Scanning Electron and Atomic Force Microscopic Analysis of Erythrocytes in a Cohort of Atopic Asthma Patients—A Pilot Study
Round 1
Reviewer 1 Report (Previous Reviewer 2)
The authors have adequately addressed all my concerns and therefore I believe that their manuscript can be accepted in its current format.
Author Response
Thank you, there are no additional comments to be addressed.
Author Response File: 
Author Response.pdf
Reviewer 2 Report (New Reviewer)
In this pilot study, Alummoottil et al. describe possible relationships between atopic asthma and changes in erythrocyte morphology and elasticity. The authors provide some informative background information in the introduction, as well as a sufficient description of the methods used. In addition to routine haematological analyses, the imaging methods LM and SEM, as well as AFM for the measurement of elastic properties and the calculation of the Young’s modulus were used. Despite the thoroughness of the study, the manuscript could benefit from some further improvements.
Despite the fact that patients and controls were stated to be matched, it isn't exactly the case, as the patient cohort had twice as many females as the healthy cohort, and vice versa for the male cohort. This might not have a significant influence on the cellular characteristics, but cannot be completely omitted. A similar issue exists for the age match (mean 53 years vs 45 years) between the two groups, which is not exactly what should be the case (might this difference even be statistically significant?).
The authors describe the use of AFM for the measurement of elastic properties of red blood cells. Various studies did the same, however, there are some doubts regarding the fixation of erythrocytes prior to analysing membrane elasticity. In imaging studies, fixation protocols are justified, but in functional assays, native cells should be used instead of fixed cells. In AFM procedures, fixed cells are much easier to manipulate, but studies show that the procedure can as well be performed without fixation.
Since AFM analyses are laborious, it might not be possible to repeat all the measurements without fixation, but maybe it would be feasible for the authors to do some “proof of principle” analyses of the same sample with and without fixation in parallel to check for possible influences of fixation on elasticity? Furthermore, a representative example of the force curves of patient and control cells would be helpful in the Results section. Did the authors also use AFM imaging to confirm the SEM images? If so, I would appreciate seeing some representative pictures here.
Minor points
Line 33: There is presumably a decimal point missing in “20 â–Ş 8 %”, please correct.
Line 35: To avoid misinterpretation, please spell out the abbreviation "SDI" when it is used for the first time.
Line 84: The authors state here “age ≥18 years” for their cohorts, while in the Results Table1 the age range of the patient’s group is 17-55. Please also see line 177 and correct.
Line 106: There is a spelling mistake: “form the study participants..” please correct to “from”
Line 107: Instead of “..one sample for each patient” it should rather be “one sample of each patient”
Line 213: Figure 1 A,B: please provide the measures (µm) for the shown scale bars.
Line 220: Do the authors mean “The Young's modulus of erythrocytes was higher in patients” instead of “the elasticity of erythrocytes was higher in patients...indicating a reduction in membrane elasticity”? Since YM is inversely proportional to elasticity, as the authors stated in the Discussion section? Please rephrase the sentence for its correct meaning. Please also change to the singular verb form “…was higher…” instead of “…were higher…” in line 220.
Line 223: Please think of rewording the heading to “…in asthma- and control subjects” or something similar. Is the "Mean" the calculated mean of Young's moduli? Please clarify the correct meaning for all the parameters stated in Table3. Note: The standard deviation seems relatively high in relation to the mean value in the patient cohort- So, from the first glance, the difference (Mean) between the cohorts appears unlikely to become statistically significant (p<0.01)? Please check.
Line 232: Figure 2: It would be nice to have the same scale for all scale bars. The authors use three different scales (200 nm, 1 µm, 2 µm) for the four pictures.
Line 239: “...slightly changed shape…”. Please correct the typing error.
Line 241: “… Erythrocyte morphology plays an integral role.” The “s” for third person singular is missing
Line 263: This should be written in plural form “Microparticles also contribute…” and please correct the following sentence to “…erythrocytes’ microparticles are associated…” as it is not only one microparticle per cell involved.
Line 266: “..the effects need to be investigated ..” The verb should be used in plural form.
Line 268: “..axial ratios of… are indicative of..” The verb should be used in plural form
Line 309: Please correct the sentence “Of note, morphologic pathology the deformability.. “ There is at least one word missing from the sentence.
Line 321: It should be “..which might contribute to the changes observed”, please correct the sentence.
Author Response
All comments were addressed, however, representative images could not be sourced from the AFM instrument. A representative image from a publication from our group was included. All editorial and language recommendations were accepted and changed accordingly.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report (New Reviewer)
I thank the authors for addressing all the comments.
No more suggestions.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
Sajee Alummoottil, Mia J. and collegues presented a study related to the Ultrastructure, morphology and elasticity of erythrocytes in a cohort study of atopic asthma patients.
Suggestions
Please rewrite the introduction, there is no clear ''pathway'' of the introduction section.
example: you describe the situation of asthma in UK and then in Sounth Africa, I dont understand why in UK? If your study conducted in Sounth Africa?
Asthma, a chronic inflammatory disorder (1), was responsible for the loss of 21.6 mil- 31 lion (95% UI 17.1–27.0) disability-adjusted life years (DALYs) in 2019, which was 20·8% 32 (17.5–24.7) of total DALYs from chronic respiratory disease (2). Death rates from asthma 33 were highest in countries of low and middle SDI, while prevalence was highest in high 34 SDI countries. Considering the prevalence, extent and duration of the episodes, asthma is 35 the fourteenth most significant chronic disease in the world (3). Currently, in the United 36 Kingdom alone, 5.4 million people receive treatment for asthma and three people die from 37 asthma on average per day (4). The prevalence of childhood asthma in southern Africa is 38 variable but has increased over the last four decades with an increase from 3.17% to over 39 21% in South Africa (5).
Important suggestions
Please only SEM images are not enough for your conclusions. Try to include a biochemical assay or a proteomic method to give strength to your result.
Author Response
Suggestions
Please rewrite the introduction, there is no clear “pathway” of the introduction section.
Example: you describe the situation of asthma in UK and then in South Africa, I don’t understand why in UK? If your study conducted in South Africa.
Response: The authors agree that the statistics from the UK confuses the reader since this study was based in South Africa. We have removed the statement and reference “Currently 5.4 million people receive treatment for asthma and three people die from asthma on average per day.”
The flow or pathway of the introduction can be understood as follows:
- The prevalence and global problem of Asthma affecting productivity of affected patients.
- The characteristics of the disease and the most well-understood causes from genetic predisposition to environmental factors.
- The role, mechanism and studies which support the complex involvement of the immune system and proinflammatory cytokines on erythrocyte health.
- How erythrocyte deformability is measured and its role in pathology of various diseases.
- This leads to the rationale of the current study.
Important suggestions
Please only SEM images are not enough for your conclusions. Try to include a biochemical assay or proteomic method to give strength to your result.
The title of the study indicates that red blood cell morphology and elasticity changes will be reported. The conclusion of the study is derived from a combination of factors from changes in axial ratio, cell morphology and erythrocyte deformability, not only SEM.
“this study demonstrated that erythrocyte cell stiffness, measured quantitatively by AFM, was altered. Since the elasticity (deformability) influences the biomechanical properties, these results also suggest that the increased stiffness due to decreased elasticity found in asthmatic erythrocyte might influence the viscosity and flow dynamics of blood. Of note, morphologic pathology the deformability of erythrocytes can also influence the elastic properties of the thrombus by modulating the fibrinolytic properties in asthma.”
In recent literature, AFM which produces a Young Modulus value, is a validated methodology which is used to support changes observed in SEM images. (1-4)
In addition, clot kinetics have been assessed in conjunction with fibrin network morphology using thromboelastography as outlined on page 8, lines 268 – 271: “The increased erythrocytes microparticle is associated with decreased clotting time due to procoagulant proteins (5), resulting in hypercoagulability in asthma (unpublished data).”
References
- Baier DM, T; Mohr, T; Windberger, U;. Red Blood Cell Stiffness and Adhesion Are Species-Specific Properties Strongly Affected by Temperature and Medium Changes in Single Cell Force Spectroscopy. Molecules. 2021;26(9):2771.
- Jeongho K HL, Sehyun S. Advances in the measurement of red blood cell deformability: A brief review. Journal of Cellular Biotechnology. 2015;1:63-79.
- Diez-Silva M, Dao M, Han J, Lim CT, Suresh S. Shape and Biomechanical Characteristics of Human Red Blood Cells in Health and Disease. MRS Bull. 2010;35(5):382-8.
- Tomaiuolo G. Biomechanical properties of red blood cells in health and disease towards microfluidics. Biomicrofluidics. 2014;8(5):051501.
- Litvinov RI, Weisel JW. Role of red blood cells in haemostasis and thrombosis. ISBT Sci Ser. 2017;12(1):176-83.
Reviewer 2 Report
In their manuscript entitled "Ultrastructure, morphology and elasticity of erythrocytes in a 2 cohort of atopic asthma patients – A pilot study" Alummoottil et al. provide original data regarding morphological and deformability alterations in RBCs of asthma patients. The manuscript is well-written and provides information in a not-so-studied subject, namely RBCs in asthma.
This Reviewer has some comments as follow:
1) Regarding the results of light microscopy, I find it important to add some micrographs to the manuscript and not show only the final results of axial ratio in a graph (Figure 1).
2) As in the case of light microscopy data, I find it important to add some images of atomic force microscopy.
3) While the morphology and elasticity results are important, I believe that a limitation of this study is that it doesn't contain measurements regarding the impact of altered morphology/deformability to lysis parameters (e.g., hemolysis, mechanical fragility). I understand that it's difficult to perform extra experiments at this stage, and especially experiments that require a new round of sample acquisition, so I do not ask for the performance of the abovementioned experiments. Nonetheless, the authors should add this as a limitation of this study.
Author Response
Comments
1) Regarding the results of light microscopy, I find it important to add some micrographs to the manuscript and not only show the final results of axial ratio in a graph (Figure 1).
The authors agree, and a representative micrograph for the control and asthma patient group has been added to Figure 1.
2) As in the case of light microscopy data, I find it important to add some images of atomic force microscopy.
This experiment was conducted at a different department and following inquiries, the images, force and distance raw data values are no longer on the system. Thus, we have included the Young modulus raw data generated from the atomic force microscope which can be made available as supplementary material to support the changes reported in this manuscript.
3) While the morphology and elasticity results are important, I believe that a limitation of this study is that it doesn’t contain measurements regarding the impact of altered morphology/deformability to lysis parameters (e.g., hemolysis, mechanical fragility). I understand that it’s difficult to perform extra experiments that require a new round of sample acquisition, so I do not ask for the performance of the abovementioned experiments. Nonetheless, the authors should add this as a limitation of this study.
The authors agree and we have included “measurements regarding the impact of altered morphology/deformability to lysis parameters (e.g., hemolysis, mechanical fragility) would provide more robust evidence to confirm the morphological and deformability changes observed in this study.” on page 9, lines 331-334. This will also be included in the protocol which is currently being developed for a larger cohort of asthma patients.
Round 2
Reviewer 1 Report
Introduction was improved removing the data for UK.
The methodology part is lacking!
I suggest conducting proteomics. SEM images aren't enough. If you stress healthy erythrocytes you will obtain the same results as for asthma RBCs. Also, there are a couple of studies analyzing microparticles.
In my opinion Scientists working with ERYTHROCYTES will not be satisfied with the methodology used to provide your conclusions!
If you can't perform proteomic analyses or other biochemical assays, I PROPOSE TO re-write the title like: SEM analysis, a new approach to evaluate RBCs deformability in asthma patients?
