T H E EFFECT OF HEPARIN ON THE PLATELET COUNT IN VITRO

W I T H PARTICULAR REFERENCE TO THE COLLECTION OF BLOOD FOR

EXTRACORPOREAL CIRCULATION

H E R B E R T A. P E R K I N S , M.D., J O H N J. OSBORN, M.D., AND F R A N K G E R B O D E , M.D.

Departments of Medicine, Pediatrics, and Surgery, Stanford University School of Medicine,
San Francisco, California

The increasing use of artificial heart-lung that the concentration of heparin required
machines and their large requirement for to prevent platelet adhesion to a foreign
heparinized donor blood has resulted for the surface was considerably higher and took
first time in the extensive employment of longer to exert its effect than that required
blood collected in heparin. Much remains to to prevent coagulation. Copley and Robb, 3
be learned about the effects of heparin as however, demonstrated that, far from having
compared with the usual acidified citrate- a protective effect on platelets, heparin
dextrose (ACD) solutions. It has taken resulted in an abrupt fall in the platelet
many years to develop our present imperfect count when added to fresh whole blood

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knowledge of the ways in which citrate is in vitro. They found the greatest loss of
best used as a blood bank anticoagulant, as platelets with the highest concentrations of
well as its effects on the stability of plasma heparin but also at levels below 1 unit per
components and on the subseciuent survival ml. Wright, on the other hand, found that
of transfused cells. with heparin, as with citrate and oxalate,
I t seems particularly important to deter- the drop in the thrombocyte count was
mine the effects of heparin on blood coagula- greater the less the anticoagulant. 8 She also
tion factors in view of the fact that a post- showed that the more the platelets dis-
operative hemorrhagic syndrome was a appeared, the more could be found coated on
major obstacle during the pioneer days of the wall of the container. She believed that
developing heart-lung machines and is still this was probably the explanation of their
a problem to man}'' of the teams in the field. removal. More in line with AVright's work
The present investigation lias concerned it- was the observation of Copley and Robb
self with the effect of heparin on platelets. that addition of citrate prevented loss of
Thrombocytopenia has been demonstrated platelets due to heparin.3 Puzzling and
to be one of the possible causes of a post- unexplained, however, was the fact that
operative hemorrhagic diathesis,6 and hep- when blood was heparinized in vivo the
arin has been reported to cause a pronounced platelet count did not drop when the blood
drop in the platelet count. was removed to a glass container, not even
Studies published to date on the effect of if more heparin was added in vitro.*
heparin on platelets have revealed a number This report will include studies done in an
of serious contradictions and have raised a effort to explain why heparin accelerates
number of unanswered questions. Best and platelet loss under some conditions but will
his associates1 made the first important prevent it under others. In addition, it will
observation in 1938 when they demonstrated include our findings on in vitro changes in
Received, May 3, 195S; revision received, May
the platelet count encountered in the course
26; accepted for publication June 9. of collecting blood by a variety of technics
Dr.. Perkins is Assistant Clinical Professor of for human heart-lung bypass.
Medicine, Dr. Osborn is Associate Professor of
METHODS
Pediatrics, and .Dr. Gerbode is Associate Professor
of Surgery. Unless otherwise stated, 0.2 ml. of hep-
This work was aided in part by grants from arin* in saline was placed in 15-ml. glass
the United States Public Health Service, Depart-
ment of Health, Education, and Welfare, and from * Upjohn heparin sodium, 1000 units per ml.,
the American H e a r t Association. was used.
397
398 PERKINS ET AL. Vol. SO

counting vials and 2 ml. of blood were TABLE 1

added. Control samples were taken simul- P L A T E L E T SURVIVAL I N D O N O R BLOOD COLLECTED
taneously in an equal volume of 10 per cent IN PLASTIC BAGS WITH SMALL QUANTITY OP
Na? EDTA. All samples of blood were taken A C I D I F I E D C I T R A T E - D E X T R O S E IN ADDITION
by venipuncture with silicone-coated TO 2000 U N I T S OP H E P A R I N

syringes from apparently normal human (all counts X 103)

volunteers and immediately added to the Control 4Hr.
counting vials. The vials were given 5
vigorous shakes to mix the blood and 196 170
anticoagulant and then allowed to stand 222 234
undisturbed at room temperature until 162 210
sampling time; at that point the vials were 214 230
shaken vigorously 100 times. Pipets were 288 210
shaken for 45 sec. in a Yankee pipet shaker.* 214 282
The platelets were allowed to settle on the 204 222
294 342

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counting chamber for 30 min. under a petri
312 258
dish containing a piece of moistened filter
282 334
paper.
All platelet counts were performed by one Average 239 249
of us (H. A. P.), using the Rees-Ecker
method7 in the early part of the study and into plastic bags that originally contained
the Brecher-Cronkite phase microscope N.I.H. formula B ACD solution.* This
method2 for the more recent determinations. solution was removed as completely as
When clumps were found that contained possible before heparin was placed in the
more than 2 or 3 platelets, so that the exact empty bag, but the construction of the
number was not certain, they were not in- bags was such that 10 to 20 ml. of ACD
cluded in the count. solution could not be removed. Two milli-
Blood from blood bank bottles and plastic liters of heparin (2000 units) were added to
bags was sampled after inverting the con- each bag immediately before the taking of
tainers 100 times. The heparinized bank blood. Table 1 indicates that there was no
blood was kept at room temperature until loss of platelets at the end of 4 hr. Table 2
it reached the operating room floor, where it lists the changes in counts that occurred in a
was placed in a 37 C. oven until sampled. series of bags that was not used for an opera-
On the average, it spent 2}/2 hr. at room tion and was stored in a refrigerator at 4 C.
temperature and V/i hr. at 37 C. Control At 24 hr., the counts had dropped greatly,
samples with EDTA were taken from the but there, were many large clumps of well-
donor at the time of venesection through defined platelets that were not included in
the same siliconed needle and plastic tube the count. At 11 days, the clumps had broken
donor set as soon as the large bottle or up, but the number of platelets was defi-
plastic bag had been disconnected. In these nitely reduced as compared to the value on
experiments, 5 ml. of blood were added to the day the blood was drawn. The platelets
0.2 ml. of EDTA in the sample vial regard- were becoming difficult to identify, and by
less of whether or not the blood was already 14 days, they could not be identified with
heparinized. any certainty.
RESULTS Empty plastic bags. The second series
(Table 3) was collected under the same
Platelet Changes in Vitro in Blood Bank conditions but into empty plastic bags (no
Bottles and Bags ACD).f Most of the platelets were still
Plastic bags with a small quantity of ACD * Saftiflex plastic bags, C u t t e r Laboratories,
solution. In our first series, donors were bled Berkeley, California.
t M T 600 plastic bags, C u t t e r Laboratories,
* Clay Adams, Inc., New York. Berkeley, California.
Nov. 1958 H E P A R I N AND P L A T E L E T COUNT 399

TABLE 2 TABLE 4
P L A T E L E T SURVIVAL IN D O N O R BLOOD COLLECTED P L A T E L E T SURVIVAL I N D O N O R BLOOD COLLECTED
IN P L A S T I C B A G S WITH SMALL Q U A N T I T Y O F I N SILICONE-COATED BOTTLES
A C I D I F I E D C I T R A T E - D E X T R O S E I N ADDITION
Control 4Hr. Procedure
TO 2000 U N I T S OF H E P A R I N

4 Hr. 1 Day 11 Days 14 Days 335 243 G r a v i t y collection

330 305 Gravity collection
486 66 112 0 205 177 Gravity collection
312 120 142 0 180 203 Gravity collection
222 90 170 0 203 205 G r a v i t y collection
312 124 276 0 252 255 Gravity collection
350 30 190 0 270 260 Gravity collection
346 50 374 0 260 21S Partial vacuum
21S 222 Partial vacuum
Average 338 SI 211 0 252 192 Vacuum
(plus (none recog- 193 17S- Vacuum

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clumps) nizable)
Average 245 220

TABLE 3
P L A T E L E T SURVIVAL I N D O N O R BLOOD COLLECTED
I N E M P T Y PLASTIC B A G S

Control 4Hr.

292
292 362
362 "1

o
352
352 190
190
456
456 332
332 "200
238
238 274
274
COUN

• 10
253 unit
312
312 253
255
255 1S3
1—
UJ
_i
Average 318
Average 266
266 UJ
1—

5100
Q.
present 4 hr. after collection, but, in con- 5
unit
trast to the first series, a small but definite 3

loss had taken place. \ — - 0 5 uni j / m l unit

Siliconed bottles. Series 3 (Table 4) was 0 units

collected in commercially available, sili-

coned glass bottles containing 1800 units of S I 2 3 4 5 6
heparin in a glucose-saline menstruum.* I HOURS OF INCUBATION
i AT ROOM TEMPERATURE
Platelet loss was negligible, particularly in 1

bottles in which the vacuum had been ex- F I G . 1. Curves illustrating the effect of various
concentrations of heparin on the platelet count
hausted before the donor set was plugged in. in vitro.
Platelet Changes in Vitro in Small Counting
Vials with a total of 91 individual platelet counts.
Two points stand out: (1) the greater part of
Effect of the concentration of heparin.
the loss occurs within the first few minutes,
Figure 1 represents the changes in the
and (2) the fall in the count is greater the
platelet count that occurred on incubation
less the heparin. The'largest decrease occurs
at room temperature with various concen-
when no anticoagulant is used.
trations of heparin per ml. of blood. The
curves represent 31 different experiments Effect of the surface of the container. The
results of paired simultaneous experiments
* Abbott Laboratories, North Chicago, Illinois. on the same blood, comparing the effect of
400 PERKINS ET AL. Vol. SO

TABLE 5
E F F E C T O F V A R I O U S SURFACES ON T H E P L A T E L E T C O U N T O F H E P A R I N I Z E D BLOOD

Minutes Hours of Incubation

Heparin Control Surface
(EDTA)
5 30 l 2 3 4 5 6

units/ml.
5 198 Glass 68 85 88 43 38
Polyethylene 38 110 45 58 45
1 318 Glass 224 98
Silicone 188 86
5 260 Glass 46 76 124 82 96
Silicone 6S 62 74 68 54
10 256 Glass 108 130 136
Silicone 222 200 178
10 318 Glass 382 136 366
Silicone 222 240 180

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TABLE 6 data in Table 6, one can see the protective
P R O T E C T I V E E F F E C T OF A C I D I F I E D CITRATE-DEX- effect of adding dilutions of ACD solution to
TROSE ON P L A T E L E T S I N P R E S E N C E the heparin. Platelet loss is decreased by
OF H E P A R I N surprisingly small quantities of the citrate
(3 u n i t s / m l . ) solution.
Heparin
- Effect of varying volume of blood in con-
Control ACD 1:20 ACD 1:200 Alone tainers of the same size. Finally, the data in
Table 7 indicate that platelet loss is slight if
290 258 242 the volume of heparinized blood is large
13S 46 66 enough to nearly fill the container. This is
324 306 222 66
true regardless of variations in the volume
338 276 134 76
324 190 136
of anticoagulant. A control sample in an
98
identical container with the same amount of
-heparin per ml. of blood but a smaller total
TABLE 7 volume of blood reveals a marked drop in
E F F E C T O F R A T I O O F VOLUAIE TO SURFACE the platelet count.
Platelet Counts X 10' 1 hr. After Mixing
Fresh Blood with Anticoagulant DISCUSSION
Heparin
Concentration
in Blood Control 15* 15* 15* In the procurement of blood for operations
1.5t 15t
(EDTA) 0.06J 0.6} ist involving extracorporeal circulation, it may
0.06J
be of importance to use technics that ensure
units/ml.
the best possible survival of the transfused
4 238 160 250 295
platelets in vivo. Technics for measuring
2.7 325 5 102 163
4 405 19 343 343
in vivo survival in human beings under these
conditions are not very satisfactory. Per-
* C o n t a i n e r size (ml.) sistence of platelets in vitro is no indication
t Volume of blood (ml.) that they will survive on subsequent injec-
% Volume of heparin (ml.) tion but can serve as a partial index of the <"
relative deficiencies of various methods of
an uncoated glass container with that of collection and storage.
siliconed glass and with polyethylene, are The in vitro survival of platelets in hepar-
listed in Table 5. No differences are found inized blood in blood bank containers was
except those to be expected within the range surprisingly good. Platelets were preserved
of error of the technic. in near normal quantities up to the time the
Effect of added ACD solidion. From the blood was added to the artificial heart-lung
Nov. 1958 H E P A R I N AND P L A T E L E T COUNT 401

machine, 4 hr. after procurement. A small valid interpretations can be made. In a few
amount of ACD solution unavoidably left experiments (not included above) the plate-
in some of the containers resulted in 100 let count rose so sharply after heparin that
per cent survival. This protective effect of fragmentation of platelets seems to be the
small amounts of ACD solution was borne only logical explanation. This happened
out by experiments with small glass vials as simultaneously to the same degree in several
reported in Table 6. In vitro platelet preser- vials, so it is not believed to be a counting
vation in commercially available, siliconed error. Obviously, fragmentation occurring to
glass bottles containing heparin in a solution a variable degree could also explain some of
of glucose-saline was better than that in the discrepancies in the counts reported by
plastic bags. The 2 sets of experiments were various authors. Another explanation is
not completely comparable, however. The suggested by the fact that Quick and his
plastic bags contained only 2000 units of associates,6 as well as Fidlar and Jaques,4
heparin in 2 ml. of saline; the bottles had found an abrupt transitory drop in the
1800 units of heparin in 60 ml. of 5 per cent platelet count with intravenous heparin in
glucose and quarter-strength saline. The 2 dogs. The latter group, however, found that

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sets of collections were obtained months some heparin products affected the platelet
apart and some differences in the technics count much more than others. Our own
of handling the blood may have occurred. experiments have never demonstrated a
Contrary to the results just mentioned, thrombocytopenia following intravenous
experiments with small counting vials con- heparin. It is possible that some of the
firmed the reports of Copley and Robb 3 earlier preparations tested were contami-
that the platelet count drops rapidly nated with something that could cause
when fresh blood is added to heparin thrombocytopenia and that this could
in vitro. Like Fidlar and Jaques, 4 we were explain the platelet drop in vitro found with
surprised to find no difference when the large amounts of heparin by Copley and
glass container was siliconed; nor did a Robb, results so different from our own and
polyethylene surface offer the platelets any those of Wright.
better protection. This thus excludes the We would like, however, to take exception
possibility that the better survival in blood to the hypothesis of Wright that the plate-
bank containers was the result of the use lets are lost by being caught on the surface of
of vessels with less harmful surfaces. Con- the container. In most cases where there is
firming the report of Helen Wright, we an abrupt fall in the platelet count, a careful
found that the loss of the platelets was search of the entire counting chamber will
greater the less the heparin. Inasmuch as disclose a few huge clumps of platelets,
platelets diminished to the greatest degree large enough to account for all those that are
when no anticoagulant was present, it is missing. Further evidence that platelets are
most likely that the drop in the platelet not necessarily lost on the surface comes
count is not the result of any direct effect of from experiments we have performed in
heparin, but is merely attributable to the which hepariiiized blood was recirculated
fact that insufficient anticoagulant is present for 30 min. through the pumps and oxy-
to protect the platelets. genators of several different types of artificial
Several explanations come to mind to heart-lung machines without an animal or
explain the discrepancies in the literature. patient in the circuit. Ninety per cent of the
Platelets tend to clump when heparin is the platelets remained in the circulating blood
anticoagulant. This happens to a greater at the end of that time. 5 Equal fall of plate-
degree with small concentrations of heparin, let counts in uncoated glass, siliconed glass,
but was encountered at all levels tested. and polyethylene containers also suggests
Because of this, even dispersion of the that adherence to the surface of the con-
platelets is impossible to achieve and the tainer is not the major cause of platelet loss.
inaccuracy of the counts is increased. Ex- This does not contradict Wright's demon-
periments need many repetitions before stration that more platelets adhere to un-
402 P E R K I N S ET AL. Vol. 30

coated glass when heparin is present in time to produce full effect. This would ex-
smaller concentration. plain why loss of platelets is minimal after y
We have shown, as did Copley and Robb, 3 the first few minutes. It would also explain
that most of the loss of platelets occurs in why blood which has had adequate time to
the first few minutes. After that, platelet react with heparin in vivo does not lose ^J
loss is relatively slight and is accompanied platelets when transferred to a glass con-
by gradual loss of the typical platelet ap- tainer, i
pearance. The granules fade away and The above findings suggest that in the |
clumps become 1 large amorphous mass collection of blood in heparin for use in an ^
before the platelets finally become indis- artificial heart-lung machine the container
tinguishable. These changes are accelerated size should be such that it is nearly filled by
by frequent shaking of the container—much the volume of blood to be taken. The con- •*•
more so if the container is shaken exces- tainer should be shaken no more than is
sively in the first few minutes after collec- absolutely necessary to mix anticoagulant
tion. and blood during the period of collection 4
The preservation of platelets in blood and for a short time thereafter.
taken into heparin is revealed to be depen-

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dent on the relation between the volume of SUMMARY AND CONCLUSIONS
blood and the size of the container in which <
it is placed. If the container is nearly filled 1. The fall in the platelet count that
with blood, loss of platelets is minimal, even occurs when fresh blood is added to heparin
at concentrations of heparin that permit a in vitro is greater the less the anticoagulant.
sharp drop in the count if a small quantity Most of the loss occurs in the first 5 min.
of blood is placed in a very large container. 2. Loss of platelets is as great and as
This suggests a high volume to surface ratio rapid in siliconed glass containers and
is beneficial because most of the contained polyethylene vials as in uncoated glass. *
platelets never come into contact with the Platelets are lost primarily by clumping and
foreign surface of the container or (perhaps disintegration rather than by adherence to
even more important) the air-blood inter- the foreign surface. i
phase. 3. Loss of platelets is minimized by hav-
ing the containers filled to near capacity.
Finally, we have confirmed the fact that
This explains why loss of platelets is slight
platelets do not drop in heparinized blood
under the conditions in which donor blood is
if citrate or EDTA is also present.3 We also
collected for extracorporeal circulation. «-
find the in vitro platelet count of blood
heparinized in vivo stable without any SUMMARIO I N INTERLINGUA
additional anticoagulant.
We believe that the following hypothesis 1. Le reduction in le numeration plachettal '
fits all the facts as outlined and is the best que occurre quando sanguine fresc es addite
explanation of what occurs. Heparin prod- a heparina in vitro es plus marcate con
ucts presently available have no direct anticoagulante diminuite. Le plus grande .<,
thrombocytopenic effect in vitro or in vivo. parte del perdita occurre durante le prime
Inadequate amounts of heparin permit 5 minutas.
changes in platelets leading to their clump- 2. Le perdita de plachettas es tanto
ing and disintegration. Contact with a marcate e rapide in receptaculos de vitro
foreign surface is necessary to initiate these revestite de silicona e in phiales de poly-
changes. Inasmuch as nonwettable surfaces ethyleno como in vitro non-revestite.
have the same action as uncoated glass, this Plachettas es perdite per conglutination e '
is apparently not mediated through the disintegration plus tosto que per adhesion a
initiation of blood coagulation. Heparin will superficies estranie.
prevent these changes if present in suf- 3. Le perdita de plachettas es reducite al -i
ficiently high concentration. The action of minimo quando le receptaculos es plenate
heparin in inhibiting platelet changes takes quasi al limites de lor capacitate. Isto
explica proque le perdita de plachettas es „
 NOV. 1958 HEPARIN AND /ATELET COUNT 403

y leve sub le conditiones de collection de san- 4. F I D L A R , E . , AND J A Q U E S , L. B . : T h e effect ol

commercial heparin on t h e platelet count.
guine de donator pro le circulation extra- J . L a b . & Clin. Med., 33: 1410-1423, 1948.
corporee. 5. P E R K I N S , H . A., O S B O R N , J . J . , AND G E R B O D E ,
F . : Platelet loss in operations with an arti-
-> REFERENCES ficial heart-lung a p p a r a t u s . Clin. R e s .
P r o c , 5: 35, 1957.
1. B E S T , C. H . , C O W A N , C , AND M A C L E A N , D . L . :
v 6. Q U I C K , A. J., SHAMBERGE, J. N., AND
Heparin and t h e formation of white thrombi.
STEFANINI, M . : T h e effect of heparin on
J . Physiol., 92: 20-31, 1938. platelets in vivo. J . L a b . & Clin. Med., 3 3 :
^ 2. B R E C H E R , G., SCHNEIDERMAN, M., AND
1424-1430, 1948.
C R O N K I T E , E . P . : T h e reproducibility and
constancy of t h e platelet count. Am. J. 7. W I N T R O B E , M . M . : Clinical Hematology. E d .
Clin. P a t h . , 23: 15-26, 1953. 2. Philadelphia: Lea & Febiger, 1946.
3. C O P L E Y , A. L., AND R O B B , T . P . : Studies on 8. W R I G H T , H . P . : T h e adhesiveness of blood
platelets. I I . T h e effect of heparin on t h e platelets in normal subjects with varying
platelet count in vitro. Am. J . Clin. P a t h . , concentrations of anticoagulants. J . P a t h .
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