Evaluation of platelet function during extended storage in additive solution, prepared in a new container that allows manual buffy-coat platelet pooling and leucoreduction in the same system

Abstract

Background

A novel and practical storage container designed for manual buffy-coat pooling and leucodepletion was evaluated to assess its filtration performance and to analyse the quality of stored leucoreduced buffy-coat-derived platelet pools.

Materials and methods.

To analyse the Grifols Leucored^® Transfer PL system, blood was collected from random donors into standard triple bag systems, and fractionated using standard procedures to obtain buffy-coats. Ten leucodepleted platelet pools were prepared each from five units of buffy-coats in additive solution. Concentrates were stored for 10 days at 22 °C on an end-over-end agitator. On days 0, 5, 7, and 10 of storage, samples were tested using standard in vitro platelet parameters.

Results

The use of this novel system for volume reduction and leucodepletion of buffy-coats resuspended in additive solution led to platelet pools that met the European requirements. pH was maintained well, declining from an initial value of 7.11±0.04 to 6.88±0.08 after 10 days. Parameters of cell lysis, response to a hypotonic stimulus and aggregation induced by agonists (arachidonic acid, ristocetin, collagen or thrombin receptor activating peptide) were also well-preserved. During storage, the quality profile of the platelet pools remained very similar to that previously reported in platelet concentrates in terms of metabolism, platelet activation (CD62, CD63, sCD62), expression of glycoproteins Ib and IIb/IIIa, capacity of glycoprotein IIb/IIIa to become activated upon ADP stimulation, and release of biological response modifiers (sCD40L and RANTES).

Discussion.

This new system allows the preparation of leucodepleted buffy-coat platelet pools in additive solution with good preservation of platelet function. The logistics of the procedure are relatively simple and it results in good-quality components, which may reduce costs and ease the process of buffy-coat pooling and leucocyte reduction in transfusion services.

Introduction

Platelets have been transfused for more than 50 years. Improvements in their collection, storage and testing for infectious diseases have encouraged more aggressive procedures during surgery, during chemotherapy, and for patients with particular bleeding disorders. Unfortunately, platelet storage is limited because of the risk of bacterial contamination¹ and decreased functionality over time, the latter often referred to as the platelet storage lesion^2,3. Despite significant advances, platelet production is still a costly process requiring a dedicated environment and the use of specially formulated plastic storage containers. The quality of platelet concentrates (PC) improved after the introduction of storage containers that are more oxygen permeable than the previous generation of containers⁴; these containers, in addition to bacterial testing and/or pathogen reduction methods have extended the shelf life of PC up to 7 days¹. PC can be obtained by apheresis or from whole blood by the platelet-rich plasma or the buffy-coat (BC) method². The predominant method in Europe is the BC one⁵, while apheresis is the method of choice in the United States⁶. Pooled PC can be produced from BC using either the pooling kit method or the chain method⁵ manually or in an automated process^7–9; platelets are then usually stored in additive solutions, thus reducing plasma-associated transfusion reactions¹⁰.

Adoption of universal leucoreduction, the use of platelet additive solutions (PAS) and, eventually, of pathogen reduction technologies are new introductions that together with the different preparation methods have led to a substantial variability of PC with regards to platelet content, residual leucocyte count, platelet preservation, and bacterial contamination.

Grifols’ Leucored^® Transfer PL system (Laboratorios Grifols S.A., Barcelona, Spain) is a new storage container for leucoreduced platelet units designed for manual BC pooling and leucocyte filtering in the same system. Through the Leucored^® Transfer PL, different BC fractions are pooled, leucoreduced and preserved without any contact with the atmosphere which guarantees maintenance of sterility. This study was performed to assess the compliance of PC produced using the Leucored^® Transfer PL with in vitro quality standards of stored platelets.

Material and methods

The Leucored^® Transfer PL container

The Leucored^® Transfer PL system is a closed system consisting of three connected plastic bags (two DEHP-free PVC transfer bags [TOTM-PVC and BTHC-PVC] and one bag for sample collection) and an ATS-BC filter (Pall Biomedical Inc., Fajardo, PR, USA) for leucoreduction of platelet pools from 4–6 BC units resuspended in either plasma or PAS. None of the parts of the process (mixture, centrifugation and filtration) compromises the closed system which preserves sterility. Details of the system, the process of transferring platelet suspensions through the bags and filtration are shown in Figure 1.

Figure 1.

The Leucored^® Transfer PL system. Four to six units containing BC from donors and a bag containing plasma or PAS are connected through a multi-tube connector sterile connection device (A) to the 600 mL PVC-TOTM transfer bag (B) where they are mixed and homogenised. After centrifugation and leucoreduction through an ATS-BC filter (C) the content is transferred to the 1,000 mL PVC-BHTC transfer/storage bag (D) where the leucoreduced platelet unit is obtained and stored. The system also incorporates a bag for sample collection (E) as well as clamps in every connector tube for opening and closing control at any moment.

Preparation of buffy-coats and platelet concentrates

Whole blood (450±50 mL) was collected from healthy volunteer donors meeting the European Guidelines¹ for blood donation, using a triple bag system containing 63 mL citrate-phosphate-dextrose as an anticoagulant in the collection container (T&B KS, Laboratorios Grifols S.A., Barcelona, Spain). According to the standard procedures of the Blood Donation Centre, immediately after collection whole blood units were placed on top of isopropanol plates at 22 °C and within 18 hours of collection the blood bags were submitted to hard spin centrifugation (4,527 × g, 15 min, 22 °C). The plasma and red blood cells were automatically derived to separate bags using a Compomat G4 device (Menarini Diagnostics, Barcelona, Spain), leaving the BC layers in the primary bag. Buffy-coat units were left undisturbed for 2 hours, and thereafter stored at 22 °C under gentle agitation until use.

For this study, ten BC pools using the Leucored^® Transfer PL system were prepared each from five units of BC and 300 mL of PAS III-M (69.3 mM NaCl, 5.0 mM KCl, 1.5 mM MgCl, 32.5 mM sodium acetate, 6.7 mM NaH₂PO₄, 21.5 mM Na₂HPO₄, and 10.8 mM sodium citrate) (Grifols), which were connected to the Leucored^® Transfer PL system using a sterile connecting device (TSCD^®-II, Terumo Europe, Leuven, Belgium). After homogeneous mixing, a 550–600 mL BC-PAS pool was obtained, with a haematocrit ranging from 18–25% and with platelet and leucocyte counts in the ranges of 500–800×10⁹/L and 10–20×10⁹/L, respectively, as assessed using a cell counter (Coulter Electronics, Izasa, Barcelona, Spain). The BC-PAS pool was centrifuged (1,718 × g, 3 min, 22 °C) using a Sorvall RC-3 Plus centrifuge (Thermo Fisher Scientific, Waltham, MA, USA) and the supernatant containing the platelet-rich fraction was then filtered in line with the Leucored^® Transfer PL filter to obtain the final leucodepleted PC, which was separated by means of a thermic tube sealer device (T-SEAL II, Terumo Europe, Leuven, Belgium). The platelet content in the PC was determined by electronic counting with a cell counter, while residual leucocytes were enumerated by flow cytometry using Leukocount^™ (Becton Dickinson, San Jose, CA, USA), according to the manufacturer’s instruction. Using these conditions, ten different leucoreduced PC from BC-PAS pools were prepared and stored in an incubator (Helmer Inc, Noblesville, IN, USA) under standard blood banking conditions (22 °C, and continuous agitation at 60 cycles/min) for up to 10 days. Throughout the storage period, PC samples were obtained on days 1 (baseline), 5, 7 and 10 for in vitro platelet quality assessments: metabolism, physical integrity, aggregation response to agonists, surface expression of membrane adhesive glycoproteins and activation markers, and quantification of pro-inflammatory markers.

Evaluation of platelet biochemical parameters

At the selected storage intervals, 5 mL of PC samples were aseptically obtained, centrifuged (1,000 × g, 10 min), and both the pellet and supernatant were recovered. The platelet pellet was gently resuspended in the appropriate volume of identical aliquots of AB fresh-frozen plasma (stored at −80°C and thawed at 37 °C just before use) in order to adjust the platelet count to 300×10⁹/L. These adjusted platelet suspensions were then used in the functional assays described below. The supernatant of the PC samples was centrifuged (1,500 × g, 10 min) and the sediment of cell fragments and impurities was rejected. The pH was measured at 22 °C with a pH-meter (M220, Corning Incorporated, Corning, NY, USA), and aliquots of the clean plasma supernatants were stored frozen at −80°C until use. Bicarbonate and lactate plasma levels were analysed in an ABL 825 Blood Gas Analyser (Radiometer, Copenhagen, Denmark). Glucose and lactate dehydrogenase (LDH) levels were measured in an Advia 1800 Chemistry Analyser (Siemens Healthcare Diagnostics, Tarrytown, NY, USA) by using glucose oxidase and pyruvate-lactate, respectively. A potentiometric method, based on selective membrane electrodes, was used for pH and pCO₂ (from which bicarbonate concentration was calculated) while an amperometric method, using a specific electrode, was used to measure lactate. All procedures were included in internal and external quality assurance programmes, and met generally accepted specifications for precision and bias.

Hypotonic shock response test

The preservation of the capacity of platelets to restore normal morphology after a hypotonic shock has long been considered a useful predictor of in vitro physical integrity and functionality, and of in vivo platelet viability of stored platelets^2,11. In order to test the hypotonic shock response, platelet samples (300×10⁹/L in AB plasma, as described above), were mixed with distilled water (1.5:1 volume ratio of PCs: water) in a spectrophotometer cuvette. The optical density (OD) at 610 nm was recorded immediately after addition of water (highest transmittance or lowest optical density) and following incubation of cells for 15 minutes, by means of a U-2000 spectrophotometer (Hitachi Ltd, Tokyo, Japan). An identical OD recording was made in parallel in platelet samples mixed with isotonic phosphate-buffered saline (PBS) instead of water. The percentage hypotonic shock response was determined by applying the formula:

$$\frac{[\left(\text{OD}\hspace{0.17em}\text{Post}\hspace{0.17em}15\text{min}\hspace{0.17em}\text{in}\hspace{0.17em}\text{water}-\text{lowest}\hspace{0.17em}\text{OD}\right)}{\left(\text{OD}\hspace{0.17em}\text{in}\hspace{0.17em}\text{PBS}-\text{lowest}\hspace{0.17em}\text{OD}\right)]\times 100}$$

Platelet aggregation assays

Platelet aggregation assays at the selected storage intervals were performed using the classical turbidimetric technique¹². Briefly, platelet samples resuspended in AB plasma (300×10⁹/L) were stimulated in aggregometer cuvettes with agonists and the aggregation response was monitored optically on an aggregometer (Aggrecorder II, Menarini Diagnostics, Florence, Italy) set at 37 °C and 1000 rpm. Results are reported as the maximum change in light transmission (%) for a total time of 5 minutes, using the initial platelet suspension as the baseline and platelet-poor plasma as 100%. The agonists and concentrations used were: 1.6 mM arachidonic acid (DiaMed, Cressier, Switzerland), 25 μM thrombin receptor activating peptide (TRAP) (Sigma-Chemical, Madrid, Spain), 1.25 mg/mL ristocetin (Sigma-Chemical) and 2 μg/mL collagen (Menarini Diagnostics).

Assessment of platelet surface glycoprotein expression by flow cytometry

Samples of PC were assessed for expression of major platelet membrane glycoproteins (GP) that support platelet adhesion and aggregation (GP Ibα, GP IIIa, and activated GPIIb/IIIa complex) and platelet activation markers (P-selectin, and granulophysin), as previously described, using a direct standard flow cytometry technique¹³ with appropriate, labelled monoclonal antibodies in a FACScalibur platform (Becton Dickinson, San Jose, CA, USA). The monoclonal antibodies (Becton Dickinson or Pharmingen) used were FITC*CD42b (GPIbα), FITC*CD61 (GPIIIa), FITC*PAC-1 (activation-specific anti-GP IIb/IIIa antibody), FITC*CD62P (P-selectin), and FITC*CD63 (granulophysin). For each sample run, data were acquired for 10,000 events gated on forward and side-angle light scatter with gains adjusted to include the platelet population. The fluorescence of stained platelets was analysed (CellQuest software, Becton Dickinson) to obtain both the percentage of positively stained cells and the mean fluorescence intensity (MFI).

Mediators of inflammation in supernatants

The concentrations of pro-inflammatory markers of platelet activation, sCD62P (soluble P-selectin), RANTES (regulated upon activation, normal T-cell expressed and secreted, also known as CCL5), and sCD40L (soluble CD40 ligand) were measured in supernatants of PC with the BD™ Cytometric Bead Array (Becton Dickinson) following the manufacturer instructions. The following kits were used for each marker: Human soluble P-Selectin (sCD62P) Flex Set, Human sCD154 (sCD40 ligand) Flex Set, and Human RANTES Flex Set. Measurements were carried out in a BD FaCScalibur™ (Becton Dickinson) flow cytometer. Concentrations of markers are expressed as pg/mL.

Statistical analysis

Results are expressed as mean and standard deviation (SD) of the mean, and minimum and maximum values when appropriate. Changes of platelet parameters through storage were analysed by means of a one-way ANOVA and Bonferroni’s correction test. Values of pre- and post- filtration/leucodepletion parameters were compared by a Student’s t test for paired data. A P value of ≤0.05 was considered statistically significant. Statistical analyses were performed using SPSS 17.0 software for Windows (SPSS Inc., Chicago, IL, USA).

Results

Platelet and leucocyte recovery

After processing the BC pools with the Leucored^® Transfer PL system, the platelet recovery in the leucodepleted PC with respect to the BC-PAS pools was 78.2±5.1% (818.0±126.7×10⁹/L final concentration of platelets) while leucocyte content was reduced to negligible levels (0.020±0.063×10⁶ cells/L). Detailed information on the volume and cell content of the BC-PAS pools before and after filtration is shown in Table I.

Table I.

Characteristics of the BC pools resuspended in PAS III-M additive solution and of the leucoreduced platelet concentrates obtained with the Leucored^® Transfer PL system.

	Buffy-coat pools Pre-filtration	Platelet concentrates Post-filtration
Total volume (mL)	580.6±8.6	368.2±9.4
Haematocrit (%)	21.7±1.3	0.1±0.1
Platelets
(x109/pool)	384±49	301±49
(x109/L)	662±87	818±127
Leucocytes
(x106/pool)	9,948±1,505	0.007±0.02
(x106/L)	17,140±2,647	0.02±0.06

Legend: Values are mean ± SD from ten different units.

Platelet count in the leucodepleted PC remained stable after 5 and 7 days of storage (804.4±135.8, and 805.2±121.7×10⁹ platelets/L, respectively), and fell slightly on day 10 (694.0±104.1×10⁹ platelets/L; P=0.049).

All platelet pools tested negative for bacterial contamination by microbiological cultures performed on day 10.

Platelet metabolism and cell integrity

The pH of leucodepleted PC on day 1 was 7.11±0.04; it rose slightly but significantly (P=0.024) on day 5 and progressively declined down to 6.88±0.08 on day 10 (P<0.001) (Table II). Nevertheless, throughout the entire storage period, the pH values were within the established range for platelet products for clinical use¹. Both glucose (149.9±5.7 mg/dL on day 1) and bicarbonate (7.2±0.5 mM on day 1) showed sharp time-dependent decreases during storage down to 10.6±14.4 mg/dL and 2.2±0.8 mM, respectively, on day 10, with the reduction being statistically significant since day 5 (P<0.001; Table II). By contrast, the lactate level increased steadily from 4.1±0.4 mM on day 1 to 17.2±1.4 mM on day 10, with this difference also being statistically significant since day 5 (P<0.001; Table II). As concerns cell integrity during storage of leucodepleted platelet pools, we observed that LDH concentration, a potential marker of cell lysis/damage, was very low on day 1 (151.2±12.2 U/L) and progressively rose to 345.6±87.1 U/L on day 5 (P<0.001), and to 446.2±187.4 U/L on day 10 (P<0.001) (Table II).

Table II.

Platelet metabolism (pH, glucose, bicarbonate and lactate levels) and integrity (LDH) parameters, and hypotonic shock response (HSR) during 10 days of storage of BC-derived leucodepleted PC obtained with the Leucored^® Transfer PL system.

Parameter	Day 1	Day 5	Day 7	Day 10
pH	7.11±0.04	7.19±0.08*	7.09±0.05	6.88±0.08*
Glucose mg/dL	149.9±5.7	95.8±12.1*	68.8±15.2*	10.6±14.4*
Bicarbonatae (mM)	7.2±0.5	5.1±0.9*	4.6±1.03*	2.2±0.8*
Lactate (mM)	4.1±0.4	8.9±1.0*	11.1±1.8*	17.2±1.4*
LDH (U/L)	151.2±12.2	345.6±87.2*	354.3±140.0*	446.2±187.4*
HSR (%)	85.2±5.5	88.9±8.2	86.3±10.7	63.0±11.6*

Legend: Values are mean ± standard deviation from ten different PC.

^*Denotes P≤0.05 with respect to the initial value at day 1.

Platelet functional status

Several indices of platelet function were evaluated during standard storage of leucodepleted PC prepared with the Leucored^® Transfer PL system. First, hypotonic shock response studies showed that the average percentage of platelets that preserved the capacity to recover shape after hypotonic stress was over 85% during 7 days of storage. At the end of storage, however, the percentage fell to 62.9±11.6% (Table II).

Mean maximum platelet aggregation with the four agonists tested ranged from 81.3% to 88.8% on day 1, and progressively decreased in the subsequent days of storage down to 35.2–64.5% on day 10 (Figure 2). As shown, the decline in the aggregation response was faster for arachidonic acid and for collagen than for the stronger agonists ristocetin and TRAP. It is worth noting that the maximal aggregation on day 7, the longest storage allowed for platelet products intended for clinical use, was still at around 70–85% with respect to the baseline values (Figure 2).

Figure 2.

Platelet aggregation response with several agonists (arachidonic acid, thrombin receptor activating peptide [TRAP], ristocetin and collagen) during 10 days of storage of leucodepleted PC obtained with the Leucored^® Transfer PL system. Values are maximal aggregation data presented as the mean ± standard deviation for ten experiments. *Denotes P≤0.05 with respect to the initial value (day 1).

The evolution of platelet membrane GP expression measured by flow cytometry is illustrated in Figure 3. The immunoreactive level of GPIbα showed a storage-promoted decrease that reached statistical significance from day 5 (P=0.021 vs. day 1). In contrast, the level of GPIIIa was relatively stable during storage. The percentage of platelets that expressed the epitope at the fibrinogen receptor of activated cells recognised by PAC-1, was low throughout storage (mean ranges: 2.0–3.6%). Stimulation of platelets with 25 μM ADP on day 1 induced a conformational change in 56.4±14.4% GPIIb/IIIa complexes as assessed by binding of PAC-1. However, the exposure of the ligand-binding site upon ADP activation decreased significantly from day 5, and at day 10 only 15.9±5.1% of activated platelets bound PAC-1 (P<0.0001). With expression of the activation markers CD62P and CD63, increased consistently during storage (14.5±5.8% CD62P and 7.7±3.6% CD63 positive platelets on day 1 vs. 55.8±6.9% CD62P and 27.0±4.4% CD63 positive platelets on day 10), showing statistical significance from day 5 (Figure 3).

Figure 3.

Platelet function evolution during 10 days of storage of leucodepleted BC-derived platelet pools obtained with the Leucored^® Transfer PL system. Mean fluorescence intensity (MFI) expression of membrane adhesive glycoproteins (GPIIIa and GPIbα), percentage of platelets expressing surface activation markers (CD62P and CD63) and percentage of platelets expressing the activated configuration of the GPIIb/IIIa complex (positive for PAC-1 antibody before and after stimulation with 25 μM ADP) are shown. Values are presented as mean ± standard deviation for ten experiments. *Denotes P≤0.05 with respect to the initial value (day 1).

Mediators of inflammation in supernatants

As shown in Figure 4, the concentration of sCD62P, RANTES and sCD40L in the BC-derived leucodepleted PC increased significantly during the first 5 days of storage (P<0.001), and remained relatively stable thereafter. The increment on day 5 was lower for sCD62P (2.3-fold vs. day 1) than for RANTES and sCD40L (7.3 and 7.9-fold vs. day 1, respectively).

Figure 4.

Evolution of the pro-inflammatory profile during 10 days of storage of leucodepleted BC-derived platelet pools with the Leucored^® Transfer PL system. Levels of sCD62P, RANTES and sCD40L are shown. Values are presented as mean ± standard deviation for ten experiments. *Denotes P≤0.05 with respect to the initial value (day 1).

Discussion

The preparation of PC from BC is a laborious procedure, facilitated by automated systems to obtain pooled and filtered units. However, these systems are expensive and their use may not, therefore, be feasible, especially in developing countries and other under-resourced nations. For this reason, new systems that ease the process and allow manual or semi-automated pooling need to be developed, and verification of adequate preservation of platelet quality during storage has to be undertaken.

This study shows that PC obtained after volume reduction and leucodepletion of BC resuspended in Grifols PAS III-M by using a Leucored^® Transfer PL system meet the quality standards established for these products by the European Guidelines¹ (>2.5×10¹¹ platelets and <106 leucocytes per unit, in more than 90% of pools). Under our processing conditions platelet recovery from BC pools and the final volumes of the PC were similar to those obtained in our centre by routine processing with the marketed automated OrbiSac system (Caridian Bct, Lakewood, CO, USA) (data not shown), and also comparable to those reported by others^4,14. Additionally, the PC obtained with this novel system were equivalent to those obtained with the automated procedure with regards to the in vitro platelet characteristics during storage for 7 days^8,9,14; the sterility of PC prepared with the Leucored^® Transfer PL system was preserved during the study.

The extent of the platelet storage lesion is usually assessed by evaluating in vitro changes in platelet parameters and/or release of bioactive compounds in PC^2,15. For example, during storage at 22 °C, the metabolism of platelets changes and the increase in the glycolytic pathway leads to accumulation of lactate, a decrease in glucose levels, and to a fall in pH, which is deleterious for platelet viability and functionality^16–20. In this study, the pH of platelet pools obtained with the Leucored^® Transfer PL system was maintained around 6.4–7.4 during storage at 22 °C for 10 days, according with the usual behaviour of platelet products stored under standard blood banking conditions^2,20,21, and complying with the quality standards required for PC¹. With regard to the other tested metabolic parameters, glucose and bicarbonate levels showed marked decreases during storage over 7 days, as expected in platelet pools with 30% of plasma carry-over. In fact, the decrease of these parameters, and the increase of lactate levels during storage were similar to those reported in other studies of BC-derived platelets in additive solutions^21–25. The use of the Leucored^® Transfer PL system was associated with low baseline LDH values, a general marker of cell integrity, reflecting the lack of severe cell damage by the leucodepletion system. The increase in LDH during storage was similar to that described in previous studies measuring this parameter in platelet products^2,21,22.

The response of platelets to hypotonic shock has been extensively used to assess in vitro the preservation of platelets in relation to their likely in vivo recoveries after infusion¹¹. In our study, the hypotonic shock test in pooled platelets stored after Leucored^® Transfer PL system filtration was above 60% after storage for 10 days (75% vs. fresh platelets), a value that likely predicts high recovery or survival of these platelets after infusion, and is comparable to that found in other recent studies of similar platelet products²⁶.

Light transmission aggregometry is still the “gold standard” for assessing platelet function and dysfunction and it has long been known that platelets stored as concentrates in blood banks progressively lose the aggregation response to weak agonists and show impaired response to strong agonists². In this study, the aggregation response of BC-derived platelets prepared with the Leucored^® Transfer PL system to four agonists remained at 75% of baseline values after 7 days of storage, which can be considered an appropriate preservation level, similar to that found by others in similar PC²⁶. Moreover, we evaluated the aggregation response of stored platelets upon resuspension in allogeneic fresh plasma, a situation mimicking the infusion of the platelet product into the bloodstream of a potential recipient²⁷.

Since the haemostatic effectiveness of platelets is related to the ability of the membrane GPIb/IX and GPIIb/IIIa complexes to interact with their respective physiological ligands (mainly von Willebrand factor and thrombin for GPIb/IX and fibrinogen for GPIIb/IIIa), an evaluation of the immunoreactive levels of these receptors in in vitro stored platelets is of great significance²⁸. In our study, GPIb and GPIIIa were found to remain relatively stable for up to 7 days of storage after Leucored^® Transfer PL system filtration, and, for GPIIIa, even up to 10 days. The detrimental storage effect on the capacity of GPIIb/IIIa to become activated after stimulation with ADP, evaluated with the PAC antibody, was somewhat expected since deterioration of responsiveness to this weak agonist in PAS has been described already²⁹.

Processing and storage-promoted activation triggers a release reaction in platelets, and enhanced expression of CD62P and CD63 has long been known to be associated with the platelet storage lesion^2,15,30, although the significance of this increased expression on the transfusion efficacy of platelets is still unknown. During storage of PC, proteolytic cleavage of CD62P releases sCD62P, the soluble form of P-selectin, which has been speculated to be a risk factor for venous thromboembolism^31,32. As expected, in our study we found that storage promoted an increase of these platelet activation markers in platelet pools prepared with the Leucored^® Transfer PL system, similar to those previously found in leucodepleted PC prepared either manually or with marketed semi-automatic systems^2,33,34.

Recent studies have demonstrated that biological response modifiers released from platelets during storage may have a clinical significance in the platelet transfusion reaction^35,36. We, and others, have previously shown that pre-storage leucocyte depletion can prevent the release of leucocyte-derived bioactive compounds but do not prevent the accumulation of other platelet-derived bioactive substances or the potential activation of plasma components^37,38. One such compound is RANTES, a proinflammatory C-C chemokine released upon platelet activation which is thought to be associated with inflammatory reactions as well as allergic reactions^39,40. Platelets are also the richest source of sCD40L, which seems to be involved in inflammation and thrombosis⁴¹. Upon transfusion of PC, the sCD40L accumulated during storage can favour both the occurrence of transfusion-related acute lung injury (TRALI) by promoting activation of polymorphonuclear leucocytes and endothelial damage⁴², and fever due to induction of cyclooxygenase-2 expression⁴³.

As the method of obtaining PC can influence the secretion of pro-inflammatory products during storage³⁴, new systems of platelet preparation for transfusion purposes should be evaluated for their impact on the accumulation of bioactive compounds. In this study we found that the concentration of RANTES and sCD40L in the leucodepleted PC prepared and stored with the Leucored^® Transfer PL system increased during the first 5 days of standard storage and remained relatively stable thereafter. However, the levels of these bioactive compounds in PC obtained with the Leucored^® Transfer PL are in the range or below those reported in other platelet products derived from whole blood fractionation or obtained by apheresis^39–50. In our centre, we have recently found slight, but significantly higher levels of sCD62P, RANTES, or sCD40L, in BC-derived PC prepared with OrbiSac and TACSI systems than those found in the platelet PC obtained with the Leucored^® Transfer PL system (data not shown).

In summary, this study shows that the Leucored^® Transfer PL system is a new option for the preparation of leucodepleted BC platelet pools in PAS III-M, which may simplify blood component preparation. Platelet concentrates prepared in this study met the European Council¹ and AABB⁵¹ requirements. Additionally, the use of the Leucored^® Transfer PL system had little impact on the in vitro quality of platelets, with integrity, functionally of platelets, and accumulation of bioactive compounds similar to those of other platelet products currently in clinical use.