completehematologylabmanual22mlh-201-230708095854-57b542f3

UNIVERSITY INSTITUTE OF APPLIED HEALTH SCIENCES
DEPARTMENT OF MEDICAL LAB TECHNOLOGY
LAB MANUAL
PROGRAM NAME: B.Sc. MLT / Sem-3 / Batch-2022
Subject Name: Hematology Practical
Subject Code: 22MLH-201
Faculty Name: Attuluri Vamsi Kumar (E13404)

Assistant professor
Ph No: 7416660584 Mail ID: attuluri.e13404@cumail.in

Department of MLT
Prepared by Verified by Approved by
Attuluri Vamsi Kumar

E13404
1
TABLE OF CONTENTS
RULES AND REGULATIONS IN HEMATOLOGY LAB ................................. 3

SAFETY INSTRUCTIONS IN HEMATOLOGY LAB ....................................... 4
Hand washing guidelines: ...................................................................................... 4
EMERGENCY GUIDELINES .............................................................................. 5
IN CASE OF NEEDLE STICK INJURY .............................................................. 6
POSSIBLE HAZARDS IN HEMATOLOGY LAB .............................................. 7
MAINTAINANCE OF RECORDS & LAB DECORUM ..................................... 8
SYLLABUS ........................................................................................................... 9
CO PO MAPPAING ............................................................................................ 10
Experiment CO Mapping ..................................................................................... 11
1. PREPARATION OF EDTA VIALS................................................................ 12
2. COLLECTION OF BLOOD SAMPLE, PLASMA SEPARATION............... 18
3. ESTIMATION OF ESR ................................................................................... 25
4. ABO BLOOD GROUPING ............................................................................. 32
5. BLEEDING TIME & CLOTTING TIME ...................................................... 37
6. TOTAL WHITE BLOOD CELL COUNT IN BLOOD .................................. 49
7. TOTAL RED BLOOD CELL COUNT IN BLOOD ....................................... 55
8. ESTIMATION OF PLATELETS COUNT IN BLOOD ................................. 62
9. HEMOGLOBIN (HB) ESTIMATION SHALI 'S METHOD ......................... 68
10. DIFFERENTIAL LEUKOCYTE COUNT (DLC) ........................................ 74
2
RULES AND REGULATIONS IN HEMATOLOGY LAB
1. He/she should wear protective clothing (laboratory coats/gowns) over normal clothing:
preferably. Wear closed shoes and not walk barefooted in the laboratory.
2. Everyone must carry their own Lab box (Lab box must contain the following things:
Cotton, Sprit, permanent marker, Lancet, Bandages, Sanitizer, Face mask, Hair mask,
Gloves, Nail cutter, Blade, Permanent marker, toothpicks, Cello tape-transparent)
3. All specimens and infected materials should be handled with care.
4. He/she should avoid eating. Drinking or chewing gum in the laboratory.
5. He/she should refrain from smoking in the working zone of the laboratory and also
refrain from applying cosmetics in the laboratory.
6. Nothing should be pipette with the mouth. Gummed labels should not be licked neither,
should pens or pencils be put in the mouth, or stuck in the hair.
7. Protective gloves or plastic aprons should be worn when collecting blood sample for
hepatitis, AIDS or viral hemorrhagic fever investigations.
8. Used needle should be inserted back into its guard immediately after use. But due to the
increased awareness of the risks of infection from needle pricks, it is no longer advisable
to recap used needles. Instead, “Sharp safe” containers should be made available for
immediate disposal of used needles. The containers are finally incinerated.
9. When handling specimens or culture containing highly infectious pathogens gloves
should be worn.
10. Process specimens or cultures containing highly infectious pathogens in the safety
cabinet.
11. Any cuts, insect bites, open sore or wounds should be covered with water-proof adhesive
dressing.
12. Finger nails should be kept short.
13. All infected or contaminated materials should be disinfected before disposal.
14. There should be a jar of disinfectant on each bench at the start of the day’s work. The
disinfectant must be changed every day.
15. In case of any spillage, disinfectant solution should be poured to cover the spilled.
16. Do not touch any instrument with out permission from lab instructor.
17. Workbench must be cleaned before and after every practical.
18. Wash your hands before and after of every practical.
3
SAFETY INSTRUCTIONS IN HEMATOLOGY LAB
1. Always wear protective clothing, including lab coat, gloves, and safety goggles.
2. Handle all samples and reagents with care, as they may contain potentially harmful
substances.
3. Follow proper disposal procedures for biohazardous materials.
4. Avoid eating, drinking, or applying cosmetics in the lab.
5. Keep the lab clean and organized to prevent accidents and cross-contamination.
6. Be aware of emergency procedures and know the location of safety equipment such as fire
extinguishers and eye wash stations.
7. Follow all instructions provided by lab supervisors and instructors.
8. Do not perform any experiment without proper training and permission.
9. Report any accident or incident to the supervisor immediately.
10. Use equipment and chemicals only for their intended purpose.
Hand washing guidelines:

Hand hygiene is a critical aspect of laboratory safety and should be strictly followed in hematology
labs. The Centers for Disease Control and Prevention (CDC) recommends the following guidelines
for hand hygiene in laboratory settings:
• Hands should be washed with soap and water before and after handling specimens and after
removing gloves.
• If hands are not visibly soiled, an alcohol-based hand sanitizer that contains at least 60%
alcohol can be used in addition to or in place of soap and water.
• Hands should be dried thoroughly with a single-use towel or air dryer after washing or
using hand sanitizer.
It is also important to avoid touching the face, hair, and clothing while working in the lab and to
avoid eating, drinking, or smoking in the lab. Additionally, gloves should be worn when handling
specimens and should be changed between handling different specimens.
4
EMERGENCY GUIDELINES
1. Call for immediate assistance: Call the dispensary unit of the University immidiatly.
2. Evacuate the area: If there is a fire, chemical spill, or other hazardous situation, safely
evacuate the area and move to a safe location.
3. Administer first aid: If someone is injured, administer first aid as necessary.
4. Secure the scene: If there is a hazardous materials spill or other situation that poses a risk
to others, take steps to secure the scene and prevent further injury or damage.
5. Report the incident: Report the incident to your supervisor or the appropriate authority,
such as your local health department, as soon as possible.
6. Follow up with cleanup and decontamination procedures: If necessary, follow established
procedures for cleaning up and decontaminating the affected area.
7. Document the incident: Document the incident, including any injuries, damage, and
actions taken, in order to facilitate the investigation and follow-up.
5
IN CASE OF NEEDLE STICK INJURY
If you experience a needle stick injury, it is important to follow these guidelines to minimize the
risk of infection:
1. Wash the wound with soap and water for at least 15 minutes.
2. Apply an antiseptic to the wound.
3. Seek medical attention immediately. You may need to receive vaccinations or treatment
to prevent infection.
4. Report the incident to your supervisor or employer.
5. Do not squeeze or suck the wound.
6. Do not try to recap the needle or remove it from the skin.
7. Do not ignore the wound or delay seeking medical attention.
8. Follow the guidance of medical professional for any other necessary steps.
It is important to note that these guidelines are general and may vary depending on the specific
situation, so it is always recommended to follow the instructions of medical professionals.
6
POSSIBLE HAZARDS IN HEMATOLOGY LAB
1. Biological hazards: Hematology labs handle blood and other bodily fluids, which can
contain pathogens that can cause infections. It is important to follow proper biosafety
procedures to minimize the risk of exposure.
2. Chemical hazards: Hematology labs use a variety of chemicals, such as fixatives and
stains, which may be hazardous if not handled properly. It is important to familiarize
yourself with the safety data sheets of these chemicals and to use them in accordance with
the manufacturer's instructions.
3. Radiation hazards: Hematology labs may use X-ray equipment, which can emit ionizing
radiation. It is important to use this equipment in accordance with the manufacturer's
instructions and to follow proper radiation safety procedures.
4. Physical hazards: Hematology labs may have equipment that can be hazardous if not used
properly, such as centrifuges and microscopes. It is important to be properly trained on the
use of this equipment and to follow the manufacturer's instructions.
5. Ergonomic hazards: Hematology lab workers may spend long hours working with
microscope or doing repetitive tasks such as pipetting, which can cause physical strain and
musculoskeletal disorders.
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MAINTAINANCE OF RECORDS & LAB DECORUM
Every record must have the following:

1. Brown sheet + printed Label + Transparent cover over label
2. All columns in Certificate in front/first page must be filled
3. Index Page – Must be filled with Date of experiment & submitted date + Page number
4. Diagrams on left side (with pencil only)
5. After writing of each experiment you must get signature from the concerned faculty
6. Viva will be taken in every lab session
7. Everyone must bring a lab box: Contents in Lab box: Bandage, Sprit, Cotton, Torniquets,
nail cutter, scissor, a pair of Gloves, Head mask, face mask, Hair band, cello tape, permanent
marker, paper cutter.
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SYLLABUS
LIST OF EXPERIMENTS
Subject Name: Hematology Practical

Subject Code: 22MLH-201
Course Objectives
To know the composition of blood and methods of estimating different components of blood
To know the basic concepts of Hematology& routine clinical investigations of Hematology

laboratory.
Course outcomes
CO1 Demonstrate the components of blood and their staining procedures
CO2 Make use of various procedures for blood count
CO3 Explain and take part in the blood banking procedures
Experiment No Experiment Name
1. Preparation of EDTA Vials
2. Collection of Blood sample, Plasma separation
3. Estimation of ESR
4. ABO Blood Grouping
5. Bleeding Time. Clotting Time,
6. Total White Blood Cell Count in Blood
7. Total Red Blood Cell Count in Blood
8. Estimation of Platelets count in Blood
9. Hemoglobin (Hb) estimation Shali 's method
10. Differential leukocyte count (DLC)
9
CO3
CO2
CO1
CO No
procedures
staining procedures
CO Statement
Explain and take part in the blood banking

Demonstrate the components of blood and their
Make use of various procedures for blood count
3
3
2
PO1: Laboratory knowledge
3
2
1
PO2: Problem analysis
10
0
0
1
PO3:Design/development of solutions
2
3
2
CO PO MAPPAING
PO4: Conduct investigations of complex

problems
1
2
2
PO5: Modern tool usage

2
2
1
PO6: The laboratory and society

1
2
1
PO7: Environment and sustainability

3
3
2
PO8: Quality Control

2
2
3
PO9: Individual or teamwork

0
3
0
PO10: Communication
2
2
1
PO11: Project management and finance

3
2
2
PO12: Life-long learning

Experiment CO Mapping
Experiment Experiment Mapped CO

No
Exp 1 Preparation of EDTA Vials CO1, CO2
Exp 2 Collection of Blood sample, Plasma seperation CO1, CO2
Exp 3 Estimation of ESR CO1,CO3
Exp 4 ABO Blood Grouping CO2
Exp 5 Bleeding Time. Clotting Time, CO2
Exp 6 Total White Blood Cell Count in Blood CO3
Exp 7 Total Red Blood Cell Count in Blood CO3
Exp 8 Estimation of Platelets count in Blood CO3
Exp 9 Hemoglobin (Hb) estimation Shali 's method CO3
Exp 10 Differential leukocyte count (DLC) CO3
Textbooks / Reference Books
1. An Introduction to medical laboratory technology,F.J.Baker et al., Butterworks and co. , London.
2. Blood Coagulation, Thomson J. CHur chill Livingstone.
3. Hematology for Medical TeCHnologists, Charles F.Sieverd.
4. Clinical Diagnosis and Management by Laboratory Methods, Todd and Sanford, W.B Saunders
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1. PREPARATION OF EDTA VIALS
Course Name/ code Hematology practical / 22MLH-201
Experiment No 1
Title Preparation of EDTA Vials
Master subject coordinator Mr. Attuluri Vamsi Kumar
Department / Institute Department of MLT/ UIAHS
Program B.Sc. MLT
Semester 3
Learning Outcomes:
By the end of this experiment, students should be able to:
1. Understand the principle of how EDTA acts as an anticoagulant in blood samples.
2. Recognize the applications of EDTA in various medical lab tests.
3. Demonstrate proficiency in preparing EDTA vials for sample collection.
4. Develop skills in accurate measurement and mixing of chemical solutions in a lab setting.
5. Apply proper sterilization techniques for laboratory equipment and vials.
6. Understand the importance of safety practices when handling chemical reagents and blood
samples.
7. Recognize when EDTA vials are contraindicated and be able to suggest suitable
alternatives.
8. Understand the implications of improperly prepared EDTA vials on the integrity of blood
samples and subsequent lab test results.
9. Enhance their problem-solving skills by understanding how to troubleshoot issues during
the preparation process and sample collection.
10. Develop a broad understanding of the role and importance of anticoagulants in medical
laboratory technology.
Introduction:
In medical laboratories, the preservation of the integrity of blood samples is crucial for accurate
test results. One of the most widely used anticoagulants for preserving blood samples is
Ethylenediaminetetraacetic acid, commonly known as EDTA. This versatile chelating agent binds
to calcium ions in the blood, preventing the coagulation process and thus allowing the sample to
be preserved for extended periods. This experiment focuses on the preparation of EDTA vials that
are commonly used for collecting and storing blood samples in medical laboratories.
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Understanding the properties of EDTA, how it functions as an anticoagulant, and how to correctly
prepare vials using this chemical is a fundamental skill for anyone working in a clinical lab setting.
Aim: To prepare EDTA (Ethylenediaminetetraacetic acid) vials used in clinical laboratories for
various hematological tests.
Principle: EDTA acts as a powerful chelating agent, binding to metal ions present in the blood
sample, thereby preventing coagulation by inhibiting the clotting process. The anti-coagulative
property of EDTA makes it an ideal choice for preserving blood samples for complete blood count
(CBC) and flow cytometry testing.
Materials Required:
1. EDTA (disodium salt or potassium salt)
2. Distilled water
3. Vials (10 ml)
4. Micro-pipettes
5. Weighing machine
6. Laboratory-grade Stirrer
7. Autoclave machine
8. Personal Protective Equipment (PPE) – lab coat, gloves, and safety glasses
Procedure:
1. Wear your Personal Protective Equipment (PPE) to ensure safety.
2. Determine the amount of EDTA needed. The typical concentration is around 1.5-2.2
mg/mL of blood. For a 10 mL vial, you would need approximately 15-22 mg of EDTA.
3. Accurately weigh the required amount of EDTA using the weighing machine.
4. Add the weighed EDTA to the vial.
5. Add distilled water to the vial, ensuring it's filled up to the 10 ml mark.
6. Stir the solution until the EDTA is fully dissolved.
7. The vials should then be autoclaved for sterility. Follow the instructions of your specific
autoclave machine to ensure proper sterilization.
8. Once the vials are cooled down, they are ready for use or storage.
Results:
……………………………………………………………………………………………………
…………………………………………………………………………………………………….
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Observation:
……………………………………………………………………………………………………
…………………………………………………………………………………………………….
Observation Table
Observation Observation Observation Result

No time & date
Results Interpretation: The success of the experiment can be gauged by the complete dissolution
of the EDTA and the sterility of the vial post-autoclave. The prepared vial should be able to
preserve the blood samples without causing coagulation, allowing for accurate hematological
analyses.
14
Indications:
1. Need for hematological tests such as complete blood count (CBC), blood film examination,
reticulocyte count, and flow cytometry.
2. Requirement for genetic studies as EDTA prevents DNA degradation.
3. For drawing blood samples that require transport or storage before analysis.
Contraindications:
1. EDTA should not be used when blood cultures for microbiological investigations are
required. The antibacterial effect of EDTA might lead to false-negative results.
2. EDTA vials are not suitable for coagulation studies, as EDTA chelates calcium ions
necessary for the clotting process.
3. It is contraindicated for serum biochemical tests and electrolyte analysis due to the same
chelating property.
4. Incorrect EDTA concentration may lead to morphological changes in blood cells, affecting
the test results. Hence, accurate weighing and preparation are crucial.
Clinical Significance of the Preparation of EDTA Vials Experiment:

The preparation of EDTA vials is a vital part of numerous clinical procedures and tests. The ability
to correctly prepare these vials directly impacts the quality of the patient care provided and the
accuracy of the diagnostic information obtained. Here's the clinical significance of this experiment:
1. Blood Analysis: The EDTA vials prepared during this experiment play a crucial role in
hematological investigations such as Complete Blood Count (CBC), blood smear
microscopy, flow cytometry, and HbA1c tests. These investigations are often the first-line
diagnostic procedures for conditions like anemia, leukemia, infections, and diabetes.
2. Genetic Studies: EDTA preserves the integrity of the DNA in a blood sample, making it
suitable for genetic and molecular studies. These studies help in diagnosing genetic
disorders, cancer mutations, and susceptibility to certain diseases.
3. Preservation of Samples: The experiment is significant in learning how to maintain the
stability of blood samples, especially when they require storage or transport before
analysis. This is crucial in scenarios where the laboratory performing the tests is located at
a considerable distance from the sample collection site.
4. Quality Control: Proficiency in preparing EDTA vials helps ensure the accuracy and
reliability of lab results. A correctly prepared EDTA vial prevents false results due to
clotted samples, while incorrect preparation may lead to cellular changes, affecting the test
results.
15
5. Patient Safety: EDTA is generally considered safe for patient use, but improper handling
or usage can lead to adverse effects. Understanding the right preparation and usage
methods reduces potential risks, contributing to patient safety.
6. Education for Best Practices: Understanding the contraindications of EDTA is equally
important. EDTA should not be used for certain tests such as blood cultures and
coagulation studies. Therefore, the experiment provides valuable education on best
practices for sample collection and storage.
Sample Viva Questions:
Question 1: What is the primary function of EDTA in blood sample vials?
Answer: EDTA acts as a powerful chelating agent in blood sample vials, binding to calcium ions
and preventing coagulation, thus preserving the sample for hematological analysis.
Question 2: In which types of tests are EDTA vials commonly used?
Answer: EDTA vials are commonly used for hematological tests like complete blood count (CBC),
blood smear microscopy, flow cytometry, and HbA1c tests. They are also used for genetic and
molecular studies.
Question 3: Why is it necessary to autoclave the vials after adding EDTA and water?
Answer: Autoclaving the vials is necessary for sterilization. It ensures that no unwanted
microorganisms are present in the vial, which could potentially interfere with the tests.
Question 4: Why can't we use EDTA vials for blood cultures or coagulation studies?
Answer: EDTA chelates calcium ions necessary for the clotting process, hence it interferes with
coagulation studies. For blood cultures, EDTA's antibacterial effect might lead to false-negative
results.
Question 5:What is the typical concentration of EDTA in a blood collection vial?
Answer: The typical concentration of EDTA in a blood collection vial is around 1.5-2.2 mg/mL of
blood.
Question 6: What could be the effect of using an incorrect concentration of EDTA in the blood
collection vials?
Answer: Incorrect EDTA concentration can lead to morphological changes in blood cells, affecting
the test results. Additionally, too much EDTA may lead to excessive chelation of calcium, making
the blood sample unsuitable for certain tests.
Question 7: Why do we need to wear Personal Protective Equipment (PPE) during this
experiment?
Answer: Wearing PPE ensures safety from potential chemical spills, splashes, or aerosols
generated during the process. It also prevents the introduction of contaminants into the vials.
Question 8: What role does EDTA play in genetic studies?
16
Answer: EDTA preserves the integrity of DNA in a blood sample, preventing DNA degradation,
which makes it suitable for genetic and molecular studies.
Question 9: What is the significance of accurately weighing the EDTA before adding it to the
vial?
Answer: Accurate weighing ensures that the correct concentration of EDTA is used. This prevents
over or under chelation of calcium ions which could interfere with test results or blood cell
morphology.
Question 10: What are the safety measures one should take while performing this experiment?
Answer: The safety measures include wearing personal protective equipment (PPE) such as lab
coats, gloves, and safety glasses, handling chemicals carefully, using lab equipment properly, and
disposing of all materials responsibly when finished.
Reference materials:
1. "Clinical Hematology: Theory and Procedures" by Mary Louise Turgeon - This textbook
provides a solid foundation in the theory and procedures of hematology, helping to
understand the principles of hematological tests and the preparation and use of EDTA in
such procedures.
2. "Clinical Laboratory Hematology" by Shirlyn B. McKenzie - This comprehensive book
provides a detailed exploration of hematology in a clinical lab setting. It offers insights into
the collection and preservation of blood samples and how various anticoagulants, including
EDTA, work.
3. "Phlebotomy Essentials" by Ruth McCall and Cathee Tankersley - This book is a useful
resource for understanding blood collection processes. It explains the importance of
anticoagulants like EDTA in preserving blood samples and discusses the methodology of
preparing EDTA vials.
17
2. COLLECTION OF BLOOD SAMPLE, PLASMA SEPARATION
Experiment No 2
Title Collection of Blood sample, Plasma separation
Program B.Sc. MLT
Semester 3
Learning Outcomes:
Upon completion of this experiment, students will be able to:
1. Understand and explain the principle behind venipuncture and the separation of plasma
and serum from blood.
2. Demonstrate the correct and safe method of blood collection through venipuncture.
3. Understand the materials required for venipuncture and the separation of serum and
plasma, and how to correctly use these materials.
4. Effectively perform centrifugation to separate serum and plasma from blood, and identify
these components visually.
5. Understand the differences between serum and plasma, and why certain tests may require
one over the other.
6. Recognize and explain the indications and contraindications for venipuncture.
7. Understand the ethical and safety considerations in blood collection and handling.
8. Apply problem-solving skills when faced with issues during venipuncture or separation
procedures.
9. Document and interpret the results of the experiment effectively.
10. Implement good laboratory practices, including safe handling and disposal of biohazardous
materials.
Introduction:
In the field of medical laboratory technology, blood tests are a critical diagnostic tool. Blood is
often analyzed to determine a patient's overall health, identify infections, screen for certain genetic
conditions, and help doctors evaluate the functionality of organs such as the liver and kidneys.
This lab experiment focuses on one of the fundamental skills a medical lab technologist needs to
master: the collection of blood samples through venipuncture and the separation of serum and
plasma from these samples.
18
Blood samples can be collected in various ways, and venipuncture is the most common method
used to obtain these samples. A vein puncher or needle is used to puncture a vein, usually in the
patient's arm, to collect the sample. Once collected, the blood can be processed to separate its
different components.
Plasma and serum are two of these components. Plasma, which constitutes about 55% of total
blood volume, is the liquid component of blood. It contains water, salts, enzymes, antibodies and
other proteins. Serum, on the other hand, is similar to plasma but lacks the clotting factors that are
present in plasma. Serum is obtained from clotted blood, separated by centrifugation.
Aim: The aim of this lab experiment is to enable the medical laboratory technology students to:
1. Understand and practice the method of blood collection by using a vein puncher.
2. Separate and distinguish serum and plasma from the collected blood samples.
Principle: Venipuncture is a common medical procedure for blood collection, where a vein
puncher or needle is used to puncture the vein. Blood collected is used for various types of medical
diagnosis.
Blood consists of plasma (the liquid component, made up of water, salts, and protein) and blood
cells. When blood clots, the fluid that remains is termed as serum. The main difference between
serum and plasma lies in the clotting factors: serum is plasma without the clotting factors.
Plasma and serum can be separated from the blood by the process of centrifugation, which uses
the principle of density-based separation. When centrifuged, the heavier components (red and
white blood cells) get pushed towards the bottom, and the lighter components (plasma or serum)
remain on top.
Materials Required:
1. Venipuncture kit (includes vein puncher/needle, collection tubes)
2. Antiseptic wipes
3. Gloves
4. Tourniquet
5. Gauze pads
6. Biohazard waste bin
7. Centrifuge
8. Micro-pipettes
9. Lab coats and eye protection
Procedure:
1. Ensure the appropriate personal protective equipment is worn.
2. Prepare the patient by asking them to sit comfortably. Assure the patient and explain the
process.
19
3. Select the appropriate vein for blood collection and apply the tourniquet.
4. Clean the area with an antiseptic wipe.
5. Perform the venipuncture using the vein puncher/needle.
6. Collect the blood in the appropriate tube: a tube with an anticoagulant for plasma and a
tube without an anticoagulant for serum.
7. Once the blood is collected, remove the tourniquet and apply pressure to the puncture site
with a gauze pad.
8. Carefully dispose of the used needle in a biohazard waste bin.
9. Centrifuge the collected blood to separate plasma/serum. Typically, blood is centrifuged at
1000-2000 g for 10 minutes. However, follow your lab's specific protocol.
10. After centrifugation, the heavier blood cells will settle at the bottom of the tube, with the
plasma or serum on top. Use a micro-pipette to carefully separate the plasma or serum.
11. Results:
……………………………………………………………………………………………………
……………………………………………………………………………………………………
12. Observation:
……………………………………………………………………………………………………
…………………………………………………………………………………………………….
Observation Table

No time & date
Results Interpretation: The top, light-yellow fluid after centrifugation is plasma or serum, which
can be used for further analysis. The presence or absence of clotting factors helps to distinguish
between plasma (contains clotting factors) and serum (does not contain clotting factors).
20
Indications: Venipuncture is indicated whenever a blood sample is required for diagnostic testing.
Contraindications:
1. Sites with a hematoma or signs of infection or inflammation.
2. Arm on the side of a mastectomy, dialysis fistula, or vascular graft.
3. Areas of scar tissue.
4. Patients with hemophilia or other bleeding disorders should be handled with caution.
5. In general, if a patient has a known allergy to any of the materials used, an alternative
method of blood collection should be considered.
Collection of blood by vein puncher
21
Clinical Significance:
The venipuncture and subsequent separation of serum and plasma are critical components of
diagnostic laboratory medicine. The skills taught and practiced in this experiment have wide-
ranging clinical significance:
Diagnostic Applications: Serum and plasma are routinely analyzed in clinical laboratories to aid
in the diagnosis of numerous diseases and conditions. They can provide insight into a patient's
metabolic status, organ function, blood cell counts, and presence of potential diseases.
Furthermore, they can be used for drug testing, therapeutic drug monitoring, and in the detection
of antibodies in immunoassays.
Monitoring of Health and Disease: Regular blood tests allow healthcare professionals to monitor
a patient's overall health, track the progress of diseases, and evaluate the efficacy of treatments.
The separation of serum and plasma plays an important role in these assessments.
Precision and Personalized Medicine: The analysis of serum and plasma can provide
personalized data on a patient's health status. This information can be used to tailor treatments to
the individual, an approach known as precision or personalized medicine.
Preventive Medicine: Regular blood tests can help in the early detection of diseases, even before
the onset of symptoms. This allows for early intervention and treatment, potentially preventing the
progression of the disease.
Therapeutic Applications: Plasma can be used therapeutically in the form of plasma transfusion.
It can provide clotting factors and other proteins to patients in need, such as those with liver
disease, severe infections, or burn victims.
Research Applications: Serum and plasma samples are often used in biomedical research to
understand disease mechanisms, develop new treatments, and advance the understanding of human
biology.
22
Sample viva questions
Question 1: What is the difference between serum and plasma?
Answer: Plasma is the liquid component of blood that contains clotting factors, while serum is
similar to plasma but lacks these clotting factors. Serum is essentially plasma from which the
clotting proteins have been removed.
Question 2: Why is centrifugation used in the separation of plasma and serum?
Answer: Centrifugation is a process that uses centrifugal force to separate the components of
blood based on their densities. The heavier blood cells are pushed to the bottom of the tube, while
the lighter plasma or serum remains on top.
Question 3: What precautions should be taken during venipuncture?
Answer: Some key precautions include: wearing personal protective equipment, properly
disinfecting the puncture site, selecting the appropriate vein, and ensuring the patient is
comfortable and informed about the procedure. After the procedure, the used needle should be
carefully disposed of in a biohazard waste bin.
Question 4: What are some contraindications for venipuncture?
Answer: Contraindications for venipuncture include sites with a hematoma, signs of infection or
inflammation, presence of a vascular graft or dialysis fistula, areas of scar tissue, and the arm on
the side of a mastectomy.
Question 5: Why are serum and plasma commonly used in clinical laboratories?
Answer: Serum and plasma are used in clinical laboratories because they contain a variety of
components (proteins, electrolytes, hormones, etc.) that can provide valuable information about a
patient's health status. They can be used to diagnose diseases, monitor treatment progress, and
assess overall health.
Question 6: What are some indications for venipuncture?
Answer: Venipuncture is indicated whenever a blood sample is required for diagnostic testing.
This can include routine health screenings, monitoring of chronic conditions, evaluation of organ
function, and testing for suspected diseases.
Question 7: What safety measures should be implemented when handling biohazardous materials
like blood?
Answer: Safety measures include wearing appropriate personal protective equipment (such as
gloves and lab coats), using safe practices for needle handling and disposal, preventing splashes
and aerosols, disinfecting surfaces, and treating all blood samples as potentially infectious.
Question 8: Why are anticoagulants used in the collection of blood for plasma separation?
Answer: Anticoagulants are used to prevent blood from clotting. For plasma separation, we need
to prevent the blood from clotting because plasma contains clotting factors. If the blood clots, these
clotting factors will be used up, essentially turning the plasma into serum.
23
Question 9: What factors could potentially affect the results of the serum and plasma separation?
Answer: Factors that could affect the results include improper sample handling, incorrect
centrifugation speed or time, contamination of the sample, and incomplete clotting in the case of
serum separation.
Question 10: What does the presence of a "buffy coat" indicate after centrifugation of blood?
Answer: The "buffy coat" is a thin, whitish layer that appears between the plasma or serum (on
top) and the red blood cells (at the bottom) after centrifugation. This layer contains white blood
cells and platelets, and its presence is normal after centrifugation of whole blood.
1. "Phlebotomy Essentials" by Ruth E. McCall and Cathee M. Tankersley. This book
provides comprehensive information on phlebotomy techniques, including venipuncture,
and would be an excellent reference for the blood collection portion of the experiment.
2. "Clinical Chemistry: Principles, Techniques, and Correlations" by Michael L. Bishop,
Edward P. Fody, and Larry E. Schoeff. This book covers the principles and methodologies
used in clinical laboratory testing, including the separation and analysis of serum and
plasma.
3. "Tietz Textbook of Clinical Chemistry and Molecular Diagnostics" by Nader Rifai,
Andrea Rita Horvath, and Carl T. Wittwer. This is a comprehensive reference that includes
information on blood collection and processing, and would be a valuable resource for
understanding the clinical significance of serum and plasma analysis.
24
3. ESTIMATION OF ESR
Experiment No 3
Title Estimation of ESR
Program B.Sc. MLT
Semester 3
Learning Outcomes:
Upon successful completion of the ESR estimation experiment, learners should be able to:
1. Understand the principles behind the ESR test and the significance of the results.
2. Demonstrate the ability to conduct the ESR test using standard lab equipment, including
pipettes, blood sample tubes, and an ESR meter.
3. Explain the factors that can influence the ESR rate, including age, gender, and disease
state.
4. Interpret the results of ESR tests in the context of potential underlying conditions, while
understanding the non-specific nature of the test.
5. Understand the correlation between ESR values and various inflammatory and non-
inflammatory conditions.
6. Discuss the potential limitations and drawbacks of ESR as a diagnostic tool.
7. Apply appropriate safety and biohazard precautions when handling and testing blood
samples.
8. Demonstrate proper data recording and reporting techniques, presenting ESR test
findings in a clear and understandable manner.
9. Understand the ethical implications of blood testing, including patient consent and
confidentiality.
10. Collaborate effectively in a laboratory setting, demonstrating teamwork, communication,
and problem-solving skills.
Introduction:
Erythrocyte Sedimentation Rate (ESR) is a common blood test that measures the rate at which
red blood cells sediment in a period of one hour. It is a non-specific measure of inflammation
25
that is commonly used in medicine as a simple marker of whether inflammation is present. The
test is performed by adding anticoagulant to a tall, thin tube of blood, and then measuring the
rate of fall (sedimentation) of erythrocytes (red blood cells) over a specified period.
Increased ESR rates might indicate inflammatory diseases such as rheumatoid arthritis, systemic
lupus erythematosus, and vasculitis, among others. Decreased ESR rates, on the other hand, can
occur with conditions like polycythemia, extreme leukocytosis, and protein abnormalities.
However, it's important to note that an elevated or reduced ESR does not confirm a specific
diagnosis, but rather indicates the presence or absence of inflammatory activity.
AIM:
To demonstrate the Erythrocyte sedimentation time by Westergren’s method
Principle
When well-mixed anticoagulated blood is placed in a vertical tube, the erythrocytes tend to fall
towards the bottom of the tube/pipette till they form a packed column in the lower part of the tube
in a given time.
Phases in ESR
ESR takes place in the following 3 phases which are carried out in one hour:
• Phase of rouleaux formation: In the initial period of 10 minutes, the process of rouleaux
formation occurs and there is little sedimentation.
• Phase of settling: In the next 40 minutes, settling of RBCs occurs at a constant rate.
• Phase of packing: In the last 10 minutes sedimentation slows and packing of the RBCs to
the bottom occurs.
• That is why ESR by all methods is expressed as mm first hour rather than per hour.
Methods of ESR
1. Westergren’s method
2. Wintrobe’s method
3. Micro ESR method
4. Automated methods
26
1. Westergren’s Method
➢ This is the most commonly used method.

➢ It is performed by using Westergren’s pipette which is a straight pipette 30 cm long
open at both ends with internal bore diameter of 2.5 mm and is calibrated from 0-
200 mm from top to bottom
EQUIPMENT:
1. Syringe
2. tornioquete
3. Westergrens tube
4. ESR stand
5. Stopwatch
6. Cotton, sprit
7. Other PPE’s
Anticoagulant:
➢ Trisodium citrate as g/dl liquid anticoagulant is used.

➢ It is used in the concentration of 1:4 i.e. four parts of blood are added to one part
of anticoagulant.
Procedure
1. The patient is advised to come in the morning fasting (as heavy protein diet affects
concentraton of plasma proteins).
2. Take 1.6 ml of patient’s blood and mix it with 0.4 ml of citrate anticoagulant already
put in a tube.
3. The test should be done within two hours of taking blood.
4. Fill the pipette upto mark O with citrated blood with the help of rubber teat by vacuum
filling and fix it in a rack vertically away from sun light or vibrations.
5. Let it stand for one hour after which reading is taken at the upper meniscus of the RBCs.
Normal values
• Males 3-5 mm 1st hour

• Females 4-7 mm 1st hour
Advantages
27
• It is a more sensitive method.
• It is easy to fill and clean the Westergren’s pipette.
Disadvantages
• Requires more amount of blood.

• Dilution of blood in anticoagulant affects ESR.
• Filling of blood by mouth pipetting should be strictly discouraged.
Observation:
……………………………………………………………………………………………………
……………………………………………………………………………………………………
……………………..
Result:
……………………………………………………………………………………………………
……………………………………………………………………………………………………
……………………
Observation Table

No time & date
The clinical significance of the Erythrocyte Sedimentation Rate (ESR) test is vast due to its ability
to reflect inflammatory activity within the body, which can be a hallmark of several diseases and
medical conditions. Below are some of its key clinical implications:
28
1. Inflammatory and Autoimmune Conditions: An elevated ESR is a common finding in
systemic inflammatory conditions such as rheumatoid arthritis, systemic lupus
erythematosus, and vasculitis. It helps in confirming inflammation and monitoring the
activity and progression of these diseases.
2. Infections: ESR can be significantly elevated in acute and chronic infections. It may be
used as a general marker to monitor the course of an infection and the effectiveness of
treatment.
3. Malignancies: Certain cancers, like lymphoma or multiple myeloma, can cause a high ESR,
and the test may be used as one of many tools in the diagnosis and monitoring of these
conditions.
4. Age-Related Conditions: ESR tends to increase with age and can be a non-specific marker
for various age-related conditions such as giant cell arteritis and polymyalgia rheumatica.
5. Monitoring Treatment Response: ESR is often used to monitor the body's response to
treatments for a variety of conditions, particularly those related to inflammation and
infection. A decreasing ESR is generally a sign that the condition is responding to
treatment.
Sample viva questions:
1. Q: What does an ESR test measure?
A: The ESR test measures the rate at which red blood cells (erythrocytes) settle at the
bottom of a test tube in one hour. It's a non-specific measure of inflammation.
2. Q: What can cause an elevated ESR?
A: Elevated ESR levels may indicate the presence of an inflammatory condition, such as
rheumatoid arthritis, systemic lupus erythematosus, or vasculitis. It can also be high in
certain infections and malignancies.
3. Q: Can the ESR test diagnose a specific disease?
A: No, the ESR test alone cannot diagnose a specific disease. It's a non-specific test that
indicates the presence or absence of inflammation. Other tests are usually required for a
specific diagnosis.
4. Q: How is the ESR test conducted?
A: The ESR test is conducted by adding an anticoagulant to a blood sample in a tall, thin
tube, and then measuring the distance the red blood cells fall in one hour.
5. Q: Are there any conditions that can result in a decreased ESR? A: Yes, conditions such as
polycythemia (a high red blood cell count), extreme leukocytosis (a high white blood cell
count), and certain protein abnormalities can result in a decreased ESR.
6. Q: Is the ESR test the same for men and women?
A: While the test procedure is the same for both sexes, the reference range can vary.
Generally, the normal range is lower for men than for women.
7. Q: How is an ESR test result interpreted?
A: A high ESR value generally indicates increased inflammation in the body, while a low
ESR can suggest the opposite. However, interpretation should be done in the context of a
patient's symptoms, clinical history, and other test results.
8. Q: Can age affect the ESR value?
A: Yes, ESR tends to increase with age. The reference range for a 'normal' ESR increases
slightly as people get older.
9. Q: How is the ESR test used in monitoring treatment response?
A: In diseases that cause inflammation, the ESR often decreases in response to treatment
that reduces inflammation. Therefore, it can be used to monitor the effectiveness of
treatment.
10. Q: Can an elevated ESR be a sign of cancer?
29
A: Yes, certain malignancies, including lymphoma and multiple myeloma, can cause an
elevated ESR. However, a high ESR alone is not sufficient to diagnose cancer; other
diagnostic tests are necessary.
References:
1. "Henry's Clinical Diagnosis and Management by Laboratory Methods" by Richard A.
McPherson MD MSc and Matthew R. Pincus MD PhD. This comprehensive reference
covers a wide range of laboratory tests, including the ESR test, and their clinical
implications.
2. "Wallach's Interpretation of Diagnostic Tests: Pathways to Arriving at a Clinical
Diagnosis" by Mary A. Williamson, MT (ASCP), PhD and L. Michael Snyder, MD. This
book serves as a guide to the selection and interpretation of commonly used diagnostic
tests, including the ESR test.
3. "Clinical Laboratory Hematology" by Shirlyn B. McKenzie and Lynne Williams. This
book provides comprehensive coverage of hematology basics, including various blood
tests such as the ESR, making it an excellent reference for understanding these diagnostic
tests in a clinical context.
30
31
4. ABO BLOOD GROUPING
Experiment No 4
Title ABO blood grouping
Program B.Sc. MLT
Semester 3
Learning Outcomes
Upon completion of the ABO Blood grouping by slide method experiment, students should be
able to:
1. Understand the fundamental concepts of the ABO blood group system, its importance,
and the role it plays in blood transfusion procedures.
2. Demonstrate the ability to safely and accurately perform the ABO blood grouping slide
method, following standard laboratory safety protocols.
3. Identify the different ABO blood groups based on the agglutination reaction observed in
the experiment.
4. Interpret and analyze the results of the blood grouping experiment, linking the presence
or absence of certain antigens to specific blood groups.
5. Recognize the implications of the results in real-world scenarios such as blood
transfusions, organ transplants, and paternity testing.
6. Appreciate the need for precision, accuracy, and attention to detail in clinical laboratory
procedures and understand the consequences of errors.
7. Develop an understanding of the immune response related to the ABO blood system and
its role in transfusion reactions and certain disease associations.
8. Apply the principles and skills learned in the experiment to other serological and
immunological techniques.
9. Communicate the results effectively, in both written and oral forms, detailing the
procedure, findings, and their interpretations.
Introduction
The ABO blood grouping system is a classification system for human blood that's based on the
presence or absence of antigens on the surface of red blood cells. It consists of four main blood
groups: A, B, AB, and O. This system is significant because it directly impacts blood
transfusions; receiving blood from an incompatible group can lead to severe reactions or even
death.
32
The ABO Blood grouping by slide method experiment is an essential practice in clinical and
medical laboratories. This simple, yet powerful method provides an opportunity for a hands-on
experience in identifying the ABO blood groups, which is crucial in various fields such as
medicine, immunology, genetics, and forensic science. The slide method is a quick and
convenient technique, using commercially available antisera, that does not require specialized
laboratory equipment. However, it must be performed with precision and care to avoid
inaccurate results.
Aim: To perform ABO bold group by Forward & Reverse blood group method
Principle:
When red cells are mixed with various reagent antiseras (soluble antibody), agglutination will
occur on the slides containing cells positive for (possessing the antigen) the corresponding
antigen. No agglutination will occur when the red cells do not contain the corresponding
antigen. One primary application of this principle is blood typing.
Material: 1. Antibody A
2. Antibody B
3. Red blood cells (#1 and #2)
4. Slides
5. Applicator sticks
6. Pipets
Procedure: 1. On the section of slide labeled anti-A place one drop of antibody A.
2. On the section of slide labeled anti-B place one drop of antibody B.
3. Place one drop of cells in each antibody containing circle.
4. Carefully mix each solution with a separate applicator stick.
5. Tilt slowly for one minute.
6. Record results.
Interpretation: Agglutination (clumping) of the red blood cells is positive. No

agglutination is negative. It is critical to read the results immediately as false positives can
occur when the mixture begins to dry on the side.
33
Observation:
Agglutination has been observed in rings anti sera A & B but no agglutination in ring Antisera D
Result:
The given sample blood group is AB Negative.
Observation Table

No time & date
Clinical Significance
The clinical significance of ABO blood grouping is that it is important to know a person's blood
type for several reasons:
34
1. Blood transfusions: It is important to know a person's blood type in order to ensure that
they receive a compatible blood transfusion. If a person receives a blood transfusion with
incompatible blood, it can cause serious complications or even death.
2. Organ transplantation: A person's blood type is also important when considering organ
transplantation. The blood type of the donor and the recipient must be compatible in
order for the transplant to be successful.
3. Pregnancy: In pregnancy, the mother's blood type may be incompatible with the blood
type of the fetus. This can lead to complications such as hemolytic disease of the
newborn, which can be serious or even fatal.
4. Other medical procedures: ABO blood typing may also be important in certain medical
procedures, such as dialysis or surgery, to ensure that there are no adverse reactions to
blood or blood products.
Sample Viva Questions
Question1: What are the four main types of the ABO blood group system?
Answer: The four main types of the ABO blood group system are A, B, AB, and O.
Question 2: Why is it crucial to match blood types in blood transfusions?
Answer: Matching blood types in blood transfusions is essential to prevent a harmful

immune response from the recipient's body. If the blood types are not compatible, the
recipient's immune system may perceive the donor blood cells as foreign and attack them,
leading to a transfusion reaction that can be life-threatening.
Question 3: What is agglutination in the context of the ABO blood grouping slide method?
Answer: Agglutination is the clumping together of red blood cells in response to a specific
antibody. In the context of the ABO blood grouping slide method, agglutination indicates the
presence of a specific antigen on the surface of the red blood cells.
Question 4: Which blood group is considered a universal donor and why?
Answer: Blood group O is considered a universal donor because it lacks A and B antigens on
the red blood cells, hence it can be safely donated to any other ABO blood group without
causing an immune response.
Question 5: Which blood group is considered a universal recipient and why?
Answer: Blood group AB is considered a universal recipient because it has both A and B
antigens on the red blood cells. Therefore, it can receive blood from any ABO blood group
without causing an immune response.
35
Question 6: If a person with blood type A receives blood from a person with blood type B,
what will happen and why?
Answer: If a person with blood type A receives blood from a person with blood type B, a
transfusion reaction would likely occur. This is because the recipient's immune system would
recognize the B antigens as foreign and produce antibodies against them, leading to
agglutination and destruction of the transfused blood cells.
Question 7: How do you determine a person's blood type using the slide method?
Answer: A person's blood type is determined using the slide method by mixing their blood
with different antisera (Anti-A, Anti-B, and Anti-D). If agglutination (clumping) occurs
when mixed with a certain antiserum, it indicates the presence of the corresponding antigen
on the person's red blood cells, hence determining their blood type.
Question 8: What safety precautions must be taken when performing the ABO blood
grouping slide method?
Answer: Safety precautions include wearing appropriate personal protective equipment (like
gloves, lab coats, and safety glasses), handling blood samples with care to prevent exposure,
disposing of used materials in biohazard containers, and disinfecting the work area before
and after the experiment.
Question 9: If no agglutination occurs in a slide test with Anti-A and Anti-B sera, what is
the blood type?
Answer: If no agglutination occurs with Anti-A and Anti-B sera, the blood type is O.
Question 10: Can you determine the Rh factor using the ABO blood grouping slide method?
How?
Answer: Yes, the Rh factor can be determined using the ABO blood grouping slide method.
This is done by mixing the blood sample with Anti-D serum. If agglutination occurs, it
indicates the presence of Rh antigen, meaning the blood type is Rh positive. If no
agglutination occurs, the blood type is Rh negative.
36
5. BLEEDING TIME & CLOTTING TIME
Experiment No 5.1
Title Bleeding time
Program B.Sc. MLT
Semester 3
Learning Outcomes: Bleeding Time

Upon successful completion of the 'bleeding time' experiment, learners should be able to:
1. Understand and explain the concept of hemostasis, its importance, and its mechanisms,
including vasoconstriction, platelet plug formation, and coagulation.
2. Demonstrate the ability to carry out the finger prick method safely and effectively,
following the established protocols to minimize risk.
3. Accurately record the time taken for bleeding to cease following the incision and interpret
these findings in the context of normal and abnormal bleeding times.
4. Explain the role and function of platelets in the clotting process, and how their quantity or
functionality can impact bleeding time.
5. Discuss potential disorders related to abnormal bleeding times, such as thrombocytopenia,
Von Willebrand disease, and hemophilia.
6. Understand the limitations and variability of the bleeding time test and recognize when and
why other diagnostic tests might be necessary or preferred.
7. Apply ethical and patient-centered considerations in performing the experiment, including
obtaining consent, ensuring patient comfort, and providing clear explanations about the
procedure.
8. Evaluate and interpret the results in the context of a patient's overall health, taking into
account factors that could influence the results, such as medications, alcohol, smoking, and
others.
9. Recognize the clinical significance of the bleeding time test in various contexts, such as
before surgical procedures, in diagnosing bleeding disorders, and in monitoring the
effectiveness of treatment.
10. Practice professional communication skills when explaining the procedure and results to
patients or colleagues.
Introduction
The 'bleeding time' experiment is a commonly used procedure in medical and clinical settings to
evaluate the efficiency of the body's hemostasis process. Hemostasis is the body's way of stopping
bleeding or blood flow via blood vessel constriction, platelet plug formation, and blood
coagulation. The bleeding time primarily measures the function of platelets, which are blood cells
that help with clotting.
37
In this experiment, a small, standardized incision is made on the fingertip, using a finger prick
method. The time taken from the point of injury to the point when bleeding stops is then recorded.
This is known as the 'bleeding time'. It's a crucial indicator of how well the platelets and
coagulation pathways interact to achieve hemostasis.
This experiment can reveal potential disorders related to platelets or blood vessels. It is also
essential in pre-surgery evaluations to assess a patient's risk of excessive bleeding during or after
a surgical procedure. However, please note that due to variability and potential for complications,
it's gradually being replaced by other tests in many clinical settings.
AIM:
To demonstrate the Bleeding Time by Finger prick method
PRINCIPLE:
A small puncture is made on the skin and the time for which it bleeds is noted. Bleeding stops
when platelet plug forms and breach in the vessel wall has sealed.
EQUIPMENT:
• Sterile lancet
• Filter paper
• Stopwatch
• Cotton, sprit
• Other PPE’s
PROCEDURE
1. Clean the tip of a finger with spirit.
2. Prick with a disposable needle or lancet.
3. Start the stop-watch immediately.
4. Start gently touching the pricked finger with a filter paper for every 30 seconds till blood
spots continue to be made on the filter paper.
5. Stop the watch when no more blood spot comes on the filter paper and note the time.
Normal values:
1 – 3 Minutes
Observation
……………………………………………………………………………………………………
……………………………………………………………………………………………………
…………………………………………………………………………………………….
Result
The bleeding time of the subject is found to be ………………………. Minutes
38
Observation Table

No time & date
Bleeding time estimation is a simple and commonly used test that measures the time it takes for
bleeding to stop after a standardized skin incision. It is used to assess platelet function and
screening for various bleeding disorders. The clinical significance of bleeding time estimation
includes:
Diagnosis of platelet dysfunction: Platelets play a crucial role in blood clotting, and a
prolonged bleeding time can indicate platelet dysfunction. This test can be used to diagnose
various platelet disorders, including von Willebrand disease, Bernard-Soulier syndrome, and
Glanzmann thrombasthenia.
Monitoring antiplatelet therapy: Antiplatelet medications, such as aspirin, clopidogrel, and
prasugrel, are commonly used to prevent heart attacks and strokes. Bleeding time estimation can
be used to monitor the effectiveness of these medications and ensure that the patient is not at risk
of excessive bleeding.
39
Preoperative evaluation: Bleeding time estimation is often used as part of a preoperative
evaluation to assess the risk of bleeding during surgery. If a patient has a prolonged bleeding
time, the surgeon may need to take special precautions during the procedure to minimize
bleeding.
Screening for bleeding disorders: Bleeding time estimation is often used as a screening test for
bleeding disorders, such as hemophilia and von Willebrand disease. If the bleeding time is
prolonged, further testing may be necessary to confirm the diagnosis.
1. Question: What is hemostasis and why is it important?
• Answer: Hemostasis is the body's process of stopping bleeding from a damaged

blood vessel. It involves vasoconstriction, platelet plug formation, and blood
coagulation. It's important to prevent excessive blood loss and ensure proper wound
healing.
2. Question: How is the bleeding time test performed?
• Answer: In the bleeding time test, a small incision is made on the finger (the finger
prick method) and the time it takes for the bleeding to stop is recorded.
3. Question: What does the bleeding time test primarily measure?
• Answer: The bleeding time test primarily measures platelet function and how
effectively the coagulation pathways work to achieve hemostasis.
4. Question: Name two disorders that can result in prolonged bleeding times.
• Answer: Thrombocytopenia (low platelet count) and Von Willebrand disease (a

bleeding disorder caused by a deficiency of the Von Willebrand factor, which is
necessary for platelet adhesion) can result in prolonged bleeding times.
5. Question: How can medications affect bleeding time?
• Answer: Some medications, such as aspirin or anticoagulants, can prolong bleeding

time by inhibiting platelet aggregation or the coagulation cascade.
6. Question: What precautions should be taken while performing the finger prick method?
• Answer: The finger prick method should be performed with clean, sterilized
equipment, and gloves should be worn. The patient should be comfortably seated
and informed about the procedure. The site of incision should be free from visible
veins and arteries.
7. Question: Why is the bleeding time test not as commonly used today?
40
• Answer: The bleeding time test has been largely replaced by other tests due to its
variability, potential for complications (such as infection), and its insensitivity to
mild or moderate platelet function disorders.
8. Question: How can abnormal bleeding times be treated?
• Answer: The treatment of abnormal bleeding times depends on the underlying

cause. It could involve stopping or changing certain medications, treating
underlying diseases, or in severe cases, blood transfusions or surgery may be
necessary.
9. Question: What factors can influence the results of a bleeding time test?
• Answer: Factors such as medications, alcohol consumption, smoking, age, sex, and
overall health status can influence the results of a bleeding time test.
10. Question: How does the body stop bleeding naturally after a cut or wound?
• Answer: When a cut or wound occurs, the body stops bleeding through a process
known as hemostasis. This involves vasoconstriction (narrowing of blood vessels)
to reduce blood flow, formation of a platelet plug to cover the wound, and the
coagulation of blood to form a stable clot.
References:
1. "Hoffbrand's Essential Haematology" by A. Victor Hoffbrand and Paul A. H. Moss - This

book provides a thorough understanding of hematology, including various tests and
procedures, such as the bleeding time test.
2. "Rodak's Hematology: Clinical Principles and Applications" by Elaine Keohane, Larry

Smith, Jeanine Walenga - This textbook presents a detailed view of hematology concepts,
tests, and procedures. The bleeding time test and its variations are covered under platelet
function tests and coagulation disorders.
3. "Wintrobe's Clinical Hematology" by John P. Greer, Daniel A. Arber, Bertil Glader, Alan
F. List, Robert T. Means Jr, Frixos Paraskevas, George M. Rodgers - This is a
comprehensive guide to hematology that includes extensive information on bleeding and
clotting disorders, including the methods and interpretation of bleeding time tests.
41
42
Experiment No 5.2
Title Clotting Time Estimation
Program B.Sc. MLT
Semester 3
Learning Outcomes
Upon completing this experiment, you should be able to:

1. Understand the concept and importance of clotting time in human health and pathology.
2. Appreciate the relevance of clotting time estimation in clinical diagnostics and patient
management.
3. Demonstrate the ability to draw a blood sample into a capillary tube safely and effectively.
4. Execute the capillary tube method to estimate clotting time accurately.
5. Understand the implications of various clotting times in terms of potential health conditions
or disorders.
6. Analyze and interpret the results of the capillary tube method.
7. Effectively communicate the results and their implications in a clear and concise manner.
8. Understand the limitations and potential sources of error in the capillary tube method and
propose ways to overcome these issues.
9. Appreciate the ethical considerations involved in blood sampling and handling.
10. Develop critical thinking skills to connect the knowledge gained from this experiment with
real-world clinical scenarios.
INTRODUCTION
The clotting estimation by capillary tube method is a diagnostic technique used to evaluate the
clotting time of blood. This is an essential measure in assessing a person's ability to form blood
clots, a critical process that prevents excessive bleeding. The test helps clinicians identify bleeding
disorders, such as hemophilia, von Willebrand disease, and other conditions that may affect blood
43
coagulation. This method involves drawing a small amount of blood into a capillary tube and
observing the time it takes for a clot to form.
In clinical practice, clotting time is an important aspect of a patient's health profile. Prolonged
clotting times may indicate a deficiency in clotting factors, while a shortened clotting time may be
indicative of an increased risk of clot formation, leading to conditions such as deep vein
thrombosis, pulmonary embolism, or stroke. Therefore, understanding and effectively estimating
clotting times are vital in both diagnosing and monitoring these conditions.
In this experiment, we will be using the capillary tube method to estimate clotting time. This is a
straightforward and inexpensive method to determine clotting time and can be performed in a
laboratory with basic equipment.
Definition
• Clotting time is defined as the time interval in between onset of bleeding and appearance
of semisolid mass i.e. Clot.
• Normal value of clotting time is 3-4 minutes.
• It is a screening test for coagulation disorders.
Methods
1. Capillary tube method

2. Lee and White method
AIM:
To demonstrate the clotting time by Capillary tube method
1. Capillary Tube Method
EQUIPMENT:
• Sterile lancet
• capillary tube
• Stopwatch
• Cotton, sprit
• Other PPE’s
44
Procedure
1. Clean the tip of a finger with spirit.

2. Puncture it upto 3 mm deep with a disposable needle.
3. Start the stop-watch.
4. Fill two capillary tubes with free flowing blood from the puncture after wiping the first
drop of blood.
5. Keep these tubes at body temperature.
6. After 2 minutes start breaking the capillary tube at 1cm distance to see whether a thin fibrin
strand is formed between the two broken ends.
7. Stop the watch and calculate the time from average of the two capillary tubes.
Observation
……………………………………………………………………………………………………
……………………………………………………………………………………………………
…………………………………………………………………………………………….
Result
The clotting time of the subject is found to be ………………………. Minutes
Observation Table

No time & date
45
Clotting time refers to the time it takes for blood to form a clot after a vessel is injured. The
clinical significance of clotting time lies in its ability to diagnose and monitor bleeding disorders
and to evaluate the effectiveness of anticoagulant therapies.
Abnormal clotting time values can indicate a bleeding disorder, such as hemophilia, von
Willebrand disease, or liver disease. Prolonged clotting time can also be a side effect of certain
medications, such as anticoagulants, antiplatelet agents, and thrombolytic agents. Therefore,
clotting time tests can help diagnose bleeding disorders and monitor the effectiveness of
treatment for these conditions.
Additionally, clotting time tests are also used to evaluate the effectiveness of anticoagulant
therapies, such as heparin and warfarin. These medications work by prolonging clotting time to
prevent blood clots, and monitoring clotting time can ensure that the dose of medication is
appropriate and safe for the patient.
In summary, clotting time is a valuable diagnostic tool for identifying bleeding disorders and
monitoring anticoagulant therapies.

1. Question: What is clotting time, and why is it important?
46
Answer: Clotting time is the duration it takes for blood to change from a liquid to a gel-
like state forming a clot. It is important as it reflects the body's ability to prevent
excessive bleeding and can help identify blood disorders or conditions affecting blood
coagulation.
2. Question: What conditions might result in a prolonged clotting time?
Answer: Conditions that could result in prolonged clotting time include hemophilia, von
Willebrand disease, liver disease, and certain deficiencies in clotting factors. Certain
medications like anticoagulants can also prolong clotting time.
3. Question: What conditions might lead to a shortened clotting time?
Answer: Conditions that might shorten clotting time include thrombophilia (a propensity
to form clots) and certain inflammatory conditions. Some medications may also decrease
clotting time.
4. Question: What is the capillary tube method in clotting time estimation?
Answer: The capillary tube method is a procedure in which a small amount of blood is
drawn into a capillary tube. The time taken for a clot to form is then measured, providing
an estimation of clotting time.
5. Question: How should the capillary tube be handled during the experiment?
Answer: The capillary tube should be handled carefully to avoid breakage. It should not
be touched directly after the blood sample is drawn into it, as this can impact the
temperature and potentially affect clotting time.
6. Question: What are some potential sources of error in this experiment?
Answer: Potential sources of error could include improper handling of the sample,
temperature fluctuations, inaccuracies in timing, or contamination of the blood sample.
7. Question: How can you ensure the accuracy of your results?
Answer: Accuracy can be ensured by careful handling and timing, maintaining a
consistent temperature, using a clean and uncontaminated sample, and repeating the test
to confirm results.
8. Question: How can the results of this experiment impact patient management?
Answer: The results of this experiment can help guide patient management by providing
information on the patient's clotting status. This can be particularly important for patients
with bleeding disorders, those undergoing surgery, or patients on anticoagulant therapy.
9. Question: What ethical considerations should be taken into account during this
experiment? Answer: Ethical considerations include obtaining informed consent for the
blood draw, ensuring patient confidentiality, and properly disposing of used materials to
prevent risk of infection.
10. Question: Can this method be used to determine the specific clotting factor deficiencies?
47
Answer: No, the capillary tube method can provide an overall estimate of clotting time
but does not differentiate between the specific clotting factors. More specialized tests
would be needed to identify specific clotting factor deficiencies.
Reference materials :
1. "Hematology: Clinical Principles and Applications" by Bernadette F. Rodak,
George A. Fritsma, and Elaine Keohane. This comprehensive book provides a detailed
explanation on various aspects of hematology including clotting time estimation and the
implications of various results.
2. "Clinical Laboratory Hematology" by Shirlyn B. McKenzie. This textbook provides
an understanding of hematologic laboratory techniques, including the capillary tube
method for clotting time estimation, along with case studies that can apply these
techniques in real-world scenarios.
3. "Henry's Clinical Diagnosis and Management by Laboratory Methods" by Richard
A. McPherson and Matthew R. Pincus. Known as the definitive book in laboratory
medicine since 1908, this reference book explains the test selection and interpretation of
results that every clinician should know, including the procedure for clotting time
estimation.
48
6. TOTAL WHITE BLOOD CELL COUNT IN BLOOD
Experiment No 6
Title Total white blood cell count
Program B.Sc. MLT
Semester 3
Learning Outcomes

1. Understand the role and importance of white blood cells in the human body and their
relevance in medical diagnostics.
2. Demonstrate knowledge of the Neubauer chamber method and its application in
hematology.
3. Prepare a diluted blood sample using the appropriate diluting fluid for WBC counting.
4. Use a microscope effectively to count white blood cells using the Neubauer chamber.
5. Calculate the total white blood cell count accurately from the counted sample, applying the
appropriate multiplication factor.
6. Interpret the results and understand the clinical significance of high or low WBC count.
7. Appreciate the need for precision and accuracy in laboratory experiments, especially in
medical diagnostics.
8. Understand the safety measures and precautions to be followed while handling human
blood samples and waste disposal.
9. Develop skills in reporting and documenting experimental results in a scientific and
professional manner.
10. Work collaboratively in a laboratory setting, demonstrating teamwork and effective
communication.
Introduction
White blood cells (WBCs) are an integral part of the body's immune system, responsible for
fighting infections and providing protection against diseases. Their number is usually a strong
49
indicator of the body's overall health condition, with abnormal counts suggesting the presence of
an infection, inflammation, or even blood disorders such as leukemia.
The total white blood cell count is a common laboratory test performed in medical diagnostics to
evaluate the body's immune response. The Neubauer chamber, a specialized type of counting
chamber used in haematology, is an instrument frequently employed for this purpose. It is
designed to facilitate the counting of cells or particles suspended in a liquid.
The Neubauer chamber method, known for its accuracy and precision, involves staining the
WBCs, placing a diluted sample into the chamber, and then counting the cells under a
microscope. The depth of the chamber is known, so you can calculate the total number of cells in
the original sample.
This experiment introduces students to the protocol of using the Neubauer chamber for counting
WBCs and understanding the clinical implications of the results.
AIM:
To estimate the total number of white blood cells in the given blood samples by visual haemocytometer
METHODS
• There are two methods:
1. Visual haemacytometer method
2. Electronic method
Visual Haemacytometer Method

Principle.
This is counting of WBCs in a calibrated chamber by diluting of blood to 1:20 dilution with diluent which
causes lysis of RBCs and stains WBCs.
Diluting fluid
Turk’s fluid is used which has the following
composition:
i. Glacial acetic acid : 3.0 ml
ii. 1% Aqueous gentian violet : 2.0 ml
iii. Distilled water : 195 ml
Materials required
1. Neubar counting chamber
2. Coverslip
3. RBC diluting fluid
4. Sterile lancet
5. Cotton, Sprit
6. Microscope
7. Other PPE’s
PROCEDURE
1. Suck anticoagulated blood or blood from finger prick up to mark 0.5 in WBC pipette
2. Wipe tip and outside of the pipette.
3. Draw diluting fluid up to mark 11 in the WBC pipette to get dilution of 1:20.
50
4. Mix well by rotating the pipette for 2-3 minutes.
5. Charge the Neubauer’s chamber (haemacytometer)after discarding 1-2 drops of the mixture
from the WBC pipette.
6. Allow the cells to settle down for 2 minutes.
7. Count the WBCs under low power (10x) in 4 large corner squares
8. Count the cells lying on left and lower lines while ignoring those on its right and upper lines.
NORMAL RANGE FOR WBC COUNT

• Adults : 4,000–11,000/μl
• Infants at birth : 10,000–26,000/μl
• Children under 1 year : 6,000–18,000/μl
CAUSES OF ABNORMAL LEUCOCYTE COUNT
• Increased leucocyte count: Leucocytosis
• Decreased leucocyte count: Leucopenia
Observation:
……………………………………………………………………………………………………………………………………………………………
……………………………………………………………………………………………………………………………………………………………
………..
Result:
……………………………………………………………………………………………………………………………………………………………
……………………………………………………………………………………………………………………………………………………………
……………………………………………………………………………………………………………………………………………………………
…………….
Observation Table

No time & date
51
4
Clinical significance
Infection: An elevated WBC count is often seen in response to infections, as the body tries to fight off
invading microorganisms.
Inflammation: WBC count may be elevated in conditions that cause inflammation, such as rheumatoid
arthritis, lupus, and other autoimmune disorders.
Leukemia: Abnormal WBC count can be seen in leukemia, which is a type of blood cancer that affects
white blood cells.
Bone marrow disorders: Abnormal WBC count can also be seen in bone marrow disorders, such as
myelodysplastic syndromes and aplastic anemia.
Allergic reactions: An increase in eosinophils (a type of WBC) is often seen in response to allergic
reactions, such as asthma and eczema.
Medications: Certain medications, such as corticosteroids, can affect the WBC count.
In summary, the total white blood cell count is an important clinical indicator that can help diagnose and
monitor a wide range of conditions, including infections, inflammatory disorders, cancers, and blood
disorders. However, it is important to interpret the WBC count in conjunction with other laboratory
tests and clinical findings to determine the underlying cause of any abnormalities.

1. What is the primary role of white blood cells in the human body?
Answer: The primary role of white blood cells (WBCs) is to fight infections and provide protection
against diseases. They are a crucial part of the body's immune system.
52
2. What is the Neubauer chamber and why is it used in this experiment?
Answer: The Neubauer chamber is a specialized type of counting chamber used in hematology. It
is used to facilitate the counting of cells or particles suspended in a liquid. In this experiment, it is
used for counting the number of white blood cells in a blood sample.
3. What could a high white blood cell count indicate?
Answer: A high white blood cell count could indicate an infection, inflammation, an immune
response, or blood disorders such as leukemia.
4. What is the diluting fluid used in the experiment, and why is it necessary?
Answer: The diluting fluid used in this experiment is typically a solution such as Turk's solution.
It lyses the red blood cells and makes the white blood cells more visible under a microscope. The
dilution is necessary to obtain a countable number of cells in the sample.
5. How do you calculate the total white blood cell count from the counted sample?
Answer: The total white blood cell count is calculated by multiplying the number of cells counted
in the sample by the dilution factor and the depth factor (usually 10^4), and then dividing by the
number of squares counted.
6. What safety measures should be taken while handling human blood samples?
Answer: Safety measures include using personal protective equipment such as gloves and lab coats,
handling samples with care to prevent spillage, and disposing of all materials that come into contact
with the sample in a biohazard container.
7. How does a low white blood cell count affect the body's immunity?
Answer: A low white blood cell count, or leukopenia, can compromise the body's ability to fight
off infections, making an individual more susceptible to diseases.
8. What is the difference between manual and automated WBC counting?
Answer: Manual counting using a Neubauer chamber requires microscopic examination and
manual calculation of the WBC count, which can be time-consuming and prone to human error.
Automated counting, on the other hand, uses machines that can provide faster and more precise
counts.
9. Why is accuracy important in WBC counting?
Answer: Accuracy in WBC counting is essential as it directly affects the diagnosis and treatment
plan. Inaccurate counts could lead to misdiagnosis, incorrect treatment, or a failure to detect a
serious condition.
10. What could cause an error in the WBC count using the Neubauer chamber method?
Answer: Potential sources of error could include improper dilution of the sample, incorrect
counting or calculation, errors in placing the sample in the chamber, and variations in the quality
of the microscope or staining process.
Reference material:
1. "Rodak's Hematology: Clinical Principles and Applications" by Elaine Keohane, Larry Smith,
Jeanine Walenga. This book provides comprehensive information about the principles and
procedures in hematology and includes in-depth details about white blood cell counting techniques.
2. "Clinical Laboratory Hematology" by Shirlyn B. McKenzie, Lynne Williams. This book offers
a complete guide to hematology in laboratory practice and explains both manual and automated
methods of blood cell counting.
53
3. "Atlas of Hematology" by Renu Saxena. This atlas covers various aspects of hematology,
including practical procedures for counting blood cells and preparing and interpreting peripheral
blood films. It provides a visual guide that can be very useful for understanding the Neubauer
chamber method.
54
7. TOTAL RED BLOOD CELL COUNT IN BLOOD
Experiment No 7
Title Total red blood cell count
Program B.Sc. MLT
Semester 3
Learning Outcomes
By the end of the practical session on total red blood cell count using the Neubauer chamber
method, learners should be able to:
1. Understand the principle behind the total red blood cell count procedure and the role of the
Neubauer chamber in this procedure.
2. Familiarize themselves with the Neubauer chamber device, its parts, and the role of each
part.
3. Demonstrate proficiency in preparing a diluted blood sample for total red blood cell count.
4. Accurately load the diluted blood sample into the Neubauer chamber without damaging
the chamber or disrupting the sample.
5. Use a microscope to visualize and count red blood cells present in a defined area of the
Neubauer chamber.
6. Calculate the total red blood cell count per cubic millimeter of blood based on the count
and the dilution factor.
7. Interpret the results and understand their clinical implications in diagnosing various blood
disorders.
8. Demonstrate the ability to follow lab safety protocols and maintain hygiene while handling
blood samples.
9. Understand the sources of error that could influence the total red blood cell count and ways
to minimize these errors.
10. Develop the skills to document and report the results clearly and accurately.
55
Introduction
The Total Red Blood Cell Count is a crucial clinical analysis method used to detect variations in
the number of red blood cells (RBCs) in a patient's body. This method has significant
implications in diagnosing and monitoring a wide variety of diseases, such as anemia,
polycythemia, and other hematologic disorders. The Neubauer Chamber Method, also known as
the Hemocytometer method, is a commonly used laboratory technique for determining the total
count of red blood cells.
The Neubauer chamber is a thick glass microscope slide with a rectangular indentation that creates
a chamber. This chamber is engraved with a laser-etched grid of perpendicular lines. The device
is designed to facilitate the counting of cells and the calculation of the concentration of cells in the
fluid under study. In the context of red blood cell count, the sample of blood is diluted and then
applied to the chamber to allow for counting under a microscope.
Aim: To estimate the total red blood cells in the given blood sample by visual haemocytometer
method.
Methods For tRBC Count

1. Visual haemacytometer method
2. Electronic method
1. Visual Haemacytometer Method
Principle.
• This is counting of RBCs in a calibrated chamber by dilution of blood to 1 in 200
dilution with a diluent which is isotonic to blood.
• The diluent used prevents clotting, clumping and rouleaux formation and does not
destroy WBCs.
Diluting fluids
Two types of diluting fluids are used for RBC counting:
• Hayem’s fluid and
• Dacie’s fluid.
Composition of Hayem’s fluid
• Mercuric chloride : 0.25 g
• Sodium chloride : 0.5 g
• Sodium sulphate : 2.5 g
• Distilled water : 100 ml
Composition of Dacie’s fluid
• 40% Formaldehyde : 5 ml
• 3% Trisodium citrate : 495 ml
56
Materials required
1. Neubar counting chamber
2. Coverslip
3. RBC diluting fluid
4. Sterile lancet
5. Cotton, Sprit
6. Microscope
7. Other PPE’s
Procedure
1. Draw anticoagulated blood or blood from finger prick up to mark 0.5 in RBC pipette
2. Wipe tip and outside of the pipette.
3. Draw diluting fluid up to mark 101 in the RBC pipette.
4. Mix well by rotating the pipette for 2-3 minutes.
5. Charge the Neubauer’s chamber after discarding 1-2 drops of mixture from the RBC
pipette.
6. Allow the cells to settle down for 2 minutes.
7. Count RBCs under high power 40X in 80 tiny squares (5 × 16 tiny squares) in the centre
of the chamber.
Calculations
• Volume of area in which RBCs counted in 5 squares
NORMAL RANGE FOR RBC COUNT

• Males : 5.0–6.0 million/μl (5.5 + 0.5 million/μl)
• Females : 4.5–5.5 million/μl (5 + 0.5 million/μl)
• Children : 4.0–5.0 million/μl (4.5 + 0.5 million/μl)
CAUSE OF DECREASED RBC COUNT
i. Anaemia
CAUSE OF INCREASED RBC COUNT
i. Polycythemia
Observation:
……………………………………………………………………………………………………
……………………………………………….
Result:
57
……………………………………………………………………………………………………
………………………………………………
Observation Table

No time & date
Anemia: A low RBC count can indicate anemia, which is a condition characterized by a
deficiency of red blood cells or hemoglobin in the blood. Anemia can cause fatigue, weakness,
shortness of breath, and other symptoms.
Polycythemia: A high RBC count can indicate polycythemia, which is a condition characterized
by an excessive number of red blood cells in the blood. Polycythemia can lead to increased blood
viscosity, which can cause blood clots, strokes, and other complications.
Chronic Kidney Disease: Chronic kidney disease can lead to decreased production of
erythropoietin hormone by the kidneys which stimulates the production of RBCs in bone
marrow, leading to a decreased RBC count.
Dehydration: Dehydration can cause an increase in RBC count as the plasma volume decreases,
causing a relative increase in the concentration of RBCs.
Other Conditions: Certain conditions like thalassemia, sickle cell disease, and some cancers can
also affect RBC count.
58
Sample Viva questions
Question 1: What is the main purpose of performing a total red blood cell count?
Answer: The main purpose of performing a total red blood cell count is to detect variations in
the number of red blood cells (RBCs) in a patient's body, helping to diagnose and monitor a wide
range of diseases such as anemia, polycythemia, and other hematologic disorders.
Question 2: What is the role of the Neubauer chamber in the total red blood cell count
procedure? Answer: The Neubauer chamber is a specialized counting chamber device used for
the microscopic counting of cells. In the context of red blood cell count, the sample of blood is
diluted and then applied to the chamber to allow for counting under a microscope.
Question 3: How does one load a sample into the Neubauer chamber?
Answer: To load a sample into the Neubauer chamber, first, clean the chamber and cover slip.
Then, mix the diluted blood sample well and draw it into a pipette. Carefully place the pipette tip
against the edge of the cover slip and gently expel the sample. The sample will be drawn into the
chamber by capillary action.
59
Question 4: How do you calculate the total red blood cell count from the count obtained from
the Neubauer chamber?
Answer: The total red blood cell count can be calculated from the count obtained from the
Neubauer chamber by multiplying the count by the dilution factor and the depth factor, and
dividing by the area of the grid square counted.
Question 5: What is the typical dilution used in the total red blood cell count using the Neubauer
chamber method?
Answer: For the total red blood cell count using the Neubauer chamber method, a typical
dilution used is 1:200 (1 part blood to 199 parts diluting fluid).
Question 6: Name a few conditions where an abnormal red blood cell count might be observed.
Answer: Conditions that might cause an abnormal red blood cell count include anemia (low
RBC count), polycythemia (high RBC count), dehydration, and conditions affecting bone
marrow such as leukemia.
Question 7: What are some safety precautions to be taken while handling blood samples?
Answer: Some safety precautions include wearing personal protective equipment (like gloves
and lab coats), handling sharps carefully, disposing of waste properly, avoiding mouth pipetting,
and washing hands before and after handling samples.
Question 8: Why is it important to thoroughly mix the blood sample and diluent?
Answer: It's important to thoroughly mix the blood sample and diluent to ensure a uniform
distribution of cells in the sample. This improves the accuracy of the cell count.
Question 9: What factors could potentially influence the total red blood cell count results?
Answer: Factors that could influence the total red blood cell count results include the precision
of the dilution, thorough mixing of the sample, accurate counting, and condition of the Neubauer
chamber and microscope.
Question 10: What should you do if the cells in your sample appear to clump together when
viewed under the microscope?
Answer: If the cells in your sample appear to clump together, this could indicate that the sample
was not mixed well enough, or that there are physical or chemical factors causing cell
agglutination. It is necessary to prepare a fresh sample, ensuring proper mixing or adjusting the
diluent or sample treatment to prevent clumping.
Reference Materials:
"Clinical Laboratory Hematology" (3rd Edition) by Shirlyn B. McKenzie - This

comprehensive book provides detailed explanations of hematology laboratory techniques,
including the Neubauer chamber method for total red blood cell count.
60
"Dacie and Lewis Practical Haematology" (12th Edition) by Barbara J. Bain, Imelda
Bates, and Mike A. Laffan - This is a widely respected text in the field of haematology. It
includes details of various techniques involved in hematological investigations, including red
blood cell counting using a hemocytometer.
"Hematology: Clinical Principles and Applications" (5th Edition) by Bernadette F. Rodak,

George A. Fritsma, and Elaine Keohane - This textbook is known for its reader-friendly style
and extensive coverage of hematological pathology, laboratory techniques, and clinical
applications. It should contain useful information on the total red blood cell count procedure.
61
8. ESTIMATION OF PLATELETS COUNT IN BLOOD
Experiment No 8
Title Estimation of Platelets count in Blood
Program B.Sc. MLT
Semester 3
Learning Outcomes:
Upon completion of this lab experiment, students should be able to:
1. Understand the function of platelets in the body and the importance of maintaining a
healthy platelet count.
2. Demonstrate knowledge of the principle behind the manual method of platelet counting
using the improved Neubauer hemocytometer.
3. Perform the correct blood dilution technique for platelet counting.
4. Accurately count platelets in the sample using a microscope.
5. Calculate the platelet count per microliter of blood using the formula derived from the
manual counting procedure.
6. Interpret the results of the platelet count and correlate with clinical conditions like
thrombocytopenia and thrombocytosis.
7. Understand the indications and contraindications for the manual method of platelet
counting.
8. Develop practical skills in hematological laboratory procedures, improving their

proficiency in laboratory diagnostics.
Introduction:
Platelets, also known as thrombocytes, are tiny blood cells that help the body form clots to stop
62
bleeding. If one of the body's blood vessels gets damaged, it sends out signals that are picked up
by platelets. The platelets then rush to the damaged site to begin the healing process by sticking
to the lining of the injured blood vessel – a process called adhesion. This platelet plug, or clot,
blocks the blood vessel's hole to prevent blood loss.
Estimating the platelet count is an essential test in clinical practice. It helps in diagnosing various
blood disorders, evaluating the risk of excessive bleeding, and monitoring the health condition of
patients undergoing treatments that might affect platelet count.
Manual counting methods like the improved Neubauer hemocytometer method have been used
for years to estimate the platelet count. Despite advances in automated methods, the manual
method still plays a crucial role in resource-constrained settings due to its simplicity,
affordability, and ease of execution.
Aim: The main objective of this experiment is to manually estimate the number of platelets in a
blood sample to evaluate a patient's coagulation status and diagnose hematological disorders.
Principle: The principle behind this experiment is the use of light microscopy to visually count
the number of platelets in a blood smear. Platelets are small cell fragments in the blood that are
involved in clotting. They appear as small, purple dots when stained with Wright's stain and
viewed under the microscope.
Method: The method used in this experiment is the improved Neubauer hemocytometer method,
which involves counting platelets in a diluted blood sample under a microscope.
Materials Required:
1. Blood sample (anticoagulated with EDTA)
2. Light microscope
3. Microscope slides and coverslips
4. Improved Neubauer Hemocytometer
5. Pipettes
6. Platelet diluting fluid (Ammonium oxalate solution)
7. Wright's stain
8. Microcentrifuge tubes
9. Distilled water
Procedure:
1. Prepare a 1:100 dilution of the blood sample with platelet diluting fluid. This is typically
done by adding 0.02 ml of blood to 1.98 ml of diluting fluid in a microcentrifuge tube.
63
2. Mix the diluted sample thoroughly.
3. Charge the hemocytometer with the diluted blood sample and let it stand for about 20
minutes to allow the platelets to settle.
4. View the hemocytometer under the microscope, using the 40x objective lens.
5. Count the platelets in the 25 small squares located at the center of the counting grid.
6. Repeat the counting for at least three grids to achieve a more accurate average count.
Results Interpretation:
The platelet count per microliter of blood can be calculated by multiplying the average count
from the grids by the dilution factor (100) and the factor for the volume represented by the
counted squares (10^4 for the 25 squares), i.e.,
Platelet count/μl = Average count * Dilution factor * Volume factor
A normal platelet count ranges from 150,000 to 450,000 platelets per microliter of blood. A
count below this range may indicate thrombocytopenia, and a count above this range may
suggest thrombocytosis.
Indications:
The platelet count test is indicated in cases of:
1. Suspected bleeding disorders, especially excessive bleeding or clotting
2. Diagnosis and monitoring of diseases that can affect platelet count, such as leukemia or
anemia
3. Monitoring the effectiveness of treatment for diseases that affect platelet counts
4. Assessment of bone marrow function
Contraindications:
This manual method is contraindicated if the patient has severe thrombocytopenia or if the
platelets are clumped together in the blood sample. The procedure may not be accurate for
extremely high platelet counts. Automated methods are recommended in these cases to provide a
more precise count.
Observation:
……………………………………………………………………………………………………
……………………………………………….
Result:
……………………………………………………………………………………………………
………………………………………………
Observation Table
64
No time & date
Clinical Significance of Estimating Platelet Count by Manual Method:

The manual estimation of platelet count is an essential diagnostic tool in hematology with
significant clinical applications:
1. Diagnosis of Hematological Disorders: Abnormal platelet counts often indicate
hematological disorders. Thrombocytopenia (low platelet count) can be seen in
conditions like leukemia, aplastic anemia, viral infections (HIV, hepatitis C), and certain
medications. Thrombocytosis (high platelet count) can occur in conditions such as
cancer, inflammation, and after spleen removal.
2. Risk Assessment for Bleeding or Clotting Disorders: Platelet count provides vital
information about a patient's risk for bleeding or clotting disorders. Patients with low
platelet counts are at increased risk of bruising and bleeding, while patients with high
platelet counts may be at a higher risk of developing blood clots.
3. Monitoring the Effectiveness of Treatments: Certain treatments, like chemotherapy or
radiation therapy, can significantly affect the bone marrow's ability to produce platelets.
Regular monitoring of platelet count helps in assessing the impact of these therapies on
the bone marrow function.
4. Pre-Surgery Evaluation: Determining platelet count is a standard part of the preoperative
assessment to evaluate the risk of excessive bleeding during and after surgery.
5. Diagnosis of Disseminated Intravascular Coagulation (DIC): DIC is a condition
characterized by widespread activation of the clotting cascade, leading to the formation
of small blood clots throughout the body. This leads to an initial increase in platelet count
followed by a rapid drop due to excessive consumption of platelets.
6. Assessing Severity of Dengue Fever: In dengue fever, the platelet count tends to drop
significantly and is an essential parameter for assessing disease severity and progression.
7. Monitoring Immune Thrombocytopenic Purpura (ITP): ITP is an autoimmune disease
where the body's immune system mistakenly targets platelets, leading to a decreased
65
platelet count. Regular monitoring of the platelet count is essential in managing this
condition
Sample viva questions:

Question 1: What is the primary function of platelets in the body?
Answer 1: Platelets, or thrombocytes, are responsible for forming clots to stop bleeding when a
blood vessel is damaged.
Question 2: Explain the principle behind the manual estimation of platelet count.
Answer 2: The principle behind the manual method of platelet counting involves counting the
platelets in a diluted blood sample under a microscope using a hemocytometer.
Question 3: What materials are required to perform a manual platelet count?
Answer 3: The materials required for a manual platelet count include an anticoagulated blood
sample, a light microscope, microscope slides and coverslips, an improved Neubauer
hemocytometer, pipettes, platelet diluting fluid (Ammonium oxalate solution), Wright's stain,
microcentrifuge tubes, and distilled water.
Question 4: What is the standard dilution used in the manual method for platelet counting?
Answer 4: A 1:100 dilution is typically used in the manual method for platelet counting. This
involves adding 0.02 ml of blood to 1.98 ml of diluting fluid.
Question 5: How is the platelet count calculated from the observations made on the
hemocytometer?
66
Answer 5: The platelet count per microliter of blood is calculated by multiplying the average
count from the grids by the dilution factor (100) and the factor for the volume represented by the
counted squares (10^4 for the 25 squares).
Question 6: What is the typical range for a normal platelet count in a healthy individual?
Answer 6: A normal platelet count ranges from 150,000 to 450,000 platelets per microliter of
blood.
Question 7: What conditions might cause thrombocytopenia?
Answer 7: Conditions that might cause thrombocytopenia (low platelet count) include leukemia,
aplastic anemia, viral infections such as HIV and hepatitis C, and certain medications.
Question 8: What conditions might cause thrombocytosis?
Answer 8: Conditions that might cause thrombocytosis (high platelet count) include cancer,
inflammation, and after spleen removal.
Question 9: In which clinical situations would a platelet count be indicated?
Answer 9: A platelet count test is indicated in cases of suspected bleeding or clotting disorders,
diseases that affect platelet count (such as leukemia or anemia), to monitor the effectiveness of
treatments affecting platelet counts, and to assess bone marrow function.
Question 10: What are the limitations of manual platelet counting methods?
Answer 10: Manual methods for platelet counting can be inaccurate if the patient has severe
thrombocytopenia or if the platelets are clumped together in the blood sample. The procedure
may also lack precision for extremely high platelet counts. In these cases, automated methods
would provide a more accurate count.
Reference Materials
"Rodak's Hematology: Clinical Principles and Applications" by Elaine M. Keohane, Larry
Smith, and Jeanine Walenga. This book provides a comprehensive overview of hematology
basics, including various manual techniques such as platelet counting.
"Clinical Laboratory Hematology" by Shirlyn B. McKenzie and Lynne Williams. This book
covers all aspects of laboratory hematology, including methodologies and clinical applications of
procedures performed in the hematology laboratory.
"Wintrobe's Clinical Hematology" by John P. Greer, Daniel A. Arber, and Bertil Glader. This
textbook provides an in-depth understanding of the pathophysiology, clinical manifestations, and
treatment protocols for diseases that affect blood cells, including methods for counting these
cells.
67
9. HEMOGLOBIN (HB) ESTIMATION SHALI 'S METHOD
Experiment No 9
Title Hemoglobin (Hb) estimation Shali 's method
Program B.Sc. MLT
Semester 3
Learning Outcomes
1. Understand the biological role of haemoglobin and its significance in human health.
2. Understand the principle behind Sahli's method, and why it is still used in certain
situations despite more modern alternatives.
3. Demonstrate the ability to prepare a blood sample for haemoglobin estimation.
4. Conduct the Sahli's method experiment accurately, including the conversion of
haemoglobin to acid haematin and colour comparison.
5. Develop skills in handling and using laboratory equipment, such as the Sahli's
haemoglobinometer.
6. Interpret the results of the Sahli's method experiment and understand their implications in
a clinical context.
7. Develop the ability to identify potential errors in the experimental procedure and suggest
appropriate solutions.
8. Understand the limitations of Sahli's method and compare it with other methods of
haemoglobin estimation.
9. Understand and follow safety protocols during the experiment, including safe handling of
blood samples and disposal of biohazardous waste.
10. Develop scientific reporting skills by accurately recording the procedure, observations,
and results of the experiment in a laboratory report.
Introduction
Haemoglobin is an iron-containing protein complex, found in red blood cells, that transports
oxygen from the lungs to the rest of the body. Estimating the haemoglobin concentration in a
blood sample is a critical diagnostic tool to detect several health conditions, most notably
anaemia.
68
Sahli's method, also known as Sahli's acid haematin method, is a manual and direct method of
measuring haemoglobin concentration. It has been in use for more than a century, thanks to its
simplicity, cost-effectiveness, and minimal equipment requirements. Despite its age, Sahli's
method remains widely used in laboratories, particularly in low-resource settings.
The method involves converting haemoglobin to acid haematin, which is a brown-coloured
substance. The intensity of this colour is directly proportional to the amount of haemoglobin in
the sample, and the measurement is done visually by matching the colour with a comparator.
Although it lacks the precision of some newer methods, Sahli's method is a fundamental
technique that every biology or medical student should learn, understand, and practice.
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
……………………………………………………………………………………………………
……………………………………………………………………………………………….
69
Observation Table

No time & date
Normal Values:
Raised Hemoglobin Content

• Polycythemia Vera
• Associated with Hypoxia
• Cyanotic Congenital Heart disease
70
• 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
Sample Viva questions:

Question 1: What is the biological role of haemoglobin in the human body?
Answer 1: Haemoglobin is a protein found in red blood cells that is responsible for carrying
oxygen from the lungs to the rest of the body. It also helps in transporting carbon dioxide from
the body's tissues back to the lungs.
Question 2: How does Sahli's method work to estimate haemoglobin concentration?
Answer 2: Sahli's method involves converting haemoglobin in a blood sample to acid haematin,
which is brown in color. The intensity of this color is directly proportional to the amount of
haemoglobin in the sample. The haemoglobin concentration is then estimated by visually
matching the color of the sample with a color comparator.
Question 3: What is the main advantage of Sahli's method?
Answer 3: Sahli's method is simple, cost-effective, and requires minimal equipment, making it a
useful technique in low-resource settings.
Question 4: What are the limitations of Sahli's method?
Answer 4: Sahli's method is less precise than some more modern techniques. The color
comparison is subjective and can be affected by ambient light and the observer's perception.
Also, it only provides an estimate of the total haemoglobin and doesn't provide information about
other blood parameters.
Question 5: How can errors be minimized in Sahli's method?
71
Answer 5: Errors can be minimized by ensuring careful sample preparation, correct measurement
of blood and acid, accurate color matching, and repeating the experiment if the results are
ambiguous. Furthermore, conducting the color comparison under consistent lighting conditions
can improve accuracy.
Question 6: Why is it important to dispose of blood samples and other biohazardous waste
safely after the experiment?
Answer 6: Safe disposal of biohazardous waste is crucial to prevent the spread of infectious
diseases and to maintain a clean and safe lab environment.
Question 7: How does anaemia affect the haemoglobin concentration in the blood?
Answer 7: Anaemia is a condition characterized by a lower-than-normal quantity of
haemoglobin in the blood. This can be due to a decreased number of red blood cells or a decrease
in the amount of haemoglobin in these cells.
Question 8: What other methods can be used to estimate haemoglobin concentration?
Answer 8: Other methods include the cyanmethemoglobin method, gasometric method,
HemoCue system, and automated haematology analyzers. These methods are generally more
accurate and precise than Sahli's method but may be more expensive and require more
sophisticated equipment.
Question 9: Why is the estimation of haemoglobin levels important in clinical diagnosis?
Answer 9: Estimation of haemoglobin levels is a critical diagnostic tool to detect several health
conditions, most notably anaemia. It can also help to monitor the health status of patients with
chronic diseases, assess nutritional status, and guide therapy.
Question 10: What safety precautions should be taken while performing the Sahli's method
experiment?
Answer 10: Safety precautions include wearing appropriate personal protective equipment (like
lab coat, gloves, and eye protection), careful handling of the blood sample to avoid spills or
splashes, using a biological safety cabinet if available, and proper disposal of the used sample
and other biohazardous waste.
1. "Dacie and Lewis Practical Haematology" by Barbara J. Bain, Imelda Bates, and Mike
A. Laffan. This is a comprehensive guide to laboratory haematology. It covers a range of
techniques used in the diagnosis and monitoring of diseases related to blood and bone
marrow.
2. "Clinical Laboratory Hematology" by Shirlyn B. McKenzie, Lynne Williams. This
textbook provides detailed information on the principles of clinical hematology,
including the measurement of haemoglobin levels.
3. "Tietz Textbook of Clinical Chemistry and Molecular Diagnostics" by Carl A.
Burtis, Edward R. Ashwood, and David E. Bruns. This is a broad reference book that
72
covers a range of topics related to clinical chemistry and molecular diagnostics, including
haemoglobin and its measurement techniques.
73
10. DIFFERENTIAL LEUKOCYTE COUNT (DLC)
Experiment No 10
Title Differential leukocyte count (DLC)
Program B.Sc. MLT
Semester 3
Learning Outcomes
Upon successful completion of the experiment, students should be able to:
1. Understand the principle and importance of the differential leukocyte count in medical
diagnostics and health monitoring.
2. Identify and differentiate between various types of white blood cells based on their
morphological characteristics.
3. Demonstrate proficiency in preparing a blood smear for microscopic analysis.
4. Use a Neubauer chamber effectively to conduct a differential leukocyte count.
5. Accurately count and calculate the percentages of each type of white blood cell present in
the blood sample.
6. Interpret the results of the differential leukocyte count and correlate them with potential
health conditions.
7. Demonstrate safe handling and disposal of biohazardous materials, including blood
samples and used lab equipment.
8. Understand the limitations and potential sources of error in the differential leukocyte count,
and ways to minimize them.
9. Develop proficiency in recording and reporting experimental data in a clear, accurate, and
comprehensive manner.
10. Critically analyze and discuss the implications of the differential leukocyte count in clinical
scenarios and case studies.
11. Work collaboratively in a lab setting, demonstrating effective teamwork, communication,
and problem-solving skills.
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
74
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
75
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
76
Morphology of White Blood cells
77
Observation:
……………………………………………………………………………………………………
……………………………………………………………………………………………………
……………………………………………………………………………………………………
…………………..
Result:
Observation Table
78
No time & date
1. Infection and Inflammation Identification: An increase in total leukocyte count
(leukocytosis), particularly neutrophils, often indicates bacterial infection or
inflammation. On the other hand, viral infections may lead to an increase in lymphocytes.
2. Allergic Responses and Parasitic Infections: An increase in the count of eosinophils
might signal allergic reactions or parasitic infections.
3. Immune Response Monitoring: An increase in monocytes can signify ongoing immune
response against pathogens or foreign substances. Chronic infections or diseases like
tuberculosis and syphilis may show a rise in monocyte count.
4. Disease Progression Tracking: The differential leukocyte count can help track the
progression of diseases such as HIV/AIDS, where a decrease in lymphocyte count over
time can indicate disease progression.
5. Leukemia Diagnosis: Abnormal increase in white blood cells or the presence of immature
or abnormal leukocytes could indicate blood disorders like leukemia.
6. Immune Disorders: Conditions such as lymphocytopenia (reduced lymphocyte count) can
suggest potential immune disorders or conditions like lupus, rheumatoid arthritis, or
corticosteroid use.
7. Therapeutic Monitoring: Monitoring changes in differential leukocyte count can also be
used to evaluate the effectiveness of certain treatments, such as chemotherapy, where an
expected decrease in WBCs should occur.
8. Stress Response: An increase in neutrophil count may indicate stress response in the body
due to conditions like surgery, burns, or trauma.
1. Question: What is a differential leukocyte count?
79
• Answer: A differential leukocyte count is a test that measures the percentage of
each type of white blood cell, or leukocyte, in a blood sample. It helps identify
various diseases and conditions.
2. Question: What are the five types of white blood cells counted in a differential leukocyte
count?
• Answer: The five types of white blood cells counted in a differential leukocyte
count are neutrophils, lymphocytes, monocytes, eosinophils, and basophils.
3. Question: What is the significance of a high neutrophil count in the differential leukocyte
count?
• Answer: A high neutrophil count, or neutrophilia, often indicates a bacterial
infection or inflammation. It may also be seen in conditions such as acute stress,
burns, or during certain medical treatments.
4. Question: Which white blood cell type is typically elevated during a viral infection?
• Answer: Lymphocytes are typically elevated during a viral infection, a condition
known as lymphocytosis.
5. Question: What is the purpose of a Neubauer chamber in this experiment?
• Answer: The Neubauer chamber is a specialized type of microscopic slide used
for counting cells in a defined volume. It's used in this experiment to perform a
differential leukocyte count.
6. Question: What might an increase in eosinophil count indicate?
• Answer: An increase in eosinophil count, or eosinophilia, might indicate an
allergic response or a parasitic infection.
7. Question: How can the differential leukocyte count help in diagnosing leukemia?
• Answer: In leukemia, there's an abnormal increase in white blood cells, and often
immature or abnormal leukocytes are present. The differential leukocyte count
can help identify these abnormalities, aiding in the diagnosis.
8. Question: Why is it important to handle biohazardous materials, like blood samples,
safely in this experiment?
• Answer: Safe handling of biohazardous materials is crucial to prevent the spread
of infectious diseases, protect the individual conducting the experiment, and
maintain a safe laboratory environment.
9. Question: In what medical scenario might you observe a decrease in the lymphocyte
count?
• Answer: A decrease in the lymphocyte count, or lymphocytopenia, may be
observed in conditions like HIV/AIDS, certain types of cancer, autoimmune
disorders, or as a side effect of steroid therapy.
80
10. Question: How can a differential leukocyte count be used to monitor the effectiveness of
treatments like chemotherapy?
• Answer: Chemotherapy often results in a decrease in white blood cells as a side
effect of treatment. Monitoring the differential leukocyte count can therefore be
used to assess the impact of the therapy on the patient's immune system.
81
Reference material
1. "Cell and Molecular Biology: Concepts and Experiments" by Gerald Karp: This book
gives a comprehensive introduction to cell biology, including detailed sections on the
blood and immune system. It's a valuable resource for understanding the biological
functions of different leukocytes.
2. "Clinical Laboratory Hematology" by Shirlyn B. McKenzie: This book provides a
complete overview of hematology in a clinical context. It's an excellent reference for
understanding the methods and significance of differential leukocyte counts in disease
diagnosis and monitoring.
3. "Hematology: Clinical Principles and Applications" by Bernadette F. Rodak, George A.
Fritsma, and Elaine Keohane: This comprehensive guide to hematology offers an in-
depth examination of the theory and applications of hematology, including the
differential leukocyte count procedure and interpretation of the results.
82

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UNIVERSITY INSTITUTE OF APPLIED HEALTH SCIENCES

DEPARTMENT OF MEDICAL LAB TECHNOLOGY

PROGRAM NAME: B.Sc. MLT / Sem-3 / Batch-2022

Subject Name: Hematology Practical

Subject Code: 22MLH-201

Faculty Name: Attuluri Vamsi Kumar (E13404)

Ph No: 7416660584 Mail ID: attuluri.e13404@cumail.in

Prepared by Verified by Approved by

Attuluri Vamsi Kumar

RULES AND REGULATIONS IN HEMATOLOGY LAB ................................. 3

Hand washing guidelines:

Every record must have the following:

Subject Name: Hematology Practical

To know the basic concepts of Hematology& routine clinical investigations of Hematology

CO2 Make use of various procedures for blood count

CO3 Explain and take part in the blood banking procedures

Experiment No Experiment Name

1. Preparation of EDTA Vials

2. Collection of Blood sample, Plasma separation

4. ABO Blood Grouping

5. Bleeding Time. Clotting Time,

6. Total White Blood Cell Count in Blood

7. Total Red Blood Cell Count in Blood

8. Estimation of Platelets count in Blood

9. Hemoglobin (Hb) estimation Shali 's method

10. Differential leukocyte count (DLC)

Explain and take part in the blood banking

Make use of various procedures for blood count

PO1: Laboratory knowledge

PO2: Problem analysis

PO4: Conduct investigations of complex

PO5: Modern tool usage

PO6: The laboratory and society

PO7: Environment and sustainability

PO8: Quality Control

PO9: Individual or teamwork

PO11: Project management and finance

PO12: Life-long learning

Experiment Experiment Mapped CO

Exp 1 Preparation of EDTA Vials CO1, CO2

Exp 2 Collection of Blood sample, Plasma seperation CO1, CO2

Exp 3 Estimation of ESR CO1,CO3

Exp 4 ABO Blood Grouping CO2

Exp 5 Bleeding Time. Clotting Time, CO2

Exp 6 Total White Blood Cell Count in Blood CO3

Exp 7 Total Red Blood Cell Count in Blood CO3

Exp 8 Estimation of Platelets count in Blood CO3

Exp 9 Hemoglobin (Hb) estimation Shali 's method CO3

Exp 10 Differential leukocyte count (DLC) CO3

Textbooks / Reference Books

1. An Introduction to medical laboratory technology,F.J.Baker et al., Butterworks and co. , London.

2. Blood Coagulation, Thomson J. CHur chill Livingstone.

3. Hematology for Medical TeCHnologists, Charles F.Sieverd.

Title Preparation of EDTA Vials

Master subject coordinator Mr. Attuluri Vamsi Kumar

Department / Institute Department of MLT/ UIAHS

Program B.Sc. MLT

Observation Observation Observation Result

Clinical Significance of the Preparation of EDTA Vials Experiment: