Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article examines the effect of hyperthermia on the morphology of peripheral blood leukocytes in Wistar rats using geometric morphometry. The authors analyze changes in the shape of neutrophils, lymphocytes, eosinophils and monocytes on digital images of blood smears and identify statistically significant changes in the shape of neutrophils and lymphocytes with increasing temperature. The work is of interest as an attempt to apply geometric morphometry to analyze changes in the shape of leukocytes in hyperthermia. The authors claim that this is the first study of its kind. However, methodological limitations significantly reduce the credibility of the conclusions. The authors do not provide the total number of cells. The number of cells of each type. At the same time, statistical analysis is carried out at the cellular level, not at the animal level. That is, 40 rats, but there is no number of cells that were included in the analysis. How did you divide morphologically into different cells? Again, you have 40 rats, and you take a swab from each one. There are many cells in the smear. That is, the cells are independent, but the cells inside the same rat are similar. And you imagine that each cell is an independent observation. What if there is no type of lymphocyte in the smears? then what was done in this case. Next, you write that you took more representative fields, but this is subjective and not standardized. And also, how did you do the smear? Did you use a special device so that the speed and pressure on the glass were the same? or do you have one person or several people doing business? Figure 4-6 is it 50 cells or 5000? There is no information about the sample size, and it is not possible to evaluate the reliability of the results.

Author Response

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Reviewer 2 Report

Comments and Suggestions for Authors

the present manuscript titled "Heat exposure associated alterations in leukocyte morphology revealed through geometric morphometrics analysis in Wistar rats" by Dervišević and collaborator is preliminary at best with figures that can be improved. The authors used a fairly large number of animal and should be able to provide reliable data to an important topic.

As it is the manuscript is not acceptable for scientific publication. The introduction needs more references especially with such strong statements being made. Starting the document by "Numerous studies have proven that..." and not providing a single reference is not acceptable. What type of "feed" was given to the animals. What is the proportion of male and female and each cohort? Any sex difference in the results? how many slides per blood sample were analyzed? how does the proportion of the difference leucocytes population change between before and after the warm bath? what are "standard laboratory procedures"? (no description and no reference) what kind of statistical test are in the software used? Which parameters weight the most in each PC? 
These a just some obvious questions that must be addressed before the manuscript can be send for review.
Please look for typos in the text.

Author Response

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Reviewer 3 Report

Comments and Suggestions for Authors

The aim of this study was to analyze morphological changes of leukocytes on peripheral blood smears of Wistar rats exposed to hyperthermia using the geometric morphometrics method. A group of forty Wistar albino rats were divided into three experimental groups according to water temperature exposure (37°C, 41°C, and 44°C). Peripheral blood smears were prepared, stained, and digitally recorded, after which selected images were analyzed using geometric morphometric programs to evaluate leukocyte shape variations. Comparative analysis demonstrated statistically significant morphological changes in neutrophil shape between the control group (37°C) and rats exposed to 41°C (p=0.009). Significant differences were also identified in lymphocyte morphology between antemortem and postmortem groups (p=0.00307). The findings indicate that exposure to elevated temperatures induces measurable alterations in white blood cell morphology, confirming that hyperthermia produces significant structural changes in neutrophils and lymphocytes detectable through geometric morphometric analysis. 

The paper is original, with a "healthy" level of plagiarism (below 10%). It is acceptable after a minor revision: two major points should be adressed:

1) The main problem with this paper is the way of presentation of results in figures and tables. Their content looks unclear and is difficult for interpretation, especially for non-professional readers. The results should be presented in a more comprehensive way. 

2) The Authors should explain in more detail how big is the putative contribution of their fresults to the research on the influence of hyperthermia on blood cell function.

 

Author Response

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Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been corrected. The edits have been made. The only comment is that I would write polymorphonuclear cells instead of neutrophils, and mononuclear cells instead of lymphocytes.

Author Response

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Reviewer 2 Report

Comments and Suggestions for Authors

 Thank you for considering a subset of the comments and adding some clarifications. The results presented in the current manuscript draft are still very preliminary and much work is needed for publication.
Basic analyses are still missing, when plotting the PCA results please add the % of variance explained by the displayed PC on the plots. In figure 4 the ellipses are still cropped.  
The analysis regarding the sex difference, which involve minimal work from the author, are still missing and should be part of this work.
"how does the proportion of the difference leucocytes population change between before and 
after the warm bath?" have not been addressed. Is each cell type affected the same way, and to which extend? the authors have the data and they should be included.
"Which parameters weight the most in each PC? " This is not answered either. Does cell shape, width, granularity, ... weigh more in PC1 than PC2?

Author Response

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Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

thank you for addressing the comments. One minor suggestion: when figure captions use acronyms and color codes those should be spelled out in the corresponding figure legend.

Author Response

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Author Response File: Author Response.pdf