COLEGIO SAN AGUSTIN-BACOLOD

College of Health and Allied Professions MLS 102 LABORATORY ACTIVITY SHEET
Medical Technology Program
LABORATORY ACTIVITY #10
BLEEDING TIME AND CLOTTING TIME

I. Desired learning outcomes

When a vessel is injured in patient with a normal coagulation function, platelets
adhered to the exposed wound and lining of vessel to form a primary platelet plug. The bleeding
time is a measure of the functional integrity of the small vessels and the ability of platelets to
form hemostatic plugs to stop bleeding. (Hoeltke, 2018)
Clotting time is the time required for a sample of blood to coagulate in vitro under
standard conditions. (clotting time, ND)

After performing this activity, students should be able to:

1. List several methods and explain the principle of tests for clotting and bleeding time.
2. Determine factors affecting clotting and bleeding time.
3. Perform clotting and bleeding time.

II. Materials
lancets 70% isopropranol/tincture of iodine
red top evacuated tube (3) Cotton balls/gauze
ETS multiple use needle Tourniquet
Filter paper Adhesive tape
Timer water bath
Blue tip capillary tubes (4) distilled water

III. Procedure
A. General procedure:
1. Prepare your 10% disinfectant. Make sure to label properly the container.
2. Clean the bench area. Attach in the right side of your working table a small plastic to
serve your trash bin.
3. Assemble all required materials, including the dummy laboratory request and ball pen
Decide among the group members who will serve as the phlebotomist and the patient
or you can invite a person to serve as patient.
NOTE:
Scarring can occur at the site of the BT and patients should be warned of this. For these
reasons, the BT is rarely performed in children.
4. Wear complete PPE.
5. Perform hand hygiene before wearing and after removing gloves.

B. Bleeding Time -Ivy method (clotting time,ND; Hoeltke, L (2018)

1. Make sure that your patient is comfortably seated.
2. Introduce your name and explain briefly the procedure to be done.
3. Identify your patient properly and verify the data in your laboratory request.
4. Assess alcohol/latex allergy.

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College of Health and Allied Professions MLS 102 LABORATORY ACTIVITY SHEET
Medical Technology Program
5. Place a blood pressure cuff on the upper arm and inflate to 40 mmHg.
6. Apply antiseptic into the intended site in an inside to outward circular motion.
7. Use a disposable lancet to make two separate cuts into the forearm usually 5-10cm
apart in quick succession.
8. A stopwatch is started immediately and every 30 seconds filter paper is used to draw off
the blood. The filter paper should not touch the edge of the clot as this may disturb the
formation of the platelet plug.
9. The time from when the incision is made until all bleeding has stopped is called the
bleeding time.
10. The test is finished when bleeding has stopped completely.

Interpretation
The test is dependent upon an adequate number of functionally active platelets that can
adhere to the endothelium to form aggregates.

Reference Ranges
The reference range for this test is between 2-7 minutes. In cases in which the BT
exceeds 20 minutes it is usual to stop at 20 minutes and report the BT as >20minutes.

C. Clotting time (Dhage, H , ND)

c.1. Capillary Tube Method:
1. Clean the tip of a finger with 70% isopropanol.
2. Puncture it up to 3 mm deep with a disposable lancet.
3. Start the stopwatch.
4. Wipe the first drop of blood.
5. Fill two capillary tubes with free flowing blood form the puncture.
6. Keep these tubes at body temperature.
7. After 2 minutes, start breaking the capillary tube at 1 cm distance to see whether a thin
fibrin stand is formed between the two broken ends.
8. Stop the watch and calculate the time from average of the tow capillary tubes.

c.1.a. Disadvantages:
1. Method is insensitive.
2. Method is unreliable.

c.1.b. Advantage:
1. It can be performed when venous blood cannot be obtained.

c.1.c. Reference values

1. Normal clotting time is 1-5 minutes.

c.2. Lee and White Method:

1. After cleaning the forearm, make a venipuncture to draw 3 ml of blood.
2. Start the stopwatch.
3. Transfer 1 ml of blood each into 3 glass tubes and incubate at 37° C in a water bath.

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College of Health and Allied Professions MLS 102 LABORATORY ACTIVITY SHEET
Medical Technology Program
4. After 3 minutes tilt the tubes one by one every 30 seconds.
5. The clotting time is taken when the tubes can be title without spilling of their contents.
6. Calculate the clotting time by average of 3 tubes.

c.2.a. Advantages:
1. More accurate and standard method.
2. Test can be run with control.

c.2.b. Disadvantages:
1. It is also a rough method.
2. There can be contamination of syringe or tube.
3. The temperature should be maintained because higher temperature accelerates
clotting.
4. The diameter of the glass tubes should be uniform because clotting is accelerated in
narrow tubes.
5. Vigorous agitation of the tubes should be avoided as it shortens the clotting time.

c.2.c. Reference values

Normal clotting time is 5-10 minutes.

c.2.d. Clotting time is prolonged in following conditions:

1. Severe deficiency of coagulation factors.
2. Afibrinogenaemia.
3. Administration of heparin.
4. Disseminated intravascular coagulation (DIC).
5. Administration of drugs such as anticoagulants.

IV. Guide Questions

1. List several methods (other than listed in this activity) and explain the principle of tests
for clotting and bleeding time.
2. What factors and explain how these factors can affect clotting and bleeding time?

Clotting time (ND) retrieved from https://www.google.com/search?rlz=1C2AVSA_enPH527PH527&ei=

IYwtXJTmE5DmwQPFhpLgCg&q=clotting+time
Dhage, H (ND). Top 2 Methods for Clotting Time | Blood Clot | Biology retrieved from http://www.biologydiscussion.com/hematology-2/blood-clot/top-2-methods-for-clotting-

time-blood-clot-biology/
Hoeltke, L (2018) The compete textbook of phlebotomy. 5 th ed. Canada: Thomson Delmar Trading

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COLEGIO SAN AGUSTIN-BACOLOD
College of Health and Allied Professions MLS 102 LABORATORY ACTIVITY SHEET
Medical Technology Program

NAME OF STUDENT: Maelyn Dujale BSMT1-C DATE PERFORMED: 02/24/20

TITLE OF LABORATORY ACTIVITY: Lab. Act. #10 BLEEDING TIME AND CLOTTING TIME DATE SUBMITTED: 03/02/20

I. Observation/Results

Bleeding time (Ivy Method)

Place a blood pressure cuff on the upper arm and inflate to 40 mmHg. Use a disposable lancet to make
two separate cuts into the forearm usually 5-10cm apart in quick succession. A stopwatch is started
immediately and every 30 seconds filter paper is used to draw off the blood. The filter paper should not
touch the edge of the clot as this may disturb the formation of the platelet plug. The test is dependent
upon an adequate number of functionally active platelets that can adhere to the endothelium to form
aggregates.

Clotting time

Puncture it up to 3 mm deep with a disposable lancet. Start the stop watch then wipe the first drop of
blood. Fill two capillary tubes with free flowing blood form the puncture. Keep these tubes at body
temperature. After 2 minutes, start breaking the capillary tube at 1 cm distance to see whether a thin
fibrin stand is formed between the two broken ends. Stop the watch and calculate the time from
average of the tow capillary tubes.

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College of Health and Allied Professions MLS 102 LABORATORY ACTIVITY SHEET
Medical Technology Program

Lee and White method

Make a venipuncture to draw 3 ml of blood. Start the stopwatch. Transfer 1 ml of blood each into 3
glass tubes and incubate at 37° C in a water bath. After, 3 minutes tilt the tubes one by one every 30
seconds. The clotting time is taken when the tubes can be title without spilling of their contents.
Calculate the clotting time by average of 3 tubes. Normal clotting time is 5-10 minutes.

II. Discussion
List several methods (other than listed in this activity) and explain the principle of tests for clotting and
bleeding time.

 Prothrombin Time Test (PT)

 The hemostasis pathway is normally activated when damage occurs to blood vessel
endothelium or to body tissue. The extrinsic pathway converts Factor X, a proenzyme to
the enzyme X, which in turn converts prothrombin to the enzyme thrombin. An enzyme
is a protein that is able to cause or accelerate changes in other substances without being
changed. Thrombin acts on fibrinogen to form fibrin monomers that make up the initial
unstable clot.

 Activated Partial Thromboplastin Time Test (APTT)

 Activated partial thromboplastin time (APTT) is a commonly used coagulation assay that
is easy to perform, is affordable, and is therefore performed in most coagulation
laboratories, both clinical and research, worldwide. The APTT is based on the principle
that in citrated plasma, the addition of a platelet substitute, factor XII activator, and
CaCl2 allows for formation of a stable clot. The time required for the formation of a
stable clot is recorded in seconds and represents the actual APTT result.

 Fibrinogen Activation Test

 A fibrinogen activity test evaluates that part of the clotting process in which
soluble fibrinogen is converted into fibrin threads. It measures the time that it takes for
a fibrin clot to form after a standard amount of thrombin is added to your blood sample
(plasma).

 PIVKA Test

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COLEGIO SAN AGUSTIN-BACOLOD
College of Health and Allied Professions MLS 102 LABORATORY ACTIVITY SHEET
Medical Technology Program
 The thrombotest or PIVKA test evaluates proteins induced by vitamin K
absence/antagonism (PIVKA).  These proteins are not activated in the presence of
vitamin K antagonists or vitamin K deficiency, resulting in a prolonged thrombotest, in
which clot formation is dependent on vitamin K dependent factors.

 D-Dimer Test
 D-Dimer proteins in the sample bind to the specific anti-D-Dimer antibody, which is
coated on latex particles, and causes agglutination. The degree of the turbidity caused
by agglutination can be measured optically and is proportional to the amount of D-
Dimer in the sample.

 Activated Coagulation Time (ACT)

 A modification of the APTT, the ACT assesses the intrinsic and common pathways in
whole blood and requires patient platelets and calcium. It is less sensitive than the
APTT but is an available quick in-house screening test.

The coagulation factors are numbered in the order of their discovery. There are 13 numerals but only 12
factors. Factor VI was subsequently found to be part of another factor. The following are coagulation
factors and their common names:

 Factor I - fibrinogen
 Factor II - prothrombin
 Factor III - tissue thromboplastin (tissue factor)
 Factor IV - ionized calcium ( Ca++ )
 Factor V - labile factor or proaccelerin
 Factor VI - unassigned
 Factor VII - stable factor or proconvertin
 Factor VIII - antihemophilic factor
 Factor IX - plasma thromboplastin component, Christmas factor
 Factor X - Stuart-Prower factor
 Factor XI - plasma thromboplastin antecedent
 Factor XII - Hageman factor
 Factor XIII - fibrin-stabilizing factor

According to (Cannon and Mendenhall 1914), this great discrepancy shows that there is no definite
coagulation time quite independent o the method used. The conditions are peculiar to any coagulation
are likely affects the time of clotting. Since to draw blood any instrument is a foreign body, all that is
required of is that the conditions of its use shall constants. The overwhelming majority of clotting factors
are manufactured principally in hepatocytes. Hepatocytes are responsible for providing the body with
clotting factors XIII, XII, XI, X, IX, VII, V, II, and I. Clotting factors VIII (antihemophilic factor A), and III
(tissue factor) originate from endothelial cells, whereas clotting factor IV (calcium ion) is freely available
in plasma. Megakaryocytes produce the body’s platelets and also contribute to the production of factor
V.

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College of Health and Allied Professions MLS 102 LABORATORY ACTIVITY SHEET
Medical Technology Program

III. Conclusion

This laboratory activity that we perform was really important to determine the time to
takes it to stop the bleeding time and this test is useful for determining the abnormalities of platelets.
When performing this test need to determine the proper sites of different kinds of venipuncture. I
determine the different factors of clotting and bleeding time even though it’s hard for us because we’re
only a beginner for this but it’s also a part for us, as a medical technology student to know the different
factors.

IV. References

Estridge,B., Reynolds,A.and Walters,N. (2020). Basic Medical Laboratory Techniques. [online] Google

Books. Retrieved from: https://books.google.com.ph/books?
id=qMgAbOHSlsMC&pg=PA245&dq=what+are+the+other+methods+except+for+clotting+and+bl
eeding+time.&hl=en&sa=X&ved=0ahUKEwioguvtj_jnAhULL6YKHZGEA9IQ6AEIODAC#v=onepage
&q=what%20are%20the%20other%20methods%20except%20for%20clotting%20and
%20bleeding%20time.&f=false. [Accessed 1 Mar. 2020].

Cannon, W. B., & Harvard Medical School. (1914, May 1). FACTORS AFFECTING THE COAGULATION TIMe
OF BLOOD. Retrieved from
https://journals.physiology.org/doi/abs/10.1152/ajplegacy.1914.34.2.225?
journalCode=ajplegacy.

Tests. (n.d.). Cornell University College of Veterinary Medicine. Retrieved from:

http://eclinpath.com/hemostasis/tests/

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