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General Approach of

Haemostasis

Lecture 3:
Coagulation Time of whole blood
Coagulation Time ( Clotting Time) CT.
 Clotting Time is the time required for blood to form
a clot in vitro.
 The basis for the test is that whole blood will form a
solid clot when exposed to a foreign surface such
as a glass tube.
 It is a rough measure of all intrinsic clotting factors
in the absence of tissue factors. Variations are
wide and the test sensitivity is limited.
 As the test is the least effective test in the diagnosis
of actual haemostasis failure, it has been replaced
by PTT.
 Clotting time was used as a screening test to
measure all stages in the intrinsic coagulation
system and to monitor heparin therapy .
 It is however, a time-consuming test, has poor
reproducibility, is sensitive only extreme factor
deficiencies. It is therefore, of limited use in
today’s laboratory .
 It is a complex process involving over 30
substances
 Conditions accompanied by increased Clotting
Time:
 Factors V, VII, VIII, IX, XI, XII Deficiencies.
 Hemorrhagic disease of Newborn
 Vitamin K deficiency.
 Heparin Therapy.
 Presence of Circulating antibodies (inhibitors)
 Anemia and leukemia.
 Afibinogenemia and Pneumonia.
 and heparin.
Methods:
 Capillary Method.

 Slide Method.

 Tube Method.
Tube Method (Lee-White method)

 Reagent & equipment


 Water bath, 37O C.
 Glass test tube (10 x 75 mm)
 Stopwatch.
 Plastic syringe and 20-gauge needle.

 Specimen
 Fresh whole blood , 4 ml .
Procedure
1. Label 3 glass test tube with patient name and
number them, 1, 2, and 3.
2. Perform a clean, Untraumatic venipucture using a
20-gauge needle and drawn 4 mL of blood.
3. Remove the needle from the syringe, and fill
each of the three tubes with 1 ml blood.
 The last 1 ml of blood may be discarded.
 Start the stopwatch as soon as the blood
enters the syringe.
4. Place the three test tubes in a 37°C water bath.
5. At exactly 3 min., Remove the first tube form
water bath and tilt gently to a 45° angle to see
whether the blood has clotted.
6. If Blood not clotted return it to the water bath
and examine it at 30 second intervals.
5. After the blood in the first tube has
clotted, examine the second tube
immediately.
6. Then examine the 3rd one.
7. Record the time it took the blood in the 3rd
test tube to clot.

Normal Range:
5 – 12 Minutes***
Discussion
1. Poor venipucture technique, causing hemolysis or
tissue thromboplastin to mix with the blood, shortens
the clotting time.
2. Bubbles entering the syringe when the blood sample is
being obtained increase the rate of coagulation.
3. Always tilt the tube in the same direction and at the
same angle so that the blood is moving in the same
pathway up the side of the tube each time.
4. Excessive agitation of blood (occur during the transfer
of the blood from the syringe to the test tube). The
blood should be allowed to flow gently down the
inside of the test tube and not forcefully squirted into
the test tube.
5. At the completion of the clotting time, one tube
should remain in the 37°C water bath to be checked
for clot retraction. Also, this same tube may be
allowed to remain in the water bath overnight and
checked the next day for clot lysis.
6. Dirty Test Tubes will affect the result.

 Coagulation will be retarded by the following:


 Temperature Below 35O C.
 Temperature above 45O C.
 Diameter of the test tube (the smaller the diameter, the
more rapid the clot formation is).
ACT (Activated clotting Time)
 Fresh whole blood is added to a tube containing
negatively charged particles and timed for the
formation of a clot.
 The type of negatively charged particle affects
the "normal" length of ACT
 Celite = Diatomaceous Earth: normal is 100-
170 seconds
 Kaolin: normal is 90-150 seconds
 Glass particles: normal 110-190 seconds
 Type of machine affects normal and therapeutic
values
 Daily calibration checks are imperativee

 Therapeutic Range: 180-240 seconds.


Hemochron®
 Tests measured
 Heparin monitoring with or without Aprotinin.

 Sample and volume requirements


 Fresh whole blood- 2mL. Elite: 200µL to fill cuvette well.

 Controls
 Electronic QC
 Liquid quality control- normal and abnormal

 Reference Ranges
 Normal range- healthy donors
 RFTCA 510 tubes: 105 – 167 seconds
 FTK-ACT tubes: 91 – 151 seconds
 Normal range- cardiopulmonary bypass patients
 HRFTCA 510 tubes: 86 - 147 seconds
 FTK-ACT tubes: 89 - 153 seconds
Clotting Time - Capillary method
Material

 Sterile disposable pricking needle or lancet.


 Stop watch
 Dry glass capillary tube (narrow diameter top 2
mm, minimum 10 cm long.)
 Cotton Swab of absorbent cotton.
 Spirit wetted, cotton swab.
 70 % v/v ethyl alcohol
PROCEDURE
1. Apply alcoholic 70 % v/v to the clean finger with cotton
swab. Allow it to dry naturally.
2. Prick the finger with usual aseptic precautions. Remove the
first drop.
3. Dip one end of the capillary into blood drop gently without
pressure. (use 3-4 capillary tubes)
4. The Timer is started when the first blood start to enter the first
capillary tube.
5. Allow to fill the capillary with blood by lowering the end of
fitted capillary. (Do not suck the blood) around ¾th of its
length undipped.
6. After every 30 seconds, using stopwatch, break a small
piece of capillary.
7. Repeat breaking at regular time intervals, till fibrin thread
appears at the broken end of capillary tube. Do not pull
away the cut pieces ling apart and bristly.
8. Record time interval between pricking finger and first
appearance of fibrin thread at the broken ends of capillary
tube. That is clotting time of blood.
9. Don't forget to dispose of the broken tube in the SHARPS
CONTAINER.
Clotting time of whole blood
Clotting Time - Slide Method

 The surface of the glass


tube initiates the clotting
process. This test is
sensitive to the factors
involved in the intrinsic
pathway
 The expected range for
clotting time is 4-10 min.
Clot retraction (Obsolete test)
 This test measures the amount of time it takes for a
blood clot to pull away from the walls of a test tube
(Shrinking).
 The edges of the blood vessel wall at the point of
injury are slowly brought together again to repair
the damage.
 It is used to evaluate and manage blood platelet
disorders, including Glanzmann's thrombasthenia
 So Clot retraction depends primarily on the number
and activity of the blood platelets.
 This test is reliable only when the concentration of
fibrinogen within normal limits.
 Normally a blood clot will begin to retract from the
walls of the tube and express serum within one hour.
Glanzmann's thrombasthenia
 Is an abnormality of the platelets
 It is an extremely rare coagulopathy in which the
platelets lack glycoprotein IIb/IIIa .
 Reduced glycoprotein IIb/IIIa causes reduced
platelet aggregation and clot retraction - no
fibrinogen bridging can occur, and bleeding time is
significantly prolonged);
Clot retraction test
 This is a measure of platelet function. The test is
done in blood to which no anticoagulant has
been added and is allowed to clot. Clot
retraction is then looked for.
 Clot retraction becomes abnormal in conditions
like
 Fibrinogen deficiency (congenital or acquired)
 Thrombocytopenia < 1ooooo
 Thrombosthenia
 Polycythaemia
 Reduced clot formation: Glanzmann
thrombasthenia , DIC, hypofibrinogenemia,
dysfibrinogenemia (small clot with increased
red blood cell “fall-out”)
Procedure
1. Obtain 2 ml of blood by venipucture. Place
one ml in a glass test tube in a 37oC water
bath.
2. Inspect clot at 1, 2, 4 and 24 hours after
clotting. Observe for retraction, quality,
and lysis of the clot.
3. Remaining 40-60% consists of serum and
red blood cell “fall-out” from clot
4. A visual examination of the clot is made.
Usually the clot retracts and expresses
serum within two hours.
Interpretation of results
 The clot will retract from the walls of the tube until
the red cell mass occupies 50% of the total volume
of blood in the tube
 There is a variable degree of retraction or there is
no retraction at all. Grades as follows:
 0 : no serum extruded
 1+ : 5-10% serum extruded
 2+ : 10-20% serum extruded
 3+ : 20-35% serum extruded
 4+ : 35-50% serum extruded
 Results are reported as the length of time it took for
the clot to retract
 2-4 hrs is reported as normal
 After 4 hrs is reported as poor
 After 24 hrs is reported as no retraction
Clot lysis test
 The whole blood clot lysis time is used to detect
increase fibrinolysis
 This test is only able to detect high increase in
fibrinolytic activity
 Clot lysis testing is a measure of the presence of the
clot after 24 hours (blood fibrinolysis).
Procedure
1. At the completion of the clotting time one tube
should remain in the 37 water bath
2. A second tube is placed in the refrigerator as soon as
clotted as a control
3. The tube is test for the disappearance of clot after 4,
8 and 24 hrs
4. If the sample becomes fluid in less than 48 hrs , the
blood is poured out onto a piece of filter paper to be
sure for clot disappearance
Interpretation
 The disappearance of clot before 48 hrs means
increase fibrinolysis
 After 48 hrs clot may be

37 C tube Refrigerated tube Comment

Disappear Disappear Low fibrinogen

Still intact Still intact Abnormal fibrinolysis


(No clot lysis after 48
hrs)
Disappear Still intact Nomal fibrinolysis
Activity

What is the test mentioned below and how w


we can do it?.

Euglobulin Clot Lysis Time


Next Lecture: Prothrombin Time (PT)