Basic Haematology practical Lab manual

LAB MANUAL
BASIC HAEMATOLOGY – (PRACTICAL)
BATCH -A & B
SUBJECT CODE: BMLS2052
WINTER_2021-22
DEPARTMENT OF MEDICAL LAB TECHNOLOGY
SMAS
FACULTY INCHARGE: Mr. A. VAMSI KUMR (Asst Prof)

SYLLABUS
List of Experiments
Experiment No Experiment Name
1.
Preparation of various anticoagulants: EDTA, Sodium Citrate, Oxalate with
Fluoride
2. Collection of blood sample for various Lab Investigations
3. Familiarization and working of routine Haematology Lab. Instruments
Microscopes
4. Demonstration of Haemocytometers
5. Demonstration of Colorimeter
6. Demonstration of Spectrophotometer
7. Demonstration of Glass pipettes & Auto pipettes Glassware
8. Demonstration of Sahli‘s Apparatus
9. Identification of Normal blood cells
10. Urine Analysis: Routine biochemistry of Urine for pH Specific Gravity
11. Routine biochemistry of Urine for Glucose
12. Routine biochemistry of Urine for Ketones bodies
13. Routine biochemistry of Urine for Bilirubin
14. Routine biochemistry of Urine for Albumin
15. Microscopic Examination of Urine
Reference Textbooks
1. Text book of Medical Laboratory Technology by Praful B. Godkar
COURSE OUTCOMES
On completion of this course the students will able to:
1.
Demonstrate usage and handling of haematology laboratory equipment and instruments
2.
Demonstrate blood collection
3.
Test urine for routine urine investigations

2. Medical laboratory Technology by K.L. Mukherjee Volume-I
3. Practical Haematology by J.B. Dacie
4. De Gruchy's Clinical Haematology in Medical Practice
5. Clinical Diagnosis &Management by Laboratory methods (20th edition) by John Bernard
Henry.
LIST OF STUDENTS BATCH – P1
S.No Student Name Admission Number Enrolment Number
1 Abdul Rehman 21SMAS1050068 40803T021GUSMAS105N066
2 ADITYA UPADHYAY 21SMAS1050038 35158T021GUSMAS105N037
3 Alam Hussain 21SMAS1050062 40092T021GUSMAS105N062
4 AMAN KUMAR 21SMAS1050001 20238T021GUSMAS105N001
5 Aman Kumar 21SMAS1050025 31977T021GUSMAS105N024
6 AMBUJ RAJ 21SMAS1050006 21745T021GUSMAS105N006
7 Ankit Kumar 21SMAS1050034 20360T021GUSMAS105N033
8 Ashish Gangwar 21SMAS1050041 35145T021GUSMAS105N040
9 Asrar Bin Yusuf 21SMAS1050072 40997T021GUSMAS105N070
10 ATUL UPADHYAYA 21SMAS1050039 35453T021GUSMAS105N038
11 Ayaz Ahmad 21SMAS1050053 37831T021GUSMAS105N051
12 DANIEL
MATAYAUNGA
21SMAS1050054 23088T021GUSBBS101N001
13 Dron Vashistha 21SMAS1050056 38654T021GUSMAS105N054
14 FAIZ SIDDIQUE 21SMAS1050027 33425T021GUSMAS105N026
15 farhan akhtar 21SMAS1050058 38273T021GUSMAS105N056
16 Gulab . 21SMAS1050050 37002T021GUSMAS105N049
17 HARSH BHATI 21SMAS1050002 20271T021GUSMAS105N002
18 HIDAYA HAMADI
HIZZA
21SMAS1050069 40836T021GUSMAS105N067
19 Himanshu yadav 21SMAS1050007 20532T021GUSMAS105N007
20 Hitesh . 21SMAS1050014 24298T021GUSMAS105N014
21 Jai Mishra 21SMAS1050005 20698T021GUSMAS105N005
22 Manish Sharma 21SMAS1050028 19507T021GUSMAS105N027
23 Manisha yadav 21SMAS1050042 35986T021GUSMAS105N041
24 MANJEET BHATI 21SMAS1050073 41192T021GUSMAS105N071
25 Md Alerashul 21SMAS1050026 33066T021GUSMAS105N025
26 Md. Faizan Ali 21SMAS1050044 36465T021GUSMAS105N043
27 Md. rafique 21SMAS1050059 38984T021GUSMAS105N057
28 Milky Gupta 21SMAS1050061 39189T021GUSMAS105N059
29 Mohd Toheed 21SMAS1050015 23297T021GUSMAS105N015

LIST OF STUDENTS BATCH – P2
S.No Student Name Admission Number Enrolment No.
1 MOHD SHAVEZ 21SMAS1050049 36967T021GUSMAS105N048
2 Monika 21SMAS1050013 23417T021GUSMAS105N013
3 Mubassir islam 21SMAS1050064 39985T021GUSMAS105N062
4 Muskan . 21SMAS1050048 24080T021GUSMAS105N047
5 Nazma khatoon 21SMAS1050047 30377T021GUSMAS105N046
6 NEETU YADAV 21SMAS1050030 33743T021GUSMAS105N029
7 Neha Negi 21SMAS1050010 22024T021GUSMAS105N010
8 Nidhi Singh 21SMAS1050043 35007T021GUSMAS105N042
9 Nikita rani 21SMAS1050024 20854T021GUSMAS105N023
10 Nishant bhati 21SMAS1050070 41045T021GUSMAS105N068
11 Nishu Rai 21SMAS1050018 23206T021GUSMAS105N018
12 Pankaj Sharma 21SMAS1050066 40377T021GUSMAS105N064
13 Pooja Gupta 21SMAS1050022 30994T021GUSMAS105N021
14 Prince Maurya 21SMAS1050004 18578T021GUSMAS105N004
15 Puneet Mishra 21SMAS1050071 41070T021GUSMAS105N069
16 Radhika Kumari 21SMAS1050003 20580T021GUSMAS105N003
17 REYAZ MAHMOOD 21SMAS1050055 38633T021GUSMAS105N053
18 Rishita Trivedi 21SMAS1050019 21720T021GUSMAS105N019
19 Rohit Sharma 21SMAS1050009 22888T021GUSMAS105N009
20 Rohit Kumar Thakur 21SMAS1050017 28435T021GUSMAS105N017
21 Ruby Yadav 21SMAS1050035 20236T021GUSMAS105N034

22 Sapna Upadhyay 21SMAS1050067 40452T021GUSMAS105N065
23 Sazid Arshad 21SMAS1050057 38515T021GUSMAS105N055
24 Shivam Mamgain 21SMAS1050040 35582T021GUSMAS105N039
25 Shreya Tiwari 21SMAS1050008 20844T021GUSMAS105N008
26 Shruti keshri 21SMAS1050011 22218T021GUSMAS105N011
27 Trisha Rawat 21SMAS1050045 36566T021GUSMAS105N044
28 Tushar gaur 21SMAS1050033 31515T021GUSMAS105N032
29 Vibha kumari 21SMAS1050023 31094T021GUSMAS105N022
30 Vijayshyam bind 21SMAS1050021 23733T021GUSMAS105N020
31 Vikas kurrey 21SMAS1050012 18726T021GUSMAS105N012
32 vishal kumar 21SMAS1050037 35523T021GUSMAS105N036
33 Yatish talan 21SMAS1050051 37369T021GUSMAS105N049
Course Name/ code Basic Hematology Practical / BMLS2052
Experiment No 1

Title Preparation of various anticoagulants: EDTA,
Sodium Citrate, Oxalate with Fluoride
Course Co-Ordinator / Faculty Mr. A. Vamsi Kumar
Department /Division/ School Paramedical/MLT/SMAS
Semester II
1. PREPARATION OF VARIOUS ANTICOAGULANTS: EDTA, SODIUM
CITRATE, OXALATE WITH FLUORIDE
Aim: To prepare EDTA, Sodium citrate & Oxalate with fluoride for haematological studies
Principle:
Materials required:
1. Physical balance
2. EDTA, Sodium, Citrate, Oxalate & Fluroid powder
3. Distilled water.
4. Conical flask
5. Measuring cylinder
6. Micropipette (40μl capacity)
7. Spatula
8. Hot air oven
9. Vials
10. Storing bottle
11. Filtter paper
Procedure:
I. EDTA (Ethylenediaminetetraacetic acid)
1. This is a chelating agent that binds the calcium, which is needed for
coagulation. Chelation prevents coagulation
2. It is effective at a final concentration of 1 to 2 mg / mL of blood.
3. This can be used as a powder or make the solution and then add to vials. Let it
dry.
4. It is used as disodium, or dipotassium, or tripotassium salt.
Solution:
5. EDTA solution of 0.1% can be prepared and used. Let it evaporate at room
temperature.
6. Or 1.5 mg/mL.
7. More than 2 mg/mL causes shrinkage of the cells.
Advantages:
8. EDTA preserves the morphology of the blood cell structure.

9. This is the anticoagulant of choice for hematocrit, Hb, and differential count.
10. This is the best anticoagulant for peripheral blood smears and studies.
11. It has little effect on the various tests.
12. They produce less shrinkage of RBCs.
13. There is less increase in the cell volume after keeping the blood.
Drawbacks:
14. It inhibits alkaline phosphatase, creatine kinase, and leucine aminopeptidase
activities.
15. EDTA is not suitable for Calcium and iron estimation.
II. Sodium Citrate
1. Citrate is used as trisodium citrate salt.
2. It is a white hygroscopic crystalline powder.
Indications:
1. Sodium citrate is widely used for coagulation studies.
2. For PT and PTT.
3. The sample can be used for ESR by the Westergren method.
Mechanism of action:
4. it is used in solution form.
5. This will chelate calcium. Inactivates Ca++
ions.
6. This will prevent the rapid deterioration of labile coagulation factors like
factor V and factor VII.
5. Solution preparation and uses:
5. Trisodium citrate= 3.2 to 3.8 g/dL (3.2% solution).
6. Mix well Trisodium citrate 3.8 grams in distle water.
7. This can be used as 0.109 mg/mL.
8. In blood, its ratio is 1:9, where 9 parts are blood, and 1 part is sodium citrate.
5. PT and PTT= Blood: Sodium citrate = 9: 1 part (blood 9 parts: sodium
citrate 1 part)
6. ESR = Blood: Sodium citrate = 4:1 (1.6 mL of blood: o.4 mL Sodium
citrate).
6. Drawbacks
5. This is used in liquid form (liquid anticoagulant).
6. This is not a good anticoagulant for a complete blood examination.
7. This is not good for the estimation of calcium.
8. It inhibits aminotransferase and alkaline phosphatase.
9. This will stimulate acid phosphatase when phenyl phosphate is used as the
substrate.
10. It has little value in clinical chemistry.
III. Potassium Oxalate
a. Potassium oxalate at a concentration of 1 to 2 mg/mL of blood is used.

b. Bulk solution: when you mix 30 grams/dL in distal water.
i. Now add a few drops to the test tube side and dry it in the oven below
100 °C.
The combination of ammonium/potassium oxalate does not lead to shrinkage of
the RBCs.
While other oxalates cause shrinkage.
IV. Sodium Fluorid
a. This is effective at a concentration of 2 mg/mL of blood along with another
anticoagulant like potassium oxalate.
b. When used alone, then more concentration than 2 mg/mL is needed.
c. This can be used in combination with oxalate as a fluoride-oxalate mixture.
Most specimens are preserved at 25 °C for 24 hours and at 4 °C for 48 hours.
Sodium fluoride is poorly soluble, so mix blood thoroughly before effective anti-
glycolysis occurs.
This is mostly used for glucose estimation.
The rate of decrease is faster in newborns because of the increased metabolic
activity of the white cells.
Reference Textbooks

Experiment No 2
Title Collection of Venous Blood
Semester II
2. Collection of Venous Blood
Aim:
To demonstrate the collection of Blood.
Introduction about Phlebotomy:
Phlebotomy is the process of making an incision in a vein with a needle.
• The procedure is known as a venipuncture.
• A person who performs phlebotomy is called a phlebotomist, although doctors, nurses &
medical laboratory scientists.
• Blood specimens are obtained through capillary skin punctures (finger, toe, heel), arterial or
venous sampling.
Introduction Venipuncture:
Venipuncture is the process of obtaining blood samples from veins for laboratory testing, It is probably
the most common procedure in the medical field, usually performed for the following reasons:
➢ To obtain blood samples in order to perform diagnostics.
➢ To collect blood for later use should the patient’s condition requires transfusions.
➢ To remove blood that was found with excessive levels of erythrocytes or iron.
Method :
Venipuncture method
Capillary blood collection method
Arterial blood collection method
Materials required:
Gloves
Syringes
Evacuated collection tubes
Alcohol wipes
Tourniquet
Venipuncture Procedure:

Explain the procedure and purpose for the patient.
Assess the patient's physical disposition (i.e. diet, exercise).
Position the patient - sitting or lying (NEVER allow the patient to sit upright on a high stool
or standing due to the possibility of syncope).
Check the requisition form for requested tests, patient information, and any special
requirements.
Select a suitable site for venipuncture.
Prepare the equipment, the patient and the puncture site.
Perform the venipuncture.
Collect the sample in the appropriate container.
While the tube fills, remove the tourniquet.
Label the collection tubes at the drawing area.
Immediately send the specimens with the requisition to the laboratory.
Order of draw
Avoid performing a venipuncture on:
➢ Arm on side of mastectomy: If drawn here, the test results could be inaccurate because of
lymph edema.
➢ Scarred or burned areas :Performing a venipuncture at these sites is more difficult due to
the scar tissue.
➢ Arm in which blood is being transfusion / IV cannula: The fluid in the IV could dilute the
specimen.
➢ A hematoma :(A hematoma is an abnormal collection of blood outside of a blood vessel. It
occurs because the wall of a blood vessel wall, artery, vein, or capillary, has been damaged
and blood has leaked into tissues ) If drawn here, could cause incorrect test results.
➢ Edematous :(Edema is swelling caused by fluid retention) should be avoid because the
accumulated fluid could alter test results.

Reference Textbooks

Experiment No 3
Title Familiarization and working of routine
Haematology Lab. Instruments Microscopes
Semester II
3. Familiarization and working of routine Haematology Lab. Instruments
Microscopes
Components of the compound Light Microscope

Experiment No 4
Title Demonstration of Haemocytometers
Semester II
4. DEMONSTRATION OF HAEMOCYTOMETERS
Aim: To demonstrate the hemocytometer
The simplest, most convenient and cheapest means of accurately determining the numbers of
cells in a sample is to use a Haemocytometer and a microscope. A Haemocytometer is a
specialized slide that has a counting chamber with a known volume of liquid.
• The Haemocytometer consists of a heavy glass slide with two counting chambers,
each of which is divided into nine large 1 mm squares, on an etched and silvered
surface separated by a trough.
• A coverslip sits on top of the raised supports of the 'H' shaped toughs enclosing both
chambers. There is a 'V' or notch at either end where the cell suspension is loaded into
the Haemocytometer. When loaded with the cell suspension it contains a defined
volume of liquid.
• The engraved grid on the surface of the counting chamber ensures that the number of
particles in a defined volume of liquid is counted.
• The Haemocytometer is placed on the microscope stage and the cell suspension is
counted.

The hemocytometer is divideded into 9 major squares of 1mm x 1mm size. The four coner
squares (identified by the red square) are further subdivided into 4 x 4 grids. The height of
the chamber formed with the cover glass is 0.1 mm, so a 1 mm x 1 mm x 0.1 mm chamber
has a volume of 0.1 mm3 or 10-4 ml.
To count cells using a hemocytometer, add 15-20μl of cell suspension between the
hemocytometer and cover glass using a P-20 Pipetman. The goal is to have roughly 100-200
cells/square. Count the number of cells in all four outer squares divide by four (the mean
number of cells/square). The number of cells per square x 104 = the number of cells/ml of
suspension.
Calculation for WBCs

Calculation for RBCs
Reference Textbooks
2. Medical laboratory Technology by K.L. Mukherjee Volume-I
3. Practical Haematology by J.B. Dacie
4. De Gruchy's Clinical Haematology in Medical Practice
5. Clinical Diagnosis &Management by Laboratory methods (20th edition) by John Bernard
Henry.

Experiment No 5
Title Demonstration of Colorimeter
Semester II
5. DEMONSTRATION OF COLORIMETER
Aim: To demonstrate the colorimeter for hematological studies
Principle:
A Colorimeter is a light and sensitive device used to measure the transmittance and absorbance
of light that passes through a liquid sample. The colorimeter device also measures the intensity
or colour concentration that develops upon introducing a particular reagent into a solution.
The Beer-Lambert law is expressed as:
A = εLc
where,
• A is the amount of light absorbed for a particular wavelength by the sample
• ε is the molar extinction coefficient
• L is the distance covered by the light through the solution
• c is the concentration of the absorbing species
Following is an equation to solve for molar extinction coefficient:
But Beer-Lambert law is a combination of two different laws: Beer’s law and Lambert law.
What is Beer’s Law?
Beer’s law was stated by August Beer which states that concentration and absorbance are
directly proportional to each other.
What is Lambert Law?
Johann Heinrich Lambert stated Lambert law. It states that absorbance and path length are
directly proportional.
Beer-Lambert Law Formula
Where,

• I is the intensity
• I0 is the initial intensity
• x is the depth in meters
• 𝜇 is the coefficient of absorption
Parts of Colorimeter
Uses of Colorimeter
• It is used in laboratories and hospitals to estimate biochemical samples such as urine,
cerebrospinal fluid, plasma, serum, etc.
• It is used in the manufacturing of paints.
• It is used in textile and food industry.
• It is used in the quantitative analysis of proteins, glucose, and other biochemical
compounds.
• It is used to test water quality.
• It is used to determine the concentration of haemoglobin in the blood.
Reference:
https://byjus.com/chemistry/colorimeter/#:~:text=A%20colorimeter%20is%20a%20device,of%2
0the%20Beer%2DLambert%20law.

Experiment No 6
Title Demonstration of Spectrophotometer
Semester II
6. DEMONSTRATION OF SPECTROPHOTOMETER
Aim: To demonstrate spectrophotometer
Principle:
The spectrophotometer technique is to measure light intensity as a function of wavelength. It
does this by diffracting the light beam into a spectrum of wavelengths, detecting the
intensities with a charge-coupled device, and displaying the results as a graph on the detector
and then on the display device.
1. In the spectrophotometer, a prism (or) grating is used to split the incident beam into
different wavelengths.
2. By suitable mechanisms, waves of specific wavelengths can be manipulated to fall on
the test solution. The range of the wavelengths of the incident light can be as low as 1
to 2nm.
3. The spectrophotometer is useful for measuring the absorption spectrum of a
compound, that is, the absorption of light by a solution at each wavelength.
Applications
Some of the major applications of spectrophotometers include the following:
• Detection of concentration of substances
• Detection of impurities
• Structure elucidation of organic compounds
• Monitoring dissolved oxygen content in freshwater and marine ecosystems
• Characterization of proteins
• Detection of functional groups
• Respiratory gas analysis in hospitals
• Molecular weight determination of compounds
• The visible and UV spectrophotometer may be used to identify classes of
compounds in both the pure state and in biological preparations.
Reference:
https://microbenotes.com/spectrophotometer-principle-instrumentation-applications/
Experiment No 7

Title Demonstration of Glass pipettes & Auto pipettes
Glassware
Semester II
7. DEMONSTRATION OF GLASS PIPETTES & AUTO PIPETTES GLASSWARE
Aim: To demonstrate various glassware used in hematology laboratory
Types of Glassware

Steps in cleaning glassware
Cleaning Basics steps are:
1. It’s generally easier to clean glassware if you do it right away.
2. When detergent is used, it can use commercially available as Liquinox or Alconox.
0. The detergent should meet the following criteria:
0. It can soften the local water supply.
1. It should be able to remove organic material at a temperature of 60 °C.
2. It should have a neutral pH after rinsing with water.
3. Glassware should be free of the microbiological organism after the
following rinsing.
3. Much of the time, detergent and tap water is neither required nor desirable.
0. You can rinse the glassware with the proper solvent.
1. Then finish up with a couple of rinses with distilled water.
2. A final rinse follows this with deionized water.
Hematological Glassware Needs Special Precautions:
• Do not use detergents because if there is a minute concentration, that may lead to
RBCs’ hemolysis.
• So for general Tubes, pipettes, and slides, wash these thoroughly under tap water. Can
use a brush to remove any leftover from the glassware.
• Keep the hematology used material in a dichromate solution for 12 to 24 hours. Then
again, wash thoroughly with tap water.
• Allow draining.
• Dry in the hot oven.
Reference:
https://labpedia.net/laboratory-glassware-cleaning-and-sterilization/

Experiment No 8
Title Demonstration of Sahli‘s Apparatus
Semester II
8. DEMONSTRATION OF SAHLI‘S APPARATUS
Aim: To estimate hemoglobin in the given sample by Sahli’s method.
Principle: Hb is converted into acid haematin with the action of dilute hydrochloric acid
(N/10 HCl). The acid haematin is brown in colour and its intensity is matched with a standard
brown glass comparator in a visual colorimeter called Sahli’s
colorimeter.
Materials required:
• Sahli’s Apparatus
o Hemoglobin pipette (0.02 ml or 20 µl capacity)
o Sahli’s graduated Hemoglobin tube
o Thin glass rod Stirrer for Hemoglobin Tube
o Sahli’s Comparator box with brown glass standard
• Spirit swab
• Blood Lancet
• Dry cotton swab
• Pasteur pipette
Procedure:
• Fill Sahli’s Hb tube up to mark 2 with N/10 HCl.
• Deliver 20 μl (0.02 ml) of blood from a Hb pipette into it.
• Stir with a stirrer and wait for 10 minutes.
• Add distilled water drop by drop and stir till colour matches
• with the comparator.
• Take the reading at upper meniscus
Observation
………………………………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………………………………
Result
…………………………………………………………………………………………………
………………………………………………………………………………………………….
Normal Values:

Raised Hemoglobin Content
• Polycythemia Vera
• Associated with Hypoxia
• Cyanotic Congenital Heart disease
• High Altitudes
• Heavy smoking
• Methemoglobinemia
• Elevated erythropoietin levels
o Tumors of Kidney, Liver, CNS, Ovary etc.
o Renal Diseases (Hydronephrosis & Vascular impairment)
• Adrenal hypercorticism
• Therapeutic androgens
• Relative causes of high hemoglobin content
o Dehydration – Water deprivation, Vomiting, Diarrhea
o Plasma loss – Burns, Enteropathy
Reduced Hemoglobin Content
Low Hemoglobin value means anemia caused by the following conditions
Leukemia
Tuberculosis
Iron deficiency anemia
Parasitic infections severely in hookworm infection
Sickle cell anemia
Thalassemia
Aplastic anemia
Hemolytic anemia
Loss of blood
Reference:
https://www.notesonzoology.com/practical-zoology/laboratory/top-3-haematological-
experiments-for-counting-blood-cells-under-microscope/3003

Experiment No 9
Title Demonstration of White Blood Cells
Semester II
9. DEMONSTRATION OF WHITE BLOOD CELLS
Introduction:
The differential white cell count is the determination of the different types of white blood cells or
leukocytes present in blood. White blood cells are classified into various groups depending on their
size, features of the nucleus and features of cytoplasm. The WBCs exist in two forms granulocytes
and agranulocytes. Granulocytes are further classified eosinophil, basophil, Neutrophils. Where as,
agranulocytes include monocytes and Lymphocytes.
Aim: The aim of the experiment is to estimate the differential white blood cell count of a given blood
sample.
Requirements:
1. Cotton
2. Spirit, needle
3. Glass slide
4. Distilled water
5. Leishman's stain
6. Light microscope with oil immersion objective (100X).
Procedure:
1. Preparing the blood smear
1. Sterilize the finger tip of the subject with a cotton swab dipped in 70% alcohol and is dried
2. Take a bold prick on the fingertip to have free flow of blood
3. Collect drops of blood on the end side of a glass slide.
4. Spread the blood drop with another glass slide (Spreader) by placing it at an angle of 45
degree and move sideways
5. Hold the spreader firmly and move it on the previous slide to the other end in a straight line
with same force and pressure.
6. Allow the glass slide to dry after formation of the smear

2. Staining the slide
1. Keep the smeared glass slide on a flat surface with the smeared surface facing upwards
2. Pour drops of leishman's stain on the glass slide to cover the smear or film
3. Keep it undisturbed for 2-5 minutes.
4. Pour drops of distilled water on the slide and leave it for 10 minutes
5. Remove the dye and water.
6. Remove the extra stain by keeping the slide under running water
7. Keep the slide aside for some time to dry
3. Observation of the glass slide and counting of cells
1. Keep the prepared glass slide under low power of compound microscope and choose a good
quality slide.
2. Then identify different types of WBC under medium power
3. Draw a table with 10 boxes both on horizontal and vertical axis on a observation notebook
4. Fix the slide on the plateform and choose a area towards the corner
5. Note the different types of WBC found on the table in an abbreviated
6. Move downwards and in chain like manner till 100 cells are observed
7. After counting 100 cells prepare the report

Morphology of White Blood cells
Observation:
……………………………………………………………………………………………………………………………………………………………
……………………………………………………………………………………………………………………………………………………………
………..
Result:
……………………………………………………………………………………………………………………………………………………………
……………………………………………………………………………………………………………………………………………………………
………..

Clinical Significance:
………………………………………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………………………………………
………………………..

Experiment No 10.1
Title Routine biochemistry of Urine for pH Specific
Gravity
Semester II
10.1 ROUTINE BIOCHEMISTRY OF URINE FOR PH SPECIFIC GRAVITY
Aim: To estimate the urine pH by litmus paper.
Principle: Red litmus contains a weak diprotic acid. Upon exposure to a base, hydrogen ions
from the acidic urine react with the base, producing a color change to blue. Blue litmus paper,
on the other hand, already contains the blue conjugate base. It reacts with an acid to change to
red.
In general, litmus paper is red below a pH of 4.5 and blue above a pH of 8.3. If the paper
turns purple, this indicates the pH is near neutral. Red paper that does not change color
indicates the sample is an acid. Blue paper that does not change color indicates the sample is
a base.
Materials required:
• Litmus paper
• Urine sample
Procedure:
• Collect a freshly voided well mixed urine.
• Tear small blue litmus paper.
• Dip the paper, in the urine and remove immediately.
• Look for color change of blue litmus paper. If the blue colors of paper change to red, it
indicates the acidity of the urine.
• Tear small red litmus paper.
• Dip the paper, in the urine and remove it immediately.
• Look for color change of red litmus paper. If the red litmus paper change to blue, it indicates
that the urine is alkaline. The blue and red litmus paper technique is a less sensitive method.
This is because it indicates only the alkalinity or acidity of urine; it does not tell the exact
quantity or figure of pH
Observation
………………………………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………………………………
Result
…………………………………………………………………………………………………
………………………………………………………………………………………………….
Normal Values:
pH 4.6 to 8.0.
Persistent alkaline urine (pH > 6) may be caused by:
▪ UTI

▪ Renal failure
▪ Vomiting
▪ Anorexia nervosa
▪ Alkalosis (metabolic or respiratory e.g. due to accumulation CO2 in our body.
▪ Alkalizing drugs i.e. during intake of drugs such as streptomycin, kanamycin
etc. eg. for UTI.
▪ It should also important to bear in mind that certain vegetables,citrus
fruits, and milk products also may cause alkaline urine, whichis not
pathological
Persistent acid urine (pH < 6) may be caused by:
▪ Diarrhea
▪ Malabsorption syndromes
▪ Diabetic ketoacidosis
▪ Dehydration
▪ Fever
▪ Starvation
▪ And also certain drugs such as – Phenacetic
▪ Here it is important to bear in mind that high protein diet may also result in
acidic urine, but this is not a pathological condition.
▪ pH measurement is also important in the management of renal stone
patients, who are being treated for renal calculi and who are frequently
given diets or medications to change the pH of the urineso that kidney
stone will not form.
▪ Calcium phosphates, calcium carbonate, and magnesium phosphatestones
develop in alkaline urine. In such instances the urine must be kept acidic
(i.e. either by diet such as meat, or medication).
▪ Uric acid, cystine, and calcium oxalate stones are precipitated in acidic
urine. Therefore, as part of treatment, the urine should be kept alkaline
(either by diet eg. leguminous plants, citrus fruits and most vegetables or by
medication)

Experiment No 10.2
Title Routine biochemistry of Urine for pH Specific
Gravity
Semester II
10.2 ROUTINE BIOCHEMISTRY OF URINE FOR PH SPECIFIC GRAVITY
Aim: To estimate specific gravity of the given urine sample by urinometer
Principle:
Urinometer is an instrument used to measure the specific gravity of urine. It is based on the principle
of BUOYANCY. Because of increased density of urine compared to that of water, the urinometer will
float higher in urine than in water.
Materials required:
• Urinometer
• PPE
• Urine sample
• Sterile container
Procedure:
Observation
…………………………………………………………………………………………………
…………………………………………………………………………………………………
…………………………………………………………………………………………
Result
…………………………………………………………………………………………………
………………………………………………………………………………………………….
Normal Values
The normal specific gravity of urine is 1.003 to 1.030.
Clinical Significance

Experiment No 11
Title Routine biochemistry of Urine for Glucose
Semester II
11. ROUTINE BIOCHEMISTRY OF URINE FOR GLUCOSE
Aim: To determine blood glucose by benedicts test.
Principle:
When boiled in an alkaline copper sulphate solution, glucose and other reducing substances
reduce (convert) the blue copper (II) in Benedict's qualitative reagent to copper (I) oxide
(Cu2O), which is orange to red in color. A positive reaction is graded as a change in color
ranging from blue to green, yellow, orange and finally red.
The overall reaction is:
Cupric ions + reducing sugar alkali
Cuprous ions + Oxidized sugar
( CuSO4 ) (eg. glucose) heat (Cu2O)(e.g.gluconic acid)
(Blue) (Orange-red )
The copper (II) ions are supplied in Benedict's qualitative reagent in the form of copper sulphate
(CuS04). In the presence of a strong alkali this is converted to copper ( I) oxide (Cu2O ). The
heat is supplied by means of a boiling-water (100O
C) bath. The tubes are brought back to room
temperature, and the results are read when convenient.
Materials Required
• Glassware
• Benedicts reagent
• Bunsen burner
• Sample
• PPE
Procedure
1. Measure 8 to 10 drops or 0.5 ml of well-mixed urine in a
testtube.
2. Add 5 ml of Benedict's qualitative reagent. Mix well.
3. Place in boiling-water bath for exactly 5 minutes (or boil in
nakedflame for
exactly 2 minutes.
4. Remove from the boiling-water bath and immediately cool to
roomtemperature in a cold water bath (about 10 minutes).
5. Observe the color change.
A positive reaction depends on the presence of a fine
yellow,orange, or brick red precipitate.
The test is then graded on the basis of the color of the mixed solution.

Grade results according to the following criteria:
Negative: No change in the blue color of the reagent or the
occurrence of a white or green precipitate from phosphates
in the urine.
Trace: Slight amount of yellow precipitate with a greenish blue to
bluish green mixed solution. (This represents less than
500mg/dl of sugar).
+ : Moderate amount of yellow precipitate with green, often
referred to as apple green, mixed solution. (Approximately
500mg/dl of sugar).
++: Large amount of yellow precipitate with a yellowish green,
often called muddy green mixed solution. (Appr. 750mg/dl
of sugar).
+++: Large amount of yellow precipitate with green yellow, or
muddy orange, mixed solution. Some blue color remains
in supernatant.
(Appr. 1000mg/dl of sugar)
++++: Large amount of yellow to red precipitate with reddish
yellow to red mixed solution. No blue remains in the
supernatant. (Appr. 2000mg/dl)

Reference:
https://www.cartercenter.org/resources/pdfs/health/ephti/library/lecture_notes/med_lab_tech_st
udents/ln_urinalysis_final.pdf

Experiment No 12
Title Routine biochemistry of Urine for Ketones bodies
Semester II
12. ROUTINE BIOCHEMISTRY OF URINE FOR KETONES BODIES
Aim: To determine urine ketone bodies by Rothera’s test
Principle: Acetoacetic acid and acetone react with alkaline solution of sodium nitroprusside
to form a purple colored complex. This method can detect above 1-5 mg/dl of acetoacetic
acid and 10-20 mg/dl of acetone. Beta-hydroxybutyrate is not detected.
Materials Required:
• Specimen: Urine
• Glassware: Test tubes, pipette.
• Rothera’s powder: Sodium nitroprusside = 0.75 gm, Ammonium sulphate = 20gm
(Mix and pulverize).
• Liquor Ammonia (Ammonium hydroxide)
Procedure:
1. Take a clean test tube and add 5 ml of urine to it.
2. Transfers 1 gm of Rothera’s powder mixture within the test tube and mix well.
3. Add 1-2 ml of concentrated ammonium hydroxide to the urine sample within the test
tube. It will create a thin layer over the urine sample.
4. Observe the pink-purple ring at the interface.
Observation:
…………………………………………………………………………………………………
………………………………………………………………………………………………….
Result:
…………………………………………………………………………………………………
………………………………………………………………………………………………….
Results Interpretation:
• Positive Test: If a purple permanganate-colored ring is immediately formed at the
interface, means Ketone bodies are present.
• Negative Test: If no permanganate colored ring is formed at the interface, means
ketone bodies are absent within the test sample.

Clinical Significance:
When the rate of formation of ketone bodies is greater than the rate
of their use, their levels begin to rise in the blood, which is called
ketonemia, and eventually in the urine, which is known as ketonuria.
These two conditions are seen most often in cases of starvation and
diabetes mellitus. Ketone bodies can be seen also in the urine during
prolonged vomiting, severe diarrhea, anesthesia, severe liver
damage, high fat intake and low carbohydrate diet.
The excessive production and accumulation of ketone bodies may
leadto ketosis.
Its physiological effect is serious because acetoacetic acid and -
hydroxybutyric acid contribute excess hydrogen ions to the blood,
resulting in acidosis - a condition that tends to lower the blood pH.
If not corrected in time this may result in death.
Another physiological effect of ketone accumulation concerns the
substance acetone and acetoacetic acid. Both have been found to be
toxic to brain tissue when present in increased amounts in the
blood. So this condition can result in permanent brain damage.
When ketones accumulate in the blood and urine, they do not occur
in equal concentrations. -hydroxybutric acid is present in the
greatest

39
concentration and acetone in the smallest concentrations. Howevermost of the tests
for ketonuria are most sensitive to the presence of acetoacetate. There are no simple
laboratory tests for -hydroxybutyric acid. Most tests react with acetone and
acetoacetate or both.
Reference:
https://laboratorytests.org/rotheras-test/

40
Experiment No 13
Title Routine biochemistry of Urine for Bilirubin
Semester II
13. ROUTINE BIOCHEMISTRY OF URINE FOR BILIRUBIN
Aim: Estimation of total urine bilirubin by modified Jendrassik and Grof’s Method
Principle:
Bilirubin reacts with diazotised sulphanilic acid to form a coloured azobilirubin compound. The
unconjugated bilirubin couples with the sulphanilic acid in the presence of a caffein-benzoate accelerator.
The intensity of the colour formed is directly proportional to the amount of bilirubin present in the sample.
Materials required:
• Test tubes
• Bilirubin reagents
• PPE
• Colorimeter
• Distilled water
• Sample
Procedure
Observation
…………………………………………………………………………………………………………………
…………………………………………………………………………………
Results:
…………………………………………………………………………………………………………………
…………………………………………………………………………………

41
Clinical Significance
If bilirubin is found in urine, it may indicate:
• A liver disease such as hepatitis
• A blockage in the structures that carry bile from liver
• A problem with liver function
A bilirubin in urine test is only one measure of liver function. If the results are abnormal, the health care
provider may order additional blood and urine tests, including a liver panel. A liver panel is a series of blood
tests that measure various enzymes, proteins, and substances in the liver. It is often used to detect liver
disease.
Reference:
https://www.beaconindia.com/admin/pages/usermanual/Bilirubin.pdf

42
Experiment No 14
Title Routine biochemistry of Urine for Albumin
Semester II
14. ROUTINE BIOCHEMISTRY OF URINE FOR ALBUMIN
Aim: To determine the presence of protein in the given urine sample by heat acetic acid method.
Principle:
This test is based on the principle that proteins get precipitated when boiled in an acidic medium.
Materials Required:
• Urine sample
• Test tubes
• Holders
• Bunsen burner
• Acetic acid
• PPE
Procedure:
1. Take 5-10ml clear urine in a test tube.
2. Boil the upper portion over a flame.
3. Compare the heated part with the lower part. Cloudiness or turbidity indicates the presence
of either proteins or phosphates/carbonates.
4. Add 2-4 drops of 10% glacial acetic acid and boil the upper portion again.
5. If turbidity is still present, protein is present in urine. If turbidity disappears, that is due to
phosphates or carbonates present in urine.
Observation:
…………………………………………………………………………………………………………………
…………………………………………………………………………………………………………………

43
Result:
……………………………………………………………………………………………………………………………………………………………………………………
……………………………………………………………………………………………………………………………………………………………………………………
Result and Interpretation:
Grade the turbidity as follows:
▪ Negative : No cloudiness
▪ Trace: Barely visible cloudiness.
▪ 1+ : definite cloud without granular flocculation
▪ 2+ : heavy and granular cloud without granular flocculation
▪ 3+ : densed cloud with marked flocculation.
▪ 4+ : thick curdy precipitation and coagulation
Reference:
https://laboratoryinfo.com/heat-and-acetic-acid-test-urine-test-of-proteinuria/

44
Experiment No 15
Title Microscopic examination of urine
Semester II
15. MICROSCOPIC EXAMINATION OF URINE
Aim: To perform the physical and microscopic examination on given urine sample.
Sample collection: The specimen should be properly collected in a clean container which should be properly labelled with
name of the patient, age, sex, identity number with date and time of collection. It
should not show signs of contamination.
Procedure:
Place a drop of urine on a clean glass slide and add a cover slip and observe under microscope
MICROSCOPIC EXAMINATION OF URINE:

45
Reference:
https://www.microscopemaster.com/urine-analysis.html
THE END

46
CONTACT DETAILS
Course Name/ code Basic Hematology
Practical/BMLS2052
Course Co-Ordinator Mr. A. Vamsi Kumar (Asst Prof)
Phone Number +91 7416660584
Lab Assistant Mr. Manoj / Mr. Raja
Lab Assistant Contact No. +91 7088801539
Department /
Division/School
Paramedical/Medical Lab
Technology/SMAS
Semester II

Basic Haematology practical Lab manual

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to Basic Haematology practical Lab manual

Similar to Basic Haematology practical Lab manual (20)

More from Vamsi kumar

More from Vamsi kumar (20)

Recently uploaded

Recently uploaded (20)

Basic Haematology practical Lab manual