PONE-D-23-00813

Cardiopulmonary bypass in a rat model may shorten the lifespan of stored red blood cells by activating caspase-3

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript a rat blood bypass model was used to observe effects of 2 hours of pumping blood through the bypass roller pump on stored and transfused red cells (RBCs). Stored red cells were labelled with FITC, infused into the animal and pumped for 2 hours. The presence of labeled RBCs in circulation was measured at 2, 24 and 48 hrs post infusion. Presence of fluoresce associated with other cells was measured at the same time points. To determine the mechanism of reduced RBC life span, plasma from animals that had been on bypass was incubated in vitro with stored, FITC -labelled RBCs and changes in the RBCs were measured at 2, 24, and 48 hours. RBCs stored in bypass plasma exhibited changes associated with apoptosis such as caspace activation, phosphatidylserine expression and ATP decline. Pre-treatment of RBCs with a caspace inhibitor prior to infusion into rats with by-pass reduced the loss of red cells. Plasma from such experiments, when incubated with RBCs did not induce the apoptosis-related changes which occurred with plasma from animals that received RBCs not treated with the caspace inhibitor. The authors concluded that reduced lifespan of infused stored red cells in animals on by-pass was due to free radical generation during the by-pass phase which activated RBCs caspace and induced these cells to go through apoptosis.

Comments

This is an interesting report on how RBCs could be protected by pretreatment with a caspase inhibitor from damage due to by-pass pumps. There are several questions that come to mind:

These experiments utilize stored red cells which are likely to have developed storage lesions and be more fragile than fresh cells. Would freshly collected red cells produce the same results in these experiments? What storage time of human red cells does 10 days of rat red cell storage represent? This could be mentioned in the discussion.

Free radicals have a very brief life span. How is it possible that plasma from by-pass animals could retain the free radicals? If the free radicals remain in the plasma it should be possible to include anti-oxidant molecule(s) that would block the free radical effect on red cells when they are incubated with the by-pass plasma. Such an experiment would strengthen the free radical argument. Alternatively, there could be other explanations such as complement activation and/or free hemoglobin release. These too could be measured in the by-pass plasma. Similarly performing the experiments with fresh cells which may be less prone to lysis would reduce the possibility that free hemoglobin is involved in the responses.

Phagocytosis of red cells

FITC labels only the outside of red cells. Physical stress/damage from the by-pass pumps likely leads to release red cell membrane fragments or microvesicles while the red cell remains in circulation. In several places it is stated that macrophages and neutrophils can internalize damaged RBCs because there is increased fluorescence associated with these cells. An alternate explanation could be that these cells are internalizing membrane fragments or microvesicles labelled with FITCs or that the fragments and microvesicles are attached to the outside of the cells. Fluorescent microscopy should be able to determine which process is going on.

The manuscript has many grammatical errors. A review by a qualified English writer would be recommended.

Reviewer #2: In this research article Han et al. examined the effects of cardiopulmonary bypass on the recovery and post-transfusion physiology of RBCs. By using a rat model of cardiopulmonary bypass to simulate the clinical procedure applied in cardiac surgery patients they found that bypass increases the erythrophagocytosis and thus, reduces the recovery of stored RBCs. Moreover, by using in vitro experiments which included incubation of stored cells with plasma isolated by bypass or sham bypass animal blood, they detected oxidative stress, caspase-3 activation, cell morphology distortions, externalization of phosphatidylserine and reduced intracellular ATP levels in reconstitutions with bypass plasma. Finally, inhibition of caspase-3 activity seems to prevent in part those negative effects, leading to prolong RBC lifespan after transfusions to rats subjected to bypass.

It is an interesting work and a well-written manuscript. I congratulate the authors especially for the Materials and Methods section which is described in detail. I highlight here, however, some concerns and points that need improvement:

Major comments

- The statement that “the function of transfused cells appears to decline over time” (L19) or “after surgery” (L46) and other similar statements present throughout the manuscript are not literally true. We know that the recovery of stored RBCs varies as a function of numerous donor- and recipient-related factors and that a percentage of transfused cells are removed from the circulation the first 24h post transfusion. However, the “survivals” circulate for a normal period in the recipient. Moreover, 24h recovery is actually the most measured property of transfused RBCs per se. In contrast, other physiological measures of transfused RBCs (deformability, morphology, fragility etc) are only indirectly assessed in vivo by measurements performed in the mixed RBC population of the recipient which consists of donor and recipient cells. We must take into consideration that these different cell populations may mutually change as a result of the transfusion event, that also includes residual donor plasma and transmittance of biological response modifiers, such as extracellular vesicles. In vitro models, like the one used by the authors, can reveal transfusion effects exclusively on donor RBCs but only those related to body temperature and recipient plasma. Despite useful and informative, these models are not equal to a “real” transfusion event.

-Figure 4 results (L242): In the basic experiment reported in the text (L239-243) stored RBC pretreated with caspase-3 inhibitor and then incubated with bypass or sham bypass plasma, while Fig 4 shows another comparison, namely that between inhibitor vs. DMSO treated RBCs with bypass plasma. First of all, DMSO seems to significantly affect the caspase activity (Fig. 2f vs. Fig. 4a). Moreover, ROS staining shown in the representative micrographs of Fig. 4b is inconsistent with the ROS bar graphs shown in Fig. 4c. And even for the comparison DMSO vs. inhibitor, one would expect lower ROS and MDA levels in the inhibitor-treated cells (vs. the DMSO-treated ones) in the context of significantly lower caspase activity (Fig. 4a). However, this is not the case as shown in Fig. 4c-f. Having these in mind, the authors cannot support that the inhibitor reversed the effects of bypass plasma on the ROS, MDA, GSH and SOD levels as reported in L242-243. On the opposite, the results shown n Fig. 5 are consistent with lower shape abnormalities and PS exposure on cells that exhibit lower caspase activation.

-according to the results L271-273 the inhibition of caspase-3 cannot reverse all the negative effects of bypass as reported in the discussion L296-297. Please rephrase.

-L341-342: Beyond doubt, there are additional pathways involved in RBC destruction (including the band-3/IgG/C3b and CD47-related surface removal signaling) and the authors should report them along with the respective citations.

Minor comments

-Introduction is too short.

- A study limitations paragraph is missing.

- In my opinion, red arrow in Fig. 3 indicates an echinocyte and not an acanthocyte.

-There is a mismatch in the a-f labeling between the Figure 4 plates/graphs and the Legend.

- The two sentences in L271-274 need rephrasing.

- “It has been shown that caspase-3 is present in red blood cells…” Another work to support this statement in stored RBCs is that of Kriebardis et al. (TRANSFUSION 2007;47:1212-1220).

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Reviewer #1: No

Reviewer #2: No

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Cardiopulmonary bypass in a rat model may shorten the lifespan of stored red blood cells by activating caspase-3

PONE-D-23-00813R1

Dear Dr. Gou,

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. Please address the minor revisions suggested by Reviewer 2 below before submitting the final version of your manuscript.

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Kind regards,

Heather Faith Pidcoke, MD, MSCI, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: Please report the specific method/kit by which the stored RBCs were labeled with FITC (surface Ab conjugated to FITC? General membrane labeling by cell tracker or another reagent?) (L. 107)

Please rephrase the paragraph of Morphology of stored RBCs (“Abnormal cells were identified as acanthocytes or spherocytes”. L138) according to the text of Fig. 3 legend (“The blue arrow indicates a spherocyte; the red arrow, an echinocyte”. L241)

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Reviewer #1: No

Reviewer #2: No

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