Al-Quds University ‫جامعت القذس‬

Faculty of Health Professions ‫كليت المهن الصحيت‬

Medical Laboratory Sciences ‫دائرة العلوم المخبريت الطبيت‬
Department

Coagulation and Hemostasis

Laboratory Manual
(0202311)

Instructor:
Dr. Montaser Haddad

Teaching Assistant:
Ms. Mai Abd Al-Salam

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Table of Contents;
Overview ______________________________________________________________________________ 2

Introduction (Revision Part)Laboratory safety ________________________________________________ 5

Lab no.1 Introduction to Coagulation Lab ___________________________________________________ 8

Lab n.2 Platelet Count: Manual and automated ______________________________________________ 13

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 Overview
General Information:

 Course title: Coagulation and Hemostasis

 Course code: 0202311
 Number of credit hours: 3 CH (Theory: 2 Cr./ Lab: 1 Cr)
 Course Classification: MLS Required Course
 Term: Spring Semester
Course Description

Through the study of this course, students will learn the laboratory methods used to
detect quantitative and qualitative abnormalities in elements of the coagulation process.
Also, it discusses the coagulation factors involved in blood clotting and the associated
diseases.
Learning goals

1. Become familiar with the “best practices” of laboratory procedures in Coagulation

lab.
2. Demonstrate adequate knowledge and application of the theory and practical skills.

Course General Objectives

1. List the anticoagulants used in venous blood collection for coagulation studies,
select the anticoagulant of choice and give reasons for your choice.
2. Describe the sources of error involved with specimen collection and handling
and explain their effect on test results.
3. Understand the coagulation cascade and platelet structure.
a. Describe thrombocytopathies and cite examples.
b. Describe the Brecher-Cronkite and Ress-Ecker methods for platelet counts.
c. Compare and contrast the two methods.
d. Correlate the platelet count with the number of platelets seen on the smear.
e. Associate platelet count results with specific clinical conditions
4.Understand the interactions between all systems involved in hemostasis.
5.Apply objectives a-f to the tests listed below:
a. Describe the principle of each procedure.
b. Outline steps in the procedure including materials and reagents needed.
c. Identify factors tested for, and recognize abnormal results.
d. Evaluate and correlate data with regard to clinical significance and possible
disease states.
e. Recommend additional tests to pinpoint specific deficiencies.
f. Identify sources of error and the QC necessary to minimize their effect.

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 Tests:
1. PT (Prothrombin Time).
2. APTT (Activated Partial thromboplastin Time).
3. TT (Thrombin Time).
4. FDP (fibrin degradation products).

Methods of Assessment

Continuous assessment is carried out all through the course. Students are subjected to
challenging questions during the lectures and are encouraged to interact by asking
questions, guess interpretations, solve problems and suggest appropriate decisions.
Students will be graded through the following means of assessment and their final grade
will be calculated from the forms of assessment as listed below:

Assessment Policy and Tools Practical

Midterm Exam 5%
-Lab Activity Worksheets and 3%
Quizzes
- Affective behavior 4%
Final Exam 8%
Total 20%

- The final exam will be a comprehensive exam including all the areas of the syllabus.
- Students are encouraged to review their Midterm Exam & Quizzes & assignments
results and discuss their mistakes.
- Make-up quizzes will only be permitted with prior arrangement or certified medical
excuse.

Laboratory Participation

1. Students must be present in lab and complete each exercise to get credit. Make-up
labs will only be permitted with prior arrangement or certified medical excuse.
2. Affective behavior is determined by the students’ adherence to safety regulations,
punctuality, etc.

Learning tasks

In order to obtain the above skills, the student should:

1. Print the experiment handout uploaded on e-class.
2. Read the experiment handout a day prior.
3. Bring the handout assigned for each lab.
4. Attend class lectures and laboratory sessions to take appropriate notes.
5. Carefully observe the instructor’s demonstrations.

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 6. Conduct the exercises as directed.
7. Participate in all Laboratory activities and complete all lab assignments on time.

References and Learning Resources

 Clinical Hematology and Fundamentals of Hemostasis by Denise Harmening

(5th edition), 2009, F.A Davis Philadelphia. (ISBN-0-8036-0135-2).
 Clinical Laboratory Hematology (ISBN-13: 978-0135137321).
 Clinical Hematology Atlas (ISBN-13:978-1455708307).
 NAACLS laboratory guidelines.
 Protocols are available in kit insert sheets.

Suggested Additional Resources

 Useful Web Resources:

 Essential Hematology. Hoffbrand. Blackwell Science. (ISBN 063-205-
1531).
 Practical Hematology. Dacie & Lewis. Churchill Livingstone (ISBN-13:
978-0702034084).
 Clinical Hematology Theory & Procedures. (ISBN 978-1-60831-076-0).
 Wintrobe’s Clinical Hematology.
 American Journal of Clinical Pathology.
 http://www.hematologyatlas.com/principalpage.htm.
 www.ascp.org http://www.ascp.org/and ASH image bank.
 www.bloodnet.comhttp://www.bloodnet.com/.
 www.cap.orghttp://www.cap.org/.

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 Introduction (Revision Part)
Laboratory Safety Techniques
Objectives

After attending a laboratory session on safety precautions, the student will:

1. Conduct professional practice according to established protocols, safety guidelines,
and existing legislation.
2. Use personal protective equipment, e.g., gloves, gowns, mask, and face shields,
aprons.
3. Utilize laboratory safety devices in a correct manner, e.g., biological safety cabinets,
fume hoods, laminar flow cabinets, safety pipetting devices, safety containers and
carriers, safety shower and eyewash stations.
4. Apply first-aid measures in response to incidents, e.g., chemical injury.
5. Apply spill containment and clean up procedures for dangerous chemicals according
to program policy.
6. Report incidents related to safety and personal injury (e.g., needle stick injuries) in a
timely manner.
7. Complete general Safety Checklist for Laboratories. Students should take the
initiative to locate or ask the instructor about the following safety items in every
laboratory they work in.

Course: Room Number:

Item: Information About:
Fire Safety:
Alarm pull stations
Fire extinguishers
Fire blankets
Emergency exit plans
Lab Waste:
Sharps containers
Biohazard waste boxes/red bags
Regular trash bins
Chemical Safety:
Emergency eyewash
Safety Shower
Flammables storagecabinets/rooms
MSDS sheets
Biological Safety:
Latex and non-latex gloves /Biohazard face shields/bench/Shields/
Disinfectants.
Other; First Aid Supplies, Hand washing sinks,
Lab coat storage.

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 Laboratory Safety Techniques

General Lab Safety Guidelines

Students should be familiar with safety procedures, follow instructions carefully and
take appropriate precautions at all times to insure the safety of every student in the lab
especially when hazardous conditions occur or hazardous materials are being used,
and they have to familiarize themselves with the emergency and fire procedures
reactions.

Personal safety rules

1. Students should dress appropriately in the lab.

(White, strong cloth, long to knees, long sleeves, tight wrist, pockets, etc.)
2. Wear suitable gloves, masks and goggles as needed.
3. Closed non-absorbent shoes must be worn (no slippers, sandals or open shoes).
4. Long hair must be tied.
5. Do not eat, drink, smoke, chew gum, apply cosmetics or remove/insert contact
lenses while in the laboratory.
6. Lessen your movement in the lab as
much as you can.
7. Avoid sudden movement and turning
around.
8. Playing around is prohibited during the
laboratory session.
9. Anyone injured in the lab, should
inform the instructor immediately and
take immediate action to reduce the
risk of further injury.

Lab. Environment
1. The lab environment should be
considered extremely dangerous,
poisonous and infectious.
2. Do not take any personal stuffs to the
lab (i.e. coats, bags, books, food, etc.).
3. All materials in the lab should be
considered infectious and dangerous;
so they must be handled carefully.
4. All samples and specimens with
biological origin (e.g., saliva, urine
and blood) or organisms, living or
dead, are infectious, even the source of
it is the student himself, his relatives or
friends, etc. should be treated with care and respect.

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 5. Students should be familiar with the evacuation plans.
6. Chemicals may be poisonous, corrosive, or flammable; no chemicals, even
chemicals known to be safe, should be tasted, ingested, inhaled, or touched unless
specifically directed to do so by your instructor.
7. Human organic must be disposed appropriately to eliminate any possibility for
contamination and the spread of disease.
8. The safe use of specific equipment and tools (e.g., microscopes, slides, scalpels,
and pipettes) will be demonstrated by the instructor during the laboratory sessions.
9. Students should remove and dispose of all trash, clean up any spills, return materials
where they belong, as instructed and disinfect the bench area.
10. After completing laboratory activities and cleaning up, students should wash their
hands to avoid contamination and hazardous chemicals.

Instructions

1. Do not do anything that you don’t know without asking.

2. Label all your samples clearly; the most dangerous substance is the one that has no
label.
3. Dispose all contaminated material in autoclave bags.
4. Do not take cultures out from lab area for any reason.
5. Immediately report all accidents and spills to the instructor.
6. It is important to know as much about a chemical as possible.
7. Do not use chipped or cracked glass wares.
8. Use hazardous chemicals under a fume hood.

Emergency Procedures for a Laboratory Fire

1. Do not use a fire extinguisher unless you are trained to use it.
2. In case of a small fire at your desk, smother with a book or piece of cloth; Not your
hands.
3. If your clothes are on fire, such as a sleeve, run water over it. If the fire has spread
beyond a small portion of clothing or appears that it will, use the fire blanket.
4. If a large fire occurs such as in a waste basket, smother the fire with the fire blanket.
5. If the fire is larger, do not attempt to put out the fire. Leave the laboratory
immediately.

Electrical Equipment

1. Do not spray or splash water to electrical equipment which is plugged in.

2. If equipment crackles, snaps or begins to smoke, call the instructor immediately.
3. In case of any worn or broken electrical cords, notify the instructor.

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 Lab no.1 Introduction to Coagulation Lab
Objectives

After attending a laboratory session on Introduction to Coagulation Lab, the student

will:
1. Understand the main role of Coagulation Screening Procedures.
2. Name disease state/deficiency when given results of coagulation tests.
3. List the most Laboratory Screening tests for detection of any abnormality
related to the coagulation system.
4. Correlate deficiency of coagulation factors with specific disease states.
5. Understand the basic requirements for accurate Coagulation tests.
6. List the anticoagulants used in venous blood collection for coagulation studies.
7. Understand the general role for performance of Coagulation Tests.
8. Describe the sources of error involved with specimen collection and handling
and explain their effect on test results.

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 Lab no.1 Introduction to Coagulation Lab

Coagulation Screening Procedures are mainly done to seek different variables, such
as; Clinical history of the patient and his family which performed by the physician,
for detection of:
1. An abnormality of primary hemostasis may present with easy bruisability (bruises,
petechiae) and or bleeding of the mucus membrane, (vascular or platelet problems).
2.Factor deficiency: generally, exhibit bleeding from the lager blood vessels.
(Hematoma, Bleeding in joints)
3.A family history of bleeding disorders may be helpful in the diagnosis of a
hereditary abnormality.
4. Drugs may induce bleeding: Aspirin, penicillin, (have been found to affect blood
coagulation).

The main Laboratory Screening tests are performed to follow any of these variables
are listed on the next table (Table 1), with mentioned the related causes.

Table 1; Shown Coagulation Laboratory Screening tests, and the evaluation of each one.
Test Name Evaluate
Examination of the blood smear Platelet morphology (giant, lack of granules ,clumping ,schistocytes
or target cells.)
Platelet count More or less than (150-400x103 /uL )

Bleeding Time (B.T.) Platelet function (primary hemostasis)

Clotting Time (C.T.) Contact factors (FXII, FXI, PreKallikrein , HMWK )

Prothrombin Time (P.T) (vitamin K dependent factors) Extrinsic coagulation system;

(FVII ,X , V, II, I, deficiency/ inhibitors)

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 Activated Partial Intrinsic coagulation system; (FXII,XI, IX, VIII, X, V, II, I ,
Thromboplastin Time (APTT) Fletcher, Fitzgerald, deficiency or inhibitors )
Quantitative tests For functional fibrinogen ( or Thrombin Time )
A factor assay May be done to determine the activity of the deficient factor .
Monitoring oral anticoagulant Either one or both PT, APTT is prolonged in the presence of a
therapy circulating anticoagulant. The PT is generally employed while in
Heparin therapy ,the APTT is employed.

 Requirements for accurate Coagulation tests should be taken into

considerations to get a represented accurate result are:

A. Collection of the blood sample

1. The anticoagulant of choice for the coagulation tests is Sodium Citrate (3.2% or 3.8%,
buffered or not). The usual ratio of blood to anticoagulant is 9:1. It is critical that the
proper amount of blood be added to the anticoagulant.

- Why do we need to use sodium citrate?

- What is the importance of the blood to anticoagulant ratio?

- What is the order of collection tubes?

2. If patient blood has a high hematocrit (Hct), will contain less plasma. So when blood is
centrifuged, plasma will contain more anticoagulant (increase Na citrate), leads to
prolonged clotting time in patients with 55% or higher, so during testing, as calcium is
added to the plasma, it will combine with the excess anticoagulant present instead of
being available for the coagulation process. Therefore: Hct > 55% or < 20%, amount of
Na citrate should be decrease. or increase. according to Hct.
Reading The following formula may be followed for determining the proper amount of
Na citrate to use, (If the final volume in the tube is 5 mL and 0.5 mL of Na citrate is
normally used).
Formula for 5.0 mL Sodium Citrate Tubes:
Anticoagulant vol. (X) = (100 – Hematocrit/595 – Hematocrit) X volume of anti-coagulated blood
required
Example: Patient hematocrit = 60%
((100 – 60) / 595 – 60) X 5.0 = .37 mL sodium citrate
*5.0 mL for standard

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3. A clean venipuncture is absolutely necessary. If any contamination of the blood with
Tissue Thromboplastin, false results will occur (shorter time). For routine coagulation
tests; vacutainer blood collection system is adequate, the coagulation tube should be the
second or third tube to be filled.
Note; When obtaining a blood sample for special coagulation procedure, a two – syringe
technique may be applied (using a butterfly system)/ (2 plastic syringes), withdraw 2 mL
into the first syringe, disconnect and discard, connect the second syring to the butterfly
and proceed. When testing for (factors XI, XII, Plt. function), plasticsyringes and tubes
(or siliconized) should be used.

B. Preparation of the plasma sample

1. Before centrifuging the blood sample, check each tube with applicator sticks for the
presence of the clot.
2. Coagulation Testing should be completed within 4 Hrs. of obtaining the sample from
the patients. The sample should be centrifuged within one hour of collection. The plasma
should be removed from the tube using a plastic pipette and placed in a stoppered tube.
Exposure of the plasma to air results in a pH change of plasma which may result in
invalidly prolonged clotting times. (CO2 changes pH).
3.To preserve the labile factors (FV, FVIII), the blood sample should be placed on ice (a
cup or tray of crushed ice) immediately after it is withdrawn.
4. Hemolysed plasma should not be used for coagulation studies. It tends to give shortened
clotting time. Also lipemic or icteric plasma should in general not be tested on instruments
designed to detect clots by Optical density methods.

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 C. Performance of Coagulation. Tests
1. All coagulation tests should be performed as soon as possible after the patient’s sample
has been received in the Lab.

2. Pipettes used should have a large bore; permitting rapid and complete pipetting.
Automatic pipettes are recommended.
3. Most coagulation tests are carried out at 37⁰c. It is essential, when required, that
the sample and the reagents reach the proper temp. of 37⁰c before proceeding with the
test. Prolonged heating may lead to destruction of some of the coagulation Factors,
and, therefore a prolonged clotting time. The temp. of the incubator should not fluctuate
morethan + 0.5⁰c.

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 Lab no.2
Platelet Count: Manual; using blood smear; and
automated
Objectives

After attending a laboratory session on Platelet Count Lab, the student will:
1. Describe the Brecher-Cronkite and Ress-Ecker methods for platelet count.
2. Compare and contrast of the two methods.
3. Perform platelet count by Rees – Ecker method
4. Correlate the platelet count with the number of platelets seen on the smear.
5. Describe and perform Electronic cell count.
6. Understand the principle of platelet count and interpretation of the results.
7. Associate platelet count results with specific clinical conditions.

Figure 1; Blood smear; RBCs & Platelets (arrows pointed on them).

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 Lab no.2
Platelet Count: Manual; using blood smear; and automated

Overview
Platelets or thrombocytes are disc-shaped cytoplasmic fragments, normally present in
the peripheral blood. Circulating platelets are produced from megakaryocytes primarily
in the bone marrow. Mature platelets, although small in size, they play an important
role in both primary hemostasis, along with the coagulation factors, and body’s
inflammatory response. There are three major methods of platelet count:
1. Rees- Ecker.
2. Brecker-Cronkite.
3. Electronic cell count.
For counting platelets blood sample is collected with EDTA, which helps to decrease
the clumping of platelets. Blood from finger, heel and toe, can be tested, but the results
are less satisfactory and significantly lower than venous blood.

Table 2; Explain the principles between the three methods of platelet count.

Method Brecker-Cronkite Rees- Ecker Electronic cell count

-Is considered as the reference -The diluting agent - Mainly done though CBC test.
method. consists of: Na - Electrical impedance proportional
to the cell volume is observed
-It's using phase microscope but citrate: 3.8 gm,
- Using this relationship between the
Principle the light microscope can be used. Formaldehyde 40% impedance signal and size of the cell

-This method uses the diluting = 0.2 ml, Brilliant / particle, Small cells – 2 to 30 fL –
relating to platelets.
agent : 1% Ammonium Oxalate cresyl blue 0.1 gm,
- A disadvantage of the electrical
,will lyse RBCs but preserve D. H2O: 100 ml. impedance method is the difficulty in
platelets , WBC , reticulocyte. -This will not lyse distinguishing large platelets from
extremely microcytic or fragmented
- Prevents the platelets RBCs .
RBCs.
aggregation.

 Experiment

TEST: Platelet count using Rees-Ecker method.

REAGENTS/SUPPLIES: Phlebotomy set, EDTA blood tube, hemocytometer,
serological test tubes, automated pipette, Na citrate: 3.8 gm, Formaldehyde 40% = 0.2
ml, Brilliant cresyl blue 0.1 gm, D. H2O: 100 ml.

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 PROCEDURE:
1. Withdraw blood, add 10 uL of blood to 1990 uL of diluent in a test tube. Mix well
by shaker or by hand (2to 3 min).
(Discard the 1st 4 drops if pipet is used)
2. Fill the two sides of the counting chamber.
3. Then count under the microscope using 40x. Count the entire center square (25
secondary squares x 16 tertiary squares) ( or 10 of the 25 secondary squares).
4. Calculate the number of plts / ul as follows:
Plts /µl = average no of plts . x dilution factor x volume correction factor
Y x 200 x 10
Plts /µl= av. no of plts. X 2000 (if 25 squares)
(Volume correction factor if 25 squares are counted: 1 x 1 x 0.1 = 0.1 µl.
so factor= 10)

Figure2; Hemocytometer (chambercount); the counting area.

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 5. To confirm Plt. Count indirect method:
A stained blood smear estimate: count plts. in 5 to 10 fields, take the average and
multiply by 20.000 (rough estimate). Normally 8 to 20 plts are found in each 100x
field (oil immersion).If RBCs are greater than or lower than 5 million: Correct the plt.
no by counting plt in 1000 RBCs and multiply by RBC count divided by 100.

INTERPRETATION OF RESULTS:
-The platelets will appear round, oval or rod shaped (smaller than RBCs), dense and
disk and light refractile (not to be confused with dirt particles) stain light blue.
-Normal platelets count = 150.000- 450.000/ µl.

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Table 3; Describe Terms& Conditions when platelet count is more/less than normal.

Thrombocytopenia Thrombocytosis
CASE (Platelet Count is < 100.000/uL) (Platelet Count is > 500.000/uL)
Thrombocytopenia Purpura Polycythemia Vera
Aplastic anemia Idiopathic Thrombocythemia
CONDIT Acute leukemia Chronic Myelogenous Leukemia
ION Pernicious anemia Following splenectomy
Radiation and chemotherapy

- Platelet Satellitosis (satellitism):

A phenomenon where a neutrophil is surrounded by platelets. (see next figure)

- Giant platelets: abnormally large platelets.

- Platelet clumping: occurs when the blood platelets responsible for coagulation stick to one
another to form clusters.

IMPORTANT NOTES;
- If clumps of platelet are present repeat the procedure. The platelets should be
evenly distributed within +/- 10 plts. difference in the primary squares in both
sides.
- If platelets < 50 in the primary square (or less than 100.000) use dilution 1:20
(or 1:100).
- If platelets > 500.000, use dilution 1:500.
- When pipette is removed from the mixer, it should not stand for more than 8 to
10 sec without remixing.
- Blood should be diluted and smear made within 5 hrs. of blood collection, or
within 24 hrs. if has been refrigerated.

- Count should be completed within 30 min to ensure no disintegration

(range of error 16 to 25).