BLOOD COMPONENTS Extended storage of buffy coat platelet concentrates in plasma or a platelet additive solution Sherrill J. Slichter,1,2 Doug Bolgiano,1 Jill Corson,1 Mary Kay Jones,1 Todd Christoffel,1 S. Lawrence Bailey,1 and Esther Pellham1

BACKGROUND: Platelet (PLT) concentrates (PCs) prepared from whole blood in the United States are made using the PLT-rich plasma method. The PCs must be made within 8 hours of blood collection and stored for only 5 days. In Europe and Canada, PCs are made using the buffy coat (BC) method from whole blood held overnight at 22°C and storage times may be up to 7 days. Our studies were designed to determine how long BC PLTs can be stored in plasma or Plasmalyte while meeting the FDA’s poststorage viability criteria. STUDY DESIGN AND METHODS: Normal subjects donated whole blood that was stored at 22°C for 22 ± 2 hours before preparation of BC PLTs. PLTs were stored for 5 to 8 days in either plasma or Plasmalyte concentrations of 65 or 80%. Radiolabeled autologous stored versus fresh PLT recoveries and survivals were assessed as well as poststorage in vitro assays. RESULTS: BC PLTs stored in either plasma or 65% Plasmalyte met FDA poststorage PLT recovery criteria for 7 days but survivals for only 6 days, while storage in 80% Plasmalyte gave very poor results. Both stored PLT recoveries and survivals correlated with the same donor’s fresh results, but the correlation was much stronger between recoveries than survivals. In vitro measures of extent of shape change, morphology score, and pH best predicted poststorage PLT recoveries, while annexin V binding best predicted PLT survivals. CONCLUSION: BC PLTs stored in either plasma or 65% Plasmalyte meet FDA’s poststorage viability criteria for 6 days.

P

latelet (PLT) concentrates (PCs) from whole blood are prepared using two different techniques: either the PLT-rich plasma (PRP) method or the buffy coat (BC) method. The major difference between the two methods is the centrifugation of the whole blood. In the PRP method, a soft centrifugation of the whole blood is performed, the supernatant PRP is transferred to a storage bag, a hard centrifugation of the PRP is performed, the majority of the PLT-poor plasma (PPP) is removed, and the PC is resuspended in 50 to 60 mL of PPP.1 In the BC method, the whole blood is hard centrifuged, the supernatant PPP is removed, the BC is transferred to another bag, the BC is soft centrifuged, and the supernatant PLTs are removed to a storage bag.2 In most blood centers, BCs from four to six whole blood collections are pooled before the soft centrifugation of the BC is performed, and the BCs are stored as prestorage pools.3 Most blood centers in Europe prepare PCs as BCs, and Canada has recently adopted this system. The United States remains the only major country that still uses the PRP method for preparing PCs. Because the PLTs are never hard centrifuged against the bottom of the bag but only against the red blood cell (RBC) layer, it

ABBREVIATIONS: BC = buffy coat; ESC = extent of shape change; HSR = hypotonic shock response; MPV = mean platelet volume; PC(s) = platelet concentrate(s); PPP = PLT-poor plasma; PRP = platelet-rich plasma. From the 1Puget Sound Blood Center and the 2University of Washington School of Medicine, Seattle, Washington. Address reprint requests to: Sherrill J. Slichter, MD, Platelet Transfusion Research, Puget Sound Blood Center, 921 Terry Avenue, Seattle, WA 98104-1256; e-mail: [email protected]. This research study was supported by a grant from the National Heart, Lung and Blood Institute, National Institutes of Health (1 P50 HL081015). Received for publication October 17, 2013; revision received January 27, 2014, and accepted January 27, 2014. doi: 10.1111/trf.12634 © 2014 AABB TRANSFUSION 2014;54:2283-2291. Volume 54, September 2014 TRANSFUSION

2283

SLICHTER ET AL.

has been assumed that the quality of BC PLTs would be better than PRP PLTs. However, when the same normal subjects donated whole blood on two separate occasions allowing a direct comparison of 7-day-stored PRP or BC PLTs from the two donations, there were no differences observed in the radiolabeled autologous PLT recoveries and survivals nor in the in vitro results between the two products.4 We have previously reported the in vivo and in vitro results of extended stored PRP PCs stored in plasma or 80% Plasmalyte.5 In this study, we report similar data for extended storage of BC PCs stored in plasma or Plasmalyte. Plasmalyte was selected for use in our studies as it was the only physiologic solution that was currently licensed for intravenous use that had previously been used for PLT storage.6

MATERIALS AND METHODS Study population Healthy subjects who met allogeneic blood donor criteria were recruited to participate in these studies, and each signed a study consent. The study protocol and consent forms were approved by the institutional review board of the University of Washington School of Medicine. Between three and 10 normal subjects participated in each of the storage studies. Fewer subjects participated in studies in which the initial data suggested the stored PLTs would not meet FDA acceptance criteria.

Preparation of stored BC PLTs Each subject donated 500 mL of blood into a blood bag (Teruflex, Terumo Corp., Tokyo, Japan) with diversion sampling arm (BB* AGQ456A2, Terumo Corp.,) using standard whole blood donation procedures. This bag is used routinely at our blood center and is made of polyvinyl chloride with a citrate plasticizer. A subject participated in each study only once. All stored PLTs met the manufacturers’ guidelines for PLT concentration, total PLT count, and storage volume. The blood was held at 22°C for 22 to 24 hours before preparation of the BC PCs as this is the routine procedure for preparing BC PLTs.3 The whole blood storage time was considered to be the first day of PLT storage. The whole blood was then sterilely docked (Terumo SCD 312, Terumo Corp., Somerset, NJ) to a Fresenius top-bottom bag with the anticoagulant removed (Fresenius Compoflex top-bottom bag, Fresenius Corp., Waltham, MA) to allow transfer of the whole blood. As this bag is not licensed in the United States, the bag was used under an FDA IND #13798. The blood was centrifuged at 3566 × g for 12 minutes and 30 seconds with standard braking (RC3 BP Thermo Scientific, Waltham, MA). An automatic blood processing device was used (Compomat G4, Fresenius Corp.) to remove the BC 2284

TRANSFUSION Volume 54, September 2014

PLTs, and 50 mL plasma was added. The anticoagulant was removed from a cord blood collection bag (Collection Set 791-88, Pall Corp., Port Washington, NY), and the cord blood bag was sterile docked onto the Fresenius system to allow transfer of the BC to the cord blood bag. The BC was allowed to sit undisturbed for 1.5 hours before centrifugation in a special holder to hold the cord blood bag upright, and centrifugation was performed at 511 × g for 5 minutes with standard braking to sediment the PLTs. A PLT storage bag (Terumo) was sterile docked to the cord blood bag, and the supernatant PLTs were transferred to the storage bag without disturbing the sedimented RBCs. The procedures used to prepare the buffy coat PLTs were determined by preliminary studies to optimize the PLT yield in the BC PLTs. For storage of PLTs in Plasmalyte, between 10 and 20 mL of plasma was left with the PC. Eight or 6.5 mL of Plasmalyte was added for every 2 or 3.5 mL of PC to give an expected final concentration of 80 or 65% Plasmalyte with 20 or 35% residual plasma, respectively. Plasmalyte contains 90 mmol/L NaCl, 5 mmol/L KCl, 27 mmol/L Na-acetate, 23 mmol/L Na-gluconate, and 3 mmol/L magnesium chloride.6 The PLTs were allowed to rest undisturbed at room temperature for 1.5 hours. After preparation, both the plasma and the Plasmalyte PLTs were placed in an incubator (Model PF96, Helmer Corp., Fort Wayne, IN) with continuous agitation at 70 cycles/ min during storage at 22 ± 2°C. For PLTs stored in Plasmalyte, albumin concentrations were determined in a sample of the donor’s plasma and in their PC after the addition of Plasmalyte. Based on the albumin dilution, the percentage of Plasmalyte in the PC was calculated.

Preparation of fresh PLTs Fresh PLTs were prepared from a 43-mL sample of whole blood drawn from the subject into 9 mL of ACD-A. The whole blood was transferred into two 50-mL conical screw-top tubes (Becton Dickinson, Franklin Lakes, NJ) and rested for 30 minutes. The tubes were centrifuged at 200 × g for 15 minutes to prepare PRP, and the PRP from each tube was transferred to another conical tube. ACD-A equal to 15% of the PRP volume was added, the PRP was centrifuged at 2000 × g for 15 minutes, and the ACD-A added was calculated to give a pH of 6.5 to allow immediate PLT resuspension.

In vitro PLT assays PLT counts were performed on the day after preparation of the PLTs and at the end of storage using a hematology analyzer (ABX Diagnostics, Irvine, CA). Also, on the last day of storage, several in vitro measurements of PLT quality were determined, that is, glucose concentration,

EXTENDED BUFFY COAT PLT STORAGE

TABLE 1. In vivo radiolabeled recoveries and survivals of BC PLTs stored in plasma* Storage time (days)

Number of observations

5 6 7 8

10 10 10 8

PLT recoveries (%) Fresh Stored 61 ± 13 67 ± 14 63 ± 17 62 ± 12

55 ± 16 54 ± 13 50 ± 14 42 ± 14

Stored recoveries % of fresh 95% LCL† 89 ± 10 80 ± 9 79 ± 6 67 ± 13

80 74 75 56

PLT survivals (days) Fresh Stored 7.7 ± 1.2 8.1 ± 1.3 8.2 ± 0.8 8.5 ± 0.6

Stored survivals % of fresh 95% LCL†

5.9 ± 1.1 5.4 ± 1.3 4.8 ± 1.1 3.9 ± 1.4

78 ± 16 67 ± 10 59 ± 16 45 ± 15

66 60 48 34

* Data reported as mean ± 1 SD. † LCL = lower confidence limit. FDA criteria for 95% LCL for mean stored as a % of fresh—recovery, ≥67%; survival, ≥58%.

pH at 37°C, extent of shape change (ESC),7 hypotonic shock response (HSR),7 annexin V binding, morphology score,8 and mean PLT volume (MPV). HSR evaluates a PLT’s ability to recover normal volume after exposure to a hypotonic solution of distilled water. ESC measures the ability of PLTs to change from discoid to spherical shapes after addition of ADP. Both of these assays use a whole blood aggregometer (Model 500-Ca, Chrono-Log, Havertown, PA) to measure the response photometrically. For the Plasmalyte-stored PLTs, residual donor plasma that had been stored at 4°C was warmed to 22°C and added to the PLTs to adjust the PLT count to 300 × 109/L before the ESC and HSR measurements were performed.9 Annexin V binds to phosphatidylserine, which becomes expressed on the PLT surface with aging. Annexin V is fluorescently labeled to allow its detection using a flow cytometer (FACScan, Becton Dickinson, San Jose, CA).

storage (days) and the storage solution (plasma or Plasmalyte). Differences between group means and other linear contrasts have been evaluated for significance. Significance levels have been assessed using the t-distribution. Scatter plots are presented to show the degree of association between stored and fresh in vivo PLT recoveries and survivals. Similarly, scatter plots of in vivo versus in vitro measurements are also presented. Backward stepwise linear regressions of stored PLT recovery and survival measurements on poststorage in vitro measurements have been fit to determine which in vitro measures may predict poststorage PLT viability.

RESULTS In vivo autologous radiolabeled PLT recoveries and survivals PLT storage in plasma

PLT radiolabeling A 43-mL aliquot of the stored PC was radiolabeled with either 111In or 51Cr, and the opposite label was used to radiolabel the donor’s fresh PLTs. Radiolabeling was performed by established techniques, and PLT recoveries and survivals were corrected for elution of the label and for residual radioactivity on Day 10 posttransfusion that was considered due to contaminating radiolabeled RBCs.10 The radiolabeled PLT recoveries and survivals were calculated using the multiple-hit COST program.11 Blood samples were drawn from the donor before infusion; at 2 hours postinfusion; and on Days 1, 2, 3, 5, 7, and 10 postinfusion. The radiolabels used for the fresh and stored PLTs were rotated with each sequential subject enrolled in a study so that, at the end of each storage experiment, equal numbers of the subjects’ fresh and stored PLTs were labeled with each isotope.

Statistical analysis Summary statistics for in vitro and in vivo measurements of the quality of stored PCs were calculated. Observations from individual units have been grouped by duration of

Thirty-eight normal subjects participated in the plasma PLT storage studies. Each subject’s PLTs were stored for 5 to 8 days with fresh PLTs drawn on the day their stored PLTs were transfused. The results are given in Table 1 and Fig. 1. There were progressive decreases in both PLT recoveries and survivals over storage time. The FDA’s poststorage PLT viability criteria are that the lower 95% confidence limits for mean PLT recoveries as a percentage of the same donor’s fresh recoveries should be at least 67% and for mean survivals should be at least 58% of fresh.12 PLT recoveries met FDA poststorage criteria for 7 days, but PLT survivals for only 6 days of storage (Table 1).

PLT storage in Plasmalyte Two different concentrations of Plasmalyte were evaluated, that is, 80% Plasmalyte with 20% residual plasma or 65% Plasmalyte with 35% residual plasma. Initial studies were performed with 80% Plasmalyte, but because of very poor results, only a few studies were done at this Plasmalyte concentration before switching to a 65% Plasmalyte concentration. In addition, 6-day storage studies were also done with PRP-prepared PLTs stored in 65% Plasmalyte and 35% residual plasma. The PRP PLTs Volume 54, September 2014 TRANSFUSION

2285

SLICHTER ET AL.

TABLE 2. In vivo radiolabeled recoveries and survivals of BC and PRP PLTs stored in Plasmalyte*

PLTs BC BC BC PRP BC BC

Plasmalyte concentration (%) 65 ± 7 79 ± 4 67 ± 5 65 ± 3 65 ± 7 84 ± 3

Storage time (days) 5 5 6 6 7 7

Number of observations 6 3 10 10 5 3

PLT recoveries (%)

Stored recoveries

PLT survivals (days)

Stored survivals

Fresh 52 ± 8 56 ± 6 61 ± 13 68 ± 12 74 ± 3 59 ± 17

% of fresh 89 ± 11 83 ± 1 80 ± 10 82 ± 6 79 ± 5 49 ± 7

Fresh 8.3 ± 0.8 7.1 ± 1.7 8.0 ± 1.5 8.1 ± 1.4 7.3 ± 1.8 7.9 ± 0.3

% of fresh 72 ± 13 53 ± 10 70 ± 14 62 ± 17 55 ± 10 32 ± 13

Stored 47 ± 10 46 ± 6 49 ± 12 55 ± 11 58 ± 3 29 ± 10

95% LCL† 76 76 72 77 73 28

Stored 5.9 ± 0.7 3.9 ± 1.7 5.5 ± 1.2 5.0 ± 1.4 4.1 ± 1.6 2.5 ± 1.0

95% LCL† 58 30 59 49 42 1

* Data reported as mean ± 1 SD. Comparisons of 80 and 65% Plasmalyte-stored BC PLTs were made and showed recoveries at 5 days of storage were the same, but survivals were significantly less for PLTs stored in 80% Plasmalyte (p = 0.02). At 7 days of storage, both recoveries and survivals of 80% Plasmalyte-stored PLTs were significantly less than 65% Plasmalyte-stored PLTs (both p = 0.01). † LCL = lower confidence limit. FDA criteria for 95% LCL for mean stored as a % of fresh—recovery, ≥67%; survival, ≥58%.

were prepared as previously described except that the whole blood was stored at 22°C for 22 ± 2 hours before preparation of the PCs.5 Thirty-seven normal subjects participated in the Plasmalyte storage studies, and the results are given in Table 2 and Fig. 1. For these studies, storage times from 5 to 7 days were evaluated. Recoveries and survivals of 65% Plasmalyte-stored PLTs were not significantly different from PLTs stored in plasma for the same storage times of 5, 6, or 7 days. In addition, 65% Plasmalyte-stored BC PLTs met FDA guidelines for 7 days for recoveries and 6 days for survivals, the same as BC plasma–stored PLTs. PRP PLTs stored in 65% Plasmalyte for 6 days did not differ from BC PLTs stored for 6 days in either plasma or Plasmalyte, and these PRP PLTs met FDA guidelines for recoveries but not for survivals. BC PLTs stored in 80% Plasmalyte for 5 days had recoveries the same as similarly stored BC PLTs in 65% Plasmalyte or in plasma and met FDA guidelines. However, survivals at 5 days of storage were significantly less than both 65% Plasmalyte and plasma-stored PC PLTs (p = 0.02) and did not meet FDA guidelines. Recoveries and survivals of 80% Plasmalyte-stored BC PLTs were markedly less than 65% Plasmalyte or plasma stored PLTs after 7 days of storage (both p = 0.01) and did not meet FDA storage guidelines.

Correlations between fresh and stored PLT recoveries and survivals Plots of stored versus fresh PLT recoveries and survivals are given in Fig. 2, respectively. The correlations between the recovery and survival variables are 0.83 and 0.37, respectively (p < 0.001 and p = 0.005).

In vitro PLT assays Results of in vitro PLT assays for PLTs stored in plasma or Plasmalyte are given in Table 3. Regardless of the method of storage (plasma or Plasmalyte) or the storage time (5 to 2286

TRANSFUSION Volume 54, September 2014

8 days), there was no significant loss of PLTs during storage. However, the initial PLT counts of the BC PCs prepared with 80% Plasmalyte averaged 5.7 × 1010 versus 8.2 × 1010 for 65% Plasmalyte- and 9.6 × 1010 for plasmaprepared PLTs, but these results were not statistically different (p > 0.5 and p > 0.25 for 80% Plasmalyte PLTs vs. 65% Plasmalyte- and plasma-stored PLTs, respectively). pH levels were all maintained above 6.3 and averaged 7.0 ± 0.1 to 7.3 ± 0.2. MPVs, morphology scores, ESC, and HSR responses were relatively constant over the varying storage times and did not differ between plasma- and Plasmalyte-stored PLTs for similar storage times. Annexin V binding increased over storage time with no differences between plasma- and Plasmalyte-stored PLTs. As expected, glucose concentrations were significantly less for Plasmalyte- versus plasma-stored PLTs at similar storage times (p < 0.001). Interestingly, the PRP PLTs stored in 65% Plasmalyte had substantially less residual glucose at the end of 6 days than similarly stored BC PLTs (74 ± 18 mg/dL vs. 174 ± 25 mg/dL, respectively; p < 0.001). However, there were no other in vitro differences between these two products. Scatter plots of in vitro variables versus stored PLT recoveries and survivals are given in Figs. 3A and 3B, respectively. Table 4 gives the bivariate correlations between the in vivo variables of PLT recoveries and survivals and the in vitro variables. These values give a numerical summary for the associations seen in Figs. 3A and 3B. The bottom section of the table gives approximate p values, adjusted for the number of correlations being evaluated.13 Thus, for example, the estimate of the correlation between morphology score and survival is 0.36, and the approximate p value, for the hypothesis that the correlation is equal to zero, is equal to 0.02. In addition to these bivariate models, we fit multivariate regression models to the in vivo variables using backward, stepwise regression methods to determine which in vitro variables were jointly associated with the in vivo variables. Results for recovery indicate that ESC and pH were jointly associated with it. When three cases with unusual values for in

EXTENDED BUFFY COAT PLT STORAGE

A

A

80

Stored PLT recovery (%)

PLT recovery (%)

65

50

35

20 2

4 6 8 PLT storage time (days)

40

10

0

40

80

120

Fresh PLT recovery (%) B

10

Stored PLT survival (days)

8 PLT survival (days)

80

0

0 B

120

6

4

2

10

8

6

4

2

0

0 0

2

4

6

8

10

PLT storage time (days) Fig. 1. Autologous radiolabeled PLT recoveries and survivals. (A) Mean PLT recoveries. PLT recoveries (%) are given based on storage time. (B) Mean PLT survivals. PLT survivals (days) are given based on storage time. (●) Plasma BC-stored PLTs; (○) BC 65% Plasmalyte; (△) BC 80% Plasmalyte; (□) PRP 65% Plasmalyte. Data are given as the mean ± 1 SE.

vitro variables (one, pH 7.79; two with morphology scores = 205) were excluded, the final model also included morphology score. Recovery tends to increase as morphology score and ESC increase, but it tends to decrease as pH increases. When the in vitro variables are considered as a group, these three variables are most strongly associated with PLT recovery. Results from fitting similar models to survival indicate that ESC and annexin V binding are most strongly associated with survival when the in vitro vari-

0

2

4

6

8

10

Fresh PLT survival (days) Fig. 2. Stored versus fresh PLT recoveries and survivals. (A) Stored versus fresh PLT recoveries. (B) Stored versus fresh PLT survivals. The regression lines for the comparisons of fresh and stored PLT recoveries and survivals are given as the dashed lines. Correlations are 0.83 (p < 0.001) and 0.37 (p = 0.005) for PLT recoveries and survivals, respectively. (●) Data for BC plasma stored PLTs; (○) BC 65% Plasmalyte; (△) BC 80% Plasmalyte; (□) PRP 65% Plasmalyte.

ables are considered as a group. When one case that had a high value of ESC (36.95) and exerted a large influence on the model fit was removed, only annexin V binding was retained, indicating that it is the in vitro variable most strongly associated with survival. As the value of annexin V binding increases, PLT survival tends to decrease. Volume 54, September 2014 TRANSFUSION

2287

SLICHTER ET AL.

ESC 23 ± 8 21 ± 5 17 ± 6 20 ± 6 21 ± 4 22 ± 8 20 ± 4 20 ± 10 10 ± 5 14 ± 4 pH 7.3 ± 0.2 7.2 ± 0.1 7.2 ± 0.1 7.1 ± 0.2 7.2 ± 0.1 7.0 ± 0.1 7.1 ± 0.1 7.2 ± 0.2 7.2 ± 0.2 7.1 ± 0.1 Test day‡ (×1010) 8.4 ± 2.7 7.2 ± 2.3 5.8 ± 1.1 9.8 ± 2.4 8.8 ± 2.7 9.0 ± 1.2 10.0 ± 4.3 7.6 ± 1.1 5.2 ± 1.3 8.2 ± 2.5

2288

* † ‡ §

Plasma

65 ± 7 84 ± 3 Plasma Plasmalyte

67 ± 6 65 ± 4 Plasma Plasmalyte

67 ± 3 79 ± 4

Solution Plasma Plasmalyte

Data are given as mean ±1 SD. Data are given for PCs made on Day 1 after whole blood collection. Test day is the last day of storage as given in Column 1. PRP-prepared PCs. All others are buffy coat-prepared PCs.

PLT counts Day 1† Donor (×1010) (×108/L) 225,000 ± 48,000 8.4 ± 2.4 239,000 ± 59,000 7.4 ± 2.2 240,000 ± 50,000 5.8 ± 1.3 269,000 ± 55,000 10.0 ± 2.5 258,000 ± 76,000 8.9 ± 2.9 254,000 ± 36,000 8.8 ± 1.2 271,000 ± 96,000 11.0 ± 3.9 264,000 ± 31,000 7.9 ± 1.3 230,000 ± 44,000 5.7 ± 1.2 238,000 ± 48,000 8.8 ± 2.6 Storage Time (days) 5 5 5 6 6 6 7 7 7 8

conditions

Plasmalyte concentration (%)

Volume of PCs (mL) 86 ± 22 75 ± 13 66 ± 12 80 ± 7 99 ± 19 93 ± 9 96 ± 11 83 ± 22 62 ± 9 99 ± 14

Number of observations 10 7 3 10 10 10§ 10 5 3 8

TABLE 3. In vitro PLT measurements*

MPV 7.6 ± 0.9 7.1 ± 0.5 7.6 ± 1.1 7.8 ± 0.6 7.6 ± 0.9 7.6 ± 1.0 7.7 ± 0.9 7.9 ± 0.8 7.4 ± 0.8 8.2 ± 0.7

MS 343 ± 32 352 ± 20 353 ± 36 344 ± 26 345 ± 19 332 ± 52 338 ± 28 332 ± 25 300 ± 17 311 ± 51

Test day†

HSR 36 ± 17 45 ± 18 33 ± 13 32 ± 12 33 ± 9 39 ± 9 42 ± 11 27 ± 7 25 ± 11 29 ± 13

Annexin V binding 11 ± 5 9±3 14 ± 12 9±3 8±3 12 ± 6 14 ± 6 16 ± 4 25 ± 5 17 ± 9

Glucose (mg/dL) 340 ± 42 157 ± 79 155 ± 36 332 ± 33 174 ± 25 74 ± 18 331 ± 54 156 ± 29 124 ± 23 349 ± 56

DISCUSSION

TRANSFUSION Volume 54, September 2014

These studies have demonstrated that plasma-stored BC PLTs meet FDA PLT recovery criteria for 7 days of storage, but PLT survivals for only 6 days. The storage time for all of our studies considered the first day of PLT storage as within the whole blood that was maintained at room temperature for 22 to 24 hours before PC preparation on the day after whole blood collection. These results are the same as we previously reported for PRP-stored PCs.5 Two different concentrations of Plasmalyte were evaluated in these BC PLT storage studies, that is, 80% Plasmalyte and 20% residual plasma and 65% Plasmalyte with 35% residual plasma. The use of 80% Plasmalyte was based on experiments using Haemonetics-collected apheresis PLTs where concentrations of Plasmalyte between 50 and 82% did not affect either autologous radiolabeled PLT recoveries or survivals for 7-day-stored PLTs.14 In our prior studies evaluating PRP PCs, the 80% Plasmalyte concentration, which was the only concentration evaluated, resulted in very poor PLT recoveries and survivals for PLTs stored for 6 days or longer compared to plasma-stored PLTs.5 For BC PLTs, we evaluated both 5 and 7 days of 80% Plasmalyte-stored PLTs. The 5-day Plasmalyte-stored PLTs had PLT recoveries the same as plasma-stored PLTs, but survivals were significantly less (p = 0.02). After 7 days of storage, both recoveries and survivals were significantly less than plasma-stored BC PLTs (both p = 0.01), and neither PLT recoveries nor survivals met FDA poststorage viability criteria. Because of these very poor results with 80% Plasmalyte stored PLTs, we evaluated the 65% PLT additive solution (PAS) concentration with 35% residual plasma that has been used extensively in Europe.15 Most of the reported studies used a different PAS than Plasmalyte, but these solutions are very similar to Plasmalyte.15 Using the 65% Plasmalyte concentration, data similar to plasmastored BC PLTs were achieved. Both PLT recoveries and survivals of 65% Plasmalyte-stored PLTs met FDA criteria for 6 days of storage similar to plasma-stored PLTs (Tables 1 and 2 and Figs. 1 and 2). Although PLT recoveries met FDA criteria for 7 days of storage, survivals did not. As the results with 80% Plasmalyte used for storing PRP PC in our prior study were also very poor, we evaluated PRP PLTs stored in 65% Plasmalyte for 6 days, which was the longest time that 65% Plasmalyte-stored BC PLTs met FDA criteria. The PRP poststorage PLT recoveries and survivals were the same as BC PLTs stored in 65% Plasmalyte or plasma for 6 days. However, these PLTs met FDA criteria for recoveries but not survivals. There is known to be a substantial amount of heterogenicity among donors, even for their fresh, autologous PLT recoveries and survivals.16 Therefore, Murphy17 suggested that, when evaluating stored PLT viability data,

EXTENDED BUFFY COAT PLT STORAGE

90

90

80

80

80

70 60 50 40 30

PLT recovery (%)

90

PLT recovery (%)

PLT recovery (%)

A

70 60 50 40 30

0

100

200

300

400

0

500

20

30

200

80

60 50 40 30

70 60 50 40 30

20

20

10

10 10

20

30

40

PLT recovery (%)

90

80

70

50 40 30

10 6.5

10 20 30 40 50 60 70 80

7

7

3 2 1

PLT survival (days)

7

PLT survival (days)

8

6 5 4 3 2 1

0

100

200

300

400

500

0

Glucose (mg/dL)

10

20

4 3 2 1 200

30

7

7

4 3 2

PLT survival (days)

7

PLT survival (days)

8

6 5 4 3 2 1

1 10

20

30

ESC (%)

40

50

0

10 20 30 40 50 60 70 80

HSR (%)

250

300

350

400

Morphology Score

Annexin V Binding (%)

5

8.0

5

8

6

7.5

6

8

0

7.0

pH

8

4

400

60

8

5

350

70

HSR (%)

6

300

20 0

50

250

Morphology Score

90

PLT recovery (%)

PLT recovery (%)

10

80

ESC (%)

PLT survival (days)

30

90

0

PLT survival (days)

40

Annexin V Binding (%)

Glucose (mg/dL)

B

50

10

10

10

60

20

20

20

70

6 5 4 3 2 1 6.5

7.0

7.5

8.0

pH

Fig. 3. In vitro assays compared to poststorage PLT recoveries and survivals. (A) In vitro assays compared to poststorage in vivo PLT recoveries. (B) In vitro assays compared to poststorage in vivo PLT survivals. (●) BC plasma stored PLTs; (○) BC 65% Plasmalyte; (△) BC 80% Plasmalyte; (□) PRP 65% Plasmalyte. Volume 54, September 2014 TRANSFUSION

2289

SLICHTER ET AL.

TABLE 4. Relationship between in vitro and in vivo PLT measurements PLTs MPV Annexin V binding (%) MS ESC (%) Correlations between PLT recovery, survival, and in vitro measures of PLT quality Recovery (%) −0.14 −0.21 0.34 0.45 Survival (days) −0.39 −0.59 0.36 0.39 Dunn-Sidak probabilities Recovery (%) 0.98 0.61 0.03 0.00 Survival (days) 0.01 0.00 0.02 0.01

HSR (%)

Glucose (mg/dL)

pH

0.28 0.10

−0.08 0.05

−0.26 0.10

0.20 1.00

1.00 1.00

0.29 1.00

MS = morphology score.

each donor should serve as his or her own control; that is, each donor’s fresh recoveries and survivals should be compared to his or her stored. When fresh and stored PLT recoveries and survivals were compared to each other, there was substantial correlations between recoveries (r2 = 0.83, p < 0.001) but less so between survivals (r2 = 0.37, p = 0.005; Figs. 2A and 2B, respectively). These correlations between each donor’s fresh and stored data suggest that this may be a valid way to determine the quality of stored PLTs. Overall, the results of storing BC PLTs in plasma or Plasmalyte were not different than storing PRP PLTs for similar times. These data are not different from those of other studies that compared radiolabeled autologous BC versus PRP PLTs in normal volunteers4,18 and patients with thrombocytopenia.19-21 These data suggest that BC PLTs would pass FDA licensing criteria for 6 days of storage and could be used in the United States interchangeably with PRP PCs. Regarding the in vitro measurements (Table 3), when outliers in the in vitro results were excluded (three observations for recoveries and one for survivals), higher values of ESC and morphology score were associated with improved recoveries, while higher pH values reduced PLT recoveries (Table 3 and Fig. 3A). Only annexin V binding was associated with PLT survivals (Table 3 and Fig. 3B) with an inverse relationship between these two measurements. We elected not to measure P-selectin levels as a prior study had demonstrated ESC and HSR assays showed better correlation with PLT recoveries and survivals than P-selectin.22 There were no significant differences between the in vitro measurements of similarly stored plasma and Plasmalyte BC PLTs except for lower glucose concentrations for the Plasmalyte-stored PLTs. Although it has been suggested that enough residual plasma needs to be present to maintain adequate glucose levels, there was no association between glucose levels and either PLT recoveries or survivals; that is, residual glucose levels did not explain the improved recoveries and survivals of PLTs stored in 65% Plasmalyte versus 80% Plasmalyte. Morphology scores, pH, MPV, ESC, and HSR values were relatively stable over storage times of 5 to 7 days in both plasma and 65% Plasmalyte. 2290

TRANSFUSION Volume 54, September 2014

ACKNOWLEDGMENTS We acknowledge the support of Kate Sivertson of Fresenius Kabi who supplied the Fresenius top-bottom bags and Compomat device to allow preparation of the buffy coat platelets. Dr Dana Devine of Canadian Blood Services, Vancouver, BC, provided information on the centrifugation conditions required to prepare the buffy coat platelets. Also, we thank Ed Nelson at Pall Corporation who supplied the cord blood bags for centrifuging the buffy coat platelets. The authors are also grateful to our administrative assistant, Ginny Knight, for her excellent assistance in preparing the manuscript including tables and figures.

CONFLICT OF INTEREST The authors report no conflicts of interest or funding sources.

REFERENCES 1. Slichter SJ, Harker LA. Preparation and storage of platelet concentrates. I. Factors influencing the harvest of viable platelets from whole blood. Br J Haematol 1976;34:395-402. 2. Pietersz RN, Reesink HW, Huijgens PC, et al. Preparation of

3.

4.

5.

6.

7.

leucocyte-poor platelet concentrates from buffy coats. Vox Sang 1988;55:129-32. Perez-Pujol S, Lozano M, Perea D, et al. Effect of holding buffy coats 4 or 18 hours before preparing pooled filtered PLT concentrates in plasma. Transfusion 2004;44:202-9. Dumont LJ, Dumont DF, Unger ZM, et al.; for the BEST Collaborative. A randomized controlled trial comparing autologous radiolabeled in vivo platelet recoveries and survivals of 7-day stored platelet-rich plasma and buffy coat platelets from the same subjects. Transfusion 2011;51: 1241-8. Slichter SJ, Bolgiano D, Corson J, et al. Extended storage of platelet-rich plasma prepared platelet concentrates in plasma or Plasmalyte. Transfusion 2010;50:2199-209. Rock G, White J, Labow R. Storage of platelets in balanced salt solutions: a simple platelet storage medium. Transfusion 1991;31:21-5. Holme S, Moroff G, Murphy S. A multi-laboratory evaluation of in vitro platelet assays: the tests for extent of shape change and response to hypotonic shock. Biomedical

EXTENDED BUFFY COAT PLT STORAGE

Excellence for Safer Transfusion Working Party of the Inter-

15. Gulliksson H. Additive solutions for the storage of platelets

national Society of Blood Transfusion. Transfusion 1998;38: 31-40.

for transfusion. Transfus Med 2000;10:257-64. 16. Rock G, Tittley P, Adams GA. Donor variables affecting sur-

8. Kunicki TJ, Tuccelli M, Becker GA, et al. A study of variables affecting the quality of platelets stored at “room temperature.” Transfusion 1975;15:414-21. 9. VandenBroeke T, Dumont LJ, Hunter S, et al.; for the Biomedical Excellence for Safer Transfusion Working Party of the International Society of Blood Transfusion. Platelet storage solution effects on the accuracy of laboratory tests for platelet function: a multi-laboratory study. Vox Sang 2004;86:183-8. 10. The Biomedical Excellence for Safer Transfusion (BEST)

vival of autologous platelets. Transfusion 1986;26:30-6. 17. Murphy S. Radiolabeling of PLTs to assess viability: a proposal for a standard. Transfusion 2004;44:131-3. 18. Cardigan R, Williamson LM. The quality of platelets after storage for 7 days. Transfus Med 2003;13:173-87. 19. Keegan T, Heaton A, Holme S, et al. Paired comparison of platelet concentrates prepared from platelet-rich plasma and buffy coats using a new technique with 111In and 51Cr. Transfusion 1992;32:113-20. 20. Shanwell A, Larsson S, Aschan J, et al. A randomized trial

Collaborative. Platelet radiolabeling procedure. Transfusion

comparing the use of fresh and stored platelets in the treat-

2006;46(Suppl 3):59S-66S.

ment of bone marrow transplant recipients. Eur J Haematol

11. Lotter MG, Rabe WL, Van Zyl JM, et al. A computer program in compiled BASIC for the IBM personal computer to calculate the mean platelet survival time with the multiple hit and weighted mean methods. Comput Biol Med 1988;18:305-15. 12. Vostal JG. Efficacy evaluation of current and future platelet

1992;49:77-81. 21. Bertolini F, Rebulla P, Riccardi D, et al. Evaluation of platelet concentrates prepared from buffy coats and stored in a glucose-free crystalloid medium. Transfusion 1989;29: 605-9. 22. Holme S, Sweeney JD, Sawyer S, et al. The expression of

transfusion products. J Trauma 2006;60(6 Suppl):S78-82. 13. Shaffer JP. Multiple hypothesis testing. Annu Rev Psychol

p-selectin during collection, processing, and storage of platelet concentrates: relationship to loss of in vivo viabil-

1995;46:561-84. 14. Slichter SJ, Corson J, Jones MK, et al. Exploratory studies of

ity. Transfusion 1997;37:12-7.

extended storage of apheresis platelets in a platelet additive solution (PAS). Blood 2014;123:271-80.

Volume 54, September 2014 TRANSFUSION

2291

Extended storage of buffy coat platelet concentrates in plasma or a platelet additive solution.

Platelet (PLT) concentrates (PCs) prepared from whole blood in the United States are made using the PLT-rich plasma method. The PCs must be made withi...
270KB Sizes 2 Downloads 3 Views