Polybrene Neutralization as a Rapid Means of
Monitoring Blood Heparin Levels
VICTOR
R. GRANN, M.D., KATHRYN HOMEWOOD, MT(ASCP)
AND WALTER GOLDEN, P H . D .
Stamford Hospital,
Stamford,
Connecticut
ABSTRACT
Grann, Victor R., Homewood, Kathryn, and Golden, Walter: Polybrene neutralization as a rapid means of monitoring blood heparin levels. Am. J. Clin.
Pathol. 58: 26-32, 1972. Polybrene neutralization is a rapid and accurate
test for determining and following heparin anticoagulation. The method and
five studies are described. In addition to its simplicity, this test offers the
added advantage of following heparin therapy at high concentrations in the
blood and in patients receiving Warfarin anticoagulants. It also permits quantification of dosage of protamine when neutralization with heparin is necessary.
the use in vitro of a specific heparin antagonist. This approach was the basis of
the protamine titration of heparin for the
purpose of determining the amount of protamine to use for in vivo neutralization of
heparin overdose.1-r
Protamine solutions have the disadvantage of very limited stability. Therefore, an
agent which would obviate the difficulty
was sought.8 Hexadimethrine bromide *
(Polybrene, Abbott Laboratories) appears
to meet this requirement. This report presents a method for assaying the activity of
heparin in vitro utilizing Polybrene as the
specific neutralizing agent. The method is
performed by adding increasing concentrations of Polybrene to separate portions of
the patient's citrated plasma. A standard
amount of thrombin is added to these mixtures. A solid clot will form within 1 min.
in those tubes in which the antithrombin
effect of heparin is blocked by the added
Polybrene.
of newer methods for
anticoagulation, heparin continues to be
used widely and effectively in the treatment of thrombophlebitis, pulmonary embolism, and other conditions. Unfortunately
no test has been available which accurately
and easily measures heparin activity, especially at high dosage levels.
DESPITE THE ADVENT
T h e Lee-White clotting time, which is
probably the most widely used test for
monitoring treatment with heparin, has serious disadvantages. It is an empirical test,
poorly reproducible because of inability to
standardize conditions of performance, and
must be performed as an individual bedside test for each patient. A more serious
objection to its continued use is that it
correlates poorly with levels of heparin at
higher dosage schedules.
A more rational method for assaying the
activity of heparin would be one based on
Received April 19, 1971; received revised manuscript July 29, 1971; accepted for publication August 18, 1971.
Presented at the Regional Meeting of the American College of Physicians at Portland, Maine, October 1G-17, 1970.
* This agent was introduced more than 10 yr.
ago for the in vivo neutralization of heparin. This
use, however, has been discontinued because of an
agglutinating effect on erythrocytes.
26
July 1972
MONITORING BLOOD HEPARIN LEVELS
Method
(A)
Equipment
1. 37 C. water bath.
2. Stopwatch.
3. Blood collection tubes, containing 0.5
ml. of 3.8% sodium citrate, evacuated
to draw 4.5 ml. of blood (B-D Vacutainer #3204W).
4. Test tubes, 10 by 75 mm. size (disposable tubes—used once).
5. Serologic pipettes, 1.0 ml. capacity.
6. Pipetting system for repetitive delivery of 0.05 ml. samples of two reagents.t
7. Centrifuge.
8. Siliconized test tube (B-D Vacutainer
#3204).
(B) Reagents
1. Thrombin, Parke-Davis, Topical Thrombin, bovine, 5,000 N.I.H. units per ampule.
T h e lyophilized vial of thrombin is reconstituted with 0.9% saline solution to
a final volume of 300 ml. Approximately
2 ml. aliquots of this solution are transferred to the siliconized test tubes and
then stored in deep freeze at —20 C. for
future use. A tube of this solution is
thawed at room temperature on the day
of use.
The activity of the thrombin solution
can be tested by the following procedure:
Three vol. of thrombin solution are diluted with 4 vol. of 0.9% saline solution.
Then 0.1 ml. of the diluted thrombin is
added to 0.2 ml. of normal control plasma
(Ortho Plasma Coagulation Control) prewarmed to 37 C. This is mixed and the
appearance of a clot is timed. This preparation should give an 11 to '12 sec. thrombin time. Our experience to date has shown
that when the processing of the thrombin
reagent is strictly adhered to, its activity
has always proven satisfactory by this test.
•f For tliis purpose wc use an Oxford Sampler
#50 with disposable plastic tips.
27
2. Polybrene
a. Stock Polybrene obtainable from Aldrich Chemical Co., Milwaukee, Wis.
b. Stock Polybrene solution in 0.9% saline solution, containing 1 mg. Polybrene per ml. Weigh 100 mg. Polybrene on the analytical balance. Dissolve and make to 100 ml. vol. Store
in refrigerator. Stable at least 1 year.
c. Polybrene (PB) working solutions.
(1) 0.01 mg. PB per ml. Dilute 1 ml.
stock to 100 ml. with saline solution.
(2) 0.04 mg. PB per ml. Dilute 4 ml.
stock to 100 ml. with saline solution.
(3) 0.08 mg. PB per ml. Dilute 8 ml.
stock to 100 ml. with saline solution.
These working solutions are stored in
the refrigerator and are stable at least
6 months.
(C) Procedure
1. Blood obtained by routine venipuncture is drawn directly into citrated collecting tubes (B-D Vac #3204W).
2. The specimens are centrifuged (within
1 hr. of collection) at 2,400 r.p.m. for
5 min. T h e supernatant plasma is
transferred to another tube.
3. For each specimen, four tubes (10 by
75 mm.) are placed in the 37 C. water
bath; 0.5 ml. of plasma is pipetted
into each of the tubes and allowed to
stand for at least 1 min. to come to
temperature (37 C ) .
4. Into the first tube 0.05 ml. of thrombin solution is pipetted directly into
plasma. This is mixed and allowed to
sit in the water bath for L min. The
presence of a firm solid clot indicates
no heparin activity in this specimen.
If no clot forms, proceed to subsequent steps.
5. Into the second tube 0.05 ml. of PB
working solution #1 (0.01 mg. PB per
28
GRANN ET
AL.
A.j.C.P.— Vol. 5S
to 1.0 units per ml. No clot formation
in tube 4 indicates a level of plasma
heparin greater than 1.0 units per ml.,
which is considered excessive according to present dosage schedules.
Operating Notes
The entire procedure can be reduced to
a two-tube test for the majority of patients
by altering the sequence of steps in the
following manner.
Initially prepare only two plasma tubes
(0.5 ml. each). With the first tube proceed
as in step 5 above. If a clot forms, proceed
with the next tube as detailed in step 4
above; if no clot forms, proceed with step 6.
Since the majority of patients to be tested
will fall in the first three designated ranges,
the test will have been completed after the
second tube when performed in this sequence.
0.1
0.2
0.4
0.6
Heparin units per ml plasma
Fie. 1. In vitro
0.8
1.0
relationship between heparin
and Polybrene.
ml.) is pipetted and mixed. After exactly 1 min., the mixture is examined.
Clot formation indicates a level of
plasma heparin of less than 0.2 units
per ml. of plasma. If a solid clot does
not form, proceed to the next step.
6. Into the third tube 0.05 ml. of PB
working solution #2 (0.04 mg. PB per
ml.) is pipetted. Again, thrombin is
added and the process exactly as described in step 5 is followed. Clot formation indicates a level of plasma
heparin greater than 0.2 units and
less than 0.6 units per ml. If a solid
clot does not form, proceed to the
next step.
7. Proceed to the fourth tube, using PB
working solution #3 and thrombin
exactly as above. Clot formation indicates a plasma heparin range of 0.6
Experimental Studies
Experiment 1. In vitro Relationship between Heparin and Polybrene. Citrated
plasma was prepared by drawing blood
from normal donors into B-D vacutainer
citrated tubes and pooling the plasma so
obtained. Heparin was added to portions
of this plasma to yield the following range
of concentrations: 0.0, 0.1, 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, and 5.0
units of heparin per ml. plasma. T h e heparin dilutions for this experiment were
prepared from a stock solution of sodium
heparin, "Trent"-labelled 10,000 units per
ml. The samples were divided into triplicate portions and coded. T h e test was then
performed on these replicate samples using
solutions of Polybrene covering a range of
concentrations up to 1.0 mg. of PB per ml.
The data are presented graphically in Figure 1. Each point represents the lowest
concentration of Polybrene capable of producing a solid clot under the standard
condition with a given concentration of
heparin.
July 1972
29
iVfONITORING BLOOD HEPARIN LEVELS
This experiment indicates an almost direct proportionality between increasing levels of heparin and the amount of Polybrene used, thereby indicating a probable
stoichiometric reaction between the two
compounds.
Experiment 2. Plasma Heparin Activity
Levels after Heparin
Administration—
Replicate Studies. Three vacutainer tubes
of blood were drawn from each of 12 patients receiving heparin. The triplicate
specimens were coded, and the levels of
heparin activity were determined. All three
tubes from each patient gave the same heparin activity value (Table 1).
Experiment 3. Heparin Activity Measured at Various Times after Drawing
Blood. Blood specimens were drawn in
triplicate from another group of 30 patients receiving heparin. One tube from
each patient was centrifuged and tested
within 1 hr. The second tube was centrifuged; the plasma was separated immediately, held at room temperature for 4 hr.,
and then tested. The third tube was allowed to stand at room temperature for 4
hr., then centrifuged and the plasma separated and tested. In 26 instances all three
tubes gave the same results. In the remaining four, the plasma not separated immediately gave a slightly lower level of heparin activity at 4 hr.
Expeirment 4. Heparin Activity and LeeWhite Clotting Time Determined after Intravenous Heparin. Heparin was given intravenously in 10 separate studies. The
doses ranged between 22 and 50 mg. and
were given in bolus form. Blood was drawn
prior to injection and at selected periods
following heparinization. Lee-White clotting times were determined in a 37 C. water
bath by one technician using the method
of Cartwright. 4 Heparin activities were determined on a duplicate portion (Fig. 2).
Although there is a general correlation between Lee-White clotting time and heparin
activity, the slope of the heparin activity
Table 1. Polybrene Neutralization with Repealed
Samples from the Same Patient
Patient
Tubel
Tube 2
Tube 3
1
2
3
4
5
6
7
8
9
10
11
12
Thrombin
0.01
0.04
0.01
0.04
Thrombin
0.01
Thrombin
Thrombin
0.04
0.01
Thrombin
Thrombin
0.01
0.04
0.01
0.04
Thrombin
0.01
Thrombin
Thrombin
0.04
0.01
Thrombin
Thrombin
0.01
0.04
0.01
0.04
Thrombin
0.01
Thrombin
Thrombin
0.04
0.01
Thrombin
shows a slightly closer resemblance to
the expected pharmacologic disappearance
curve for heparin (Fig. 3). The clotting
time seems less sensitive to alterations in
levels of heparin. In two instances the 60
min. and in one instance the 120 min. values were higher than the preceding values.
Experiment 5. Heparin Activity and Clotting Time Determined Simultaneously in
Blood from Patients Receiving
Heparin
Therapy. During a 2-month period all
Lee-White clotting times performed in our
routine hospital laboratory were correlated
with heparin activity. In 12 studies performed with blood from patients who had
not yet received heparin, the clotting times
ranged from 10 to 40 min. No heparin activity was shown in the blood of these patients. In blood from patients receiving
heparin, the heparin activity was reproducible in each instance. The clotting times
showed no such consistency. It should be
reemphasized that Lee-White clotting times
were not done in the ideal way as in the
previous study, but were performed by regular technicians during a busy hospital
schedule.
Discussion
One of the difficulties with heparin therapy in the past has been the lack of an
appropriate test to monitor anticoagulant
effect. The test we have developed utilizing
30
GRANN ET AL.
A.J.CP.—Vol.
58
80
LEE WHITE CLOTTING TIME
AND POLYBRENE NEUTRALIZATION
70
.07
6 50
.c
J 40
.04
I 30
.03
3
20
.02
10
5 .01
40
60
80
100
120 140
0
20
Minutes after Heparin injection
FIG. 2. Comparison of decay curve of heparin measured by Lee-White clotting time
and Polybrene neutralization.
Polybrene neutralization is rapid and simple to perform. Multiple studies can be
performed simultaneously. The results are
consistent, and blood, once drawn and separated, can be tested as long as 4 hr. later
with no loss of accuracy. The solutions
themselves are stable and can be used for
more than a year with no alterations.
Other tests for determining heparin have
been described. The Lee-White clotting
time is extremely time-consuming and the
results are frequently neither reproducible
nor representative of heparin blood levels.6
The thrombin time performed with platelet-poor plasma (Rappaport and Ames 0 ) is
easily performed but only separates levels
of heparin into ranges greater than or
less than 0.2 units of heparin per ml. of
plasma.10
The partial thromboplastin time, the activated partial thromboplastin time, and
the plasma recalcification time 2> "• " are not
very sensitive. In fact, they may give normal values when the Lee-White clotting
time is three or four times normal, which
is a range commonly employed in clinical
practice.
Two additional methods of activating
whole blood have been described. Hattersley 6 employed whole blood which is acti-
vated by drawing the blood directly into
an evacuated diatomite tube prewarmed to
37 C. This test, although rapid, is still difficult for the routine laboratory and requires
technicians to do the test at the bedside.
It is also fraught with much of the difficulty of the Lee-White clotting time.
Blakely 3 uses platelin and kaolin to activate whole blood. Again, there is tremendous overlap in the therapeutic range of
heparin.
Our test has many advantages over those
described. By using prepared solutions of
Polybrene, we have simplified the most accurate method of heparin measurement,
i.e., the thrombin time. It is also the only
test outside of the thrombin time test or
protamine neutralization which measures
heparin in patients receiving oral anticoagulants, such as Coumadin.
Polybrene concentrations of 0.01, 0.04,
and 0.08 mg. per ml. were selected, since
they reflect heparin levels encountered in
clinical practice. The O.OL mg. solution of
Polybrene neutralizes a heparin concentration that prolongs the clotting time to two
to three times normal. The 0.04 mg. solutions neutralize a heparin concentration
that prolongs the clotting time to two to
six times normal and the 0.08 solution pro-
July 1972
31
MONITORING BLOOD HEPARIN LEVELS
Fie. 3. Comparison of Lee-White .§ 50
clotting time and Polybrene neu- a>
tralization in three patients.
20
40
60
80
100
120
M0
Minutes after heparin injection
longs the time to four to eight times normal (Table 2). Further levels of heparin
can be determined by preparing additional
Polybrene solutions, but this was considered unnecessary for routine anticoagulation. As previously noted, there is a
straight-line correlation between heparin
level and Polybrene concentration.
At Stamford Hospital we report the concentration of heparin neutralized by thrombin or Polybrene according to the following formula. The concentration neutralized by thrombin alone is reported as no
heparin activity, the concentration neutralized by 0.01 mg. of Polybrene as minimal heparin activity, the concentration
neutralized by 0.04 mg. of Polybrene as
moderate heparin activity, the concentra-
tion neutralized by 0.08 mg. of Polybrene
as high heparin activity, and the concentration neutralized by more than 0.08 mg.
of Polybrene as excessive heparin activity.
Initially, we recommend measuring heparin levels 1 hr. after giving an intravenous
close and thereafter daily 1 hr. prior to the
next dose. The determination 1 hr. after
administration of heparin acts as a guide
for therapeutic levels of heparin and monitoring 1 hr. before administration prevents
excessive heparin accumulation.
Perhaps one of the major advantages of
Polybrene neutralization is that it allows
us to monitor heparin levels that cannot
be tested with any of the previously described methods. Heparin can be measured
at levels greater than 0.6 units per ml. of
32
GRANN ET
Table 2. Comparative Value for Lee-White Clotting
Time and Polybrene Neutralization
Polybrene Concentration
Clotting Time
(min.)
None added (no heparin detected)
10-20
0.01 mg. per ml. (<0.2 U. per
ml. of heparin)
12-25
0.04 mg. per ml. (0.2-0.6 U.
per ml. of heparin)
25-40
0.08 mg. per ml. (0.6-1.0 U.
per ml. of heparin)
40-80
plasma. At this concentration the LeeWhite clotting time is more than an hour
or indeterminate. This provides added
safety and allows a more aggressive approach to therapy with heparin, especially
in life-threatening situations. An additional
benefit of this test is that the proper
strength of protamine solution can be extrapolated from the Polybrene concentration. This tells us the protamine neutralization dose necessary to reverse bleeding
due to excessive dosage or following bypass
procedures.
AL.
A.J.CP.— Vol. 5S
AcknowledgmeiUs. Mrs. Marjorie Giachetto, Mrs.
Jadwiga Bellos, and Doctors Edward Breakell, Robert Erichson, and Walter Richar helped and advised in the preparation of this manuscript.
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