Cryopreserved Platelets in a Non-Toxic DMSO-Free Solution Maintain Hemostatic Function In Vitro

Round 1

Reviewer 1 Report

In this manuscript, Ehn et al presented data comparing platelet functions after cryopreserving using cryoprotectant agent (CPA) with DMSO and without DMSO (only saline). CPA without DMSO appeared to exhibit improved recovery with a higher number of platelets in the final product. With controlled freezing, the viability of platelets preserved with CPA without DMSO was comparable to that of established DMSO-containing CPA. These results are promising.

 

Comments:

# line 4, please check the names for the 2nd and 3rd author.

# fig.2, no error bar was shown. The authors wrote “A two-tailed t-test was performed between the DMSO and DMSO-free groups”, but asterisks were shown both for DMSO and DMSO-free groups, please double check this.

# fig 4, could the authors show representative curves of TEG tracings? This could help readers understand the message. Separate clotting time, clot formation time and MCF into 3 panels may help visualize the differences between groups.

# fig.5, please indicate the length of the bar.

# line 428, please indicate the name of company.

# line 478, please also indicate what the error bars represent, SD or SEM.

# line 487, It seems redundant to have this conclusion section.

# How does the expression of platelets look like if controlled freezing (CF) was used with DMSO-free CPA? I assume that the data in fig 2 represents data obtained without CF?

# line 328-334, it seems to me that the higher platelet concentration is the only difference between platelet concentrate resuspended in 50mL or 100mL, I don’t quite get what argument the authors are trying to convey, please further clarify.

Author Response

First, we would like to thank the associated editor and the reviewers for their valuable times and prompt responses. All in attempt to improve this paper. The following are our point-by-point responses to reviewers ‘comments whereby each comment is copied and followed by our responses. All relevant revisions are highlighted, in the revised manuscript for reviewers´ convenience. 

Please see the attachment for our response.

Author Response File: Author Response.docx

Reviewer 2 Report

Ehn et al., report a novel process for manufacturing cryopreserved platelets, using saline rather than DMSO as a cryoprotectant. The study design and experimental methods are appropriate to address the research question, although additional rationale and explanation for some of the subsequent experimental stages would be valuable. The manuscript is well written and adds value to the growing body of literature surrounding cryopreserved platelets. The below revisions should be made to improve clarity.

 

 

Introduction

* Page 2, line 46: It should be made clearer what ‘recover’ refers to. Perhaps “50-70% of platelets are recovered”, or “an in vitro recovery of 50-70%”

* Page 2, line 59: Consider including reference to more recent attempts of using DMSO adjuvants, such as ice recrystallisation inhibitors or alternatives to remove DMSO prior to freezing, as described by Waters et al., 2020. doi: 10.1016/j.cryobiol.2020.07.003; Dumont et al., 2023 doi: 10.1111/trf.17464; Yi et al., 2023 doi: 10.1111/vox.13483. 

 

Results

Page 4, line 138: Mention GPVI in the text

Page 4, line 150-151: state ‘data not shown’ for activation data

Page 4, line 152-155: provide p-value for ‘significant’ difference between fresh and frozen for PAC-1+ platelet populations.

Page 5, line 176: Did the authors look at markers other than JC-1 in the UCRF vs CRF experiments? Please comment.

Page 5, line 195: why was the clot formation time of the fresh platelets so much shorter than cryopreserved platelets? This does not fit with the current literature, not with the fact that as per above you are adding more platelets to the frozen groups than the fresh. Please comment on the variation observed in the CFT in DMSO-free platelets (Figure 4, green bar)

Page 5, line 198: because of the flow of the manuscript (with the methods at the end), it is unclear at this point what JC-1+ 200x10^9/L means. Perhaps included an explanatory sentence, including why this approach was taken. Based on the JC-1 results you would be ‘loading’ twice as many DMSO-free platelets into the thromboelastometry experiments than the DMSO group. Presumably the majority of these platelets would be PS positive and therefore likely to contribute a procoagulant surface.

Page 5, line 201: Suggest changing ‘increased’ to ‘higher’

Page 6, line 213: Figure 1 doesn’t seem to be the correct figure. Perhaps 6.

 

Discussion

Page 9, line 303-318: Given that the DMSO-free platelets show reduced MCF and glycoproteins, beyond the reduction already seen with the standard DMSO method, can the authors provide their opinion as to whether this is an acceptable trade off?

 

Page 9, line 325: Consider revising the statement regarding DMSO toxicity and the need for dilution in large volumes of plasma. Given that the platelets are hyperconcentrated prior to freezing, there is relatively little DMSO content remaining in the product.

 

While on this point, the authors should also discuss the rationale for centrifugation of the DMSO-free group. If saline isn’t toxic is it necessary to concentrate the platelets and then resuspend them? This adds complexity to the process, both pre and post freeze/thaw

 

Page 9, line 331: state that the reference used to support the ‘leveling out’ of the association between the platelet concentration and MCF was not referring to cryopreserved platelets.

Page 9, line 349-350: the penultimate and final sentence appear to be replicated. The authors should also consider including the recent publication by Johnson et al., 2023 (DOI: 10.1038/s41598-023-28352-2) describing platelet subpopulations in cryopreserved platelets.

Greater rationale for the experiments performed with less plasma (100 vs 50mL) should be included. Obviously having a higher concentration of platelets may translate to faster clotting and a stronger clot when measured in this in vitro setting. However, how will this translate to an effect upon transfusion when the bag of platelets is diluted in the larger blood volume anyway.

 

Please discuss the mechanism by which saline may be acting as a cryoprotectant. Can the authors speculate on whether the cryoprotective effect of saline is expected to be suitably long-term? They have tested platelets out to 4 months, but there is new data suggesting that DMSO platelets could be stored at -80 for 12 years.   

 

Methods

Page 10, line 375: Please include the volume and type of solution used for standard reconstitution, given that this detail is important to interpret stage 2 and stage 3 manufacture.

Page 11, line 396: Correct reference to Figure 1, presumably it should be 6.

Page 11, line 428: add concentration and company details for ADP and collagen

Page 11, line 438-441: Please provide details of staining buffer used for PMP analysis. Given that very low Annexin-V binding was observed in all treatment groups, please confirm that sufficient calcium was present in the buffer.

 

Figure 1.

Perhaps re-order the bars on the graph to match the order of mentioning in the text.

 

Figure 2.

Include error bars on the graph.

 

Figure 4.

Suggest separating the different parameters into 3 graphs, so that the axis can be more appropriately applied to each parameter. While the MCF is statistically different, it is difficult to appreciate due to the height of the axis.

More details are required in the legend to understand what the nomenclature of the groups represent to ensure the figure is standalone and does not require reference to the methods section.

 

Figure 5.

Do the authors have TEM for fresh platelets to demonstrate the morphology/proportion of the normal platelet morphology. Check ‘assessed’ in legend. Is this the correct word?

 

Figure 6.

Add the volume of plasma typically used for reconstitution to stage 1 and 2. Add CRF or UCRF (as relevant) to stage 3.

Author Response

First, we would like to thank the associated editor and the reviewers for their valuable times and prompt responses. All in attempt to improve this paper. The following are our point-by-point responses to reviewers ‘comments whereby each comment is copied and followed by our responses. All relevant revisions are highlighted, in the revised manuscript for reviewers´ convenience. 

Please see the attachment for our response.

Author Response File: Author Response.docx

Reviewer 3 Report

In this paper the authors demonstrate a novel way to freeze platelets using freezing media a non-toxic DMSO-free solution  while maintaining hemostatic function in vitro. This is a solid paper from a respected researchers in the field and the paper is well written an structured

 

Minor comments.

Figure 1. Data could be presented as number/ mL instead of per unit.

Figure 2. Error bars are missing form the graphs

Figure 1, figure 3 and figure 4. What the errorbars are depicting is missing from the figure text.

Author Response

First, we would like to thank the associated editor and the reviewers for their valuable times and prompt responses. All in attempt to improve this paper. The following are our point-by-point responses to reviewers ‘comments whereby each comment is copied and followed by our responses. All relevant revisions are highlighted, in the revised manuscript for reviewers´ convenience. 

Please see attachment for our response.

Author Response File: Author Response.docx