Adverse effects of microparticles on transfusion of stored red blood cell concentrates

Abstract

Background: Systemic and pulmonary coagulopathy and inflammation are important characteristics of transfusion-related acute lung injury (TRALI). Whether microparticles that accumulate in transfused red blood cell concentrates (RBCs) have proinflammatory and procoagulant potential and contribute to adverse reactions of RBC transfusions is unclear.

Aim: To investigate the ability of microparticles in stored RBCs to promote thrombin generation and induce human pulmonary microvascular endothelial cell (HMVEC) activation and damage.

Methods: The number and size of microparticles were determined by flow cytometric and nanoparticle tracking analyses, respectively. Thrombin generation and the intrinsic coagulation pathway were assayed by a calibrated automated thrombogram and by measuring activated partial thromboplastin time (aPTT), respectively. The expression of ICAM-1 and the release of cytokines by endothelial cells were detected by flow cytometric analyses. HMVEC damage was assessed by incubating lipopolysaccharide-activated endothelial cells with MP-primed polymorphonuclear neutrophils (PMNs).

Results: The size of the microparticles in the RBC supernatant was approximately 100-300 nm. Microparticles promoted thrombin generation in a dose-dependent manner and the aPTT was shortened. Depleting microparticles from the supernatant of RBCs stored for 35 days by either filtration or centrifugation significantly decreased the promotion of thrombin generation. The expression of ICAM-1 on HMVECs was increased significantly by incubation with isolated microparticles. Furthermore, microparticles induced the release of interleukin-6 (IL-6) and interleukin-8 (IL-8) from HMVECs. Microparticles induced lipopolysaccharide-activated HMVEC damage by priming PMNs, but this effect was prevented by inhibiting the PMNs respiratory burst with apocynin.

Conclusion: Microparticles in stored RBCs promote thrombin generation, HMVEC activation and damage which may be involved in TRALI development.

Keywords: Microparticles; Procoagulant; Proinflammatory; Stored red blood cell concentrates; Transfusion-related acute lung injury.

Figure 1

Nanoparticle tracking analysis of microparticles (MPs) in supernatant obtained from red blood cells (RBCs) centrifuged at 1850 × g for 20 min. (A) Microparticles in supernatant of RBCs stored for 35 days. (B) Microparticles in supernatant of RBCs stored for 35 days after filtration with a 0.1 μm filter.

Figure 2

Flow cytometric analysis of microparticles (MPs) in red blood cell (RBC) supernatant. (A) RBC MPs, platelet MPs and leukocyte MPs in RBC supernatant were double-labeled with mouse anti-human CD235a, mouse anti-human CD61 and mouse anti-human CD45 respectively, combined with 10 µM of carboxyfluorescein succinimidyl ester (CFSE). The amount of MPs in pre- and post-filtration of RBC supernatant (0.1 μm filter) stored for 35 days were counted with a BD TruCOUNT tube by flow cytometric analysis (n = 3). (B) RBC MPs accumulated in RBCs during storage for 35 days were double-labeled with mouse anti-human CD235a combined with 10 µM of CFSE and counted by the same method (n = 5). * p-value <0.05.

Figure 3

Calibrated automated thrombogram (CAT) assay showing that serially diluted red blood cell (RBC) microparticles (MPs) promote thrombin generation in plasma (n = 5). RBC supernatant was centrifuged at 4 °C and 20,000 × g for one hour and washed with phosphate-buffered saline (PBS) containing 0.3% citric acid. They were prepared as a 10-fold concentrated suspension in PBS buffer before dilution. Thrombin generation was recorded by computer software. The data are expressed as nanomoles of thrombin generated per unit time (ETP, nM/min), lag time (min), peak of the thrombin concentration (nM) and time to peak (min). * p-value <0.05,**p-value<0.01,***p<0.001,****p<0.0001; compared with the group of 500 RMP counts/μl.

Figure 4

Effect of filtration or centrifugation on the generation of thrombin by microparticles from the supernatant of red blood cells stored for 35 days (*p-value <0.05,****p-value<0.0001; n = 5).

Figure 5

Effects of microparticles (MPs) on partial thromboplastin time/activated partial thromboplastin time (PT/aPTT). The PT and aPTT were assayed with an ACL7000 hemostasis testing system. Samples were prepared by serially diluting red blood cell (RBC) MPs with platelet poor plasma. (A) Result of aPTT and PT (n = 7). (B) aPTT analysis for the pre- and post-filtration (0.1 μm filter) supernatant from RBCs stored for 35 days (n = 7). .

Figure 6

Flow cytometric analysis of human pulmonary microvascular endothelial cells (HMVECs) during 24 h of incubation. (A) ICAM-1 expression on HMVECs (n = 3). After the incubation of endothelial cells with microparticles (MPs) for 2, 6, 12 or 24 h, the cells were detached and labeled with mouse anti-human CD54 before flow cytometric analysis. Interleukin-6 (IL-6) (B) and interleukin-8 (IL-8) (C) released from HMVECs (n = 3). After incubation of HMVECs with MPs for 2, 6, 12 or 24 h, the medium was collected, centrifuged at 12,000 × g for five minutes to remove dead cells or debris. The levels of IL-6 and IL-8 were simultaneously determined using a human cytokine cytometric bead array (CBA Kit, BD Pharmingen) according to the instructions provided by the manufacturer. * p-value <0.05,**p-value<0.01,***p-value<0.001,****p-value<0.0001; compared with the contol group at same time point.

Figure 7

Red blood cell (RBC) microparticles (MPs) induced lipopolysaccharide (LPS)-activated human pulmonary microvascular endothelial cell (HMVEC) damage. The cells were treated with 200 ng/mL LPS for six hours. Polymorphonuclear neutrophils (PMNs) were added to endothelial cells at an effector cell:target cell ratio of 10:1. After settling, the PMNs were exposed to MPs or buffer for 30 min. The numbers of viable cells were counted over a 1-mm surface area by fluorescence microscopy after staining with a LIVE/DEAD Cell Imaging Kit. The PMN respiratory burst was inhibited by incubating PMNs with 300–1200 μM apocynin for 15 min at 37 °C before adding them to HMVECs. (A) Effect of serially diluted RBC MPs on HMVEC damage (n = 5). (B) Effect of treating PMNs with apocynin on RBC MP-induced HMVEC (3 × 10⁴/μL) damage (n = 5). * p-value <0.05,**p-value<0.01,***p-value<0.001,****p-value<0.0001.