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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of Interest
  8. References

Background

Pathogen reduction technologies (PRTs) can induce platelet (PLT) lesions that reduce PLT survival and recovery from circulation and may be associated with acute lung injury (ALI).

Study Design and Methods

Human PLTs (hPLTs) in plasma with or without single or multiple Mirasol PRT treatments were assessed in vitro by aggregation and percentage of P-selectin expression. In vivo studies included PLT recovery in SCID mice and assessment of ALI in a two-event mouse model in which the sensitizing event was lipopolysaccharide injection and the second event was infusion of Mirasol-treated hPLTs.

Results

A single-dose Mirasol treatment (5 J/cm2) did not induce any change in aggregation in response to adenosine 5′-diphosphate (ADP) while a five-times-repeat Mirasol treatment (5×) increased aggregation response to low concentration of ADP. Mirasol PLTs (1×-5×) had increased percentage of P-selectin–positive PLTs after treatment and decreased aggregation with TRAP as the agonist. In vivo recovery in SCID mice was reduced extensively with Mirasol treatments (1× and 5×). In the two-event model of ALI, only the 5× Mirasol PLTs accumulated in the lung and this was not accompanied by changes in lung histology or increases in MIP-2 levels in bronchoalveolar lavage fluid.

Conclusions

Mirasol PRT treatment induced PLT activation and reduced in vivo recovery in a SCID mouse model of transfusion. In our two-event mouse model of ALI, the 5× Mirasol hPLTs accumulated in the lung, but did not cause signs of ALI. The 1× Mirasol treatment did not lead to PLT lung accumulation or ALI in this model.

Abbreviations
ALI

acute lung injury;

BALF

bronchoalveolar lavage fluid;

hPLT(s)

human platelet(s)

LPS

lipopolysaccharide

PRT(s)

pathogen reduction technology(-ies)

Approximately two million platelet (PLT) unit equivalents are transfused per year in the United States alone.[1] Despite advances in bacterial detection methods and devices, identification of contaminated PLT units is only partially effective and a risk of bacterial sepsis from PLT transfusion remains. Approximately 30 to 100 cases of sepsis are associated with PLT transfusions annually in the United States,[2, 3] and one to five deaths from transfusion of bacterially contaminated PLTs have been reported annually to the FDA in recent years.[4] PLT transfusion products can also contain new and emerging pathogens that may not be detected with the standard testing approach which has prompted a proposal of an alternate paradigm for minimizing the risk of transfusion-transmitted disease with the use of pathogen reduction technologies (PRTs).[5]

Current PRTs are based on ultraviolet (UV) light plus chemical or UV light alone processes that target nucleic acids to inactivate a broad spectrum of pathogens in blood transfusion products. In the past two decades, three major systems using PRTs for PLTs have been developed, namely Intercept system from Cerus Corp,[6] Mirasol system[7] from Terumo BCT, and THERAFLEX UV system (UVC) from MacoPharma.[8]

Despite the potential benefits of PRTs, clinical trial data of PRT PLTs have raised safety concerns in pulmonary adverse events. It is known that PRT processes can damage PLTs as is evident from their reduced in vivo recovery and survival in circulation of healthy volunteers[9, 10] and reduced corrected count increments in patients.[7, 9] In the SPRINT trial on PRT PLTs (UVA-amotosalen HCl treatment of apheresis PLTs), there was a significant increase in the frequency of acute respiratory distress syndrome cases reported in the PRT PLT arm (5/318, 1.6%) versus the control PLT arm (0/327, 0%). A retrospective reanalysis of the pulmonary data in selected patients with clinically serious pulmonary adverse events identified a total of 12 cases (12/78) of acute respiratory distress syndrome in the PRT PLT arm and five cases (5/70) in the control arm,[11] although this difference did not reach significance. In a similar clinical trial, the Mirasol Clinical Evaluation (MIRACLE) of Mirasol PLTs, the PRT PLTs also showed higher number of adverse events per patient in the respiratory, thoracic, and mediastinal category.[7] We recently reported that in a two-event SCID mouse model of transfusion where the primary event was lipopolysaccharide (LPS) administration, PLTs directly illuminated with UVB light (at 2.4 J/cm2) accumulated in lungs and caused acute lung injury (ALI). This model adds mechanistic plausibility to an ALI mediated by transfusion of UV-exposed PLTs.[12-14] However, due to the limitations of the clinical trials and of the transfusion model the issue of whether PRT PLTs can contribute to respiratory adverse events in transfused patients remains unresolved.[15]

Among the commercially available PRT systems, the Mirasol PRT system (Terumo BCT, Lakewood, CO) uses UV light (270–360 nm) that is mostly in the UVB spectrum, at 5 J/cm2, to activate the photosensitizer riboflavin. The Mirasol system is currently approved in European countries for pathogen reduction of PLTs in plasma. In this study, we evaluated in vitro properties of Mirasol PRT PLTs, as well as their in vivo properties in the SCID mouse model, with an emphasis on modeling potential pulmonary adverse events.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of Interest
  8. References

Antibodies and other reagents

Monoclonal antibodies (MoAbs) and reagents used for immunostaining include anti-human CD41 (HIP8; ABBIOTEC, San Diego, CA), anti-CD68 (Clone FA-11, BD Abcam, Cambridge, MA), Alexa Fluor 568–conjugated, goat anti-mouse IgG1 (Invitrogen, Carlsbad, CA), and Hoechst 33342 (Invitrogen). All MoAbs used for flow cytometry were purchased from BD Biosciences (San Diego, CA) and these include anti-human CD41–fluorescein isothiocyanate (FITC; Clone HIP8), anti-human CD62P–phycoerythrin (PE; Clone AK-4), as well as matched isotype controls.

Animals

SCID mice, 6 to 8 weeks old, were obtained from NCI/DCT (Bethesda, MD). LYS-eGFP mice (on C57BL/6 background) were the gifts from Dr D. Sacks (NIAID, Bethesda, MD)[16, 17] upon approval from Dr T. Graf. SCID mice were crossed with LYS-eGFP mice to obtain double homozygous mice (SCID/SCID; LYS-eGFPki/ki), hereafter referred to as SCID/LYS-eGFP mice. Genotypes of LYS-eGFP mice and SCID mice were determined as published.[16, 18]

All mice were kept in a pathogen-free facility and animal protocols were in compliance with guidelines provided by the Center for Biologics Evaluation and Research Animal Research Advisory Committee.

PLT products

Leukoreduced hPLTs were collected by apheresis[12, 14] at the National Institutes of Health Division of Transfusion Medicine, under full institutional review board approval. PLTs were allowed to rest for at least 2 hours on a PLT agitator (Helmer, Noblesville, IN) to reduce the mechanical activation induced by the collection protocol.

PRT treatment of hPLTs

Mirasol PRT treatment was performed 2 to 20 hours after collection. PLTs from the same apheresis unit were used for both control and Mirasol arms of the study. Briefly, 55 mL of apheresis hPLTs in plasma (approx. 1 × 106/μL) were transferred to Mirasol illumination and storage bags designed for research purposes only, with the same plastic as used in the commercial PRT system (Terumo BCT). Riboflavin was added to a final concentration of 50 μM, and the products were illuminated in the Mirasol system (Terumo BCT) at 6.2 J/mL (approx. 5 J/cm2), following the manufacturer's instructions. The mean illumination time was approximately 3 to 4 minutes. To study the safety margin for the treatment, we repeated Mirasol illuminations (2× to 5× per unit) on some units. To do this, we overrode the safety features of the Mirasol system by manually changing the product name. UVB irradiation of hPLTs was performed as previously described.[12] The total UVB dose per sample was calculated to be 2.4 J/cm2.

Cell counts

Complete blood counts were performed on a clinical hematology analyzer (Cell Dyn 3700, Abbott Diagnostics, Santa Clara, CA).

Transfusion of hPLTs and mouse whole blood collection

hPLTs (approx. 1 × 109 PLTs in 100 μL phosphate-buffered saline [PBS] or PLT resuspension buffer) were infused into mice via tail veins as previously described.[12, 19] Mouse whole blood was collected via tail vein bleeds. SCID mice were injected intraperitoneally with 3 mg/kg LPS (diluted in 100 μL PBS) from Escherichia coli 0111:B4 (Sigma, St Louis, MO) or an equal volume of PBS 2 hours before injection of PLTs.

Antibody-induced ALI

An established mouse model of antibody-mediated ALI[20, 21] that mimics human transfusion-related acute lung injury (TRALI) was also examined as a positive control in this study. ALI was induced in male SCID mice by an intraperitoneal injection of LPS (0.1 mg/g) 24 hours before intravenous infusion of the anti-major histocompatibility complex Class I (anti-MHC) 1 MoAb, (H2Kd; IgG2a, κ) at 2 mg/kg. Two hours later the animal was euthanized followed by bronchoalveolar lavage or lung tissue sample collection.

Bronchoalveolar lavage fluid collection and assays to measure MIP-2 and total protein concentrations

Mice were euthanized with CO2 inhalation 2 hours after PLT injection, and the bronchoalveolar lavage fluid (BALF) was collected as previously described.[14] The supernatants of BALF were analyzed for MIP-2 concentrations, using a mouse CXCL2/MIP-2 enzyme-linked immunosorbent assay kit (DuoSet, R&D Systems, Minneapolis, MN). Total protein concentration in BALF was measured by bicinchoninic acid assay kit (QuantiPro, Sigma).

Lung histology and immunofluorescent staining

Paraffin and cryosections of the mouse lungs were prepared as previously described.[14] Lung paraffin sections (5 μm) were stained with hematoxylin and eosin and imaged with a microscope (Nikon Eclipse E800, Nikon Co., Ltd, Tokyo, Japan). Lung cryosections (10 μm) were postfixed with methanol, blocked in 3% goat serum (Sigma) with 1% bovine serum albumin in PBS/1%Triton ×100, followed by incubation with monoclonal mouse anti-human CD41 (1:100) or anti-CD68 (1:200) antibodies overnight in 3% goat serum/PBS/1% Triton ×100. An Alexa Fluor 568–conjugated, goat anti-mouse IgG1 (1:500) was used as the secondary antibody. All sections were stained with Hoechst 33342 (Vector Laboratories, Burlingame, CA), mounted in medium (VECTASHIELD, Vector Laboratories), and photographed using a confocal microscope (Zeiss LSM710, Carl Zeiss, Inc., Jena, Germany). Three serial lung sections from at least four mice of each treatment group were used for analysis. We have confirmed that the anti-hCD41 we used to stain hPLTs in the lung are specific to hPLTs and do not cross-react with mPLTs.[14]

PLT aggregation studies

PLT aggregation in PRP was measured on an aggregometer (PAP8E, Bio/Data Corp., Horsham, PA), according to the manufacturer's instructions. TRAP (Sigma) and adenosine 5′-diphosphate (ADP; Bio/Data Corp.) were used as representatives of strong and weak agonists, respectively. The maximum aggregation was monitored for the duration of 10 minutes.

Flow cytometric analysis

Mouse whole blood or apheresis hPLTs were incubated with the antibodies for 20 minutes at room temperature in the dark, and cells were washed once (5 min at 1000 × g, with 1 μg/mL prostaglandin E1) and resuspended in 500 mL of PBS containing 0.3% bovine serum albumin. Flow cytometry was performed on a flow cytometer equipped with its accompanying software (FACSCalibur and CellQuestPro, respectively, Becton Dickinson, San Jose, CA). PLT recovery was defined as the percentage of anti-hCD41-FITC–positive events in 5 μL of mouse whole blood. Control hPLT recovery 30 minutes after transfusion was designated as 100% recovery for all subsequent time points. Flow cytometric data were analyzed using computer software (FlowJo, Version 7.6.3, http://www.flowjo.com/download/).

Image analysis

Quantitative measure for the fluorescence signal (number of pixels in the selected channel) was obtained using computer software (Adobe Photoshop CS3, Adobe Systems, Inc., San Jose, CA), as previously described.[12] Three serial lung sections per mouse from at least four mice of each treatment group were used for analysis. Mean and standard deviation (SD) were calculated for each treatment group.

Confocal imaging of the unfixed lungs

The lungs were imaged under a confocal microscope (LSM710, Carl Zeiss, Inc.), as previously described.[14]

Statistical analysis

A t test was performed for statistical analysis with computer software (Windows Excel, Microsoft Corp., Redmond, WA) program with significance set at p values of less than 0.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of Interest
  8. References

Mirasol treatment does not induce spontaneous PLT aggregation

Apheresis hPLTs (0.9 × 1012–1.5 × 1012/L) in plasma with 50 μmol/L riboflavin were illuminated in the Mirasol illuminator system. The level of spontaneous PLT aggregation and activation was evaluated on PLT smear slides stained with anti-hCD41. After the Mirasol treatment, PLTs showed normal morphology and no aggregates (Fig. 1F), similar to untreated PLTs (Fig. 1A) or PLTs treated with only riboflavin (Fig. 1E). Multiple sequential Mirasol illumination treatments (2×-5×) of the same PLTs caused PLT aggregation in a dose-dependent manner. The 3× Mirasol treatments caused small PLT aggregates (<10 PLTs/aggregate), with approximately 1 aggregate per field of view (Zeiss 40x/NA 0.45 Plan Fluor objective; Fig. 1G). The 5× Mirasol treatment caused larger PLT aggregates (≥10 PLTs), with two to three aggregates per field (Fig. 1H). We previously reported that UVB illumination can cause significant PLT aggregation,[12-14] and this was confirmed in this study (Fig. 1B). Adding the photosensitizer riboflavin (50 μmol/L), the same concentration as that used in the Mirasol system, did not protect the PLTs from UVB-induced aggregation (Fig. 1C). Interestingly, UVB illumination of PLTs contained in the Mirasol illumination and storage bag, at 2.4 J/cm2 (the same dosage that caused significant PLT aggregation in Fig. 1B), caused only minor aggregation (Fig. 1D).

figure

Figure 1. Mirasol PRT treatment does not cause spontaneous PLT aggregation in vitro. Five microliters of PLTs from different treatments was smeared onto a glass slide and stained with anti-hCD41. (A) Untreated PLTs; (B) PLTs after UVB illumination; (C) PLTs with 50 μmol/L riboflavin that underwent UVB illumination; (D) PLTs in Mirasol illumination and storage bag after UVB illumination; (E) untreated PLTs with 50 μmol/L riboflavin; (F) PLTs after Mirasol treatment; (G) PLTs after 3× Mirasol treatments; (H) PLTs after 5× Mirasol treatments. UVB illumination, 2.4 J/cm2; Mirasol treatment, 5 J/cm2, with 50 μmol/L riboflavin. The composite images shown were representative of four independent experiments. All images were taken using a laser scanning confocal microscope (Zeiss 710), with a 40×/NA1.0 Plan-Apochromat water objective.

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Mirasol PRT-treated PLTs show higher percentage of P-selectin expression

P-selectin expression was 23.5 ± 4.8% in untreated PLTs and 23.4 ± 4.5% in PLTs treated with 50 μmol/L riboflavin. Mirasol treatment at 1×, 2×, 3×, 4×, and 5× doses resulted in P-selectin expression of 32.2 ± 5.9, 33.5 ± 5.7, 35.7 ± 0.7, 38.1 ± 4.3, and 38.6 ± 4.2%, respectively, which were all significantly higher than untreated PLTs (p < 0.05) or PLTs treated only with riboflavin (n = 4, p < 0.05). There was no significant difference in P-selectin expression percentage induced by various doses of Mirasol treatments (Fig. 2).

figure

Figure 2. Percentage of P-selectin–positive PLTs significantly increases in Mirasol PRT-treated PLTs. hPLTs were stained with CD41-FITC and CD62P (P-selectin)-PE, and samples were analyzed by flow cytometry for percentage of P-selectin–positive PLTs. The data present mean ± SD (error bars) from four independent experiments. *p < 0.05.

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Mirasol PRT PLTs show altered in vitro aggregation properties

We characterized PLT aggregation of Mirasol PRT PLTs (1× and 5×) with an aggregometer, by using ADP and TRAP as representatives of weak and strong agonists, respectively. The ADP-induced PLT aggregation was not altered in 1× Mirasol PRT-treated PLTs compared to untreated PLTs (Fig. 3A), but was potentiated significantly at low ADP concentrations in 5× Mirasol PRT-treated PLTs (Fig. 3B). TRAP-induced PLT aggregation was mildly inhibited in 1× Mirasol PRT PLTs (Fig. 3C) and strongly inhibited in 5× Mirasol PRT PLTs (Fig. 3D).

figure

Figure 3. Mirasol PRT PLTs exhibit altered in vitro aggregation properties in response to agonists. (A) In vitro PLT aggregation assay, in response to 5 to 160 μmol/L ADP, was performed with untreated (□) and Mirasol-treated (■) PRP. (B) In vitro PLT aggregation assay, in response to 5 to 160 μmol/L ADP, was performed with untreated (□) and 5× Mirasol-treated (■) PRP. (C) In vitro PLT aggregation assay, in response to 2.5 to 25 μmol/L TRAP, was performed with untreated (□) and Mirasol-treated (■) PRP. (D) In vitro PLT aggregation assay, in response to 2.5 to 25 μmol/L TRAP, was performed with untreated (□) and 5× Mirasol-treated (■) PRP. The data present mean ± SD (error bars) from three independent experiments. **p < 0.01.

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Mirasol PRT treatment reduces posttransfusion recovery in SCID mice

The damage to hPLTs induced by Mirasol PRT treatment can be detected by reduced PLT recovery in the SCID mouse model.[19] Using the percentage of control hPLTs in the acquisition gate at 30 minutes after transfusion as 100% recovery, the recovery of hPLTs was evaluated at 2, 4, 6, and 24 hours for control PLTs (n = 9), Mirasol PRT (1×) PLTs (n = 5), and Mirasol PRT (5×) PLTs (n = 3) in SCID mice (Fig. 4). The initial recovery of Mirasol hPLT (1× and 5×) was also expressed as a percentage of the mean control hPLT recovery at 30 minutes. At 30 minutes, Mirasol (1×) PLTs showed a slightly higher recovery (106.7 ± 14.4%), although this was not significant from the control PLTs (100%). Thereafter, Mirasol (1×) PLTs showed significantly lower recovery at 2 hours (Mirasol 1×, 55.7 ± 2.08%; control, 66.7 ± 6.5%; p < 0.01) and 6 hours (Mirasol 1×, 36.0 ± 4.6%; control, 48.2 ± 6.3%; p < 0.01). At 4 hours, Mirasol (1×) PLT recovery was not significantly different from control PLTs (Mirasol 1×, 44.5 ± 7.9%; control, 55.2 ± 5.7%; p > 0.05). Mirasol (5×) PLTs showed severely reduced recovery at all time points examined; at 30 minutes (Mirasol 5×, 20.6 ± 4.9%; control, 100%; p < 0.01), at 2 hours (Mirasol 5×, 10.5 ± 5.3%; control, 66.7 ± 6.5%; p < 0.01), at 4 hours (Mirasol 5×, 3.2 ± 0.2%; control, 55.2 ± 5.7%; p < 0.01), and at 6 hours (Mirasol 5×, 1.6 ± 0.8%; control, 48.2 ± 6.3%; p < 0.01). At 24 hours after transfusion, all percentage recoveries were approaching zero and no significant difference among the three groups. The approximate t1/2 of hPLTs in SCID mice, defined as the time to reach 50% of recovery was 6 hours for control PLTs and 3 hours for Mirasol (1×) PLTs.

figure

Figure 4. Mirasol treatment causes reduced posttransfusion recovery in SCID mice. A total of 1 × 109 hPLTs were infused into SCID mice, followed by serial blood sampling and flow cytometry. Using the percentage of control hPLTs in the acquisition gate at 30 minutes after transfusion as 100% recovery, the recovery of hPLTs was evaluated at 2, 4, 6, and 24 hours for control PLTs (n = 9, □), Mirasol PRT (1×) PLTs (n = 5, ■), and Mirasol PRT (5×) PLTs (n = 3, ▴) in SCID mice. The recovery of Mirasol PLTs (1× and 5×) was expressed as a percentage of the mean recovery of control PLTs at 30 minutes. **p < 0.01.

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Accumulation of Mirasol PRT-treated hPLTs in the lung of LPS-primed mice is dose dependent

We previously reported that UVB-treated hPLTs were sequestered in the lungs of LPS-primed mice and mediated ALI.[12, 14] In this study, we investigated if Mirasol PRT-treated hPLTs also accumulate in the SCID mouse lung. In healthy SCID mice, we did not observe significant accumulation of Mirasol PLTs (5×) in the lung (Figs. 5A-b and 5B), although the 5× Mirasol PRT-treated PLTs were significantly aggregated (Fig. 1H). In LPS-primed mice, when 1× Mirasol PRT PLTs were transfused, the number of hPLTs in the lung (Fig. 5A-f) was not significantly different from that of the control PLT group (Fig. 5A-d). When 3× Mirasol PRT-treated hPLTs were transfused, a few hPLTs or small hPLT aggregates were observed in the lungs of LPS-treated SCID mice (Fig. 5A-g), but the hPLT-associated fluorescent intensity was not significantly higher than that for control hPLTs (Fig. 5B). The 5× Mirasol PRT-treated hPLTs, however, accumulated significantly in the lungs of LPS-treated SCID mice (Figs. 5A-h and 5B), mostly as small PLT aggregates. This shows that pulmonary accumulation of Mirasol PRT hPLTs is dose dependent. These data are also consistent with the two-event mouse model we previously proposed, where the sensitizing event with LPS was necessary for pulmonary accumulation of UVB-illuminated PLTs.[12, 14] However, the lung accumulation of Mirasol PLTs was not as significant as that of UVB PLTs (Figs. 5 and 5B), in which larger aggregates were observed (Fig. 5A-e).

figure

Figure 5. Mirasol PRT treatment does not cause hPLT accumulation in the lungs of LPS-injected SCID mice. The panel shows confocal images of lung sections of healthy (a, b) and LPS-injected (c to h) SCID mice infused with control hPLTs (Ctrl PLT), UVB-treated hPLTs (UVB PLT), or Mirasol PRT-treated hPLTs at different doses (f to h). The composite images shown were representative of at least three independent experiments. In healthy mice, there are very few hPLTs in the lung in mice transfused with either Ctrl PLTs (a) or Mirasol (5×) PLTs (b). Mirasol (1×) (f) and Mirasol (3×) (g) PRT-treated hPLTs do not accumulate in the lung of LPS-injected SCID mice, while higher number of Mirasol (5×) PRT-treated hPLTs are sequestered in the lung of LPS-injected SCID mice (h), although the accumulation is not as significant as the UVB PLTs (e). Lung cryosections were stained with anti-hCD41 (red). Blue fluorescence represents Hoechst 33342–stained nuclei. All images were taken using a laser scanning confocal microscope (Zeiss 710), with a 63×/NA1.4 Plan-Apochromat oil objective. (B). Quantification of the fluorescence intensity of anti-hCD41 staining on lung cryosection. Data are expressed as mean ± SD; n = 3 for Mirasol-treated groups and n ≥ 6 for remaining groups. **p < 0.01.

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Mirasol PRT hPLT infusion does not cause histologic changes or more inflammatory infiltrates in the lungs of LPS-primed SCID mice

Histologic examination of the lungs from Mirasol PRT hPLT infusion showed that LPS injection caused inflammatory infiltrates of neutrophils (Fig. 6D). The number of neutrophils in the lungs was increased when hPLTs were injected, regardless of whether it was control PLT (Fig. 6E) or Mirasol PRT PLTs (Figs. 6F and 6G). This observation was confirmed with the use of confocal microscopy with LYS-eGFP;SCID mice, in which neutrophils were genetically labeled with e-GFP (Figs. 7A and 7A'). There were no other signs of ALI. To characterize the level of the immune response, we used a well-characterized antibody-based model of TRALI as a positive control for evaluation of lung injury in mice. In this model an infusion of a monoclonal anti-MHC Class I antibody 34–1-2s produced histologic evidence of lung injury with inflammatory infiltrates, septal thickening, and an increase in intraalveolar proteinaceous fluid (Fig. 6H).

figure

Figure 6. Mirasol PRT treatment does not cause histologic changes in the lung of SCID mice. Lung histology of healthy and LPS-injected (3 mg/g, intraperitoneally) SCID mice after infusion of PBS, control PLTs, or Mirasol-treated PLTs (1× or 5×). Lung histology of mice treated with LPS (0.1 mg/kg, intraperitoneally) plus MHC I MoAb (2 mg/kg) served as a positive control for ALI, in which the arrow points to protein rich alveolar exudates. All sections were hematoxylin and eosin stained. Composite images A through G are representative of three independent experiments and image H is representative of two independent experiments. All transmitted light images were taken with a microscope (Nikon Eclipse E800, Nikon Co.), using a 20×/NA 0.45 Plan Fluor objective.

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figure

Figure 7. Mirasol PRT treatments does not cause neutrophil or macrophage sequestration in the lung (A). Representative confocal images of unfixed lungs from LYS-eGFP;SCID mice. In these mice, neutrophils are labeled with eGFP (green; A'). Quantification of the fluorescence intensity of LYS-eGFP in (A). (B) Representative confocal images of frozen lung sections stained with anti-CD68 (red), a marker for macrophages. (B') Quantification of the fluorescence intensity of anti-CD68 antibodies in (B). Data are expressed as mean ± SD. n = 3; *p < 0.05.

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Recently it was also shown that macrophages were an important player in mediating the pathologic pulmonary changes in this antibody-based model of ALI[22] and thus we visualized macrophages in the lungs of mice infused with Mirasol PRT-treated hPLTs by using a CD68 antibody. LPS alone induced macrophage accumulation in the lung (Figs. 7B-d to 7B-f), but transfusion of Mirasol PRT(1×) hPLTs into LPS-injected mice did not cause additional macrophage accumulation in the lung (Fig. 7 and 7B').

Mirasol PRT hPLT infusion does not increase the level of MIP-2 in BALF

SCID mice were injected with LPS followed by transfusion of Mirasol PRT (5×) hPLTs. Three hours later, we collected BALF and measured in it the levels of a potent neutrophil chemotactic cytokine, MIP-2 (murine equivalent to human IL-8; Fig. 8). Mirasol PRT (5×) hPLTs did not induce MIP-2 production in the alveolar fluid as has been reported in various models of ALI.[5, 13, 23, 24] The MIP-2 concentration in the BALF from the 5× Mirasol-treated hPLT group (808 ± 113 pg/mL) is slightly higher on average than, but not significant from, the control hPLT group (716 ± 108 pg/mL). We used the MHC Class I antibody–induced TRALI mouse model[21, 22] as a positive control and assayed the BALF from these mice for MIP-2 levels. In the antibody-treated mice the MIP-2 level in BALF was 2199 ± 384 pg/mL, which was approximately 2.7-fold higher than from Mirasol PLT-infused mice. BALF from low LPS- (0.1 mg/kg) primed mice showed similar MIP-2 level (91 ± 33 pg/mL) as that of PBS-injected control mice (92 ± 28 pg/mL), which was significantly lower than that of high LPS- (3 mg/kg) injected mice (543 ± 213 pg/mL). We also measured the total protein content in BALF and found that Mirasol PRT hPLT infusion (up to 5×) did not increase the protein content in BALF (data not shown).

figure

Figure 8. Infusion of Mirasol PRT PLTs does not induce higher level of MIP-2 in BALF in SCID mice. BALF MIP-2 concentration (pg/mL) was measured in different experimental conditions as indicated. LPS, 3 mg/kg, intraperitoneally; L-LPS, 0.1 mg/kg, intraperitoneally. n = 3 L-LPS and n = 6 for all other groups. All data are expressed as mean ± SD. **p < 0.01; *p < 0.05.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of Interest
  8. References

In this study, we investigated the effects of Mirasol PRT treatment on PLTs in plasma in vitro, as well as the in vivo responses to infused Mirasol PRT-treated PLTs by using our two-event SCID mouse model (BALB scid, CB 17 strain). We used this model previously to evaluate UVB-exposed hPLT products.[12-14, 19] These SCID mice have a severe combined immunodeficiency affecting both B and T lymphocytes, which mimics the condition in many transfusion patients with impaired immune systems. The SCID mice have normal granulocytes, macrophages, and NK cells, as well as normal complement activities (http://www.criver.com/EN-US/PRODSERV/BYTYPE/RESMODOVER/RESMOD/Pages/Fox_Chase_SCID_Mice.aspx). This is relevant because other animal studies have suggested that PLT–neutrophil interactions may play critical roles in the pathology of ALI[25] and that macrophages and activation of complement were involved in the induction of ALI.[22] We have also shown that the SCID mice were very sensitive to MHC-I antibody–induced TRALI, with higher MIP-2 (Fig. 8) and protein concentrations (unpublished data) in BALF than what has been reported in BALB/c mice.[22] This is consistent with other recently published reports.[5, 26] In our two-event SCID mouse model of transfusion we employed an LPS injection as the first event to mimic the active inflammatory condition of some transfusion patients (sepsis, surgery, chemotherapy). The second event was the infusion of hPLTs (Mirasol PRT PLTs or untreated PLTs). Mice have a much higher PLT count per volume of blood with approximately three times more PLTs compared to human blood and the range of PLT counts varies greatly among different mouse strains.[27, 28] The PLT count in our SCID mice averaged 809 × 109 ± 149 × 109/L (unpublished data). In this study, we infused 1 × 109 hPLTs per mouse, which is equivalent to approximately 85% of the entire PLT content in mouse circulation, and mimics repeated PLT transfusions in which a large percentage of circulating PLTs in the thrombocytopenic patient come from transfusions.

The Mirasol PRT treatment resulted in PLT activation as evidenced by an increase in expression of the PLT activation marker, CD62P (P-selectin), from 23% in control PLTs to 32% in Mirasol PRT-treated PLTs (p < 0.05). With multiple Mirasol PRT treatments, up to 5×, there was a slight dose-related trend of increasing percentage of P-selectin–positive PLTs, although the differences did not achieve significance.

Mirasol PRT-treated PLTs did not exhibit any morphologic changes or spontaneous aggregation and only exhibited minimal changes in aggregation to agonists. Multiple doses of illumination produced spontaneous PLT aggregation in a dose-dependent manner, but this was not as severe as what we had reported previously with UVB-illuminated PLTs in an open illumination system.[13, 14] It is of interest that PLTs illuminated in Mirasol illumination and storage bags by the same UVB source and the same dosage (2.4 J/cm2) did not exhibit potentiation of aggregation and morphologically resembled control PLTs. This indicates that the PLTs treated in the illumination and storage bag were damaged less than those exposed in an open system, which could be the combinatorial result of a lower illumination dose delivered to the PLTs through the bag plastics and decreased access to air which may lead to less reactive oxygen species production. Both aspects could alleviate cellular injury. Mirasol PRT PLTs also showed reduced in vivo recovery at 2 hours (−11%) and 6 hours (−12%) in SCID mice, which is similar to the 15% reduction in recovery of Mirasol PRT-treated PLTs in a crossover clinical study with human volunteers.[10]

The absence of lung sequestration of Mirasol PRT (1×) PLTs and ALI is different from what we observed in SCID mice infused with UVB-treated PLTs, which showed lung sequestration of hPLTs and signs of ALI.[12-14] The differences between the Mirasol PRT system and the UVB illumination system, such as the use of illumination and storage bag, temperature-controlled illumination chamber, and the presence of riboflavin, could contribute to the improved in vitro and in vivo properties of Mirasol PLTs, when compared with directly UVB-illuminated PLTs as published previously.[12-14]

To evaluate the safety margins of the Mirasol system, we illuminated some units of PLTs multiple times (up to 5×). We want to emphasize that there are built-in safety features in the Mirasol system that prevents accidental overdosing. Multiple illuminations can be carried out only by intentionally overriding these safety features. We show that a higher dosage of Mirasol treatment did result in more PLT injury. This was demonstrated by dosage-dependent spontaneous PLT aggregation at 3× to 5×. At 5×, we also observed altered aggregation response to agonists, lung sequestration of PLTs, and significantly reduced PLT recovery. It is of interest that the 5× Mirasol PLTs also accumulated in the lungs (although the accumulation was not as significant as the UVB-exposed PLTs), but were not associated with changes in lung histology or an increase in MIP-2 levels or protein levels in BALF. This is in contrast to UVB-exposed PLTs, which accumulated in the lung and were associated with ALI.[12-14] The UVB and 5× Mirasol PLTs share some characteristics such as spontaneous aggregation and pronounced reduction in circulation survival but also differ in that Mirasol 5× treatments did not increase P-selectin expression significantly in a dose-dependent manner and did not significantly potentiate ADP-induced aggregation. These differences may indicate that lung accumulation of PLTs and development of ALI may be based on PLT activation mechanisms possibly associated with potentiation of ADP-induced responses.

Overall our data indicate that in the two-event SCID mouse model of PLT transfusion the safety margin of Mirasol system is approximately five repeated doses. Overdosing of Mirasol treatment should be prevented to avoid PLT injury evidenced by rapid clearance from circulation and increased accumulation of PLTs in the lung.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of Interest
  8. References

The authors thank the National Institutes of Health Blood Bank for collection of apheresis platelets.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of Interest
  8. References
  • 1
    US Department of Health and Human Services. The 2009 national blood collection and utilization survey report. Washington, DC: US Department of Health and Human Services, Office of the Assistant Secretary for Health; 2011.
  • 2
    Eder AF, Kennedy JM, Dy BA, Notari EP, Weiss JW, Fang CT, Wagner S, Dodd RY, Benjamin RJ. Bacterial screening of apheresis platelets and the residual risk of septic transfusion reactions: the American Red Cross experience (2004–2006). Transfusion 2007;47:1134-1142.
  • 3
    Corash L. Bacterial contamination of platelet components: potential solutions to prevent transfusion-related sepsis. Expert Rev Hematol 2011;4:509-525.
  • 4
    US Food and Drug Administration. Fatalities reported to FDA following blood collection and transfusion: annual summary for fiscal year 2011. Silver Spring, MD: US Food and Drug Administration; last updated 2012 May 8 [cited 2013 Mar 10]. Available from: http://www.fda.gov/BiologicsBloodVaccines/SafetyAvailability/ReportaProblem/TransfusionDonationFatalities/ucm302847.htm.
  • 5
    Semple JW, Kim M, Hou J, McVey M, Lee YJ, Tabuchi A, Kuebler WM, Chai ZW, Lazarus AH. Intravenous immunoglobulin prevents murine antibody-mediated acute lung injury at the level of neutrophil reactive oxygen species (ROS) production. Plos ONE 2012;7:e31357.
  • 6
    Snyder E, McCullough J, Slichter SJ, Strauss RG, Lopez-Plaza I, Lin JS, Corash L, Conlan MG. Clinical safety of platelets photochemically treated with amotosalen HCl and ultraviolet A light for pathogen inactivation: the SPRINT trial. Transfusion 2005;45:1864-1875.
  • 7
    Mirasol Clinical Evaluation Study Group. A randomized controlled clinical trial evaluating the performance and safety of platelets treated with MIRASOL pathogen reduction technology. Transfusion 2010;50:2362-2375.
  • 8
    McCullough J. Pathogen inactivation: a new paradigm for preventing transfusion-transmitted infections. Am J Clin Pathol 2007;128:945-955.
  • 9
    Snyder E, Raife T, Lin L, Cimino G, Metzel P, Rheinschmidt M, Baril L, Davis K, Buchholz DH, Corash L, Conlan MG. Recovery and life span of 111indium-radiolabeled platelets treated with pathogen inactivation with amotosalen HCl (S-59) and ultraviolet A light. Transfusion 2004;44:1732-1740.
  • 10
    AuBuchon JP, Herschel L, Roger J, Taylor H, Whitley P, Li J, Edrich R, Goodrich RP. Efficacy of apheresis platelets treated with riboflavin and ultraviolet light for pathogen reduction. Transfusion 2005;45:1335-1341.
  • 11
    Corash L, Lin JS, Sherman CD, Eiden J. Determination of acute lung injury after repeated platelet transfusions. Blood 2011;117:1014-1020.
  • 12
    Gelderman MP, Chi X, Zhi L, Vostal JG. Ultraviolet B light-exposed human platelets mediate acute lung injury in a two-event mouse model of transfusion. Transfusion 2011;51:2343-2357.
  • 13
    Zhi L, Chi X, Gelderman MP, Vostal JG. Activation of platelet protein kinase C by ultraviolet light B mediates platelet transfusion-related acute lung injury in a two-event animal model. Transfusion 2013;53:722-731.
  • 14
    Chi X, Zhi L, Gelderman MP, Vostal JG. Host platelets and, in part, neutrophils mediate lung accumulation of transfused UVB-irradiated human platelets in a mouse model of acute lung injury. Plos ONE 2012;7:e44829.
  • 15
    US Food and Drug Administration, Center for Biologics Evaluation and Research. Blood Products Advisory Committee Meeting, November 16, 2009 [Internet transcript]. [cited 2013 Mar 10]. Available from: http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/BloodVaccinesandOtherBiologics/BloodProductsAdvisoryCommittee/UCM193385.pdf.
  • 16
    Faust N, Varas F, Kelly LM, Heck S, Graf T. Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages. Blood 2000;96:719-726.
  • 17
    Peters NC, Egen JG, Secundino N, Debrabant A, Kimblin N, Kamhawi S, Lawyer P, Fay MP, Germain RN, Sacks D. In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies. Science 2008;321:970-974.
  • 18
    Sealey AL, Hobbs NK, Schmidt EE. Molecular genotyping of the mouse scid allele. J Immunol Methods 2002;260:303-304.
  • 19
    Piper JT, Gelderman MP, Vostal JG. In vivo recovery of human platelets in severe combined immunodeficient mice as a measure of platelet damage. Transfusion 2007;47:1540-1549.
  • 20
    Looney MR, Su X, Van Ziffle JA, Lowell CA, Matthay MA. Neutrophils and their Fc gamma receptors are essential in a mouse model of transfusion-related acute lung injury. J Clin Invest 2006;116:1615-1623.
  • 21
    Looney MR, Nguyen JX, Hu Y, Van Ziffle JA, Lowell CA, Matthay MA. Platelet depletion and aspirin treatment protect mice in a two-event model of transfusion-related acute lung injury. J Clin Invest 2009;119:3450-3461.
  • 22
    Strait RT, Hicks W, Barasa N, Mahler A, Khodoun M, Kohl J, Stringer K, Witte D, Van Rooijen N, Susskind BM, Finkelman FD. MHC class I-specific antibody binding to nonhematopoietic cells drives complement activation to induce transfusion-related acute lung injury in mice. J Exp Med 2011;208:2525-2544.
  • 23
    Kubota Y, Iwasaki Y, Harada H, Yokomura I, Ueda M, Hashimoto S, Nakagawa M. Role of alveolar macrophages in Candida-induced acute lung injury. Clin Diagn Lab Immunol 2001;8:1258-1262.
  • 24
    Tsujimoto H, Ono S, Mochizuki H, Aosasa S, Majima T, Ueno C, Matsumoto A. Role of macrophage inflammatory protein 2 in acute lung injury in murine peritonitis. J Surg Res 2002;103:61-67.
  • 25
    Zarbock A, Singbartl K, Ley K. Complete reversal of acid-induced acute lung injury by blocking of platelet-neutrophil aggregation. J Clin Invest 2006;116:3211-3219.
  • 26
    Fung YL, Kim M, Tabuchi A, Aslam R, Speck ER, Chow L, Kuebler WM, Freedman J, Semple JW. Recipient T lymphocytes modulate the severity of antibody-mediated transfusion-related acute lung injury. Blood 2010;116:3073-3079.
  • 27
    Chenaille PJ, Steward SA, Ashmun RA, Jackson CW. Prolonged thrombocytosis in mice after 5-fluorouracil results from failure to down-regulate megakaryocyte concentration. An experimental model that dissociates regulation of megakaryocyte size and DNA content from megakaryocyte concentration. Blood 1990;76:508-515.
  • 28
    Levin J, Ebbe S. Why are recently published platelet counts in normal mice so low? Blood 1994;83:3829-3831.