60, 80 and 100g/l by
adding the appropriate amount of water. The total volume of each should be 10ml.
Principle
The Biuret method, which is the most widely used method relies on the
complexation of Cu
+2
by the function groups involved with the peptide bond. A minimum of
two peptides bonds is needed for the complexation to occur. Upon complexation, a violet
color is observed. The absorbance of the Cu
+2
protein complex is measured at 540nm and
compared to a standard curve.
Procedure
1. Pipette 5.0ml of the biuret reagent into a each of 7 test tubes.
2. Pipette 5.0ml of biuret reagent blank into a each of 7 test tubes.
3. Prepare a reagent series by adding 100 L of each of the protein standards to 5
separate test tubes filled with a biuret reagent.
4. Prepare reagent blank by adding 100 L of water to a 6
th
test tube different tets tube
filled with biuret reagent.
5. Prepare the serum unknown by adding 100 L of serum to a 7
th
test tube filled with
biuret reagent. Mix each tube.
6. Prepare a reagent blank series by adding 100 L of protein standards to 5 separate test
tubes filled with a biuret blank reagent.
7. Prepare reagent blank by adding 100 L of water to a 6
th
test tube different test tube
filled with biuret blank reagent.
8. Prepare the serum unknown by adding 100 L of serum to a 7
th
tets tube filled with
biuret blank reagent. Mix each tube.
9. Allow the cuvettes to stand at room temperature for 30min.
10.Using the reagent series blank, Zero the Spec 20 at 540nm and measur e the
absorbance of the reagent series including the serum unknown.
11.Using the blank series blank, re zero the Spec 20 at 540nm and measure the
absorbance of the reagent series including the serum unknown.
12.Conduct the blank subtraction by subtracting the absorbance of the blank series
counterpart. Plot the new absorbance vs. concentration, perform a least squares fit to
the standard curve and determine the concentration of the unknown.
Observation & Calculations

~ For given Serum Sample:-

S.No.

Protein standard solutions
(ml)
Biuret
Reagent
(ml)
Incubation
time
O.D.
(at 540nm)
BSA conc.
(ml)
Amount of
Distilled
water

Std 1.
0
(Blank)

1

5

30mins.

0
Std 2. 0.2ml 0.8 5 30mins. 0.10
Std 3. 0.4ml 0.6 5 30mins. 0.20
Std 4. 0.6ml 0.4 5 30mins. 0.29
Std 5. 0.8ml 0.2 5 30mins. 0.36
Std 6. 1ml 0 5 30mins. 0.52
7.
Serum
sample
Sample
Conc.
0.5ml

0.5

5

30mins.

0.43

Result
Amount of protein present as calculated from the graph are:
In Serum sample = 0.81


1. Aim quantitative determination of csf and urinary proteins by turbidimetry
method
Normal range
1) Csf proteins : 15-45 mg/dl.
2) Urinary proteins : chemically detectable proteins are absent in normal time .
Clinical significance
* CSF proteins
CSF proteins are increased in the different type of meningitis , in
polyneuitis and in tumors.
Requirements:
1) Test-tubes 15x 125 mm.
2) 5.0 ml. 0.2 ml serological pipettes.
3) 1.0 ml volumetric pipettes.
4) Photometer.
Reagents
1) 3g/dl. Sulfosalicylic acid.
2) Normal saline (0.85 g/dl sodium chloride )
3) 6.0 g/dl (stock protein stanadard).
Preparation of working standards
a) 60 mg/dl : it is a prepared by mixing 0.1 ml of the standard and 9.9 ml of normal
saline . (this standard is used for the urine protein determinati on)
Stability of the reagent
y CSF
Procedure
now pipette in the tube labeled as follows :
Test
CSF
Std 60 Std
120
Blank
1)3g/dl sulfoslicylic acid ml 4.0 4.0 4.0 4.0
2)CSF ,ml 1.0
3)working std 60 mg/dl ml 1.0
4)working stk 120 mg/dl 1.0
5)distilled water, ml 1.0

Mix thoroughly and keep at room temperature for exactly 5 minutes. Measure the
intensities fo the test and standard by setting blank at 100% t, by using 640 nm(red filter).
Calculations
CSF proteins, mg/dl = O.D test/O.D std. x 60 x 10
Test o.d= .01
Test std = .31
Csf, mg/dl = 19.354

















Quantitative analysis of lipids
1.Aim- quantitative determination of total lipids by sulfo-phosphovanillin
Test principle
Lipids react with vanillin in the presence of sulfuric and phosphoric acid to form a pink
colored complex.
Samples material
Serum(fasting)
Normal values
400-1000 mg/dl
Reagents
1) Total lipid standard : 1000 mg/dl. It is
prepared by dissolving 1.0 g of live oil in chloroform.
2) Color reagent ( phosphovanillin). It is prepared by mixing
a) 0.61 gm/dl vanillin : 350 ml
b) orthophosphoric acid : 600
ml c) distilled water :
50 ml
3) Concentrated sulfuric acid (AR).
Stability of the reagents
reagents 1 and 2 at 2-8 degree C in amber colored bottles and
reagent 3 is stable at room temperature.
Procedure
pipette in the tubes labeled as follows.
Table
Test Std
1)solution 1, ml 0.05
2)serum, ml 0.05
3)reagent 3 ml 2.0 2.0

Mix thoroughly , plug with cotton wool, keep in a boiling waterbath for 10 minutes. Then
cool in a cold waterbath and again pipette into dry test tubes as follows :
Table
Test Std Blank
1)Solution 1, ml 0.10 0.10
2)Serum, ml 0.05 0.10
3)Reagents 3 , ml 2.5 2.5 2.5

Mix thoroughly and keep at room temperature for 15 minutes. Read absorbance of test and
standard against blank in a dry cuvette at 546 nm(green filter, 520 -560 nm).
Calculations
Serum total lipids mg/dl = O.D test/O.D std x 1000
o.d test = .002
o.d std = 1.9
serum, mg/dl = 1.028
Precautions
1) Reagents 2 &3 are corrosive, handle with care.
2) Detergents & fats interface with the determination.
3) Keep solutions away from open falmes & sources of heat.









2.Aim: - determination of total cholesterol by leiberman burchard method
Principle : the Lieberman burchard or acetic anhydride test is used for the detection of
cholesterol. The formation of a green or green blue colour after a few minutes is positive.
Lieberman burchard is a reagent used in a colorimetric test to detect cholesterol, which
gives a deep green color. This colour begins as a purplish, pink color and progresses through
to a light green then dark green color. The colour is due to the hydroxyl group (-OH) of
cholesterol reacting with the reagents and increasing the conjunctions of the un -saturations
in the adjacent fused ring. Since this test uses acetic anhydride and sulfuric acid as reagents,
caution must be exercised so as not to receive severe burns.
Requirements :-
1. Cholesterol.
2. Glacial acetic acid A.R.
3. Ferric chloride
4. Sulphuric acid.
Preparation of solutions :
1. Cholesterol stock standard : dissolve 1.0 mg of cholesterol in 1.0 ml of distilled
water.
2. Cholesterol working solution. Dilute 1.0 ml of s tock standard to 5 ml with glacial
acetic acid.
3. Ferric chloride 10%
4. Color reagents : - Add 0.5 ml of 10% FeCl3 in a 50 ml flask / cylinder and then add
conc. H2SO4 upto the mark.

Test sample :-
1. Take 6ml of glacial acetic acid in a test tube.
2. To this add 0.5 ml of tissue homogenate or sample.
3. Now add 4 ml of color reagent.
4. Mix and leave the tubes to be cooled.
STANDARD :-
1. Take 6 ml of glacial acetic acid in a test tube.
2. To this add 0.5 ml of cholesterol working solution.
3. Now add 4 ml of color reagent.
4. Mix and leave the tubes to be cooled.
BLANK: -
1. Take 6 ml of glacial acetic acid in a test tube.
2. To this add 0.5 ml of distilled water.
3. Now add 4 ml of color reagent.
4. Mix and leave the tubes to be cooled.
Read at 559 nm (green yallow) against blank.
CALCULATION : -
Reading of unknown/reading of standard X 0.1 X 1000/tissue or
sample taken .
Unit : mg cholesterol / gm of tissue.
Std o.d = 0.118
Test o.d = 1.493


Quantitative analysis of enzymes
1.Aim :- determination of lactic dehydrogenase by worblewslis method.
Principle : wroblewskli and ladue published the first UV kintic method for the determination
of LDH activity in serum in 1955. Their method was based on the classic Kubowitz and Ott
assay (1943) utilizing the pyruvate to lactate reaction. In 1956 , wacker at all describe a
procedure that followed a lactate to pyruvate reaction became the preferred reaction , even
though the slower of the two , because of a wider linear range and no pre -incubation
requirement. The present method f ollows the forward reaction and has been optimized for
greater sensitivity and linearity.
Principle ::-
LDH
L- Lactate + NAD + .pyruvate + NADH + H+
Lactate dehydrogenase catalyzes the oxidation of lactate to pyruvate with simultaneous
reduction of NAD to NADH. The rate of NAD reduction can be measured as an proportional
to LDH activity in serum.
Reagents :
1. Dipotassium hydrogen phosphate.
2. Pyruvic acid / sodium pyruvate.
3. Phosphoric acid.
4. Dinitrophenyl hydrazine reagent.
5. Chloroform.
6. NADH/ DPNH.
7. Hydrochloric acid.
8. Sodium hydroxide.
Preparation of solution : --
1. Phosphoric acid (0.3 M) 2ml of 85% phosphoric acid sp. Gravity 1.7, made upto
100 ml with GDW.
2. Substrate : dissolve 10 gm K2HPO4.3H2O(OR 7.7 GM K2HPO4) in 500 ml of GDW
ad 200 mg pyruvic acid with mixing , add 8 ml of 0.3M phosphoric acid. Adjust pH
7.5 7.7. make upto 1 liter and add a drop of chloroform to preserve. This is
pyruvate buffer.
Note :-
y 253 mg of sodium pyruvate may be used in place of free acid.
y On the day of use add 10 ml of pyruvate buffer to 10 mg NADH / DPNH. Mix well.
3. Dinitrophenyl hydrazine reagent : 220 mg. 2,4 dinitrophenyl hydrazine +85 ml
concentrated HCL. Make up the volume to one liter with GDW.
4. NaOH -0.4 N.
Procedure :
1. Calibration curve
Pyrurate buffer 9ml Water (ml) LDH unit
1. 0.5 0 00
2. 0.4 0.1 300
3. 0.3 0.2 700
4. 0.2 0.3 1000
5. 01 0.4 1500
6. 0.05 0.45 2000

1. To each tube add 0.5 ml dinityolphenyl hydrazine.
2. Mix and wait for 20 minutes.
3. Add 5 ml of .4 N NaOH.
Read against blank at 464 nm in the spectrophotometer.
2. Sample :
1. Add 0.1 ml of homogenate into 0.5 ml of substance.
2. Mix and incubate at 37 degree C for 45 minutes.
3. After incubation immediately add 0.5 ml 2.4 dinitrophenyl.
4. Nix and allow it to stand for 20 minutes at room temperature.
5. Add 5 ml of 0.4 N NaOH.
6. Mix and allow standing for 30 minutes at room temperature.
7. Blank : distilled water.
8. Read against blank at 464 nm in spectrometer.
Calculation :
Reading from graph/ tissue taken x 1000 x 60/5.
Unit : LDH unit / gm of tissue / hr.


AIM : Estimation of serum amylase by the amylolytic method of somgyi.
PRINCIPLE:
Amylase is a digestive enzyme which hydrolyses starch into maltose. It is present in saliva
and pancreatic juice where it is secreted by parotid glands and pancrea ses respectively.
Amylase is measured by allowing the serum enzyme to act upon starch and measuring the
rate of hydrolysis of starch. The rate of reaction is proportional to the amylase content of
the serum.

CALCULATION:

Serum amylase (S.U./100 ml) = Ac-(Au/Ac)*800

Where Ac = Absorbance of control (initial)
Au = Absorbance of unknown

REQUIREMENT:
Test tubes, test tubes holders, conical flask etc.

REAGENTS:

1. Starch Solution:
a. 1gm of starch is added to 20-25 ml of water and shaken well. 9gm of NaCl is
added to 60 ml of water and heated for few minutes. Starch solution is added
to is slowly.
b. After boiling for a few minutes, the solution is cooled to room temperature
and diluted to 100 ml with water.
c. It is stored in refrigerator and di luted 1 in 10 before use.

2. Phosphate buffer:
a. In a 1 Litre volumetric flask, 1.736 gm of Na
2
HPO
4
and 1.059 gm

KH
2
PO
4
are
dissolved in and diluted to 1 Litre with water.

3. Buffered starch:
a. 4 ml of starch is mixed with 5 ml of phosphate buffer.

4. Iodine solution:
a. In a 1 Litre volumetric flask, 3gm of KI and 13gm of iodine are dissolved in and
diluted to 1 Litre with water.
b. Before use, 1 ml of this solution is diluted to 10 ml of water.


PROCEDURE:

1. Label two test tubes Unknown and Control, add 0.9 ml of buffered starch to each.
2. Dilute serum 1 in 10 with water and add 0.1 ml to unknown.
3. Incubate both the tubes at 37
0
C for 15 min.
4. Add 0.4 ml of iodine solution and 8.6 ml of water to both and 0.1 ml of diluted serum
to control.
5. Mix and read against water at 670 nm or using a red filter.

PRECAUTIONS:

1. Serum sample must be handled carefully.
2. Blank must be set before taking readings.

OBSERVATION TABLE:

S. NO. Absorbance of
control
(Ac)
Absorbance of unknown
(Au)
Serum Sample
(S.U./ 100 ml)


Serum amylase (S.U./100 ml) =((Ac-Au)/Ac )*800
Ac= Absorbance of control
Au= Absorbance of unknown



Other quantitative analysis
1.Aim : quantitative determination of serum urea nitrogen by biacetyl monoxime method.
Clinical significance elevated levels of urea are observed in pre renal , renal and post renal
conditions.
Test principle : urea reacts with diacetyl nonoxime in hot acidic medium an in the presence
of thiosemicarbazide and ferric ions to form a pink colored compound which can be
measured on green filter.
Requirements ::
1. Test tubes
2. Pipette
3. Measuring cylinder
4. Water bath
5. Photometer
6. Pipette
Reagents
y Reagent 1 : it consists of 0.2 g/dl diacetyl monoxime in distilled water.
y Reagent 2 : it contains 40 mg/dl thiosemicarbazide in distilled water.
y Reagent 3: it contains 60 ml of conc. Sulfuric acid , 10 ml of orthophosphoric acid
and 10 ml of 1 g/dl of ferric chloride in orthophosphoric acid in one liter of the
reagent prepared in distilled water.
y Urea nitrogen standard : 20 mg/dl : it contains 42.8 mg of urea in 100 ml of
saturated benzoic acid .
y Working reagent : it prepared fresh by mixing one part of reagent 1 amd one part
of reagent 2 and two part of reagent 3.
Specimen-serum
Procedure-
1. Pipette in the tubes
2. Mix the contents of the tube thoroughly and keep in the water boiling bath for
15 minutes.
3. Cool the tube to room temperature and read absorbance at 530 nm after 5
minutes.

Observations :-
Test Standard Blank
Working reagent 5.0 5.0 5.0
Diluted serum 0.05
Urea nitrogen std 0.05
Distilled water 0.05

Calculations
Serum urea nitrogen Mg/dl= O.D. of test / O.D. of standard x 20
Test o.d = .02
Std o.d = .05
Result= 8 mg/ml



Serum Analysis
Quantitative Assessment
Determination of Creatinine in Serum by Alkaline Picrate Method
Clinical Significance
Serum Creatinine is increased in renal failure. Increased serum Creatinine
concentration above 1.5 to 2.0mg/dl is virtually diagnostic of renal disease. Elevated values
are also observed in certain other conditions like congestive heart failure, shock and
mechanical obstruction of the urinary tract.
Principle
Creatinine reacts with picric acid in alkaline medium to form a reddish yellow
complex, intensity of which is directly proportional to the concentration of Creatinine in the
specimen and can be measured at 520nm.
Requirements
1. Test tubes
2. Pipettes
3. Centrifuge tubes
4. Photometer
Reagents
1. Picric acid reagent : 0.91gm/dl (0.04M)
2. 10gm/dl Sodium Hydroxide
3. Working Creatinine standards. 1mg/dl, 5mg/dl and 10mg/dl
4. Sulfosalicylic acid : 3gm/dl
Specimen Serum

Procedure
1. Pipette 1ml of serum in a test tube.
2. Now pipette in the tubes labelled as follows:


Test Standard
Distilled Water 3.0 4.0
Serum 1.0
Standard 1mg/dl, ml
1.0
2/3N Sulfuric acid 0.5
10mg/dl Sodium
tungstate

0.5


3. Centrifuge the contents in the test and clear filterate. Pipette in the tubes as follows:
Test Standard Blank
Distilled Water 3.0 3.0 5.0
Filterate 2.0
Standard 1mg/dl, ml
undiluted

2.0

Alkaline picrate
reagent, ml

1.0

1.0

1.0

Observations & Calculations

Standard

Urine Sample

O.D. (at 520nm)

0.5

0.1

Result
Amount of Serum Creatinine present in given serum sample is 20mg/dl.


Determination of Serum Uric Acid by Henry Caraways Method
Clinical Significance
Uric acid is the end product of nucleorotein metabolism. It is allow threshold
excretory product. The serum uric acid level is often raised in gout. The determination has
diagnostic value in differentiating gout from non gout.
Principle Uric acid in the protein free filterate reacts with phosphotungstic acid reagent in
the presence of sodium carbonate to form a blue colored complex. The intensity of the colour
is measured at 660nm.
Requirements
1. Centrifuge tubes
2. Test tubes
3. Pipettes
4. Photometer
Reagents
5. Deproteinizing Reagent: 10gm/dl Sodium Tungstate 50ml; 2/3N sulphuric
acid 50ml; Orthophosphoric acid - 1drop; Distilled water 800ml. Mix well
10gm/dl sodium carbonate dissolve 10gm in 100ml of water.
6. Stock Phosphotungstic acid reagent: sodium tungstate 50gm; orthophosphoric
acid 40ml; distilled water 400ml. Mix and reflux for two hours, cool and make
final volume to 500ml.
7. Stock uric acid standard: heat about 80ml of distilled water in 250ml beaker to
60C. Add 60mg lithium carbonate and mix well. Add 100mg uric acid and mix
thoroughly. Add 2ml of formalin and then, slowly with shaking 1ml acetic acid.
Mix well and make final volume to 100ml by adding distilled water.
Specimen Serum
Procedure
1. Dilute the reagent 3, stock phosphotungstic acid 1:10. Mix well.
2. Dilute stock uric acid standard 1:200. Pipette 19.9ml of distilled water in a test
tube and add 0.1ml of stock standard. Mix well.
3. Now pipette in the tubes, labelled as follows: Deproteinizingagent : 4-5ml; serum :
0.6ml. Mix and centrifuge at 3000rpm for 10minutes.
1. Mix thoroughly,keep in the dark for exactly 10mins. and read intestine at 660nm.
Test Standard Blank
Diluted Urine 3.0
Diluted Standard 3.0
Distilled Water 3.0
10gm/dl Sodium
carbonate
1.0 1.0 1.0
Diluted
Phosphotungstic
Acid reagent
1.0 1.0 1.0

Observations & Calculations

Standard

Sample

O.D. (at 660nm)

0.1

0

= 0mg/dl
Result
Presence of Uric acid in given serum sample was not detected i.e. amount of uric acid in
given serum sample is zero(0)mg/dl.





Total Leukocyte Count by Hemocytometer
Clinical significance
Increase in total count of more than 10,000/cu mm is known as leukocytosis and decrease of
less than 4,000 cu. mm as leucopenia.
Specimen EDTA blood or capillary blood
Principle
The glacial acetic acid lyses the RBC while the gentian violet slightly stains the nuclei of the
leukocytes. The blood specimens is diluted 1:20 in a WBC pipette with the diluting fluid and
the cells are counted under low power of the microscope by using a counting chamber. The
number of cells in undiluted blood is reported per cu.mm of whole blood.
Requirements
y Microscope
y Improved Neubauer chamber
y WBC pipette
y WBC diluting fluid : Glacial acetic acid 2.0ml; 1% (w/v) gentian violet 1.0ml and
distilled water 97ml.
Procedure
1. Draw blood up to 0.5mark of WBC pipette.
2. Carefully wipe excess blood outside the pipette by using cotton.
3. Mix the contents in the pipette and after five minutes by discarding few drops, fill the
counting chamber and allow the cells to settle for 2-3minutes. Make a dilution of
blood by adding 20 l of blood to 0.38ml of diluting fluid in a glass tube. Fill the
Neubauercounting chamber by means of a Pasteur pipette or glass capillary.
4. Focus on one of the W marked areas (each having 16small squares) by turning
objective to low power(10X).
5. Count cells in all four W marked corners.
Calculations
(per cu.mm of whole blood)
=



Where,
Dilution = 20; area counted = 41 sq.mm = 4sq.mm; depth of fluid = 0.1mm
Hence,

cumm



III uI IIuuu



Blood Cell Count
Introduction
The technique of counting of the blood cells is known as haemocytometer. This involves
manual counting of the cells with the help of a microscope after diluting blood in respective
diluting fluids. The cells most often counted by this technique are red cells, white cells, and
platelets and eosinophils.
Total Erythrocyte Count by Hemocytometer
Introduction
The RBC count is important in diagnostic hematology. It permits the MCV and MCH values
to be calculated. The manual method preferred for RBC count is an automated method.
Clinical significance
At birth the total erythrocyte count varies from 6.5millions/cu mm to 7.5millions/cu mm.
There is steady decline after a few hours and at the end of the 15 days to one month there is
slow rise to normal adult levels. An increase in total count is observed in conditions such as
hemoconcentrations due to burns, cholera, etc. in central cyanotic states as seen in chronic
heart disease, conditions of decreased lung functions and in polycythemia. Decrease in count
is observed in old age, in pregnance, etc.
Specimens EDTA blood or capillary blood
Principle
The blood specimen is diluted 1:200 with the RBC diluting fluid cells are counted under high
power (40 X objective) by using a counting chamber. The number of cells in undiluted blood
are calculated and reported as the number of red cells per cu mm of whole blood.
Requirements
y Microscope
y Improved Neubauer Chamber
y RBC pipette
y RBC diluting fluid Sodium citrate 3g ; Formalin 1ml; distilled water 100ml.
Procedure
1. Mix the anticoagulated blood carefully by swirling the bulb.
2. In the case of capillary blood the lancet stab should be sufficiently deep to allow free
flow of blood.
3. Draw blood up to 0.5mark.
4. Carefully wipe the excess blood outside the pipette by using cotton or a gauze.
5. Draw diluting fluid up to 101mark.
6. The pipette is rotated by keeping it horizontal during mixing.
7. After 5minutes, by discarding few drops from the pipette and holding it slightly
inclined small volume of the fluid is introduced under the cover slip which is placed
on the counting chamber.
8. Allow the cells to settle for 2-3minutes.
9. Place the counting chamber on the stage of the microscope.
10. Switch to low power (10X objective). Adjust light and locate the large square in the
centre with 25small squares.
11. Now switch to high power (40X objective).
12. The res blood cells in the four corner squares and in the centre square are counted.
13. Use following formulae for the calculation or red blood cells :


OR










Wh
BiluIiun
Aia countu sqmm
Sinc clls ai countu in Iiggi squais anu such squai is Iuithi uiviuu into
small squais
Each small squai sqmm
Bnc aia of such aia sqmm sqmm
Bpth of fluiu mm
NumIi of iu clls countu N
Bnc

cumm
Determination of Platelet count
Clinical significance
Determination of platelets is required in the investigation of bleeding
disorders. Thrombocytopenia (decreased platelet count) is often associated with prolonged
bleeding and poor clot reaction. It also occurs in aplastic anemia, megaloblasticanemia,
hypersplenism, acute leukemia and in immune thrombocytopenia. Thrombocytosis (increased
platelet count) is found in polycythemiavera, following splenectomy and in chronic
myelogenousleukemia.
Speecimen EDTA anticoagulated blood
Requirement
a) Microscope
b) Improved Neubauer counting chamber
c) RBC pipette
d) Platelet diluting fluid Procrainehydrocjloride 3.0g; sodium chloride 10g; distilled
water 100ml. Filter it through Whatmann No. 44 filter paper and store in a clean and
dry plastic container. It is stable at 2-8 C
Procedure
1. Mix the blood specimen carefully.
2. By using RBC pipette draw blood up to 0.5mark.
3. Wipe the excess blood on the outside of the pipette.
4. The diluting fluid is drawn up to the mark 101 (blood is diluted 1:200).
5. Mix the contents in the bulb thoroughly.
6. After 5minutes, discard the first drop, then transfer a small drop on one side of the
counting chamber.
7. Place the filed mounted counting chamber under a petri dish with a moist filter paper.
Let it stay undisturbed for 15minutes.
8. Place the counting chamber carefully on the stage of the microscope. Under the low
power magnification focus red cell counting area. Move to view the corner square of
the red cell area and change to high power objective.
9. Keep the condenser down and reduce the light by adjusting the diaphragm. The
platelets will appear like highly refractile particles.
10. Count the platelets in all 25 small squares. The area covered by 25 squares is
equivalent to 1sq. mm.




Calculations





Where,
Dilution = 200
Volume of fluid = 1 0.1 = 0.1cu mm.
Hence,




















BLOOD FILM

AIM:
Preparation of blood film

REQUIREMENTS:
1. Spreader
2. A smooth slide
3. Blood sample

PROCEDURE:
1. The slide should be clean. Place a small drop of blood about 1 -2 cm from one end.
2. Without delay place a spreader at an angle of 45
o
from the slide and move it back to
make comtact with the
drop.
3. The drop should spread out quickly along the line of contact of spreader with the
slide.
4. The moment this occurs, spread the film by rapid smooth forward movement of the
spreader.
5. The film should be 3-4 cm in length.
6. The end from where the spread has ended is called tail end.
7. The ideal zone to examine the blood film is the areas between tail and body.
8. if the film is too thin or a rough edged spread is used many leucocytes accumulate in
edges and at tail
9. DLC should not be attempted on such slide.

CHARACTERISTICS OF AN IDEAL BLOOD SMEAR:
1. It should be in the central 2/3 of slide.
2. It should have straight lateral border and short tongue shaped tail.
3. It should not be too thick or too thin.



PRECAUTIONS:
1. Angle should be maintained at 45
o
.
2. Blood drop should of proper size.
3. Spreaders edges should be smooth and it should be smaller than the slide on
which smear is being made.
4. Pressure applied should be proper.
5. Preparation should be in one single stroke.

Determination of Erythrocyte Sedimentaion Rate(ESR
Specimen: Fresh fasting EATA anticogulated and undiluted blood.(It is necessary to
draw at last 2 to 3 ml of blood )
Principal: The mixture blood is draw into a wintrode tube upto the zero mark and the
tubeset upright (vertical position) in a stand.
The level of the top of the red column is read at the end of 1 hour.
Requirements
1) Wintrobe tube
2) Wintrobe tube stand
3) Pasteur pipette or 2 ml syring with needle.
4) Timer or watch.
Procedure
1. Mix the blood carefully.
2. Fill the wintroble tube to the zero mark by using a Pasteur pipette or by using a syring .
3. Place the tube in exact vertical position in the stand. Set timer for 1 hour.
4. At the end of one hour note the level of erythrocytes colume in term of mm after 1
st
hour.
Normal Range
Male 0-9 mm / after 1 hour.
Female 0-20 mm /after 1 hour.
RESULT
18 mm
Precautions
1. Wash the tube under running tap water by introducing a thickwire in the tube repeatedly
to remove the packed cells completely . Afterwards dry the tubes in the incubater (40 C to
50 C
2. In the case of infants and if blood quantity insufficient the LANDAU method .

DETENMINATION OF BLEEDING TIME
Clinical Significance
Determination of bleeding time helps to detect vascular defect & platelet disorder:
Prolonged bleeding time is generally associated with thrombocytopenia.In case of vol
willebrands disease,bleeding time is high with a normal platelet count.It caused by a
platelet defect comblined with factor 7 deficiency.
Name of the Method: Dukes method
Specimen: Blood collected by esrlobe or finger puncture.
Principle: A 1mm deep incision is made on the ear lob or finger of the patienit. The length of
time required for bleeding to cease is recorded.
Normal Range: 1-5 minutes
Requirements
1. Sterile lancet
2. Spirit or 70% alcohol
3. Circular filter paper
4. Stopwatch

Procedure
1. Clean the ear lobe (or finger) with spirit or alcohol by using a piece of cotton. Allow
to dry.
2. Puncture the ear lobe (or finger) deeply (about 1 mm) by using steril lancet, Start the
stopwatch . The blood should flow freely , without squeezing the lobe (o r finger).
3. After 30 seconds collect the drop of blood at one corner of filter paper. Do not touch
the skin with the paper.
4. Repeat step no.3 after every 30 seconds.
5. When bleeding ceases, stop the stopwatch.
6. Note the time on the watch.

RESULT:
4 min

Determination of Hemoglobin by Sahli Method
Clinical significance
A decrease in haemoglobin blow the normal range is an indication of anaemia.
An increase in haemoglobin concentration occurshemoconcentration due to loss of body fluid
in severe diarrheoa and vomiting. High values are also observed in congenital heart diseases
in emphysema and also in polycythemia. Haemoglobin concentration drops during pregnancy
due to hemodilution.

Principle
When blood is added to 0.1N HCl haemoglobin is converted to brown coloured acid hematin.
The resulting colour after dilution is compared with standard brown glass reference blocks of
sahlihemoglobinometer.

Specimen
Capillary blood or thoroughly mixed anticoagulant (EDTA) venous blood.
Requirements
Sahlihemoglobinometer : it consists of
1. A standard brown glass mounted on a comparator.
2. A graduated tube.
3. Hb pipette
0.1N HCl
Distilled water
Pasteur pipettes
Procedure
1. By using Pasteur pipette add 0.1N HCl in the tube up to the lowest mark.
2. Draw blood up to 20 l mark in the Hb pipette. Adjust the blood column carefully
without bubbles. Wipe excess of the blood on sides of the pipette by using a dry piece
of cotton.
3. Transfer blood to the acid in the graduated tube, rinse the pipette well. Mix the
reaction mixture and allow the tube stand for atleast 10minute.
4. Dilute the solution with distilled water by adding few drops at a time carefully and by
mixing the reaction mixture, until the colour matches the glass plate in the comparator.
5. The matching should be done only against natural light.

RESULT:Amt of haemoglobin- 13.3

BLOOD CLOTTING TIME

AIM:
Determination of blood clotting time

CLINICAL SIGNAFICANCE:
This method is generally useful in severe clotting disorders.

METHOD:
Capillary method

NORMAL RANGE:
4 to 9 minutes

PRINCIPLE:
The blood is collected in acapillary tube after a finger prick and the stop watch is started.
The formation of fibrin
String is noted by breaking the capoillary tube at regular intervals. The time is noted at the
first appearance of the
fibrin string.
REQUIREMENTS:
1. Sterile lancet
2. Capillary tubes
3. Cotton
4. Spirit of 70% alcohol
5. Stop watch

PROCEDURE:
1. By using a piece of cotton, apply spirit to patients fingertip.
2. Make a deep (1mm) incision with asterile lancet and start the stop watch.
3. Wipe off the first blood drop and collect blood in the capillary up to 2/3 of its
length.
4. After every half minute, break off about 1cm of the capillary to find out whether
fibrin has formed.
5. when fibrin string appears, stop the stop watch and n ote the time.

ADDITIONAL INFORMATION:
Lee-White method is more reliable than the capillary method with venous blood.

OBSERVATIONS:
Time passed in stop watch- 4 min

RESULTS:
Clotting time is 4 minutes.




Determination of Hematocrit (PCV) by Macro hematocrit
Packed cell volume is the amount of packed red blood cells following centrifugation,
expressed as percentage of the total blood volume.
Clinical significance
Fall of hematocrit values are observed in Anemias, Hydremia. Increases in
values are observed in Polycythemia, Dehydration emphysema, Congenital heart disease.
Principle
When anticoagulant blood is centrifuged in a hematocrit tube at high speed,
the erythrocyte sediment at the bottom. The red cell column is called as PCV of hematocrit
(cell volume percent).
Specimen EDTA Blood or Capillary blood
Requirements
1. Wintrobehematocrit tube: it is 110mm long with a 3mm internal bore. It is
graduated from 0 up to 100mm. The scale with the descending order is used for PCV
determination.
2. Pasteur pipette
3. Centrifuge
Procedure
1. Mix the blood sample carefully.
2. Label a Wintrobe tube.
3. Fill the tube by using Pasteur pipette up to the mark 100.
4. Place the tube in a centrifuge and centrifuge for 30min at 3000rpm.
5. Note the reading. Multiply by 100 for vol ume percent. If the blood is above the 10
mark, calculate the cell pack by dividing the height of the packed erythrocytes by the
total height of the cell column and plasma.
6. Note the following observations:
Colour capacity Expected condition
Yellow may be jaundice
Milky lipemia
Cloudy multiple myeloma
Reddish hemolysis
Buffy layer normally it is 0.5 to 1nm each 0.1ml = 1000cells/cu mm.
RESULTS:
Pcv= 44%

BLOOD GROUP TYPING

Principle: A blood type (also called a blood group) is a classification of blood based on the
presence or absence of inherited antigenic substances on the surface of red blood cells
(RBCs). These antigens may be proteins, carbohydrates, glycoproteins, or glycolipids,
depending on the blood group system. Some of these antigens are also present on the
surface of other types of cells of various tissues. Several of these red blood cell surface
antigens that stem from one allele (or very closely linked genes), collectively form a blood
group system. Blood types are inherited and represent contributions from both parents. The
ABO system is the most important blood-group system in human-blood transfusion. The
associated anti-A antibodies and anti-B antibodies are usually "Immunoglobulin M",
abbreviated IgM, antibodies. ABO IgM antibodies are produced in the first years of life by
sensitization to

environmental substances such as food, bacteria, and viruses. The "O" in ABO is often called
"0" (zero/null) in other languages.
Phenotype Genotype
A AA or AO
B BB or BO
AB AB
O OO
The Rh system is the second most significant blood-group system in human-blood
transfusion with currently 50 antigens. The most significant Rh antigen is the D antigen
because it is the most immunogenic of the five main rhesus antigens. It is common for D-
negative individuals not to have any anti -D IgG or IgM antibodies, because anti -D antibodies
are not usually produced by sensitization against environmental substances. However, D-
negative individuals can produce IgG anti-D antibodies following a sensitizing event: possibly
a fetomaternal transfusion of blood from a fetus in pregnancy or occasionally a blood
transfusion with D positive RBCs. Rh disease can develop in these cases.
Materials Provided: Blood, Anti -A, Anti-B, Anti-D
Other : Beaker, droper, slides, ethanol, cotton
Procedure:
y Finger was sterilized with cotton and ethanol.
y Finger was pricked with a lancet.
y Three different drops of blood were placed on slide.
y Anti-A was added to first drop.
y Anti-B was added to second drop.
y Anti-D was added to third drop.
y Properly mixed.
y Observation was taken



Observations: Observe the antigen antibody reactions which form clumps of red blood cells.








Anti- A Anti -B Anti -D
Interpretation:
Clumping in Anti -A indicates the presence of Antigen A in the blood, hence Blood
group A determined.
Clumping in Anti -B indicates the presence of Antigen B in the blood, hence Blood
group B determined.
Clumping in Anti -D indicates the presence of Rh- Antigen, Blood group is Rh positive.

Result: A POSITIVE