Distribution of Red Blood Cells Deformability: Study on Density-Separated Cell Subpopulations

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors of the manuscript entitled 'Distribution of red blood cells deformability: Study on density-separated cell subpopulations.' investigated the deformability distribution of RBC groups with different densities separated by Percoll linear density gradient. They observed a strong correlation between MCHC and the RBC deformability. The study design is reasonable, the data analysis is clear, and the obtained conclusion is authentic. However, this reviewer has a few concerns.

 

1. Authors investigated the RBC deformability with their own cell flow properties analyzer. There are many modalities of deformability measurements reported, i.e., ektacytometry, micropipette technique, optical tweezer, rheoscope, filtration technique, and so on. In this reviewer's personal opinion, the most standard technique is ektacytometry, which is standardized, and commercially available, allowing elongation index measurement under the steady state with a wide range of shear rates. The methodology adopted in this manuscript may be explained in another article (doi: 10.1111/micc.12296), how are the sensitivity and the reproducibility of this technique? This technique seems to observe the morphology of RBCs adhered (not flowing!?) to the microfluidic glass surface and elongated in parallel to the shear (3Pa) flow direction. RBC behaviors in microchannels are so complicated that they behave as tumbling, bending, tank-tread, and polylobe deformation by using high-speed video camera enabling cell tracking (doi: 10.1016/j.bpj.2019.05.022). The authors should explain their own techniques applied specifically to this protocol.       

2. The authors demonstrated a clear relationship between MCHC and several indices of deformability obtained by their technique. How was the relationship between the MCV and MCHC, and that between MCV and the aforementioned indices? Cell aging increases MCHC and reduces MCV due to cell dehydration and shrinkage. Readers may be eager to know these additional relationships even in the Supplementary Files.

3. Furthermore, the cell-to-cell variation of MCV relates to the red cell distribution width (RDW). RDW is deeply involved in the red cell deformability, i.e., higher RDW (anisocytotic RBCs) indicates impaired deformability (doi: 10.7754/Clin.Lab.2022.211260., doi: 10.1007/978-1-4614-4989-8_29.). This field of consideration enriches the Discussion of this manuscript. 

 

Author Response

Reviewer 1

The authors of the manuscript entitled 'Distribution of red blood cells deformability: Study on density-separated cell subpopulations.' investigated the deformability distribution of RBC groups with different densities separated by Percoll linear density gradient. They observed a strong correlation between MCHC and the RBC deformability. The study design is reasonable, the data analysis is clear, and the obtained conclusion is authentic. However, this reviewer has a few concerns.

1. Authors investigated the RBC deformability with their own cell flow properties analyzer. There are many modalities of deformability measurements reported, i.e., ektacytometry, micropipette technique, optical tweezer, rheoscope, filtration technique, and so on. In this reviewer's personal opinion, the most standard technique is ektacytometry, which is standardized, and commercially available, allowing elongation index measurement under the steady state with a wide range of shear rates. The methodology adopted in this manuscript may be explained in another article (doi: 10.1111/micc.12296), how are the sensitivity and the reproducibility of this technique? This technique seems to observe the morphology of RBCs adhered (not flowing!?) to the microfluidic glass surface and elongated in parallel to the shear (3Pa) flow direction. RBC behaviors in microchannels are so complicated that they behave as tumbling, bending, tank-tread, and polylobe deformation by using high-speed video camera enabling cell tracking (doi: 10.1016/j.bpj.2019.05.022). The authors should explain their own techniques applied specifically to this protocol.

We agree with the reviewer that many tools are available to characterize cell deformability. Each of the listed methods has its advantages and disadvantages. The ektacytometry mentioned by the reviewer as a "standardized and commercially available" method also has several disadvantages, which include: 1. Ektacytometry is the non-direct method of RBC deformation characterization; 2. For measurements, a high concentration of PVP is used to increase the viscosity of the buffer up to 30 cP. This condition dramatically changes the ratio of intercellular and extracellular viscosity. Under physiological conditions, intracellular viscosity is higher than extracellular viscosity. Still, in the LORRCA system, the extracellular viscosity is higher, significantly changing cell deformation conditions in the flow. 3. Light scattering is correct only in the case of a symmetrical cell shape, which is not always valid for pathology cases.

Regarding CFA. The first version of the device was developed and manufactured by us in 1994 to visualize RBC aggregation, deformation, and adhesion.

The main advantage of the CFA is the direct visualization of RBC deformation and ability, which allows us to characterize not only the average value of RBC deformability but also the distribution of this indicator in the large cells population. We demonstrated that the weight of the undeformable cells subpopulation in many situations is more critical than the average value of deformability, which LORRCA measures.

Moreover, we record the deformation (change in shape) of adherent (immobile) cells, which allows us to:

1. Evaluate the deformation of a large population of cells (more than 10,000) under the influence of the same and constant shear stress;
2. Record for each cell the value of ER with high accuracy.

Taking the reviewer's recommendation, we added the relevant paragraph and illustration to the manuscript's text (Page 4).

       

2. The authors demonstrated a clear relationship between MCHC and several indices of deformability obtained by their technique. How was the relationship between the MCV and MCHC, and that between MCV and the aforementioned indices? Cell aging increases MCHC and reduces MCV due to cell dehydration and shrinkage. Readers may be eager to know these additional relationships even in the Supplementary Files.

Thanks to the reviewer's comment, we have added relevant information to the edited manuscript (see Line 47 – 50 and paragraph "Limitation").

3. Furthermore, the cell-to-cell variation of MCV relates to the red cell distribution width (RDW). RDW is deeply involved in the red cell deformability, i.e., higher RDW (anisocytotic RBCs) indicates impaired deformability (doi: 10.7754/Clin.Lab.2022.211260., doi: 10.1007/978-1-4614-4989-8_29.). This field of consideration enriches the Discussion of this manuscript. 

We agree with the reviewer's comment but, in this manuscript, intend to focus on the correlation between MCHC and cell deformability.

We added the corresponding comment to the edited manuscript in the "Limitations" section (Lines 315-320).

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Some paragraphs are slightly redundant and a general improvement of the flow could positively impact this work, but the manuscript is interesting and is appropriate for the Journal.

Comments on the Quality of English Language

the manuscript could benefit of a small revision of flow and readability but is overall well written

Author Response

Some paragraphs are slightly redundant and a general improvement of the flow could positively impact this work, but the manuscript is interesting and is appropriate for the Journal.

Comments on the Quality of English Language

the manuscript could benefit of a small revision of flow and readability but is overall well written

Thanks to the reviewer for his positive assessment of our manuscript. We hope that the new version looks more readable.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

In this manuscript, the researchers compared the distribution of red blood cell deformability from cell fractions characterized by different densities. I consider the topic and results relevant to the scientific community. However, the novelty of the work is unclear, information is missing from the methodology, and I have serious concerns about the consistence, presentation and interpretation of the results and the discussion. My main observations are the following:

 

1. The novelty of the study needs to be clarified. What does the study of the distribution add to the previous studies that relate the deformability with the density and age of RBC? 

 

2. Subsection 2.3 must include the information on which hematological indexes determined by the XP-300 protocol will be relevant for this study. This is not mentioned anywhere in the methods, however assessment on such parameters are used to support the discussion. For example, line 267 reads, "The connection between MCHC and cell deformability we have demonstrated in this study…" However, it is not mentioned how MCHC was measured anywhere in the manuscript. Therefore, technically, the connection has not been demonstrated in this study. 

 

3. The criteria for the shear stress selection should be clarified. How did the authors decide for 3.0 Pa to set the criteria of ER<1.1 as un-deformable? Wouldn't the stiffer cells deform at higher shear stresses showing ER>1.1, or more deformable cells change their shape at lower shear stresses?

 

4. Why did the authors show the results using a cumulative distribution? I am not convinced that the cumulative distribution is the most straightforward option for interpreting the results presented. I suggest adding the histogram; this is a better choice to represent the distribution.

 

5. The plots in Figures 1 and 2 are not explained or discussed. The results and their relevance need to be interpreted.

 

6. In Table 3, the authors compared the deformability with the MCHC. However, there is no mention of the measurements of MCHC in the methods, nor how they related MCHC to the density of the cell fractions. MCHC as a parameter is also mentioned in the discussion and the conclusions, for example:

"Our study demonstrated that a decrease in deformability of RBCs, occurring as a part of cells in-vivo aging, is correlated with an elevation of MCHC (see Table 3). Thus, alteration of RBC deformability, to a large extent, can be related to the changes in viscosity of the cytosol (determined by the concentration of intracellular hemoglobin)."

However, the information needed to make this assessment is missing. According to the information given in the Material and Methods and Results sections, MCHC was not measured.

 

7. The results shown in Table 3 are not explained. If a parameter is shown in a table, it should be explained. Why are those values relevant to interpret and draw conclusions from the data analyzed? 

In the discussion, line 257 mentions``Our study demonstrated that a decrease in deformability of RBCs, occurring as a part of cells in-vivo aging, is correlated with an elevation of MCHC (see Table 3)." 

In line 283 ``From the data presented in Table 3, we can conclude that while we do observe a pronounced dependence of the RBC's deformability on their density, additional factors certainly affect deformation."

 

These statements can not be deduced from the information presented in Table 3. Statistical data and values need to be contextualized. 

 

8. In line 300 ``Moreover, we demonstrated a strong correlation between RBC deformability and intracellular Hb concentration."

 

This asseveration and others, are not supported by the information presented in the manuscript. The discussion is inconsistent with the results and methods. 

 

Minor remarks:

1. There is a type error in line 102; I think it should be "heavy."

2. The sentence between lines 115 and 118 is confusing; I recommend rephrasing it.

3. The word "device" is missing after microfluidic in lines 133, 136 and 137.

 

4. In line 185, describing control experiments, the authors declare, "the Percoll gradient does not alter cell features." However, they only analyzed deformability; this sentence should specify that it does not alter the deformability. If other properties were analyzed and were not affected by the Percoll, these should be mentioned in the manuscript.

 

 

Comments on the Quality of English Language

1. There is a type error in line 102; I think it should be "heavy."

2. The sentence between lines 115 and 118 is confusing; I recommend rephrasing it.

3. The word "device" is missing after microfluidic in lines 133, 136 and 137.

Author Response

Reviewer 3

In this manuscript, the researchers compared the distribution of red blood cell deformability from cell fractions characterized by different densities. I consider the topic and results relevant to the scientific community. However, the novelty of the work is unclear, information is missing from the methodology, and I have serious concerns about the consistence, presentation and interpretation of the results and the discussion. My main observations are the following:

 We are grateful to the reviewers for their positive assessment of our study.

1. The novelty of the study needs to be clarified. What does the study of the distribution add to the previous studies that relate the deformability with the density and age of RBC? 

 Thanks to the reviewer for his comment; the corresponding change has been made to the new version of the manuscript (Lines 105 -111).

2. Subsection 2.3 must include the information on which hematological indexes determined by the XP-300 protocol will be relevant for this study. This is not mentioned anywhere in the methods, however assessment on such parameters are used to support the discussion. For example, line 267 reads, "The connection between MCHC and cell deformability we have demonstrated in this study…"However, it is not mentioned how MCHC was measured anywhere in the manuscript. Therefore, technically, the connection has not been demonstrated in this study. 

Thanks to the reviewer for his comment; the corresponding change has been made to the new version of the manuscript (Lines 129 -133).

3. The criteria for the shear stress selection should be clarified. How did the authors decide for 3.0 Pa to set the criteria of ER<1.1 as un-deformable? Wouldn't the stiffer cells deform at higher shear stresses showing ER>1.1, or more deformable cells change their shape at lower shear stresses?

Thanks to the reviewer for his comment; the corresponding change has been made to the new version of the manuscript (Lines 181-183 and 197 – 200).

1. Why did the authors show the results using a cumulative distribution? I am not convinced that the cumulative distribution is the most straightforward option for interpreting the results presented. I suggest adding the histogram; this is a better choice to represent the distribution.

 

Thanks to the reviewer's comment, the corresponding change has been made (see Fig. 4).

5. The plots in Figures 1 and 2 are not explained or discussed. The results and their relevance need to be interpreted.

 Thanks to the reviewer for his comment; the corresponding change has been made to the new version of the manuscript (Lines 148-176 and 279-280).

6. In Table 3, the authors compared the deformability with the MCHC. However, there is no mention of the measurements of MCHC in the methods, nor how they related MCHC to the density of the cell fractions. MCHC as a parameter is also mentioned in the discussion and the conclusions, for example:

"Our study demonstrated that a decrease in deformability of RBCs, occurring as a part of cells in-vivo aging, is correlated with an elevation of MCHC (see Table 3). Thus, alteration of RBC deformability, to a large extent, can be related to the changes in viscosity of the cytosol (determined by the concentration of intracellular hemoglobin)."

However, the information needed to make this assessment is missing. According to the information given in the Material and Methods and Results sections, MCHC was not measured.

  Thanks to the reviewer for his comment; the corresponding change has been made to the new version of the manuscript (Lines 137-139 and 263-272).

 

7. The results shown in Table 3 are not explained. If a parameter is shown in a table, it should be explained. Why are those values relevant to interpret and draw conclusions from the data analyzed? 

Thanks to the reviewer for his comment; the corresponding change has been made to the new version of the manuscript (Lines 304-320).

 

In the discussion, line 257 mentions``Our study demonstrated that a decrease in deformability of RBCs, occurring as a part of cells in-vivo aging, is correlated with an elevation of MCHC (see Table 3)." 

In line 283 ``From the data presented in Table 3, we can conclude that while we do observe a pronounced dependence of the RBC's deformability on their density, additional factors certainly affect deformation."

 These statements can not be deduced from the information presented in Table 3. Statistical data and values need to be contextualized. 

 Thanks to the reviewer for his comment; the corresponding change has been made to the new version of the manuscript (Lines 352-354).

8. In line 300 ``Moreover, we demonstrated a strong correlation between RBC deformability and intracellular Hb concentration."

 This asseveration and others, are not supported by the information presented in the manuscript.

This reviewer's remark seems strange to us. The data in Table 3 clearly indicate the presence of a linear correlation between the value of MCHC (intracellular hemoglobin concentration) and the parameters describing cell deformability. For this reason, we do not see any problem with what we said on line 300 (the original version of the manuscript).

The discussion is inconsistent with the results and methods. 

 Following the reviewer's comments, we have adjusted the new text version.

Minor remarks:

1. There is a type error in line 102; I think it should be "heavy."
2. The sentence between lines 115 and 118 is confusing; I recommend rephrasing it.
3. The word "device" is missing after microfluidic in lines 133, 136 and 137.

We corrected all type errors.

4. In line 185, describing control experiments, the authors declare,"the Percoll gradient does not alter cell features." However, they only analyzed deformability; this sentence should specify that it does not alter the deformability. If other properties were analyzed and were not affected by the Percoll, these should be mentioned in the manuscript.

 Thanks to the reviewer for his comment; the corresponding change has been made to the new version of the manuscript (Line 231).

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Dear Editor, dear Authors

 

please, find below my comments about the paper entitled “Distribution of red blood cells deformability: Study on density-separated cell subpopulations.”. By Gregory Barshtein et al.

 

The paper concerns with an evaluation of the cell deformability in erythrocytes isolated , by Percoll gradient, at different step of their physiological aging. The main result is a correlation between cell deformation and Hb concentration.

 

The subject of the investigation is interesting, and the experiment is well designed. The introduction section contains the information important to understand the rest of the paper and the English is readable and understandable, with a small number of sentences to be checked.

The control experiments were properly designed and the interpretation of the data is straightforward and well described.

 

Despite my overall positive feeling about the paper, there are still some points to clarify and understand before the paper can be considered for publication. 

Below are listed my questions and doubts.

 

1) The authors performed significant experiments measuring the deformability of RBCs in a microfluidic chamber. However, I didn’t find a sketch of the chamber nor details on the shape, size and characteristics of the system used. I know the authors already employed this approach, yet I believe the reader of the present manuscript should be provided with the fundamental information on the most relevant methods and apparatus employed here. 

A sketch of the microfluidic chamber, explaining the deposition and the method to induce and measure the shear stress is required.

  

2) In the controlled deformation experiments, the authors expose the cell to significant shear stress and observed deformation up to, roughly, twice their original size. I wonder why the cells did not detach under these applied forces as RBCs, normally, do not adhere tightly to surfaces. 

- How large is the fraction of cells that adhere to the microfluidic chamber? 

- Did the authors perform any treatment to improve the adhesion of RBCs in the microfluidic chamber? 

- Did the authors observe (significant, negligible, rare) cell detachment during the experiments? 

 

Overall, this is an important point to explain to help understanding the significance of the data, and I recommend the authors to comment this point in the text.  I would be very surprised if, for instance, 99% of such a special class of cell adhered strongly to an untreated surface and massively endure the significant deformation reported. Possibly, more information on the data collection would be appreciated.

 

- Eventually, for the same purpose as before, can the author explain how strong is the interaction between cell and surface compared to the induced stress?

Please add comments on this point in the text, as I believe a better understanding of the force endurance can certainly help understanding the data.

 

3) The interpretation of the data the authors provided is clear, and I do not disagree with it. Yet, to give proper sense to variation of just a few points in percentage, I need to know more about the statistics and about the experiment replicas. For instance:

- How many times have been repeated the Percoll separation and the consequent experiments (for each donor)? 

- Did the authors observe differences in the various density separation profiles among different donors?

Please add information and comment in the text

 

4) What about the difference observed between different donors?  My question requires comments both in terms of Percoll separation profiles and, especially, in terms of the behavior of the subpopulation of cells among donors? Noting has been discussed about this issue in the text and it is such a relevant topic that deserve some considerations.

 

5) About figure 2: I suggest redrawing figure 2 using larger size symbols as, in the current version, they are barely distinguishable each other.

 

6) Other minor points:

- The sentence at lines 115-118 sounds a bit convoluted, please make it simpler and more clear for the reading. 

 

- Concerning the sentence at line 247 “reduced deformability of cells is the reason for their removal…”, I would rather have more cautions in the statement. Current knowledge suggest that there would be no single determinant for cell removal although, certainly, reduced deformability is a fundamental agent. 

I recommend, if the authors agree, changing the sentence as follow: “reduced deformability of cells is one of the major factor causing their removal…”

 

 

- Please, check whether the brand of the CFA is properly reported

Comments on the Quality of English Language

The English is good enough. Changes on a few sentences has been suggested in the "comments to the authors"

Author Response

Reviewer 4

Dear Editor, dear Authors please, find below my comments about the paper entitled “Distribution of red blood cells deformability: Study on density-separated cell subpopulations.”. By Gregory Barshtein et al.

 

The paper concerns with an evaluation of the cell deformability in erythrocytes isolated , by Percoll gradient, at different step of their physiological aging. The main result is a correlation between cell deformation and Hb concentration.

 

The subject of the investigation is interesting, and the experiment is well designed. The introduction section contains the information important to understand the rest of the paper and the English is readable and understandable, with a small number of sentences to be checked.

The control experiments were properly designed and the interpretation of the data is straightforward and well described.

 We are grateful to the reviewers for their positive assessment of our study.

 Despite my overall positive feeling about the paper, there are still some points to clarify and understand before the paper can be considered for publication. 

Below are listed my questions and doubts.

 

1) The authors performed significant experiments measuring the deformability of RBCs in a microfluidic chamber. However, I didn’t find a sketch of the chamber nor details on the shape, size and characteristics of the system used. I know the authors already employed this approach, yet I believe the reader of the present manuscript should be provided with the fundamental information on the most relevant methods and apparatus employed here. 

A sketch of the microfluidic chamber, explaining the deposition and the method to induce and measure the shear stress is required.

 

Thanks to the reviewer for his comment; the corresponding change has been made to the new version of the manuscript (Lines 146 - 200).

2) In the controlled deformation experiments, the authors expose the cell to significant shear stress and observed deformation up to, roughly, twice their original size. I wonder why the cells did not detach under these applied forces as RBCs, normally, do not adhere tightly to surfaces. 

- How large is the fraction of cells that adhere to the microfluidic chamber? 

As can be seen from the comparison of Fig. 2B and 2C (which we added to the edited version of the manuscript), only single cells detached from the slide under a shear stress of 3.0 Pa. It should be emphasized that these two figures are obtained for one field, where the figure B is captured when shear stress is equal 0.0 Pa, while the figure C, under the action of a shear stress of 3.0 Pa. As follows from the comparison of these figures, not a single cell was detached under the flow application.

- Did the authors perform any treatment to improve the adhesion of RBCs in the microfluidic chamber? 

No, the surface of the slide has not been treated (Line 160 in the revised version).

- Did the authors observe (significant, negligible, rare) cell detachment during the experiments? 

 Based on our estimate, only a few cells are detached from the slide surface under a shear stress of 3.0 Pa. Please compare the number of attached cells in Figs. 2B and 2C for illustration.

Overall, this is an important point to explain to help understanding the significance of the data, and I recommend the authors to comment this point in the text.  I would be very surprised if, for instance, 99% of such a special class of cell adhered strongly to an untreated surface and massively endure the significant deformation reported. Possibly, more information on the data collection would be appreciated.

 Please see the information that we added to a revised version of the manuscript (Lines 134 - 206).

- Eventually, for the same purpose as before, can the author explain how strong is the interaction between cell and surface compared to the induced stress?

Please add comments on this point in the text, as I believe a better understanding of the force endurance can certainly help understanding the data.

 The idea of ​​using adherent cells to measure their deformability was expressed earlier in the works of Hochmuth et al. [1] and Artmann [2]. Wang and colleagues [3] used a microfluidic system (similar to CFA) to study the deformability of adherent RBC. We did not assess the critical cell detachment wall shear stress and the distribution of this value in the cell population. However, experimentally, we chose the magnitude of the applied wall shear stress corresponding to the conditions under which the deformation of the RBCs becomes significant, but their detachment from the glass remains insignificant.

3) The interpretation of the data the authors provided is clear, and I do not disagree with it. Yet, to give proper sense to variation of just a few points in percentage, I need to know more about the statistics and about the experiment replicas. For instance:

- How many times have been repeated the Percoll separation and the consequent experiments (for each donor)? 

The Percoll separation occurred for one sample of blood from each donor. Cell deformability was measured for a cell population of 10,000 to 15,000 in each case.

- Did the authors observe differences in the various density separation profiles among different donors?

The profiles for all six donors were similar. Here, we must consider that we used blood taken from non-smoking young, healthy donors (18-25 years old), male, and one blood group O+ (Lines 113-115).

Please add information and comment in the text

4) What about the difference observed between different donors?  My question requires comments both in terms of Percoll separation profiles and, especially, in terms of the behavior of the subpopulation of cells among donors? Noting has been discussed about this issue in the text and it is such a relevant topic that deserve some considerations.

The profiles for all six donors were similar (see Fig. 1 in corrected version of the manuscript).

5) About figure 2: I suggest redrawing figure 2 using larger size symbols as, in the current version, they are barely distinguishable each other.

We corrected the relevant figure in the revised manuscript.

 

6) Other minor points:

- The sentence at lines 115-118 sounds a bit convoluted, please make it simpler and more clear for the reading. 

 We corrected this sentence (see Lines 124-126).

- Concerning the sentence at line 247 “reduced deformability of cells is the reason for their removal…”, I would rather have more cautions in the statement. Current knowledge suggest that there would be no single determinant for cell removal although, certainly, reduced deformability is a fundamental agent. 

I recommend, if the authors agree, changing the sentence as follow: “reduced deformability of cells is one of the major factor causing their removal…”

 Thanks to the reviewer for his recommendation. We corrected this sentence (see Lines 327).

- Please, check whether the brand of the CFA is properly reported

CFA is the equipment that is designed and produced in the laboratory of Prof. S. Yedgar.

Comments on the Quality of English Language

The English is good enough. Changes on a few sentences has been suggested in the "comments to the authors"

1. Hochmuth, R.M.; Mohandas, N.; Blackshear, P.L., Jr. Measurement of the elastic modulus for red cell membrane using a fluid mechanical technique. Biophys J 1973, 13, 747-762, doi:10.1016/S0006-3495(73)86021-7.
2. Artmann, G.M. Microscopic photometric quantification of stiffness and relaxation time of red blood cells in a flow chamber. Biorheology 1995, 32, 553-570, doi:10.1016/0006-355X(95)00032-5.
3. Wang, Y.; You, G.; Chen, P.; Li, J.; Chen, G.; Wang, B.; Li, P.; Han, D.; Zhou, H.; Zhao, L. The mechanical properties of stored red blood cells measured by a convenient microfluidic approach combining with mathematic model. Biomicrofluidics 2016, 10, 024104, doi:10.1063/1.4943861.

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 4 Report

Comments and Suggestions for Authors

Dear Editor, dear Authors

 

I've read the revised version of the manuscript by G. Barshtein et al. and I found the text significantly improved in clarity and readability compared to the previous version.

All my previous criticisms have been addressed with the help of comments in the text or figures.

 

I still have a few minor points to underline:

 

- Please check the sentence at lines: 147-150 as they need an English review (“demonstrated” should probably be “shown”; the data, I suppose, “are” presented; please, specify what is a “station” and so on)

 

- Please check that in the discussion there are still recall to H-fraction (which should have been updated to D-fraction

Comments on the Quality of English Language

The Witten English is understandable