Download some in vitro effects of various concentrations of disodium

SOME IN VITRO EFFECTS OF VARIOUS CONCENTRATIONS OF DISODIUM
ETHYLENEDIAMINE TETRACETATE, POTASSIUM OXALATE,
AND SODIUM CITRATE ON COAGULATION OF BLOOD
J. F. MUSTARD, M.D., P H . D .
Department of Veterans Affairs, Sunnybrook Hospital, and Department of Medicine, University of Toronto,
Toronto, Ontario, Canada
Knowledge of the effect of various concentrations ' of the anticoagulant salts on
blood coagulation is essential in the collection and also in the preservation of samples
of blood for the study of disorders of coagulation and in the collection and preparation of blood products for treating disorders
of coagulation. Too little anticoagulant fails
to prevent coagulation, and there is some
evidence that too much anticoagulant impairs the activity of clotting factors, such
as Factor-V. 6 ' u The concentrations of the
anticoagulant salts required for maximum
effectiveness in stabilizing the coagulation
mechanism are not well known. The results
of a study of this problem are reported in
this communication.
The effects of the various concentrations
of disodium ethylenediamine tetracetate
(EDTA), potassium oxalate, and trisodium
citrate, on blood coagulation were assessed
in the following manner: (1) the minimum
concentration of anticoagulant necessary to
prevent blood from clotting; (2) the effect
on the thrombin fibrinogen reaction; (3) the
effect on the 1-stage prothrombin time; (4)
the effect on Factor-V and antihemophilic
globulin (AHG) activity. Some characteristics of the effect of excessive amounts of
anticoagulant were studied. The practical
importance of variations in the concentration of citrate in acid citrate dextrose (ACD)
solutions on Factor-V and AHG activity was
also investigated.
BLOOD
SAMPLES
Samples of blood were collected, using
clean petrolatum (paraffin jelly) coated glass
Received, May 26, 1958; revision received,
June 20; accepted for publication August 18.
Dr. Mustard is Fellow, Department of Medicine, University of Toronto, and Research Fellow,
Department of Veterans Affairs, Sunn3'brook
Hospital.
syringes and 18-gage needles. The samples
of blood were placed in silicone-coated glass
centrifuge tubes in the proportion of 9 parts
of blood to 1 part of anticoagulant solution.
With the exception of the study using the
ACD solutions, the concentration of anticoagulant referred to in the test is that into
which the sample of blood was taken.
EDTA. The EDTA solutions used are
outlined in Table 1.
Trisodium citrate. The following citrate
solutions were used: 50 mM, 75 mM, 100
mM, 160 mM, 200 mM, 300 mM, and
600 mM.
Potassium oxalate. The following oxalate
solutions were used: 10 mM, 20 mM, 30
mM, 50 mM, 100 mM, 200 mM, 300 mM,
and 600 mM.
Calcium chloride and barium chloride.
These salts were made up in solutions of
various concentrations.
Imidazole buffer pH 7.8. 1.72 Gm. of
imidazole were dissolved in 90 ml. of 0.1
N HC1 and diluted to 100 ml. with distilled
water. '
Brain thromboplastin. This was prepared
as described by Biggs and Macfarlane.5
Factor-V and antihemophilic globulin. Solutions of these factors were prepared by the
method described by Biggs and Macfarlane.5
Thrombin. Thrombin solutions in 0.85
per cent saline were made from dried
thrombin prepared and supplied by the
Lister Institute, London, England. The
strength used was 25 units per ml.
One-stage prothrombin test and quantitative
lest for Factor-V activity. These were performed as described previously.5
Dialysis. Plasma samples were dialyzed
against 500 times as much 0.85 per cent
saline at 4 C. for 12 hr.
Thromboplastin generation tests. These were
performed as described by Biggs and
Douglas,4 with the following modifications:
49S
Dec. 195S
BLOOD
499
COAGULATION
TABLE 2
TABLE 1
Molarity
EDTA
NaCI
Distilled Water
ffJAf
Gm.
Gm.
ml.
12
15
18
24
30
60
0.4
0.5
0.6
0.8
1.0
2.0
0.74
0.72
0.69
0.64
0.59
0.33
100
100
100
100
100
100
1. Brain extract prepared by the method
of Bell and Alton2 was used instead of
platelet suspensions.
2. The aluminum hydroxide-adsorbed
plasma (Al(OH)s-treated plasma) was diluted 1 in 5, using various proportions of
buffer, saline, EDTA, and calcium chloride
solutions. The quantities used are given with
the results for each experimental study.
AUG activity. This was determined by the
method which has been described.8
pH. The p l i of plasma samples and test
materials were determined by using a glass
electrode universal pH meter (W. G. Pye
Ltd., Cambridge).
Blood stored in ACD solutions. Samples
of blood were taken from a donor by a sterile
technic and added to various ACD solutions
containing from 1.5 to 4.25 Gm. disodium
citrate per 120 ml. ACD solution. Four parts
were added to 1 part ACD solution. The
blood anticoagulant mixture was stored at
4 C. for 7 days. Baseline values for the coagulation tests were determined on blood
taken into the various anticoagulants on
the first day.
RESULTS
Minimum Concentration of Anticoagulant
Necessary to Prevent Gross Evidence
of Coagulation
The minimum concentrations of the anticoagulants necessary to prevent gross evidence of coagulation were as follows: concentration of anticoagulant into which blood
sample taken (1 part anticoagulant to 9
parts blood)
EDTA: between 12 mM and 15 mM
Oxalate: between 20 mM and 30 mM
Citrate: between 50 mM and 75 mM
THROMBIN FIBRINOGEN REACTION
Time after Blood and Anticoagulant Mixed*
Anticoagulant
1 hr.
6 hr
12 hr.
Clotting time
0.1 ml. of thrombin added to 0.1 ml. of plasma
sec.
EDTAt
15 mM
30 mM
60 mM
Oxalate
30 mM
100 mM
300 mM
Citrate
100 mM
150 mM
300 mM
3.0
10.0
19.0
3.0
30.0
65.0
4.2
100.0
+ 120.0
6.0
+ 120.0
+ 120.0
4.4
4.4
5.2
4.4
4.2
5.4
4.2
4.6
6.0
4.S
4.7
7.S
3.S
4.2
5.S
3.7
4.2
6.0
4.0
.4.S
10. S
4.3
5.6
22.0
* Plasma samples were left standing a t room
temperature.
f Ethylenediamine t e t r a c e t a t e .
Thus, in terms of molality, approximately
twice as much oxalate and 5 times as much
citrate as EDTA were necessary to prevent
gross coagulation.
Thrombin fibrinogen reaction. Plasma prepared from blood taken into the minimum
requirement of EDTA (15 mM) showed
little change in the thrombin clotting time,
whereas plasma prepared from blood taken
into a greater concentration showed progressive prolongation of the thrombin clotting time (Table 2). This was also true for
excess citrate, but the effect was much less
marked than that observed with EDTA.
Excess oxalate caused some prolongation,
but the effect was less than that of citrate.
The degree of change seemed to be related
to the concentration of anticoagulant used
and the length of time the anticoagulant
was mixed with the plasma.
Plasma pH values. The pH values of the
plasma samples studied were observed to be
similar. The tests were performed by using
imidazole buffer pH 7.3, which kept the pH
within a range of 7.28 to 7.35 during experiments which required the mixing of various
materials.
One-stage prothrombin test. The greater
the amount of EDTA and oxalate above the
500
Vol. SO
MUSTARD
TABLE 3
TABLE 4
E F F E C T ON T H E PROLONGED 1-STAGE PROTHBOMBI N
ONE-STAGE PROTHROMBIN T I M E
T I M E OF PLASMA SAMPLES CONTAINING E X C E S S
Time after Blood and Anticoagulant Mixed*
Anticoagulant
30 min.
4 hr.
8 hr.
PLASMA,
OR
CACL2
OF
GREATER
STRENGTH
One-stage prothrombin time
EDTAf
15 mM
30 mM
60 mM
Oxalate
30 mM
100 mM
200 rail
Citrate
100 mM
150 mM
300 mM
ANTICOAGULANT OF ADDING S E R U M OR A L ( O H ) 3 TREATED
sec.
sec.
sec.
13.0
15.0
16.0
12.S
16.0
16.5
12.4
17.5
20.0
14.5
15.0
15.0
14.5
16.0
20.0
14.5
19.0
28.0
14.0
13.5
16.0
13.0
13.0
17.0
11.5
12.8
19.5
Material Added to Plasma
Calcium
Anticoagulant
M/10
M/20
M/40
§1
<
One-stage prothrombin time
sec.
EDTA,* 60 mM 25.0
Oxalate, 200
28.0
mM
Citrate, 300
24.0
mM
sec.
sec.
sec.
sec.
22.0
26.0
19.0
26.0
15.0
17.0
19.0
27.0
21.0
18.0
15.0
IS.5
* Ethylenediamine t e t r a c e t a t e .
* Plasma samples were left standing a t room
temperature.
| Ethylenediamine t e t r a c e t a t e .
minimum required to prevent gross coagulation, the more unstable was the 1-stage
prothrombin (Table 3). The concentration
of anticoagulant used and the length of time
the anticoagulant was mixed with the
plasma were factors in determining the degree of prolongation of the 1-stage prothrombin time.
Samples of plasma prepared from blood
taken into citrate concentration slightly
greater than the minimum necessary to prevent gross coagulation revealed acceleration
of the prothrombin time, rather than prolongation. If greater amounts of citrate
were used, however, the prothrombin times
for the plasma samples were prolonged
(Table 3).
The prolonged prothrombin times of
samples of plasma containing excess anticoagulant were not accelerated by the use
of calcium solutions of greater strength.
The prolonged prothrombin times were accelerated by the addition of 20 per cent of
normal Al(OH) 3 -treated citrate (3.8 per
cent) plasma, but not by the addition of 20
per cent of normal serum (Table 4).
Activity of Al{OH) ^-treated plasma in the
thromboplastin generation test. The greater
the concentration of EDTA used in comparison to the minimum requirement, the
less was the activity of the Al(OH) 3 -treated
plasma in the thromboplastin generation
test (Fig. 1). This was also true for oxalate.
Blood taken into the minimum concentration of citrate, however, lost activity. For
citrate there appeared to be an optimum
concentration that prevented loss of activity. This was approximately twice the
minimum concentration necessary to prevent gross coagulation. Citrate concentration greater than the optimum caused a loss
of activity (Fig. 1).
The decrease in activity of A1(0 Untreated plasma containing excess anticoagulant was related to the length of time
the anticoagulant was mixed with the
plasma. Thus, after standing for 12 hr. at
room temperature, Al(OH) 3 -treated plasma
prepared from blood taken into 30 mM
EDTA (twice minimum), 300 mM citrate
(4 times minimum and twice optimum),
and 100 mM oxalate (3 times minimum)
had very little activity in the thromboplastin generation test (Fig. 2).
The addition of a Factor-V solution to
these plasma samples 12 hr. after preparation produced some improvement in their
thromboplastin activity. Following the addition of an AHG solution there was considerable improvement in the activity of
these plasma samples. This suggests that
the change in activity is due, in part, to
loss of Factor-V and AHG activity.
Dec. 1958
BLOOD
501
COAGULATION
CITRATE
INCUBATION
TIME -
MINUTES
F I G . 1. Thromboplastin activity of Al(OH) 3 -treated plasma prepared
from blood taken into various amounts of E D T A , oxalate, and citrate. T h e
tests were performed S hr. after the blood was collected. The control test in
each instance is indicated by the curve O
O . The Al(OH) 3 -treated
plasma used in this test was prepared from blood taken into 3.S per cent
citrate. The treated plasma was diluted as follows: Al(OH) 3 -treated plasma,
0.06 ml.; buffer pH 7.3, 0.06 ml.; 0.S5 per cent saline solution, 0.1S ml. T h e
Al(OH) 3 -treated plasma prepared from the blood samples taken into the
various anticoagulants were used in place of the 3.8 per cent citrate plasma
in the test system. The concentration of anticoagulant into which each of
the blood samples was taken is indicated with the curve for the corresponding Al(OH) 3 -troated plasma sample •
•.
INGIBATION
TIME
-
MINUTES
F i d . 2. Change in the thromboplastin activity of Al(OH) 3 -treated plasma
prepared from blood taken into excess amounts of E D T A , oxalate, and
citrate during a period of 12 hr. The Al(OH) 3 -treatcd plasma samples were
diluted as described for Figure 1.
The effect of adding solutions of AHG and Factor-V to the Al(OH) 3 treated samples of plasma after 12 hr. is also indicated O
O . When
adding Factor-V or the AHG solutions, the Al(OH) 3 -treated samples of
plasma were prepared for the tost as follows : Al(OH) 3 -treated plasma (12-hr.
sample), 0.06 ml.; buffer pH 7.3, 0.06 ml.; Factor-V or AHG solution, 0.06
nil.; 0.85 per cent saline solution, 0.12 ml.
502
Vol. SO
MUSTARD
Some Characteristics of the Effect of Excess
Anticoagulants on the Thrombin Fibrinogen
Reaction and the Thromboplastin Activity of
Al(OH)3-treated Plasma
TABLE 5
F A C T O R - V AND A H G * ACTIVITY
Anticoagulant
EDTAf
15 mM
30 mM
60 mM
Oxalate
30 mM
100 mM
300 mM
Citrate
100 mM
150 mM
300 mM
Factor-V and AHG Activity 12 Hr.
after Blood and Anticoagulant Mixedf
Factor-V activity
AHG activity
per cent
per cent
70
15
18
100
18
10
75
4S
5
95
40
12
50
90
20
65
100
40
Thrombin fibrinogen reaction. The addition of a sufficient quantity of calcium or
barium chloride to the plasma samples accelerated the prolonged clotting times of
plasma prepared from blood taken into
excess citrate and EDTA (Table 6).
In the case of oxalate the addition of
calcium chloride formed a precipitate and
did not accelerate the prolonged thrombin
clotting time. Dialysis of plasma samples
against normal saline also accelerated the
thrombin clotting time. This was true for
plasma samples stored for at least 7 days.
Thromboplastin activity of Al(OH)t-treated
plasma. The diminished activity of Al(OH)3treated plasma samples containing excess
anticoagulant was not increased by the addition of more calcium to the generation
mixture. If the same amount of calcium was
incubated with the plasma sample for 10
min. at 37 C., however, before being tested
in the thromboplastin generation test, there
was some improvement in activity. The
plasma sample had to be incubated with an
adequate concentration of calcium. Amounts
less than that necessary produced proportionately less effect (Fig. 3).
* Antihemophilic globulin.
f Allowed to stand a t room temperature.
t Ethylenediamine t e t r a e e t a t e .
Factor-V and AHG activity. There was
a progressive loss of Factor-V and AHG
activity in plasma samples as the concentrations of EDTA and oxalate used were
increased (Table 5). In the case of citrate,
concentrations less than optimal, but greater
than that needed to stop gross coagulation,
did not prevent loss of Factor-V and AHG
activity. Citrate concentrations greater than
the optimal requirement produced a loss of
Factor-V and AHG activity (Table 5).
Dialysis of deteriorated plasma samples
against normal saline also improved the
activity of Al(OH) 3 -treated plasma in the
TABLE 6
T H E E F F E C T OF D I A L Y S I S OR THE ADDITION OF D I V A L E N T C A T I O N S ON THE T H R O M B I N
CLOTTING T I M E
Thrombin Clotting Time—Seconds
Anticoagulant
Divalent cations added*
Saline
added*
CaCls
11/10
EDTA,t 30 mM
Oxalate, 300 mM
Citrate, 300 mM
+ 120
13
20
M/20
M/40
M/10
M/20
M/40
6.5
5.0
6.0
7
6
7
110
6
6
7
• 9
90
7
6
0
* One p a r t plasma + 1 p a r t of solution,
t Ethylenediamine t e t r a e e t a t e .
Dialysis
BaCIs
13
5
6
Dec. 1958
BLOOD COAGULATION
thromboplastin generation test. However,
after a variable period of time (approximately 8 to 24 hr. at room temperature),
neither dialysis nor incubation with calcium
improved the activity of the samples of
plasma.
503
amounts of citrate were determined. When
the concentration of citrate is greater or
less than the optimum, the stability of
Factor-V and AHG activity is impaired
(Fig. 4).
DISCUSSION
Some Practical Aspects of the Effect of Variations in the Citrate Concentrations of ACD
Solutions on Factor-V and AHG Activity
during Blood Storage
The Factor-V and AHG activity of
samples of blood after 7 days' storage at
4 C. in ACD solutions containing various
CONTROL
»MCACLO
ZO NO
N O \K
INCUBATION
INCUBATION
TIME-MINUTES
F I G . 3. Thromboplastin activity in thromboplastin generation test of a sample of Al(OH) 3 treated plasma prepared from blood taken into a
30-mM. solution of EDTA after incubation with
CaClj solutions of various strength for a period of
5 min. The Al(OH) 3 -treated sample of plasma was
diluted as follows, and incubated at 37 C. for 10
min. prior to being added to the generation mixt u r e : Al(OH) 3 -treated plasma, 0.06 ml.; buffer
pH 7.3, 0.06 ml.; CaCI 2 (M/20, M/40, or M/S0),
0.06 ml.; 0.85 per cent saline solution, 0.12 ml.
T h e activity of the Al(OH) 3 -treated sample of
plasma, made up as above with M/20 CaCl 2 ,
b u t not incubated before testing, is indicated by
•
• . The degree of activity is about the same
as the plasma when there is no added CaClj
•
• . The control (3.8 per cent solution of
citrate), Al(OH) 3 -treated plasma activity is
indicated by O
O, and the activity after
incubation with CaCl» X
X . (The amounts of
CaCl; used are illustrated with corresponding
curve.)
Concentration of Anticoagulant Salt Necessary
to Stabilize the Coagulation Mechanisms
EDTA. A 15-mM concentration of this
anticoagulant is sufficient to prevent blood
from clotting when 9 parts of blood are
taken into 1 part of anticoagulant. Although
amounts greater than this prevent gross
coagulation, there is considerable deterioration of the coagulation mechanism, particularly a loss of Factor-V and AHG
activity. Triantaphyllopoulous, Quick, and
Greenwalt13 found a 13- to 15-mM concentration of EDTA was necessary to inhibit
the coagulation mechanism, and that plasma
prepared from blood taken into this concentration of EDTA had a normal 1-stage prothrombin time. Their findings and the findings for the coagulation indices reported in
this communication suggest that when the
optimal amount of EDTA is used, the coagulation mechanism is reasonably stable.
Oxalate. Approximately 30 mM of potassium oxalate is sufficient to stabilize the
coagulation mechanism when 1 part anticoagulant is used to 9 parts blood. If this
concentration is used, Factor-V activity is
stable. When excess anticoagulant is used,
however, Factor-V activity is unstable.
Fahey and his associates,6 and Stefanini"
observed that Factor-V was progressively
less stable with increasing concentrations
of oxalate. It would therefore appear that
if the optimal concentration of oxalate is
used, Factor-V activity is comparatively
stable, rather than unstable, as is usually
stated.
Excess oxalate also produces a loss of AHG
activity. Spaet and Garner10 regarded AHG
activity to be very unstable in oxalated
plasma. They attributed this instability of
AHG in oxalated plasma to the action of an
AHG inactivator. Possibly the poor stability
of AHG observed in their study was caused,
in part, by the fact that the concentration
of anticoagulant used was 33^ times the
504
Vol. SO
MUSTARD
CONCENTRATION
OF
DISOOIUM
CITRATE
G/120 ML A.CD.
FIG. 4. The AHG and Factor-V activity of samples of blood
taken into ACD solutions of various concentrations of citrate
after 7 clays' storage at 4 C. The activity for each of the samples
at 7 clays is expressed in per cent of the activity found on the day
of collection of blood in samples taken into the corresponding
amounts of anticoagulant.
optimal value found in the present study.
Spaet and Garner comment that the stability
of AHG in oxalate plasma is improved by
dialysis, whether oxalate is, or is not, removed from the plasma. The crucial experiment for this statement was that dialysis of
an oxalate plasma sample against an oxalate
saline solution improved AHG stability.
The concentration of oxalate in their oxalate
saline solution, however, was approximately
two-thirds that in the oxalated plasma
(saline; saline 9 parts, 100 mM oxalate 1
part = final oxalate concentration of 10
mM: plasma; 9 parts blood to 1 part 100
mM oxalate = oxalate concentration in
plasma (assuming hematocrit 45 per cent)
of 14.4 mM). Dialysis of the plasma against
the oxalate saline, therefore, should reduce
the plasma concentration of oxalate, the
degree depending upon the volume of plasma
and dialysis solution used. Inasmuch as the
concentration of oxalate in the plasma was
being diminished, dialysis should have improved AHG stability, which it seems to
have done.
Citrate. Concentration of citrate near the
minimum required to prevent gross coagula-
tion did not stabilize the activity of factors
such as Factor-V and AHG. However, if
optimum amounts were used (approximately
twice the minimum to prevent clotting),
Factor-V and AHG activity were stable.
Amounts of citrate greater than the optimum
impaired Factor-V and AHG activity in a
similar manner to excess EDTA and oxalate.
It would seem that the range for the optimal
concentration of citrate necessary to stabilize
the coagulation mechanism is wider than
that for EDTA and oxalate.
Nature of the Effect of Excess A nticoagulants
on the Coagulation Mechanism
There are certain differences between
the effect of excess EDTA, citrate, and oxalate on Factor-V and AHG activity and their
effect on the thrombin fibrinogen reaction.
The prolonged thrombin fibrinogen clotting
times can be accelerated by the addition of
some divalent cations or by dialysis of the
plasma samples for a period of at least
7 days. In contrast, the activity of Al(OH)3treated plasma prepared from blood containing excess anticoagulant can be improved
only by incubation with calcium or dialysis
Dec. 1958
505
BLOOD COAGULATION
for a short period of time. The effect of
excess anticoagulants on the thrombin
fibrinogen reaction, therefore, appears to
be reversible, whereas the effect on the
thromboplastin activity of Al(OH) 3 -treated
plasma may be irreversible.
The effect on the thrombin fibrinogen
reaction could be caused by increasing the
ionic charge and/or ionic strength of the
electrolytes, as suggested by Astrup 1 and
Mommaerts. 7 If it were caused by ionic
charge, however, oxalate should have been
as effective as EDTA, which it was not.
Furthermore, the increase in ionic strength
brought about by the greatest concentration
of EDTA used was approximately onetenth that of citrate and oxalate salts.
Inasmuch as EDTA was much more effective than citrate and oxalate, therefore, it
would seem that the prolonged thrombin
clotting times were not specifically caused
by change of ionic charge or ionic strength,
or a combination of both.
Stormorken12 has suggested that changes
in the reactivity of plasma to thrombin may
be caused, in part, by denatured fibrinogen.
In the case of EDTA, the amount necessary just to ensure stability of the coagulation mechanism was theoretically sufficient
to react with approximately 10.8 mg. of
calcium per 100 ml. of plasma. An increase
in the amount of EDTA from 15 m l to 18
mM produced a considerable loss of AHG
and Factor-V activity. The latter concentration of EDTA is theoretically sufficient to
combine with 13.0 mg. of calcium per 100
ml. of plasma. It would seem that when
there is more than enough EDTA to react
with the plasma calcium, there is impairment of the stability of the coagulation
mechanism. In view of this, the speculation
made by some investigators that excess of
the anticoagulant salts may interfere with
protein-bound divalent cations does not
seem unreasonable.11
Practical Considerations
Laboratory tests. Obviously the variations
in the concentration and type of anticoagulant salt used by different laboratories
are of some importance. Regarding coagulation of blood, it would seem that citrate has
a wider range for optimal effectiveness, but
if EDTA and oxalate are used in critical
concentrations, the coagulation mechanism
is just as stable as with citrate. In view of
the fact that 0.1 M oxalate is almost universally used in clinical laboratories, this
might be regarded as an important difference and that 0.03 M oxalate should be used.
For routine work, however, where hematocrits vary widely, the 0.1 M oxalate value
still seems best, especially as most tests are
standardized to this amount of oxalate,
and the coagulation changes are not too
important between the usual time of blood
collecting and testing.
Blood transfusion. Variations in the concentration of citrate in ACD solutions
clearly affect the stability of the coagulation
mechanism. Concentration below the optimum seems to allow slow activation of
the coagulation mechanism,9 whereas excess concentration destroys the activity of
such factors as Factor-V and AHG. The
use of an optimal concentration of citrate,
coupled with care in collection of blood,
should ensure good stability of the coagulation mechanism.
Another possibly important aspect of
excess anticoagulant is the effect of EDTA
on platelet stability. The concentration of
EDTA normally used for preparing platelets from blood (30 mM, i.e., twice the
optimal concentration) rapidly destroys
Factor-V and AHG activity. The available
evidence indicates that platelets are involved
in a reaction with AHG during clotting.3
It is reasonable, therefore, to speculate that
anything that rapidly destroys AHG activity and binds calcium should make
platelets stable.
SUMMARY
When the optimum concentration of the
salts, ethylenediamine tetracetate (EDTA),
citrate, and oxalate are used as anticoagulants for collection of samples of blood, the
coagulation mechanism is stable. With
EDTA, this is the concentration that will
just prevent the blood from clotting, that
is, 1 part 15 mM EDTA to 9 parts of blood.
This also seems to be true for oxalate. A
concentration of 30 mM is adequate under
506
Vol. SO
MUSTARD
the conditions of this study. With citrate,
the amount necessary to ensure stability
of the coagulation mechanism seems to be
twice the minimum concentration necessary to prevent gross clotting, that is,
approximately 150 mM.
Anticoagulant concentrations greater than
the optimum requirement prolong the
thrombin clotting time, and cause a loss
of Factor-V and AHG activity. The effect
of excess anticoagulant on the thrombin
fibrinogen reaction is reversible by dialyzing
the plasma sample against saline, or by the
addition of divalent cations such as calcium
and barium. After a short period of time,
the effect of the excess anticoagulants on
AHG and Factor-V activity seems to be
nonreversible.
These observations indicate that the
amount and type of anticoagulant salts are
of importance in stabilizing the coagulation
mechanism in stored blood, in preparing
platelets for transfusion, and in ensuring
uniformity in the study and interpretation
of studies on coagulation disorders.
SUMMAKIO I N IN'TERLIiVGUA
Quando concentrationes optimal del sales
ethylenediaminotetraacetato (EDTA), citrato, e oxalato es usate como anticoagulantes in le collection de specimens de
sanguine, le mechanismo coagulatori es
stabile. In le caso de EDTA, isto es le
concentration que justo preveni le coagulation, i.e., 1 parte EDTA de 15 mM in 9
partes de sanguine. Le mesmo pare esser ver
etiam pro oxalato. Un concentration de 30
mM es adequate sub le conditiones de iste
studio. In le caso de citrato, le quantitate
necessari pro assecurar stabilitate del
mechanismo de coagulation pare esser duo
vices le concentration minimal necessari pro
prevenir coagulation grassier, i.e. approximativemente 150 mM.
Concentrationes de anticoagulante in
excesso del requirimento optimal prolonga le
tempore de coagulation a thrombina e causa
un perdita in activitate de Factor V e de
globulina antihemophilic (GAH). Le effecto
de excessos de anticoagulante super le reaction de thrombina e fibrinogeno es reversibile
per dialysar le specimen de plasma contra
solution salin o per adder cationes divalente,
como per exemplo calcium e barium. Post
un breve periodo de tempore, le effecto del
excesso de anticoagulantes super le activitate
de GAH e de Factor V es apparentemente
non-reversibile.
Iste observationes indica que le quantitate
e le typo de sales anticoagulante es de importantia in stabilisar le mechanismo coagulatori in sanguine immagasinate, in preparar
plachettas pro le transfusion, e in assecurar
uniformitate in le studio e le interpretation
de disordines del coagulation.
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