BLOOD COMPONENTS In vitro variables of buffy coat–derived platelet concentrates with residual plasma of down to 10% are stably maintained in new-generation platelet additive solutions Ute Gravemann,1 Thorsten Volgmann,1 Kyungyoon Min,2 Roman Philipp,3 Bernd Lambrecht,1 € ller,1 and Axel Seltsam1 Thomas H. Mu
BACKGROUND: Platelet additive solutions (PASs) facilitate plasma recovery and may reduce the risk of plasma-associated adverse transfusion reactions. Whereas current apheresis techniques are able to produce platelet concentrates (PCs) with reduced residual plasma volumes, it is still technically challenging to prepare PCs with plasma levels less than 20% from whole blood. This study aimed to evaluate the feasibility of producing buffy coat (BC)-derived platelets (PLTs) with as little as 10% residual plasma and to test the ability of PASs to preserve PLT quality under these conditions. STUDY DESIGN AND METHODS: A pool-and-split design was used to evaluate the in vitro quality of semiautomatically prepared BC-derived PCs stored for 7 days in either SSP1 (PAS-IIIM) or Intersol-G (glucosecontaining PAS) with 10% to 30% residual plasma in three phases: 1) 30% plasma (SSP1, Intersol-G), 2) 20 and 15% plasma (SSP1, Intersol-G), and 3) 10% plasma (Intersol-G). RESULTS: The different plasma-reduced PAS-PC types were comparable in volume, PLT concentration, and PLT yield and met all regulatory requirements. The in vitro quality variables of PLTs suspended in 20% or less residual plasma were comparable to those of PLTs stored in 30% plasma for both PAS types. PLT activation was slightly higher with SSP1 than with Intersol-G. The quality of PCs suspended in Intersol-G with 10% plasma was well maintained during storage. CONCLUSIONS: Preparation of BC-derived PCs with minimal plasma concentrations as low as 10% is feasible. Late-generation PASs satisfactorily preserve the in vitro quality of such PCs during storage.
P
latelets (PLTs) are collected from whole blood donations or via apheresis. These blood products are traditionally stored as PLT concentrate (PC) in 100% plasma. However, different types of PLT additive solution (PAS) have been shown to be suitable for the storage and preservation of plasma-reduced PCs.1,2 Optimized synthetic storage media may have the potential to counteract PLT storage lesions and may allow more plasma to be recovered for fractionation or clinical use. Moreover, they may reduce acute hemolytic reactions due to ABO-incompatible plasma and potentially lower the risk of transfusion reactions, particularly allergic reactions and transfusion-associated acute lung injury, in which plasma proteins and antibodies have been implicated.3-6 Pooled buffy coat (BC)-derived PLTs stored in PAS have been used for almost three decades in Europe.7,8 The composition of PASs has gradually evolved to incorporate compounds designed to minimize morphologic, metabolic, and functional changes observed in PLTs during storage. Currently, most commercially available PASs require the carryover of substantial amounts of plasma (approx. 30%) for successful storage of PCs. Newer formulations containing potassium and magnesium should ABBREVIATIONS: BC(s) 5 buffy coat(s); MPV(s) 5 mean PLT volume(s); PC(s) 5 platelet concentrate(s); TRAP 5 thrombin receptor–activating peptide. From the 1German Red Cross Blood Service NSTOB, Institute Springe, Springe, Germany; 2Fresenius Kabi USA, Lake Zurich, Illinois; and 3Fresenius Kabi Germany, Bad Homburg, Germany Address reprint requests to: Prof. Dr med Axel Seltsam, MD, MHBA, German Red Cross Blood Service NSTOB, Institute Springe, Eldagsener Strasse 38, 31832 Springe, Germany; e-mail: [email protected]. Received for publication October
1,
2014;
revision
received December 4, 2014; and accepted December 4, 2014. doi:10.1111/trf.13000 C 2015 AABB V
TRANSFUSION 2015;55;1700–1709 1700 TRANSFUSION Volume 55, July 2015
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improve PLT function and allow further reduction of residual plasma to 20%.9,10 Recent research activities designed to further optimize PASs have resulted in the development of newgeneration glucose-containing formulations that may allow for additional reduction of residual plasma. Glucose was added as metabolic substrate because most glucose is removed from genuine plasma during processing. These new PASs, marketed as PAS-G (Pall Corp., Covina, CA) and M-Sol,11 BRS-A,12 PAS-5, and Intersol-G (Fresenius Kabi, Bad Homburg, Germany), may support additional reduction of residual plasma to 20% or below while maintaining in vitro PLT quality at levels equivalent or superior to those of PLTs stored in plasma with non–glucosecontaining PASs, such as SSP1 (Macopharma, Tourcoing, France).13-16 Glucose is important for maintaining ATP levels and NADPH-reducing equivalents and was shown to be essential for the maintenance of PLTs stored as concentrates with minimal volumes of plasma.15,17-19 In the past, the development of glucose-containing PASs has been limited by standard steam sterilization techniques that caramelize sugars at pH ranges greater than 5.5. Intersol-G and SSP1 are among the recently developed PASs. Both contain magnesium and potassium, but Intersol-G has less phosphate and additionally contains calcium and 16.8 mmol/L glucose (Table 1). The glucose in Intersol-G is maintained at a low pH during steam sterilization and is kept separate from the other PAS components to prevent caramelization. Intersol-G, which does not contain bicarbonate unlike other glucose-containing PASs, is provided in single-use bags as a ready-to-use solution, allowing for standardized PC preparation. The aim of this study was to evaluate whether IntersolG is able to preserve the in vitro properties of BC-derived PLTs with residual plasma levels as low as 10%. The results were compared to those of PLTs suspended in SSP1, which has been the standard PAS for preparation of pooled BCderived PCs in our blood transfusion service for more than a decade. Manual preparation of pooled BC-derived PCs with less than 20% plasma is technically challenging and requires the adjustment of production variables. Previous studies have used apheresis procedures or an additional centrifugation step to obtain pooled BC-derived PCs with minimal residual plasma.12,13,15,21,22 In this study, we were able to establish a semiautomated process for the preparation of pooled BC-derived PCs with less than 20% residual plasma under routine production conditions.
MATERIALS AND METHODS Study design A pool-and-split design was used to compare the in vitro quality of BC-derived PCs suspended in either SSP1 or Intersol-G with a final plasma concentration of 10% to 30%
TABLE 1. Composition of the two PASs (mmol/L) Constituent NaCl Na-acetate Na3 citrate NaH2PO4/Na2HPO4 KCl MgCl2/MgSO4 CaCl2 Glucose
SSP1*
Intersol-G†
69.3 32.5 10.8 28.2 5.0 1.5
69.9 30.0 10.0 9.4 5.0 1.5 1.0 16.8
* Alternate names: PAS-IIIM and PAS-E according to the “Standard Terminology for Platelet Additive Solutions” proposed by Ashford and colleagues.20 † Alternate name: PAS-G according to the “Standard Terminology for Platelet Additive Solutions” proposed by Ashford and colleagues20 plus CaCl2.
in three study phases: 1) 30% plasma (SSP1, Intersol-G), 2) 20 and 15% plasma (SSP1, Intersol-G), and 3) 10% plasma (Intersol-G). PLT quality data for the different groups of PCs were collected over a storage period of 7 days.
PC preparation All donations were from eligible volunteers. On Day 0, whole blood units (450-500 mL) were collected in 70 mL CPD into top-and-bottom bags (Fresenius Kabi; Macopharma) and stored overnight at 22 C until processing. On the next day (Day 1), BCs were separated from plasma and red blood cells (RBCs) by high-speed centrifugation (Roto Silenta 630 RS/63 RS, Hettich, Tuttlingen, Germany) and automated blood component separation (Macopress Smart, Macopharma). BCs were stored for 2 hours at room temperature before further processing. Up to 16 ABO-matched BCs were mixed and split into two or four pools of similar weight, each of which was supplemented with SSP1 (specific gravity, 1.016 g/mL) or Intersol-G (specific gravity, 1.009 g/mL) to yield the target plasma/ PAS ratio and then mixed and centrifuged under “softspin” conditions (Table 2). When transferring PLTs to the storage bag (Pall ELX, Pall, Ascoli Piceno, Italy), the PLTrich supernatant was leukoreduced by filtration using a blood component separator (Optipress II, Fresenius Kabi). PCs were stored under agitation at 22 6 2 C for 7 days. The specific centrifugation and BC variables for production of the different types of PCs are shown in Table 2. Fourteen PC pairs were prepared in a pool-and-split design and stored in Intersol-G or SSP1 at plasma-to-PAS ratios of approximately 30% to 70% for 7 days. With routine processing variables, PCs with 30% residual plasma were prepared from leukoreduced pools of four BCs (mean volume, 50 mL; mean hematocrit [Hct], 46%) in 250 mL of PAS. ABO-compatible BCs were pooled and split into four pools supplemented with 285 mL of PAS (SSP1 or Intersol-G) plus 15 mL plasma or with 300 mL PAS (SSP1 or Volume 55, July 2015 TRANSFUSION 1701
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TABLE 2. Production variables for centrifugation, BCs and PASs PC groups* 30% plasma Variable Hard-spin centrifugation BC volume (mL) BC Hct (%)† Soft-spin centrifugation PAS volume added (mL)
SSP1
Intersol-G
50 6 2 46 6 2 527 3 g, 7.5 min 250
20% plasma SSP1
Intersol-G
15% plasma SSP1
4000 3 g, 10 min 36 6 4 56 6 4 722 3 g, 5.5 min 285 1 15 mL plasma 300
Intersol-G
10% plasma Intersol-G 43 6 2 80 6 5 300
* The estimated percentage plasma carryover was calculated using the equation: estimated percentage plasma carryover 5 volume of plasma/(volume of plasma 1 volume of PAS) 3 100, whereby the volume of plasma was calculated as follows: volume of plasma 5 number of BCs 3 volume of BC 3 (1 – Hct of BC). † Measured in BC pools.
Intersol-G) to yield a residual plasma concentration of 20 or 15%, respectively. Compared to PCs with 30% plasma, BCs with a lower volume (mean, 36 mL) and a higher Hct (mean, 56%) were used for the production of PCs with 20 and 15% plasma. In addition, the BC pools were centrifuged for a shorter time but at a higher g-force according to an empirically established in-house protocol for better separation of RBCs and white blood cells (WBCs) from the PLT-rich supernatant. Only Intersol-G was used for preparation of PCs with 10% residual plasma, as SSP1 was specified as not being suitable for storage of PCs with such a low plasma concentration. Eleven PCs of this type were prepared and studied for 7 days. To achieve a final plasma concentration of 10%, four ABO-compatible BCs with high Hct levels (up to 85%) and volumes of approximately 43 and 300 mL PAS were used for pooling. The same soft-spin centrifugation protocol described for preparation of PCs with 20 and 15% plasma was employed. Samples for laboratory analysis were drawn aseptically on Days 1, 5, and 7. All sampling was carried out by sterile connection of sampling bags to the respective containers. The compositions of the PASs (SSP1 and IntersolG) are shown in Table 1.
Laboratory analysis PLT counts, RBC counts, Hct levels, and mean PLT volume(s) (MPVs) were determined using an automated cell counter (XS1000i, Sysmex, Norderstedt, Germany). PLT content (Table 3) was calculated as the product of PLT concentration and volume (weight of unit/specific gravity of PAS) measured on the same day. pH was measured using the pH meter (Seven Easy, Mettler-Toledo, Inc., Columbus, OH). Glucose and lactate concentrations were determined by spectrophotometry with a chemistry system (Vitros DT 60II, Ortho-Clinical Diagnostics, Rochester, NY). The protein concentration was determined spectrophotometrically in the PC supernatant using a protein assay based on Coomassie blue staining (Pierce, Rockford, IL). The residual plasma content in PCs was cal1702 TRANSFUSION Volume 55, July 2015
culated from the protein concentration of the PC supernatant using the protein concentration of a plasma pool as reference. PLT aggregation was monitored using an € Lab, Langenfeld, Germany). aggregometer (PAP 8, Mo Aggregation was induced by collagen (Nycomed Pharma GmbH, Unterschleissheim, Germany) at a final concentration of 10 lg/mL. The extent of PLT activation was measured in freshly stained samples by flow cytometry (FACSCalibur, BD Biosciences, Heidelberg, Germany) with fluorescein isothiocyanate–labeled anti-CD62P and phycoerythrin-labeled PAC-1 monoclonal antibodies (MoAbs) and their isotype controls (all from BD Biosciences). The percentage of positive PLTs in total PLTs expressing the activation marker CD62P above the background level determined by the mouse immunoglobulin (Ig)G1 isotype control was recorded. The PAC-1 MoAb recognizes a conformational epitope on the glycoprotein GPIIb/IIIa complex appearing as a result of PLT activation. PLT samples were unstimulated or maximally stimulated with 17 mmol/L thrombin receptor–activating peptide (TRAP-6, Bachem, Weil am Rhein, Germany) at room temperature for 10 minutes to activate the GPIIb/IIIa receptor and then stained with 10 mL of PAC-1/CD62P antibody mix. PAC-1 binding results were expressed as mean fluorescence intensity (MFI) ratios calculated by dividing the MFI for the samples by the MFI of the TRAP-activated control to correct for any PAS-mediated effect that could influence PAC-1 binding (matrix effect). For calculation of the percentage of activated PLTs, TRAP-6 activated samples were set to 100% (= positive control).
Statistical analysis Results are expressed as mean 6 standard deviation (SD) unless otherwise indicated. Determination of means, SDs of experimental values, and performance of analysis of variance (ANOVA) with repeated measures or t test was carried out using commercially available software (Prism 6, Version 6.04, GraphPad Software, Inc., La Jolla, CA).
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TABLE 3. PC specifications by plasma concentration and PAS type* PC group
Specifications PLT concentration (3108/mL) PLT count (31011/unit) Volume (mL) Plasma fraction (%)
30% plasma (n 5 14)
20% plasma (n 5 12)
SSP1
Intersol-G
SSP1
Intersol-G
SSP1
15% plasma (n 5 12) Intersol-G
10% plasma (n 5 11) Intersol-G
10.0 6 1.1 3.1 6 0.3 313 6 8 30.6 6 1.1
10.1 6 1.2 3.2 6 0.4 295 6 8† 31.7 6 1.2†
9.7 6 1.1 2.9 6 0.4 297 6 8‡ 17.3 6 1.5‡
10.2 6 0.7† 2.9 6 0.2 283 6 18† 18.2 6 1.3†
9.4 6 1.1 2.8 6 0.3 299 6 9‡ 13.1 6 1.5‡
10.1 6 0.6 2.9 6 0.2 282 6 14† 14.2 6 1.1†§
10.0 6 1.5 2.7 6 0.5§ 272 6 15§ 9.2 6 1.6§
* Data are presented as mean 6 SD. † p < 0.05 paired t test, Intersol-G versus SSP1 with the same plasma concentration levels. ‡ p < 0.05 ANOVA, SSP1 with 15 or 20% plasma versus SSP1 with 30% plasma. § p < 0.05 ANOVA, Intersol-G with 10, 15, or 20% plasma versus Intersol-G with 30% plasma.
Differences were considered significant at the level of p < 0.05.
RESULTS Phase 1 (30% plasma) Fourteen PC pairs were prepared and stored in Intersol-G or SSP1 at plasma-to-PAS ratios of approximately 30 to 70% for 7 days. There were slight but significant volume differences between the final products (313 6 8 mL SSP1 vs. 295 6 8 mL Intersol-G, p < 0.05), which suggests that the type of PAS influenced the sedimentation behavior of pooled BCs. PLT yields and concentrations (Table 3) as well as MPVs (data not shown) were similar for both PC types. The plasma ratio was approximately 30%. The pH measured at 22 C was slightly but significantly higher in Intersol-G PCs on Days 5 and 7 and was higher than 7.0 at all sampling times in all groups (Fig. 1). Despite the higher glucose concentration in Intersol-G PCs, lactate concentrations did not differ between the PAS-PC groups over storage. The aggregation response to collagen (10 mg/mL) was significantly better in Intersol-G PCs than in SSP1 PCs. PLT activation markers (CD62P expression and PAC-1 antibody binding) were significantly lower on Intersol-G PCs than on SSP1 PCs throughout the 7-day period.
Phase 2 (20 and 15% plasma) A total of 12 PC pairs per plasma concentration and PAS were prepared and stored for up to 7 days. The final plasma level in the PCs was slightly lower than intended in both PAS-PC groups, whereby the mean plasma concentrations in the Intersol-G PCs were slightly higher than those of the SSP1 PCs. Similar to the 30% plasma PCs, 20 and 15% plasma PCs suspended in SSP1 had slightly higher volumes than those in Intersol-G (Table 3). Since Intersol-G PCs had higher PLT concentrations, PLT yields were similar in both PAS-PC pairs. There was no difference in MPV between Intersol-G and SSP1 PCs (data not shown). At 22 C the pH was higher than 7.0 in all PC products throughout storage. Glucose concentrations con-
stantly decreased over storage in all PC-PAS groups. In the Intersol-G group, glucose levels remained high and were still higher than 11.0 mmol/L at the end of storage, independent of plasma concentration, whereas those in the SSP1 group were dependent on residual plasma concentration and reached the limit of detection on Day 7. Lactate levels increased during storage and did not differ between PAS-PC groups. In addition, the aggregation response to collagen stimulation was similar for all PASPC groups during storage. CD62P expression and PAC-1 antibody binding values gradually increased during storage in all groups, but were significantly higher in SSP1 PCs than in Intersol-G PCs. This difference in activation marker levels was related to PAS type rather than to residual plasma concentration (Fig. 2).
Phase 3 (10% plasma) Eleven PCs with 10% residual plasma were prepared and studied for 7 days. PLT concentrations, PLT yields, and PC volumes were similar to those in PCs with higher plasma levels. The mean plasma concentration of the final products was 9.2%. Glucose levels in the PCs remained high during storage (12 mmol/L on Day 7). Lactate concentrations increased continuously, peaking at 7.1 mmol/L on Day 7. During storage, pH remained stable and was always greater than 7.0. PLT aggregability showed a slight but significant decrease during storage. CD62P expression on PLTs significantly decreased until Day 5 of storage, and then increased until Day 7, reaching a level similar to that measured at the beginning of storage. PAC-1 antibody binding increased slightly during storage and was significantly higher on Day 7 than on Day 1 of storage (Fig. 3).
DISCUSSION Our study shows that it is feasible to produce BC-derived PCs with residual plasma levels as low as 10% using a semiautomated preparation process. In vitro data suggest that glucose is not required in new-generation PASs to preserve the quality of PCs stored in PAS with at least 15% residual plasma for 7 days. Good in vitro PLT quality is Volume 55, July 2015 TRANSFUSION 1703
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Fig. 1. In vitro quality of BC-derived PCs with 30% residual plasma. Metabolic, functional, and activation variables of PLTs derived from PCs resuspended in 70% SSP1 (䊉) or Intersol-G (䊏) over 7 days of storage. Data are presented as mean 6 SD (n 5 14). *Significant differences between groups on the same storage day: p < 0.05 (multiple t test).
even obtained in PCs with 10% residual plasma when using glucose-containing Intersol-G; however, it still remains to be determined whether glucose is a critical component in PASs for maintaining PLT storage quality at this minimal plasma level. In general, PASs can be used for both apheresis and BC-derived PCs. The storage medium is normally composed of a mixture of plasma (generally 20%-40%) and PAS (60-80%). With apheresis technology, hyperconcen1704 TRANSFUSION Volume 55, July 2015
trated PLT units can be collected with either manual or automated addition of PAS without additional centrifugation or washing.23,24 This technique has been used to produce plasma-reduced PCs with plasma concentrations as low as 5%.13,25,26 In contrast, the precision of the BC method becomes critical if the percentage of plasma carryover is less than 20%. Modern software-controlled separation devices use sensitive volume and Hct sensors that allow for the preparation of “dry” BCs with very little
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Fig. 2. In vitro quality of BC-derived PCs with 20 or 15% residual plasma. Metabolic, functional, and activation variables of PLTs derived from PCs resuspended in 80 or 85% SSP1 and Intersol-G, respectively, over 7 days of storage. Data are presented as mean 6 SD (n 5 12). Significant differences (p < 0.05, two-way repeated-measures ANOVA) between groups on the same storage day are indicated: *85% Intersol-G (W) versus 85% SSP1 (䉲); †80% Intersol-G (䊉) versus 80% SSP1 (䉱).
residual plasma and optimal PLT recovery while avoiding any additional contamination by WBCs and RBCs in the final blood products. In this study, we established a process for the production of PCs with plasma concentrations as low as 10%, which is suitable for use in routine opera-
tions. A key step was adaption of the centrifugation settings to the different PAS/plasma ratios in the BC pools to achieve optimal PLT recovery.17 PASs have specific gravities similar to that of water, while the soluble proteins in plasma increase its specific gravity. Thus, alteration of the Volume 55, July 2015 TRANSFUSION 1705
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Fig. 3. In vitro quality of BC-derived PCs with 10% residual plasma. Metabolic, functional, and activation variables of PLTs derived from PCs resuspended in 90% Intersol-G over 7 days of storage. Data are presented as mean 6 SD (n 5 11). *Significant differences compared to Day 1: p < 0.05 (one-way repeated-measures ANOVA).
PAS/plasma ratio results in a change in the specific gravity of the storage medium, which influences the sedimentation behavior of the blood cells. The fact that the mean volumes of the Intersol-G PCs were consistently lower than those of the SSP1 PCs at all plasma concentrations studied despite the use of identical source materials and preparation procedures probably reflects the difference in specific gravity between the two PASs. As the MPVs were not different between Intersol-G and SSP1 PCs, the different sedimentation behaviors of PLTs cannot be explained 1706 TRANSFUSION Volume 55, July 2015
by swelling or shrinking of the PLTs due to a difference in PAS compositions. This finding demonstrates that the optimal production variables critically depend on the characteristics of the individual product components. SSP1 and Intersol-G, two of the latest-generation PASs, are designed to partially replace plasma in the preparation and storage of BC-derived or apheresis PCs. The recommended ratio is up to 80% PAS to 20% plasma. These solutions enable PLT storage at 22 6 2 C for up to 7 days after collection. We evaluated the ability of both PASs
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to maintain PLT quality after being used for the preparation of PCs with even lower plasma ratios. Intersol-G differs from SSP1 in that it additionally contains calcium and glucose, which have been proposed as key constituents for the maintenance of in vitro PLT quality in PCs with a low percentage of plasma carryover.12-14,22,27 This study of PLT quality revealed significant differences in the activation markers CD62P and PAC-1 between the two PASs. While plasma concentration had a consistent but differential effect on the two activation markers within each PAS-PC group, activation levels were generally higher in SSP1 PCs than in Intersol-G PCs. This suggests that the difference in activation is related to PAS type rather than to residual plasma concentration. It remains to be determined whether the presence of calcium, which is reportedly involved in several control functions and membranemodifying effects in PLTs,28,29 may contribute to the lower activation signals in Intersol-G PLTs. A previous study did not detect significant differences between calciumcontaining and calcium-free PAS using PCs with 20% plasma carryover.22,27 The difference in PAC-1 binding between the two PASs was not substantial. Moreover, the clinical significance of PLT surface expression of CD62P has been questioned and the data appear to be inconsistent.30-32 Therefore, in vivo studies are needed to show whether the reduced activation of PLTs stored in IntersolG will translate into any clinical benefits. The importance of glucose for in vitro storage of PLTs also remains to be adequately explained. While other substrates, such as acetate and fatty acids, also act as energy sources for PLTs,15,33,34 approximately 25% of the PLT energy requirement is supplied by glucose, which is considered crucial for maintaining the viability of PLTs in vitro and in vivo.32,35 Although glucose concentrations reached very low levels in SSP1 PCs with 15 and 20% plasma by the end of storage (Day 7), metabolic, aggregation, and activation markers still indicated that PLT quality was well preserved and comparable to that of Intersol-G PCs stored in an excess of glucose. It is still a matter of debate whether residual glucose is required and if acetate can substitute glucose to maintain PLT viability.32,35-38 Both PASs used in this study contain approximately 30 mmol/L acetate. Given that PLTs metabolize 2 mmol/L acetate per day of storage,39 this amount should easily be sufficient to maintain PLT viability for 7 days. In this study, PCs stored in 10% plasma plus 90% Intersol-G showed good PLT stability. Although SSP1 was not used for PCs with 10% plasma in this study, the results obtained for PCs with 15 and 20% residual plasma suggest that glucose-free lategeneration PASs can also be used for the preparation and storage of PCs with plasma levels below 20%. This study has some weaknesses. One is that the conclusions made based on the comparative analyses would have been stronger if all plasma concentration categories in both PAS groups had been incorporated in a single
pool-and-split design study. Another limitation is that only a small number of in vitro PLT quality variables were investigated. Additional in vitro storage variables such as pCO2, pO2, lactate dehydrogenase, hypotonic stress response, morphology, and ATP as well as in vivo studies are warranted for further investigation of poststorage PLT viability in PCs with minimal residual plasma. There is increasing evidence that the use of PASs may prevent acute transfusion reactions to apheresis PLTs and that plasma-reduced PAS apheresis PLTs may be financially and clinically superior to apheresis PLTs stored with 100% plasma.40-44 Until now, the preparation of plasmareduced PLTs was thought to be dependent on the apheresis technology. Our data demonstrate that it is feasible to prepare BC-derived plasma-reduced PCs with residual plasma concentrations far below 30%, the level commonly used under routine blood bank conditions. As plasma has clearly been identified as the major cause of transfusionrelated adverse reactions to PLT transfusions, minimizing the plasma concentration in PCs may help to further minimize or prevent these reactions.5 This study provides the technical perspective needed for the widespread use of PCs with minimal plasma concentrations, independently of the techniques used for their preparation.
ACKNOWLEDGMENTS The authors thank Elke Heide and Udo Grundmann (German Red Cross Blood Service NSTOB, Institute Springe, Springe, Germany) for their technical assistance. UG designed the study, performed the in vitro experiments, analyzed the data, and cowrote the manuscript; TV prepared the plasma-reduced PCs; KM analyzed and interpreted the data; RP analyzed the data; BL analyzed and interpreted the data; THM analyzed and interpreted the data and edited the manuscript; AS designed the study, interpreted the data, and cowrote the manuscript; and all authors read and approved the final manuscript.
CONFLICT OF INTEREST KM and RP are employees of Fresenius Kabi. The other authors have disclosed no conflicts of interest.
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on platelets stored in reformulated PAS supplied by a 20% plasma carry-over. Vox Sang 2010;98:415-22.
3406-17. 7. Rock G, Swenson SD, Adams GA. Platelet storage in a plasma-free medium. Transfusion 1985;25:551-6. 8. de Wildt-Eggen J, Schrijver JG, Smid WM, et al. Platelets stored in a new-generation container: differences between plasma and platelet additive solution II. Vox Sang 1998;75: 218-23. 9. Gulliksson H, AuBuchon JP, Vesterinen M, et al. Storage of platelets in additive solutions: a pilot in vitro study of the effects of potassium and magnesium. Vox Sang 2002;82:131-6. 10. Gulliksson H, AuBuchon JP, Cardigan R, et al. Storage of platelets in additive solutions: a multicentre study of the in vitro effects of potassium and magnesium. Vox Sang 2003;85:199205. 11. Hirayama J, Azuma H, Fujihara M, et al. Storage of platelets in a novel additive solution (M-sol), which is prepared by mixing solutions approved for clinical use that are not especially for platelet storage. Transfusion 2007;47:960-5. 12. Oikawa S, Sasaki D, Kikuchi M, et al. Comparative in vitro evaluation of apheresis platelets stored with 100% plasma versus bicarbonated Ringer’s solution with less than 5% plasma. Transfusion 2013;53:655-60. 13. Radwanski K, Wagner SJ, Skripchenko A, et al. In vitro variables of apheresis platelets are stably maintained for 7 days with 5% residual plasma in a glucose and bicarbonate salt solution, PAS-5. Transfusion 2012;52:188-94. 14. Gyongyossy-Issa MI, Zhang JG, et al. Novel system for storage of buffy-coat-derived platelet concentrates in a glucosebased platelet additive solution: parameters and metabolism during storage and comparison to plasma. Vox Sang 2009;97: 102-9. 15. Saunders C, Rowe G, Wilkins K, et al. Impact of glucose and acetate on the characteristics of the platelet storage lesion in platelets suspended in additive solutions with minimal plasma. Vox Sang 2013;105:1-10. 16. Gulliksson H. Platelet storage media. Vox Sang 2014;107:20512. 17. Zhang JG, Carter CJ, Culibrk B, et al. Buffy-coat platelet variables and metabolism during storage in additive solutions or plasma. Transfusion 2008;48:847-56.
23. Ringwald J, Haager B, Krex D, et al. Impact of different hold time before addition of platelet additive solution on the in vitro quality of apheresis platelets. Transfusion 2006;46:9428. 24. Ringwald J, Walz S, Zimmermann R, et al. Hyperconcentrated platelets stored in additive solution: aspects on productivity and in vitro quality. Vox Sang 2005;89:11-8. 25. Dumont LJ, Cancelas JA, Graminske S, et al. In vitro and in vivo quality of leukoreduced apheresis platelets stored in a new platelet additive solution. Transfusion 2013;53:972-80. 26. Morrison A, McMillan L, Radwanski K, et al. Storage of apheresis platelet concentrates after manual replacement of >95% of plasma with PAS 5. Vox Sang 2014;107:247-53. 27. Wagner SJ, Skripchenko A, Myrup A, et al. Calcium is a key constituent for maintaining the in vitro properties of platelets suspended in the bicarbonate-containing additive solution M-sol with low plasma levels. Transfusion 2010;50:1028-35. pez JJ, Pariente JA, et al. Intracellular calcium 28. Jardin I, Lo release from human platelets: different messengers for multiple stores. Trends Cardiovasc Med 2008;18:57-61. 29. Weis-Fogh US. The effect of citrate, calcium, and magnesium ions on the potassium movement across the human platelet membrane. Transfusion 1985;25:339-42. 30. Berger G, Hartwell DW, Wagner DD. P-selectin and platelet clearance. Blood 1998;92:4446-52. 31. Michelson AD, Barnard MR, Hechtman HB, Valeri CR, et al. In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function. Proc Natl Acad Sci U S A 1996;93:11877-82. 32. Holme S, Sweeney JD, Sawyer S, et al. The expression of pselectin during collection, processing, and storage of platelet concentrates: relationship to loss of in vivo viability. Transfusion 1997;37:12-7. 33. Cesar J, DiMinno G, Alam I, et al. Plasma free fatty acid metabolism during storage of platelet concentrates for transfusion. Transfusion 1987;27:434-7. 34. Murphy S. The oxidation of exogenously added organic anions by platelets facilitates maintenance of pH during their storage for transfusion at 22 degrees C. Blood 1995;85: 1929-35.
18. Gulliksson H. Platelet storage media. Transfus Apher Sci 2001;24:241-4.
35. Gulliksson H. Platelet additive solutions: current status. Immunohematology 2007;23:14-9.
19. Holme S, Heaton WA, Courtright M. Platelet storage lesion
36. Guppy M, Whisson ME, Sabaratnam R, et al. Alternative
in second-generation containers: correlation with platelet ATP levels. Vox Sang 1987;53:214-20. 1708 TRANSFUSION Volume 55, July 2015
fuels for platelet storage: a metabolic study. Vox Sang 1990; 59:146-52.
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37. Whisson ME, Nakhoul A, Howman P, et al. Quantitative
41. Tobian AA, Fuller AK, Uglik K, et al. The impact of platelet
study of starving platelets in a minimal medium: mainte-
additive solution apheresis platelets on allergic transfusion
nance by acetate or plasma but not by glucose. Transfus Med 1993;3:103-13.
reactions and corrected count increment (CME). Transfusion 2014;54:1523-9; quiz 1522.
38. Hornsey VS, McColl K, Drummond O, et al. Extended storage of platelets in SSP1 platelet additive solution. Vox Sang 2006;91:41-6. 39. Murphy S, Shimizu T, Miripol J. Platelet storage for transfusion in synthetic media: further optimization of ingredients and definition of their roles. Blood 1995;86:395160. 40. Kacker S, Ness PM, Savage WJ, et al. The cost-effectiveness of platelet additive solution to prevent allergic transfusion reactions. Transfusion 2013;53:2609-18.
42. de Wildt-Eggen J, Gulliksson H. In vivo and in vitro comparison of platelets stored in either synthetic media or plasma. Vox Sang 2003;84:256-64. 43. van Rhenen DJ, Gulliksson H, Cazenave JP, et al. Therapeutic efficacy of pooled buffy-coat platelet components prepared and stored with a platelet additive solution. Transfus Med 2004;14:289-95. 44. Slichter SJ, Bolgiano D, Corson J, et al. Extended storage of buffy coat platelet concentrates in plasma or a platelet additive solution. Transfusion 2014;54:2283-91.
Volume 55, July 2015 TRANSFUSION 1709
BACKGROUND: Platelet additive solutions (PASs) facilitate plasma recovery and may reduce the risk of plasma-associated adverse transfusion reactions. Whereas current apheresis techniques are able to produce platelet concentrates (PCs) with reduced residual plasma volumes, it is still technically challenging to prepare PCs with plasma levels less than 20% from whole blood. This study aimed to evaluate the feasibility of producing buffy coat (BC)-derived platelets (PLTs) with as little as 10% residual plasma and to test the ability of PASs to preserve PLT quality under these conditions. STUDY DESIGN AND METHODS: A pool-and-split design was used to evaluate the in vitro quality of semiautomatically prepared BC-derived PCs stored for 7 days in either SSP1 (PAS-IIIM) or Intersol-G (glucosecontaining PAS) with 10% to 30% residual plasma in three phases: 1) 30% plasma (SSP1, Intersol-G), 2) 20 and 15% plasma (SSP1, Intersol-G), and 3) 10% plasma (Intersol-G). RESULTS: The different plasma-reduced PAS-PC types were comparable in volume, PLT concentration, and PLT yield and met all regulatory requirements. The in vitro quality variables of PLTs suspended in 20% or less residual plasma were comparable to those of PLTs stored in 30% plasma for both PAS types. PLT activation was slightly higher with SSP1 than with Intersol-G. The quality of PCs suspended in Intersol-G with 10% plasma was well maintained during storage. CONCLUSIONS: Preparation of BC-derived PCs with minimal plasma concentrations as low as 10% is feasible. Late-generation PASs satisfactorily preserve the in vitro quality of such PCs during storage.
P
latelets (PLTs) are collected from whole blood donations or via apheresis. These blood products are traditionally stored as PLT concentrate (PC) in 100% plasma. However, different types of PLT additive solution (PAS) have been shown to be suitable for the storage and preservation of plasma-reduced PCs.1,2 Optimized synthetic storage media may have the potential to counteract PLT storage lesions and may allow more plasma to be recovered for fractionation or clinical use. Moreover, they may reduce acute hemolytic reactions due to ABO-incompatible plasma and potentially lower the risk of transfusion reactions, particularly allergic reactions and transfusion-associated acute lung injury, in which plasma proteins and antibodies have been implicated.3-6 Pooled buffy coat (BC)-derived PLTs stored in PAS have been used for almost three decades in Europe.7,8 The composition of PASs has gradually evolved to incorporate compounds designed to minimize morphologic, metabolic, and functional changes observed in PLTs during storage. Currently, most commercially available PASs require the carryover of substantial amounts of plasma (approx. 30%) for successful storage of PCs. Newer formulations containing potassium and magnesium should ABBREVIATIONS: BC(s) 5 buffy coat(s); MPV(s) 5 mean PLT volume(s); PC(s) 5 platelet concentrate(s); TRAP 5 thrombin receptor–activating peptide. From the 1German Red Cross Blood Service NSTOB, Institute Springe, Springe, Germany; 2Fresenius Kabi USA, Lake Zurich, Illinois; and 3Fresenius Kabi Germany, Bad Homburg, Germany Address reprint requests to: Prof. Dr med Axel Seltsam, MD, MHBA, German Red Cross Blood Service NSTOB, Institute Springe, Eldagsener Strasse 38, 31832 Springe, Germany; e-mail: [email protected]. Received for publication October
1,
2014;
revision
received December 4, 2014; and accepted December 4, 2014. doi:10.1111/trf.13000 C 2015 AABB V
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improve PLT function and allow further reduction of residual plasma to 20%.9,10 Recent research activities designed to further optimize PASs have resulted in the development of newgeneration glucose-containing formulations that may allow for additional reduction of residual plasma. Glucose was added as metabolic substrate because most glucose is removed from genuine plasma during processing. These new PASs, marketed as PAS-G (Pall Corp., Covina, CA) and M-Sol,11 BRS-A,12 PAS-5, and Intersol-G (Fresenius Kabi, Bad Homburg, Germany), may support additional reduction of residual plasma to 20% or below while maintaining in vitro PLT quality at levels equivalent or superior to those of PLTs stored in plasma with non–glucosecontaining PASs, such as SSP1 (Macopharma, Tourcoing, France).13-16 Glucose is important for maintaining ATP levels and NADPH-reducing equivalents and was shown to be essential for the maintenance of PLTs stored as concentrates with minimal volumes of plasma.15,17-19 In the past, the development of glucose-containing PASs has been limited by standard steam sterilization techniques that caramelize sugars at pH ranges greater than 5.5. Intersol-G and SSP1 are among the recently developed PASs. Both contain magnesium and potassium, but Intersol-G has less phosphate and additionally contains calcium and 16.8 mmol/L glucose (Table 1). The glucose in Intersol-G is maintained at a low pH during steam sterilization and is kept separate from the other PAS components to prevent caramelization. Intersol-G, which does not contain bicarbonate unlike other glucose-containing PASs, is provided in single-use bags as a ready-to-use solution, allowing for standardized PC preparation. The aim of this study was to evaluate whether IntersolG is able to preserve the in vitro properties of BC-derived PLTs with residual plasma levels as low as 10%. The results were compared to those of PLTs suspended in SSP1, which has been the standard PAS for preparation of pooled BCderived PCs in our blood transfusion service for more than a decade. Manual preparation of pooled BC-derived PCs with less than 20% plasma is technically challenging and requires the adjustment of production variables. Previous studies have used apheresis procedures or an additional centrifugation step to obtain pooled BC-derived PCs with minimal residual plasma.12,13,15,21,22 In this study, we were able to establish a semiautomated process for the preparation of pooled BC-derived PCs with less than 20% residual plasma under routine production conditions.
MATERIALS AND METHODS Study design A pool-and-split design was used to compare the in vitro quality of BC-derived PCs suspended in either SSP1 or Intersol-G with a final plasma concentration of 10% to 30%
TABLE 1. Composition of the two PASs (mmol/L) Constituent NaCl Na-acetate Na3 citrate NaH2PO4/Na2HPO4 KCl MgCl2/MgSO4 CaCl2 Glucose
SSP1*
Intersol-G†
69.3 32.5 10.8 28.2 5.0 1.5
69.9 30.0 10.0 9.4 5.0 1.5 1.0 16.8
* Alternate names: PAS-IIIM and PAS-E according to the “Standard Terminology for Platelet Additive Solutions” proposed by Ashford and colleagues.20 † Alternate name: PAS-G according to the “Standard Terminology for Platelet Additive Solutions” proposed by Ashford and colleagues20 plus CaCl2.
in three study phases: 1) 30% plasma (SSP1, Intersol-G), 2) 20 and 15% plasma (SSP1, Intersol-G), and 3) 10% plasma (Intersol-G). PLT quality data for the different groups of PCs were collected over a storage period of 7 days.
PC preparation All donations were from eligible volunteers. On Day 0, whole blood units (450-500 mL) were collected in 70 mL CPD into top-and-bottom bags (Fresenius Kabi; Macopharma) and stored overnight at 22 C until processing. On the next day (Day 1), BCs were separated from plasma and red blood cells (RBCs) by high-speed centrifugation (Roto Silenta 630 RS/63 RS, Hettich, Tuttlingen, Germany) and automated blood component separation (Macopress Smart, Macopharma). BCs were stored for 2 hours at room temperature before further processing. Up to 16 ABO-matched BCs were mixed and split into two or four pools of similar weight, each of which was supplemented with SSP1 (specific gravity, 1.016 g/mL) or Intersol-G (specific gravity, 1.009 g/mL) to yield the target plasma/ PAS ratio and then mixed and centrifuged under “softspin” conditions (Table 2). When transferring PLTs to the storage bag (Pall ELX, Pall, Ascoli Piceno, Italy), the PLTrich supernatant was leukoreduced by filtration using a blood component separator (Optipress II, Fresenius Kabi). PCs were stored under agitation at 22 6 2 C for 7 days. The specific centrifugation and BC variables for production of the different types of PCs are shown in Table 2. Fourteen PC pairs were prepared in a pool-and-split design and stored in Intersol-G or SSP1 at plasma-to-PAS ratios of approximately 30% to 70% for 7 days. With routine processing variables, PCs with 30% residual plasma were prepared from leukoreduced pools of four BCs (mean volume, 50 mL; mean hematocrit [Hct], 46%) in 250 mL of PAS. ABO-compatible BCs were pooled and split into four pools supplemented with 285 mL of PAS (SSP1 or Intersol-G) plus 15 mL plasma or with 300 mL PAS (SSP1 or Volume 55, July 2015 TRANSFUSION 1701
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TABLE 2. Production variables for centrifugation, BCs and PASs PC groups* 30% plasma Variable Hard-spin centrifugation BC volume (mL) BC Hct (%)† Soft-spin centrifugation PAS volume added (mL)
SSP1
Intersol-G
50 6 2 46 6 2 527 3 g, 7.5 min 250
20% plasma SSP1
Intersol-G
15% plasma SSP1
4000 3 g, 10 min 36 6 4 56 6 4 722 3 g, 5.5 min 285 1 15 mL plasma 300
Intersol-G
10% plasma Intersol-G 43 6 2 80 6 5 300
* The estimated percentage plasma carryover was calculated using the equation: estimated percentage plasma carryover 5 volume of plasma/(volume of plasma 1 volume of PAS) 3 100, whereby the volume of plasma was calculated as follows: volume of plasma 5 number of BCs 3 volume of BC 3 (1 – Hct of BC). † Measured in BC pools.
Intersol-G) to yield a residual plasma concentration of 20 or 15%, respectively. Compared to PCs with 30% plasma, BCs with a lower volume (mean, 36 mL) and a higher Hct (mean, 56%) were used for the production of PCs with 20 and 15% plasma. In addition, the BC pools were centrifuged for a shorter time but at a higher g-force according to an empirically established in-house protocol for better separation of RBCs and white blood cells (WBCs) from the PLT-rich supernatant. Only Intersol-G was used for preparation of PCs with 10% residual plasma, as SSP1 was specified as not being suitable for storage of PCs with such a low plasma concentration. Eleven PCs of this type were prepared and studied for 7 days. To achieve a final plasma concentration of 10%, four ABO-compatible BCs with high Hct levels (up to 85%) and volumes of approximately 43 and 300 mL PAS were used for pooling. The same soft-spin centrifugation protocol described for preparation of PCs with 20 and 15% plasma was employed. Samples for laboratory analysis were drawn aseptically on Days 1, 5, and 7. All sampling was carried out by sterile connection of sampling bags to the respective containers. The compositions of the PASs (SSP1 and IntersolG) are shown in Table 1.
Laboratory analysis PLT counts, RBC counts, Hct levels, and mean PLT volume(s) (MPVs) were determined using an automated cell counter (XS1000i, Sysmex, Norderstedt, Germany). PLT content (Table 3) was calculated as the product of PLT concentration and volume (weight of unit/specific gravity of PAS) measured on the same day. pH was measured using the pH meter (Seven Easy, Mettler-Toledo, Inc., Columbus, OH). Glucose and lactate concentrations were determined by spectrophotometry with a chemistry system (Vitros DT 60II, Ortho-Clinical Diagnostics, Rochester, NY). The protein concentration was determined spectrophotometrically in the PC supernatant using a protein assay based on Coomassie blue staining (Pierce, Rockford, IL). The residual plasma content in PCs was cal1702 TRANSFUSION Volume 55, July 2015
culated from the protein concentration of the PC supernatant using the protein concentration of a plasma pool as reference. PLT aggregation was monitored using an € Lab, Langenfeld, Germany). aggregometer (PAP 8, Mo Aggregation was induced by collagen (Nycomed Pharma GmbH, Unterschleissheim, Germany) at a final concentration of 10 lg/mL. The extent of PLT activation was measured in freshly stained samples by flow cytometry (FACSCalibur, BD Biosciences, Heidelberg, Germany) with fluorescein isothiocyanate–labeled anti-CD62P and phycoerythrin-labeled PAC-1 monoclonal antibodies (MoAbs) and their isotype controls (all from BD Biosciences). The percentage of positive PLTs in total PLTs expressing the activation marker CD62P above the background level determined by the mouse immunoglobulin (Ig)G1 isotype control was recorded. The PAC-1 MoAb recognizes a conformational epitope on the glycoprotein GPIIb/IIIa complex appearing as a result of PLT activation. PLT samples were unstimulated or maximally stimulated with 17 mmol/L thrombin receptor–activating peptide (TRAP-6, Bachem, Weil am Rhein, Germany) at room temperature for 10 minutes to activate the GPIIb/IIIa receptor and then stained with 10 mL of PAC-1/CD62P antibody mix. PAC-1 binding results were expressed as mean fluorescence intensity (MFI) ratios calculated by dividing the MFI for the samples by the MFI of the TRAP-activated control to correct for any PAS-mediated effect that could influence PAC-1 binding (matrix effect). For calculation of the percentage of activated PLTs, TRAP-6 activated samples were set to 100% (= positive control).
Statistical analysis Results are expressed as mean 6 standard deviation (SD) unless otherwise indicated. Determination of means, SDs of experimental values, and performance of analysis of variance (ANOVA) with repeated measures or t test was carried out using commercially available software (Prism 6, Version 6.04, GraphPad Software, Inc., La Jolla, CA).
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TABLE 3. PC specifications by plasma concentration and PAS type* PC group
Specifications PLT concentration (3108/mL) PLT count (31011/unit) Volume (mL) Plasma fraction (%)
30% plasma (n 5 14)
20% plasma (n 5 12)
SSP1
Intersol-G
SSP1
Intersol-G
SSP1
15% plasma (n 5 12) Intersol-G
10% plasma (n 5 11) Intersol-G
10.0 6 1.1 3.1 6 0.3 313 6 8 30.6 6 1.1
10.1 6 1.2 3.2 6 0.4 295 6 8† 31.7 6 1.2†
9.7 6 1.1 2.9 6 0.4 297 6 8‡ 17.3 6 1.5‡
10.2 6 0.7† 2.9 6 0.2 283 6 18† 18.2 6 1.3†
9.4 6 1.1 2.8 6 0.3 299 6 9‡ 13.1 6 1.5‡
10.1 6 0.6 2.9 6 0.2 282 6 14† 14.2 6 1.1†§
10.0 6 1.5 2.7 6 0.5§ 272 6 15§ 9.2 6 1.6§
* Data are presented as mean 6 SD. † p < 0.05 paired t test, Intersol-G versus SSP1 with the same plasma concentration levels. ‡ p < 0.05 ANOVA, SSP1 with 15 or 20% plasma versus SSP1 with 30% plasma. § p < 0.05 ANOVA, Intersol-G with 10, 15, or 20% plasma versus Intersol-G with 30% plasma.
Differences were considered significant at the level of p < 0.05.
RESULTS Phase 1 (30% plasma) Fourteen PC pairs were prepared and stored in Intersol-G or SSP1 at plasma-to-PAS ratios of approximately 30 to 70% for 7 days. There were slight but significant volume differences between the final products (313 6 8 mL SSP1 vs. 295 6 8 mL Intersol-G, p < 0.05), which suggests that the type of PAS influenced the sedimentation behavior of pooled BCs. PLT yields and concentrations (Table 3) as well as MPVs (data not shown) were similar for both PC types. The plasma ratio was approximately 30%. The pH measured at 22 C was slightly but significantly higher in Intersol-G PCs on Days 5 and 7 and was higher than 7.0 at all sampling times in all groups (Fig. 1). Despite the higher glucose concentration in Intersol-G PCs, lactate concentrations did not differ between the PAS-PC groups over storage. The aggregation response to collagen (10 mg/mL) was significantly better in Intersol-G PCs than in SSP1 PCs. PLT activation markers (CD62P expression and PAC-1 antibody binding) were significantly lower on Intersol-G PCs than on SSP1 PCs throughout the 7-day period.
Phase 2 (20 and 15% plasma) A total of 12 PC pairs per plasma concentration and PAS were prepared and stored for up to 7 days. The final plasma level in the PCs was slightly lower than intended in both PAS-PC groups, whereby the mean plasma concentrations in the Intersol-G PCs were slightly higher than those of the SSP1 PCs. Similar to the 30% plasma PCs, 20 and 15% plasma PCs suspended in SSP1 had slightly higher volumes than those in Intersol-G (Table 3). Since Intersol-G PCs had higher PLT concentrations, PLT yields were similar in both PAS-PC pairs. There was no difference in MPV between Intersol-G and SSP1 PCs (data not shown). At 22 C the pH was higher than 7.0 in all PC products throughout storage. Glucose concentrations con-
stantly decreased over storage in all PC-PAS groups. In the Intersol-G group, glucose levels remained high and were still higher than 11.0 mmol/L at the end of storage, independent of plasma concentration, whereas those in the SSP1 group were dependent on residual plasma concentration and reached the limit of detection on Day 7. Lactate levels increased during storage and did not differ between PAS-PC groups. In addition, the aggregation response to collagen stimulation was similar for all PASPC groups during storage. CD62P expression and PAC-1 antibody binding values gradually increased during storage in all groups, but were significantly higher in SSP1 PCs than in Intersol-G PCs. This difference in activation marker levels was related to PAS type rather than to residual plasma concentration (Fig. 2).
Phase 3 (10% plasma) Eleven PCs with 10% residual plasma were prepared and studied for 7 days. PLT concentrations, PLT yields, and PC volumes were similar to those in PCs with higher plasma levels. The mean plasma concentration of the final products was 9.2%. Glucose levels in the PCs remained high during storage (12 mmol/L on Day 7). Lactate concentrations increased continuously, peaking at 7.1 mmol/L on Day 7. During storage, pH remained stable and was always greater than 7.0. PLT aggregability showed a slight but significant decrease during storage. CD62P expression on PLTs significantly decreased until Day 5 of storage, and then increased until Day 7, reaching a level similar to that measured at the beginning of storage. PAC-1 antibody binding increased slightly during storage and was significantly higher on Day 7 than on Day 1 of storage (Fig. 3).
DISCUSSION Our study shows that it is feasible to produce BC-derived PCs with residual plasma levels as low as 10% using a semiautomated preparation process. In vitro data suggest that glucose is not required in new-generation PASs to preserve the quality of PCs stored in PAS with at least 15% residual plasma for 7 days. Good in vitro PLT quality is Volume 55, July 2015 TRANSFUSION 1703
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Fig. 1. In vitro quality of BC-derived PCs with 30% residual plasma. Metabolic, functional, and activation variables of PLTs derived from PCs resuspended in 70% SSP1 (䊉) or Intersol-G (䊏) over 7 days of storage. Data are presented as mean 6 SD (n 5 14). *Significant differences between groups on the same storage day: p < 0.05 (multiple t test).
even obtained in PCs with 10% residual plasma when using glucose-containing Intersol-G; however, it still remains to be determined whether glucose is a critical component in PASs for maintaining PLT storage quality at this minimal plasma level. In general, PASs can be used for both apheresis and BC-derived PCs. The storage medium is normally composed of a mixture of plasma (generally 20%-40%) and PAS (60-80%). With apheresis technology, hyperconcen1704 TRANSFUSION Volume 55, July 2015
trated PLT units can be collected with either manual or automated addition of PAS without additional centrifugation or washing.23,24 This technique has been used to produce plasma-reduced PCs with plasma concentrations as low as 5%.13,25,26 In contrast, the precision of the BC method becomes critical if the percentage of plasma carryover is less than 20%. Modern software-controlled separation devices use sensitive volume and Hct sensors that allow for the preparation of “dry” BCs with very little
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Fig. 2. In vitro quality of BC-derived PCs with 20 or 15% residual plasma. Metabolic, functional, and activation variables of PLTs derived from PCs resuspended in 80 or 85% SSP1 and Intersol-G, respectively, over 7 days of storage. Data are presented as mean 6 SD (n 5 12). Significant differences (p < 0.05, two-way repeated-measures ANOVA) between groups on the same storage day are indicated: *85% Intersol-G (W) versus 85% SSP1 (䉲); †80% Intersol-G (䊉) versus 80% SSP1 (䉱).
residual plasma and optimal PLT recovery while avoiding any additional contamination by WBCs and RBCs in the final blood products. In this study, we established a process for the production of PCs with plasma concentrations as low as 10%, which is suitable for use in routine opera-
tions. A key step was adaption of the centrifugation settings to the different PAS/plasma ratios in the BC pools to achieve optimal PLT recovery.17 PASs have specific gravities similar to that of water, while the soluble proteins in plasma increase its specific gravity. Thus, alteration of the Volume 55, July 2015 TRANSFUSION 1705
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Fig. 3. In vitro quality of BC-derived PCs with 10% residual plasma. Metabolic, functional, and activation variables of PLTs derived from PCs resuspended in 90% Intersol-G over 7 days of storage. Data are presented as mean 6 SD (n 5 11). *Significant differences compared to Day 1: p < 0.05 (one-way repeated-measures ANOVA).
PAS/plasma ratio results in a change in the specific gravity of the storage medium, which influences the sedimentation behavior of the blood cells. The fact that the mean volumes of the Intersol-G PCs were consistently lower than those of the SSP1 PCs at all plasma concentrations studied despite the use of identical source materials and preparation procedures probably reflects the difference in specific gravity between the two PASs. As the MPVs were not different between Intersol-G and SSP1 PCs, the different sedimentation behaviors of PLTs cannot be explained 1706 TRANSFUSION Volume 55, July 2015
by swelling or shrinking of the PLTs due to a difference in PAS compositions. This finding demonstrates that the optimal production variables critically depend on the characteristics of the individual product components. SSP1 and Intersol-G, two of the latest-generation PASs, are designed to partially replace plasma in the preparation and storage of BC-derived or apheresis PCs. The recommended ratio is up to 80% PAS to 20% plasma. These solutions enable PLT storage at 22 6 2 C for up to 7 days after collection. We evaluated the ability of both PASs
PLASMA-REDUCED PLT UNITS
to maintain PLT quality after being used for the preparation of PCs with even lower plasma ratios. Intersol-G differs from SSP1 in that it additionally contains calcium and glucose, which have been proposed as key constituents for the maintenance of in vitro PLT quality in PCs with a low percentage of plasma carryover.12-14,22,27 This study of PLT quality revealed significant differences in the activation markers CD62P and PAC-1 between the two PASs. While plasma concentration had a consistent but differential effect on the two activation markers within each PAS-PC group, activation levels were generally higher in SSP1 PCs than in Intersol-G PCs. This suggests that the difference in activation is related to PAS type rather than to residual plasma concentration. It remains to be determined whether the presence of calcium, which is reportedly involved in several control functions and membranemodifying effects in PLTs,28,29 may contribute to the lower activation signals in Intersol-G PLTs. A previous study did not detect significant differences between calciumcontaining and calcium-free PAS using PCs with 20% plasma carryover.22,27 The difference in PAC-1 binding between the two PASs was not substantial. Moreover, the clinical significance of PLT surface expression of CD62P has been questioned and the data appear to be inconsistent.30-32 Therefore, in vivo studies are needed to show whether the reduced activation of PLTs stored in IntersolG will translate into any clinical benefits. The importance of glucose for in vitro storage of PLTs also remains to be adequately explained. While other substrates, such as acetate and fatty acids, also act as energy sources for PLTs,15,33,34 approximately 25% of the PLT energy requirement is supplied by glucose, which is considered crucial for maintaining the viability of PLTs in vitro and in vivo.32,35 Although glucose concentrations reached very low levels in SSP1 PCs with 15 and 20% plasma by the end of storage (Day 7), metabolic, aggregation, and activation markers still indicated that PLT quality was well preserved and comparable to that of Intersol-G PCs stored in an excess of glucose. It is still a matter of debate whether residual glucose is required and if acetate can substitute glucose to maintain PLT viability.32,35-38 Both PASs used in this study contain approximately 30 mmol/L acetate. Given that PLTs metabolize 2 mmol/L acetate per day of storage,39 this amount should easily be sufficient to maintain PLT viability for 7 days. In this study, PCs stored in 10% plasma plus 90% Intersol-G showed good PLT stability. Although SSP1 was not used for PCs with 10% plasma in this study, the results obtained for PCs with 15 and 20% residual plasma suggest that glucose-free lategeneration PASs can also be used for the preparation and storage of PCs with plasma levels below 20%. This study has some weaknesses. One is that the conclusions made based on the comparative analyses would have been stronger if all plasma concentration categories in both PAS groups had been incorporated in a single
pool-and-split design study. Another limitation is that only a small number of in vitro PLT quality variables were investigated. Additional in vitro storage variables such as pCO2, pO2, lactate dehydrogenase, hypotonic stress response, morphology, and ATP as well as in vivo studies are warranted for further investigation of poststorage PLT viability in PCs with minimal residual plasma. There is increasing evidence that the use of PASs may prevent acute transfusion reactions to apheresis PLTs and that plasma-reduced PAS apheresis PLTs may be financially and clinically superior to apheresis PLTs stored with 100% plasma.40-44 Until now, the preparation of plasmareduced PLTs was thought to be dependent on the apheresis technology. Our data demonstrate that it is feasible to prepare BC-derived plasma-reduced PCs with residual plasma concentrations far below 30%, the level commonly used under routine blood bank conditions. As plasma has clearly been identified as the major cause of transfusionrelated adverse reactions to PLT transfusions, minimizing the plasma concentration in PCs may help to further minimize or prevent these reactions.5 This study provides the technical perspective needed for the widespread use of PCs with minimal plasma concentrations, independently of the techniques used for their preparation.
ACKNOWLEDGMENTS The authors thank Elke Heide and Udo Grundmann (German Red Cross Blood Service NSTOB, Institute Springe, Springe, Germany) for their technical assistance. UG designed the study, performed the in vitro experiments, analyzed the data, and cowrote the manuscript; TV prepared the plasma-reduced PCs; KM analyzed and interpreted the data; RP analyzed the data; BL analyzed and interpreted the data; THM analyzed and interpreted the data and edited the manuscript; AS designed the study, interpreted the data, and cowrote the manuscript; and all authors read and approved the final manuscript.
CONFLICT OF INTEREST KM and RP are employees of Fresenius Kabi. The other authors have disclosed no conflicts of interest.
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