Hypotonic Shock Response Research Articles

Platelet storage in Fresenius/NPBI polyolefin and BTHC‐PVC bags: a direct comparison

New platelet storage systems, such as changes in the plastic of the storage bags, require validation. In this study, pooled buffy coat platelets stored in Fresenius/NPBI polyolefin bags were compared with those stored in Fresenius/NPBI butyryl-trihexyl citrate (BTHC) plasticized polyvinyl chloride (PVC). The CompoSelect thrombocyte polishing filter system (1000 mL polyolefin bag) and the CompoStop F730 system (1300 mL BTHC-PVC bag) were used to prepare paired, plasma-suspended, buffy coat platelet concentrates. Samples were taken up to day 7 for in vitro analysis. In a separate experiment, 12 units were prepared using the CompoStop F730 system and samples taken after leucofiltration for FXIIa assay. By day 7, platelet concentrates stored in BTHC-PVC demonstrated significantly higher pH levels (7.32 +/- 0.05 vs. 7.26 +/- 0.05) and a greater degree of cell lysis as shown by increased lactate dehydrogenase levels (497 +/- 107 vs. 392 +/- 81 U L(-1)). The supernatants contained higher concentrations of soluble P-selectin and the chemokine 'regulated on activation, normal T-cell expressed and presumably secreted', which are released from the alpha-granules during activation. The ATP concentrations were significantly lower in BTHC-PVC. Platelet counts, mean platelet volume and hypotonic shock response were similar for both bags. FXIIa antigen concentrations were 0.6 +/- 0.2 ng mL(-1) indicating that activation of the contact factor pathway had not occurred. Although the CompoStop F730 leucoreduction filter did not activate the contact system, platelets stored in 100% plasma in BTHC-PVC bags demonstrated different in vitro characteristics from those stored in polyolefin. Further work is required to demonstrate whether these differences will affect in vivo recovery and survival.

The effect of gamma irradiation on the quality of apheresis platelets during storage for 7 days

This study compares the quality of gamma-irradiated versus nonirradiated platelet (PLT) concentrates (PCs) during storage for 7 days as assessed by various in vitro methods. A new technique, free oscillation rheometry (FOR), which measures clotting time and coagulum elasticity, was also used to evaluate the PLT function. Single-donor PLTs were collected by apheresis technique (n = 20). The PLTs from each donor were divided into two PCs, one gamma-irradiated with 25 Gy and the other used as a nonirradiated control. Blood gases, metabolic variables, and swirling were analyzed from Day 0. Samples taken on Days 1, 5, and 7 were also analyzed for hypotonic shock response (HSR), P-selectin, and glycoprotein (GP)Ib expression by flow cytometry and coagulation by FOR. Swirling, HSR, and the percentage of GPIb-expressing cells were well maintained for 7 days of storage. pH was always within accepted range (6.4-7.4). Glucose decreased and lactate increased during the storage period (p < 0.05). P-selectin expression increased during storage (p < 0.05). The FOR clotting time remained constant, whereas the build-up of elasticity was slower after storage (p < 0.05). No difference was found between irradiated and nonirradiated PCs. The results indicate a well-preserved quality of gamma-irradiated apheresis PLTs during storage for 7 days as assessed by in vitro methods, with no difference compared to nonirradiated PLTs.

Effects of Mirasol PRT treatment on storage lesion development in plasma‐stored apheresis‐derived platelets compared to untreated and irradiated units

The aim of this study was to examine the effects of a new riboflavin-based pathogen reduction technology (PRT), the Mirasol PRT process (Navigant Biotechnologies) on platelet (PLT) storage lesion development. A three-arm in vitro study was conducted comparing cell quality of apheresis PLTs (n = 12 each) treated with Mirasol PRT (M) to untreated (C) and gamma-irradiated units (X) collected from the same donors and stored for up to 7 days under equal conditions. PLT count, lactate dehydrogenase, and K+ release of M units were not significantly different from C units, indicating retention of cell integrity during storage. The immediate effect (Day 1) of PRT treatment was a significant decrease in hypotonic shock response (M, 80.6 +/- 7.8% vs. 89.2 +/- 8.3%) and aggregation (M, 85.7 +/- 15.2%/min vs. 111.8 +/- 31.5%/min) as well as a significant acceleration of mitochondrial membrane depolarization (M, 1.43 +/- 0.44% vs. 0.91 +/- 0.27%) and P-selectin expression (M, 38.4 +/- 13.8% vs. 15.8 +/- 7.7%) resulting in lower swirl scores on Day 5 (1.5 +/- 0.7 vs. 2.7 +/- 0.4). Significantly higher glucose consumption (60 +/- 13 nmol/10(12) cells/hr vs. 31 +/- 9 nmol/10(12) cells/hr) and lactate production rates (82 +/- 17 nmol/10(12) cells/hr vs. 40 +/- 8 nmol/10(12) cells/hr) caused higher acidity in treated units (pH on Day 5, 6.97 +/- 0.15 vs. 7.42 +/- 0.10). After PRT treatment, oxidative metabolism was still active and, from calculation of oxygen consumption (1.09 +/- 0.23 nmol/min/10(9) PLTs), appeared to be up regulated relative to controls (0.76 +/- 0.27 nmol/min/10(9) PLTs). Although storage variables clearly showed the effects of PRT treatment, apheresis PLTs treated with Mirasol PRT retained cell quality during 5 days of storage without loss of mitochondria-based oxidative respiration.

Cold storage of pooled, buffy‐coat‐derived, leucoreduced platelets in plasma

This study was designed to determine which in vitro assays would be most useful for studying the effects of cold storage on platelet concentrates and to establish an in vivo model for platelet recovery and survival. Paired, plasma-suspended, leucoreduced, buffy-coat-derived platelet concentrates were stored either at 22 or 4 degrees C. Prior to storage and after 18 h, 5 days and 7 days, samples were taken and various assays were performed. On day 6, in vivo studies were carried out using a model system. Galactosylation of the platelets, prior to cold storage, was also tested. Hypotonic shock response, collagen-induced aggregation, RANTES and P-selectin binding site measurements demonstrated differences between platelets stored at 22 and 4 degrees C. The glycocalicin assay was able to demonstrate microvesicle formation at 4 degrees C. The in vivo model showed that there was at least a 50% decrease in recovery and survival when the platelets were stored in the cold. Galactosylation did not improve these results. Several assays, both in vitro and in vivo, were able to detect differences in platelet-storage characteristics and in vivo recovery and survival in a model system. Galactosylation did not correct these cold-induced changes.

Survival and recovery of apheresis platelets stored in a polyolefin container with high oxygen permeability

Oxygen permeability is important in platelet storage media. We compared a new polyolefin container with enhanced oxygen permeability (PO-80; Kawasumi, Tokyo, Japan) to a widely used alternative (PL2410; Baxter Healthcare, Deerfield, IL, USA). In vitro characteristics of paired platelet concentrates (PCs; mean 4.2 x 10(11)/250 ml plasma/bag) stored in PO-80 or PL2410 were assessed through 9 days of storage. In vivo recovery and survival of 7-day-old autologous PCs were assessed according to the Murphy method. Laboratory assessment of platelet quality favoured PO-80 during 9 days of storage with statistically significant differences in glucose consumption (2.75 vs. 4.93 mmol/10(12)/24 h in the interval 120-168 h), lactate generation (4.37 vs. 8.11 mmol/10(12)/24 h in the interval 120-168 h), pressure of oxygen (pO(2)) (59.3 vs. 38.1 mmHg at day 1), and HCO(3)(-) (14.7 vs. 13.4 mmol/l at day 1). Statistically significant differences were not seen in aggregation, hypotonic shock response or pH. In vivo assessment of autologous platelets stored 7 days in the PO-80 container revealed that recovery was 82.1% and survival was 81.0% of fresh control. Seven-day stored PCs in PO-80 were shown in vivo to be non-inferior to fresh platelets, with upper confidence limits (UCL(95)) in recovery and survival of stored PCs below the maximum acceptable difference (MAD); 15.3% UCL(95) < 20.4% MAD and 2.1 days UCL(95) < 2.1 days MAD. The in vitro characteristics of PCs stored in a highly oxygen-permeable container were stable at least 7 days. The in vivo study supports the suitability of PO-80 for 7-day platelet storage.

Buffy‐coat platelet variables and metabolism during storage in additive solutions or plasma

Buffy-coat processing allows for the use of platelet additive solutions (PASs). PASs reduce plasma-associated transfusion reactions and conserve plasma for transfusion or fractionation. Platelet (PLT) storage in plasma was compared to storage in three commercially available PASs compared to assess their influence on in vitro laboratory variables. Platelet concentrates (PCs) were prepared from leukoreduced pools of four buffy coats (BCPs) suspended in autologous plasma or one of PASs (Composol, Fresenius-Kabi; T-Sol, Baxter Corp.; or SSP+, MacoPharma). On Days 1, 2, 3, 5, and 7 of storage, samples were tested for PLT concentration, mean PLT volume (MPV), CD62P, morphology, pO2, pCO2, glucose, lactate and total protein concentration, pH, extent of shape change (ESC), and hypotonic shock response (HSR). Data were analyzed by analysis of variance (ANOVA) with repeated measures and t tests. PLT recoveries from BCPs were higher (p < 0.05) with plasma than any PAS. Storage medium and duration did not affect PLT concentration or MPV over time. CD62P expression and morphology were significantly different among PCs pooled with different media. ANOVA showed (p < 0.05) differences among the rates of change of pCO2, pH, glucose consumption, lactate production, and ESC; PASs such as Composol and SSP+ offered excellent maintenance of pH and low rates of glucose consumption. PAS performed poorly in ESC and HSR compared to plasma. Correlation studies reveal far more significant correlations between variables of PLTs in PAS than in plasma. Newer PASs, for example, SSP+ and Composol, can maintain PLT integrity and moderate metabolism similarly to plasma but offer consistently lower PLT recoveries and limited osmotic balance.

Comparison of a novel viscous platelet additive solution and plasma: preparation and in vitro storage parameters of buffy‐coat‐derived platelet concentrates

We developed a viscous platelet additive solution (PAS) based on MacoPharma's SSP+ but containing hydroxyethyl starch to address the poor osmotic balance and low yield associated with conventional PAS for the storage of buffy-coat platelet concentrates (PC). Pools of four buffy-coats were made into leucoreduced PCs (n = 5) suspended either in plasma or viscous PAS. After determination of platelet recoveries, the PCs were stored under standard conditions. On days 1, 2, 3, 5, 7 and 9, PCs were tested for mean platelet volume, platelet concentration, soluble protein concentration, CD62 expression, platelet morphology, partial pressure of oxygen and partial pressure of carbon dioxide, glucose and lactate concentration, pH, extent of shape change, and hypotonic shock response (HSR). Platelets were prepared with greater ease using the viscous PAS and had improved platelet yield. PCs stored in either plasma or viscous PAS displayed similar storage characteristics to day 9. On days 7 and 9 of storage, platelets stored in viscous PAS displayed significantly lower (P < 0.05) CD62 expression and higher HSR scores than those stored in plasma. Alteration of the viscosity of PAS improves platelet recovery during processing and may prolong platelet quality at the later stages of storage.

The quality of platelet concentrates produced by COBE Spectra and Trima Accel cell separators during storage for 7 days as assessed by in vitro methods

The quality of platelet (PLT) concentrates (PCs) can be evaluated with various in vitro methods. A new technique, free oscillation rheometry (FOR), can be used to monitor coagulation properties of PCs and gives information on clotting time and coagulum elasticity. This study compared the quality of apheresis PCs produced by the COBE Spectra and Trima Accel cell separators (both Gambro BCT) during storage for 7 days with in vitro tests including FOR. Apheresis PCs were collected with the COBE Spectra (n = 10) and Trima Accel (n = 10) cell separators. Swirling, blood gases, and metabolic variables were analyzed on Day 0. Samples taken on Days 1, 5, and 7 were also analyzed for hypotonic shock response (HSR), P-selectin, and glycoprotein (GP)Ib expression and evaluation of coagulation by FOR. Swirling, HSR, and percent GPIb-expressing PLTs were well maintained for 7 days, whereas glucose decreased and lactate increased significantly during storage for both Spectra and Trima PCs. Percent P-selectin-expressing cells increased to the same extent in both types of PCs during storage. pH increased between Day 0 and Day 1 but then decreased. The clotting time remained constant throughout the storage period whereas the development of elasticity was reduced on Days 5 and 7 compared to Day 1 (p < 0.05) for both types of PCs. The results indicate that the PLT quality after storage for 7 days is well preserved, although activation of PLTs occurs during storage as assessed by in vitro tests. No difference in PLT quality was observed between Spectra- and Trima-produced PCs.

In vitro function of buffy coat‐derived platelet concentrates stored for 9 days in CompoSol, PASII or 100% plasma in three different storage bags

The aim of the study was to compare the in vitro quality of buffy coat-derived platelet concentrates (PC) during extended storage in plasma or additive solution in three different storage bags. A pooled and split design was chosen so that identical PCs were produced in either 100% plasma, 70% PASII : 30% plasma or 70% CompoSol : 30% plasma (n = 6 each). This was repeated for three different manufacturers' platelet storage bags (Fresenius, Baxter and Pall). PCs were sampled on days 1, 5, 7 and 9 of storage and tested in vitro using a variety of tests of platelet function. For each bag type, storage in PASII or Composol was compared with plasma (data taken across the entire storage period), and differences occurring with time were analysed for all storage media. The pH of all PCs was > 6.8 at day 9 of storage. In vitro platelet function, as assessed by markers of platelet activation and metabolism, of PCs stored in CompoSol appeared to be similar to that of PCs stored in plasma over 9 days of storage. In contrast, PCs stored in PASII tended to have significantly higher levels of platelet activation (almost a twofold increase in % platelets positive for CD62P by day 5) and lower hypotonic shock response (approximately 40%, by day 7) compared to either PCs stored in 100% plasma or 70% CompoSol. The magnitude of the differences observed between platelet storage media appeared to be dependent on the type of platelet storage bag with the highest degree of platelet activation and lowest hypotonic shock response values being observed in Fresenius bags in combination with PASII. The maintenance of platelet function in vitro during extended storage of PCs in platelet additive solutions is dependent on the combination of type of additive solution and type of platelet storage bag. For all bag types studied, storage in PASII resulted in poorer platelet function in vitro.

In vitro and in vivo effects of potassium and magnesium on storage up to 7 days of apheresis platelet concentrates in platelet additive solution

Prolonged storage of platelets up to 7 days provides improved availability, logistical management and decreased wastage. Beside methods of bacterial detection, addition of magnesium and potassium to the platelet storage solution (SSP+) may further improve the quality of platelets with extended storage. Apheresis platelets from 10 donors were divided and stored in two different platelet additive solutions (PAS) (Intersol and SSP+) for a paired comparison. A variety of in vitro platelet function and metabolic assays were performed both on day 1 and after 7 days of storage. For in vivo study, platelets were labelled with either (111)Indium or (51)Chromium after 7 days of storage and were injected into the corresponding donor. Serial blood samples were drawn for recovery and survival measurements. In vitro parameters for SSP+ showed significantly reduced glycolysis (lower glucose consumption and decreased production of lactate), a higher hypotonic shock response (HSR) and the extent of shape change reactivity and a lower degree of platelet activation by means of RANTES (regulated on activation, normal, T cell-expressed, and secreted), CD62p and CD63 expression. Platelet recovery on day 7 was higher for Intersol as compared to SSP+, 65 +/- 11 vs. 53 +/- 13% (P = 0.023), and survival showed no difference 4.2 +/- 1.9 vs. 3.6 +/- 1.4 days. In vitro characteristics of platelets stored in PAS with addition of potassium and magnesium indicated higher quality, but this could not be verified by the in vivo parameters by means of recovery and survival.

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

To reduce adverse reactions due to platelet (PLT) transfusion, medical solutions on the market, such as saline and ACD-A, are used to replace the plasma of PLT concentrates in Japan; however, they are not strongly preservative. Here, an attempt was made to develop a novel additive solution (M-sol) having the ability to preserve PLTs stably, with only approved solutions for clinical use. M-sol is a mixture of solutions for medical use, which consists of 77 mmol per L NaCl, 3 mmol per L KCl, 1 mmol per L CaCl2, 21 mmol per L Na acetate, 15 mmol per L glucose, 9.4 mmol per L Na3 citrate, 4.8 mmol per L citric acid, 44 mmol per L NaHCO3, and 1.6 mmol per L MgSO4. The in vitro variables of PLTs stored in M-sol, Seto-sol, PASIIIM, or 100 percent plasma were compared during 14 days of storage. The in vitro parameters (pH, P-selectin, %hypotonic shock response, %disk, mean PLT volume, aggregability) of PLTs were better maintained in M-sol containing 3 percent plasma than in 100 percent plasma, PASIIIM with 31 percent plasma, and Seto-sol with 3 percent plasma during 14 days of storage. The 2-week storage of PLTs in M-sol is feasible in terms of the in vitro PLT function. Our results here show that the additive solution, with a high ability to preserve PLTs, can be prepared by mixing solutions approved for clinical use that are not specifically for PLT storage.

In vitro evaluation of prestorage pools consisting of mixed A and O platelet concentrates

Current FDA regulations allow the prestorage pooling of whole blood-derived platelet concentrates (PCs) of identical ABO type in a recently cleared platelet (PLT) pooling bag (Acrodose, Pall Medical). It is unclear how ABO-mixed PC pools would store if pooled before storage. Pools consisting of ABO-identical PLTs and mixed A and O PLTs in varying proportions were evaluated on Days 1, 5, and 7 of storage with measures of the PLT storage lesion, lymphocyte activation, and activation of the complement and coagulation system. Data were analyzed by analysis of variance. Pools did not differ on Day 7 in pH (p = 0.63), hypotonic shock response (p = 0.25), extent of shape change (p = 0.26), morphology score (p = 0.18), or white cell count (p = 0.79), but surface P-selectin expression was more evident in the ABO-mixed pools (p = 0.02). Small microscopic clumps of PLTs were observed in all pools, but were more prominent in the ABO-mixed pools (p < 0.01). PLT counts, however, did not differ between pools (p = 0.93), indicating that only a small proportion of PLTs were clumped. Surface A-antigen expression was proportional to the number of A PCs in each pool and did not vary between study days. Anti-A(1) titers were either unchanged or decreased by one dilution. Complement and coagulation activation markers did not differ between pools. Pooling A and O PCs is associated with evidence of increased microscopic PLT clumping and activation, but these differences are not exacerbated with 7-day storage. Other major measures of PLT quality do not differ, and there was no evidence of a mixed lymphocyte reaction.

Comparison of 7-Day Storage of Buffy-Coat Derived Platelet Concentrates in an Osmotically Balanced, Interface Stabilizing Platelet Storage Solution and in Plasma.

The use of platelet additive solutions (PAS) for the storage of buffy-coat derived platelet concentrates (PC) could conserve plasma resources and reduce post-transfusion reactions. However, PASs are difficult to handle in a production environment where their relative low viscosity produces an unstable interface between the platelet rich plasma and the red cell pellet. In the current study, a comparison is drawn between buffy-coat derived PCs (4 buffy-coats per pool) stored over 7 days either in plasma or in a novel Viscous PAS that contains 26 % plasma as a by-product of the buffy-coat pooling process. The increased viscosity (1.18 cp at 37°C) of the PAS results in a stable interface during processing. Similar, though more consistent platelet recoveries from buffy-coat pools (BCP) are achieved by the Viscous PAS (77.0 ± 2.6%) as compared to plasma (73.7 ± 5.9 %). These are significantly higher (p<0.05) than recoveries using conventional PASs (64.5 ± 2.1 %). On day 7, platelet concentration, mean platelet volume (MPV), and soluble protein concentration in PCs stored using Viscous PAS (782 ± 87 x 109 cells/L, 9.1 ± 0.5fL, 15.0 ± 1.6 mg/L, respectively) and plasma (840 ± 104 x 109 cells/L, 9.0 ± 0.7fL, 56.09 ± 9.2 mg/L, respectively) were not significantly different from their corresponding day 1 parameters (837 ± 84 x 109 cells/L, 9.1 ± 0.3 fL, 13.5 ± 0.2 mg/L) respectively for Viscous PAS and (830 ± 73 x 109 cells/L, 8.3 ± 0.5 fL, 56.6 ± 6.5 mg/mL) and for plasma. This indicates that Viscous PAS and plasma maintain platelet integrity to a similar degree during 7-day storage. Both storage media maintain pH around 7.20 until day 7. However, the rate of glucose consumption and lactate production of PCs stored in Viscous PAS (0.3365 mmol/L.day, and 0.7591 mmol/L.day) was roughly half that of the rate exhibited by PCs stored in plasma (0.8447 mmol/L.day, and 1.4258 mmol/L.day). As lactate production and glucose consumption are correlated with in vivo platelet recovery (Goodrich et al., 2006) this augurs well for platelets stored in Viscous PAS. Monitoring the extent of shape change (ESC) and morphology scores for PCs in either of the storage media showed no significant difference on day 7. However, CD62 surface expression and the hypotonic shock response (HSR) were significantly better at day 7 (p<0.05) for platelets stored in the Viscous PAS (37.43 ± 3.31% and 75.76 ± 10.95%, respectively) than stored in plasma (58.37 ± 11.71% and 49.79 ± 15.34%, respectively). We have developed a PAS that is easy to handle in a production environment and because of its osmotic balance and realtive viscosity exhibits equivalent or better storage parameters over a 7 day period than does plasma.

New methods showing in vitro changes of platelets

Introduction In vitro methods for assessing stored platelets are time consuming and variably correlate with in vivo survival. We used the ADVIA 120 and the Thromboelastogram (TEG) to assess changes during storage to determine a correlation of the results with those of other commonly used measures. Methods Platelet concentrates were tested at 1 and 5 days of storage. In vitro test included platelet count, aggregation response, the hypotonic shock response (HSR), The Kunicki morphology score (KMS) and platelet activation using CD62. The ADVIA ® was used to measure the platelet density and the granule content. The TEG measured the kinetics of clot formation. Results The platelet count and HRS did not change during storage. Aggregation decreased 50% by day 5. The KMS dropped from 307 at day 2–170. The MPC fell below 200 g/l. There was slight platelet activation; the rate of clot formation decreased. Conclusions Standard measures of in vitro platelet function indicated alterations during storage. A MPC of less than 200 corresponded to a Kunicki morphology score of less than 200. The TEG results correlated will with the KMS. These measurements may prove useful in predicting platelet survival and recovery.

YS05 In Vitro Evaluation of Platelet Concentrates Prepared Using the Automated Gambro OrbiSac System

Introduction The Gambro OrbiSac device is designed to automate the preparation of platelet concentrates (PC) from buffy coats (BC). The aim of this study was to evaluate the in vitro function of PC produced using the OrbiSac system and stored in plasma.Study design Ten PC were produced by automated pooling of four ABO matched BC and a single unit of plasma. The ring‐shaped pooling bag was centrifuged and the platelet rich supernatant automatically expressed, through a leucodepletion filter, into a Gambro ELP storage bag. All PC were stored at 22°C with agitation and sampled on days 1, 5, 7 and 9. Samples were evaluated for platelet count and standard markers of platelet quality. Parameter OrbiSac PC Specification (Manual PC) Volume (mL) 366 (327–396) 150–350 mL (Local) Platelet Content (×109 U−1) 297 (251–419) &gt;240 (UK) Median (range) n = 10. The assay results were compared to platelets produced by manual methods and stored in a Pall ELX bag in a previous study. Parameter OrbiSac PC Manual PC Day 1 Day 5 Day 7 Day 9 Day 1 Day 5 Day 7 Day 9 pH (22 °C) 7.23 7.45 7.38 7.29 7.13 7.36 7.32 7.24 pO2 (kPa) 9.1 7.1 7.2 8.6 6.3 5.4 5.8 6.2 pCO2 (kPa) 4.5 1.8 1.7 1.7 5.9 2.6 2.5 2.4 HSR (%) 61.8† 54.3† 55.6 58.1 76.3 71.4 64.5 70.6 % +ve CD62P 25.5 21.9 28.9 36.2 22.1 16.6 31.3 43.1 % +ve CD63 12.9 18.5 15.9 16.9 9.4 11.3 14.2 17.2 Median n = 10. † P &lt; 0.001 versus manual (T‐test). Results Hypotonic shock response (HSR) values were found to be significantly lower in this study when compared to manually produced PC. This was due to increased red cell contamination in these units. HSR was further investigated during a larger scale trial and all units (n = 25) showed improved day 1 values (78%, range: 61–93%). Values from these additional PC were comparable with those from manually produced PC.Conclusion PC produced with the OrbiSac system achieved the local specification for platelet count. However, these units were higher in volume than those produced by manual methods, with 70% of units exceeding the locally specified maximum volume of 350 mL. This problem could be addressed during later trials. OrbiSac PC have acceptable pH, blood gases, HSR levels and platelet activation markers and results are broadly comparable to data generated in a previous study of manually produced PC. These results suggest that platelets produced with the Gambro OrbiSac system are comparable to those produced using the current manual methods.

Characterization of blood components prepared from whole‐blood donations after a 24‐hour hold with the platelet‐rich plasma method

The preparation of platelet (PLT) concentrates (PCs) from PLT-rich plasma (PRP) requires that whole blood (WB) be processed within 8 hours of collection. Increasing WB storage time to 24 hours would be logistically attractive. This study compares the in vitro quality of blood components prepared from WB stored for 8 and 24 hours at room temperature before processing with the PRP method. WB units were collected from ABO-matched blood donors. To reduce individual variations, paired donations were drawn in parallel, pooled, and split back in the collection bag. One unit was held for 6 to 8 hours and the other for 22 to 24 hours at 20 to 24 degrees C. Prestorage leukoreduced components were prepared with the PRP as intermediate product and analyzed during storage. RBC units prepared after an 8- or 24-hour hold were comparable in terms of hemolysis, sodium, pH, and ATP levels. RBC 2,3- diphosphoglycerate (2,3-DPG) was significantly lower in RBCs prepared from 24-hour hold donations immediately after processing but not after 20 days of storage. Residual white blood cells were approximately fivefold higher (p < 0.05) in 24-hour RBC units. For PCs, measurements for glucose, ATP, lactate, pH, extent of shape change, hypotonic shock response, and CD62p activation were similar. No differences were observed in the von Willebrand factor, factor (F)V, FVIII, and fibrinogen content of fresh-frozen plasma. The decrease in FVIII and RBC 2,3-DPG can be acceptable as a compromise to improve blood component logistics, but leukoreduction efficiency must be improved before considering the adoption of an overnight storage of WB before PRP processing.

Paired in vitro and in vivo comparison of apheresis platelet concentrates stored in platelet additive solution for 1 versus 7 days

To improve clinical access to platelet concentrates (PCs), prolonging the storage period is one alternative, provided that they are free from bacteria. The quality of platelets (PLTs) stored for 1 versus 7 days was compared by in vitro analyses and in vivo recovery and survival in blood donors. Apheresis PCs from 10 donors were divided and stored in PLT additive solution in 2 equal units for a paired comparison. PLTs in one unit were (111)In-labeled at 1 day of storage, and PLTs in the other unit were labeled after 7 days of storage. PLTs were injected into the donor after labeling and samples were drawn after 30, 60, and 150 minutes and thereafter once a day for 14 days for recovery and survival measurements. PLT recovery on Day 7 was 80 percent of the recovery on Day 1 (p<0.05), and the survival on Day 7 was 65 percent of survival on Day 1 (p<0.005). No significant differences were seen regarding mean PLT volume (MPV), pH, pCO2, pO2, bicarbonate, or hypotonic shock response. Lactate increased and lactic dehydrogenase increased slightly, whereas glucose and ATP decreased, but not to a critical level. A significant increase in RANTES (110.7+/-76.6 vs. 277.6+/-50.8 pg/10(6) PLTs [p<0.005]) and PLT factor 4 (19.9+/-9.6 vs. 59.8+/-7.5 IU/10(6) PLTs [p<0.0001]) was noticed during storage. Recovery and survival of PCs stored for 7 days decreased, but met suggested criteria. Analyzed in vitro parameters showed acceptable results. Randomized patient transfusion studies will provide additional verification of the suitability of 7-day storage of PLTs.

Storage of buffy coat–derived platelets in additive solutions: in vitro effects of storage at 4°C

The aims of this in vitro study were to compare the storage of platelets (PLTs) at 4 degrees C with those stored at 22 degrees C and to determine the in vitro effects of preincubation at 37 degrees C for 1 hour before the analysis on the basis of the maintenance of PLT metabolic and cellular integrity. PLT concentrates (PCs) were prepared from pooled buffy coats (BCs) for paired studies (total eight pools from 160 BCs). Each pool was divided into four PCs and stored under different conditions: at 20 to 24 degrees C on a flatbed agitator, at 20 to 24 degrees C on a flatbed agitator and with incubation of the samples at 37 degrees C for 1 hour before the analysis, at 4 degrees C, and at 4 degrees C and with incubation of the samples at 37 degrees C for 1 hour before the analysis. Storage of PLTs at 4 degrees C resulted in reductions in the rate of glycolysis and better retention of pH after Day 10 than in PCs stored at 22 degrees C (Day 14, 7.003 +/- 0.047 vs. 7.201 +/- 0.146). Hypotonic shock response and extent of shape change were higher at 22 degrees C than at 4 degrees C and in preincubated PCs stored at 22 degrees C than in reference PCs stored at the same temperature (Day 5, 45.6 +/- 2.7 vs. 36.5 +/- 3.9 and 24.1 +/- 2.0 vs. 15.5 +/- 1.8). The concentration of RANTES was higher in PCs stored at 22 degrees C than at 4 degrees C (Day 7, 179 +/- 25 vs. 79 +/- 32). PLTs stored at 4 degrees C without agitation maintain metabolic and cellular characteristics to a great extent during 21 days of storage. These studies confirm the view that PLTs lose their discoid shape and that this loss with storage at 4 degrees C is associated with reductions in metabolic rate and in their release of alpha-granule content.

Storage of platelet‐rich plasma–derived platelet concentrate pools in plasma and additive solution

Prestorage pooling of platelet (PLT)-rich plasma (PRP)-derived PLT concentrates (PCs) and storage in either plasma (PS) or an additive solution (AS) is logistically feasible and would result in a product similar to buffy-coat or apheresis PLTs. On Day 0, PS PRP PCs were pooled with a sterile connecting device into a new 1.3-L storage container (ELX, PALL Medical). AS-PCs were prepared by addition of a new low-pH glucose-containing AS to the PLT sediment. AS-PCs were pooled into a 1.3-L ELX bag containing four tablets of NaHCO3. PC pools were sampled on Days 1, 5, and 7. PS pools containing 5 units had a mean PLT yield similar to the AS pools (39 x 10(10) +/- 3 x 10(10) vs. 37 x 10(10) +/- 6 x 10(10); p = 0.25). All pools had WBC counts of less than 1 x 10(6). pH and HCO3 decreased in PS pools with storage, but either increased or remained constant in the AS pools. On Day 7, no differences were seen in morphology score or extent of shape change. Hypotonic shock response was better preserved in the plasma pools (71 +/- 12% vs. 56 +/- 13%, p < 0.01); however, surface P-selectin was expressed less in the AS pools (6 +/- 4% vs. 18 +/- 10%, p < 0.01). Manufacture and storage of PRP-PCs in pools either in plasma or in a glucose-containing AS in this new container are feasible, and there is good preservation of PLT quality to Day 7.

Correlation of in vitro platelet quality measurements with in vivo platelet viability in human subjects

Changes in in vitro platelet quality parameters during platelet storage are associated with a decrease of in vivo platelet viability after platelet transfusion. Many attempts have been made to identify the most predictable in vitro parameters for in vivo performance. We used a riboflavin-based ultraviolet (UV) light treatment process designed to inactivate pathogens and white blood cell (WBC) contaminants in blood products as a model system in which to study the correlation of in vitro cell quality with in vivo viability. Platelet products (n = 18) were collected by a standard Trima apheresis procedure and treated with one of three dose levels of UV light (0, 7.2 or 12.4 J/ml) in the presence of 50 microm riboflavin. Lactate production, glucose consumption and P-selectin expression, pH, pCO(2), pO(2), hypotonic shock response and swirl were measured during 5 days of platelet storage post-UV/RB treatment. Aliquots of these products were radiolabelled on day 5 of storage and were subsequently used to determine platelet recovery and survival time in autologous subjects. The responses of in vitro cell quality were observed to occur in a UV dose-dependent manner. Lactate production and pH were identified as the parameters most strongly correlated with platelet in vivo recovery, which ranged from 5 to 82%. The correlation coefficients (r) for lactate production and pH with in vivo recovery in human subjects were 0.9090 and 0.8831 with P-values of 0.007 and 0.031, respectively. Lactate production and pH were also found to be correlated with platelet survival time, with correlation coefficients of 0.8063 and 0.8384 (the P values were 0.01 and 0.001, respectively). Using conditions of riboflavin-based UV light treatment, lactate production and pH were identified as having the highest correlations with recovery and survival of radiolabelled platelets in healthy subjects.