Coagulation time test
and bleeding time test
• Submitted to:
• Mam Arooj
• Submitted by:
• Maira Naeem
• Hijab Fatima
Introduction
• The coagulation time test is a fundamental
laboratory procedure designed to measure the
duration required for blood to clot. This
assessment offers valuable information regarding
the overall functionality of the coagulation
cascade, which encompasses a complex
interplay of clotting factors, platelets, and
vascular elements. Monitoring coagulation time is
essential for diagnosing bleeding disorders,
assessing the impact of anticoagulant
treatments, and detecting possible liver
dysfunctions or vitamin K deficiencies. While
more advanced and specific tests, such as PTT,
aPTT, capillary tube method are frequently
employed, coagulation time continues to be a key
concept in the study of hemostasis and is still
applied in certain clinical and educational
contexts.
Methods of coagulation time test
❑ Activated Partial Thromboplastin Time Test (APTT)
❑ Partial Thromboplastin Time Test (PTT)
❑ Capillary Tube Method
Activated Partial Thromboplastin Time Test
(APTT)
• Definition:
The Activated Partial Thromboplastin Time (aPTT) test is a
laboratory blood test that assesses the duration required for
blood to clot through the intrinsic and common coagulation
pathways. This test evaluates the functionality of several
clotting factors, specifically factors I (fibrinogen), II
(prothrombin), V, VIII, IX, X, XI, and XII.Normal time for aptt is 25
to 35 seconds.
Purpose
❑ To identify bleeding disorders such as hemophilia A and B.
❑To oversee heparin treatment, particularly with unfractionated
heparin.
❑ To examine deficiencies in coagulation factors or the presence
of inhibitors, including lupus anticoagulants.
❑ To assess liver function and vitamin K levels, given that
numerous clotting factors are produced in the liver.
Materials • Laboratory Equipment:
• Centrifuge
a. Blood Collection: • Test tubes or reaction cuvettes
• Sterile syringe or vacutainer system • Water bath or incubator (set at 37°C)
• Tourniquet • Stopwatch or timer
• Alcohol swabs • Pipettes (manual or automatic)
• c. Reagents:
• Gloves
• aPTT reagent (contains surface activator and
• Light blue-top tube (3.2% sodium
phospholipids)
citrate)
• 0.025 M Calcium chloride (CaCl₂) solution
• Gauze and adhesive bandage
• Normal and abnormal control plasma
Sample Collection: Collect venous blood using a citrate
anticoagulant tube (light blue top). Ensure the blood-to-
Procedure anticoagulant ratio is maintained at 9:1.
Plasma Preparation: Centrifuge the blood sample at a force
of 1500–2000g for a duration of 10–15 minutes to separate
platelet-poor plasma. Carefully extract the plasma, taking
care not to disturb the buffy coat.
Pre-Warming: Pre-warm the plasma and reagents to a
temperature of 37°C for 3–5 minutes.
Test Reaction: In a test tube, combine: 0.1 mL of test plasma
0.1 mL of aPTT reagent Incubate the mixture at 37°C for 3
minutes. To initiate clotting, add 0.1 mL of pre-warmed
0.025M CaCl₂.
Timing: Start the stopwatch immediately after adding CaCl₂.
Record the time taken for a visible clot to form (end-point).
Controls: Conduct the test using both normal and abnormal
control plasma to verify accuracy.
Capillary Tube Method (for clotting time)
• Introduction:
The capillary tube method, commonly referred to as Wright’s method, is a straightforward and frequently
employed laboratory technique for measuring the clotting time of blood. This test acts as a fundamental
screening mechanism for evaluating the functionality of the intrinsic coagulation pathway and aids in
identifying bleeding disorders, including hemophilia and conditions that impact platelet function.
In this procedure, blood is collected into a slender, sterile capillary tube through capillary action following
a skin puncture. The tube is subsequently broken at intervals until a fibrin thread is detected between the
fractured ends, indicating the moment of clot formation. Normal clotting time in this method is 2 to 8
minutes.
Materials
• Sterile lancet or needle (for finger prick)
• Capillary tubes (thin, disposable glass tubes)
• Stopwatch or timer
• Cotton or gauze
• Spirit or alcohol swabs
• Gloves
• Warm water bath or hand warmer (optional, to maintain 37°C)
Procedure
• Preparation:
• Clean the fingertip with an alcohol swab and allow it to dry.
• Wear gloves and prepare all materials.
• Blood Collection:
• Prick the fingertip using a sterile lancet.
• Wipe away the first drop of blood.
• Allow a second large drop of blood to form.
• Filling the Tube:
• Fill 2–3 capillary tubes (about 5–7 cm each) by touching them to the blood drop.
• Start the stopwatch as soon as blood enters the first tube.
• Testing for Clotting:
• Keep the tubes at body temperature (ideally 37°C).
• After 1–2 minutes, gently break off a small section (0.5–1 cm) of the tube every 30 seconds.
• Observe for the appearance of a fibrin thread between the broken ends of the tube.
• End Point:
• Stop the timer when the first fibrin strand is seen.
• Record the clotting time.
Partial Thromboplastin time test (PTT)
• Introduction:
The Partial Thromboplastin Time (PTT) test assesses the duration required for blood to clot by examining the
intrinsic and common pathways of the coagulation cascade. Its applications include:
• Identifying bleeding disorders
• Monitoring heparin treatment
• Assessing deficiencies in clotting factor
• Investigating cases of unexplained bleeding or bruising
When an activator, such as kaolin or silica, is introduced, the test is known as the Activated Partial
Thromboplastin Time (aPTT), which enhances its sensitivity and makes it a frequent choice in clinical practice.
Materials
• Laboratory Equipment:
• Centrifuge
• . Blood Collection: • Test tubes or reaction cuvettes
• Sterile syringe or vacutainer system • Water bath or incubator (set at 37°C)
• Tourniquet • Stopwatch or timer
• Alcohol swabs • Pipettes (manual or automatic)
• Gloves • c. Reagents:
• Light blue-top tube (3.2% sodium • aPTT reagent (contains surface
citrate) activator and phospholipids)
• Gauze and adhesive bandage • 100 ul Neoplastin Cl Plus
• Normal and abnormal control plasma
Procedure
• Place cuvette in the incubation well for at least 3 minutes for prewarming at 37°C.
• Dispense one steel ball in the cuvette.
• Add 50 ul of patient citrated plasma in the cuvette.
• Start the timer and incubate for 60 seconds.
• Once the instrument start beeping, transfer the cuvette to measuring area and add 100 ul of SR (Start
Reagent i.e. Neoplastin Cl Plus).
• Steel ball starts vibrating under magnetic effect and timer starts simultaneously.
• Clot formation arrest the steel ball and stops its vibration.
• Time for clot formation is recorded automatically by the instrument
• Normal time is 12 to 16 seconds.
Bleeding time by Duke's Method
• Definition of bleeding time:
Bleeding time is a diagnostic procedure utilized to evaluate primary hemostasis, which refers
to the capacity of blood vessels and platelets to halt bleeding following a vascular injury. One of the
earliest and most straightforward methods for determining bleeding time is Duke’s Method. This
technique involves creating a standardized puncture on the fingertip or earlobe and timing the
duration until the bleeding ceases.
Materials
• Sterile lancet or needle
• Filter paper or blotting paper
• Stopwatch or timer
• Spirit/alcohol swab
• Cotton and gloves
PROCEDURE • Clean the earlobe or fingertip with an alcohol swab and let it
dry.
• Puncture the site with a sterile lancet deep enough to cause
continuous bleeding.
• Start the stopwatch immediately.
• Blot the blood every 30 seconds using clean filter paper,
without touching the wound.
• Be gentle to avoid disturbing clot formation.
• Continue blotting until there is no more blood stain on the
paper.
• Record the time from puncture until bleeding stops — this is
the bleeding time.
•
• Normal Range:
• 2 to 5 minutes
