SMILE
Johns Hopkins University
Baltimore, MD USA
Author:
Heidi Hanes
Review History
Document Number:
Pro62-06
Effective (or
Post) Date:
29 August 2008
Date of last
review:
6 May 2010
Reviewed by:
Heidi Hanes
Comment: Procedure should be used as an example only.
SMILE Comments: This document is provided as an example only. It
must be revised to accurately reflect your lab’s specific
processes and/or specific protocol requirements. Users are
directed to countercheck facts when considering their use in
other applications. If you have any questions contact SMILE.
(Laboratory Name)
DEPARTMENT OF PATHOLOGY
HEMATOLOGY
PROTHROMBIN TIME
FIBROMETER METHOD
PRINCIPLE:
This procedure is designed to detect a deficiency of those factors
involved in Phase II of the coagulation scheme, i.e. prothrombin
conversion. This test can be used to monitor comadin/warfarin
therapy. Tissue or extrinsic thromboplastin is an "incomplete"
thromboplastin.
It is capable of converting prothrombin to
thrombin only when it acts in combination with certain plasma
factors--Factors V, VII, and X. In the one-stage prothrombin time
plasma is reacted with a tissue thromboplastin reagent containing
an optimum concentration of calcium and the time for a fibrin clot
to form is recorded.
PRINCIPLE OF FIBROMETER:
When the instrument is activated, a timing device starts, and a
probe arm drops into the plasma-reagent mixture.
The probe
consists of two electrodes--one stationary and one moving.
The
moving electrode alternately descends and lifts in a sweeping
motion to seek and sense initial clot formation.
Formation of
this insoluble fibrin network serves to complete the electrical
circuit which amplifies the signal to stop the timer.
SPECIMEN:
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A Blue top vacutainer containing 3.2% sodium citrate should be
used. The sample must be obtained by non-traumatic venipuncture.
Citrate tubes should be drawn after the serum tubes and before
EDTA or Heparin containing tubes. There should be a one to nine
ratio of anticoagulant to blood in the tube. Clotted or hemolyzed
samples are not acceptable. Specimens are spun at 2000 RPMs for 10
minutes or at a speed and time that will produce plasma with
platelet concentration less than 10,000. Tests should be run as
soon as possible or up to 24 hours at room temperature. Samples
should not be refrigerated as this can lead to loss of specific
coagulation proteins. Plasma can be removed and frozen at -20oC
for up to two weeks and at -70oC for up to 12 months. Samples
should be thawed in 37oC water bath and gently mixed to ensure all
proteins are distributed in the sample.
Supplies and Equipment:
Lint free tissue
Deionized or distilled water
Fibrometer
Fibrometer heating block
Fibrometer cups
Fibrometer tips
12 X 75 mm plastic tubes
Wooden applicator sticks
REAGENTS:
1. THROMBOPLASTIN reagent.
(Include any
reconstitution, storage and expiration.)
2. Controls: (Include any instructions
storage, and expiration.)
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PROCEDURE:
1. If test plasma(s), and/or Thromboplastin are at refrigerator
temperature, they should be placed on an aliquot mixer
(reagents) or in a test tube rack until they reach room
temperature.
2. Mix the normal control thoroughly and pipet a small amount
(approximately 0.3 ml.) into a 12 X 75 mm. test tube placed
in the Fibrometer heating block. Allow to warm at 37C for
at least 1 minute but no longer than 5 minutes.
3. Mix the Thromboplastin thoroughly.
With the switch on and
the Fibrometer automatic pipet in the OFF position, pipet 0.2
ml. Thromboplastin into a pre-warmed labeled fibrocup. Set a
timer and allow to warm for 1 minute.
4. Move the fibrocup into the reactor well position.
5. Replace the tip on the automatic pipet and then fill with
0.1 ml. normal control.
Depress pipet to dispense
sample
into the fibrocup containing the Thromboplastin while
depressing Timer bar to start timer.
6. The probe arm will drop into place in the reaction well.
When a fibrin network has formed, electrode and timer stop,
and the result is registered on the digital readout in
seconds and tenths.
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7. Record the time; depress
position registers, dip
water, and wipe with
reposition the probe arm
the readout reset button until zero
the probe electrode into deionized
a lint-free absorbent tissue and
to resting position.
8. Repeat procedure on normal control until duplicate results
that agree within 1 second are obtained. Record the average
of the two results as the normal control value for the
Prothrombin Time.
(Answer should be rounded off to the
nearest second or 0.5 of a second).
9. Test the abnormal control in the same manner (Steps 2 through
7).
10. Results for controls (both normal and abnormal) should fall
within the expected range.
11. Test patient plasma(s) in the same manner. Step 2 through 7.
PROCEDURE NOTES:
1. If a patient result is less than 8 seconds, obtain a new
sample and repeat procedure. (Note: Values should reflex
laboratory’s low critical range for Pt)
2. If patient result is greater than or equal to 18 seconds
and the patient is not on anticoagulant therapy (coumadintype), or broad-spectrum antibiotic therapy (mandol and
related antibiotics), or does not have liver disease,
obtain a new sample and repeat procedure. (Note: Values
should reflex laboratory’s low critical range for Pt.)
3. If result in situations 1 or 2 (above) do not change on
repeat, show result to supervisor before reporting.
4. If inquiry about a patient's condition or therapy
justifies a prolonged PT, notify physician if result is
greater than 35 seconds. (Note: Value should reflex
laboratory’s critical range on therapy.)
5. If no fibrin network has formed at the end of 100 seconds,
discontinue test and report as >100 seconds.
6. Be sure probe is calibrated for 0.3 ml. Volume
7. If the patient has a hematocrit greater than 55%, the PT
may be falsely prolonged due to excess anticoagulant. Run
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the sample first. If PT is not prolonged, then report out
these results. If prolonged, follow this procedure. The
following correction formula is taken from the September
1975 issue of Laboratory Medicine:
A mean normal
hematocrit determination of 40% is used for both men and
women for purposes of determining anticoagulant ratios.
This leaves a normal plasmacrit determination of 60%. The
0.5 ml. citrate is left constant, and amount of whole
blood to be added is calculated.
FORMULA:
60
X 4.5 = ml. whole blood to be
100 - Hematocrit determination
added to 0.5 ml. citrate
NORMAL RANGES:
(Laboratory normal range)
INTERPRETATION:
Prolonged values are associated with deficiencies of Factors V,
VII, and X and severe prothrombin deficiency.
REFERENCES:
Sirridge, Marjorie S.: Laboratory Evaluation of Hemostasis, 2nd
edition, Philadelphia, 1974, Lea & Febiger.
Instruction sheets accompanying from reagents.
Instructions and Technical Information for the Fibrometer
Precision Coagulation Timer, BBL, Cockeysville, Maryland.
Lab Medicine, September 1975.
CLIS H21-A5, Collection, Transport and Processing of Blood
Specimens for Testing Plasma-Based Coagulation Assays and
Molecular Hemostasis Assays, 5th Edition, Volume 16 Number 5,
January 2008.
Brown A.,Barbara: Hematology: Principles and Procedures, Wiliams
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and Wilkins, 6th Edition, 1993
Related Documents:
Platelet-Poor Centrifuge SOP
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