UPH- Dr. Jose G.

Tamayo Medical University

COLLEGE OF MEDICAL TECHNOLOGY

Activity #9: Prothrombin Time (ProTime)

Eder Maen M. Licudan, RMT, MS CLS(c)

Learning Objectives
Discuss the role of prothrombin in blood
coagulation
Explain the major use of prothrombin
time test.
Identify the sources of error for the
prothrombin time
Introduction
Prothrombin Time (PT) Or “Pro-time”
o is one of the most frequently performed coagulation test.
o it evaluates the function of the extrinsic and common
pathways of hemostasis.
o it is used not only as a coagulation screening test, but
also to monitor oral anticoagulant therapy.
o it measures the clotting ability of factors I, II, V, VII,
and X.
o is especially effective for monitoring patients receiving
coumarin and can be used to guide the physician in
regulating the patient’s anticoagulant dose.
Prothrombin Time (PT) Or “Pro-
time”
The test was developed by Dr. AJ Quick
who named it “prothrombin time” because
he thought it measured only prothrombin,
even though it was discovered that the test
actually measures prothrombin plus
additional factors.
Prothrombin Time (PT) Or “Pro-
time”
May be reported in three ways:
1. in seconds (NV: 10-12 secs)
2. as the prothrombin ratio (PT ratio)
-obtained by dividing the patient’s
prothrombin time by the PT of the normal
control.
3. as the International Normalized Ratio
(INR).
 ISI & INR

 Developed to help standardize the difference in sensitivity in

individual thromboplastin reagents and the effect on PT assays
 - the more sensitive the thromboplastin reagent, the longer the
resulting PT
 - the less sensitive the reagent, the shorter the resulting PT
 International Sensitivity Index (ISI)

• Calibration parameter that defines the responsiveness of the reagent relative to

WHO International Reference Preparation, which by definition has an ISI of
1.0
• ISI <1.0 – MORE SENSITIVE THROMBOPLASTIN
• ISI value is critical for calculation of INR because it is the exponent in the
formula
International Normalized Ratio (INR)
Was developed to incorporate the ISI values and
attempt to make prothrombin results uniformly useable
RECOMMENDED FOR MONITORING ORAL
ANTICOAGULANT THERAPY
Mathematical calculation that corrects for the
variability in PT results caused by variable sensitivities
(ISI) of the thromboplastin agents used by laboratories

INR = PT of patient(ISI)
Normal PT
International Normalized Ratio (INR)
INR of 2.0 to 3.0 – recommended for
most indications (e.g., treatment or
prophylaxis of deep venous thrombosis, or
prevention of further clotting in patients
who have had a myocardial infarction)
INR of 2.5 to 3.5 – recommended for
patients with prosthetic heart valves
INR of 3.0 – target INR for pulmonary
embolism treatment
Specimen collection and processing
3.2% BUFFERED SODIUM CITRATE –
standard anticoagulant for coagulation studies
- stabilizes blood pH of samples and
increases stability of labile clotting factors
- correct ratio: ONE PART
ANTICOAGULANT TO NINE PARTS OF
WHOLE BLOOD (1:9) – an excess in the
anticoagulant can alter the expected dilution of
blood and produce errors in the results
(prolonged coagulation tests)
Specimen collection and processing
Coagulation tests are more sensitive to EXCESS OF
CITRATE in the plasma (occurs with high
hematocrit) than to EXCESS CALCIUM (occurs
with low hematocrit or overfilled tubes)
Effect of pH:
- Changes in the pH of samples PROLONG clotting times
Samples should remained in UNOPENED TUBES if testing
is not done immediately
Effect of temperature:
- V & VIII deteriorate at room temperature
- VII & XI tend to be prematurely activated at 4°C
Materials
1. Test tubes
2. Graduated cylinder
3. Timer
4. Reagents: thromboplastin Ca-Cl2
Procedure
MANUAL METHOD

1. Blood must be drawn with minimal trauma to the vein and surrounding
tissue to prevent the release of tissue thromboplastin into the sample
2. Blood is drawn into a tube or syringe containing an anticoagulant
compatible with the commercial reagent system being used (usually 3.8%
solution of sodium citrate) and must be centrifuge as soon as possible and
the plasma transferred to a clean tub for assay. The ratio should be 9 part
bloods with 1 part anticoagulant.
NOTE: The plasma should be assayed within four hours of blood
collection and should not stand at 37°C for more than five minutes before
being tested.
3. Centrifuge the specimen at 1,500xg for 15 minutes.
4. Separate plasma after centrifuging and place in plastic or siliconized
glass tube.
NOTE: Use plasma within 4 hours, otherwise store frozen and thaw just
prior to use.
Procedure
MANUAL METHOD

STABILITY TO PLASMA:
4h at 18-26°C
8h at 2-8°C
14 days at -20°C
6 months at -70°C
5. Incubate the PT reagent at 37°C for at least 10 minutes.
6. Pipette 25uL of sample into a test cuvette. Incubate at
37°C for 1-2 minutes.
7. Add 50uL of PT liquid reagent (37°C) and
simultaneously start test
8. Record the clotting time in seconds
Procedure
AUTOMATED METHOD

1. Turn on instrument. If using the instrument’s pipette, be certain it

is turned off.
2. Label desired number of sample cups and place in heat block
(patient sample should be run in duplicate)
3. Pipet 0.2mL of thromboplastin – CaCl 2 into cups, following
manunfacturer instructions.
4. Pipette sufficient patient plasma and control (0.4-0.5mL) into
separate cups to allow fr duplicate testing of the patient and controls.
5. Allow all component to warm the prescribed amount of time.
Place one sample cup with measured thromboplastin – CaCl 2 into the
center of fibrometer.
6. Draw up 0.1mL of Patient Plasma
7. Turn pipette “ON”
Procedure
AUTOMATED METHOD

8. Expel plasma into center cup containing 0.2mL

thromboplastin – CaCl2. The timer will start automatically
when the plunger is depressed if using the instrument pipet.
9. Wait for the timer to stop, signaling the formation of a
clot
10. Record the time in seconds
11. Gently wipe probe wires with laboratory tissue between
determinations
12. Repeat steps 5-11 using the patient plasma
13. Repeat steps 5-11 for controls
14. Record the time for the controls
15. Turn off the instrument
Reporting of Results in Laboratory
• Reporting the patient and control specimens in seconds
• Percentage of patient activity (% activity) calculated as:
• % Activity of Patient: Control time
x100
(seconds) Patient’s time
(seconds)
• INR calculated as:
• INR = Patient’s time (seconds) IS
I

Mean PT of the normal

range
 Sample Computations

• For Percent Activity

 Patient’s Time : 12.0
seconds
 Control Time : 12. 3
seconds
 12.3 / 12.0 x 100 = 102. 5 %
• For INR:
 Patient’s time : 12.0 seconds
 Mean PT of normal range: 12.4
seconds
 ISI of reagent: 1.21

 12.0 1.21

12.4
=
0.96
 Common causes of prolonged
Prothrombin time:

• Administration of oral anticoagulant drugs

• Liver disease (obstructive jaundice)

• Vitamin K deficiency
INTERPRETATIO
• Disseminated Intravascular Coagulation (DIC)
N
• Deficiency or defect of factors II, V, VII, or X
Summary
PROTHROMBIN TIME
- detects EXTRINSIC & COMMON
PATHWAY factors deficiency
- Platelet poor plasma is prepared by
centrifuging citrated blood at 2000xg for 10
minutes
(PLATELET POOR PLASMA + thromboplastin +
CaCl2 rgt)
- Begin timing after addition of CaCl2 reagent
- NV: 10-12 secs