Development of a new cell isolation device FlowMagicTM

Tomoyuki Yoshida; Yoshiaki Sakamoto; Aya Tsuruta; Risa Kimura; Nozomi Shiozawa; Takeyuki Kotaka

doi:10.1371/journal.pone.0334936

. 2025 Oct 22;20(10):e0334936. doi: 10.1371/journal.pone.0334936

Development of a new cell isolation device FlowMagic^TM

Tomoyuki Yoshida ^1,^*,^#, Yoshiaki Sakamoto ^1,^#, Aya Tsuruta ¹, Risa Kimura ¹, Nozomi Shiozawa ², Takeyuki Kotaka ^1,^*

Editor: Jeffrey Chalmers³

PMCID: PMC12543197 PMID: 41124154

Abstract

Isolation of human peripheral blood mononuclear cells (PBMCs) from blood typically involves a density gradient medium during density centrifugation. The problem of increasing red blood cell (RBC) and granulocyte (GRA) contamination during PBMC isolation as the elapsed time after blood collection increases remains unresolved. As a countermeasure against RBC contamination, hemolysis treatment is available; however, these extra steps are laborious, time-consuming, and could introduce artifacts. To overcome this challenge, we developed a novel isolation device, FlowMagic™, which features a proprietary two-layer insert structure designed to prevent RBC and GRA contamination during PBMC isolation from blood. The efficacy of this method was evaluated by isolating PBMCs from donors and analyzing immune cell populations by flow cytometry. Compared to SepMate (median (Q50) = 11.0, interquartile ranges (IQR): 8.8–19.5; p < 0.01) and Lymphoprep methods (Q50 = 9.3, IQR: 6.6–13.5; p < 0.01), FlowMagic™ achieved significantly greater reduction in RBC contamination to below detectable limits (Q50 = 0.0, IQR: 0.0–0.0), with sustained efficacy observed up to 72 hours post-collection. Additionally, the FlowMagic™ method (Q50 = 2.5, IQR: 0.5–3.4, at 48 hours, median = 4.5, IQR: 2.1–10.3, at 72 hours, respectively) significantly reduced GRA contamination compared with the SepMate (Q50 = 12.0, IQR: 7.8–25.5, at 48 hours, Q50 = 27.5, IQR: 12.3–29.0, at 72 hours, respectively; p < 0.01) and Lymphoprep methods (Q50 = 10.5, IQR: 6.9–19.8, at 48 hours, Q50 = 17.5, IQR: 13.3–23.5, at 72 hours, respectively; p < 0.01) at 48 and 72 hours after blood collection. Furthermore, the recovery rates of CD3 + , CD4 + , CD8 + , CD19 + , and CD16/56 + cells in the FlowMagic™-isolated PBMCs (Q50 = 8.6, 5.9, 2.5, 1.3, and 1.9, respectively) were significantly improved compared to those in SepMate- (Q50 = 2.2, 1.5, 0.7, 0.4, and 0.5, respectively; p < 0.01) and Lymphoprep-isolated PBMCs (Q50 = 2.4, 1.5, 0.8, 0.6, and 0.8, respectively; p < 0.01), even at 48 hours after blood collection. These findings suggest that the PBMC isolation method using FlowMagic™ is advantageous in preventing RBC and GRA contamination for research, diagnostic, and clinical applications.

Introduction

The isolation of human peripheral blood mononuclear cells (PBMCs) from whole blood in laboratory tests, clinical trials, and basic scientific research is a crucial preprocessing step for various assays, such as enzyme-linked immunosorbent spot (ELISPOT) assays [1,2], proliferation assays [3], flow cytometry [4], and cytometry by time-of-flight (CyTOF) [5]. In these assays, isolated cells should exhibit high viability and be substantially free from RBC, granulocyte and platelet contamination.

PBMCs are primarily isolated by density gradient centrifugation [6]. While the standard method for PBMC isolation is Ficoll-Paque gradient centrifugation, other PBMC isolation devices have recently become commercially available, due to their faster and simpler processes. These devices have a shorter centrifugation time with a brake, which is shorter than the Ficoll-Paque technique. The BD Vacutainer cell preparation tube (CPT) is an evacuated tube containing an anticoagulant and a cell separation medium containing a polyester gel and a density gradient liquid [7,8]. The Greiner Bio-One LeucoSep tube has a porous membrane frit that isolates the density gradient from the whole blood sample [8,9]. In addition, as a second-generation cell isolation device, the STEMCELL Technology SepMate tubes contain an insert that creates a barrier between the density gradient medium and blood, thus eliminating the need for careful blood layering and allowing mononuclear cells to be easily harvested with a simple pour [7,8].

Though there are many technologies and techniques that increase the ease of PBMC isolation, these methods are plagued by low level of contaminating RBCs [10]. Moreover, when PBMCs are isolated on a large scale, as with most ex vivo adoptive immunotherapy methods, the amount of contaminating RBC increases even further. The presence of RBC contamination in a PBMC sample may lead to inaccurate PBMC concentration measurements, which can be detrimental to downstream experiments performed with these cells. To resolve this issue, an RBC lysis protocol can be used to reduce RBC contamination, however, these extra steps are laborious, time-consuming, and could be a source of artifacts.

Granulocyte (GRA) is also a major cause of contamination during PBMC processing. GRA usually pellet with RBCs. However, prolonged storage of whole blood at room temperature for 24–48 hours prior to processing, due to transportation or facility limitations, has been shown to increase GRA contamination [11]. Hence, delaying PBMC processing influences the sample quality, which affects downstream assays. Prolonged storage-induced GRA contamination has been shown to correlate with reduced T cell function, decreased interferon-γ ELISPOT spot counts, and altered T cell metabolic pathway activities [11–13]. Thus, it is important to avoid RBC and GRA contamination during PBMC processing.

The purpose of this study is to report on the performance of a novel cell isolation device, FlowMagic™, which uses proprietary technology to prevent RBC and GRA contamination during PBMC isolation from blood. We evaluated the PBMC recovery rates and purity using an automated cell counter and flow cytometry.

Materials and methods

PBMC isolation

Venous blood was collected from healthy volunteers into sodium-heparin tubes (Termo Corporation) after obtaining written consent in accordance with guidelines for biomedical research involving human subjects. The study protocol was approved by the Institutional Review Board of the Ethical Review Committee of H.U. Group Holdings, Inc. (Approval number: 24-015-00, 24-015-02). The research period extended from 15/04/2024, to 31/03/2026. Written informed consent was obtained from volunteer participants prior to each blood collection procedure. Recruitment of volunteer participants for this study commenced on 25/04/2024 and concluded on 15/11/2024.

Fresh whole blood samples were incubated at room temperature (20–24°C) immediately after collection. Parallel PBMC isolations were performed by three different techniques within 24, 48, and 72 hours after blood collection from the same volunteers. Isolation with Lymphoprep (density of 1.077 g/mL; STEMCELL Technology) with FlowMagic™ tubes (in-house development), Lymphoprep with SepMate tubes (STEMCELL Technology), and Lymphoprep (STEMCELL Technology) alone in 50 mL polypropylene tubes (Wako) were performed according to in-house protocols and the manufacturer’s instructions.

FlowMagic™ 3D CAD images, FlowMagic™ isolation workflow and the three isolation protocols are summarized in Figs 1 and 2. For the FlowMagic™ technique, 15 mL of Lymphoprep was added to the FlowMagic™ tube, and centrifuged (1,200 g, 10 min, brake on) for 5 minutes. Heparinized blood was diluted twice with phosphate-buffered saline (PBS; FUJIFILM Wako Pure Chemical Corporation) containing 2% fetal bovine serum (FBS) and injected into the nozzle hole on upper insert of FlowMagic™. After centrifugation (1,200 g, 10 min, brake on), and pressing down the floating upper insert with a plastic tip, PBMCs are collected by pouring the supernatant into a 50 mL polypropylene tube (Wako). For the SepMate technique, heparinized blood was diluted twofold with PBS containing 2% FBS and layered on top of Lymphoprep. After centrifugation (1,200 g, 10 min, brake on), PBMCs were collected by pouring the supernatant into a 50 mL polypropylene tube. For the Lymphoprep technique, heparinized blood is diluted twofold with PBS containing 2% FBS and layered on top of Lymphoprep in the 50 mL polypropylene tube. After centrifugation (1,200 g, 20 min, no brake), PBMCs were collected using a plastic pipette and stored in a 50 mL polypropylene tube. Collected PBMCs were washed twice with PBS (FUJIFILM Wako Pure Chemical Corporation) containing 2% FBS (500 g, 10 min and 5 min, brake on). Cell counts were performed using a Microsemi LC-710 (HORIBA) automated cell counter.

Fig 1 — (a) Cross-sectional view of FlowMagic™. The components of inserts in FlowMagic™ are divided into two parts. (b) The oblique cross-sectional view of the floating upper insert and the fixed lower insert in FlowMagic™. (c) The oblique cross-sectional view of the inserts. (d) Top view of the upper insert. (e) Top view of the lower insert. (f) Bottom view of the upper insert. (g) Bottom view of the lower insert.

Fig 2 — (a) 15 mL of Lymphoprep was added to the FlowMagic™ tube. Whole blood is diluted twofold with PBS, mixed with a density gradient medium, and compressed with a pipette using the floating upper insert. The mixture is then centrifuged for 10 minutes. PBMCs are collected by decantation in the tube. (b) Comparison of between FlowMagic^TM, SepMate and Lymphprep PBMC isolation methods. Centrifugation time and speed was indicated prior to collection of PBMCs. PBMCs are decanted for FlowMagic^TM and SepMate while PBMC was collected for Lymphoprep by pipetting. The orange area for SepMate and Lymphprep indicates the PBMC layer.

Immunophenotyping of PBMC populations

Immunophenotyping was performed by flow cytometry. PBMC samples were stained using Multitest 6-Color TBNK staining kit (Becton Dickinson). Samples were measured with a BD FACSLyric system (BD Bioscieneces) and analyzed with FlowJo software v10.

Statistical analysis

To assess the differences between the FlowMagic™ method and two commercially available methods, pairwise comparisons were conducted: FlowMagic™ versus SepMate and FlowMagic™ versus Lymphoprep. Wilcoxon signed-rank test with Bonferroni correction was used for multiple comparisons as a non-parametric alternative. p-values less than of equal to 0.05 were considered statistically significant for all statistical tests. All statistical analyses were performed using Python version 3.13.5.

Results

FlowMagic™ reduces RBC and GRA contamination during PBMC isolation

Three isolation methods were performed: FlowMagic™ tube with Lymphoprep, denoted as FlowMagic™; SepMate tube with Lymphoprep, denoted as SepMate; and normal tube with Lymphoprep, denoted as Lymphoprep. Fig 1 shows the patent-pending 3D CAD images of FlowMagic™, which consists of two inserts in the 50 mL polypropylene tube. It differs from previous cell isolation devices in that the upper insert is designed to float in the tube during centrifugation. Fig 2 shows FlowMagic™ isolation workflow and a comprehensive schematic comparison of the three protocols regarding centrifugation time and speed, and collection protocol. First, we examined the recovery of PBMCs from whole blood from ten volunteers by using these three different isolation methods. The state of fractionation from whole blood collected at 24, 48, and 72 hours after blood collection using each method is shown in Fig 3. The upper Layer contains plasma, isolation medium, and PBMCs. The middle Layer (interface) contains a few RBCs and some GRAs in FlowMagic™. The lower Layer predominantly contains RBCs and GRAs in FlowMagic™.

Fig 3 — Whole blood was isolated using Lymphoprep in a FlowMagic™ tube, SepMate tube, and a normal tube. Sample A, B, and C refer to three different donors. Different layers were labelled. The upper Layer contains plasma, isolation medium, and PBMCs in FlowMagic™. The middle Layer (Interface) contains a few RBCs and some GRAs in FlowMagic™. The lower Layer predominantly contains RBCs and GRAs in FlowMagic™. (a) 24 hours after blood collection. (b) 48 hours after blood collection. (c) 72 hours after blood collection.

Fig 4a–c shows changes in white blood cell (WBC), RBC, and GRA counts, respectively, from whole blood samples processed at 24, 48, and 72 hours using three isolation methods: FlowMagic™, SepMate, and Lymphoprep. At 24 hours after blood collection, no RBC contamination was observed in all three methods (Figs 3a and 4b).

FlowMagic™ achieved significantly greater reduction in RBC contamination to below detectable limits even up to 72 hours after collection (Q50 = 0.0, IQR: 0.0–0.0) compared with SepMate (Q50 = 11.0, IQR: 8.8–19.5; p < 0.01) and Lymphoprep methods (Q50 = 9.3, IQR: 6.6–13.5; p < 0.01) (Fig 4b). Additionally, the FlowMagic™ method (Q50 = 2.5, IQR: 0.5–3.4 at 48 hours, Q50 = 4.5, IQR: 2.1–10.3 at 72 hours) significantly reduced GRA contamination compared with the SepMate (Q50 = 12.0, IQR: 7.8–25.5 at 48 hours, Q50 = 27.5, IQR: 12.3–29.0 at 72 hours; p < 0.01, p < 0.01, respectively) and Lymphoprep methods (Q50 = 10.5, IQR: 6.9–19.8 at 48 hours, Q50 = 17.5, IQR: 13.3–23.5 at 72 hours; p < 0.01, p < 0.01, respectively) at 48 and 72 hours after blood collection. (Fig 4c). A summary of the main PBMC composition data is provided in S1 Table. These results suggest the efficacy of FlowMagic™ to reduce RBC and GRA contamination.

Next, we evaluated the purity and composition of PBMCs isolated from whole blood the three different isolations methods (Fig 5). PBMCs isolated with FlowMagic™ (Q50 = 67.2, IQR: 61.5–69.6, p < 0.01) exhibited significantly higher lymphocyte purity rates compared to those isolated using SepMate (Q50 = 56.6, IQR: 50.0–62.2, p < 0.01) and Lymphoprep (Q50 = 56.0, IQR: 49.0–59.4, p < 0.01) at 72 hours after blood collection (Fig 5a). PBMCs isolated with FlowMagic™ also yielded significantly more monocytes than SepMate and Lymphoprep at 72 hours after blood collection (Fig 5b). The contamination rate of GRA in the isolated PBMC was significantly reduced when FlowMagic™ isolation method was used at 48 hours and 72 hours after blood collection (Fig 5c). A summary of the lymphocyte composition data is provided in S2 Table.

Population composition

We then assessed the composition of leukocytes that are of interest to functional assays in the isolated PBMC fractions, namely: T-cells (CD3⁺), helper T-cells (CD4⁺), cytotoxic T-cells (CD8⁺), B-cells (CD19⁺), and NK cells (CD16/56⁺) by flow cytometry. The composition of the isolated PBMC populations varied between volunteers, but in general the percentage of CD3⁺, CD4⁺, CD8⁺ CD19⁺, and CD16/56⁺ cells isolated from FlowMagic™ were greater than from SepMate and Lymphoprep (Fig 6a-6e) up to 72 hours after blood collection. This improvement in enrichment of leukocytes from FlowMagic™ when compared to SepMate and Lymphoprep was more significant at 48 hours and 72 hours after blood collection (Fig 6a-6e). There is a notable drop though in the percentage of leukocytes at 48 hours and 72 hours after blood collection, which may be due to the accumulation of a small amount of GRA contamination (Fig 5c). A summary of the lymphocyte and leukocyte composition data is provided in S2–S3 Tables respectively. Overall, these results suggest the efficacy of the FlowMagic™ isolation method, for enrichment of leukocytes from blood.

Fig 6 — Cell counts of (a) CD3⁺, (b) CD4⁺, (c) CD8⁺, (d) CD19⁺, and (e) CD16/56⁺ cells are shown at 24, 48, and 72 hours after collection. PBMCs were isolated and stained with Multitest 6-Color TBNK consisting of anti-CD45 (leukocytes), -CD3 (T-cells), -CD4 (helper T-cells), -CD8 (cytotoxic T-cells), -CD20 (B-cells), and -CD16/56 (NK-cells). Box plots show median (center line), interquartile ranges (boxes, 25th to 75th percentiles), whiskers extending to 1.5 × IQR, and outliers as individual dots. Statistical significance was assessed using the Wilcoxon signed-rank test (*p < 0.05, **p < 0.01, and n.s., not significant). N = 10. PBMC: peripheral blood mononuclear cell; IQR: interquartile ranges.

Discussion

In this paper, we reported on the efficacy of the new cell isolation device, FlowMagic™. Three PBMC isolation techniques were evaluated, focusing on cell recovery and population composition. The techniques comprised the isolation by the FlowMagic™ device with Lymphoprep, isolation by SepMate tubes with Lymphoprep, and only Lymphoprep. Lymphoprep isolation is the most attractive approach from a cost perspective, but in practice, this technique is relatively laborious and takes longer than the other two methods, and is plagued by RBC and GRA contamination. SepMate increases the yield of WBC and reduces processing time, but has problems with RBC and GRA contamination. This contamination becomes more pronounced during extended delays in blood processing. Thus, we propose that FlowMagic™ is a superior alternative method because it yields significantly lower RBC and GRA contamination than SepMate and Lymphoprep isolation methods even on isolated PBMCs processed at 48 and 72 hours after blood collection. Moreover, the recovery rates of CD3⁺, CD4⁺, CD8⁺, CD19⁺, and CD16/56⁺ cells in FlowMagic™-isolated PBMCs were significantly improved compared to SepMate- and Lymphoprep-isolated PBMCs even at 48 and 72 hours after blood collection. Based on these findings, FlowMagic™ has the potential to address RBC and GRA contamination that occurs over lengthy delays of sample processing after blood collection, particularly at 48–72 hours post-blood collection.

In overseas clinical trials, the transportation time after blood collection can take 24–48 hours, and long transportation time can degrade the quality of PBMCs [11,12]. Even in such cases, FlowMagic™ can be an effective solution for isolating PBMC with high purity. Overall, the FlowMagic™ method demonstrated exceptional efficacy, achieving complete removal of RBC contamination and a substantial reduction of GRA contamination, surpassing the performance of commonly used isolation techniques. Further studies are needed to evaluate how PBMCs isolated using the FlowMagic™ device affect cell function assays that are commonly plagued by RBC contamination.

During PBMC isolation using SepMate method and Lymphoprep method, whole blood is layered on top of the density gradient medium, hence the whole blood and the density gradient medium are not mixed [7,8]. In contrast, the FlowMagic™ isolation method mixes the density gradient medium and whole blood due to the lowering of the upper insert before centrifugation (Fig 2a). Due to this step in the FlowMagic™ isolation protocol, one disadvantage of FlowMagic™ is that it cannot simultaneously separate plasma during PBMC isolation. The inability of FlowMagic™ to isolate plasma simultaneously may be relevant for translational settings. Functional downstream assays (e.g., cytokine release, ELISPOT) are not yet presented and should be acknowledged as needed future work.

Conclusion

We propose that FlowMagic™ is a useful tool for isolating PBMCs with low levels of RBC and GRA contamination from samples with processing delays of 24–72 hours after blood collection compared to Lymphoprep alone or other commercial cell isolation devices such as SepMate. Our results have reported the efficacy of PBMC isolation from whole blood, however further extensive studies using in vitro cell functional assays are clearly needed. The present study suggests that the newly developed cell isolation device, FlowMagic™, may be useful in fields such as laboratory testing and cell culture systems.

Supporting information

S1 Table. Summary of PBMC composition isolated by three different methods from whole blood after 24-hour storage.

Data from 10 volunteers. Values are shown as means ± standard deviation. The Wilcoxon signed-rank test was used with Bonferroni correction for multiple comparisons as a non-parametric alternative. A significance level of 0.05 was used for all statistical tests. PBMC: peripheral blood mononuclear cell; WBC: white blood cell; RBC: red blood cell; GRA: granulocytes.

(TIF)

pone.0334936.s001.tif^{(116.4KB, tif)}

S2 Table. Summary of PBMC composition isolated by three different methods from whole blood after 48-hour storage.

(TIF)

pone.0334936.s002.tif^{(146.4KB, tif)}

S3 Table. Summary of PBMC composition isolated by three different methods from whole blood after 72-Hour storage.

(TIF)

pone.0334936.s003.tif^{(138.8KB, tif)}

Acknowledgments

The authors would like to give special thanks to the members of the Institutional Review Board of the Ethical Review Committee of H.U. Group Holdings, Inc. We appreciate Dr. Kazuya Omi, Dr. Hiroshi Hayashi and Dr. John Clyde Co Soriano for their critical review of this paper. We are also grateful to all volunteer blood donors. We also appreciate Mr. Masatoshi Mori, Mr. Takanori Endo, Mr. Daichi Takizawa, Ms. Yuko Sekiya, Mr. Yuta Takeda, Dr. Ryo Negishi, and Dr. Kyohei Yoshimitsu for their technical advice and assistance.

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

The author(s) received no specific funding for this work.

References

1.Filbert H, Attig S, Bidmon N, Renard BY, Janetzki S, Sahin U, et al. Serum-free freezing media support high cell quality and excellent ELISPOT assay performance across a wide variety of different assay protocols. Cancer Immunol Immunother. 2013;62(4):615–27. doi: 10.1007/s00262-012-1359-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Smith JG, Joseph HR, Green T, Field JA, Wooters M, Kaufhold RM, et al. Establishing acceptance criteria for cell-mediated-immunity assays using frozen peripheral blood mononuclear cells stored under optimal and suboptimal conditions. Clin Vaccine Immunol. 2007;14(5):527–37. doi: 10.1128/CVI.00435-06 [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Aziz N, Margolick JB, Detels R, Rinaldo CR, Phair J, Jamieson BD, et al. Value of a quality assessment program in optimizing cryopreservation of peripheral blood mononuclear cells in a multicenter study. Clin Vaccine Immunol. 2013;20(4):590–5. doi: 10.1128/CVI.00693-12 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Macchia I, Urbani F, Proietti E. Immune monitoring in cancer vaccine clinical trials: critical issues of functional flow cytometry-based assays. Biomed Res Int. 2013;2013:726239. doi: 10.1155/2013/726239 [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Newell EW, Sigal N, Bendall SC, Nolan GP, Davis MM. Cytometry by time-of-flight shows combinatorial cytokine expression and virus-specific cell niches within a continuum of CD8+ T cell phenotypes. Immunity. 2012;36(1):142–52. doi: 10.1016/j.immuni.2012.01.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Böyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl. 1968;97:77–89. [PubMed] [Google Scholar]
7.Grievink HW, Luisman T, Kluft C, Moerland M, Malone KE. Comparison of three isolation techniques for human peripheral blood mononuclear cells: cell recovery and viability, population composition, and cell functionality. Biopreserv Biobank. 2016;14(5):410–5. doi: 10.1089/bio.2015.0104 [DOI] [PubMed] [Google Scholar]
8.Muise A, White L, Badowski M, Harris DT. Analysis of high-throughput processing methods for peripheral blood cell isolation. Biopreserv Biobank. 2022;20(3):302–5. doi: 10.1089/bio.2021.0100 [DOI] [PubMed] [Google Scholar]
9.Nilsson C, Aboud S, Karlén K, Hejdeman B, Urassa W, Biberfeld G. Optimal blood mononuclear cell isolation procedures for gamma interferon enzyme-linked immunospot testing of healthy Swedish and Tanzanian subjects. Clin Vaccine Immunol. 2008;15(4):585–9. doi: 10.1128/CVI.00161-07 [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Song S, Goodwin J, Zhang J, Babbitt B, Lathey JL. Effect of contaminating red blood cells on OKT3-mediated polyclonal activation of peripheral blood mononuclear cells. Clin Diagn Lab Immunol. 2002;9(3):708–12. doi: 10.1128/cdli.9.3.708-712.2002 [DOI] [PMC free article] [PubMed] [Google Scholar]
11.McKenna KC, Beatty KM, Bilonick RA, Schoenfield L, Lathrop KL, Singh AD. Activated CD11b+ CD15+ granulocytes increase in the blood of patients with uveal melanoma. Invest Ophthalmol Vis Sci. 2009;50(9):4295–303. doi: 10.1167/iovs.08-3012 [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Yi P-C, Zhuo L, Lin J, Chang C, Goddard A, Yoon OK. Impact of delayed PBMC processing on functional and genomic assays. J Immunol Methods. 2023;519:113514. doi: 10.1016/j.jim.2023.113514 [DOI] [PubMed] [Google Scholar]
13.Qu H-Q, Kao C, Garifallou J, Wang F, Snyder J, Slater DJ, et al. Single cell transcriptome analysis of peripheral blood mononuclear cells in freshly isolated versus stored blood samples. Genes (Basel). 2023;14(1):142. doi: 10.3390/genes14010142 [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS One. 2025 Oct 22;20(10):e0334936. doi: 10.1371/journal.pone.0334936.r001

Author response to Decision Letter 0

6 May 2025

Attachment

Submitted filename: renamed_cd3c8.docx

pone.0334936.s004.docx^{(14.7KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0334936.r002

Decision Letter 0

Jeffrey Chalmers

10 Jul 2025

Dear Dr. Yoshida,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please address before the issues raised by reviewers...

Please submit your revised manuscript by Aug 24 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Jeffrey Chalmers, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We note that your Data Availability Statement is currently as follows: [If the data are all contained within the manuscript and/or Supporting Information files, enter the following: All relevant data are within the manuscript and its Supporting Information files.]

Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition).

For example, authors should submit the following data:

- The values behind the means, standard deviations and other measures reported;

- The values used to build graphs;

- The points extracted from images for analysis.

Authors do not need to submit their entire data set if only a portion of the data was used in the reported study.

If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories.

If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access.

3. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

4. Thank you for stating the following in the Competing Interests section:

[The authors have declared that no competing interests exist.]

We note that one or more of the authors are employed by a commercial company: H.U. Group Research Institute, H.U. Cells, Inc.

1. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately

indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared

Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

5. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please delete it from any other section.

Additional Editor Comments:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: No

Reviewer #2: I Don't Know

Reviewer #3: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

Reviewer #1: This manuscript addresses an important and timely topic—developing a novel centrifugation-based technique to isolate viable PBMCs from whole blood with minimal WBC and RBC contamination. The authors provide a clear description of current commercial techniques, particularly density gradient centrifugation, and highlight the limitations associated with these existing methods. The manuscript compares the performance of the proposed method against two widely used commercial technologies and includes preliminary data suggesting improved performance. However, several major revisions are necessary before the manuscript can be considered for publication.

1. The authors state that their method performs better than the current technologies, but the abstract and conclusion sections do not include any quantitative data to support this claim. Specific numerical results should be presented to demonstrate the comparative performance clearly.

2. The conclusions are somewhat overstated given the scope and depth of the current data. The authors should discuss in more detail the limitations of their study and future research that should be conducted.

3. While the manuscript provides some quantitative data, there is no statistical analysis presented to validate the claim that their technology is superior. Mean values and standard deviations should be reported for all comparative data, and appropriate statistical tests (e.g., ANOVA, t-tests) should be used to evaluate the significance of differences between groups.

4. Some suggestions for modifying the figures:

Figure 2: A schematic diagram comparing the protocols of FlowMagic, Lymphoprep, and SepMate would enhance clarity and help readers understand the procedural differences.

Figure 3: The upper, middle, and lower layers should be clearly labeled.

Figure 4 and 5: Data should include mean ± standard deviation, and statistical significance between groups (if any) should be indicated.

Reviewer #2: The article describes the development and evaluation of FlowMagic™, a new two-layer insert device for isolating human peripheral blood mononuclear cells (PBMCs) from whole blood, aiming to reduce red blood cell (RBC) and granulocyte (GRA) contamination commonly encountered in conventional methods. The device was compared with existing techniques (SepMate and Lymphoprep). The study addresses a significant technical challenge in the field and could be a useful tool for both research and medical laboratories, especially given the ongoing issue of RBC and GRA contamination when delayed processing is necessary due to sample transport or logistical factors.

Major Points for Improvement and Clarification:

• The experiments seem to include five volunteer donors, which is a reasonable starting point for a proof-of-concept study. However, the manuscript should clearly state whether any statistical analyses were performed, and if so, specify which tests were used, exact p-values, and confidence intervals, particularly given the sample size. This would strengthen the validity of the findings.

Specific comment:

o Was any statistical test performed for the comparisons described in the results (e.g., lines 140–141 and 148–153 regarding granulocyte contamination)? If so, please specify the tests and results in the text or figure legends.

• While the referenced figures (e.g., Fig. 4 and 5) demonstrate improvements, including actual numerical data, summary tables in the Results section would facilitate understanding and independent assessment. Mean values ± standard deviation, exact cell counts, and percentage improvements or fold-reduction in contamination would make the benefits more explicit.

Specific comments:

o In lines 136–138, it is stated that RBC contamination for all three methods increased after 24 and 72 hr, but Fig. 4d–f does not show an increase for FlowMagic™. How was the increase in contamination for FlowMagic™ determined? Please clarify.

o From lines 138–140, the manuscript mentions that the FlowMagic™ method excludes RBC contamination. Please clarify how this was determined, especially in light of the previous statement about increased contamination.

• More information on the time required and ease of use of the FlowMagic™ device would be helpful. A brief comparison with the workflow of existing devices would also be valuable for readers considering adopting this technology.

Minor Comments:

• In line 94, the question mark should be removed, and the parenthesis with “brand” should be replaced with the actual brand name.

Reviewer #3: Dear Authors,

Thank you for submitting your manuscript entitled “Development of a New Cell Isolation Device FlowMagic™” for consideration. Your work presents a timely and relevant advancement in the field of peripheral blood mononuclear cell (PBMC) isolation, particularly addressing the well-known challenges of red blood cell (RBC) and granulocyte (GRA) contamination in delayed blood processing. I commend your efforts in developing the FlowMagic™ device, and your comprehensive side-by-side comparison with SepMate and standard Lymphoprep techniques is a valuable contribution to the field.

Below, I offer a detailed critique aimed at enhancing the clarity, rigour, and impact of your manuscript.

Major Comments

1. Statistical Analysis and Rigor

While your data are promising, all findings are currently presented without formal statistical analysis. This is a significant limitation. For a manuscript to meet the standards of PLOS ONE, particularly when making comparative claims, appropriate statistical testing (e.g., ANOVA with post-hoc tests, paired t-tests, or non-parametric equivalents) must be applied.

Action: Please include appropriate statistical analyses across timepoints and isolation methods for all quantitative data (e.g., cell recovery, contamination levels, flow cytometry results). Report p-values and define significance thresholds in the methods section.

2. Sample Size and Justification

The study includes five donors, but no rationale is given for the chosen sample size, and inter-donor variability is not discussed in detail.

Please provide justification for the sample size, or consider increasing the number of donors. Additionally, clarify whether the same individuals were used across the three timepoints to control for inter-subject variability.

3. Figures 4 and 5 – Resolution and Readability

Thank you for including Figures 4 and 5, which provide critical support for your conclusions. However, both figures currently suffer from low resolution, limiting their interpretability:

Axis labels and tick marks are small and difficult to read.

No error bars or indicators of statistical variance are shown.

Legends do not consistently include units or definitions of abbreviations.

Data points are plotted but lack context (e.g., no median or confidence intervals).

Action: Please resubmit Figures 4 and 5 in high resolution (≥300 DPI). Ensure all panels include:

Clearly labeled axes (with units),

Error bars where applicable,

Statistical significance annotations (e.g., p < 0.05),

Expanded legends to enhance clarity.

Additionally, consider using consolidated bar graphs or boxplots instead of multiple dot plots, which may improve readability and facilitate comparisons.

4. Device Limitations and Overstatements

Your discussion makes strong claims regarding the superiority and novelty of FlowMagic™. While your data are promising, such claims should be more cautiously framed unless supported by broader benchmarking or independent validation.

Please temper phrases such as “this has not been resolved until now” and consider including limitations more explicitly. For example:

The inability of FlowMagic™ to isolate plasma simultaneously may be relevant for translational settings.

Functional downstream assays (e.g., cytokine release, ELISPOT) are not yet presented and should be acknowledged as needed future work.

5. Language and Editing

The manuscript would benefit from language polishing to improve clarity, precision, and readability. There are occasional grammatical issues and ambiguous terms (e.g., “corning tubes?” on pg. 8) that should be corrected.

I recommend engaging a professional scientific editor or fluent English speaker to review the entire text.

Minor Comments and Recommendations

Methods: Clarify if the same donor samples were used for all three timepoints across isolation methods.

Figures 1–2: The schematic CAD illustrations are useful; however, inclusion of dimensions would enhance technical comprehension.

References: The citations are appropriate and up to date. However, you may wish to add references to microfluidic PBMC isolation methods, which are emerging in this space and would provide valuable context for FlowMagic™'s niche.

Ethics: Approvals are clearly presented in both the manuscript and translated documents. Thank you for providing these in full.

Conclusion and Recommendation

Your study introduces a potentially impactful innovation in PBMC isolation, especially for delayed blood processing in clinical or field settings. The technical approach and device design are commendable, and your experimental comparisons are thoughtfully conceived.

That said, the manuscript currently requires major revisions to meet the scientific and editorial standards of PLOS ONE. In particular, the addition of statistical analysis, high-quality figures, and a more balanced discussion of strengths and limitations will substantially improve the manuscript’s impact and readability.

Once revised accordingly, I believe the work would make a valuable contribution to the literature on cell isolation technologies.

Thank you again for your submission and your thoughtful work in this important area.

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Dr Panicos Shangaris

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

PLoS One. 2025 Oct 22;20(10):e0334936. doi: 10.1371/journal.pone.0334936.r003

Author response to Decision Letter 1

8 Sep 2025

Response to reviewer 1

Reply: We thank the reviewer for their comment. We have included specific numerical data, presenting median values with corresponding interquartile ranges, to provide clear evidence of comparative performance (Fig. 4-6, Table S1-S3). The results demonstrate that FlowMagic™ achieved significantly better outcomes than both SepMate and Lymphoprep methods, with statistical significance established at p < 0.01. (Line: 23-38)

Reply: We thank the reviewer for this valuable suggestion. We have addressed the above issue in the abstract section of our manuscript. We have revised it as follows: These findings indicate that the PBMC isolation method using FlowMagic™ is advantageous in the isolation process for research and laboratory tests to prevent contamination by RBCs and GRAs. (Lines: 39-41)

Reply: We increased the sample size to 10. For the data from a total of ten volunteers presented in Figures 4, 5, and 6, the Wilcoxon signed-rank test was used with Bonferroni correction for multiple comparisons as a non-parametric alternative. All data were visualized using box plots. A significance level of 0.05 was used for all statistical tests. In response to your recommendation, we have included detailed summary tables in the Supporting Information (Tables S1-S3) that present mean values ± standard deviation. (Figs 4, 5, and 6, table S1-S3)

4. Some suggestions for modifying the figures:

Figure 2: A schematic diagram comparing the protocols of FlowMagic, Lymphoprep, and SepMate would enhance clarity and help readers understand the procedural differences.

Reply: We thank the reviewer for this valuable suggestion. We have addressed this concern by incorporating a comprehensive schematic comparison of the three protocols in Figure 2b. This diagram clearly illustrates the procedural differences between FlowMagic�, Lymphoprep, and SepMate protocols, including the key steps of density gradient centrifugation, and cell collection phases. The visual representation in Figure 2b provides readers with an immediate understanding of the methodological distinctions between these three approaches, thereby enhancing the clarity and accessibility of the comparative analysis as requested by the reviewer (Fig 2).

Figure 3: The upper, middle, and lower layers should be clearly labeled.

Reply: We thank the reviewer for this important suggestion regarding layer identification. In response to this comment, we have added clear labels for the upper, middle, and lower layers in Figure 3a, 3b, and 3c. These labels now provide precise identification of each density gradient layer, facilitating better understanding of the spatial distribution of different cell populations and debris throughout the separation process. This enhancement improves the interpretability of the results and allows readers to more effectively correlate the visual observations with the quantitative data presented in the manuscript. (Fig 3)

Figure 4 and 5: Data should include mean ± standard deviation, and statistical significance between groups (if any) should be indicated.

Reply: For the data from a total of ten volunteers presented in Figures 4, 5, and 6, the Wilcoxon signed-rank test was used with Bonferroni correction for multiple comparisons as a non-parametric alternative. All data were visualized using box plots. A significance level of 0.05 was used for all statistical tests. In response to your recommendation, we have included detailed summary tables in the Supporting Information (Tables S1-S3) that present mean values ± standard deviation and p-value. (Figs 4, 5, and 6, table S1-S3).

Response to reviewer 2

Major Points for Improvement and Clarification:

Specific comment:

• Was any statistical test performed for the comparisons described in the results (e.g., lines 140–141 and 148–153 regarding granulocyte contamination)? If so, please specify the tests and results in the text or figure legends.

Reply: An additional validation study was conducted using samples from five volunteers to increase the sample size to a total of 10. For the data from ten volunteers presented in Figures 4, 5, and 6, Wilcoxon signed-rank test was used with Bonferroni correction for multiple comparisons as a non-parametric alternative. All data were visualized using box plots. A significance level of 0.05 was considered statistically significant for all statistical tests. In response to this comment, we have added the above information in the figure legends. (Lines: 182-231, Figs 4 and 5.)

Reply: Thank you for this valuable suggestion. We agree that providing comprehensive numerical data would greatly enhance the clarity and interpretability of our findings. In response to your recommendation, we have included detailed summary tables in the Supporting Information (Tables S1-S3) that present mean values ± standard deviation, exact cell counts, and percentage improvements for all three isolation methods. These tables provide the quantitative data necessary for independent assessment of the methodological improvements and contamination reduction achieved with each approach. The inclusion of these comprehensive datasets in the Supporting Information directly addresses your concern about facilitating understanding while maintaining the flow of the main Results section. We believe these additional data will allow readers to fully evaluate the magnitude of improvements demonstrated in our comparative analysis. (Lines: 182-260, Table S1-S3)

Specific comments:

• In lines 136–138, it is stated that RBC contamination for all three methods increased after 24 and 72 hr, but Fig. 4d–f does not show an increase for FlowMagic™. How was the increase in contamination for FlowMagic™ determined? Please clarify.

Reply: We thank the reviewer for this valuable suggestion. The aforementioned description has been modified as shown below. We recognize that the previous statement may have caused confusion. At 48 and 72 hours after blood collection, the results showed an increase in the amount of RBC contamination among the two methods except FlowMagic� (Figs 3b-c and 4b). (Lines: 182-204)

• From lines 138–140, the manuscript mentions that the FlowMagic™ method excludes RBC contamination. Please clarify how this was determined, especially in light of the previous statement about increased contamination.

Reply: We thank the reviewer for this valuable suggestion. The aforementioned description has been modified as shown below. We recognize that the previous statement may have caused confusion. Although FlowMagic� yielded less WBC than other methods for blood samples that were incubated for 48-72 hours, it was able to exclude RBC contamination and greatly reduce granulocyte contamination (Fig. 4b). (Lines: 182-204)

Minor Comments:

• In line 94, the question mark should be removed, and the parenthesis with “brand” should be replaced with the actual brand name.

Reply: Thank you very much for your comments. We have already addressed this issue. (Lines: 106)

Response to reviewer 3

Reviewer #3: Dear Authors,

Below, I offer a detailed critique aimed at enhancing the clarity, rigour, and impact of your manuscript.

Major Comments

1. Statistical Analysis and Rigor

Reply: An additional validation study was conducted using samples from five volunteers. For the data from a total of ten volunteers presented in Figures 4, 5, and 6, the Wilcoxon signed-rank test was used with Bonferroni correction for multiple comparisons as a non-parametric alternative. All data were visualized using box plots. A value of 0.05 was considered significant for all statistical tests. In response to your recommendation, we have included detailed summary tables in the Supporting Information (Tables S1-S3) that present mean values ± standard deviation, exact cell counts, and percentage improvements for all three isolation methods. (Figs 4, 5, and 6, table S1-S3)

2. Sample Size and Justification

The study includes five donors, but no rationale is given for the chosen sample size, and inter-donor variability is not discussed in detail.

Reply: An additional validation study was conducted using samples from five volunteers to increase the sample size to ten. For the data from a total of ten volunteers presented in Figures 4, 5, and 6, Wilcoxon signed-rank test was used with Bonferroni correction for multiple comparisons as a non-parametric alternative. All data were visualized using box plots. A significance level of 0.05 was used for all statistical tests. Parallel PBMC isolations were performed by three different techniques within 24, 48, and 72 hours after blood collection from the same volunteers. (Line: 102-103)

3. Figures 4 and 5 – Resolution and Readability

Thank you for including Figures 4 and 5, which provide critical support for your conclusions. However, both figures currently suffer from low resolution, limiting their interpretability:

Axis labels and tick marks are small and difficult to read.

No error bars or indicators of statistical variance are shown.

Legends do not consistently include units or definitions of abbreviations.

Data points are plotted but lack context (e.g., no median or confidence intervals).

Action: Please resubmit Figures 4 and 5 in high resolution (≥300 DPI). Ensure all panels include:

Clearly labeled axes (with units),

Error bars where applicable,

Statistical significance annotations (e.g., p < 0.05),

Expanded legends to enhance clarity.

Additionally, consider using consolidated bar graphs or boxplots instead of multiple dot plots, which may improve readability and facilitate comparisons.

Reply: We thank the reviewer for this valuable suggestion. We have addressed this concern through improvements in Fig 4, 5, and 6. An additional validation study was conducted using samples from five volunteers. For the data from a total of ten volunteers presented in Figures 4, 5, and 6, the Wilcoxon signed-rank test was used with Bonferroni correction for multiple comparisons as a non-parametric alternative. All data were visualized using box plots. A significance level of 0.05 was used for all statistical tests.

4. Device Limitations and Overstatements

Please temper phrases such as “this has not been resolved until now” and consider including limitations more explicitly. For example:

Reply: We thank the reviewer for this valuable suggestion. We have already deleted the above phrase from our manuscript.

The inability of FlowMagic™ to isolate plasma simultaneously may be relevant for translational settings.

Functional downstream assays (e.g., cytokine release, ELISPOT) are not yet presented and should be acknowledged as needed future work.

Reply: We thank the reviewer for this valuable suggestion. We have already added the above phrase into the discussion s

Attachment

Submitted filename: 250908 Rebuttal letter for Reviewer.docx

pone.0334936.s005.docx^{(32.3KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0334936.r004

Decision Letter 1

Jeffrey Chalmers

6 Oct 2025

<p>Development of a New Cell Isolation Device FlowMagicTM

PONE-D-25-20717R1

Dear Dr. Yoshida,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support .

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Jeffrey Chalmers, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: The authors have addressed the reviewers' comments well. The reviewers have edited the figures and included appropriate statistical analysis to backup their claims.

Reviewer #2: (No Response)

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: No

Reviewer #2: No

**********

PLoS One. doi: 10.1371/journal.pone.0334936.r005

Acceptance letter

Jeffrey Chalmers

PONE-D-25-20717R1

PLOS ONE

Dear Dr. Yoshida,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

* All references, tables, and figures are properly cited

* All relevant supporting information is included in the manuscript submission,

* There are no issues that prevent the paper from being properly typeset

You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps.

Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing.

If we can help with anything else, please email us at customercare@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Jeffrey Chalmers

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Summary of PBMC composition isolated by three different methods from whole blood after 24-hour storage.

(TIF)

pone.0334936.s001.tif^{(116.4KB, tif)}

S2 Table. Summary of PBMC composition isolated by three different methods from whole blood after 48-hour storage.

(TIF)

pone.0334936.s002.tif^{(146.4KB, tif)}

S3 Table. Summary of PBMC composition isolated by three different methods from whole blood after 72-Hour storage.

(TIF)

pone.0334936.s003.tif^{(138.8KB, tif)}

Attachment

Submitted filename: renamed_cd3c8.docx

pone.0334936.s004.docx^{(14.7KB, docx)}

Attachment

Submitted filename: 250908 Rebuttal letter for Reviewer.docx

pone.0334936.s005.docx^{(32.3KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting information files.

[pone.0334936.ref001] 1.Filbert H, Attig S, Bidmon N, Renard BY, Janetzki S, Sahin U, et al. Serum-free freezing media support high cell quality and excellent ELISPOT assay performance across a wide variety of different assay protocols. Cancer Immunol Immunother. 2013;62(4):615–27. doi: 10.1007/s00262-012-1359-5 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0334936.ref002] 2.Smith JG, Joseph HR, Green T, Field JA, Wooters M, Kaufhold RM, et al. Establishing acceptance criteria for cell-mediated-immunity assays using frozen peripheral blood mononuclear cells stored under optimal and suboptimal conditions. Clin Vaccine Immunol. 2007;14(5):527–37. doi: 10.1128/CVI.00435-06 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0334936.ref003] 3.Aziz N, Margolick JB, Detels R, Rinaldo CR, Phair J, Jamieson BD, et al. Value of a quality assessment program in optimizing cryopreservation of peripheral blood mononuclear cells in a multicenter study. Clin Vaccine Immunol. 2013;20(4):590–5. doi: 10.1128/CVI.00693-12 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0334936.ref004] 4.Macchia I, Urbani F, Proietti E. Immune monitoring in cancer vaccine clinical trials: critical issues of functional flow cytometry-based assays. Biomed Res Int. 2013;2013:726239. doi: 10.1155/2013/726239 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0334936.ref005] 5.Newell EW, Sigal N, Bendall SC, Nolan GP, Davis MM. Cytometry by time-of-flight shows combinatorial cytokine expression and virus-specific cell niches within a continuum of CD8+ T cell phenotypes. Immunity. 2012;36(1):142–52. doi: 10.1016/j.immuni.2012.01.002 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0334936.ref006] 6.Böyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl. 1968;97:77–89. [PubMed] [Google Scholar]

[pone.0334936.ref007] 7.Grievink HW, Luisman T, Kluft C, Moerland M, Malone KE. Comparison of three isolation techniques for human peripheral blood mononuclear cells: cell recovery and viability, population composition, and cell functionality. Biopreserv Biobank. 2016;14(5):410–5. doi: 10.1089/bio.2015.0104 [DOI] [PubMed] [Google Scholar]

[pone.0334936.ref008] 8.Muise A, White L, Badowski M, Harris DT. Analysis of high-throughput processing methods for peripheral blood cell isolation. Biopreserv Biobank. 2022;20(3):302–5. doi: 10.1089/bio.2021.0100 [DOI] [PubMed] [Google Scholar]

[pone.0334936.ref009] 9.Nilsson C, Aboud S, Karlén K, Hejdeman B, Urassa W, Biberfeld G. Optimal blood mononuclear cell isolation procedures for gamma interferon enzyme-linked immunospot testing of healthy Swedish and Tanzanian subjects. Clin Vaccine Immunol. 2008;15(4):585–9. doi: 10.1128/CVI.00161-07 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0334936.ref010] 10.Song S, Goodwin J, Zhang J, Babbitt B, Lathey JL. Effect of contaminating red blood cells on OKT3-mediated polyclonal activation of peripheral blood mononuclear cells. Clin Diagn Lab Immunol. 2002;9(3):708–12. doi: 10.1128/cdli.9.3.708-712.2002 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0334936.ref011] 11.McKenna KC, Beatty KM, Bilonick RA, Schoenfield L, Lathrop KL, Singh AD. Activated CD11b+ CD15+ granulocytes increase in the blood of patients with uveal melanoma. Invest Ophthalmol Vis Sci. 2009;50(9):4295–303. doi: 10.1167/iovs.08-3012 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0334936.ref012] 12.Yi P-C, Zhuo L, Lin J, Chang C, Goddard A, Yoon OK. Impact of delayed PBMC processing on functional and genomic assays. J Immunol Methods. 2023;519:113514. doi: 10.1016/j.jim.2023.113514 [DOI] [PubMed] [Google Scholar]

[pone.0334936.ref013] 13.Qu H-Q, Kao C, Garifallou J, Wang F, Snyder J, Slater DJ, et al. Single cell transcriptome analysis of peripheral blood mononuclear cells in freshly isolated versus stored blood samples. Genes (Basel). 2023;14(1):142. doi: 10.3390/genes14010142 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Development of a new cell isolation device FlowMagicTM

Tomoyuki Yoshida

Yoshiaki Sakamoto

Aya Tsuruta

Risa Kimura

Nozomi Shiozawa

Takeyuki Kotaka

Roles

Abstract

Introduction

Materials and methods

PBMC isolation

Fig 1. 3D CAD images of FlowMagic™.

Fig 2. FlowMagic™ isolation workflow and schematic presentation of the three isolation techniques.

Immunophenotyping of PBMC populations

Statistical analysis

Results

FlowMagic™ reduces RBC and GRA contamination during PBMC isolation

Fig 3. Time-dependent changes in the state of isolation of blood.

Fig 4. Comparative performance of cell isolation methods (FlowMagic™, SepMate, Lymphoprep) following extended whole blood storage.

Fig 5. Comparative recovery rates of cell isolation methods (FlowMagic™, SepMate, Lymphoprep) following extended whole blood storage.

Population composition

Fig 6. PBMC composition by flow cytometry of cell isolation methods (FlowMagic™, SepMate, Lymphoprep) following extended whole blood storage.

Discussion

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Author response to Decision Letter 0

Decision Letter 0

Jeffrey Chalmers

Roles

Author response to Decision Letter 1

Decision Letter 1

Jeffrey Chalmers

Roles

Acceptance letter

Jeffrey Chalmers

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases

Development of a new cell isolation device FlowMagic^TM