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On 2015 Sep 23, Angelo Gaitas commented:

The entire response appears in the PLOS1 comment section under response: http://www.plosone.org/article/comments/info:doi/10.1371/journal.pone.0127219

In two recent articles [1, 2] two techniques for removing or inactivating blood borne pathogens were introduced. The initial experiments were performed in vitro under simplified conditions. First, the primary achievement of the PDT work deserves clarification [1]. PDT is a powerful therapeutic modality, but its clinical application has been hampered by the inability of light to penetrate deep layers of the tissue, which is mainly due to hemoglobins in the blood readily absorbing photons. Utilizing a millimeter- diameter transparent tube for extracorporeal blood circulation allows PDT to function well despite the presence of hemoglobins in blood. Another point that deserves clarification is that the tube capturing device is not a microfluidic device [2]. This technique can be adapted using existing medical tubing without the need for complicated microfluidics and micro-fabrication. The device is a medical tube that has been chemically modified using simple steps to adapt the internal surface for cell capturing. 

We would like to take this opportunity to respond to concerns brought up in [3]. We start off by addressing concern (1), which speculates about the possibility of overheating during the use of near IR light. Our control data (Fig.3 and Fig.4 of [3]), confirmed that controls illuminated without photosensitizer-antibody conjugates did not undergo cell death, whereas those with photosensitizer-antibody conjugates underwent significant cell death under identical conditions. Thus it is clear from our data that temperature did not affect the outcome. It has been shown that 660 nm irradiation is safe and effective [4-6]. 

Moving on to concern (2) part (a) that brings up the problem of using the CD-44 antigen as a target. Limitations of antibody specificity are common knowledge and not unique to CD-44, but to all antibodies. To our knowledge, a targeting method that exclusively binds only to cancer cells does not yet exist, making the use of such a compound an unreasonable standard for publication. We used CD-44 antibody to demonstrate feasibility. As targeting methodologies advance and better selectivity to target cells becomes available, this technique will have improved selectivity. Our experiments were designed to avoid non-specific damage to other cells by pre-staining pure cancer cells with the photosensitizer-antibody conjugates and subsequently removing extra free conjugates before spiking into blood (described in detail in [1]). This elimination of the possibility of side effects due to undesired binding to other blood cells and excess free photosensitizer-antibody conjugates precluded the need for a toxicity study, particularly because we were at the proof-of-principle stage.

Part (b) of concern (2) suggests that we may have caused non-specific damage to non-cancerous cells by ROS' convection in the blood stream. We believe that this is highly unlikely. One of the authors has been conducting research focusing on ROS and PDT for years, in collaboration with other researchers [7-15]. This research demonstrated that PDT is extremely selective to targeted cells [13]. 

 Part (c) of concern (2) states that we should have used additional cytotoxicity assays, such as Annexin V, TUNEL, and MTT. However, because none of these techniques are cell-type specific, they would be useless for the particular objective they were suggested. Once our line of investigation reaches a more mature stage, we plan to undertake more useful studies, such as applying separate fluorescent tags, or radio labels, in addition to a cell viability assay and analyzing cell death with a cell sorting technology, such as FACS, MACS, density gradient centrifugation, etc.  

Concern (3) is that the capturing work [2] lacked purity confirmation concerning non-specific capturing of blood cells. Though purity confirmation is critical in diagnostic testing, our work was strictly limited to in vitro conditions, using spiked pure PC-3 cells as a model. To visualize and quantify PC-3 cells in the presence of whole blood, PC-3 cells were pre-labeled using a fluorescence tag (Calcein AM) and the extra free dye was subsequently removed before spiking PC-3 cells into blood. Because only PC-3 cells can have fluorescence in the blood mixture, and because quantification was based on fluorescing cells, false-positive results from other blood cells can be reasonably excluded. Furthermore, if other blood cells were captured but not identified by our detection method our data would then indicate that the simple tube captured cancer cells despite being blocked by other blood cells. If our technique were applied to CTC diagnosis, independent isolation procedures could be used to ensure the purity of captured cells. In contrast, if used for therapy, the purity of captured cells would not be as critical, provided that CTCs are effectively removed. If, by chance, capturing is hampered by accumulation of non-specific binding in filtering the entire blood volume, this issue can be addressed with strategies such as scaling up the tube and carefully determining the tube dimensions, flow rate, frequency of tube replacements, etc. 

Finally, concern (4), points out that the experimental conditions were not translatable to clinical applications. Part (a) regards scaling up the system to show high throughput. The concept of extracorporeal cleansing of the entire blood volume has been used for years in cases such as hemodialysis. We already are working on optimizing the technique for larger blood volume processing. Part (b) of concern (4) discusses the static no-flow condition as being unrealistic. This issue was brought up during the review process, and we provided with our results showing data under constant flow conditions by peristaltic pump (to be published in future publication). The reviewers agreed that the use of a no-flow condition as a conservative approach during a proof-of-concept stage was appropriate.

Despite its preliminary nature, we believe that our work communicates novel ideas, an important objective of research and publication. Given the number of research articles dealing with diagnostics and microfluidics, perhaps a further point of confusion came about by thinking of our work in those terms. We want to clarify that diagnostics were not the primary objective in our work. Furthermore, as it becomes evident by this response our experimental design was carefully devised to minimized unnecessary interferences. We hope that this response mitigates any confusion and addresses the concerns raised. 

1. Kim G, Gaitas A. PloS One. 2014;10(5):e0127219-e.
2. Gaitas A, Kim G. PLoS One. 2015;10(7):e0133194. doi: 10.1371/journal.pone.0133194.
3. Marshall JR, King MR. DOI: 101007/s12195-015-0418-3. 2015;First online.
4. Ferraresi C, et al. Photonics and Lasers in Medicine. 2012;1(4):267-86.
5. Avci P, et al. Seminars in cutaneous medicine and surgery; 2013.
6. Jalian HR, Sakamoto FH. Lasers and Light Source Treatment for the Skin. 2014:43.
7. Ross B, et al. Biomedical Optics, 2004
8. Kim G, et al. Journal of biomedical optics. 2007;12(4):044020--8.
9. Kim G, et al Analytical chemistry. 2010;82(6):2165-9.
10. Hah HJ, et al. Macromolecular bioscience. 2011;11(1):90-9.
11. Qin M, et al. Photochemical & Photobiological Sciences. 2011;10(5):832-41.
12. Wang S, et al. et al. Lasers in surgery and medicine. 2011;43(7):686-95.
13. Avula UMR, et al.Heart Rhythm. 2012;9(9):1504-9.
14. Kim G, et al. R. Oxidative Stress and Nanotechnology, 2013. p. 101-14.
15. Lou X, et al. E. Lab on a Chip. 2014;14(5):892-901.
16. https://www.roswellpark.org/patients/treatment-services/innovative-treatments/photodynamic-therapy.
17. Yin H, et al. Artificial organs. 2014;38(6):510-5.
18. Yin H, et al. Journal of Photochemistry and Photobiology B: Biology. 2015.

On 2015 Aug 29, Michael King commented:

Our recent commentary discusses this paper:

http://link.springer.com/article/10.1007/s12195-015-0418-3

In cancer research, the discovery and study of circulating tumor cells (CTCs) have seemed to open a world of possibilities. We now have the potential to gain cellular and molecular understanding of individual cases of metastatic cancer without invasive procedures. This area of research is, however, not without some basic pitfalls. In this commentary, we address some of these pitfalls by considering two recent examples in the published literature and discuss ways to overcome their limitations with the hope of informing those who may be entering the growing field of CTC research. Careful research design should always be followed to prevent incomplete or misleading studies from entering the literature, and thereby avoid setting back this burgeoning field.

On 2015 Aug 29, Michael King commented:

Our recent commentary discusses this paper:

http://link.springer.com/article/10.1007/s12195-015-0418-3

In cancer research, the discovery and study of circulating tumor cells (CTCs) have seemed to open a world of possibilities. We now have the potential to gain cellular and molecular understanding of individual cases of metastatic cancer without invasive procedures. This area of research is, however, not without some basic pitfalls. In this commentary, we address some of these pitfalls by considering two recent examples in the published literature and discuss ways to overcome their limitations with the hope of informing those who may be entering the growing field of CTC research. Careful research design should always be followed to prevent incomplete or misleading studies from entering the literature, and thereby avoid setting back this burgeoning field.