EXPERIMENT 1

Blood Collection: Venipuncture Procedure

PRINCIPLE: The method is based on obtaining a sample of blood from a vein for
diagnostic testing or medical purposes. This involves using a needle
to puncture a vein, typically in the arm, and collecting the blood into a
collection tube or container.

AIM: To collect Blood sample

Syringe, Needle, Tourniquet, Alcohol wipes, Gauze sponges, Gloves,

MATERIALS:
Adhesive bandages/ tapes.

Venipuncture Site: Median Cubitan & Cephalic Vein (Most Frequently)

Basilic vein and doral hand veins also acceptable

NB: - Apply Tourniquet 3-4 Inches above selected puncture site

- Do not place too tightly or leave on more than 2 minutes

- Needle should form a 15-30 degree angle with the surface of the arm
- Bevel facing up
The Breakdown of red blood cells,
Localized swelling or collection of
releasing their contents into
blood outside blood vessels, usually
surrounding fluid.
caused by injury or trauma

To Prevent Hematoma: To prevent Hemolysis:

1. Puncture only uppermost wall of vein 1. Mix tubes with anticoagulant additves
2. Remove Tourniquet before needle 2. Avoid draing blood from a hematoma
3. Use major superficial veins 3. Avoid drawing plunger to forcefully, avoid
4. Make sure needle penetrates uppermost frothing of sample
layer of vein 4. Make sure venipuncture site is dry
5. Apply pressure to the Venipuncture site 5. Avoid a probing traumatic venipuncture
Needle
X Vein
L

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EXPERIMENT 2

To Dertermine the Number of RBC

PRINCIPLE: The Mehod is based on counting the Number of RBC’s in a known

small volume of accurately diluted Blood.
Blood is diluted exactly 1:200 with Hayem’s solution in a red cell pi-
pette and the RBC’s in a small volume of the diluted blood are counted
in a counting Chamber

AIM: To determine number of Red Blood Cells per cubic millimeter of Blood.

MATERIALS: Sterile disposable lancet, RBC Pipette, Counting Chamber,

Hayem’s Diluting Fluid, Microscope

Composition: 1. Sodium Chloride (NaCl) 0.5gm - Maintains Isotonicity

2. Sodium Sulphate (Na2SO4) 2.5gm - Prevents Rouleaux formation
3. Mecury Chloride (HgCl2) 0.35gm - acts as a Preservative
4. Distilled water 100ml - Used for Dilution
NB: Soln is Isotonic with Blood. It prevents Rouleaux formation

Dilution Factor: 1:200

Drops expelled: 5-6 Drops R1 R2

Examined using Low Power at 10X,

Magnifications:
Counted using High power at (40X) objective
R5
No. of Squares: 5 each containing 16 smaller Squares (Total- 80) R4 R3

Distribution Error: 20 Cells

Presence of Oestrogen;
Normal RBC Count: Males: 4.5 - 6 million/ cubic mm of blood A depressant to bone marrow
Females : 4.0 - 5.5 million/ cubic mm of blood

Calculation: 1 Cubic mm of Blood Contains = X x 10,000 Cells

where X = (R1 + R2 + R3 + R4 + R5)

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 Variations
Physiological (Natural variations in a biological process, non-indicative of disease)
A. Polycythemia: Increase in RBC Count

1. Age: higer in newborn babies, reduces as we grow older

2. Sex: It is Higher in males and females of non-reproductive age
3. Hypoxia: decreased partial pressure of oxygen as a result of:
i. Intense Physical activity
ii. High altitude: 10,000 ft above sea level
stimulates secretion of Erythropoietin which stimulates production of more RBC in the Bone
marrow.

4. Emotional Conditions: Increased sympathetic activity such as anxiety increases RBC

5. Increase in Temperature: Higher temperatures typically increase all body activities thus,
RBC is increased.t

B. Anemia: Decrease in RBC Count

1. High Barometric Pressure

2. Durings Sleep
3. Pregnancy

Pathological (Abnormal variations in a biological process, indicative of a disease or

dysfunction.
A. Polycythemia: Persistent Increase in RBC Count

1. Polycythemia Vera: Occurs in myeloproliferative disorders like malignancy of bone marow.

2. Secondary Polycythemia: Secondary to some pathological disease such as: Respiratory

disorders (Such as Emphysema), congenital heart disease, repeated mild hemorrhages.

B. Anemia: Blood disorder characterized by the reduction in RBC Count.

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EXPERIMENT 3

To Determine the Number of WBC

TLC: Total Leucocyte Count

PRINCIPLE: The Mehod is based on counting the Number of WBC’s in a known

small volume of accurately diluted Blood.
Blood is diluted exactly 1:20 with Turk’s solution in a White cell pipette
and the WBC’s in a small volume of the diluted blood are counted in a
counting Chamber. It should be done under low power

AIM: To determine number of White Blood Cells per millimeter of Blood.

MATERIALS: Sterile disposable lancet, WBC Pipette, Counting Chamber,

Turk’s Solution, Microscope

Composition: 1. 1% Glacial Acetic (Acid) - Destroys the Red cells, makes WBC
more prominent.
2. 2ml of 1% Genitian Violet (Dye)- Stains the white cell nuclei

Dilution Factor: 1:20

Drops expelled: 2-3 Drops W1 W2

Focused and counted under Low Power at 10X

Magnifications:

No. of Squares: 4 each containing 16 smaller Squares W4 W3

Distribution Error: 10 Cells

Nigerian TLC Count: 4,000 - 11,000 cells/ cubic mm of blood

Calculation: 1 Cubic mm of Blood Contains = X x 50 Cells

where X = (W1 + W2 + W3 + W4)

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 Variations
Physiological
Increase in WBC Count

1. Age: higer in infants and children than adults

2. Sex: It is slightly Higher in males than females
3. Pregnancy
4. During Exercise
3. Diurnal Variation: Highest in the afternoon
4. During Menstruation
5. During parturition

Vice versa for decrease

Pathological
A. Increase in WBC

1. Leucocytosis: Increase in TLC that occursin conditions such as infections, allergy,

common cold e.t.c.
2. Leukamia (aka Blood Cancer) : Diseased condition characterized by increase in WBC up to
1 million cells/ cu mm of blood

B. Decrease in WBC

1. Leukopenia: Decrease in TLC, occurs in Anaphylactic shick, liver cirrhosis, Spleen disor-
ders, viral infections, pernicious anemia, typjoid e.t.c
2.Bone marrow disorders
3.Autoimmune Diseases
4.

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 Key Differences between RBC & WBC Count

RBC WBC
Dilution Rate 1:200 1:20

Diluting Fluid Hayem’s Solution Turk’s Solution

Distribution Error 20 Cells 10 cells

Calculation X * 10,000 Cells X * 50 Cells

Pipette 0.5 - 101 0.5 - 11

Bigger Bulb White bead in bulb

Squares 5 containing 16 4 containing 16

Normal Range Male: 4.5 - 6 Nigerian Value:

million / cu mm 4,000- 11,000 million
Female: 4.0- 5.5 cells/ cu mm
mm/ cu mm

Drops Expelled 5- 6 Drops 2-3 drops

APPLIED QUESTIONS (PAST Q 2020/2021)

1. In an experiment 10 determine Red blood cell count of a female pharmacy student in physiology lab, the
following cells were counted and recorded for the subject; 1st Square= 60 cells, 2nd Square= 70 cells, 3rd
Square= 62 cells, 4th Square= 79 cells and 5th Square = 80 cells

a. What is the Subject’s RBC count

b. Mention the name of the dilution fluid used and state the function of its constituents
C. Mention the objective lens magnification used during counting RBCs
d. Comment on the result obtained
e. Mention any two (2) precaution necessary for this experiment.

2. During an experiment to determine the white blood cell (WBC) count of a 22-year old Male Medical
Laboratory Science student in the physiology laboratory, four small boxes each containing Sixteen (16) 6
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smaller
boxes were counted and recorded for white blood cells as follows; 1st Box= 12 cells, 2nd Box= 14 cells, 3rd
Box= 11
cells, and 4th Square= 10 cells.

a. Calculate the total white blood cell count of the student

b. Mention the name of the WBC diluting fluid and its constituents used during the experiment.
C. Comment on the physiological and pathological factors that might have possibly affected the results ob-
tained.
d. What is the normal field distribution error during WBC count?

ANSWER
1. a. To calculate the subject’s Red Blood Cell (RBC) count, you can use the following
formula:
RBC count = (R1 + R2 + R3 + R4 + R5) X 10,000 CELLS
= (60+70+62+79+80) X 10,000 CELLS
= 351 x 10,000 cells = 3,510,000 cells = 3.5 million cells/ cu mm of Blood

b. The dilution fluid used in this experiment is “Hayem’s Solution.” It consists of the
following constituents:
1. Sodium Chloride (NaCl) 0.5gm - Maintains Isotonicity
2. Sodium Sulphate (Na2SO4) 2.5gm - Prevents Rouleaux formation
3. Mecury Chloride (HgCl2) 0.35gm - acts as a Preservative
4. Distilled water 100ml - Used for Dilution

c. The objective lens magnification used is 40x magnification.

d. The result obtained is a calculated RBC count of 3,510,000 for the subject. The normal
range for RBC count in females of reproductive age is typically around 4.0 to 5.5 million
cells/cu mm. Thus, Subject is Anemic
e. Precautions necessary for this experiment include:
1. It was ensured that double counting was avoided.
2. It was ensured that the puncture site was sterilized with swab before puncture.
3. It was ensured that Precise and consistent pipetting was done
4. It was ensured that 5 drops from the pipette was expelled.
5. It was ensured that first drop of blood after puncture was cleaned with swab.
7

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2. a. To calculate the total white blood cell (WBC) count of the student, you can use the
following formula:

WBC count = (W1 + W2 + W3 + W4) × 50

=(12 + 14 + 11 + 10) *50
= 47 * 50 = 2,350 cells/ cu mm

b. The WBC diluting fluid used in this experiment is “Turk’s Solution”.

Turk’s Solution consists of the following constituents:

- Acetic acid: Acts as a lysis agent, facilitating the breakdown of red blood cells and leav-
ing behind the white blood cells.
- Gentian violet: Stains the white blood cells, making them more visible and easier to
count.
- Distilled water: Provides a suitable medium for the dilution and suspension of the
blood sample.

c. Physiological and pathological factors that might have possibly affected the results
obtained in WBC counting include:

- Physiological factors:
1. Certain medications such as chemotherapy drugs or immunosuppressants can sup-
press the bone marrow’s ability to produce an adequate number of White Blood cells.
2. Nutritional deficiencies such as defie=ciencies in vitamin Bi2, folate or copper can neg-
atively affect the production of white blood cells and lead to a decreased WBC Count

- Pathological factors:
1.Underlying medical conditions like infections, autoimmune diseases, leukemias, or
bone marrow disorders can significantly affect the WBC count.
2. Infections such as HIV/AIDS, tuberculosis, or severe bacterial infections can contribute
to a decreased WBC Count.

d. The normal field distribution error during WBC Count is no more than 10 cells

Precautions during HB estimation

It was ensured:
1. Air bubbles were prevented when blowing the
pipette
2.The blood collected was up to 3/4 of the
heparinized capillary tube
3. The blood was centrifuged for 5 minutes
8 4.The puncture site was sterilized before puncture,
EXPERIMENT 4

Haemoglobin Estimation
PRINCIPLE: The principle behind sahli’s method is based on the conversion of hemoglo-
Sahli’s Method bin to a stable compound called hematin and the use of sahli’s hwmoglobi-
nometer which consists of a graduated tube and a color comparison scale.
(Breakdown procedure)

AIM: To determine the oxygen carrying capacity of blood of a subject

MATERIALS: 0.1 N HCL, Sahli’s standard comparator, haemoglobin pipette, lancet, swab

CALCULATION eg. Value obtained = 70%

100%= 14 g/dl 70 x 14 = X
70% = X 100
(Cross multiply)
70 x 14 = 100X X = 9.8g/dl
(Divide Both sides by 100) Hb = 9.8 g/dl of blood

Variations
Physiological
1. Age: higer in infants and growing children than adults
2. Sex: It is Higher in males then females of reproductive age
3. Environmental factor: High altitude of 10,000 ft above sea level
stimulates secretion of Erythropoietin which stimulates production of more RBC in the Bone
marrow, thus hemoglobin
4. Nutrition: Food rich in irons and proteins increase blood protein which is synthesized to
produce hemoglobin.
5. Mild dehydration

Pathological -Any diseased or dysfunctional condition that affects production of RBC

1. Anemia: Iron deficiency anemia, sickle cell anemia
2. Polycythemia
3. Excessive hemorrhage
4. Chronic kidney disease: can affect the prod. of erythropoietin, this can lead to low Hb.
5. Bone marrow disorders: Leukamia, myelodysplastic syndrome can lower hb level
6. Hemolytic disorders: conditions in which RBC are destroyed prematurely 9
7. Severe dehydration: can lead to increase in Hb levels
 EXPERIMENT 5

Estimating Packed Cell Volume

(PCV) or Haematocrit (HCT)
PRINCIPLE: The Principle of estimating PCV using a micro haematocrit reader is
based on the seperation of blood components by centrifugation
(Proceed to describe procedure)

AIM: To determine blood Packed Cell volume of a Subject

MATERIALS: Heparinized capillary tube, Micro-haematocrit reader, plasticine,

centrifuge
CALCULATIONS:
Height of Packed red cells x 100
HCT=
Height of packed RBC and Plasma

eg = 45 x 100 = 45%
100

HCT= 45%
(Volume correction factor
For actual Value, Multiply HCT by 0.96
for trapped plasma)
45% X 0.96 = 43.2
PCV = 43.2%
Normal Values: Male: 40% - 50% Average= 45%
Female: 37- 47% Average= 42%

Hematological Indices
MCV MCH MCHC
Mean Corpuscular Volume= Mean Corpuscular Haemoglobin= Mean Corpuscular Haemoglobin Concemtration=
key
PCV X 10 HB X 10 HB X 100 Difference

RBC RBC PCV

eg: 43.2% X 10 eg: 9.8 g/dl X 10 eg: 9.8 g/dl X 100

3.5 3.5 43.2%
MCV = 123 fl Macrocytic
MCH = 28 pg Normal MCHC = 22% Hypochromic

Unit: fl Unit: Pg Unit: % pg

Greater than 100 fl = Macrocytic Cell Range: 27- 32 pg Range= 30%- 38%
Less than 100 fl = Microcytic cell Less than 30% = Hypochromic RBC
Equal to 100 fl = Normocytic cell 30%- 38% = Normochromic RBC

Indicates size of an average RBC Indicates average Haemoglobin Indicates average Haemoglobin
10 content in an average RBC content per column of blood
 Variations in PCV
Physiological
A decrease in PCV may indicate anemia which could be as a result of:

1. Excessive bleeding
2. Hemorrhage
3. Malnutrition
4. Nutritional Deficiency
5. bone disorders such as aplastic anemia
6. Kidney disease
7. Liver cirrhosis e.t.c

An Increase in PCV may be indicative of Polycythemia which could be as a result of

1.Dehydration: plasma level decreases leading to a higher proportion of RBC in blood

2.Hemolysis: Breakdown of red blood cells can lead to falsely elevated PCV levels
3.Altitude: Hypoxia
4. Medical conditions such as Polycythemia vera or chronic lung disease can lead to elevated PCV
values.

Calculations PAST Q 2020/2021

1. A 20-year old Female Radiography student’s Packed Cell volume (PCV) was evaluated in the
laboratery, A PCV reading of 33% was obtained from a Micro-hoeinatocrit reader. In reviewing her
records, it was noted at that time her Hemoglobin estimation was 12g/al while her RBC count was
3.0x10-6 cells/ mm3 of blood.
Comment on the following

a. What is the actinal PCV of the student

b. Mention any two (2) Physiological and Pathological factors that night have possibly affected the
obtained result

C. Calculate the Hematological Indices (MCH, MCHC and MCV) of the subject.

2. During as experimiment to estimate the Haemoglobin concentration of a Male Nursing Student

using the sahli’s technique at the Physiology Lab, A value of 60%, was obtained from the dilution
tube
a. Calculate the haemoglobin concentration of the subject.
b. Comment under the following leadings;
i. title of experiment
ii. Aim of experiment
iii.Materials used
iv. Mention any two (2) Plysiological and Pathological factors that could have possibly affocted the
result.
c. Mention the name of the substance formed after mixture of blood with the O.IN HCL 11
 ANSWERS

1.
Data:
PCV = 33% Hb = 12g/dl RBC = 3.0 x 10-6

a. Actual PCV of the Student:

PCV x 0.96 (Volume correction factor for trapped plasma)
33% x 0.96
Actual PCV = 31.68 (This is below Normal range for female)
b. Check pg 11 for Physiological and pathological factors that lead to low PCV Count

c. Hematological Indices:
MCV = PCV X 10
RBC
= (31.68/3.0) x 10
= 105.6 Fl (Macrocytic cell)

MCH = HB X 10
RBC
= (12/3.0) X 10
= 40 pg (Above normal range)

MCHC = HB X 100
PCV
= (12/31.68) x 100
= 37.9 = 38% (Normochromic RBC)
2.a b.
Data: i. Title of Experiment: Haemoglobin Estimation
Dilution tube reading = 60% Hb ?
ii. Aim: To determine oxygen carrying capacity of blood
of male nursing student
100% = 14 g/dl
iii. 0.1 N HCL, Sahli’s standard comparator,
60% = x (Cross Multiply)
haemoglobin, pipette, lancet, swab
iv. Check pg 11 for physiologic and pathological
60 x 14 =8.4 g/dl
factors that could cause a low PCV Value
100
C. Name of subt. formed after mixing blood with 0.1
12 NHCL:
Acid Hematin (Brownish coloured solution
EXPERIMENT 6

Hemostasis: Bleeding & Clotting

time
Hemostasis: The process of stopping bleeding after an injury.
The main steps involved in hemostasis are:

i. Vasoconstriction: When a vessel is damaged, the vessels contract,

causing the vessell to narrow. This reduces blood flow to the area which helps
slow down bleeding

ii. Platelet plug formation: Platelets (Disc shaped cells) are activated and
begin to stick to the damaged area, forming a plug. This plug helps to seal the
damaged blood vessel and stop bleeding.

ii. Blood clotting (Coagulation): Blood clotting factors are activated,

leading to formation of a clot. Clotting factors work together to conver fibrin-
ogen (Soluble protein) to fibrin (insoluble) that forms a mesh like clot. The
clot helps reinforce the platelet plug and futher seal the damaged vessel
Bleeding Time: Start of Bleeding - Stoppage of Bleeding (Normal Range: 1-3 Minutes)

Clotting Time: Time that passes between start of bleeding and formation of clot
(Normal Time at 37o C: 4 mins, can vary b/w 3-10 Mins)
Significance: They can be used to help diagnose disorders such as von willebrand dis-
ease, hemophilia (deficiencies in specific clotting factors) or
thrombocytopenia.
The test can also help predict the risk of bleeding during surgery or other
medical procedurs. This helps doctors determine effectiveness of
Calculation: treatment.
BT= CT=
No of blots No of Lines
2
Answer is given in minutes 2
NB: Blood clotting or coagulation happens via three (3) processes:
1. Activation of Clotting Factor
2. Conversion of prothrombin to thrombin
3. Conversion of fibrinogen to fibrin

13
 Factors that Influence Bleeeding
& Clotting time
Prolonged Bleeding Time:
1. Thrombocytopenia: Low platelet count
2. Medications such as anticoagulant
3. Platelet disorder such as Von willebrand disease
. Purpura

Prolonged Clotting Time:

1. Conditions like:
-Hemophilia
-Liver disease,
can affect production or activity of coagulation factors
2. Hypofibrinogema: Insufficient levels of fibrinogen
3. Insufficient Vitamin K

The 12 Coagulation/ Clotting Factors

The process of blood clotting, or coagulation, involves a series of complex interactions between various
clotting factors. These factors work together to form a stable blood clot and prevent excessive bleeding.
1. Factor I (Fibrinogen)
2. Factor II (Prothrombin)
3. Factor III (Tissue Factor)
4. Factor IV (Calcium)
5. Factor V (Proaccelerin)
6. Factor VI (Unassigned) NB: Factor 6 is unassigned, so there are
12 clotting factors
7. Factor VII (Proconvertin)
8. Factor VIII (Antihemophilic factor)
9. Factor IX (Christmas factor)
10. Factor X (Stuart-Prower factor)
11. Factor XI (Plasma thromboplastin antecedent)
12. Factor XII (Hageman factor)
13. Factor XIII (Fibrin-stabilizing factor)
14
EXPERIMENT 7

Determination of Blood Group

Golden rules concerning Blood Groups

If an Antigen (aka Agglutinogen) exist, corresponding antibodies will be absent.
During Blood transfusiom, reciepient blood must not contain antibodies (agglutinins)
against donor’s RBC’s, otherwise Agglutination of RBC may occur.

Agglutination:
This refers to the clumping of cells whic involves binding of antibodies to antigens on the
surface of cells.
Breakdown
Basically, if Agglutination with an Anti serum occurs, the corresponding Antibody is present
in the Blood sample and the subject is positive for that Blood Group.
If no Agglutination occurs, then the corresponding antigen is missing in the blood of the
subject thus, the subject is not positive for that Blood Group

Systems of Blood Grouping

1. ABO Blood Group System:
- The ABO system is based on the presence or absence of two antigens: A and B.
- People with blood type A have the A antigen on their red blood cells and produce
antibodies against the B antigen.
- People with blood type B have the B antigen on their red blood cells and produce
antibodies against the A antigen.
- Individuals with blood type AB have both A and B antigens on their red blood cells and do
not produce antibodies against either antigen.
- People with blood type O lack both A and B antigens on their red blood cells and produce
antibodies against both A and B antigens.
- The ABO system is essential for blood transfusions to ensure compatibility. For example,
individuals with type A blood can receive blood from type A or type O donors but not from
type B or AB donors.

15
2. Rh Blood Group System:
- The Rh system is based on the presence or absence of the Rh antigen (also known as the
D antigen).
- If an individual has the Rh antigen on their red blood cells, they are considered Rh
positive (Rh+). If they lack the antigen, they are Rh negative (Rh-).
- Rh status is particularly significant during pregnancy. If an Rh- woman is carrying
an Rh+ fetus, there is a risk of Rh sensitization, where the mother’s immune system can
produce antibodies against the Rh antigen. This can lead to complications in subsequent
pregnancies if the fetus is Rh+.

It’s important to note that blood typing and cross-matching are crucial steps in blood
transfusions to ensure compatibility between blood donors and recipients. A mismatch can
lead to serious immune reactions and harm the recipient.

EXAMPLE PAST Q 2020/2021

1 During an experiment to determine the blood group of a pharmacy student using the cross matching
technique in a tile at the Physiology Lab, There was agglutination of the subject’s blood with Anti sera B, AB
and D only,

a. What is the Principle for blood group determination?

b. Using the appropriate diagram, indicate the reactions observed
c. What blood group does the subject belongs to?
d. Mention two (2) possible compatible donors and recipients to/from a subject with blood group AB
Negative.

a. The principle for Blood Group determination is based on the presence or absence of specific antigens on
the surface of Red blood cells. There are four blood groups: A, B, AB and O. The presence of A na d B deter-
mines the blood group, while the presence or absence of the Rh factor ( positive or negative) futher classifies
blood type. The principle involves mixing a subject’s blood sample with specific antibodies that will react with
the corresponding antigens leading to visible agglutination. This reaction helps identify the Subject’s blood
group.

b.

No Agglutination
Agglutination

C. Subject is B+
NB:
If there was no agglutination in D
It would be B-
Rh positive individuals can recieve from Rh Negative and positive of
their same blood group and O

D. Compatible Donors: B+, B-, O-,O+,

16
 Universal
Donor DONOR

O+ O- A+ A- B+ B- AB+ AB-
O+
O-
A+
RECIPIENT

A-
B+
B-
Universal
Recipient AB+
AB-

BLOOD DONATION CHART

PRECAUTIONS

1.It was ensured that the site was disinfected with cotton swab
2. It was ensured that the amount of blood used for the test did not exceed the amount of
antiserum.
3. It was ensured that the same stirrer was not used to stir all the blood samples

17
 BONNE CHANCE!!!
Wubba lubba dub dub!!

NB:
This is a Summary and not a substitute for Physiology Manual
provided by the Physiology Department.

Any complaints, mistakes or corrections should be made

known to coursemates and creator of material via the contact info
below.

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