Successful transfusion of platelets cryopreserved for more than 3 years

Abstract

To determine the duration of storage for cryopreserved platelets, 14 transfusions of random-donor, pooled platelets, stored in the vapor phase of liquid nitrogen for a mean period of 1157 days (range 1060-1240), were analyzed. Twelve of these transfusions were compared in a paired fashion with fresh, random-donor, pooled platelets given within a few days to the same thrombocytopenic recipients. Platelets had been frozen using 5% dimethylsulfoxide as a cryoprotective agent either at a controlled rate of -1 degrees C/min to -80 degrees C or by simply placing them in the vapor phase (-120 degrees C) of a liquid nitrogen freezer. The mean freeze-thaw loss for the 14 transfusions was 22%, and the mean corrected 1-hr increment in platelet count was 12,600/microliter. In the 12 paired observations, the mean corrected 1-hr increment for frozen platelets was 11,800/microliter and 25,900 for fresh platelets, giving a frozen/fresh recovery of 46%. Random donor platelets can be cryopreserved by these methods for greater than 3 yr with satisfactory post-transfusion increments. This suggests that a reservoir of frozen platelets, either random-donor for emergency transfusion or of known HLA-type for transfusion to alloimmunized patients, can be established and stored for at least 3 yr.

Full text

1979 54: 1023-1027
PA Daly, CA Schiffer, J Aisner and PH Wiernik
Successful transfusion of platelets cryopreserved for more than 3 years
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Blood, Vol. 54, No. 5 (November), 1979 1023
Successful Transfusion of Platelets Cryopreserved
for More Than 3 Years
By Peter A. Daly, Charles A. Schiffer, Joseph Aisner, and Peter H. Wiernik
To determine the duration of storage for
cryopreserved platelets. 14 transfusions of
random-donor, pooled platelets. stored in
the vapor phase of liquid nitrogen for a
mean period of 1157 days (range 1060-
1240), were analyzed. Twelve of these
transfusions were compared in a paired
fashion with fresh, random-donor, pooled
platelets given within a few days to the
same thrombocytopenic recipients. Plate-
lets had been frozen using 5% dimethylsulf-
oxide as a cryoprotective agent either at a
controlled rate of -1 C / mm to -80’C or
by simply placing them in the vapor phase
(-1 20CC) of a liquid nitrogen freezer. The
mean freeze-thaw loss for the 14 transfu-
sions was 22%, and the mean corrected
1-hr increment in platelet count was
12,600/tI. In the 12 paired observations,
the mean corrected 1-hr increment for
frozen platelets was 11 ,800/.d and 25,900
for fresh platelets, giving a frozen/fresh
recovery of 46%. Random donor platelets
can be cryopreserved by these methods for
greater than 3 yr with satisfactory post-
transfusion increments. This suggests that
a reservoir of frozen platelets. either
random-donor for emergency transfusion
or of known HIA-type for transfusion to
alloimmunized patients, can be established
and stored for at least 3 yr.
MANY STUDIES have shown that human platelets can be cryopreserved using
dimethylsulfoxide (DMSO) as the cryoprotective agent, and that, after
thawing, they can be effectively transfused to thrombocytopenic patients.’6 In the
majority of instances, such transfusions have been given prophylactically, but there
is also now a sizeable number of reported patients who have shown control of
bleeding and shortening of bleeding times following such transfusions.’2’4’5 Autolo-
gous frozen platelet transfusions have become an important part of the supportive
care of leukemic patients at this institution during maintenance and reinduction
therapy when alloimmunization is frequently present.’-2 When frozen autologous
platelet transfusions are given to patients with acute leukemia there is generally a
short period of storage because of the nature of the disease. The concept, however,
of establishing a reservoir of frozen platelets, either random-donor for emergency
use or H LA-matched for transfusion to ailoimmunized recipients, is now a distinct
possibility, and prolonged storage may be necessary before a need to use such
platelets arises. To study the duration of storage, we analyzed transfusions of
platelets cryopreserved for >3 yr and compared posttransfusion recovery with that
obtained following transfusion of fresh platelets to the same recipients.
Patient Population
MATERIALS AND METHODS
All patients were adults receiving intensive chemotherapy for a variety of malignant neoplasms.
Informed consent was obtained prior to all frozen platelet transfusions. Nonalloimmunized patients
From the Cell Component Therapy Section, Baltimore Cancer Research Program. National Cancer
Institute, at University of Maryland Hospital. Baltimore, Md.
Submitted March 26. /979; accepted July 5. 1979.
Address reprint requests to Charles A. Schiffer, M.D.. Baltimore Cancer Research Program. 22
South Greene Street, Baltimore, Md. 2/20/.
©1979 by Grune & Stratton, Inc. 0006-4971/79/5405-0007$0!.00/0
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1024 DALY ETAL.
were sought who were either receiving their first platelet transfusion or who had recently shown a good
response to fresh random-donor pooled platelets. Only one patient was bleeding prior to frozen platelet
transfusion. He received two transfusions of frozen platelets, but his upper gastrointestinal bleeding had
been controlled with fresh platelets before he received the frozen transfusions.
Platelet Freezing and Thawing
The techniques used were similar to those previously described.’3 Units of platelet concentrate were
prepared by a standard serial centrifugation plateletpheresis technique, using acid citrate dextrose as the
anticoagulant. Whole blood was centrifuged at I 600 gfor 5 mm at 25#{176}Cin a Sorvall RC-3 centrifuge to
obtain platelet-rich plasma. This was then centrifuged at 6975 gfor 10 mm, the plasma was removed,
and the platelet concentrate retained in 40-50 ml of plasma. Three to five units of the same ABO type
were pooled for freezing. The mean number of platelets per freezing was 2.4 x 10” (range I .3-4.0).
The pooled platelets were concentrated by centrifugation at 5000 gfor 6 mm at 25#{176}C.The
supernatant plasma was then removed and the platelets resuspended in a final volume of either 30 or 50
ml, depending on the number of units to be frozen. They were then transferred either to 100-mI or
200-mI polyolefin freezing bags ((-lemoflex 1000-2 or 2030-2, Union Carbide, Chicago, III.), and an
equal volume (30 or 50 ml) of 10% DMSO in ABO-matched plasma was slowly added. The final DMSO
concentration was therefore 5%. The 10% DMSO-plasma mixture had been allowed to cool prior to its
addition to allow for dissipation of heat generated by the addition of DMSO to plasma. Six of the 14
transfusions were then frozen to -80#{176}Cat a controlled rate of -I #{176}C/minas previously described,23 and
the remaining 8 preparations were merely put in metal containers and placed horizontally in the vapor
phase (-I 20#{176}C)of a liquid nitrogen freezer.’ All platelets were stored in the vapor phase of liquid
nitrogen at approximately -I 20#{176}C.
Thawing was accomplished by immersion in a 37#{176}Cwater bath for 4-5 mm. One-hundred milliliters
ofABO-matched plasma and 10 ml ofacid citrate dextrose were added slowly over 15 mm. The platelets
were then transferred to a polyvinyl chloride bag (TA-2, Fenwal Corporation, Morton Grove, Ill.) for
centrifugation at 4400 gfor 6 mm at 25#{176}C.The supernatant plasma containing most of the DMSO was
removed and the platelets resuspended in 100 ml ofABO-matched plasma for transfusion. Samples were
taken for platelet counts, performed electronically, to determine the loss during the freeze-thaw
procedure and also for morphological evaluation by phase microscopy.
Transfusions
The equivalent of 4-8 U of platelets were given per transfusion, administered within I hr of the
thawing procedure through standard blood filters over a period of I 5-30 mm. Platelet counts, using a
Coulter Thrombocounter (Coulter Electronics, Hialeah, Fl.) were done prior to transfusion, I hr
following completion of the transfusion, and in most cases at 18-24 hr. Stable afebrile patients without
infection, bleeding, or splenomegaly were chosen for transfusions. The only exception to this was the
patient with bleeding described earlier who was febrile (>101#{176}F) during the time he had all 4
transfusions (2 frozen and 2 fresh). Three of the patients had been splenectomized. To standardize
results for the body surface area (BSA) of recipients and the number of platelets transfused, increments
were expressed as corrected count increments (Cl) where:
Cl -(Posttransfusion -pretransfusion platelet count) x BSA (sq m)
-Platelets transfused (x 10’’)
The paired transfusions were administered within a period of I wk in all but one patient. In that patient
they were separated by a period of 3 mo without any intervening transfusions. All patients were
markedly thromobocytopenic (<25.00Od) at the time of transfusion.
RESULTS
The mean number of platelets administered in the frozen transfusions was 4.5 x
10” (range 2.8-7.4), which is equivalent to approximately 6-7 “units” of platelet
concentrate. The mean freeze-thaw loss was 22% (range 4%-47%).
Thirteen patients received 14 frozen platelet transfusions (Table 1). The mean
1-hr CI was I 2,600/uI (range 5-25,000). Two patients received frozen transfusions
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PLATELET CRYOPRESERVATION 1025
Table 1. Posttransfusion Platele tCount Increments
No. of Transfusions
Corrected Coun t Increment
1 hr 18-24 hr
Frozen platelets (total)
Fresh platelets
Frozen platelets
(paired observations)
14 (13 patients)
12 (11 patients)
12 (11 patients)
12.600/RI
(5-25.000)
25.900/Ml
(14-56.000)
11 ,800/l
(5-19.000)
8, 1O0/tI
(0-19,000)
22,70O/l
(6-60.000)
7.200/pl
(0-19,000)
Values are expressed as means with ranges in parentheses. The percent recovery for the frozen/fresh paired
observations was 46% at 1 hr and 32% at 18-24 hr.
without paired fresh transfusions. There were therefore I 2 paired observations at I
hr. The mean CI at 1 hr for the 12 frozen transfusions was I I8OO/ul (range
5-19,000) and for fresh 25,900 (range 14-56,000). The mean percent recovery
frozen/fresh at I hr was 46% (range 23%-83). In 10 transfusions, paired
observations were available at I 8-24 hr. The mean Cl at I 8-24 hr for frozen
platelets was 7200/ul (range 0-19,000) and for fresh 22,700 (range 6-60,000).
This gave a frozen/fresh percent recovery at I 8-24 hr of 32%, suggesting a
decreased survival for the frozen platelets. In 2 of the 3 splenectomized patients,
however, recovery at I 8-24 hr was the same as that at I hr posttransfusion.
Nine preparations of thawed platelets were examined by phase microscopy.
Three preparations with good preservation of normal discoid morphology (‘-.50%)
without microscopic clumping gave good posttransfusion increments. Three others
gave poor increments when discoid morphology had been lost (<20% discs), and
pseudopods and clumping were present. Two poor preparations, however, gave
adequate increments, whereas one with good preservation of morphology was
associated with a poor increment.
No hemorrhage occurred in patients receiving frozen transfusions, and gastroin-
testinal bleeding remained under control in the patient previously mentioned. There
were no reactions to the residual DMSO, and patient acceptance was excellent. No
febrile transfusion reactions occurred.
DISCUSSION
Two recent studies from this institution have shown the efficacy of autologous
and allogeneic frozen platelet support in patients with leukemia.”2 In the most
recent study, the mean duration of storage for frozen autologous platelets was I I 8
days with a range of I 3-400,’ but transfusions have been administered successfully
since then after storage periods as long as 823 days (unpublished observation).
There was no correlation between storage duration and posttransfusion recovery in
these earlier reports. Most patients with acute leukemia undergoing maintenance
or reinduction therapy need their own platelets within a short period, so studies of
prolonged storage are impractical in this setting at this time. However, as survival
and treatment -improve in this disease, such studies may become feasible. In other
circumstances, long-term storage may be important for use during times of platelet
storage and in establishing a reservoir of platelets from donors with known HLA
phenotypes.
The platelets used in these transfusions were frozen during 1975. At that time, it
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1026 DALY ETAL.
was unclear whether controlled-rate freezing was necessary, and there was a
suggestion that results were comparable if the platelets were merely placed in a
freezer at -80#{176}C.7Hence, some of the platelets were frozen at a controlled rate and
some by placing them directly at -I 20#{176}Cin the vapor phase of liquid nitrogen. The
platelet concentration necessary for optimal results was also unclear at that time, so
that some were suspended in 60 ml and placed in I00-ml bags and some suspended
in 100 ml and stored in 200-mi containers. Similarly, in the preparation of platelet
concentrate, our understanding of variables such as centrifugation speeds and
methods of storage has improved in recent years. It must be noted that the
centrifugation speeds used in the preparation of these units of platelet concentrate
were not optimal, particularly in the light of the study by Slichter and Harker,8 and
that some damage may have been suffered by the platelets prior to freezing. With
better handling of platelets there has been an improvement in posttransfusion
recovery in successive studies with frozen platelets over the past few years.’
In the most recent reported study of autologous frozen transfusions,’ none of the
patients were splenectomized, and the mean 1 -hr posttransfusion CI was I 37OO/ul.
In another study,2 the mean I-hr CI was I28OO/ul. and here, a comparison with
fresh transfusions in 16 patients gave a frozen/fresh recovery of 65%. The results
obtained with this group of transfusions, while clinically satisfactory, were some-
what inferior. Hopefully, with correction of some of the variables alluded to above,
improvement in recovery should be possible. It appears from this study that
duration of storage, at least at -120#{176}C,is not critical in determining posttransfu-
sion recovery. Whether this is true for platelets stored at -80#{176}C remains to be
seen.
Even though satisfactory increments were obtained in the majority of patients,
only 4 had a rise in absolute count to SOOOO/ul. which would be likely to produce
significant shortening of the bleeding time.9 In the light of the low absolute
increment, the concomitant granulocytopenia and the risk of infection bleeding
time estimations were not done on these patients and therefore the functional
capacity of the platelets was not fully assessed. No bleeding complications occurred
in these patients, and control of upper gastrointestinal hemorrhage in a single
patient persisted following transfusion of frozen platelets.
The poorer recovery at 18-24 hr would indicate that a higher percentage of the
frozen platelets were damaged and therefore removed from the circulation during
the first 18-24 hr following transfusion. This is further suggested by the fact that in
2 of 3 patients studied who had been splenectomized, the recovery at 18-24 hr was
identical with that at I-hr posttransfusion. This was true in the absence of
spontaneous marrow recovery, as demonstrated by a fall in platelet count over
succeeding days. The absence of a spleen in these patients probably allowed for
longer circulation of abnormal platelets.
The shelf-life of frozen platelets, once thawed, has been shown to be short,’#{176}and
it is recommended that they be transfused within a few hours of thawing. It is not a
realistic prospect then to thaw frozen platelets and hold them in reserve for possible
random use. The thawing procedure takes approximately 45 mm, and it is similarly
less than optimal to rely on frozen platelets in the unusual circumstance of
immediately life-threatening emergencies. A more realistic and exciting possibility
is the establishment of a reservoir of transfusions from donors of known HLA
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PLATELET CRYOPRESERVATION 1027
phenotypes for use in alloimmunized recipients. This has obvious advantages in
terms of donation at the donor’s convenience and more immediate availability when
needed, provided a sufficiently representative spectrum of transfusions could be
stored. Presently, we are cryopreserving platelets from donors known to produce
good increments in specific H LA-matched recipients as well as defined homozy-
gotes for common HLA haplotypes who are more likely to serve as good “universal”
donors.
REFERENCES
1. Schiffer CA, Aisner J, Wiernik PH: Frozen
autologous platelet transfusion for patients with
leukemia. N EngI i Med 299:7-12, 1978
2. Schiffer CA, Aisner J, Wiernik PH: Clinical
experience with transfusion of cryopreserved
platelets. Br J Haematol 34:377-385, 1976
3. Schiffer CA, Buchholz DH, Aisner J, Wolff
JH, Wiernik PH: Frozen autologous platelets in
the supportive care of patients with leukemia.
Transfusion 16:321-329, 1976
4. Kim BK, Baldini MG: Biochemistry, func-
tion and hemostatic effectiveness of frozen human
platelets. Proc Soc Exp Biol Med 145:830-835.
1974
5. Slichter SJ, Harker LA: Cryopreservation of
viable and functional platelet concentrates. Clin
Res2O:571, 1972
6. Murphy 5, Sayar SN, Abdou NL, Gardner
FH: Platelet preservation by freezing. Use of
dimethylsulfoxide as cryoprotective agent. Trans-
fusion 14:139-144, 1974
7. Valeri CR, Feingold H, Marchionni LD: A
simple method for freezing human platelets using
6% dimethylsulfoxide and storage at -80#{176}C.
Blood 43:131-136, 1974
8. Slichter SJ, Harker LA: Preparation and
storage of platelet concentrates. Transfusion
16:8-12, 1976
9. Harker LA, Slichter Si: The bleeding time
as a screening test for evaluation of platelet func-
tion. N EngI J Med 287:155-159, 1972
10. Kim BK, Tanoue K, Baldini MG: The
shelf-life of previously frozen human platelets, in:
Platelets: Recent Advances in Basic Research and
Clinical Aspects (International Congress Series
No. 357). Amsterdam, Excerpta Medica, 1974,
pp 457-461
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