SAINt paul university

school of medicinE

Exercise 1 - Cell Physiology

Group 3
CRISTOBAL-LAGUA-MADRIAGA-MARIBBAY-NATIVIDAD
A. Red Cell Fragility
MATERIALS

Test tube rack with 5 mL test tubes-

10 pieces
0.9% NaCl solution
Distilled water
Blood sample, 5mL heparinized
Pipettes- 5mL, 1mL glass pipette with
rubber band
Parafilm
A. Red Cell Fragility
PROCEDURE

1. Make serial dilution of the NaCl solutionn in the ten test tubes as follows.

TEST TUBE NO. 0.9% NaClNO. DISTILLED WATER

1 5.0 mL 0 mL

2 4.5 mL 0.5 mL

3 4.0 mL 1 mL

4 3.5 mL 1.5 mL

5 3.0 mL 2.0 mL
A. Red Cell Fragility
PROCEDURE

1. Make serial dilution of the NaCl solutionn in the ten test tubes as follows.

TEST TUBE NO. 0.9% NaClNO. DISTILLED WATER

6 2.5 mL 2.5 mL

7 2.0 mL 3.0 mL

8 1.5 mL 3.5 mL

9 1.0 mL 4.0 mL

10 0.5 mL 4.5 mL
A. Red Cell Fragility
PROCEDURE

2. To each test tube, add 2 drops of whole blood

and mix by inverting the tube after covering with
Parafilm.

3. Centrifuge all the tubes.

4. Write down observations as to: supernatant

(color and clarity) cells at the bottom of the tube
A. Red Cell Fragility
PROCEDURE

This was the appearance of

the test tubes before they
were placed in the
centrifuge machine.
A. Red Cell Fragility
RESULTS

TEST TUBE NO. 0SUPERNATANT PRECIPITATE/ RESIDUE

1 - ++++

2 - ++++

3 - ++++

4 - ++++

5 - ++++
A. Red Cell Fragility
RESULTS
RESULTS

TEST TUBE NO. 0SUPERNATANT PRECIPITATE/ RESIDUE

6 + +++

7 +++ ++

8 ++++ +

9 ++++ -

10 ++++ -
A. Red Cell Fragility
RESULTS
OBSERVATION

Tubes 1-5 has a clear Tubes 6 and 7 Tubes 8-10 shows

supernatant fluid and demonstrate partial deep red coloration
a well formed hemolysis, exhibiting a with little to no
precipitate. pink tinge in precipitate, indicating
coloration. complete hemolysis.
A. Red Cell Fragility
QUESTIONS

1. Examine the hemolysis in this experiment.

Osmosis is the process of net movement of water from a high concentration to a low
concentration of water molecules. Hence, when the RBCs were placed in a hypotonic solution,
it caused the water to enter the cell where there was a lower concentration. This resulted in
the swelling of the cell, disrupting its membrane and eventually causing hemolysis releasing its
contents into the extracellular medium.

2. At what concentration did you note the point of completed hemolysis and initial
hemolysis?
Initial hemolysis happened at test tube no. 6 where there was a 0.45% NaCl. While the
completed hemolysis was noted on test tube no. 8 with 0.27% NaCl.
 A. Red Cell Fragility
QUESTIONS

3. What is the normal fragility index of RBC in man?

The normal red blood cell fragility index falls between an initial hemolysis of 0.45% NaCl
solution to the final or complete hemolysis of 0.30%-0.35% NaCl.

4. In what condition/s is fragility affected clinically?

Conditions affected by osmotic fragility includes:
Hereditary Elliptocytosis
Thalassemia
Iron Deficiency Anemia
Sickle Cell Anemia
Polycythemia vera
A. Red Cell Fragility
QUESTIONS
5. What is an isotonic salt solution?
Isotonic salt solution is a solution consisting of 0.9% sodium chloride, it has a concentration
similar to that of the blood, thus showing no net movement.

6. What could possibly be the untoward effects in intravenous administration of

hypotonic solution to a normal person? Is it safe to give hypertonic solution
intravenously? Why?
Administration of hypotonic IV solution may cause hypovolemia, hypotension, nausea, and
cerebral edema due to the fluid shifting into the intracellular space, which can be life
threatening. Yes, it is safe to administer hypertonic IV solution given that the patient is
suffering from metabolic alkalosis and severe hyponatremia. It should not be given to patients
with heart failure and renal failure because it might aggravate these conditions.
B. Experiment on Diffusion
Materials & Procedure

Methylene blue powder

Beaker
Water

Drop a pinch of Methylene blue powder

into a beaker of water
B. Experiment on Diffusion
Result

Before addition 30 seconds 5 minutes 60 minutes

after addition after addition after addition

Observation
Upon adding a pinch of methylene blue into a beaker of water, it immediately spreads
throughout the water in a random motion.
C. Experiment on Osmosis
PART I: Materials & Procedure

1. Fill the bulb of a Thistle tube with 5% glucose

Thin ice bags solution. Fill by holding the tube with the bulb
Beaker of water up and placing your finger over the small
opening.
5% glucose sol’n 2. Wet the membrane with water and with a rubber
Methylene blue dye band tie it over the large end of the thistle tube.
Thistle tube 3. Invert the Thistle tube into a 250 mL beaker of
water and clamp it securely to an iron stand.
Burette clamp The fluid level in the beaker and the tube must
Membrane (Japanese paper) be identical.
5% NaCl 4. Add a drop of Methylene Blue dye to the beaker
of water.
Boiled egg 5. Examine the glass tubing at intervals. What
AgNO3 happens and why?
C. Experiment on Osmosis
PART I: Observation

FINAL
LEVEL
INITIAL
LEVEL

Level of the water

in the thistle
funnel increases.

(A) At start (B) 45 mins after standing

C. Experiment on Osmosis
PART II: Procedure

Preparation 1 Preparation 2 Preparation 3

Place 30 mL of 5% NaCl Place one piece of boiled Place 30 mL of 5%

into a plastic bag. Tie the egg to a beaker of water glucose solution into a
bag with a rubber band (50 mL). Add one drop of plastic bag. Tie the bag
and place it on a beaker Methylene Blue to the with a rubber band and
with distilled water of water. place it o a beaker with
the same quantity. 5% NaCl solution of the
same quantity.
C. Experiment on Osmosis
PART II: Procedure
At the end of the lab period, for preparation 1 &
3, test the two liquids for presence or absence For preparation 2, peel off the shell
of NaCl. For example AgNO3 will react with of the egg. Record your
Sodium Chloride to form a white precipitate of observation.
Silver Chloride.

PREPARATION 1 PREPARATION 2
 PART II: Observation

Preparation 1:

There was no reaction or appearance of a

white precipitate. Thus, sodium chloride is not
present in the distilled water.
 PART II: Observation

Preparation 2:

Upon peeling the egg, a noticeable

tinge blue color was observed.
 PART II: Observation

Preparation 3:

There’s formation of white precipitate

upon addition of silver nitrate.
 QUESTIONS

1. What are the two general classification of cellular transport system and give the
transport systems under each category?

PASSIVE TRANSPORT - no ACTIVE TRANSPORT - requires

expenditure of energy. Transport expenditure of energy in the form
along the electrochemical of ATP. Transport against the
gradient. Also known as the electrochemical gradient. Also
“DOWNHILL” transport. known as the “UPHILL” transport.

TYPES: TYPES:
Osmosis Primary Active Transport
Diffusion Secondary Active Transport
Filtration
2. Differentiate Osmosis from Diffusion.

OSMOSIS
the passive movement of water
across a semi-permeable membrane
from a low solute concentration to a
region of a high solute concentration.

DIFFUSION
movement of molecules from a high
concentration to a region of a low
concentration.
3. Discuss and explain your results for Preparation 1-3.

Preparation 1: This experiment is designed to demonstrate the concept

of osmosis. The result of this experiment demonstrates that water
molecules move from an area of lower solute concentration to an area of
higher solute concentration , which is the fundamental principle of
osmosis.
3. Discuss and explain your results for Preparation 1-3.

Preparation 2: Placing a piece of boiled egg in a beaker of water with methylene

blue will explore how the eggshell membrane interact with a colored solution.
The eggshell is porous and contains microscopic pores that allow substances,
such as water and certain molecules, to pass through.

In this experiment, the methylene blue, which is a water-soluble dye, is likely to

permeate through the eggshell's membrane and enter the egg. This
experiment illustrates that some substances can move through the semi-
permeable eggshell membrane while others cannot, highlighting the concept of
selective permeability.
3. Discuss and explain your results for Preparation 1-3.

Preparation 3: Placing the 5% glucose solution in a plastic bag and

immersing it in a beaker containing a 5% NaCl solution, water molecules
from the glucose solution will move through the semi-permeable membrane
into the NaCl solution, where the concentration of solute is higher, until
equilibrium is reached. This is a classic example of osmosis and selective
permeability in action.
 REFERENCES
Costanzo, L. S. (2018). BRS Physiology (6th ed.). Lippincott Williams and Wilkins.

Hall, J. E. (2015). Guyton and hall textbook of medical physiology (13th ed.). W B Saunders.
 Saint Paul University Philippines
SCHOOL OF MEDICINE

A CASE PRESENTATION

September 2023
pathophysiology
HE results from defects in the protein scaffolding of the erythrocyte
membrane, which decrease the deformability and resilience of the
RBCs. Normal RBCs are 7 microns in diameter and assume the shape
of a biconcave disk with central pallor. They are rugged cells and can
survive in the circulation for 120 days as they repeatedly and
momentarily assume an elliptical shape to negotiate through
capillaries as small as 2-3 microns in diameter.
After passing through the microcirculation, normal RBCs can regain their
discoid shape because of their elastic recoil; however, the RBCs in HE fail to do
so. This failure to regain their discoid shape eventually produces the fixed
characteristic morphology of elliptocytes with a decreased surface-to-volume
ratio. These elliptocytes are not as deformable as normal RBCs and are
eventually trapped and removed by the spleen. This process of premature
destruction (ie, cells surviving < 120d) is the basis of the extravascular
hemolysis that clinically defines these disorders.
SIGNS & SYMPTOMS
Vary from very mild to severe fatigue, shortness of breath, gallstones, and yellowing of
the skin and eyes (jaundice).

In hereditary elliptocytosis, clinical features are similar to those of hereditary

spherocytosis but tend to be milder; splenomegaly may be present.
DIAGNOSIS
The evaluation of Hereditary elliptocytosis begins with a complete blood count.
Testing for hemolysis should be carried out.
Increased reticulocyte count
Increased lactate dehydrogenase
Increased indirect bilirubin
Decreased haptoglobin level.
A peripheral blood smear should be done, which typically shows that 15% to 100%
of the RBCs are elliptocytes.
Individuals with intermittent hemolysis or anemia may require blood transfusions
when the patient is either symptomatic, or the hemoglobin level drops below the
threshold required for their age. (1-6 years: 9.5-14 g/dL. 6-18 years: 10-15.5
g/dL. Adult men: 14-18 g/dL. Adult women: 12-16 g/dL)

Splenectomy is reserved for patients with severe anemia that is life-threatening.

Since splenectomy is associated with an increased risk of infection with
encapsulated organisms, vaccination against pneumococcus, meningococcus, and
Haemophilus influenza is required prior to the surgery.
REFERENCES
Braunstein, E., (2022). Hereditary Spherocytosis and Hereditary Elliptocytosis. Retrieved from
https://www.msdmanuals.com/professional/hematology-and-oncology/anemias-caused-by-
hemolysis/hereditary-spherocytosis-and-hereditary-elliptocytosis

Jha. S., & Vakar. S., (2022 November) Hereditary Elliptocytosis. Retrieved September 9, 2023 from
https://www.ncbi.nlm.nih.gov/books/NBK562333/#:~:text=Hereditary%20elliptocytosis%20is%20a
%20group,and%20should%20be%20managed%20accordingly.
Thank you!