Activity 6

RED CELL FRAGILITY, BLOOD TYPING, BLEEDING TIME AND CLOTTING TIME AND HYPEREMIA

Introduction
In clinical practice, hematological information accumulated from a series of blood tests conducted
on a small volume of blood - even a single drop - can be of great diagnostic and prognostic value.
There are certain molecules on the surfaces of all cells in the body that can be recognized as
foreign by the immune system of another individual. These molecules are known as antigens. As part of
the immune response, particular lymphocytes secrete a class of proteins called antibodies that bind in a
specific fashion with antigens. The specificity of antibodies for antigens is analogous to the specificity of
enzymes for their substrates, and of receptor proteins for neurotransmitters and hormones.
Materials and Methods
A. RED CELL FRAGILITY
Ten (10) test tubes were prepared in serial dilution of NaCl solution in different concentrations
with distilled water. A representative was asked to sterile his pointer finger with alcohol in gauze pad.
Using a lancet pen, the finger was pricked. Two drops of blood was added to each test tube
concentration. Each test tube was covered with parafilm stretching up to the mouth. The blood with salt
concentration was mixed in a slow inverting manner of the tube using the middle and thumb finger. Finally
the 10 test tubes were all subjected for centrifugation at 1500xg for 2 minutes.
B. BLOOD TYPING
Obtain a clean microscope slide. Using a glass-marking pencil, mark one end A and the other end
B. Lance your finger to obtain blood. Clean the palmar surface of the third or fourth finger with a sterile
gauze pad soaked with 70% ethanol. With a sterile lancet or needle, make a puncture on a fingertip. Wipe
off the first drop. Place one drop of blood on each of the marked slide. Add one drop of anti-A serum to
the A side. Add one drop of anti-B serum to the B side. With a toothpick, using a different toothpick for
each side. Spread each mixture over an area of about 0.75 in diameter. Observe the slide for any
agglutination of red cells.
C. BLEEDING TIME AND CLOTTING TIME
Sterilize the fingertip using rectified spirit and allow to dry. Make a sufficiently deep prick using a
sterile lancet, so that blood comes out freely without squeezing. Mop the blood by touching the fingertip
with a filter paper. This is repeated every 15 seconds, each time using a fresh portion of the filter paper, till
bleeding stops. Note the time. It is seen that the blood stains on the filter paper get smaller to disappear
finally when bleeding stops. Under sterile precautions make a sufficiently deep prick in the fingertip. Touch
the blood drop at the fingertip using one end of the capillary tube kept tilted downwards. The tube gets
easily filled by capillary action. After about two minutes start snapping off small lengths of the tube, at
intervals of 15 seconds, each time noting whether the fibrin thread is formed between the snapped ends.
Note the time when the fibrin thread is first seen.
D. HYPEREMIA
Wind a rubber band in the chosen finger. Wait for several minutes to note for difference in size
and color of the finger. Prepare a 500-ml tap water in a beaker then put it in water bath set at 45 degrees
C. Immerse the finger in the beaker for a few minutes to note for sensations other than warmth that the
finger was exposed to.

Results
A. Red Cell Fragility
Tube
Number
1
2
3
4
5
6
7
8
9
10

Color of Supernatant
Clear
Clear
Clear
Clear
Clear
Reddish
Reddish
Reddish
Reddish
Reddish

Observations
Presence of RBCs at the bottom of tube
+
+
+
+
+
-

2. If a cell that is normally in osmotic equilibrium is transferred to a more dilute solution, water will enter
the cell, the cell volume will increase, and the solute concentration of the cytoplasm will be reduced.
3. If the cell is transferred to a more concentrated solution, water will leave the cell, the cell volume will
decrease, and the solute concentration of the cytoplasm will increase.
B. Blood typing, bleeding time and clotting time
Your Sample

Anti-A Serum
Agglutinate

Bleeding time
Clotting time

Anti-B Serum
Agglutinate

Blood Type
AB

1 minute
45 sec

4. ABO typing:
If your blood cells stick together when mixed with:

Anti-A serum, you have type A blood

Anti-B serum, you have type B blood
Both anti-A and anti-B serums, you have type AB blood
If your blood cells do not stick together when anti-A and anti-B are added, you have type O blood.

5. Assessing blood clotting and bleeding factor is very important if you are about to have a surgical
procedure. Surgery or an injury of any kind increases the risk of a blood clot. Thats because the clotting
process is stimulated as your body attempts to stop the bleeding and close the surgical wound. A clot is
normally formed by the blood cells and clotting factors working together to create a protective scab over a
healing wound. The surgical procedure may stimulate clots to form in error in blood vessels, which then
may block the normal flow of blood.

B.2 Cross-Matching
Serum Sample

Red Cell Suspension

Agglutination
Donor: Type AB
Recipient: Type A

6. Cross matching is a must before blood transfusion to determine if the donors blood is compatible with
the blood of an intended recipient. Transfusion errors that result in such agglutination can lead to
blockage of small blood vessels and cause hemolysis (rupture of red blood cells), which may damage the
kidneys and other organs.
C. Hyperemia
Observations
7.1

Size: swell
Color: dark-purple color

By winding the rubber band on the finger the oxygen level decreases and swells due to blood vessel
dilation.

7.2

Size: (+) crease, shrink

Color: reddish

An increase metabolic rate requires a greater flow of blood thus the vessels dilate.

8. Hyperemia
Hyperemia is when there is an increase in blood flow to the tissues and organs. Active or
functional hyperemia is an increase in blood flow associated with increased metabolic activity. A greater
blood flow is required to increase oxygen delivery and enhance the removal of carbon dioxide and/or
lactate from these metabolically active tissues. Reactive hyperemia on the other hand is an increase in
blood flow to the tissues when they have been deprived of oxygen.

Discussion
Cell membranes are semipermeable barriers, and osmotic gradients are established between
intracellular and extracellular fluids which can cause water to flow into and out of the cells. The amount of
osmotic pressure depends upon the difference between the concentrations of non-diffusible ions on each
side of the membrane.
Osmotic fragility is a test to detect whether red blood cells are more likely to break down. The
intracellular fluid of erythrocytes is a solution of salts, glucose, protein and hemoglobin. A 0.9% NaCl
solution is said to be isotonic: when blood cells reside in such a medium, the intracellular and extracellular
fluids are in osmotic equilibrium across the cell membrane, and there is no net influx or efflux of water. In
the performed experiment, 0.9% NaCl wasnt exactly used and instead a higher concentration of NaCl
was given.

Figure 1
When subjected to hypertonic media (experiment Tube 1-5), the cells lose their normal biconcave
shape, undergoing collapse (leading to crenation) due to the rapid osmotic efflux of water.
On the other hand, in a hypotonic environment (Tube 7-10 with greater volume of distilled water),
an influx of water occurs: the cells swell, the integrity of their membranes is disrupted, allowing the
escape of their hemoglobin (hemolysis) which dissolves in the external medium.

Figure 2. Crenated sample (Tube 1-5)

Figure 3. Hemolyzed sample (Tube 6-10)

If as little as 0.5% of the red blood cells are hemolyzed, the released hemoglobin will cause the
serum or plasma to appear pale red or cherry red in color. Note that the hemolyzed sample is transparent,
because there are no cells to scatter light. On the other hand, the crenated sample after centrifugation
formed a cell fractionation where the resulting supernatant is clear and the pellet visible at the bottom
looks very pale.

The blood groups refer to the presence on human red blood cells of certain antigens, the blood
group factors. One very important group of factors present on the red blood cells is the ABO system. The
ABO group of a person depends on whether his/her red blood cells contain one, both, or neither of the 2
blood group antigens A and B. There are, therefore, 4 main ABO groups: A, B, AB and O. The presence or
absence of agglutinations will determine the blood type found. This agglutination reaction, which is very
important in determining the safety of transfusions is due to a mismatch of genetically determined blood
types.

Antibodies (agglutinins) for the antigens A and B exist in the plasma and these are termed anti-A
and anti-B. The corresponding antigen and antibody are never found in the same individual since, when
mixed, they form antigen-antibody complexes, effectively agglutinating the blood.
Agglutination (clumping) of red blood cells occurs when cells with A-type antigens are mixed with
anti-A antibodies and when cells with B-type antigens are mixed with anti-B antibodies. No agglutination
would occur with type O blood.
People with type A blood have type A antigens on their red blood cells and antibodies in their
plasma against the type B antigen. People with type B blood have type B antigens on their red blood cells
and antibodies in their plasma against the type A antigen. Therefore, if red blood cells from one blood type
are mixed with antibodies from the plasma of the other blood type, an agglutination reaction occurs. In
this reaction, red blood cells stick together because of antigen-antibody binding.
Hemostasis (literally - blood halting) depends
on three interrelated and overlapping sets of events:

Constriction of the blood

formation of a platelet "plug"
Blood clotting
Clot retraction

vessels

and

Bleeding time is the interval between the moment

when bleeding starts and the moment when bleeding
stops. Normal bleeding time is to 4 minutes.
Measures the time taken for blood vessel constriction
and platelet plug formation to occur. No clot is
allowed to form, so that the arrest of bleeding
depends exclusively on blood vessel constriction and platelet action. It is used to evaluate platelet
function and is typically performed, along with a platelet count, in people with a personal or family history
of bleeding disorders, or as a preoperative safety measure before a scheduled surgery.

Clotting time is the interval between the moment when bleeding starts and the moment when the
fibrin thread is first seen. Bleeding time and clotting time are not the same. Bleeding time depends on the
integrity of platelets and vessel walls, whereas clotting time depends on the availability of coagulation
factors. In coagulation disorders like haemophilia, clotting time is prolonged but bleeding time remains
normal. The normal range for clotting time is 3-10 mins.
In order for blood to clot, the enzyme thrombin must be generated from the plasma precursor
prothrombin. Thrombin then converts soluble fibrinogen into insoluble fibrin. Generation of thrombin
involves the sequential activation of a number of other plasma clotting factor, this process is also being
assisted by Ca++ and by factors released by platelets and damaged tissues . The time taken for blood to
clot mainly reflects the time required for the generation of thrombin in this manner. If the plasma
concentration of prothrombin or of some of the other factors is low (or if the factor is absent, or
functionally inactive), clotting time will be prolonged. Clotting time is also prolonged in conditions like
vitamin K deficiency, liver diseases, disseminated intravascular coagulation, overdosage of anticoagulants
etc.
Cross-matching blood, in transfusion medicine, refers to the complex testing that is performed
prior to a blood transfusion, to determine if the donors blood is compatible with the blood of an intended
recipient, or to identify matches for organ transplants. Cross matching is a must before blood transfusion
to determine if the donors blood is compatible with the blood of an intended recipient

Figure 4. Cross-matching. A type AB donor mixed with the blood of a type A recipient.
When transfusing blood, it is important to remember that the donor's blood must not contain red
blood cells that the recipient's antibodies can agglutinate. Theoretically, then, individuals belonging to
blood group O are universal donors, while those of blood group AB are universal recipients. If the types
do not match if the donor is type A, for example, and the recipient is type B the recipients antibodies
attach to the donors red blood cells and form bridges that cause the cells to clump together, or
agglutinate. Transfusion errors that result in such agglutination can lead to blockage of small blood
vessels and cause hemolysis (rupture of red blood cells), which may damage the kidneys and other
organs.
Hyperemia is when there is an increase in blood flow to the tissues and organs. When blood flow
is reduced temporarily, the skin becomes pale. If pressure is applied to the skin, the blood in the vessels
of the skin stagnates. Oxygen in the hemoglobin is quickly used by the tissue in the area, and the
hemoglobin becomes darker as a result of deoxyhemoglobin formation.
When you obstruct blood flow by tying a rubber band around a finger, it swells and the color of the
skin gives a bluish hue, termed cyanosis. Once removed, the skin turns fiery soon after the occlusion is
removed. This is known as reactive hyperemia. Reactive hyperemia is an increase in blood flow to the
tissues when they have been deprived of oxygen. When blood flow to an area is restricted, the arterioles
in that area dilate as a result of the release of chemicals (products of metabolism) by the oxygen-deprived
cells. When blood flow is no longer restricted, blood rushes into the dilated blood vessels as the oxygen
level increase.

Active or functional hyperemia is an increase in blood flow associated with increased metabolic
activity of an organ or tissue. During the immersion of one finger in a warm water there was an increased
in the metabolic activity, where the skin dilated and turned red in color, showing an increase in the blood
flow. A greater blood flow is required to increase oxygen delivery and enhance the removal of carbon
dioxide and/or lactate from these metabolically active tissues. After few minutes upon immersion a short
painful sensation other than warmth was felt which later on adapted on the surrounding temperature.
Another good example of active hyperemia is the increase in blood flow that accompanies muscle
contraction, which is also called exercise or functional hyperemia in skeletal muscle. Blood flow increases
because the increased oxygen consumption of during muscle contraction stimulates the production of
vasoactive substances that dilate the resistance vessels in the skeletal muscle.

Conclusion
The primary function of blood is to supply oxygen and nutrients as well as constitutional elements
to tissues and to remove waste products. Blood also enables hormones and other substances to be
transported between tissues and organs. Blood is also involved in maintaining homeostasis problems with
blood composition or circulation can lead to downstream tissue malfunction, thats why, assessing your
blood can greatly prevent health problems.

Literature cited
Sdmesa. Vascular. Retrieved 13 September 2015 from http://classroom.sdmesa.edu/bbrothers/Docs
%20235/vascular.pdfa

Health communities. Blood clotting tests. Retrieved on 13 September 2015 from

http://www.healthcommunities.com/blood-tests/blood-clotting-tests.shtml
McGill. ABO System. Retrieved on 13 September 2015 from
http://www.medicine.mcgill.ca/physio/vlab/bloodlabABO_n.htm
Wikipedia. Hemolysis. Retrieved on 13 September 2015 from
https://en.wikipedia.org/wiki/Hemolysis#Outside_the_body
Encyclopedia of Ayurvedic Medical Plants. Bleeding time & clotting time. Retrieved on 13 September
2015 from http://www.indianmedicinalplants.info/articles/BLEEDING-TIME.html
CVPhsyiology. Blood Flow. Retrieved on 12 September 2015 from http://www.cvphysiology.com/Blood
%20Flow/BF005.htm