SMILE
Johns Hopkins University
Baltimore, MD USA
Author:
Heidi Hanes
Review History
Document Number:
Pro62-07
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
ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT)
FIBROMETER METHOD
Principle:
This procedure is designed to detect a deficiency of those
factors involved in Phase I of the coagulation scheme, i.e.
thromboplastin generation by the intrinsic system. It is also used
to monitor Heparin therapy. When Factor XII comes into contact
with a foreign surface (e.g. glass), it is activated and in turn
causes activation of Factor XI to form the so-called activation
product.
This activation product in the presence of adequate
platelets and calcium, causes activation of Factors X, IX, VIII
and V to form intrinsic thromboplastin. Partial thromboplastin
reagent is a phospholipid substance which acts as a substitute for
platelet Factor 3. To this reagent are added activator substances
which bring about maximal activation of the contact system
(Factors XII and XI), and this reduces variations in results due
to differences in exposure of plasma to glass surfaces. To the
plasma and activated thromboplastin mixture, calcium chloride is
added 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
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Johns Hopkins University
Baltimore, MD USA
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:
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. The test can be run as
soon as possible or up to four 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. APtt reagent name: (Include any instructions, storage, and
expiration information.)
2. CaCl2: (Include any instructions and expiration information.)
3. Controls: (Include any instructions, storage, and expiration
information.)
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Procedure:
1.If test plasma(s) and/or APtt reagent are at refrigerator
temperature, they should be placed on aliquot mixer in their
bottle or in a properly labeled test tube in a rack until
they reach room temp.
A tube of 0.02 M. CaCl2 should be
maintained at 37C.
2.With the switch on the Fibrometer automatic pipet in the OFF
position, pipet 0.1 ml. APTT reagent into a prewarmed
fibrocup and allow to incubate for at least 1 minute, but no
more than 5 minutes.
3.Change the fibrometer tip and mix the normal control
thoroughly.
With the switch on the Fibrometer automatic
pipet still in the OFF position, pipet 0.1 ml. normal control
into the prewarmed APTT reagent. Mix well with an applicator
stick and allow to incubate for exactly 3 minutes.
4.Move the fibrocup into the reactor well position
5.Using a new fibrometer tip, fill the automatic pipet with 0.1
ml. prewarmed 0.02 M CaCl2. Immediately following incubation
period, dispense into fibrocup containing APTT reagent-normal
control mixture.
6.Activate timing mechanism manually as you dispense CaCl2 into
the fibrocup. The probe arm will drop into place in the
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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.
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 distilled
a lint-free absorbent tissue, and
to resting position.
8.Repeat procedure on normal control until duplicate results
that agree within 3 seconds are obtained. Record the average
of the two results as the normal control value for the APTT.
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 (Steps 2 through
7). Patient answers should be rounded off to the nearest
whole number.
Procedure Notes:
1. If result obtained is under 15 seconds, procure a new sample
and repeat procedure. If answer is the same, show results to
Supervisor before reporting.
(Note: Values should reflex laboratory’s low
critical range for APtt)
2. If result obtained is greater than or equal to 45 seconds,
and the patient has no known defect, is not on anticoagulant
therapy, or does not have liver disease, procure a new sample
and repeat procedure.
If answer is the same proceed to
perform an APTT on a solution of 50% patient plasma and 50%
normal plasma. Show result to Supervisor before reporting.
(Note: Values should reflex laboratory’s high
critical range for APtt)
3. If no fibrin network has formed at the end of 200 seconds,
discontinue test, and report as 200 seconds plus.
4. If the patient has a hematocrit greater than 55%, the APTT
may be falsely prolonged due to excess anticoagulant.
Run
the sample first. If PTT is not prolonged, then report out
these results.
If prolonged, follow this procedure.
The
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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
5. If inquiry about patient's condition or therapy justifies a
prolonged APTT, notify physician of the initial result. As
long as the results remain high they do not need to be
recalled but attached with code that critical was previously
called.
If results go below critical range and rise again
they should be recalled.
6. Be sure to use the correct probe. Probe should be calibrated
for 0.3 ml. Volume
Interpretation:
Prolonged values are associated with deficiencies of Factors V,
VIII, IX, X, XI and XII.
Prolonged values are also associated
with the presence of coagulation inhibitors and heparin therapy.
Normal Ranges:
(Enter laboratory normal range.)
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.
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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
and Wilkins, 6th Edition, 1993
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