# Donor platelets stored for at least 3 days can elicit activation marker expression by the recipient's blood mononuclear cells: an in vitro study

Fabrice Cognasse, Hind Hamzeh-cognasse, Sandrine Lafarge, Sophie Acquart, Patricia Chavarin, Rémi Courbil, Patrick Fabrigli, Olivier Garraud

► To cite this version:

Fabrice Cognasse, Hind Hamzeh-cognasse, Sandrine Lafarge, Sophie Acquart, Patricia Chavarin, et al. Donor platelets stored for at least 3 days can elicit activation marker expression by the recipient's blood mononuclear cells: an in vitro study. Transfusion, 2008, 49 (1), pp.91 - 98. 10.1111/j.1537-2995.2008.01931.x. [https://doi.org/10.1111/j.1537-2995.2008.01931.x](https://doi.org/10.1111/j.1537-2995.2008.01931.x). [hal-04994601](https://hal.science/hal-04994601v1)

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# Donor platelets stored for at least 3 days can elicit activation marker expression by the recipient's blood mononuclear cells: an in vitro study

Fabrice Cognasse,* Hind Hamzeh-Cognasse,* Sandrine Lafarge, Sophie Acquart, Patricia Chavarin, Rémi Courbil, Patrick Fabrigli, and Olivier Garraud

**BACKGROUND:** Recent studies have demonstrated that infused platelets (PLTs) can promote inflammation. The objective of this study was to evaluate the impact of storage of transfusion-grade PLTs on the peripheral blood mononuclear cells (PBMNCs) of the recipient.

**STUDY DESIGN AND METHODS:** An in vitro cell model system was established to measure the degree of activation of donor PLTs during 5 days of their storage and then to measure immune cell activation by detecting marker expression in coculture experiments.

**RESULTS:** The level of soluble CD62p increased significantly by Day 3, and membrane expression of CD62p increased significantly from Day 2, indicating some degree of PLT activation over time during storage (p < 0.05). Donor PLTs and PBMNC subsets (monocytes, B cells, and T cells) from recipients were cocultured for 48 hours. The number of PLT-PBMNC subset doublets detected by flow cytometry was correlated with the PLT storage time after Day 3 (p < 0.05), indicating consistent binding of PLTs to PBMNCs. The results of these experiments showed that there was a consistent and significant increase in expression of conventional activation markers of T cells, B cells, and monocytes compared with appropriate controls (p < 0.05 to <0.01).

**CONCLUSION:** The results of this study indicate that, from Day 3 onward, activation markers are consistently expressed on PLTs. From these results, we conclude that activated PLTs may affect PBMNC interactions in recipients.

**P**latelets (PLTs) are highly reactive nonnucleated cells and are fundamental for hemostasis, which has long been considered as their only function. However, these nonnucleated cellular elements have recently been shown to participate in the inflammatory process by releasing various substances that interact with circulating white blood cells (WBCs) and vascular endothelial cells.¹⁻³ PLT transfusion causes secondary effects, the symptoms of which resemble those of inflammation and toxicity. These effects are presumably linked to PLT physiology but they may also result from the PLTs' manufacturing process and storage of PLTs for as long as 5 days, which is the maximum duration currently authorized in many transfusion systems, such as in France.⁴

We have recently presented evidence that stored PLTs contain immunomodulatory molecules, such as cytokines and chemokines, and secrete them differentially over time during storage before their transfusion.⁵ When a patient is transfused with PLTs, these molecules, as well as others, are also infused.⁵,⁶ Some of these mediators are thought to be associated with the relatively frequently observed immediate minor incidents, such as chills and fever.⁷ We

**ABBREVIATIONS:** BC = buffy coat; PMC(s) = polymor-
phonuclear cell(s).

From EFS Auvergne-Loire and Faculté de Médecine, GIMAP-
EA3064, Université de Saint-Etienne, Saint-Etienne, France.

*Address reprint requests to:* Professor O. Garraud,
GIMAP-EA 3064, Université de Saint-Etienne, Faculté de Méde-
cine, 15 rue Ambroise Paré, 42023 Saint-Etienne cedex 2,
France; e-mail: olivier.garraud@efs.sante.fr.

<footnote>*These authors contributed equally to this work.</footnote>

<footnote>The study was funded in part by EFS Auvergne-Loire.</footnote>

<footnote>Received for publication April 8, 2008; revision received
July 18, 2008; and accepted August 6, 2008.</footnote>

<footnote>doi: 10.1111/j.1537-2995.2008.01931.x</footnote>

<footnote>**TRANSFUSION** 2009;49:91-98.</footnote>
and others have previously demonstrated that PLTs that are stored for longer than 3 days start to release mediators⁵,⁶ that are known to be involved in inflammation and may potentially interact with both vascular endothelial cells⁸ and circulating WBCs⁹ of the recipient. We have observed that most PLT transfusions are delivered at least 3 days after preparation of the PLT concentrate. The current study was designed to investigate whether the use of 3- to 5-day-old PLTs had any biologic effects on the major subsets of peripheral blood mononuclear cells (PBMNCs), that is, T lymphocytes, B lymphocytes, and monocytes. The PBMNC subsets were chosen as the targets of the study because these cells, along with polymorphonuclear cells (PMNCs), are exposed (and potentially activated) after their immediate contact with transfused PLTs. In addition, PBMNCs can be rapidly activated and induced to differentiate after an encounter with a cognate antigen for either T cells or B cells. In the present investigation, we explored only the latter mechanism.

To study potential PLT-derived effects on circulating PBMNCs, we performed coculture of homologous PLTs and PBMNCs for 48 hours, which is the time required to increase the PBMNC marker of expression in vitro. We focused our investigation on the influence of 3-day-old PLTs on PBMNC maturation/activation as deduced from the expression of activation markers. Based on the experimental data, we conclude that—in vitro—there is an interaction between stored PLTs and PBMNCs.

# MATERIALS AND METHODS

## Preparation of peripheral blood cells and PLT concentrates for transfusion and in vitro assays

Whole blood was collected from healthy human donors who had given their written consent at the Auvergne-Loire Regional Blood Bank. The blood was collected into collection bags (Optipack, Reference DGR7542, Baxter, Maurepas, Belgium) for preparation of buffy coat (BC) samples, as described previously.⁵,¹⁰ After cell isolation, PBMNCs were then prepared from BCs, after removal of the PLTs, by gradient-density centrifugation using separation medium (Lymphoprep, Abcys, Paris, France), as described previously.¹¹

The production of PLT concentrates for transfusion is a highly standardized procedure and is carried out according to good manufacturing practice.⁴ It was performed in the current study, as previously described.⁵ The Baxter OptiPure PLT DGR7012 device (Baxter, Maurepas, Belgium) allows the in-line preparation of mixtures of PLT concentrates suitable for blood transfusion starting from BC samples. Packs of 470 mL of anticoagulant citrate-phosphate-dextrose-adenine-uncoagulated whole blood were stored at 20 ± 2°C for 16 to 18 hours and then centrifuged (800 × g, 20°C). The extraction of BCs was performed using an automated blood component extractor

(Optipress II, Baxter). The BC samples (approx. 53 mL) were stored for a minimum of 2 hours without agitation in a temperature-regulated glass chamber (20 ± 2°C). The packs were pooled aseptically by mixing five BC samples with storage solution (T-Sol, RDB7846, Baxter). After centrifugation, the PLT-rich supernatant was extracted using an automated blood component extractor (Optipress II, Baxter) and the WBCs were removed during this procedure. Clinical-grade PLT concentrates containing a minimum of 3.5 × 10¹¹ PLTs per transfusion unit were then resuspended in approximately 70 percent protein-free buffer T-Sol and approximately 30 percent autologous donor plasma. The residual red blood cell (RBC) count was fewer than 10⁶ per PLT pool; the PLT pool pH at the time of using for in vitro study (the same time of delivery to the patient) ranged from 6.4 to 7.4. The PLTs were counted using an automated blood cell counter (ABX-ARGOS, Abx Diagnostics, Montpellier, France), and the number of residual WBCs was counted by flow cytometry using a WBC counting kit (LeucoCOUNT, Becton Dickinson, Le Pont de Claix, France). In this study, the residual WBC count was 0.025 × 10⁶ ± 0.023 × 10⁶ per PLT pool that contained 4.1 ± 0.5 × 10¹¹ PLTs.

Pooled clinical-grade PLT concentrates were prepared for transfusion; they will be referred to herein as transfusion-grade PLTs. Samples of 10 mL were taken from each PLT concentrate without volume replacement on Days 0, 1, 2, 3, and 5 for analysis.⁵ PLTs (1- or 2-day-old) were considered as fresh.

In this study, transfusion-grade PLTs and also, for example, the PLTs to be tested in vitro consisted of mixtures of PLTs that were prepared from the blood of five different blood donors. This means that the residual WBCs that were mainly PMNCs (according to our unpublished data obtained for quality control purposes) originated from human leukocyte antigen (HLA)-mismatched donors. To compensate the effects possibly due to HLA mismatch, in a coculture of homologous PLTs and PBMNCs, controls induced dilutions of BC-originating WBCs (equivalent to 0.025 × 10⁶ ± 0.023 × 10⁶/PLT pool containing 4.1 × 10¹¹ ± 0.5 × 10¹¹ PLTs) to culture medium.

## Measurement of endotoxin levels in PLT concentrates

To ensure that bacterial contamination could not influence the observed in vitro biologic effects, PLT samples from each pool were tested for bacterial growth using conventional hemoculture techniques. In addition, endotoxin levels were measured using a limulus amebocyte lysate kit (QCL-1000, Cambrex Bio Science, Walkersville, MD) according to the manufacturer's instructions. Briefly, the stored plasma was heated at 70°C for 10 minutes and then diluted in pyrogen-free water (1:20). Samples were at first
<table><caption>TABLE 1. MoAbs used for studying cell surface markers</caption><thead><tr><th>Cell type</th><th>MoAb cell marker</th><th>Fluorochrome-conjugated</th><th>Clone</th></tr></thead><tbody><tr><td rowspan="3">B cell</td><td>CD19</td><td>Allophycocyanin</td><td>HIB19 (BD Biosciences)</td></tr><tr><td>CD69</td><td>PE</td><td>FN50 (BD Biosciences)</td></tr><tr><td>CD80</td><td>FITC</td><td>2D10.4 (eBioscience)</td></tr><tr><td rowspan="3">T cell</td><td>CD3</td><td>Allophycocyanin</td><td>UCHT1 (BD Biosciences)</td></tr><tr><td>CD69</td><td>PE</td><td>FN50 (BD Biosciences)</td></tr><tr><td>CD25</td><td>FITC</td><td>M-A251 (BD Biosciences)</td></tr><tr><td rowspan="3">Monocyte/macrophage</td><td>CD14</td><td>Allophycocyanin</td><td>MφP9 (BD Biosciences)</td></tr><tr><td>CD86</td><td>PE</td><td>IT2.2 (eBioscience)</td></tr><tr><td>CD80</td><td>FITC</td><td>2D10.4 (eBioscience)</td></tr></tbody></table>

incubated with limulus amebocyte lysate for 10 minutes at 37°C, to which the chromagen was then added and incubated for an additional 6 minutes before reading the absorbance at 405 nm. The endotoxin levels were expressed as International Units (IU) per mL.

## Soluble P-selectin detection in supernatants of PLT concentrates

Production of soluble PLT-activating factor, sCD62p, by PLTs in the concentrates over time (0 to 5 days of storage) was quantified by a commercial enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems Europe Ltd, Lille, France) according to the manufacturer's instructions. Absorbance at 450 nm was determined with an ELISA reader (Multiskan EX, Labsystem, Helsinki, Finland).

## Flow cytometry

PLT samples were incubated with an appropriate monoclonal antibody (MoAb) for 30 minutes at 4°C in the dark and washed twice in Tyrode's buffer (Sigma-Aldrich, Saint-Quentin Fallavier, France). PLTs were stained with a fluorescein isothiocyanate (FITC)-conjugated MoAb against CD41 (BD Biosciences, Le Pont de Claix, France) for gating, because CD41 is consistently expressed by activated, as well as nonactivated, PLTs.<sup>3,12,13</sup> To determine whether the PLTs were activated, they were also labeled with either an allophycocyanin or a phycoerythrin (PE)-conjugated MoAb against human membrane CD62p (BD Biosciences).

Combinations of MoAbs were chosen to generically label T cells, B cells, and monocytes/macrophages to assess the expression of specific markers of their activation (Table 1). PBMNCs were then prepared from BCs, after removal of the PLTs, by gradient-density centrifugation and cocultured for 48 hours with samples of 1- to 5-day-old stored PLT concentrates. For this purpose, we used a predetermined number of PLTs and PBMNCs (ratio, 20:1), as described previously by Steiner et al. for a study concerning the effect of abciximab on monocyte tissue factor expression in thrombin receptor activator-stimulated whole blood in vitro. To avoid fetal calf serum-

induced PLT activation as described by Hilf and coworkers,<sup>14</sup> 2 × 10<sup>5</sup> PBMNCs were cocultured in 2 mL of minimal medium, RPMI 1640 supplemented with L-glutamine (Abcys), for 2 more days with homologous PLTs (PBMNC: PLT ratio, 1:20) in a six-well culture plate (Corning Costar/Fisher Bioblock Scientific, Illkirch, France) at 37°C and 5 percent CO<sub>2</sub> without agitation.<sup>15</sup> PBMNCs were stained with appropriate markers for 45 minutes at 4°C. The PBMNCs cocultured with PLTs were then washed in phosphate-buffered saline containing 10 percent endotoxin-free fetal bovine serum (Abcys). Flow cytometry was performed to analyze membrane receptor expression on the following day using a flow cytometer with its accompanying software (FACSvantage SE and CellQuestS-Pro, BD Biosciences).

## Statistical analysis

The differences in the storage-dependent amounts of sCD62p between groups measured over the time course of the experiment were evaluated using one-way analysis of variance (ANOVA). A Dunnett's posttest was used to compare pairs of group means because one column represents control data, and we wish to compare all other columns to that control column but not to each other. Interexperiment comparisons for expression of a given activation marker at the different storage times were analyzed using the Wilcoxon paired samples test because they were not normally distributed<sup>16</sup> (Statistica-StatSoft, Maisons-Alfort, France). Many of the results were repeated measures with 10 different PLTs over storage time.

# RESULTS

## Transfusion-grade PLTs are activated and express the CD62p activation marker over time

PLT concentrates were prepared in the identical manner when prepared for their transfusion, with the exception that small volumes were sampled on Days 0, 1, 2, 3, and 5 for the various assays. The pH of the PLTs over storage are maintained within an acceptable range of pH (6.8-7.4) according to French standards (data not shown).
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Phenotypic expression of PLT surface markers and release of soluble CD62p during storage. (A) Production of soluble CD62p by PLT concentrates during 5 days of their storage. Day 5 is the last day of storage on which PLTs can be used for transfusion in France. Production of soluble CD62p was quantified by ELISA. Background levels have been subtracted. Data are expressed as the mean ± SD of 10 experiments and are expressed in ng per mL. (B) Flow cytometry representations of CD62p/CD41 surface labeling of PLTs during 5 days of their storage. The percentage of CD62p/CD41 represents activated PLTs. Transfusion-grade PLTs acquired low levels of activation markers over time. Data are expressed as the mean ± SD of 10 experiments. \*Significant difference (*p* < 0.05; ANOVA test).</figcaption></figure>

After preparing pooled PLT concentrates, we first measured the amount of sCD62p secreted by the PLTs during storage (Fig. 1A). The concentration of sCD62p varied significantly over time in the PLT concentrates (p = 0.027; ANOVA) and the concentrations from Day 3 onward were significantly greater than the baseline concentrations compared with baseline (Day 0; p = 0.034; ANOVA). Second, membrane CD62p was weakly expressed by fresh PLTs, and its expression increased linearly and significantly by Day 2 onward (p = 0.041; ANOVA) during the 5-day storage period (Fig. 1B). Transfusion-grade PLTs are thus activated and increase expression of the activation marker CD62p over time. This effect was not due to endotoxin because no endotoxin in pooled PLT concentrates was detected by the limulus amebocyte lysate assay (data not shown).

<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Storage increases the capacity of PLTs to bind non-stimulated MNCs. Binding of CD41 transfusion-grade PLTs to purified B cells (■), monocyte/macrophages (□), and T cells (■) during 5 days of storage. PLT surface expression of CD41 and various MNCs were assayed by flow cytometry. The percentages of double-labeled CD41+/CD19+, CD41+/CD14+, and CD41+/CD3+ cell doublets are displayed. Data are expressed as the mean ± SD of 10 experiments. \*Significant difference (ANOVA test).</figcaption></figure>

## Interactions of PLTs and monocytes/macrophages, B cells, and T cells: an in vitro study using experimental coculture assays

We observed that the degree of binding of PLTs to PBMNCs clearly paralleled the age of the PLTs. The results from flow cytometry showed that the amount of PLT-PBMNC doublets increased significantly (p = 0.024; ANOVA) with time (Fig. 2). For example, we noted that the number of PLT-B cell doublets increased from less than 1 percent using fresh and 1-day-old PLTs to 5.2 ± 2.3 percent with 3-day-old PLTs and attained a level of 14.1 ± 3.8 percent with 5-day-old PLTs (p = 0.004; ANOVA). We observed the same significant result concerning the number of PLT-monocyte/macrophage or T cells during PLT storage (Fig. 2). To ensure that this observation was not the result of a random adherence phenomenon, we verified that PBMNCs did not express membrane CD41 molecules and that the PLTs did not express CD19, CD3, or CD14 surface molecules. The cellular consequences of the cell-cell interactions detected were then analyzed in greater detail.

## Transfusion-grade PLTs can modulate the phenotypic characteristics of monocytes/macrophages and B and T cells in an in vitro model

To determine whether freshly isolated PBMNC subsets can express activation markers when cocultured with 1- to 5-day-old PLTs for 48 hours, we studied conventional activation marker expression for B cells, monocytes/macrophages, and T cells (Table 1) using flow cytometry.
Figure 3A displays cytometry plots of a representative experiment, showing no significant increase in the B cell, T cell, and monocyte activation markers no matter the absence or presence of 1-day-old PLTs (Fig. 3). In contrast, when exposed to 3- or 5-day-old PLTs, those tested immune cells displayed a significant increase of activation markers; activation markers were significantly higher in those sorted populations than in control PMNCs stored for 3 to 5 days, without PLT exposure (Wilcoxon paired test), indicating the role of PLTs in marker activation acquirement. A substantial number of CD19+ B cells became positive for CD69: 39.2 ± 2.9 percent (p = 0.007) and 38.1 ± 3.1 percent (p = 0.015; respectively for contact with 3- or 5-day-old PLTs); further, 19.7 ± 2.2 percent (p = 0.042) and 22.3 ± 3.5 percent (p = 0.046) of CD19+ B cells expressed CD86 after contact with 3- or 5-day-old PLTs, respectively (Figs. 3B and 3C). A total of 12.2 ± 2.5 percent (p = 0.005) of monocytes/macrophages expressed CD80 after contact with 3-day-old PLTs and this percentage was 12.9 ± 2.8 percent in the presence of 5-day-old PLTs (p = 0.039); 96.6 ± 8.95 percent (p = 0.048) and 92.6 ± 10.1 percent (p = 0.039) of monocytes became CD86+ after exposure to 3- or 5-day-old PLTs, respectively (Figs. 3D and 3E). A total of 9.2 ± 2 percent (p = 0.04) and 8.5 ± 2.1 percent (p = 0.039) of T cells expressed CD69 after exposure to 3- or 5-day-old PLTs, respectively, whereas 26.8 ± 4.1 percent (p = 0.04) and 28.2 ± 4.9 percent (p = 0.04) of T cells expressed CD25 under the same circumstances (Figs. 3F and 3G). It is noteworthy that none of the CD19, CD14, and CD3 markers are known to label PBMNC subsets other than the one for which it is characteristic. For example, all CD19+ cells should be B lymphocytes. Further, we observed no early changes in the expression of cell surface markers in the relevant PBMNC subset after its activation in vitro. Therefore, we assumed that, in vitro, the measured percentages reflected the actual level of activation of the predefined PBMNC subsets after 48 hours of exposure to 3-day-old transfusion-grade PLTs.

DISCUSSION

The systematic leukoreduction procedure that is applied to all types of labile blood products in France leads to a maximum number of residual WBCs of 10⁶ per product (on average this figure is 10-fold lower in our hands). The aim of this procedure is to minimize the symptoms of chills and fever reactions that are frequently observed during or after transfusion of RBCs or PLTs. The remaining discomfort observed after PLT transfusion is frequently an adverse reaction of the HLA system that is usually attributed to residual donor WBCs and patient's preformed antibodies in cases of multiple transfusions or more likely due to the presence of inflammatory cytokines and chemokines in the PLT concentrates.¹⁷ The aim of this

study was 1) to investigate the expression of PLT activating markers and PLT/PBMNC interactions resulting from the aging of PLTs during the authorized duration of storage and 2) to determine, in vitro, whether the storage time of PLTs is a factor in the activation of immune cells after a PLT infusion into a patient. The cells involved in this reaction are blood WBCs, which include the three most numerous PBMNC subsets, T cells, B cells, and monocytes. Another objective of this study was thus to investigate the effect of transfusion-grade PLTs on circulating immune cells (PBMNCs) of the recipient using an in vitro cell model system. This study focused on PLTs which were collected and prepared 3 days before the experiment for the following reasons: 1) storage of PLTs for 3 days is commonly used in current transfusion protocols in France and 2) we have previously demonstrated that this duration of storage permits some degree of PLT activation and, importantly, secretion of cytokines, chemokines, and related immunomodulators.⁵

The results of the current investigation suggest that a physical interaction occurs between PLTs and PBMNC subsets, as deduced from detection of doublets by flow cytometry. We have shown that physical interactions result in mutual activation of PLTs and B cells.¹⁸ Based on our study, storage of PLTs for varying durations has two consequences: 1) there is a strong and significant up regulation of PLT membrane CD62p expression and shedding during 3 days of storage and 2) PLTs stored for 3 days are capable of triggering the expression of early and late activation markers on PBMNC subsets, such as T and B cells and monocytes when cocultured. We supposed that PLTs stored for 3 days could possibly hasten the migration of activated and even matured/differentiated PBMNC subsets into tissues, lymph nodes, or spleen. Confirmation of this latest suggestion would require the use of experimental animals. PLT-derived microparticles support primary hemostasis by their ability to mediate PLT-PLT and PLT-subendothelium interactions; moreover, in a thrombocytopenic model, the relevance of PLT-derived microparticles in stored PLT concentrates for the hemostatic response was described.¹⁹ Microvesicles in stored PLT concentrates may also play a role in maintaining normal hemostasis by initiating and propagating blood coagulation.²⁰ A partial role of PLT-derived microparticles during storage cannot thus be ruled out in our model of PLT-induced MNCs activation. But indirect support for the latter hypothesis comes from clinical and experimental evidence that PLTs are important in attracting WBCs to sites of injury where they adhere to damaged and inflamed epithelial and endothelial surfaces.¹,²¹,²²

Recent studies have focused on the effect of immunomodulatory factors released by PLTs and their effect on posttransfusion complications.²³ Nevertheless, the direct impact of PLTs and/or the influence of PLT storage on PBMNCs is well understood. Our data⁵,²⁴ give information
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on direct and possibly indirect consequences of storage of PLTs for 3 days on PBMNC activation. The use of 1- or 2-day-old PLTs, which allows the minimum time for processing and testing for biologic qualification according to

good manufacturing practice,⁴ enables 1) a reduction in the amount of cytokines and chemokines released by the infused PLTs during storage; 2) reduced, in vitro, adhesion of infused PLTs to PBMNC subsets; and, possibly, 3)
<figure><img src="image_11.png" /></figure>

Fig. 3. Percentage of activation surface markers expression in the presence (■) or absence (□) of PLTs. (A) Flow cytometry plots gated on CD19+, CD14+, or CD3+ cells cocultured with or without PLTs collected on Day 3. It is representative of one of five independent experiments. (B-G) represent the mean values ± SD of five independent experiments on CD19+, CD14+, or CD3+ cells cocultured with or without PLTs collected on Days 1, 3, or 5. CD69 (B = B cell activation marker; F = T cell activation marker), CD86 (C = B cell activation marker), CD80 and CD86 (D and E = monocyte/macrophage activation marker), and CD25 (G = T cell activation marker) were measured with or without PLTs. Data shown were obtained using PLT concentrates (n = 5) collected on Day 3. PLTs were washed before their coculture with MNCs. Data are expressed as the mean ± SD of five experiments. *Significant difference (Wilcoxon paired test).

minimal, in vitro, activation of PBMNCs. Activated PBMNCs and dendritic cells that act as antigen-presenting cells are prone to rapid migration. Activated antigen-presenting cells are able to present PLT antigens, which results in alloimmunization of the recipient's immune system. Although the latter proposal is highly speculative, attention should be paid to this possibility in future research.

## ACKNOWLEDGMENTS

The authors acknowledge the technical help of Ms S. Danini and P. Damien and Mr Philip Lawrence for revising the manuscript. We thank Dr A. Ros and Professor B. Pozzetto (Department of Microbiology, Laboratory of Bacteriology-Virology, CHU de Saint-Etienne, Saint-Etienne, France) for performing the endotoxin measurements and advice on the manuscript and Dr H. Odent-Malaure for hemovigilance data. We also thank Dr Laurence Corash (Medical Affairs, Cerus Corp., Concord, CA) for critical reading of the manuscript.

## REFERENCES

1. Elzey BD, Tian J, Jensen RJ, Swanson AK, Lees JR, Lentz SR, Stein CS, Nieswandt B, Wang Y, Davidson BL, Ratliff TL. Platelet-mediated modulation of adaptive immunity. A communication link between innate and adaptive immune compartments. Immunity 2003;19:9-19.

2. Klinger MH. Platelets and inflammation. Anat Embryol (Berl) 1997;196:1-11.

3. Weyrich AS, Zimmerman GA. Platelets: signaling cells in the immune continuum. Trends Immunol 2004;25:489-95.

4. Council of Europe. Guide to the preparation, use and quality assurance of blood components. Strasbourg: Council of Europe; 2005.

5. Cognasse F, Boussoulade F, Chavarin P, Acquart S, Fabrigli P, Lamy B, Garraud O. Release of potential immunomodulatory factors during platelet storage. Transfusion 2006;46:1184-9.

6. Apelseth TO, Hervig TA, Wentzel-Larsen T, Bruserud O. Cytokine accumulation in photochemically treated and gamma-irradiated platelet concentrates during storage. Transfusion 2006;46:800-10.

7. Phipps RP, Kaufman J, Blumberg N. Platelet derived CD154

(CD40 ligand) and febrile responses to transfusion. Lancet 2001;357:2023-4.

8. Henn V, Slupsky JR, Grafe M, Gräfe M, Anagnostopoulos I, Förster R, Müller-Berghaus G, Kroczek RA. CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells. Nature 1998;391:591-4.

9. Khan SY, Kelher MR, Heal JM, Blumberg N, Boshkov LK, Phipps R, Gettings KF, McLaughlin NJ, Silliman CC. Soluble CD40 ligand accumulates in stored blood components, primes neutrophils through CD40, and is a potential cofactor in the development of transfusion-related acute lung injury. Blood 2006;108:2455-62.

10. Cognasse F, Hamzeh H, Chavarin P, Acquart S, Genin C, Garraud O. Evidence of toll like receptor molecules on human platelets. Immunol Cell Biol 2005;83:196-8.

11. Cognasse F, Chavarin P, Acquart S, Sabido O, Beniguel L, Genin C, Richard Y, Garraud O. Differential downstream effects of CD40 ligation mediated by membrane or soluble CD40L and agonistic Ab: a study on purified human B cells. Int J Immunopathol Pharmacol 2005;18:65-74.

12. Harrison P. Platelet function analysis. Blood Rev 2005;19:111-23.

13. George JN. Platelets. Lancet 2000;355:1531-9.

14. Hilf N, Singh-Jasuha J, Schwarzmaier P, Gouttefangeas C, Rammensee HG, Schild H. Human platelets express heat shock protein receptors and regulate dendritic cell maturation. Blood 2002;99:3676-82.

15. Steiner S, Seidinger D, Huber K, Kaun C, Minar E, Kopp CW. Effect of glycoprotein IIb/IIIa antagonist abciximab on monocyte-platelet aggregates and tissue factor expression. Arterioscler Thromb Vasc Biol 2003;23:1697-702.

16. Langford EJ, Wainwright RJ, Martin JF. Platelet activation in acute myocardial infarction and unstable angina is inhibited by nitric oxide donors. Arterioscler Thromb Vasc Biol 1996;16:51-5.

17. Heddle NM, Klama L, Singer J, Richards C, Fedak P, Walker I, Kelton JG. The role of the plasma from platelet concentrates in transfusion reactions. N Engl J Med 1994;331:625-8.

18. Cognasse F, Hamzeh-Cognasse H, Lafarge S, Chavarin P, Cogné M, Richard Y, Garraud O. Human platelets can activate peripheral blood B cells and increase production of immunoglobulins. Exp Hematol 2007;35:1376-87.
19. Jy W, Horstman LL, Arce M, Ahn YS. Clinical significance of platelet microparticles in autoimmune thrombocytopenias. J Lab Clin Med 1992;119:334-45.

20. Keuren JF, Magdeleyns EJ, Govers-Riemslag JW, Lindhout T, Curvers J. Effects of storage-induced platelet microparticles on the initiation and propagation phase of blood coagulation. Br J Haematol 2006;134:307-13.

21. Jurk K, Kehrel BE. Platelets: physiology and biochemistry. Semin Thromb Hemost 2005;31:381-92.

22. Elzey BD, Grant JF, Sinn HW, Nieswandt B, Waldschmidt TJ, Ratliff TL. Cooperation between platelet-derived CD154

and CD4+ T cells for enhanced germinal center formation. J Leukoc Biol 2005;78:80-4.

23. Silliman CC, Boshkov LK, Mehdizadehkashi Z, Elzi DJ, Dickey WO, Podlosky L, Clarke G, Ambruso DR. Transfusion-related acute lung injury: epidemiology and a prospective analysis of etiologic factors. Blood 2003;101: 454-62.

24. Cognasse F, Lafarge S, Chavarin P, Acquart S, Garraud O. Lipopolysaccharide induces sCD40L release through human platelets TLR4, but not TLR2 and TLR9. Intensive Care Med 2007;33:382-4.