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EXERCISE 7

THE BLOOD

Hannah Rachel Jane Serdan

2015-05107 Date Submitted: April 22, 2019

Introduction

Blood is a special tissue that supplies oxygen and nutrients to the tissue. The blood travels

through the blood vessels which is found all over the body of an organism. The main components

of blood are the plasma, erythrocytes, leucocytes and platelets. The plasma is identified as the

straw-colored component of the blood that carries cells and proteins throughout the body. The

blood cells that are produced in the bone marrow which contains the hemoglobin and supplies the

oxygen throughout the body are the erythrocytes. While the leucocytes are the white blood cells

which are the main defense of our body against foreign organisms that may enter our system.

The blood also has an antigenic property, this is due to the presence of antigens on the red

blood cell’s membrane. The human blood can be classified into grouping system based on the

presence or absence of the specific antigens, it can be classified as group A, B, AB and O, based

on the ABO system.

When injured the body has its own way of preventing much or more danger from

happening. Haemostasis is one of the mechanisms of the body that prevents it from losing blood

when injured. Prolonged bleeding and abnormal blood clotting may denote an interference in the

haemostasis process of the body. The clotting time of the blood must be not so slow to prevent

losing too blood that may be later on cause more serious damages to the injured person.
Materials and Method

Hemocytometer

Hemocytometer counting chamber is the standard technique in cell counting. It is a 9 mm

square which is divided to nine 1 mm2 squares. The center square of the hemocytometer is also

divided into 25 smaller squares which is also subdivided into much smaller 16 1/25 mm2 squares

which are used for counting the red blood cells in the given blood samples.

To examine the hemocytometer and to proceed with the cell counting, it was done under

the microscope. The LPO was first used to locate the center square and then changed it to HPO to

focus on the much smaller squares. Cells touching or overlapping on the upper and left border of

the small squares was not counted, only the right and lower borders was included in the count.

Hayem’s Red Blood Cell Diluting Fluid

Using the 200-cc distilled water the 0.5 grams mercuric chloride, 5 grams of sodium sulfate

and 1 gram of sodium chloride was dissolved. It was filtered, and few millimeters of the solution

were placed in the clean and dry watch glass just before the cell counting.

Before the main part of the exercise, clean and dry glass slides, sterile blood lancets, cotton,

70% alcohol, toothpicks, needles, anti-A and anti- B serum were prepared. This was done in

preparation for the drawing of blood which was used in the other part of the exercise.
A. Red Blood Cell Count

Blood was extracted in this part of the exercise. The finger tip of the subject was

wiped and clean using the 70% isopropyl alcohol and a cotton. The finger was then pricked with

a lancet, the first few drops of blood that comes out from the small wound was discarded or wiped

off while the next drops were collected.

The rubber suction tube was attached to the upper end of the red blood cell pipette making

sure it was in a horizontal position while the mouthpiece was placed in the lips. The tip of the

pipette was placed in the drop of the blood making sure it stays at all times for the solid blood

column to fill the pipette. The rubber tube was sucked gently to draw blood until the 0.5 mark. The

procedure was done carefully to make sure that the pipette tip to never be detached from the blood

in order to avoid the formation of the air bubble inside the pipette. The tongue was placed against

the mouthpiece to hold the blood column and the excess blood was wiped out from the tip of the

pipette, the blood should be exactly at the 0.5 mark of the RBC pipette. The same pipette was used

to suck in the Hayem’s diluting fluid until the 101 mark. The rubber tubing was then removed and

both end of the pipette were covered by the thumb and the index finger to keep the liquid inside

the pipette intact while it was mixed in a circular motion for two minutes. Then the diluted blood

was poured into the clean and dry counting chamber, the first few drops of the blood solution was

discarded to avoid contamination. In pouring the diluted blood to the counting chamber, the cover

slip was first placed in the counting area, the tip of the pipette was placed in a position where its

tip is touching the edge of the cover slip on the hemocytometer. The fluid from the pipette was

released carefully by using the index finger to regulate the flow of the solution to the slip. After

two minutes of settling period the cell counting started with the counting using the microscope.
The counting starts at the cells in the 80 small squares on the five 1/25 mm2. The average

number of red blood cells per cubic millimeter of the whole blood was obtained and was identified

if it falls under the normal range of red blood cell count.

B. Agglutination

ABO System of Blood Typing

The finger of the subject was cleaned using the alcohol and cotton before it was punctured

with a sterile lancet. The first few drops of blood that came out from the wound was discarded

while the next following drops were collected in a clean and dry glass slide. The glass slide was

divided into the left and right portion. The left side labelled as A and the right side labelled as B.

Drops of blood was placed in both sides of the glass slide and anti-A serum was added in the blood

in the A side of the slide while anti-B serum was added in the B side of the slide. The blood and

the serum were mixed and observed.

For people with blood type A, coagulation should show after the anti-A serum was mixed

with the blood sample. Coagulation is also expected to form with the blood mixed with the anti-B

serum for people with a blood type B. coagulation should form when blood is both mixed with

anti-A and anti-B serum for people with a blood type-AB and no coagulation with both anti-A and

anti-B serum to people with blood type O.

After the blood was mixed with the serum, coagulation was observed, and the blood type

of the subject was identified.


C. Clotting Time

A drop of blood was again collected from the same finger that was punctured for other

procedures. The blood was placed at the center of the clean and dry glass slide taking note of the

time that the blood was collected from the punctured finger. Using the tip of the lancet that was

used to puncture the finger of the subject was used to draw blood to the periphery to observe if any

fibrin clings to the tip of the lancet. The time was noted when fibrin formation was observed, and

it was identified as the clotting time.

D. Factors Affecting Coagulation

Rats were put inside the container with a cotton with some drops of chloroform. Nine test

tubes were labeled A to I and was placed in the test tube rack accordingly. Blood were collected

from the already dead rats using a 2-cc syringe and a drop of blood was distributed to the nine

labelled test tubes. Test tube A contained only the blood sample and the clotting time was

determined. Test tube B was lined up with paraffin in which drop of blood was added. Test tube

C had cotton fibers at the bottom and sides of the test tube and again blood from the rat was also

placed in the test tube. Test tube D was placed in an ice bath right after the blood was dropped in

the tube while test tube E was placed in the water bath. 5 drops of 0.1% heparin was placed in test

tube F, small amount of 1% sodium oxalate in test tube G and small amount of 1% sodium citrate

in test tube H. The chemicals were dissolved in test tubes F, G and H when blood was added in the

tubes. Additional 1% calcium chloride was also mixed in the test tubes F, G and H, it was stirred

until the suspension of fine threads was observed. The test tube I with the blood sample was

continuously stirred, the clotting time of all the test tubes were identified except for test tubes F,

G and H.
Results and Discussion

Table 1. Blood type classification using the ABO blood typing system

Name Blood Type (ABO system)


Caparro Type B
Serdan Type O
The data above shows the blood type based on the result that was seen during the exercise.

Agglutination was observed when the blood sample was mixed with the anti-B serum. Blood type

O was also identified when there was no agglutination that happened when blood was mixed with

anti-A serum and anti-B serum.

3.333333333, 3%
20, 20%
A
B
AB
63.33333333, 63% 13.33333333, 14%
O

Figure 1. Blood type distribution of the whole class

The table above shows that most of the people in class has a blood O and only a few

percentages are blood type AB.

The antigen-antibody interactions are the result of the agglutination which is the clustering

of the red blood cells in the liquid plasma. Antigens which are found in the red blood cell’s surface

is a biological signature of an individual’s blood type. The antigens are specific and are different
from other blood type. The antibodies in the blood recognize foreign blood types, which is why it

is necessary to test the blood’s compatibility before blood transfusion. When the antibodies

recognize the foreign antigens clumping of red blood cells occurs or the so-called agglutination

(O’Neil 1999).

Table 2. Clotting time of Human and Rat

Subject Clotting Time (min)


Human 2.13 min
Rat 1 min
Clotting time may also differ based on the organisms. Environmental factors may also

affect the agglutination of the blood.

Blood clotting occurs when the body tissue is damaged, it happens to prevent more

complicated damages, prevents severe physiological damages like blood loss. When the body

starts to bleed the coagulation cascade initiates the sequence of the clotting factor activities. The

clotting is mainly the function of the plasma and is mainly dependent on the interaction of the

plasma proteins, phospholipid, Ca++ and on the final stage it involves the interaction with the

thrombin which over all results to the conversion of soluble plasma protein fibrinogen to insoluble

fibrin which is what we observed in the laboratory. The blood contains receptors that binds with

the fibrin and later on attracts more platelets and fibrin to be able to form a clot. (Randall et all.

2002)
Figure 2. Theoretical Clotting Time in Different Conditions

The coagulation time is expected to be longer at the test tube lined with the paraffin wax,

compared to the empty test tube. One of the regulators of the conversion of prothrombin to

thrombin is released during the breakdown of the platelets to thromboplastin. The rate of the

coagulation depends on the said process and the coagulability and fluidity of blood is dependent

on the concentration of the said substances in the blood circulating the body (Donovan et al. 1949).

On the other hand, glass is identified as an activator of blood plasma which is also said to contribute

to the higher rate of coagulation in a glass surface. It is also said that thromboplastin increases in

a glass container this helps explain the longer time for the test tube lined with the paraffin (Lozner

et al. 1942). The activation of thromboplastin contributes to the higher coagulability of the blood

(Donovan et. 1949).

The blood clots also relatively faster in test tube with cotton fibers. The presence of the

fibers and some exposed glass surface agitates and activates the coagulation of blood (Tocantins

et al. 1951).
Stirring is said to make the clotting time slower or longer compared to set up where it was

just left to stand to clot. When stirring the fibrin adhere to the stirring rod thus preventing

coagulation (Fa 2009).

Sodium citrate is one of the anticoagulants that prevents the coagulation of blood without

it altering the composition of the plasma. The sodium citrate removes calcium ions in the blood.

The calcium ion is essential in the coagulation process of the blood. The sodium citrate makes the

blood inactive thus no clotting will be observed. The sodium oxalate functions the same way with

the sodium citrate, it binds with the calcium in the blood thus forming insoluble calcium oxalate,

in this way calcium is not available thus clotting is not possible (Pal et al. 2005).

The blood clotting mechanism is much faster in higher temperature than in lower

temperature. Low temperature inhibits the effects on procoagulant enzymes which makes the

platelet disfunction which results to reduced platelet aggregation and this have a great impact on

the blood coagulation (Thorsen et al. 2011). On the other hand, blood agglutinates into clumps,

making it almost impossible to flow at high temperature (Addis 1908).

References:

O’Neil, D. (1999). Blood Components.Retrieved

fromhttp://anthro.palomar.edu/blood/blood_components.htm.

Randall, D., Burggren, W., & French, K. (2002). Eckert Animal Physiology, 5th edition. New

York: W.H. Freeman and Company.


Donovan, T.J., Zimmermann, B. (1949). The effect of artificial surfaces on blood coagulabiity,

with special reference to polyethylene. Blood 4 (12): 1310-1316.

Lozner, E.L., Taylor, F.H.L., & MacDonald,H. (1942). The effect of foreign surfaces on blood

coagulation. The Journal of ClinicalInvestigation 21 (2): 241-245.

Tocantins, L.M., Carroll, R.T., & Holburn,R.H. (1951).The clot accelerating effect of dilution on

blood and plasma. Relation to the mechanism of Coagulation of normal and hemophilic

blood.Blood 6 (8): 720-739.

Fa, J.T. (2009). Lecture 6&7: Blood Utilization.

Pal G.K., & Pal, P. (2005).Textbook of Practical Physiology, Second Edition.Chennai, India:

Orient Longman PrivateLtd.

Thorsen, K., Ringdal, K.G., Strand, K.,Soreide, E., Hagemo, J., & Soreide, K.(2011).Clinical and

cellular effects of hypothermia, acidosis ad coagulopathy inmajor injury.British Journal of Surgery

98(7): 894-907.

Addis, T. (1908).The coagulation of blood in man. Quarterly Journal of Experimental Physiology

1: 305-334.