A Point-of-Care Diagnostic Method Using Desiccation Patterns of Blood Sessile Droplets

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. The line 214 and Eq. 1 - the discussion is about evaporation flow rate for water drop in the references 19-21. Actually, a drop of blood is non-Newtonian liquid. Could yo expalin whay you can use this equation for Newtonian liquid?
2. Page 5 - Fig. 2 b - What does it mean teta/grad. - the symbol "/" - is better to remove?
3. The reference 22 - maybe is better to write also the publisher name Wiley VCH?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Referee's Report

Manuscript ID: colloids-3613537

Journal: Colloids and Interfaces

Title:  A Potential Platform for Diagnosis Using Blood Desiccation Patterns

Recommendation: Major Revision

Overview and general recommendation:

This work compares the morphology of two sessile blood drops during drying, one from a healthy donor and the other from an anemic donor. The authors showed that the sessile blood drop from anemic patients with an abnormally low hematocrit exhibits a different behavior than that from a healthy donor.

On the one hand, the article is topical and fits within the scope of the journal.  On the other hand, several aspects of this article require further details and clarification.

Therefore, I recommend that a major revision is warranted. I explain my concerns in more detail below.

 Comments:

1. The title is informative, but very general. It should be revised to include the original aspect of the study and other keywords.
2. I suggest using the term "sessile droplet" in the title and/or keywords.
3. The term "coffee ring" in the keywords list appears only in one place in the text (line 232) in order to interpret Figure 4. This term describes a very interesting phenomenon and helps explain the particle deposition following the disappearance of the drop. I invite authors to consider this phenomenon (coffee ring or inverse coffee ring) to explain the patterns observed following the evaporation of the drops.
4. The introduction provides a useful bibliographic summary. I also encourage authors to include other recent references. In fact, the most recent reference (only one) dates from 2022.
5. In paragraph 2, it is advisable to add a photo or descriptive image of the experimental device.
6. In figures 3 and 4. Does the evolution of the drops take place as a function of time? If so, it is essential to add on the figures, the instants of the screenshots, specifying the initial instant.
7. How does sessile drop evaporate? It is important to specify the evaporation mode: pinned, unpinned, or stick-slip.

Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Summary of the Work

This study explores the diagnostic potential of blood desiccation patterns for red blood cell (RBC)-related diseases, such as anaemia. Using a standardized substrate, the authors compared blood samples from five healthy donors (normal haematocrit: 0.39–0.45) and five anaemia patients (low haematocrit: 0.22–0.27). Real-time optical microscopy was employed to observe the morphological evolution of blood sessile drops during evaporation. The results revealed that anaemic samples follow distinct evaporation dynamics and form noticeably different cracking patterns compared to healthy samples, with these differences attributed to variations in haematocrit levels affecting material distribution within the drop.

Main Findings

i) A semi-qualitative correlation was established between desiccation patterns and haematocrit levels.

ii) Blood drops with low haematocrit initiate cracks in the central region, spreading outward. Blood drops with normal haematocrit initiate cracks in the peripheral region, spreading inward.

iii) The length of the coronal region and the central deposit area increase with higher haematocrit levels. Healthy samples show larger coronal regions and more extensive central deposits than anaemic samples. Morphological differences are linked to haematocrit effects on evaporation and material distribution.

General Considerations

- The English should be double-checked; several typos were found.

- The study presents an original and low-cost method for differentiating haematocrit levels based on blood desiccation patterns, which could have practical applications in resource-limited settings.

- The observed morphological differences between healthy and anaemic blood drops are consistently linked to haematocrit levels, providing a promising visual marker for disease screening.

- The analysis focuses solely on physical measurements, overlooking the significant biochemical and compositional variations (e.g., protein content, ionic concentration) that also influence desiccation behavior and are tightly coupled with haematocrit.

- The study is based on only ten samples (five per group), which may not capture the variability across different age groups, sexes, or types of anaemia, potentially limiting the generalizability of the findings.

- While a semi-qualitative correlation is established, the study lacks a robust quantitative model or statistical analysis to predict haematocrit values or classify pathological states with confidence.

- The specificity of the desiccation patterns to anaemia is not thoroughly addressed; similar morphological features could potentially arise from other blood-related conditions or variations in red cell deformability, viscosity, or hydration state.

More specifically, two aspects left me somewhat perplexed: to have neglected the biochemical factors were neglected, and the results could potentially overlap with other conditions. The following suggestions are intended to help the authors dispel these strong objections.

- Finally, the list of references is not exhaustive and should be completed, maybe with the help of the following suggestions.

Suggestions

1) The authors' model assumed that all the evaporating solvent is water; the local evaporation flux (𝐽𝑟) over the edge of the blood sessile drop could be approximated by Eq. (1) on page 7 of the manuscript (Hu, H et al. and Weon, B et al.). However, we know that blood is a complex suspension of cells, proteins, and solutes, exhibiting non-Newtonian behavior and compositional heterogeneity. The formula assumes a single-component, Newtonian fluid (pure water), failing to capture how varying haematocrit levels affect viscosity, flow, and internal redistribution during evaporation. The authors are asked to clarify this issue.

2) We may object that the model ignores biochemical interactions (e.g., cell aggregation, protein adsorption) and dynamic changes in surface tension or Marangoni flows induced by gradients in solute concentration. These effects become more pronounced at different haematocrit levels and significantly influence the evaporation flux and resulting desiccation pattern. The authors are asked to dispel this possible objection.

3) How do the authors account for the potential influence of plasma protein concentration (e.g., albumin, fibrinogen) on the evaporation-driven flow and final desiccation pattern, given their role in colloidal interactions and Marangoni stresses?

4) Have you performed any compositional analysis (e.g., via mass spectrometry or Raman spectroscopy) of the dried blood residues to confirm that observed morphological differences are solely due to haematocrit variations and not confounded by biochemical differences (e.g., metabolic by-products, oxidative stress markers)?

5) Considering that red blood cell (RBC) deformability and intracellular viscosity vary with pathophysiological states, how do you isolate the effect of haematocrit from potential alterations in cytoplasmic hemoglobin concentration or membrane rigidity that might also affect droplet dynamics?

6) Given that diseases such as diabetes mellitus or infections can alter plasma osmolarity and RBC morphology independently of haematocrit, how can your method differentiate between anaemia-specific patterns and those induced by other systemic conditions?

7) Have you considered how variations in electrolyte composition (e.g., Na⁺, K⁺, Ca²⁺) or pH, which can influence zeta potential and interfacial tension, might modify the flow fields during evaporation and thus affect the pattern formation independently of haematocrit?

8) Could shear-thinning behavior and non-Newtonian properties of whole blood, modulated by biochemical parameters like fibrinogen concentration or erythrocyte aggregation tendency, play a role in shaping the desiccation pattern beyond what is captured by your purely physical characterization?

Conclusions

While the study presents a promising method for diagnosing haematocrit-related conditions using blood desiccation patterns, it requires stronger scientific grounding. Specifically, the model should extend beyond simple water evaporation to encompass blood’s complex biochemical and rheological properties. Biochemical factors influencing haematocrit must be considered, the specificity of the patterns related to anaemia should be clarified, and quantitative, statistically validated analyses are necessary to support diagnostic claims. Authors are strongly encouraged to consider the suggestions outlined above.

Comments on the Quality of English Language

The English should be double-checked; several typos were found.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have considered the comments raised and made the necessary changes to improve the paper. The document can be accepted in this form.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have answered all the questions raised in my previous report satisfactorily. In my opinion, this version of the manuscript deserves publication.