Transfusion Medicine Reviews 28 (2014) 212–225

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Transfusion Medicine Reviews journal homepage: www.tmreviews.com

Review of In Vivo Studies of Dimethyl Sulfoxide Cryopreserved Platelets Sherrill J. Slichter a,b,⁎, Melinh Jones a, Janet Ransom c, Irena Gettinger a, Mary Kay Jones a, Todd Christoffel a, Esther Pellham a, S. Lawrence Bailey a, Jill Corson a, Doug Bolgiano a a b c

Puget Sound Blood Center Seattle, WA University of Washington School of Medicine, Seattle, WA Fast-Track Drugs & Biologics, LLC, North Potomac, MD

a r t i c l e

i n f o

Available online 21 September 2014 Keywords: Platelets Cryopreservation Dimethyl sulfoxide Platelet transfusions

a b s t r a c t A literature review was conducted to assess the efficacy and safety of dimethyl sulfoxide (DMSO) cryopreserved platelets for potential military use. In vivo DMSO cryopreserved platelet studies published between 1972 and June of 2013 were reviewed. Assessed were the methods of cryopreservation, posttransfusion platelet responses, prevention or control of bleeding, and adverse events. Using the Department of Defense's preferred 6% DMSO cryopreservation method with centrifugation to remove the DMSO plasma before freezing at −65°C and no postthaw wash, mean radiolabeled platelet recoveries in 32 normal subjects were 33% ± 10% (52% ± 12% of the same subject's fresh platelet recoveries), and survivals were 7.5 ± 1.2 days (89% ± 15% of fresh platelet survivals). Using a variety of methods to freeze autologous platelets from 178 normal subjects, mean radiolabeled platelet recoveries were consistently 39% ± 9%, and survivals, 7.4 ± 1.4 days. More than 3000 cryopreserved platelet transfusions were given to 1334 patients. There were 19 hematology/oncology patient studies, and, in 9, mean 1-hour corrected count increments were 11 100 ± 3600 (range, 5700-15 800) after cryopreserved autologous platelet transfusions. In 5 studies, bleeding times improved after transfusion; in 3, there was either no improvement or a variable response. In 4 studies, there was immediate cessation of bleeding after transfusion; in 3 studies, patients being supported only with cryopreserved platelets had no bleeding. In 1 cardiopulmonary bypass study, cryopreserved platelets resulted in significantly less bleeding vs standard platelets. In 3 trauma studies, cryopreserved platelets were hemostatically effective. No significant adverse events were reported in any study. In summary, cryopreserved platelets have platelet recoveries that are about half of fresh platelets, but survivals are only minimally reduced. The platelets appear hemostatically effective and have no significant adverse events. © 2014 Elsevier Inc. All rights reserved.

Contents Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Autologous Cryopreservation Studies in Normal Subjects . . . . . . . . . . . . . . . . . . Valeri Prefreeze DMSO Removal Method . . . . . . . . . . . . . . . . . . . . . . . In Vivo Platelet Recovery and Survival Studies Comparing CPP to Autologous Fresh Platelets Adverse Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In Vitro Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results of Previously Reported Autologous Radiolabeled Cryopreserved Platelet Studies . . In Vivo Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Radiolabeled Autologous Platelet Recoveries and Survivals . . . . . . . . . . . Adverse Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Source of funding/support: This research study was supported by the US Army Medical Research and Materiel Command under contract number W81XWH-07-D-0016. The views, opinions, and/or findings contained in this report are those of the authors and should not be construed as an official Department of the Army or Navy position, policy, or decision unless so designated by other documentation. The US Army Medical Research and Materiel Command was not involved in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; and/or in the decision to submit the manuscript for publication. Administrative support: The authors gratefully acknowledge the assistance of Ginny Knight in preparing this manuscript. Conflict of interest: The authors certify that they have no affiliation with or financial involvement in any organization or entity with a direct financial interest in the subject matter or materials discussed in this manuscript. Competing interests: The authors certify that they have no competing interests. ⁎ Corresponding author at: Sherrill J. Slichter, MD, Director, Platelet Transfusion Research, Puget Sound Blood Center, 921 Terry Ave, Seattle, WA 98104-1256. E-mail address: [email protected] (S.J. Slichter). http://dx.doi.org/10.1016/j.tmrv.2014.09.001 0887-7963/© 2014 Elsevier Inc. All rights reserved.

S. J. Slichter et al. / Transfusion Medicine Reviews 28 (2014) 212–225

In Vivo Platelet Function Studies . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In Vivo Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adverse Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . Patients Transfused With Cryopreserved Platelets . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In Vivo Platelet Responses . . . . . . . . . . . . . . . . . . . . . . . . . . Hematology/Oncology Patients . . . . . . . . . . . . . . . . . . . . Hemostatic Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adverse Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cardiopulmonary Bypass Patients (1 Study) . . . . . . . . . . . . . . . . . . Posttransfusion Platelet Responses, Blood Product Utilization, and Hemostasis Adverse Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . Trauma Patients (3 Studies) . . . . . . . . . . . . . . . . . . . . . . . . . Hemostatic Efficacy . . . . . . . . . . . . . . . . . . . . . . . . . . Adverse Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

The short 5-day shelf life of platelets severely limits platelet availability at far forward combat facilities. Because of the limited platelet supply, the US Department of Defense is supporting studies to develop cryopreserved platelets. As part of this effort, a thorough review of the English language literature from 1972 to June 2013 to evaluate the safety and effectiveness of dimethyl sulfoxide (DMSO) cryopreserved platelets based only on in vivo studies was performed. Studies included autologous cryopreserved radiolabeled platelet recovery and survival measurements in normal subjects and outcomes after cryopreserved platelet transfusions in thrombocytopenic patients with hematologic/ oncologic disorders or in surgical or trauma patients. To use cryopreserved platelets, they must be approved by the US Food and Drug Administration (FDA). The FDA has expressed concern about the potential prothrombotic phenotype of cryopreserved platelets. In vitro studies of cryopreserved platelets have demonstrated a significant increase in the number of microparticles after platelet thawing as well as increases in the expression of activation markers such as pselectin and annexin V [1–4]. However, in vitro platelet assays do not necessarily correlate with posttransfusion platelet viability, hemostatic effectiveness, or thrombotic potential [5–8]. Therefore, this review will report only the results of in vivo studies, and any in vitro data provided in the same article will be given in Supplementary Appendices I and II. Background In the mid 1970s, Dr Robert Valeri, with the support of the US Office of Naval Research, began development of a potential substitute for standard room temperature stored platelets that could be used on the battlefield. His original method involved freezing platelets with 6% DMSO at −80°C, thawing, followed by centrifugation with washing to remove most of the DMSO, and platelet resuspension in plasma [9]. However, the washing step that required centrifugation would be difficult to achieve in battlefield conditions, and so, a second method was developed to centrifuge the platelets to remove the DMSO-containing supernatant plasma before freezing [10]. Cryopreserved platelets, according to Valeri's second method, will be termed CPP in this report and using other methods as “cryopreserved platelets.”

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recovery and survival measurements of CPP in 12 normal subjects. Results from 2 centers (nos. 1 and 2) have been previously reported [11], and the radiolabeling data from all 3 centers are provided here to give the latest comprehensive results. In Vivo Platelet Recovery and Survival Studies Comparing CPP to Autologous Fresh Platelets Apheresis platelets were suspended in 6% DMSO, concentrated by centrifugation, all except 10 to 15 mL of the supernatant DMSO plasma was removed, and the platelets were placed in a less than or equal to −65°C freezer for 7 to 11 days. In 1 center (no. 1), platelets were cryopreserved within 8 hours of collection, and in the other 2 (nos. 2 and 3), apheresis platelets were stored for up to 57 hours at 22°C followed by 2500 cGy irradiation before cryopreserving. The extra time before cryopreserving would likely be needed in routine blood center operations. On the day of reinfusion, cryopreserved CPP were thawed for 10 to 15 minutes at 37°C and resuspended in 25 mL of 0.9% NaCl. Fresh autologous platelets were also collected on the day of reinfusion. Resuspended CPP and fresh platelets from the same donor were radiolabeled with either 51Cr or 111In, combined and reinfused into their autologous donor. Radiolabeling was performed using the Biomedical Excellence for Safer Transfusion method [12]. Blood samples were collected over the next 12 days to determine recovery and survival of each product using the multiple-hit regression method [13]. Combining the results of the 32 evaluable subjects, mean CPP in vivo recoveries were 33% ± 10% vs 63% ± 9% for fresh recoveries (52% ± 12% of fresh) (P b .0001), and CPP survivals were 7.5 ± 1.2 days vs 8.6 ± 1.1 days for fresh (89% ± 15% of fresh) (P b .0001). There were no differences among the laboratories (Table 1). According to FDA's platelet acceptance criteria, the 95% lower confidence limit of the test platelet (CPP) recoveries should be greater than or equal to 67% of the same subject's fresh recoveries, and survivals should be greater than or equal to 58% of fresh survivals [14]. Although CPP recoveries did not meet FDA acceptance criteria, survival criteria were met. Adverse Events

Autologous Cryopreservation Studies in Normal Subjects Valeri Prefreeze DMSO Removal Method Three centers are collaborating with the Department of Defense to evaluate CPP. Each center conducted radiolabeled autologous platelet

Adverse events in the 36 subjects in these microdosing studies (one-tenth of an apheresis unit of CPP) were 1 subject had transient (2 hours) metallic taste and mild chest tightness, 1 subject had a sinus headache, 1 subject had an upset stomach/diarrhea, and 1 subject had mild hypertension after transfusion.

214

S. J. Slichter et al. / Transfusion Medicine Reviews 28 (2014) 212–225

Table 1 Radiolabeled autologous CPP platelet recoveries and survivals by laboratory Laboratory

1 2 3 Total

n

12 9 11

Platelet recovery (%)

Platelet survival (d) P

% of fresh

Fresh

CPPa

34 ± 8 35 ± 9 31 ± 12

b.0001 b.0001 b.0001

54 ± 10 52 ± 12 52 ± 14

9.1 ± 0.4 7.7 ± 1.7 8.7 ± 0.4

8.0 ± 1.5 6.7 ± 1.1 7.7 ± 0.7

.0158 .0305 .0004

87 ± 16 90 ± 20 89 ± 7

33 ± 10

b.0001

52 ± 12

8.6 ± 1.1

7.5 ± 1.2

b.0001

89 ± 15

Fresh

CPP

64 ± 5 68 ± 7 60 ± 13 63 ± 9

a

P

% of fresh

Data are reported as mean ± 1 SD. a Cryopreserved platelets prepared according to second method of Valeri and the method under current development for FDA approval.

Results of Previously Reported Autologous Radiolabeled Cryopreserved Platelet Studies

In Vitro Assays In vitro qualitative and functional assays of the same donor's fresh platelets, after 22°C storage for 45 to 57 hours followed by γ-irradiation and after thawing were done at center 3 (Table 2). The following assays decreased over time and as result of cryopreservation: swirl test positive units (an indication of discoid morphology), morphology scores (change from discoid appearance to spherical appearance), aggregation responses to platelet agonists, hypotonic shock response, extent of shape change, and pH. The following assays showed an increase: platelet-associated p-selectin expression (an activation marker), annexin V binding (an apoptotic marker), and platelet microparticles. These data were further analyzed to show potential correlations among the in vitro assays and in vivo measurements of CPP recoveries and survivals (Figure). The only statistically significant relationship was an inverse correlation between glucose concentration and CPP recoveries (P = .01). Although both platelets stored at 22°C and cryopreserved demonstrate increased activation markers, decreased aggregation in vitro, and evidence of damage (morphology and microparticles), these deficits appear to be reversible after transfusion and do not correlate with platelet in vivo viability. Summary These data demonstrate substantial decreases in posttransfusion CPP platelet recoveries, but of those platelets that circulate, survivals are very similar to fresh. Furthermore, storing the platelets for 2 days under standard blood center conditions followed by γ-irradiation before freezing does not appear to affect postthaw platelet results making CPP practical for routine blood center operations.

The purpose of this review was 2-fold: (1) to identify the effects of freezing conditions on in vivo platelet recoveries and survivals and (2) to determine if there was any evidence of thromboembolic complications in subjects with normal hemostatic systems who might be very susceptible to thromboembolic complications when transfused with even small numbers of activated platelets. A complete summary of the data is given in Supplementary Appendix I. In Vivo Data Radiolabeled Autologous Platelet Recoveries and Survivals Eleven studies of autologous radiolabeled cryopreserved platelets given to normal subjects from 1972 to 2005 were identified with 9 coming from the laboratory of Dr Valeri (coauthors Drs Handin, Spector, Vecchione, and Melaragno) [9,10,15–23] (Table 3). Another study by Valeri et al [24] was identified, but the radiolabeled data appeared to be the same as previously reported [17]. Some studies had concurrent fresh as well as cryopreserved radiolabeled platelet recovery and survival data, but the fresh and cryopreserved platelets were not from the same subjects. Although we report the results of these fresh compared with cryopreserved platelet data, the results are provided only for information as FDA acceptance criteria requires fresh and cryopreserved data from the same subject. Mean cryopreserved platelet recoveries ranged between 29% and 63% in 11 studies involving 150 subjects, with an overall mean of 39% ± 9% (± 1 SD) [9,10,15–23]. In the 5 studies that reported fresh data in 41 subjects, mean fresh recoveries ranged between 60% and 73%,

Table 2 In vitro characteristics of the same subject's fresh platelets, after storage for 45 to 57 hours at 22°C followed by γ-irradiation, and after thawing Parameter

Fresh platelets

Platelets stored at 22°C for 45-57 h followed by γ-irradiation

Thawed platelets

No. of CPP units evaluateda pH (22°C) pO2 (mm Hg) pCO2 (mm Hg) Glucose concentration (mg/dL) Lactate concentration (mmol/L) Extent of shape change (%) Hypotonic shock response (%) P-selectin expression (%) Platelet microparticles (%) Morphology by light microscopy Platelet swirl (2, positive; 1, intermediate; 0, negative) Platelet aggregation (ADP + EPI) (%) Platelet aggregation (collagen) (%) Annexin V binding (%) CPP yield relative to fresh platelets (%) CPP yield relative to platelets stored for 45-57 h (%)

12 7.2 ± 0 120 ± 17 73 ± 8 387 ± 28 1.9 ± 0.9 24 ± 6 62 ± 12 12 ± 10 1.9 ± 0.8 377 ± 10 2±0 92 ± 3 93 ± 3 3±2 NA NA

12 7.4 ± 0.1 110 ± 18 31 ± 5 353 ± 2539 5.3 ± 1.9 24 ± 5 66 ± 13 21 ± 9 2.1 ± 0.6 356 ± 13 2±0 63 ± 28 77 ± 13 5±4 NA NA

12 6.6 ± 0.1 63 ± 24 51 ± 7 122 ± 19 7.3 ± 1.1 3±3 20 ± 6 84 ± 6 22.9 ± 6.5 189 ± 29 0±0 26 ± 15 19 ± 13 64 ± 12 65 ± 3 74 ± 5

Abbreviations: NA, not applicable. Data are reported as mean ± 1 SD. a In vitro data from center 3.

S. J. Slichter et al. / Transfusion Medicine Reviews 28 (2014) 212–225

215

Figure. Relationship between in vivo and in vitro assays. (A) Platelet recoveries versus in vitro assays. Radiolabeled autologous recovery of CPP vs in vitro assays. There is a statistically significant inverse correlation between glucose concentrations and in vivo recoveries (P = .01). There are no other significant correlations. (B) Platelet survivals vs in vitro assays. Radiolabeled autologous survivals of CPP are plotted against in vitro assays. There are no statistically significant correlations. Abbreviations: MPV, mean platelet volume; ESC, extent of shape change; HSR, hypotonic shock response.

with an overall mean of 65% ± 4% [9,15–17,19,21,22]. Cryopreserved recoveries as a percent of fresh recoveries reported in the same manuscript ranged between 42% and 95%, with an overall mean of 69% ± 16% [9,15–17,19,21,22]. Mean cryopreserved platelet survivals ranged from 3.1 to 8.8 days, with an overall mean of 7.4 ± 1.4 days [9,10,16–22]. For the studies with fresh data, platelet survivals ranged between greater than or equal to 7 and 8.9 days with a mean of 8.4 ± 0.4 days [9,15–17,19,21,22]. Cryopreserved platelet survivals were between 99% and 100% of fresh [9,16,17,19,21,22]. These studies used a variety of freezing conditions including DMSO concentrations of 4% to 6%, 2% DMSO with Thrombosol, freezing bags (polyvinyl chloride vs polyolefin), freezing rates that were controlled at 1°C/min or uncontrolled at 2 to 3°C/min, storage at − 80°C in a mechanical freezer or storage at − 150°C in the vapor phase of liquid nitrogen, storage times of 1 day to 235 days, and a variety of methods of adding and removing the DMSO. Because of small numbers and other uncontrolled variables between the studies, it

was not apparent that any of these factors made any consistent difference in the radiolabeled recoveries and survivals of autologous cryopreserved platelets. Mean freeze/thaw platelet loss for 16 different cryopreserved platelet products was 25% ± 12%, with a range of 6% to 45% (Table 3). Adverse Events In 4 studies that involved 43 subjects who received autologous cryopreserved platelets, no adverse events were reported [9,10,15,16]. In the other studies, no information was given on adverse events. In Vivo Platelet Function Studies In thrombocytopenic patients, the hemostatic function of cryopreserved platelets is easily measured as almost all of their circulating platelets would be from the transfusion. However, in normal subjects, a strategy of evaluating the function of the subjects' transfused autologous cryopreserved platelets needed to be developed. The method developed was to collect a unit of whole blood from the subject, prepare

S. J. Slichter et al. / Transfusion Medicine Reviews 28 (2014) 212–225

250

CPP Platelet Survival (hr)

CPP Platelet Survival (hr)

B

200 150 100 50 0 40

50

60

70

250

CPP Platelet Survival (hr)

216

200 150 100 50 0

80

2.5

150 100 50 0

200 150 100 50 0 100

0 7

150

200

200 150 100 50 0 10

8

250 200 150 100 50 0 10

15

ESC (%)

20

25

HSR (%)

9

10

11

30

40

250 200 150 100 50 0

250

0

30

10

20

ANNEXIN V BINDING (%) CPP Platelet Survival (hr)

250

5

50

MORPHOLOGY SCORE CPP Platelet Survival (hr)

CPP Platelet Survival (hr)

GLUCOSE (mgm/dl)

0

100

MPV

250

90 100 110 120 130 140 150 160

-5

150

4.0

CPP Platelet Survival (hr)

CPP Platelet Survival (hr)

CPP Platelet Survival (hr)

200

3.5

200

TOTAL PLT (x 10 11 )

STORAGE VOLUME (mls) 250

3.0

250

250 200 150 100 50 0 6.3

6.4

6.5

6.6

6.7

6.8

pH

Figure (continued).

a platelet concentrate, and cryopreserve these platelets for subsequent transfusion. The subject was then given 650 mg of aspirin (ASA) to render their autologous normal circulating platelets dysfunctional as determined by a prolongation of their bleeding time (BT) at 24 hours post-ASA compared with their pre-ASA BT. Their autologous cryopreserved platelets were then transfused, and posttransfusion BTs were measured at 2 and 24 hours to determine if the frozen platelets had improved the subjects' ASA-induced prolonged BT, thus demonstrating that the cryopreserved platelets were functional. In the 4 reported studies (all from Dr Valeri's laboratory) [16–18,20], aspirinated BT measurements were performed pretransfusion and posttransfusion (Table 4). However, the aspirinated BT data reported in Valeri et al [16] apparently provided data from the same subjects reported in Valeri [17]. At 2 hours posttransfusion, none of the cryopreserved platelet transfusions gave a statistically significant correction of the aspirinated BT compared with the aspirinated BT in control nontransfused subjects. At 24 hours posttransfusion, 27 subjects given 4% to 6% DMSO cryopreserved platelets stored at − 80°C for up to 142 days had a significant correction in their BT [17,18]. When 6% DMSO cryopreserved platelets were stored for 63 days at −80°C, the BTs were shortened at 24 hours

posttransfusion, but there were no controls so a statistical assessment could not be made [20]. These data may suggest that correction of the aspirinated BT can be achieved even with prolonged storage of cryopreserved platelets [18]. However, fresh platelets, platelets stored at 22°C for 24 hours, and 5% DMSO cryopreserved platelets did not correct the aspirinated BT at any time point. In contrast, platelets stored at 4°C for 24 hours, and the previously discussed 6% DMSO cryopreserved platelets had significant corrections in BTs at 24 hours posttransfusion. Valeri concluded that 4°C stored and 6% DMSO cryopreserved platelets were activated resulting in BT improvements compared with the other products. It is not clear why the 5% DMSO cryopreserved platelets would not also have been activated resulting in BT corrections. Unfortunately, there may be problems using the aspirinated BT to evaluate platelet hemostatic efficacy: 1. The mean baseline BTs were 4.8 ± 1.0 minutes in 115 normal subjects and, at 24 hours post-ASA, were only 11.2 ± 1.4 minutes, that is, approximately 2.3× baseline but with an absolute difference of only 6.4 minutes (Table 4). With such a narrow response range, it may be very difficult to show a statistically significant change after a transfusion, particularly with small subject numbers.

Table 3 Literature review of autologous radiolabeled cryopreserved platelets Author

Subjects (fresh/cryopreserved)

Freezing method

Handin and Valeri [9]

10/13

Kim and Baldini [15]

NI/5

Platelet recoveries (%) Fresh

Cryopreserved

% of Fresh

Platelet survivals (d) Fresh

Cryopreserveda

% of fresh

Comments

45

65 ± 5

47 ± 3

72

8.9 ± 0.3

8.8 ± 0.3

99

38

66 (52-85)

49 (37-59)

74

7.0-10.4

1.8-4.4

Fresh and cryopreserved platelet studies done in different subjects. No information about fresh subjects.

11

66 (52-85)

63 (46-71)

95

7.0-10.4

5.2-7.6

23 ± 8

65 ± 8

46 ± 11

71

8.5

8.5

100

Fresh and cryopreserved platelet studies done in different subjects. Fresh platelets from allogeneic donors. Fresh platelets from allogeneic donors.

Valeri et al [16]

10/12

5% DMSO stored for up to 1 mo at −150°C 5% DMSO stored in PVC bags at −79°C for 24 h 5% DMSO stored in polyolefin bags at −79°C for 24 h 6% DMSO stored at −80°C for 24 h

Valeri [17]

8/12

6% DMSO stored at −80°C for 24 h

24 ± 11

60 ± 11

46 ± 11

67

8.0

8.0

100

8/10

5% DMSO stored at −150°C for 24 h

30 ± 14

60 ± 11

40 ± 10

77

8.0

8.0

100

Vecchione et al [19]

ND/8 ND/14 9/8

4% DMSO stored at −80°C for 138 d 5% DMSO stored at −80°C for 142 d 6% DMSO stored at −80°C for a mean of 11 d

24 ± 16 36 ± 16 22

ND ND 62 ± 18

34 ± 6 33 ± 7 32

– – 52

ND ND 7

N8 N8 7

– – 100

Vecchione et al [20]

ND/6

6% DMSO stored at −80°C for a mean of 55 d in polyolefin bags 6% DMSO stored at −80°C for a mean of 68 d in PVC bags 5.5%-7.5% DMSO stored at −80°C (storage time not given)

26 ± 11

ND

40 ± 15



ND

8



19 ± 13

ND

42 ± 13



ND

8



20

73 ± 10

31 ± 9

42

8

8

100

NI/7

Spector et al [18]

ND/12 Melaragno et al [21]

4/9

Melaragno et al [22]

ND/28

6% DMSO stored at −80°C for up to 3 y

23

ND

30



ND

8



Currie et al [23]

ND/16

Thrombosol/2% DMSO stored at −80°C for 7-10 d 6% DMSO stored at −80°C for 7-10 d

0-6

ND

40 ± 12



ND

6.9 ± 1.6



20

ND

29 ± 10b (P b .005)



ND

6.3 ± 2.0b (P = .09)



6% DMSO added and spun to remove DMSO before freezing at −80°C and stored at least 2 mo

6±2

ND

30



ND

7.0



23 ± 11

65 ± 4

39 ± 9

69 ± 16

8.4 ± 0.4

7.4 ± 1.4

100

ND/16

Valeri et al [10]

Summary results

ND/2

Cryopreserved platelet recovery, 32%; rest of data are estimated from Figure 2, page 398.

Cryopreserved platelet recoveries; 31%. Rest of values estimated from Figure 1, page 323. PLTs isolated by mechanical apheresis or manual centrifugation. Apheresis collection split in 2 and ½ cryopreserved with Thrombosol with 2% DMSO and ½ cryopreserved with 6% DMSO. P values are for the paired transfusions given to the same subject.

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F/T/W Platelet Loss (%)

Results reported mean ±1 SD.

Abbreviations: ND, not done; NI, no information; F/T/W, post–freeze/thaw/wash platelet loss. a To determine the cryopreserved summary survival data, the following data were used: for studies that reported only ranges the mid-point in the range was used as the mean survival and for survivals reported as greater than 8 days, a value of 8 days was assigned to the results. b P values are for comparison of Thrombosol/2% DMSO to 6% DMSO.

217

218

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Table 4 Aspirinated BT measurements pre–platelet transfusion and post–platelet transfusion Author

Subjects

Platelet product transfused

BT (min) Baseline

Valeri [17]a 8 6 6 10 12 Spector et al [18]

51 6 9

Vecchione et al [20] Summary resultsb

7

1 U of platelets given 24 h post 650 mg ASA. (a) fresh platelets. (b) platelets stored for 24 h at 22°C. (c) platelets stored for 24 hours at 4°C. (d) 5% DMSO cryopreserved platelets stored at −150°C for 24 h. (e) 6% DMSO cryopreserved platelets stored at −80°C for 24 h. No transfusion post 650 mg ASA. 1 U of platelets given 24 h post 650 mg ASA. (a) 4% DMSO cryopreserved platelets stored at -80°C for 138 ± 56 days. (b) 5% DMSO cryopreserved platelets stored at -80°C for 142 ± 60 d. 1 U of 6% DMSO cryopreserved platelets stored at −80°C for a mean of 63 d given 24 h post 650 mg ASA.

Comments Post-ASA (Time) Pretransfusion (24 h post-ASA)

2h posttransfusion

24 h posttransfusion

5-6 4 6-7 5

13-14 11 13 10

12 9-10 9 10

11 (P N .05) 9 (P N .05) 7 (P b .05) 9 (P N .05)

5

11

9

8 (P b .01)

5.4 ± 1.4

11.6 ± 3.1



10.6 ± 2.8

4.6 ± 1.4

11.1 ± 3.1



3.1 ± 0.6

9.1 ± 2.0



4.5 ± 1.9

10.2 ± 4.1

8.9 ± 4.9

8.7 ± 3.8 (P b .002) 6.2 ± 2.7 (P b .05) 7.4 ± 2.3

4.4 ± 0.8

10.3 ± 0.8

9.3 ± 0.6

7.9 ± 1.1

In all of these studies, the same subjects received ASA on 2 occasions with and without a subsequent platelet transfusion (P values for paired results).

Control group P values compared to nonconcurrent control group. P values compared with nonconcurrent control group. No P values given.

In the Valeri et al [17], BTs were estimated from figures because no actual BT results were given. The same subjects had BTs before and after ASA administration on 2 occasions either without a transfusion to determine the time for spontaneous correction of their BT or the BT correction after transfusion. In the 2 other studies [18,20], BT results were given, but there were no paired aspirinated BT measurements in the same subjects with and without a transfusion. a Bleeding time values estimated from figures. No numerical values were given in the article. b Data only from subjects given cryopreserved platelets.

2. There was no improvement in the BT with any of these platelet products at 2 hours posttransfusion and only in some of them at 24 hours posttransfusion. This is in marked contrast to BT studies in thrombocytopenic patients who received allogeneic donor transfusions where an immediate correction of the BT was usually observed. If the BT correction was not immediate, it may have been because the platelet donor had inadvertently taken an ASA-containing drug [25]. 3. The transfusion of just the supernatant plasma from 4°C stored platelets is able to correct the aspirinated BT [26]. 4. Neither fresh nor 22°C stored platelets shortened the aspirinated BT in normal volunteers, whereas 4°C stored platelets did [17,27]. These results are in contrast to studies in thrombocytopenic patients where fresh and 22°C stored donor platelets immediately correct the BT [25,28], whereas 4°C platelets do so only transiently, if at all. 5. Finally, the most appropriate way to test hemostasis after a platelet transfusion is in thrombocytopenic patients. At platelet counts of less than 20 000/μL, the normal BT is prolonged to more than 30 minutes allowing measurable changes in posttransfusion BTs to be easily documented [29].

showed that recoveries were better for platelets cryopreserved with Thrombosol/2% DMSO than with 6% DMSO alone (40% ± 12% vs 29 ± 10%, P b .005). In 1 study, freezing results appeared to be better using a polyolefin vs a polyvinyl chloride bag [15], whereas in another study, the results were comparable [20]. One study compared − 80°C storage vs − 150°C storage for 24 hours with no difference [17] and 4% DMSO vs 5% DMSO with no difference [18]. Most of the other studies evaluated DMSO concentrations between 4% and 6%, storage times between 24 hours to 142 days, and freezing at controlled rates in liquid nitrogen with final storage in the vapor phase of liquid nitrogen or a − 80°C freezer. These studies generally showed no apparent differences between these variables, and long-term storage for 4 to 5 months is possible. In vivo hemostatic function as assessed by correction of the aspirinated BT when 1 autologous cryopreserved platelet concentrate was given to a normal subject showed improvement in the BT at 24 hours but not at 2 hours posttransfusion. As discussed, there are many problems evaluating hemostasis based on aspirinated BTs and delayed correction of the BT is unacceptable for actively bleeding patients.

In summary, measuring platelet function by using an aspirinated BT approach in normal subjects may not provide relevant information as to the hemostatistic effectiveness of cryopreserved platelets when given to thrombocytopenic patients.

Adverse Events No serious adverse events were reported after the transfusion of autologous DMSO cryopreserved platelets given to normal subjects, and no thromboembolic complications were reported.

Summary

Patients Transfused With Cryopreserved Platelets

In Vivo Results Publications of radiolabeled autologous cryopreserved platelet recovery and survival measurements are compromised by several factors. The number of participants was small with a range of 2 [10] to 16 subjects [23]. The study by Currie et al [23] was well designed, as the same apheresis platelet collection was divided in half and clearly

A literature review of cryopreserved platelets given to patients assessed multiple factors including the platelet product used for freezing (apheresis or whole blood derived platelet concentrates), freezing conditions, postthaw removal of DMSO, transfusion indication, transfusion monitoring, number of transfused patients, number of transfusions, adverse events, in vitro activation and functional platelet assays, and

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219

Table 5 Cryopreserved platelet transfusions given to patients with hematologic/oncologic disorders Author

Slichter and Harker [30]

Platelet product

Autologous multiple PC

Cryopreservation conditions

• 10% DMSO

F/T/W Platelet Loss (%)

Cryopreserved platelets

Response

Transfusions Patients Transfusion (no.) (no.)

Hemostatic

NI

7

7

BT in 3 tested patients shortened appropriate for increase in posttransfusion platelet count

NI

11

11

• Controlled rate freezing • Storage: vapor phase liquid nitrogen • No DMSO removal • Storage time: 8-28 d

Kim and Baldini [37]

Allogeneic PC Mean 6.3 × 1010 platelets/unit (range, 4.6-7.6 × 1010) 8 patients received 4 U/transfusion; 3 patients received 2 U/transfusion

• 5% DMSO

Allogeneic PC 4.4 × 1011 platelets/ transfusion (range, 2-7.1 × 1011)

• 5% DMSO

Cryopreserved platelet recovery maximal at 4 h Radiolabeled recovery: Cryopreserved platelet Auto: 48% ± 9% Fresh Auto: 58% ± 13% % of Fresh: 83% Radiolabeled survival: Cryopreserved platelet Auto: 6.2 ± 1.5 d Fresh Auto: 6.7 ± 1.4 d % of Fresh: 93% NI

• Controlled rate freezing

• BT pretransfusion 19-30 min (11 patients)

• Storage: −80°C

• BT 3 h posttransfusion

• Gradual DMSO removal

mean 10 min (11 patients)

• Resuspend plasma

• BT 24 h posttransfusion

• Storage time: 1-4 d

mean 19 min (8 patients); N25 min (3 patients)

Schiffer et al [33]

21%

40

23

• Controlled rate freezing • Storage: vapor phase liquid nitrogen • Washed and resuspend in

Mean 1-h CCI of cryopreserved platelets: 12.8 × 109/L (range: 0-29.5 × 109/L) Mean recovery of cryopreserved platelets was 65 ± 9% (SE) (range, 0%-160%, n = 16) of fresh platelets

• BT b14 min at platelet counts N85 000/μL (n = 6) • BT 18 min at platelet count of 95 000/μL (n = 1) • BT N20 min at platelet counts 55 000-60 000/μL

plasma • Storage time: mean 63 d

(n = 2)

(range, 3-201 d) Schiffer et al [34]

Schiffer et al [35]

Autologous apheresis platelets 5.1 × 1011 platelets/transfusion (range, 1.4-13.8 × 1011/platelets/ transfusion)

• 5% DMSO

36%

107

36

Mean 1-h CCI: 6400 ± 540 (SE) (range, 600-19 000) (n = 73 transfusions)

BT after 17 individual transfusions corrected to b20 min with platelet counts N40 000/μL after 11 transfusions

13%

91

25

Mean 1-h CCI: 13.7 ± 6.7 × 103 (range, 1.9-26 × 103) In 18 patients after 38 transfusions, 70% of platelets still circulating at 18-24 h posttransfusion. Recovery: 53% ± 3% (range, 8%116%) after 8 transfusions stored for N250 d. No correlation between duration of storage and clinical results.

2 patients oral bleeding and 1 patient heavy uterine bleeding; all bleeding controlled posttransfusion

22% (4%-47%)

14

13

Mean 1-h CCI: 12 600 (range, 5000-25 000) Paired comparison of fresh vs cryopreserved platelets from different donors (n = 12) Mean 1-h CCI: Cryopreserved platelets: 11 800 Fresh: 25 900 % of Fresh: 46% Mean 18-24 h CCI: Cryopreserved platelets: 7200 Fresh: 22 700 % of Fresh: 32%

No bleeding in patients who received cryopreserved platelets

35%

6

3

Radiolabeled recovery: 31% ± 15% (n = 3) Radiolabeled survival: 2.7 ± 1.4 d (n = 3)

BT decreased after 4 of 5 transfusions

• Controlled rate freezing • Storage: vapor phase liquid nitrogen • Washed and resuspend in

plasma Autologous multiple • 5% DMSO PC • Non-Cr freezing 3-3.5 × 1011 platelets/ • Storage: vapor phase liqtransfusion uid nitrogen • Supernatant DMSO plasma removed • Resuspended in autologous plasma • Storage time: mean 118 d (range: 13-400 d)

Daly et al [36]

Allogeneic pooled PC or apheresis 4.5 × 1011 platelets/ transfusion (range, 2.8-7.4 × 1011)

• 5% DMSO • Placed in vapor phase of liquid nitrogen and stored for a mean of 1157 d (n = 8) • Controlled rate freezing to −80°C (n = 6) then stored in the vapor phase of liquid nitrogen • Spun, supernatant removed, and resuspended in plasma

Zaroulis et al [38]

Allogeneic pooled PC 3-14 U/transfusion

• 5% DMSO • Controlled rate • Stored at -80°C

(continued on next page)

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Table 5 (continued) Author

Platelet product

Cryopreservation conditions

F/T/W Platelet Loss (%)

Cryopreserved platelets

Response

Transfusions Patients Transfusion (no.) (no.)

Hemostatic

14%

12

6

1 patient—baseline, BT N15 min, posttransfusion CR—Cryopreserved or fresh platelets, BT b6 min Post non-CR, BT 10 min

NI

708

155

All transfusions to a patient were either fresh or cryopreserved from the same donor 1-h CCI: Fresh: 22 000 ± 2300 (SE) Cryopreserved platelets controlled freezing (CR): 22 900 ± 5100 Cryopreserved platelets noncontrolled freezing (non-CR): 7600 ± 900 20 h CCI: Fresh: 14 800 ± 1500 Cryopreserved platelets CR freezing: 12 500 ± 1600 Cryopreserved platelets non-CR freezing: 2600 ± 1000 1-h mean CCI: 12 400 (range, 0-36 800)

19%

16

6

No bleeding observed

• Pooled

22

15

10

9

42 (24 nonwashed and 18 washed)

12

Mean 1-h CCI: Fresh allo: 19 800 ± 9500 (range, 4100-30 900) Cryopreserved platelet auto: 11 000 ± 6500 (SD) (range, 12 200-22 100) (56% of fresh) Mean 24 h cryopreserved platelet CCI was 55% of 1-h cryopreserved CCI 10 paired cryopreserved and fresh transfusions from the same donor 1-h mean CCI: Fresh: 25 000 ± 9000 Cryopreserved platelets: 12 900 ± 10 000 % of Fresh: 52% 1-h mean recoveries (%): Fresh: 62% ± 23% Cryopreserved platelets: 30% ± 22% % of Fresh: 48% Cryopreserved auto platelets vs fresh allogeneic apheresis platelets 1-h mean CCI: Fresh: 13 600 ± 3100 Cryopreserved platelets: 5700 ± 1300 % of Fresh: 42% 24-h mean CCI: Fresh: 11 900 ± 2100 Cryopreserved platelets: 3200 ± 1800 % of Fresh: 27% Washed vs nonwashed cryopreserved platelets-no differences so data combined 1-h increments: Fresh allogeneic: 30.6 ± 16.1 × 103/µl Cryopreserved auto platelets: 15.2 ± 10.1 × 103/µl (P b .0005) % of Fresh allogeneic: 50% Interval between transfusions (d): Fresh allogeneic: 2.3 ± 1.0

• Spun, supernatant removed, resuspended in plasma • Storage time: 24 h or 10 wk Lazarus et al [31]

Apheresis HLA identical sibling

• 10% DMSO • Paired units cryopreserved at: noncontrolled or controlled rate freezing in vapor phase of liquid nitrogen • No DMSO removal postthaw

Schiffer et al [39]

Autologous apheresis 3-5 × 1011 platelets/ transfusion

• 5% DMSO • Storage: vapor phase liquid nitrogen • Spun, supernatant removed, and resuspended in auto plasma • Storage time: 151 d (range, 13-1237 d)

van Imhoff et al [40]

Autologous apheresis

• 5% DMSO • Storage: vapor phase liquid nitrogen • Spun, supernatant removed, and resuspended in auto plasma

Melaragno et al [22]

Pooled allogeneic PC (n = 15 patients) Autologous apheresis (n = 8 patients)

• 4%-6% DMSO • Storage: −80°C freezer • Spun, supernatant removed, and resuspended in auto plasma • Storage time: ≤36 mo

Towell et al [41]

Autologous apheresis Mean 5.3 × 1011 platelets/transfusion

• 5% DMSO

PCs 15% ± 14% • Apheresis PCs 22% ± 8%

37%

• Storage: -80°C or -95°C • Spun, supernatant removed and re-suspended in auto plasma • Storage time: mean 15 days (range: 9-29 days)

Mulder et al [32]

Autologous apheresis Mean 247 × 109 (range, 144-523 × 109) washed to remove DMSO (n = 18) Mean 308 × 109 (range 90-645 × 109) no DMSO removal

• 5% DMSO • Non-CR freezing • Storage: vapor phase of liquid nitrogen • Transfusion not washed (n = 24)

Transfused cryopreserved platelets produced immediate cessation of bleeding, if present

BT measured after 7 auto cryopreserved platelet transfusions and 4 fresh allogeneic transfusions with wide range of results regardless of posttransfusions platelet count

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221

Table 5 (continued) Author

Platelet product

Cryopreservation conditions

F/T/W Platelet Loss (%)

Cryopreserved platelets

Response

Transfusions Patients Transfusion (no.) (no.) Cryopreserved auto platelets: 2.3 ± 1.2 (P, NS) % of Fresh allogeneic: 100%

• Spun, supernatant removed, and resuspended Angelini et al [45]

Autologous apheresis

in auto plasma (n = 18) Compared 2 different cryopreservation methods

• Cryoprotectant: 6% DMSO;

Hemostatic

25% with DMSO 46% with DMSO/HES

15, DMSO 11, DMSO/ HES

8, DMSO 8, DMSO/ HES

24-h CCI for DMSO: 5600 ± 1000 24-h CCI for DMSO/HES: 2000 ± 1100

3 episodes of oral mucosal bleeding controlled

15% ± 4%

78

8

Mean 1-h CCI: 11 000 ± 5000 (range, 0-26 000) (n = 71 transfusions) Mean 24-h CCI: 8000 ± 4000 (range: 0-20 000) (n = 70 transfusions) Mean 1-h recovery: 29% ± 12% Interval between transfusions: 3.5 d

BT pretransfusion N15 min 1-h post – 7.5 ± 0.9 (n = 7) Hemorrhage prevented in all patients

37%

28

28

Mean 1-h CCI: 8700 (range, 2400-19 000)

Hemorrhage not recorded in any patient

37% (range, 19%-56%)

25

25

Adequate 1-h CCI N7.5 × 109/μL in 21 patients (84% of patients)

2 patients required allogeneic transfusions due to nosebleed 24-48 h after cryopreserved platelet transfusion despite platelet count N20 000/μL

5% DMSO/6% HES • Non-CR freezing • Storage: −80°C freezer • Diluted 4% HSA, spun, and re-suspended in plasma • Storage time: 2-8 mo Funke et al [42]

Autologous apheresis Mean 5.0 × 1011 platelets/transfusion (range, 1.9-10.7 × 1011)

• 5% DMSO • Irradiated 30 Gy • CR freezing • Stored: vapor phase of liquid nitrogen • Spun, supernatant removed, and resuspended in plasma • Storage time: ≤3 mo

Pedrazzoli et al Autologous apheresis [43] 6.6 × 1011 platelets/ transfusion (range, 4.8-12.2 × 1011)

• 5% DMSO • CR-freezing • Storage: liquid nitrogen • Spun, remove supernatant, and resuspend in auto

Torretta et al [44]

plasma Autologous apheresis • 5% DMSO 7.7 ± 1.7 × 1011 • CR-freezing platelets/transfusion 11 (range, 4.8-12.2 × 10 ) • Storage: liquid nitrogen • Spun, supernatant removed, and resuspended in autologous plasma

Pedrazzoli et al Autologous apheresis [46]

At time of transfusion, most patients had oral or GI mucositis that was controlled by transfusion

• 2% DMSO/Thrombosol • Controlled rate freezing in liquid nitrogen • Non-CR freezing and stored in -80°C freezer

55% 51%

7 7

7 7

Mean 1-h CCI: 9200 ± 5400 Mean 1-h CCI: 2700 ± 1400

26%

63

19

Auto cryopreserved platelets vs allogeneic fresh platelets Mean 1-h CCI: Fresh: 24 800 Cryopreserved: 15 800 % of Fresh: 64% Mean 24-h CCI: Fresh: 21 200 Cryopreserved: 13 900 % of Fresh: 66%

• Spun, remove supernatant, and resuspend in Plasmalyte Vadhan-Raj et al [47]

Autologous apheresis

• 2% DMSO/Thrombosol • Non-CR freezing • Storage: -80°C freezer • Spun, remove supernatant, and resuspend in autologous plasma

No bleeding episodes

Abbreviations: Auto, autologous; CR, controlled rate freezing; F/T/W, freeze/thaw/wash; HSA, human serum albumen; NI, no information; NS, not significant; PC, platelet concentrates.

in vivo posttransfusion platelet responses. As might be expected, many studies did not have all of the desired information included in their reports (see Supplementary Appendix II for detailed description of the results of the literature review of cryopreserved platelets given to thrombocytopenic patients). Particular emphasis was placed on any reports of thrombotic events.

Results Twenty-three studies were identified in which DMSO cryopreserved platelets were given, and the results are summarized in Tables 5 and 6. Mean freeze/thaw platelet loss was 28% ± 12% with a range of 13% to 55% in 18 studies.

222

Table 6 Cryopreserved platelet transfusions given to patients undergoing cardiopulmonary bypass or with trauma Author

Khuri et al [49]

Platelet product

Cardiopulmonary Allogeneic pooled PC or bypass allogeneic apheresis

Trauma/ITP

Allogeneic apheresis 3.8 ± 0.7 × 1011 platelets/unit

Cryopreservation conditions

F/T/W Platelet Loss (%)

Cryopreserved Platelets Transfusions (no.)

30% ± 18% Cryopreserved platelets: 4.5 ± 2.1 × 1011 platelets/ patient in 24 patients • Spun, remove supernatant, Standard platelets: 6.9 ± resuspend in plasma 3.9 × 1011 platelets/patient in 29 patients (P = .008 for • Storage time: 1-24 mo platelets/patient) • 6% DMSO

• Storage: -80°C freezer

• 5% DMSO

17% ± 17% 2

Response Patients (no.)

Transfusion

Cryopreserved platelet increments: 30 min posttransfusions: 0.8 ± 1.1 × 109/μL 24 h posttransfusions: 0.7 ± 1.2 × 109/μL Standard platelet increments: 30 min posttransfusions: 2.5 ± 2.2 × 109/μL 24 h posttransfusions: 1.8 ± 1.6 × 109/μL (P = .02) Platelet survivals did not differ (P = .07)

• Preoperative and postop-

2

• Centrifuged

Hemostatic

erative BT did not differ between the groups • Median postoperative blood loss for cryopreserved platelets, 1721 mL, vs standard platelets, 2299 mL (P = .007) • Total blood products used less for cryopreserved platelets vs standard platelets (P = .008) Almost no change in platelet count

Persistent bleeding controlled in both patients within b20 min

• Supernatant plasma removed • Storage: -80°C freezer • Resuspend in AB plasma

Noorman et al [52]

N90% of patients trauma

Allogeneic apheresis

• 5% DMSO

595a

533

Effective support for trauma patients

26% ± 12% 1082

333

Massive transfused patients (N10 U RBC/24 h) had better survivals when transfused with higher numbers of CPP (n = 34; 5 ± 2 CPP transfusions/ patient; survival, 74%) vs low numbers of CPP platelets (n = 12; 1 CPP platelet transfusion/patient; survival, 50%). With nonmassively transfused patients number of CPP units transfused/patient did not affect survival.

NI

• Centrifuged • Supernatant plasma removed • Storage -80°C freezer • Resuspend AB plasma

Noorman and Badloe [53]

Trauma

Allogeneic apheresis 3.0 ± 0.6 × 1011 platelets/unit

• 5% DMSO • Centrifuged • Supernatant plasma removed • Storage: -80°C freezer • Resuspend AB plasma

Abbreviations: F/T/W, freeze/thaw/wash; TTP, thrombotic thrombocytopenic purpura. a Units of cryopreserved platelets given. Not number of transfusions.

S. J. Slichter et al. / Transfusion Medicine Reviews 28 (2014) 212–225

Lelkens et al [51]

Patient population

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In Vivo Platelet Responses

223

and allogeneic platelets are not that different from 5 to 7 day 22°C stored platelets given to thrombocytopenic patients [48].

Hematology/Oncology Patients Posttransfusion Corrected Count Increment and/or Platelet Recoveries and Survivals. In 14 (74%) of 19 studies, autologous platelets were obtained from patients in remission in an attempt to support them through their next course of chemotherapy (Table 5). Most of these patients had been documented to be alloimmune platelet refractory during a prior course of treatment, and HLA-compatible platelets were frequently not available. In the first reported study, 7 patients received both autologous radiolabeled cryopreserved and fresh platelet concentrates [30]. Platelets were cryopreserved in 10% DMSO at a controlled rate and stored in the vapor phase of liquid nitrogen for 8 to 28 days, and after thawing, the DMSO was not removed. Platelet recoveries were 83% of fresh, and survivals were 93% of fresh. A second study used the same freezing conditions but compared controlled rate to noncontrolled rate freezing using platelets from an HLA-identical sibling. One-hour corrected count increments (CCIs) were 104% of the same donor's fresh platelets with controlled rate freezing and only 35% of fresh with noncontrolled rate freezing. Corrected count increments at 24 hours were 84% and 18% of fresh for controlled vs noncontrolled rate freezing, respectively [31]. One other study evaluated the transfusion of autologous platelets cryopreserved with 5% DMSO in the vapor phase of liquid nitrogen that either had the DMSO removed after thawing (n = 18) or not (n = 24), and no differences in posttransfusion CCIs or intervals to next transfusion were observed [32]. Because of the very strong patient's exhaled sulfur odor associated with the administration of DMSO, most later studies centrifuged the platelets after thawing to remove the supernatant plasma with the platelets resuspended in either autologous or allogeneic plasma. Predominantly, 5% DMSO [37–44] was used with or without controlled rate freezing, with storage in the vapor phase of liquid nitrogen or at −80°C for 1 day to 3 years. Because of the heterogenicity in these studies, it is very difficult to determine if the freezing variables made any significant differences in the posttransfusion results. However, 1 study using autologous apheresis platelets showed a clear difference in 24-hour CCIs between 6% DMSO cryopreserved platelets vs freezing in 5% DMSO/6% Hepes buffer (HES) with 1-hour CCIs of 5600 ± 1000 vs 2000 ± 1100, respectively [45]. In 11 studies that evaluated CCI responses to cryopreserved autologous platelets, the cumulative mean CCI at 1 hour posttransfusion was 10 400 ± 4000 (range, 2700-15 800) [32,34,35,39–43,46,47]. Another study reported that 84% of the patients transfused had a 1-hour CCI of greater than 7500, which is considered an acceptable response [44]. Only 5 studies reported a 24-hour CCI, and the mean 6500 ± 4200 (range, 3200-13 900) [40–42,45,47]. Three studies also calculated percent platelet recoveries for autologous cryopreserved platelets, and the mean was 37% ± 14% [22,35,42] with 1 study reporting an interval between transfusions of 3.5 days [42]. Only 2 studies reported CCI responses to allogeneic cryopreserved platelets with mean 1-hour CCIs of 12 800 and 12 600, respectively [33,36]. There was 1 allogeneic radiolabeled cryopreserved platelet study that reported recoveries of 31% ± 15% and survivals of 2.7 ± 1.4 days [38]. Six studies reported fresh transfusion data along with the cryopreserved platelet data. One study had both auto fresh and cryopreserved data from the same subject with cryopreserved recoveries that were 48% of fresh and 1-hour CCIs that were 52% of fresh [22]; 2 studies had both fresh and cryopreserved allogeneic data from different donors with cryopreserved CCIs of 65% and 46% of fresh CCIs at 1 hour [33,36] and 32% of fresh at 24 hours [36]; and 3 studies had cryopreserved autologous donors compared with fresh allogeneic donors with 1-hour CCIs of 56%, 42%, and 64% of fresh, respectively [40,41,47] and 27% and 66% of fresh at 18 to 24 hours posttransfusion, respectively [41,47]. Overall, these data suggest responses to cryopreserved both autologous

Hemostatic Responses Hemostatic efficacy was reported in 17 of the 19 studies with 5 showing a decrease in BTs after a cryopreserved platelet transfusion [30,31,33,37,42], and in 2 others, there was either no improvement [34] or a variable response to both fresh and cryopreserved platelet transfusions regardless of the posttransfusion platelet count [41]. In 4 studies, there was immediate cessation of active bleeding [22,35,38,46]; in 3 studies, no hemorrhagic events were recorded in any patients being supported by cryopreserved platelets [36,40,47]; and in another study, 2 patients required a standard platelet transfusion 24 to 48 hours after a cryopreserved platelet transfusion due to persistent nosebleeds [44]. These data demonstrate that cryopreserved platelets can provide effective hemostasis. Adverse Events In 6 studies involving 101 patients given 181 cryopreserved transfusions, no adverse events were reported [35,36,38,40,43,44]. In 4 studies involving 72 patients who received 181 cryopreserved transfusions, either a bad odor or bad taste (metallic) was noted that was likely related to residual DMSO; however, the frequency of these events was not given [22,32,34,41]. In another study involving 19 patients who received 63 cryopreserved transfusions, a bad taste and smell occurred after a single transfusion in 2 patients [47]. Other adverse events noted were 1 episode of urticaria and 1 of slight nausea in 8 patients who received 78 cryopreserved transfusions [42]. In 7 studies involving 232 patients who received 818 cryopreserved transfusions, no information was provided on adverse events [30,31,33,37,39,45,46], with the presumption being there were not likely to have been any serious adverse events. Cardiopulmonary Bypass Patients (1 Study) Posttransfusion Platelet Responses, Blood Product Utilization, and Hemostasis One cardiopulmonary bypass study used cryopreserved platelets (Table 6). The most comprehensive study involved 53 patients who were randomized to receive either cryopreserved (n = 24) or standard platelets (n = 29) [49]. The platelets were cryopreserved in 6% DMSO, stored in a − 80°C freezer for 1 to 24 months, thawed, centrifuged, and resuspended in plasma. The patients given cryopreserved platelets received a total of 4.5 ± 2.1 × 1011 platelets/patient vs 6.9 ± 3.9 × 10 11 platelets/patient for standard platelets (P = .008); median postoperative blood loss was 1721 mL per patient in those who received cryopreserved platelets vs 2299 mL per patient for those who received standard platelets (P = .007), and total blood products given was less for the patients who received cryopreserved platelets (P = .008). In a study of baboon cryopreserved platelets, 2 equal subpopulations of normal or reduced GP1b expressing platelets were observed, similar to human cryopreserved platelets. The GP1b-reduced platelets were rapidly removed from circulation, whereas the GP1b normal platelet population had recoveries of 48% and survivals of 6 days. It was postulated that the rapid removal of the GP1b-reduced platelet population was used to maintain endothelial integrity accounting for the improved hemostatic effectiveness of cryopreserved compared to standard platelets in the CPB study [50]. Adverse Events There were no transfusion reactions observed after the transfusion of either the cryopreserved or standard platelets [49]. In addition, the incidence of thromboembolic or infectious complications (wound or

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pneumonia) did not differ statistically between the 2 groups, but the frequency of these events was not given. In summary, hemostasis was better maintained with cryopreserved platelets than with standard platelets. Trauma Patients (3 Studies) Hemostatic Efficacy There were 3 studies in trauma patients that involved 868 patients who received 1679 cryopreserved platelet transfusions (Table 6) [51–53]. These transfusions were mainly given in battlefield situations by the Netherlands military. All studies reported that effective hemostasis was achieved, and mortality was less in massively-transfused patients, defined as receiving more than 10 U of red blood cells in a 24-hour period, when a higher number of platelet transfusions were given per patient; that is, in 34 massively transfused patients given an mean of 5 ± 2 U of cryopreserved platelets/patient, survival was 74% vs, in 12 patients given 1 cryopreserved platelet transfusion per patient, survival was only 50% [53]. Unfortunately, the studies with the largest number of patients were only reported as abstracts limiting the data available [52,53]. Adverse Events There were no adverse events or transfusion reactions reported. Discussion Early interest in the use of cryopreserved platelets during the period from 1972 to 2002 was predominately for the management of alloimmunized platelet-refractory patients for whom HLA-compatible donors could not be identified because of the small size of HLA-typed apheresis platelet donor registries. In most of these studies, hematology/ oncology patients donated multiple units of autologous platelets during remission that were cryopreserved to provide platelet support during subsequent courses of chemotherapy or bone marrow transplant–induced thrombocytopenia. There were a variety of cryopreservation methods evaluated for their effects on radiolabeled autologous platelet recovery and survival measurements. The parameters that were assessed were concentration of DMSO, the freezing rate, storage bag, storage in either the vapor phase of liquid nitrogen or −80°C freezer, and storage duration. There were very limited numbers of studies and participants to allow definitive conclusions to be reached on the optimum method of cryopreservation. However, most investigators used 5% DMSO with noncontrolled rate freezing in a −80°C freezer for up to 2 years. In general, cryopreserved platelets have approximately 50% lower platelet recoveries/CCIs than fresh or 5-day 22°C stored platelets. Furthermore, the mean freeze/thaw/wash platelet loss was 28% ± 12%. However, of the platelets that circulate, their survival was very similar to fresh platelets. In addition, hemostasis was effective, and adverse events were minor consisting mainly of abnormal breath odors or metallic taste. Recently, there has been renewed interest in cryopreserved platelets to meet military needs. Because of the very short shelf life of standard room temperature stored platelets, it has been extremely difficult to supply platelets for forward-combat surgical facilities. Advancing the availability of cryopreserved platelets has been the studies of Dr Valeri that have demonstrated that platelets can be centrifuged before freezing to remove the plasma containing DMSO [10]. This has accomplished 2 critical requirements for utilization of cryopreserved platelets in combat situations: (1) the elimination of centrifuges on the battlefield to remove the DMSO postthawing and (2) the small volume of each cryopreserved apheresis platelet unit means that fewer freezers are needed to accommodate large numbers of platelets. Cryopreserved platelets used by the Dutch military are resuspended in plasma as opposed to CPP being developed by the US Army that will be resuspended

in saline. However, cryopreserved platelets as produced and used by the Dutch military by anecdotal observation and reporting appear to provide satisfactory hemostasis with no reported serious side effects [52,53]. As additional evidence that cryopreserved platelets can provide effective hemostasis even in patients who do not have an intact vascular system, 1 cardiopulmonary bypass study has been performed with the original Valeri method of centrifuging the platelets after freezing to remove the DMSO containing plasma [49]. In this study, the cryopreserved platelets provided hemostasis that was superior to standard platelets while using fewer platelets as well as other blood products [49]. Conclusion • Approximately 28% of platelets are lost during processing and cryopreservation. • Immediate posttransfusion autologous platelet responses (platelet recoveries or CCIs) after transfusion of DMSO cryopreserved platelets ranged from 52% to 69% of those observed with fresh platelets (Tables 1 and 5, respectively). • Platelet survivals are very similar to fresh platelets. • Platelets cryopreserved in DMSO may prevent hemorrhagic events in thrombocytopenic patients with similar efficacy to that of standard platelets. • Hemostasis after the transfusion of cryopreserved platelets in surgical or trauma patients is at least equivalent to, if not better than, that observed with standard platelets. • Dimethyl sulfoxide at the concentrations used in platelet cryopreservation is safe and well tolerated even in patients who have received multiple transfusions. • Infusion of DMSO cryopreserved platelets does not appear to be associated with clinical risks greater than that seen with standard platelets. • Specifically, there were no reports of thromboembolic complications after the infusion of DMSO cryopreserved platelets. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.tmrv.2014.09.001. References [1] Valeri CR, Macgregor H, Ragno G. Correlation between in vitro aggregation and thromboxane A2 production in fresh, liquid-preserved, and cryopreserved human platelets: effect of agonists, pH, and plasma and saline resuspension. Transfusion 2005;45:596–603. [2] Freyssinet JM. Cellular microparticles: what are they bad or good for? J Thromb Haemost 2003;1:1655–62. [3] Hornsey VS, McMillan L, Morrison A, Drummond O, Macgregor IR, Prowse CV. Freezing of buffy coat–derived, leukoreduced platelet concentrates in 6 percent dimethyl sulfoxide. Transfusion 2008;48:2508–14. [4] Böck M, Schleuning M, Heim MU, Mempel W. Cryopreservation of human platelets with dimethyl sulfoxide: changes in biochemistry and cell function. Transfusion 1995;35:921–4. [5] Rothwell SW, Maglasang P, Reid TJ, Gorogias M, Krishnamurti C. Correlation of in vivo and in vitro functions of fresh and stored human platelets. Transfusion 2000;40:988–93. [6] Rinder HM, Smith BR. In vitro evaluation of stored platelets: is there hope for predicting posttransfusion platelet survival and function? Transfusion 2003;43:2–6. [7] Miyaji R, Sakai M, Urano H, Nakata K, Sakamoto H, Shirahata A. Decreased platelet aggregation of platelet concentrate during storage recovers in the body after transfusion. Transfusion 2004;44:891–9. [8] Rinder HM, Snyder EL, Tracey JB, Dincecco D, Wang C, Baril L, et al. Reversibility of severe metabolic stress in stored platelets after in vitro plasma rescue or in vivo transfusion: restoration of secretory function and maintenance of platelet survival. Transfusion 2003;43:1230–7. [9] Handin RI, Valeri CR. Improved viability of previously frozen platelets. Blood 1972; 40:509–13. [10] Valeri CR, Ragno G, Khuri S. Freezing human platelets with 6 percent dimethyl sulfoxide with removal of the supernatant solution before freezing and storage at −80 degrees C without postthaw processing. Transfusion 2005;45:1890–8. [11] Dumont LJ, Cancelas JA, Dumont DF, Siegel AH, Szczepiorkowski ZM, Rugg N, et al. A randomized controlled trial evaluating recovery and survival of 6% dimethyl sulfoxide– frozen autologous platelets in healthy volunteers. Transfusion 2013;53:128–37.

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Review of in vivo studies of dimethyl sulfoxide cryopreserved platelets.

A literature review was conducted to assess the efficacy and safety of dimethyl sulfoxide (DMSO) cryopreserved platelets for potential military use. I...
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