HEMATOLOGY 2 LAB

FINALS

ACTIVITY 9: COAGULATION TIME

2) Make a clean venipuncture and start the time at which blood

INTRODUCTION enters the syringe (h ub), and draw 4mL of blood.
● Also known as clotting time 3) Carefully dispense 1mL to tube 3, then 1 mL to tube 2, and 1mL
● While it is advised that other coagulation tests, like PT and APTT, to tube 1.
are performed, the performance of clotting time will still provide - Dispensing = 3 → 2 → 1
useful information about the hemostatic condition of our patient. 4) Incubate all tubes in the water bath at 37°C (+/– 0.5°C)
PRINCIPLE 5) At precisely five minutes, tilt tube number 1 to a 45° angle and
● The coagulation time of whole blood is the length of time required observe clotting (Manual tilt method). Repeat every 30 seconds
for a measured amount of blood to clot under certain specified until the tube can be inverted entirely without spilling contents.
conditions. Note the time of clotting.
- If there is no clot at 30 seconds, return it to the water
Notes: bath and wait for another 30 seconds. (Do not check
⇒ If the venous blood makes contact with a foreign tubes 2 and 3 until tube 1 is clotted)
substance (e.g., glass), it will eventually form a solid 6) After 30 seconds, tube 1 is clotted, proceed to tube 2, and repeat
clot → the time it will take will be the measure of the the above procedure–repeat procedure with tube 3.
overall intrinsic and common pathways of - Tilting = 1 → 2 → 3
coagulation. 7) Record coagulation time as the time elapsed between withdrawal
⇒ Certain specified conditions: of blood and the completion of coagulation in tube 3.
○ Non-traumatic collection of blood
○ Maintain stable temperature (37°C) ● Endpoint = the time at which tube 3 has clotted.
● Example reporting:
Tube 1 = 5 mins.
PURPOSE
Tube 2 = 5 minutes 30 seconds
● Gives a clue if the patient has a risk of excessive bleeding
Tube 3 = 6 minutes
○ Especially if the patient will undergo a surgery or
Clotting Time (tube mx) → 6 minutes
dental procedure.
● Linearity: 1 minute
● Monitor heparin therapy
● Reporting should be done per tube but final reporting should be
○ Heparin → inhibits the action of thrombin
the time it took for tube 3 to clot.

ADVANTAGES
METHODS
● Can be performed at the bedside of the patient.
SLIDE METHOD ● No reagents are used.
● Aka Micro method
● Normal Value: 2 to 7 minutes (2-8 minutes) LIMITATIONS
● Test for intrinsic pathway (glass + blood → intrinsic pathway ● Low sensitivity
activated) ○ Even if we have a normal result, it is possible that the
● Method of collection: Skin puncture patient has certain deficiencies.
● No QC measure
1) Do a finger puncture. Start the stopwatch at the time of ○ No means to check the accuracy and precision of the
appearance of blood from puncture. test.
● Perform skin puncture → wipe off first drop of blood ● Results are easily altered by agitation
(this eliminates tissue factor contamination)
● Obtain 3 drops of equal volume. Drop of blood must be
dome-shaped in appearance (approximately 10-20uL)
CLOT RETRACTION TEST
- Do NOT throw away the feather lancet immediately. ● Modified McFarlane method
2) Place in a clean glass slide three (3) separate drops of blood. ● Test for adequacy of platelet function
3) Allow to stand for 2 minutes at room temperature. ● Method of collection: syringe venipuncture
4) Check clot formation by drawing blood with a needle or lancet ● Materials:
(bent tip) and observe for thread formation. (check fibrin threads ○ Conical tube
in the sides of the blood drop) ○ Cotton
- If there is still no fibrin thread formed, wait for another ○ Applicator stick
30 seconds before checking again. Until such a time
that the endpoint is reached.
5) Record clotting time from start to fibrin thread formation.

LEE & WHITE METHOD

● Aka Tube or Macro method
● Normal values = 5 to 15 minutes
● Test for intrinsic pathway
● Method of collection: venipuncture (syringe method)
● Materials:
○ Water bath (37 C) → intended to mimic body
temperature
○ 13 x100 mm test tubes Di ko alam bat ang labo huhu, mata ko lang ba

1) Label three (3) uniform sized tubes “1”, “2”, and “3”.
PROTHROMBIN TIME (HIGH TECHNOLOGY, INC)
 HEMATOLOGY 2 LAB
FINALS

● PRINCIPLE: Used as a screening tool for coagulation factors in

the extrinsic and common pathways. It is especially well suited for
the induction and monitoring of oral anticoagulant therapy. It also
used for checking the synthesis performance of the liver in
hepatic diseases.
● Reagents:
○ 0.2% Sodium Azide (buffer)
○ Recombinant hTF
○ 0.5% BSA (optional)
○ 0.015 M CaCl2
● Testing:
○ 50uL of plasma + 100 uL of PT reagent (37 C for 3
minutes)
○ Principle of bimagnetism
○ Setup: Two electromagnets between the cuvette and
then the cuvette will contain the steel ball
○ After adding the plasma and the reagent, it forms a clot
○ Endpoint: steel ball can no longer move
○ Normal value: 10-14
○ INR: 0.8 - 1.2

ACTIVATED PARTIAL THROMBOPLASTIN TIME (HIGH

TECHNOLOGY, INC)
● PRINCIPLE: It is used as a screening tool to evaluate coagulation
abnormalities in the intrinsic and common pathway. It is also used
to monitor effectiveness of heparin therapy.
● Reagents:
○ 0.1% Ellagic acid (activator) → alternative: micronized
silica, kaolin
○ 0.1% Sodium Azide (buffer)
○ 0.2% Phospholipids (Reagent 1)
○ 0.5% BSA (optional)
○ 0.025 M CaCl2 (Reagent 2)
● Testing:
○ 50 uL plasma + 50 uL APTT reagent (37 C for 3
minutes) + 50 uL CaCl2
● N.V.: 26-36 seconds