Extracellular Vesicles in Autologous Cell Salvaged Blood in Orthopedic Surgery
Round 1
Reviewer 1 Report
This was an interesting study that I am glad to have had the pleasure of reviewing.
Summary statement: The authors used flow cytometry to measure and characterize EV's from cell salvage technology in elective orthopaedic surgery patients -- finding minimal amounts of EV's from platelets, myeloid and monocytic origin, but high and variable amounts of EV's from erythrocytes.
Pros and cons: Well done study using flow cytometry. Clinical significance of erthrocyte EVs is not clear, even less understood in elective orthopaedic surgery.
Major comments:
I'm unsure of the specific style of the journal requests/permits the current study's structure of "Materials and Methods" after the Discussion. I was looking for the paragraph after the Introduction, personally.
How long were patients followed for their clinical course when examining complications?
The biggest comment I have is the discussion of significance of erythocyte EV's and their high amounts. How does this compare to the literature? Is there a link to higher amounts of erythocyte EV's in allogeneic transfusions? Improving the discussion with more references to clarify the significance of their findings would greatly improve the paper.
Minor comments:
Page 2;Line 41-3: "But as in allogeneic RBC...". This grammar is awkward. Would help to reword for clarity.
Page 4; Line 88-90: "Characteristics..." Grammar awk. needs an "and"
Page 6;Line 170-1: "From the remnant...". Grammar awk.
Page 6; Line 174-5: "To avoid swarm..." Grammar awk.
Author Response
This was an interesting study that I am glad to have had the pleasure of reviewing… Well done study using flow cytometry...
We would like to thank the reviewer for the positive feedback.
I'm unsure of the specific style of the journal requests/permits the current study's structure of "Materials and Methods" after the Discussion…
By mistake we applied the sequence of sections (results, materials&methods, discussion) from other MDPI journals‘ guidelines. In the revised manuscript the sequence has been corrected.
How long were patients followed for their clinical course when examining complications?
Patients were followed until hospital discharge. This information was included in the methods section.
The biggest comment I have is the discussion of significance of erythocyte EV's and their high amounts. How does this compare to the literature? Is there a link to higher amounts of erythocyte EV's in allogeneic transfusions? Improving the discussion with more references to clarify the significance of their findings would greatly improve the paper.
As mentioned in our response to the comments from reviewer #1 above the question on a comparison to allogeneic blood transfusion is not of high relevance in a clinical field where the evidence-based concept of patient blood management (PBM) is recommended. Therefore, we did not further discuss this issue in the revised manuscript. In the original discussion section we, however, discuss our results with previous data from first-generation cell salvage machines: „Despite differences in methodology of flow cytometry, our non-red blood cell EV counts from platelets and monocytes appear markedly lower compared to cell salvage from a first-generation cell saver machine CATS (Fresenius AG, Bad Homburg, Germany) [16].“
In the discussion section on page 5 we report that „by carrying phospholipids and tissue factor, EVs can promote hemostasis, specifically by increasing platelet adhesion, platelet aggregation, and thrombin generation.“ We believe that we clearly state one significant aspect of our findings: „Our results of very low EVs from these cell origins show no safety signal in terms of exaggerated microparticle load and support the use of fresh autologous salvaged blood in major orthopedic surgery“.
In the discussion section on page 6, second paragraph we report that „In critically ill patients erythrocyte-derived microparticles had a microcirculatory impact [22,23]. EVs isolated from the plasma of animals with sepsis significantly decreased deformability of erythrocytes ex vivo [24]. EVs in allogeneic red blood cell concentrates have been linked to transfusion-related immunomodulation [20,25].“ Overall, our study may prompt further research on the clinical relevance of EVs from salvaged red blood cells in not critically ill patients undergoing major orthopedic surgery.
Page 2;Line 41-3: This grammar is awkward.
We reworded this sentence. It now reads: „Similar to allogeneic RBC preparation, also autologous cell salvage involves centrifugation and washing procedures which may activate and/or destroy the patient’s blood cells,
Page 4; Line 88-90: "Characteristics..." Grammar awk. needs an "and"
We replaced the word „characteristics“ by „patterns“ at its first appearance.
Page 6;Line 170-1: "From the remnant...". Grammar awk.
We added the word „blood“ after remnant. We did not delete the word remnant because it is important for ethical reasons that we did not deprive our patients from retransfusate for study purposes but took material which would have been discharded within the lines and filters.
Page 6; Line 174-5: "To avoid swarm..." Grammar awk
We could not find another term for „swarm effects“. In this sentence we explain why we diluted cell salvaged blood: in ordert o avoid swarm effects.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
In this study the authors assessed the appearance of extracellular vesicles in autologous cell savaged blood. Though I found the topic interesting, this study is very preliminary and doesn't demonstrate why it is important and if the use of autologous cell savage demonstrates any benefit over allogenic savage.
My concerns/questions are below:
- In the introduction, authors mentioned that markers for cell damage can be helpful in defining quality and safety of re-transfused blood. It will improve the quality of the manuscript if authors can elaborate on what those markers are and what makes looking into extracellular vesicles unique.
- The results obtained should be a part of result section and not discussion - for Figure 1.
- There is no inclusion/discussion of results obtained in Figure 2 in the manuscript except the Figure 2 legend. This part needs to be added in results and then discussed in the discussion section.
- For methodology, it is known that generic markers do not detect all EVs, and detection is also dependent on the sensitivity of the flow cytometer used which may inhibit repetition of results at other institutions. Why more specific markers for EVs (such as tetraspanins for both exosomes and microvesicles) were not chosen and other techniques like SP-IRIS or western blotting not incorporated for consilience.
- EVs content from RBCs is high. How does it correlate with EVs content from allogenic blood. This comparison is needed or atleast discussed in more details even for a preliminary study to demonstrate whether autologous cell savage is better than allogenic sources.
- Is EV content from allogenic myeloid, platelets and monocytes higher than autologous sources? A comparison or discussion comparing published studies will be helpful.
Author Response
„.. the study is very preliminary …“
Our study is indeed one of the first reports of extracellular vesicles (EV) in perioperative cell salvaged blood! This is important because our study may prompt further studies, e.g. comparing new generations of cell salvage machines. Even if our study is one of the first reports, it is not preliminary in a methodological sense. We included this aspect of our flow cytometric methods used for future research in the modified discussion section. It reads:
“… Our aim was on the one hand to document the cellular origin of the vesicles as much as possible and on the other hand to use the simplest and fastest routinely applicable method that could represent a further step towards standardization.”
„…and doesn’t demonstrate why it is important and if the use of autologous cell salvage demonstrates any benefit over allogeneic salvage …“
It was not the aim of the present study to compare the clinical use of autologous cell salvage with allogeneic blood transfusion. Importantly, allogeneic transfusions are – according to the evidence-based concept of patient blood management (PBM) - highly recommended to be avoided perioperatively whenever possible. Donated blood transfusions are not a recommended routine. Surgeons and anaesthesiologists are required to manage severely bleeding patients rather with the patients‘ own, autologous blood. Therefore, the aim of our PBM-oriented study was to investigate autologous blood only. This aspect is important to readers of SURGERY. We highlighted this aspect of PBM in the first paragraph of the original introduction section.
- In the introduction, authors mentioned that markers for cell damage can be helpful in defining quality and safety of re-transfused blood. It will improve the quality of the manuscript if authors can elaborate on what those markers are and what makes looking into extracellular vesicles unique.
In the second paragraph of the original introduction section we elaborate on the EV markers:„… preparation of autologous cell salvage involves centrifugation and washing procedures which may activate and/or destroy the patient’s blood cells, resulting in the generation of cell-derived EVs.“ The better the quality of a cell salvage procedure the less cell damage and EV detritus is produced. We further explain: „Considering the increasing clinical use of cell salvage and innovations in cell salvage technology, markers for cell damage can be helpful in defining quality and safety of retransfused blood.“
We did not state that looking into EVs is unique and the only option to assess cell damage. But looking into EVs is interesting because as discussed in the discussion section „by carrying phospholipids and tissue factor, EVs can promote hemostasis, specifically by increasing platelet adhesion, platelet aggregation, and thrombin generation.“Our results of very low EVs from these cell origins „show no safety signal in terms of exaggerated microparticle load and support the use of fresh autologous salvaged blood in major orthopedic surgery“.
- The results obtained should be a part of result section and not discussion - for Figure 1.
By mistake we applied the sequence of sections (results, materials&methods, discussion) from other MDPI journals‘ guidelines. In the revised manuscript the sequence has been corrected.
- There is no inclusion/discussion of results obtained in Figure 2 in the manuscript except the Figure 2 legend. This part needs to be added in results and then discussed in the discussion section.
Between figure 1 and figure 2 an explanatory text is in the manuscript: „A typical pattern of labeling is shown in Figure 2.“ We increased the distance to the legend of figure 1 in order to increase visibility of this sentence.
- For methodology, it is known that generic markers do not detect all EVs, and detection is also dependent on the sensitivity of the flow cytometer used which may inhibit repetition of results at other institutions. Why more specific markers for EVs (such as tetraspanins for both exosomes and microvesicles) were not chosen and other techniques like SP-IRIS or western blotting not incorporated for consilience.
We agree with the reviewer that the detection of the markers and/or EVs is currently still extremely incomplete and very dependent on the sensitivity of the flow cytometer used and the detection method used.
In our study we selected a sensitive flow cytometer with avalanche photodiodes, which shows excellent sensitivity in the red excitation and emission area. Especially for this determination we used lactadherin which was conjugated for our purpose with AF647, which can be excited with a 638nm laser and has an emission maximum at approx. 670nm wavelength. In addition to the detection of phosphatidylserine (unfortunately PS is only about 30-40% expressed on the "classic" microvesicles), we stained our samples with calcein AM which was converted to a fluorescent substance by intravesicular esterases. This enabled us to detect the non-PS positive events and thus detect a significantly larger number of EVs carrying the cellular specific markers.
It is true that we could have determined tetraspanins as well. However, our goal was on the one hand to document the cellular origin of the vesicles as far as possible and, on the other hand, to do this with the simplest and most quickly routinely applicable method.
We included this information in the discussion section of the revised manuscript on page 6.
- EVs content from RBCs is high. How does it correlate with EVs content from allogenic blood. This comparison is needed or atleast discussed in more details even for a preliminary study to demonstrate whether autologous cell savage is better than allogenic sources.
As mentioned in our response above the question on a comparison to allogeneic blood transfusion is not of high relevance in a clinical field where the evidence-based concept of PBM is recommended. Therefore, we did not further discuss this issue in the revised manuscript. In the original discussion section we, however, discuss our results with previous data from first-generation cell salvage machines: „Despite differences in methodology of flow cytometry, our non-red blood cell EV counts from platelets and monocytes appear markedly lower compared to cell salvage from a first-generation cell saver machine CATS (Fresenius AG, Bad Homburg, Germany) [16].“
- Is EV content from allogenic myeloid, platelets and monocytes higher than autologous sources? A comparison or discussion comparing published studies will be helpful.
Apart from our response above (to point 5) on the limited value of a comparison between allogeneic and autologous blood the comparison of EV content in ours and previous published studies is also not very useful due to the very different methodologies used, which can hardly be compared. We would like to justify this as follows: as already mentioned in the answer to point 4, we used a very sensitive flow cytometer and a custom-made lactadherin (first made especially for us - it is now used by a large number of groups), which detects phosphatidylserine very efficiently. The additional staining with calcein detects even more EVs, so that we probably detect significantly more events. In addition, our flow cytometer shows the measured events per µl. However, after the plasma had received a very high dilution, between 200 - 400 fold dilution (minimizing swarm effects), this dilution had to be corrected for the actual number and then related to the total amount of plasma per ml of starting material. The given numbers therefore correspond at least to the events "visible" to us in the autologous cell salvage.For these reasons, a comparison with earlier work does not seem useful to us, but we have included a paragraph in the discussion on page 6.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
All my comments were appropriately addressed by the authors. Well done.
Reviewer 2 Report
The authors have addressed the concerns raised by me.
