Ten healthy donors were recruited by Héma-Québec for two GC donations (the first after prednisone stimulation and the second after G-CSF stimulation) separated by a 6-months wash out period prior to leukapheresis with SpectraOptia (Terumo) according to Héma-Québec guidelines. All donations were collected in blood collection bags containing sodium citrate (46.7%) and hydroxyethyl starch (HESPAN^® 6% B. Braun Medical Inc.) to enhance red blood cell sedimentation at a ratio of 13:1 (product:anticoagulant solution). GCs were sent by the Globule Laval collection center to Québec City Héma-Québec for irradiation at 25 Gy prior to delivery to our laboratory for analysis on the day of collection (D1) as well as 24 h (D2) and 48 h (D3) post-collection. GCs were stored at room temperature without agitation. A pre-leukapheresis blood sample was drawn from donors in acid-citrate-dextrose (ACD) (BD Vacutainer) tubes. Blood sample and GC composition was determined at the Héma-Québec collection center with a cell counter (Ac•T 5diff hematology analyser, Beckman Coulter). One donation was excluded from the functional analysis because of a change in the sedimentation agent during the first GC donation. For comparison, blood donations from unstimulated frequency matched healthy donors was drawn in ACD tubes by the Clinical Research Platform at the CHU de Québec-Laval University and were used as control.

HES was purchased from Braun Medical Inc. (US). Fura-2- acetoxymethyl ester (Fura-2AM), CM-H2DCFDA, calcein-AM, the 7AAD viability staining solution and the pHRodo Red Zymosan A Bioparticles kit were obtained from Thermo Fisher (ON, Canada); dextran T500, cytochrome C, Hemacolor ® Rapid staining kit from Sigma-Aldrich (ON, Canada); lymphocyte separation medium, RPMI-1640, BSA (bovin serum albumin) and FBS (fetal bovine serum) from Wisent Bioproducts (QC, Canada); and the ChemoTx microplates (101–8) from NeuroProbe (MD, USA).

Pre-leukapheresis circulating neutrophils were isolated by density gradient from peripheral blood at room temperature under sterile conditions as described in Fernandes et al. (23). Neutrophils were resuspended in Mg²⁺-free HBSS containing 1.6 mM CaCl₂ at 10 x 10⁶ ml⁻¹. Neutrophils isolated from GCs did not require dextran sedimentation as they already contain hydroxyethyl starch, a sedimenting agent. The mononuclear cell layer was harvested after density gradient separation, washed and resuspended in PBS at 10 x 10⁶ ml⁻¹.

A LSRII flow cytometer was used for immunophenotyping with a twelve antibody panel and for fluorescent non-opsonized zymosan phagocytosis analysis. A CantoII flow cytometer was used for viability and intracellular ROS production assays. Cells were always used at 10 x 10⁶ ml⁻¹, stained at room temperature and kept on ice until analysis. All assays are explained in detail in the Supplementary Material.

Ten healthy donors that met the exclusion and inclusion criteria in Table 1 were recruited to donate GCs. Inter-donor variability was minimized by harvesting from all donors a prednisone GC followed by a G-CSF GC with a wash out period of 6 months between GC donations. The average blood volume filtrated from prednisone-stimulated donors during leukapheresis was 4,964 ml (4211-6001 ml) and 4,831 ml (3,611–6,039 ml) for G-CSF-stimulated donors (Table 2). Ten healthy, age and sex frequency-matched donors were also recruited for comparative purposes.

Since G-CSF mobilizes neutrophils more efficiently than glucocorticoids, we analyzed the leukocyte content in both types of GCs. Prednisone stimulation consistently generated GCs of a similar or significantly lower leukocyte concentration than G-CSF (62.9 x 10⁹.L⁻¹ and 109.5 x 10⁹.L⁻¹ leukocytes, respectively; Table 2, Figure 1A). The minimal ANC required per transfusion of 10¹⁰ neutrophils was thus observed in 6/9 (67%) of prednisone GCs compared to 10/10 G-CSF GCs (Figure 1). Neutrophils were 1.5 to 4-fold more abundant in G-CSF than prednisone GCs and comprised an average of 75.5% of all leukocytes in G-CSF GCs compared to 56.8% in prednisone GCs.

While neutrophils are the most abundant cells in GCs, other leukocytes are also present in this cellular product. Notably, lymphocytes represented 30% of all leukocytes in prednisone GCs and 16% in G-CSF GCs (Table 2). A substantial number of monocytes are also detected, 11% in prednisone GCs and 7% in G-CSF GCs. Minor leukocytes populations in these GCs included basophils and eosinophils. Together, these findings indicate that G-CSF is more efficient at mobilizing neutrophils for GCs than prednisone, and that GCs contain a significant proportion of lymphocytes and monocytes in addition to neutrophils.

To characterize the effect of prednisone and G-CSF stimulation on neutrophils in vivo, we compared their viability, maturity and cell-surface marker expression prior to leukapheresis. Spontaneous apoptosis and necrosis was not significantly altered in G-CSF GC neutrophils compared to neutrophils of non-stimulated healthy donors (Figure 2A).

Since G-CSF induces the release of immature neutrophils from the bone marrow (24), we stained G-CSF mobilized neutrophils with the maturity marker CD10. G-CSF induced the release of both mature (60% of CD10^high) and immature (40% of CD10^low) neutrophils into the circulation (Figures 2C,D). The presence of neutrophils with a mature or immature nuclear morphology confirmed this observation (Figure 2B). Contrary to low-density, immature neutrophils that increase in numbers in some disease states, G-CSF GC immature neutrophils have a similar density as mature neutrophils as they pellet to the bottom of the density gradient. In contrast, prednisone only mobilized CD10^high neutrophils with multi-lobular nuclei, a typical feature of mature neutrophils (Figures 2B,C) (25). An additional distinctive feature of G-CSF GC immature neutrophils is their significantly lower expression of CD16 (CD16^low) compared to their CD16^high mature counterparts. The expression of the other cell-surface markers tested in prednisone and G-CSF-mobilized neutrophils was not altered (Figure 2D). Together, these data indicate that prednisone and G-CSF mobilized neutrophils had similar viability but differed in their maturation stage.

Since neutrophils are the main source of antimicrobial activity in GCs, we assayed the key neutrophil functions required to fight infections in GC neutrophils 6 h post-leukapheresis. For comparative purposes, the assays were also performed on neutrophils from unstimulated, healthy donors. Of these neutrophil responses, the function that was significantly altered in prednisone GC neutrophils was the phagocytosis of non-opsonized zymosan. While considerable variability in the phagocytosis was observed amongst non-stimulated healthy donors, prednisone leveled this response to 90% phagocytosis in all donors (Figure 3). A diminution in fMLF-induced chemotaxis was also observed but did not reach significance. Likewise, no significant difference in the LPS-induced production of IL-8 and the fMLF and PMA-induced ROS production, or increase in cytoplasmic calcium was observed between prednisone or G-CSF GC and non-stimulated, healthy donor neutrophils (Figure 3).

G-CSF GC neutrophils also exhibited functional differences with non-stimulated, healthy donor neutrophils albeit for different functions (Figure 3). Chemotaxis induced by fMLF was significantly downregulated in G-CSF GC neutrophils whereas LPS-induced production of IL-8 was significantly enhanced (Figure 3). No significant differences in the other functional assays were observed. Together, these data reveal a mobilizing agent-dependent alteration in GC neutrophil function.

To determine whether leukapheresis contributes to the functional alterations observed in GC neutrophils, their responses were compared before and after leukapheresis. A significant diminution in the fMLF-induced increase in cytoplasmic calcium (Figure 4A) and an increase in PMA, but not fMLF, -induced intracellular ROS production in prednisone GC neutrophils was observed after leukapheresis (Figures 4B,C). In contrast, G-CSF GC neutrophils were unaffected by this procedure (Supplementary Figure 1). Together, these data indicate that prednisone GC neutrophils are more affected by leukapheresis than G-CSF GC neutrophils.

While the negative effect of storage on G-CSF GC neutrophil viability and function was reported in several studies (18, 26, 27), the impact of storage on prednisone GC neutrophils remains unknown. Storage negatively affects neutrophils as we were not able to isolate the predicted quantity of neutrophils from these GCs based on the neutrophil concentration in the GC prior to isolation. The number of neutrophils isolated from prednisone GCs after 24 and 48 h of storage declined by 20 and 40%, respectively, compared to the neutrophil yield on the day of collection (Figure 5A). Apoptosis of the isolated neutrophils harvested from prednisone GCs after 24 h of storage significantly increased by 8–10% in 4/9 donors (Figure 6A). As for cell-surface marker expression, CD66b expression significantly increased after 48 h of storage suggesting an increased degranulation (Supplementary Figure 2).

As observed for prednisone GC neutrophils, G-CSF GC neutrophils were also affected by storage. The most striking effect was the difficulty in isolating neutrophils from these GCs. The neutrophil yield dropped by 40% after 24 h storage and 80%, 48 h post-leukapheresis (Figure 5A). The functional data on these GCs is thus shown as supplementary data (Supplementary Figures 3, 4). Storage had no significant effect on cell-surface marker expression of G-CSF GC neutrophils (data not shown).

A change in metabolite concentration and/or pH could explain the observed negative effects of storage on GCs. Data for two prednisone GC donations and for all G-CSF GCs was generated as the metabolite measurements began later in the study. We consistently observed a decrease in pH (7,25 to 6,82) 24 h post-collection in G-CSF GCs as well as glucose with a concomitant 5 to 8-fold increase in lactate concentration during storage (Figure 5B). Together, these data indicate that the decrease in GC neutrophil viability is highly likely due to rapid changes in pH and a significant change in metabolite concentration.

A diminution in most neutrophil responses including calcium mobilization, fMLF-induced chemotaxis and the phagocytosis of non-opsonized zymosan by prednisone GC neutrophils was observed after 24 h storage but did not reach significance (Figures 6B–D). In contrast, a significant increase was observed in both PMA-induced superoxide production and spontaneous release of IL-8 but not in intracellular ROS production (Figures 6E–G). The increase in superoxide production is suggestive of neutrophil priming during storage. Since the number of neutrophils that could be isolated from the collection bag diminished considerably with storage, functional data on prednisone GC neutrophils after 48 h storage and G-CSF GC neutrophils after 24 and 48 h storage may not be representative of all neutrophils in GCs (Supplementary Figures 3, 4). Together, these data reveal that G-CSF GC neutrophils rapidly deteriorate with storage and that storage differentially affects prednisone GC neutrophil effector functions.