Increase of plasma concentration to 10% improves a number of in vitro storage parameters of apheresis platelets suspended in a bicarbonate-containing additive solution and stored with a 24-hour interruption of agitation

Abstract

Background

A previous study demonstrated several pH failures during 7-day storage of platelets suspended in 5% plasma/95% PAS-5 following a 24-hour interruption of agitation. The aim of this study was to investigate whether pH control improves in platelets stored in PAS-5 with 10% plasma following interruption of agitation.

Materials and methods

Four aliquots were prepared from a single unit of apheresis platelets: two each with 5% and 10% plasma. After resting for 1 hour, the aliquots were placed on an agitator. On day 2, agitation of one aliquot with 5% plasma and another with 10% plasma was interrupted for 24 hours before the aliquots were returned to agitator. The two control aliquots remained on the agitator. An array of platelet parameters was measured on days 2, 5 and 7.

Results

On day 7, aliquots containing 10% plasma and subjected to interruption of agitation had a significantly higher mean pH compared to those of similarly treated aliquots containing 5% plasma (6.80±0.54 vs 6.41±0.57, p≤0.05). Platelets containing 10% plasma/95% PAS-5 subjected to interruption of agitation had a greater hypotonic stress response, greater shape change, higher mitochondrial membrane potential, decreased glucose utilisation and lower CD62P levels compared to those of similarly treated platelets suspended in 5% plasma.

Discussion

Increasing plasma concentration to 10% improves pH control and some in vitro platelet properties during 7 days of storage of platelets suspended in PAS-5 after a 24-hour interruption of agitation compared to those of similarly treated platelets suspended in 5% plasma/95% PAS-5.

Introduction

The use of platelets suspended in additive solution has led to a reduction of the frequency and/or severity of a number of adverse reactions attributable to plasma^1–3. Platelets prepared in current generation additive solutions typically contain from 30 to 40% plasma⁴. Further reduction in plasma to levels substantially less than 20% can be achieved with recently developed platelet additive solutions (PAS) that contain bicarbonate and glucose, which are present to provide necessary buffering capacity and energy from glycolysis and reducing power via the pentose-phosphate pathway⁵. Two such bicarbonate- and glucose-containing PAS are M-Sol and PAS-5, which have been used to store platelets containing 5% or less plasma for 7 days or more at 20–24 °C with continuous agitation and with adequate maintenance of key in vitro storage properties^6,7.

With the consolidation of some blood centres, shipping of platelets has become more frequent and this blood component is transported over longer distances. Platelets are not, however, continuously agitated during shipment. Agitation is important for gas exchange and maintaining an optimal rate of glycolysis. With the interruption of agitation for periods of 24 or 30 hours, glycolysis is stimulated leading to increased lactate levels, bicarbonate neutralisation, and a decrease in pH⁸. Several studies have demonstrated small but acceptable decrements in platelet in vitro storage properties after a 24-hour interruption of agitation (IA) and storage for 5 to 7 days in units suspended in 100% plasma or 65% PAS/35% plasma^8–10. However, platelets stored in small amounts of plasma (95% PAS-5/5% plasma) have marked decrements in a number of platelet quality measures. For example, pH values on day 7 were ≤6.2 in seven of 12 units subjected to IA for 24 hours¹¹.

Plasma may provide stored platelets with some factors or constituents that protect them from extended periods without agitation beyond the constituents or concentration of constituents present in PAS-5. The aim of this study was to evaluate the impact of increasing plasma levels from 5 to 10% for platelets suspended in PAS-5 which were subjected to IA for 24 hours.

Materials and methods

Materials

PAS-5 constituents were purchased from Sigma Aldrich (St Louis, MO, USA) except for 8.4% sodium bicarbonate (Hospira, Inc. Lake Forest, IL, USA). PAS-5 solution was filter-sterilised (Corning Inc., Corning, NY, USA). Platelet aliquots were stored in CF-250 polyolefin containers (Charter Medical Ltd., Winston-Salem, NC, USA). ADP and collagen were obtained from Chrono-Log Corp. (Havertown, PA, USA). Human serum albumin was provided by the American Red Cross (Washington, DC, USA). CD61-FITC, CD62P-PE, CD42b-PE, mouse IgG₁-PE, mouse IgG₁-FITC, and annexin V-PE were purchased from Becton Dickinson (Immunocytometry Systems, San Jose, CA, USA). MitoProbe JC-1 kits containing dihydroethidium (DHE) were purchased from Molecular Probes (Life Technologies, Grand Island, NY, USA) along with 5-(and -6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H₂DCFDA).

Platelet collection and study design

A single unit of apheresis platelets was collected on an Amicus separator from consenting donors with a target yield of 4.2×10¹¹ with concurrent plasma. Following a resting period of 1–4 hours and 1 hour of agitation, approximately 60 mL aliquots were transferred into four CF-250 containers and PAS-5 was added to yield a 33% plasma suspension of ~180 mL. Aliquots were centrifuged at 3,800 rpm for 15 minutes (5.5×10⁷ ACE) and as much supernatant as possible was expressed. Plasma and PAS-5 were added to make four aliquots of ~59 mL; two each with 5 and 10% plasma. After a 1 hour period of rest, the aliquots were placed on an agitator. On day 2, one aliquot with 5% plasma and another with 10% plasma were placed in a stationary shipping container at 20–24 °C for 24±1 hours and then returned to the agitator while the two control aliquots remained on the agitator. Samples were taken from aliquots for an array of platelet assays on day 2 prior to the IA, and on days 5 and 7. The study was repeated 14 times.

In vitro platelet assays

Aliquots were sampled by syringe (~3.8 mL) using a CBS needle-free spike (OriGen Biomedical, Austin, TX, USA). Platelet counts were measured by a Sysmex XE 2100D haematology analyser (Sysmex Corp., Lincolnshire, IL, USA) and the total platelet content was determined by multiplying the platelet count by the volume of the aliquot. Percent plasma was determined using a Coomassie blue staining protein assay (Bio-Rad Laboratories Inc., Hercules, CA, USA)

Extracellular platelet pH was measured using a bench top meter (Orion; Thermo Scientific, Beverly, MA, USA) and pH electrode (Accu-pHast; Fisher Scientific, Pittsburgh, PA, USA) at room temperature. Glucose and lactate concentrations and partial pressures of carbon dioxide (CO₂) and oxygen were measured using a blood gas analyser (Cobas b221; Roche Diagnostics, Indianapolis, IN, USA). The analyser determined bicarbonate concentrations from pH and CO₂ partial pressures. Glucose utilisation, lactate production and bicarbonate neutralisation were calculated from values obtained on days 2 and 7.

Platelet morphology was measured by phase microscopy as the percentage of discoid platelets. Platelet aggregation was measured turbometrically (model 600 aggregometer, Chrono-log Corp.) using the simultaneous addition of ADP (10 μM) and collagen (10 μg/mL), as previously described⁹. The extent of shape change (ESC) and hypotonic stress response (HSR) were also measured turbometrically (Chrono-Log SPA 2000, Chrono-Log) as previously described¹².

Platelet p-selectin (CD62P, BD Biosciences) and GPIbα (CD42b, BD Biosciences) expression, phosphatidylserine exposure (annexin V binding, BD Biosciences), mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) generation were measured by flow cytometry as previously described¹¹. Freshly collected, unfixed platelet samples were diluted to 1×10⁶ platelets/mL in phosphate-buffered saline containing 0.1% human serum albumin and incubated with saturating concentrations of CD61-FITC and CD62P-PE (p-selectin) or CD42b or annexin V for 15, 20, or 15 minutes, respectively. Saturated concentrations of platelet isotype controls served as negative controls. Colour compensation between FITC (530 nm bandpass filter) and PE (585 bandpass filter) fluorescence and daily quality controls were facilitated with standard, three-color beads (Calibrite^TM 3; BD Biosciences). Platelets were gated by forward and side scattering and binding of CD61. A Mitoprobe JC-1 Assay Kit (Molecular Probes; Life Technologies Corp., ThermoFischer Scientific, Grand Island, NY, USA) was used to measure changes in MMP using 1 μmol/L JC-1. The fluorescence resulting from the oxidation of 5 μmol/L of one of the intracellular probes, DHE or CM-H2DCFDA (Molecular Probes, ThermoFisher Scientific), was utilised to measure ROS generation.

Statistics

The means, standard deviations, and analysis of variance with repeated measures (ANOVA) were calculated using standard software (Instat; GraphPad Software, San Diego, CA, USA). For ANOVA of the four experimental conditions, p-values <0.0009 were considered statistically significant based on the repeated measurements for 18 platelet assays and three testing days. For ANOVA of a platelet assay with a statistically significant result (p<0.0009), statistical differences between pair values of the four platelet aliquots reported in the results section were determined by Tukey-Kramer post hoc tests, with p-values <0.05 being considered statistically significant. Fisher’s exact test was used to analyse the number of units with pH values above and below 6.4 on day 7.

Results

The characteristics of the platelet aliquots are presented in Table I. Aliquots had comparable total platelet content, count and volume. Two aliquots had mean plasma contents of 5.5% and the remaining aliquots had mean contents of 10.7%.

Table I.

Characteristics of the platelet aliquots (means, n=14).

Parameter	5% plasma, CA	10% plasma, CA	5% plasma, IA	10% plasma, IA
Platelet content (×1010)	9.23±0.74	9.09±0.65	9.13±0.66	8.98±0.85
Platelet count (platelets ×103/μL)	1,561±86	1,540±79	1,551±87	1,522±124
Aliquot volume (mL)	59.0±2.5	59.0±2.6	59.0±2.3	59.0±2.7
Plasma content (%)	5.5±0.5	10.7 ±1.0	5.5±0.6	10.7±0.9

CA: continuous agitation; IA: 24-hour interruption of agitation.

Metabolic parameters for the aliquots are presented in Figure 1. Continuously agitated aliquots maintained a mean pH above 7.2 during the 7 days of storage. In contrast, units subjected to a 24-hour IA had significantly lower pH values on days 5 and 7 compared to those of the continuously agitated (CA) aliquots. On days 5 and 7, aliquots subjected to IA containing 10% plasma had significantly greater mean pH levels (p<0.05) than those of similarly treated aliquots containing 5% plasma, with mean pH levels of aliquots with 10% plasma and IA averaging 0.39 pH units higher than those with 5% plasma and IA. In addition, seven of 14 aliquots with 5% plasma subjected to IA had pH values ≤6.4 on day 7, compared to two of 14 similarly treated aliquots with 10% plasma. This difference did not reach statistical significance (p=0.1). On day 5, all units containing 5% plasma and subjected to IA had pH values ≥6.45; all units containing 10% plasma and subjected to IA had pH values ≥6.69. Rates of glucose utilisation, lactate production and bicarbonate neutralisation significantly increased with aliquots subjected to IA compared to those with CA (p<0.001). In addition, the rate of glucose utilisation in aliquots containing 10% plasma and subjected to IA was significantly less than those of similarly treated aliquots containing 5% plasma (p<0.05).

Figure 1.

Platelet metabolic properties.

Solid black bars: 5% plasma with continuous agitation; solid unfilled bars: 10% plasma with continuous agitation; dark grey bars: 5% plasma with interruption of agitation; light grey bars: 10% plasma with interruption of agitation. Experiments were repeated 14 times. Error bars denote standard deviation. Asterisks provide results for the Tukey-Kramer post hoc test; ***: p<0.001; ** p<0.01; *: p<0.05. No asterisks: p>0.05.

Figure 2 presents the functional properties of the platelet aliquots. On days 5 and 7, aliquots subjected to IA had lower HSR and ESC values than those of aliquots with similar levels of plasma and CA (p<0.05). Aliquots subjected to IA containing 10% plasma had significantly higher mean HSR (day 7) and ESC (Days 5 and 7) values than similarly treated aliquots containing 5% plasma (p<0.05). On days 5 and 7, aliquots stored with CA had significantly greater aggregation amplitude than those of aliquots containing similar plasma levels but subjected to IA (p<0.05). No differences in aggregation amplitude were observed between aliquots containing 5 or 10% plasma and subjected to IA (p>0.05).

Figure 2.

Platelet functional properties.

Solid black bars: 5% plasma with continuous agitation; solid unfilled bars: 10% plasma with continuous agitation; dark grey bars: 5% plasma with interruption of agitation; light grey bars: 10% plasma with interruption of agitation. Experiments were repeated 14 times. Error bars denote standard deviation. Asterisks provide results for the Tukey-Kramer post hoc test; ***: p<0.001; ** p<0.01; *: p<0.05. No asterisks: p>0.05. HSR: hypotonic stress response; ESC: extent of shape change; amp: amplitude.

Platelet structural properties are presented in Figure 3. On days 5 and 7, aliquots subjected to IA had lesser discoid morphology, lower CD42b values and greater CD62P and annexin V binding than those stored with CA (p<0.05). Aliquots containing 10% plasma and subjected to IA had significantly lower mean CD62P values on day 7 than those of similarly treated aliquots containing 5% plasma (p<0.05).

Figure 3.

Platelet structural properties.

Solid black bars: 5% plasma with continuous agitation; solid unfilled bars: 10% plasma with continuous agitation; dark grey bars: 5% plasma with interruption of agitation; light grey bars: 10% plasma with interruption of agitation. Experiments were repeated 14 times. Error bars denote standard deviation. Asterisks provide results for the Tukey-Kramer post hoc test; ***: p<0.001; ** p<0.01; *: p<0.05. No asterisks: p>0.05. Morph. (%disc.): morphology (% discoid); MFI: mean fluorescence intensity.

Figure 4 shows the results of assays measuring mitochondrial properties and the generation of ROS. Aliquots with 5% plasma subjected to IA had lower MMP and higher DHE and CM-H₂CDFDA values on days 5 and 7 than those of CA aliquots with 5% plasma (p<0.01). Aliquots with 10% plasma subjected to IA had lower MMP and higher DHE on day 7 than those of aliquots with 10% plasma and CA (p<0.05). On day 7, aliquots containing 10% plasma subjected to IA had significantly higher MMP and lower DHE values than those of similarly treated aliquots containing 5% plasma.

Figure 4.

Mitochondrial membrane potential and reactive oxygen species generation.

Solid black bars: 5% plasma with continuous agitation; solid unfilled bars: 10% plasma with continuous agitation; dark grey bars: 5% plasma with interruption of agitation; light grey bars: 10% plasma with interruption of agitation. Experiments were repeated 14 times. Error bars denote standard deviation. Asterisks provide results for the Tukey-Kramer post hoc test; ***: p<0.001; ** p<0.01; *: p<0.05. No asterisks: p>0.05. MMP: mitochondrial membrane potential; DHE: dihydroethidium; CM-H₂DCFDA: 5-(and -6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester.

Discussion

The use of additive solutions to store platelets with 30% to 40% residual plasma has been associated with a reduction in the frequency of allergic and febrile non-haemolytic transfusion reactions attributable to plasma and decreased anti-A and anti-B titres in group O platelets, which may be important in transfusion across ABO barriers^1,2,13. Efforts have been made to minimise levels of plasma in new generation additive solutions in the hope of further diminishing plasma-associated transfusion reactions. An early glucose- and bicarbonate-containing PAS sought to mimic several of the ionic constituents present in plasma, and platelet suspensions of this PAS with 7.1% plasma had superior maintenance of in vitro and in vivo platelet properties during 7 days of storage in a 5% CO₂ incubator compared to those of platelets suspended in 100% plasma and stored under standard conditions in air¹⁴. Subsequently, similar PAS formulations containing glucose and bicarbonate, such as M-sol and PAS-5, have been studied in platelets containing 2–5% levels of residual plasma with good maintenance of in vitro platelet properties for periods of at least 7 days under standard storage conditions^6,7. However, platelets suspended in 5% plasma/95% PAS-5 that were subjected to 24 hours IA, designed to mimic the maximum time allowed for platelet shipment, resulted in seven of 12 units having a pH <6.2 on day 7¹¹. With Amicus platelets suspended in 100% plasma and subjected to 24 hours IA during storage in standard containers, one of 12 units had a pH value <6.2 on day 7 compared to 0 of 12 that did not undergo IA⁸.

This study sought to evaluate whether a small increase in plasma content, an additional 5% plasma, ameliorated the poor storage characteristics of platelets suspended in 5% plasma/95% PAS-5 when subjected to a 24-hour IA. Like previously published results, platelets containing 5% plasma/95% PAS-5 that were subjected to 24 hours of IA had increased glycolysis and substantial declines in pH compared to controls under CA for 7 days of storage. Significant decrements were also noted in the functional (HSR), structural (morphology, ESC, CD42b), activation (CD62P, annexin V binding), mitochondrial (MMP) and ROS (DHE, CM-H₂CDFDA) properties of platelets suspended in 5% plasma/95% PAS-5 following IA in this study and in the previous study compared to those of CA controls. For aliquots stored with IA, suspension of platelets in 5% additional plasma (total plasma content 10.7%) led to significant improvements of pH, HSR, ESC, MMP and decreased glucose utilisation and CD62P levels compared to those of similarly treated platelets suspended in 5% plasma/95% PAS-5. Compared to a 24-hour IA of Amicus platelets stored in standard containers and suspended in 100% plasma⁸, the average day 7 decrement (the absolute difference between the mean values for IA and CA relative to the mean value obtained for CA) for Amicus platelet aliquots suspended in 90% PAS-5/10% plasma was 44.5% vs 29.7% (100% plasma) for ESC, 25.7% vs 22.0% (100% plasma) for HSR, 36.0% vs 12.5% (100% plasma) for CD62P, and 28.6% vs 20.6% (100% plasma) for percent discoid morphology. The current study was performed with apheresis platelet aliquots that underwent multiple manipulations, including an additional centrifugation, to prepare 5% and 10% platelet suspensions that were stored in containers suitable for the storage of whole blood-derived platelets. Additional studies would be needed to confirm the results of this study using entire apheresis units stored in standard containers.

A study with apheresis platelets washed in M-sol containing 2.35% residual plasma maintained pH above 6.95 and retained other key platelet storage properties during 7 days of storage following an IA for 24 hours¹⁵. These differing results compared to those of this and our previous study may be due to higher bicarbonate levels in M-sol. Alternatively, results may be dependent on the platelet count. This study and the previous PAS-5 study were performed with a relatively high platelet count (~1.5×10⁶/μL) for stringent testing of pH control during storage following IA.

Another formulation of PAS-5 containing 20 mM instead of 10 mM bicarbonate has been shown to maintain platelet pH levels between 7.6 and 7.8 during 14 days of storage¹⁶. No IA studies to mimic platelet shipment have been reported with this formulation.

Conclusions

The observation of improved storage characteristics of platelets subjected to IA for 24 hours if residual plasma levels are increased from 5 to 10%, representing a very small increase of 1 mM more bicarbonate and 1 mM more glucose, suggests that there is a component (or more than one) in plasma, not present or optimised in PAS-5, which protects platelets from activation, glucose utilisation leading to pH decline and decrements in some other storage parameters following periods without agitation. Whether that component is an ion or protein constituent is not currently known, but if identified, could lead to the development of a new PAS formulation that, when used in platelet suspensions with plasma levels <10%, would provide improved platelet storage characteristics compared to those of platelets suspended in PAS-5 and 5% plasma following a 24-hour IA.