(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 2nd 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.

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.

Serum is the yellow watery part of blood that is left after blood has been allowed to clot and blood cells have been removed.

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.

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 600,000×g are called Ultracentrifuges.

RCF = R×(RPM)2×118×10-7

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-Lambert’s 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 Solutions2. Molar Solutions3. Percent Solutions4. Buffer Solutions5. Buffered Substances6. Indicators7. Primary Standards8. 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 =No .ofgramequivalentsofsolute

No . oflitresofsolution

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 = No . of moles of solute

No .of litresof solution

3. Percent Solutions – concentrations of solution is expressed as:

W /V type – the percent is calculated from the weight of solute in gram divided by the volume(in ml).

V /V 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 Heller’s test for proteins.

PRINCIPLE:

Alkaloid reagents precipitate the protein Heller’s 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 layer3. 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 Benedict’s method.

PRINCIPLE:

The Benedict’s 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 citrate2. Anhydrous sodium carbonate3. Potassium thiocynate4. Distilled water5. Copper sulphate: 18 gm6. 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

Result–55.55

(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 35 ° C to 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 (30 °−40° ) 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 solution4. Add 5 ml of 1% starch solution5. Add 2-3 drops of iodine solution6. 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 blood 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

Preparation and stability of the reagents.

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 pipette4). Gucose standard : 100 mg/ dl5.) Bunsen burner or hotplate6.) Water bath7.) Stows

8.) Centrig

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 2-8oC in an amber colored bottle. It may solidify if stored cold. Allow it to thaw at room temperature before use. If stored cold. 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 for one year at 2-8o C.

Additional Reagent

3) 5G/DL trichloroacetic acid. This reagent is stable at room temperature (25oC+5oC) 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 icteric, an alterative method is used.

Quantitative Assessment of Proteins

(A) Quantitative determination of total serum proteins by Biuret Method

Reagents–NaOH, 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 100µL of each of the protein standards to 5

separate test tubes filled with a biuret reagent.4. Prepare reagent blank by adding 100µL of water to a 6 th test tube different tets tube

filled with biuret reagent.5. Prepare the serum unknown by adding 100µL of serum to a 7th test tube filled with

biuret reagent. Mix each tube.6. Prepare a reagent blank series by adding 100µL of protein standards to 5 separate test

tubes filled with a biuret blank reagent.7. Prepare reagent blank by adding 100µL of water to a 6 th test tube different test tube

filled with biuret blank reagent.8. Prepare the serum unknown by adding 100µL of serum to a 7thtets 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 measure 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. 0Std 2. 0.2ml 0.8 5 30mins. 0.10Std 3. 0.4ml 0.6 5 30mins. 0.20Std 4. 0.6ml 0.4 5 30mins. 0.29Std 5. 0.8ml 0.2 5 30mins. 0.36Std 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 determination)

Stability of the reagent

CSF

Procedurenow 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 mixinga) 0.61 gm/dl vanillin : 350 ml

b) orthophosphoric acid : 600 ml c) distilled water :

50 ml3) Concentrated sulfuric acid (AR).

Stability of the reagentsreagents 1 and 2 at 2-8 degree C in amber colored bottles and

reagent 3 is stable at room temperature.

Procedurepipette 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 chloride4. 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 stock 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 worblewsli’s 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 follows 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 :-

253 mg of sodium pyruvate may be used in place of free acid. 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 pancreases 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 diluted 1 in 10 before use.

2. Phosphate buffer:a. In a 1 Litre volumetric flask, 1.736 gm of Na2HPO4 and 1.059 gm KH2PO4 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 370 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 )*800Ac= Absorbance of controlAu= 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 tubes2. Pipette3. Measuring cylinder4. Water bath5. Photometer6. Pipette

Reagents –

Reagent 1 : it consists of 0.2 g/dl diacetyl monoxime in distilled water. Reagent 2 : it contains 40 mg/dl thiosemicarbazide in distilled water. 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.

Urea nitrogen standard : 20 mg/dl : it contains 42.8 mg of urea in 100 ml of saturated benzoic acid .

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 tubes2. 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 Hydroxide3. Working Creatinine standards. 1mg/dl, 5mg/dl and 10mg/dl4. 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 4.0Serum 1.0

Standard 1mg/dl, ml1.0

2/3N Sulfuric acid 0.510mg/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 5.0Filterate 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

SerumCreatinine , mg /dl=O . D. testO . D . std

×100

¿ 0.10.5

×100=20 mg /dl

Result –

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

Determination of Serum Uric Acid by Henry Caraway’s 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 tubes2. Test tubes3. Pipettes4. 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 60ºC. 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 BlankDiluted Urine 3.0Diluted Standard 3.0

Distilled Water 3.010gm/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

SerumUric acid , mg /dl=O . D. testO . D . std

×5

¿ 00.1

×5 = 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 –

Microscope Improved Neubauer chamber WBC pipette 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 20µl 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 –

No . of white cells (per cu.mm of whole blood)

= No .of white cells counted × Dilution

Area counted × Depthof fluid

Where,

Dilution = 20; area counted = 4×1 sq.mm = 4sq.mm; depth of fluid = 0.1mm

Hence,

No . of white blood cells per cumm of whole blood

¿ No .of cells counted ×204 ×0.1

¿ No . of cellscounted × 50

= 182 × 50 = 9100/cu.mmNo . of leukocyte counted per liter of blood

¿ No . of cells/cu mm (ml ) ×10⁶

= 9100 × 10 /litre of⁶ blood

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 –

Microscope Improved Neubauer Chamber RBC pipette 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 :

Total Red Blood Cells ( per litre of blood )=RBCs /cumm ( µl )× 10 ⁶

OR

RBC count per litre=Number of cell countedV olumecounted (µl)

×dilution ×10⁶

Total red blood cells per cumm ( µl )

¿ Number of red blood cells counted × dilutionArea counted × Depthof fluid

Where,(1) Dilution = 1:200(2) Area counted = 80/400 = 1/5 sq.mmSince cells are counted in 5 bigger squares and such square is further divided into 16 small squares.Each small square = 1/400 sq.mmHence, area of (5×16) = 80 such area = 80/400 sq.mm = 1/5 sq.mmDepth of fluid = 1/10mmNumber of red cells counted = N = 217Hence,

Total red blood cells , cumm= N ×2001/5 ×1/10

=N ×200×50=N ×10000

= 217 × 10,000 = 2.17×106/cu.mm

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) Microscopeb) Improved Neubauer counting chamberc) RBC pipetted) 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 –

Platelets per cumm= Number of platelets×dilutionVolumeof fluid

Where,

Dilution = 200

Volume of fluid = 1× 0.1 = 0.1cu mm.

Hence,

Platelets per cumm= No . of platelet s× 2000.1

=Number of platelets×2000

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 45o from the slide and move it back to

make comtact with thedrop.

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 tail9. 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 45o.2. Blood drop should of proper size.

3. Spreader’s 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 stand3) 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 1st 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 willebrand’s 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: Duke’s 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 lancet2. Spirit or 70% alcohol3. Circular filter paper4. 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 (or 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 20µl 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 patient’s 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 note 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 pipette3. 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 volume 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:

Finger was sterilized with cotton and ethanol.

Finger was pricked with a lancet.

Three different drops of blood were placed on slide.

Anti-A was added to first drop.

Anti-B was added to second drop.

Anti-D was added to third drop.

Properly mixed.

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