Characterization of Extracellular Vesicle-Enriched Populations in B-Cell Acute Lymphoblastic Leukemia from Peripheral Blood

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this manuscript, the authors aim to phenotype extracellular vesicles derived from plasma membrane budding in B-ALL patients. The authors have chosen not to ultracentrifuge the vesicles. Their work mainly relies on nanoscale flow cytometry analysis.

Although the authors’ hypothesis is likely correct, the methods used do not allow it to be properly addressed in the manuscript in its current form.

Major points

A nanoscale flow cytometry approach requires proper calibration, or at least a precise understanding of the size of the vesicles being studied. This is all the more important given that the authors are not analyzing all vesicles, but only microvesicles, typically ranging from around 150 nm to larger sizes, originating from membrane budding. Unfortunately, no data are provided in the manuscript regarding vesicle size, and nowhere are these calibrations shown.

The authors repeatedly refer to vesicle quantification; however, no such quantification has actually been performed.

Cell budding is a multifactorial process; it may just as well result from treatments rather than tumor activity. These vesicles could also originate from transfusions, from immune activation potentially induced by vaccination, or from another condition such as a simple infection.

Regarding the presence of CD3⁺CD19⁺ vesicles, this does not in any way demonstrate that they originate from leukemic cells. One would at least expect associated cellular phenotyping performed at the same time.

In my opinion, the microscopy does not provide reliable information. The issue of aggregate analysis also arises: how can one be certain that these aggregates are not generated by the sample preparation protocol? No data from healthy donors are provided.

A substantial revision of the manuscript is needed, and a short communication format might be more appropriate. Including a larger number of patients would likely provide more robust statistical data. At present, these data appear too preliminary for publication.

Author Response

Reviewer 1

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files. We appreciate your comments, which we have taken on account, and would like to provide the following information:

Comments 1:

A nanoscale flow cytometry approach requires proper calibration, or at least a precise understanding of the size of the vesicles being studied. This is all the more important given that the authors are not analyzing all vesicles, but only microvesicles, typically ranging from around 150 nm to larger sizes, originating from membrane budding. Unfortunately, no data are provided in the manuscript regarding vesicle size, and nowhere are these calibrations shown.

Response 1

Thank you for pointing this out. We agree with this comment. We added supplementary materials figure S2

To estimate the size range of the EVEPs, a calibration was performed using different types of beads with diameters provided by the suppliers. The following reference beads were used: 100 nm beads (Cytek YG100 nm, P/N: B7-10012, Batch: F-102423-05; concentration: 3.6 × 10⁷ particles/mL), 1000 nm beads (Fluorobead T lymphocyte-100, REF: 1-100, Lot: 157), 4500 nm beads (Life Screen Deluxe Beads Kit, Lot: 3013358, REF: 405567) and 10,000 nm beads (SONY Precision Count Beads, Part: 2724510, Lot: 251751). All beads were analysed using the same instrumental parameters as the EVEP samples.

The size range was determined using the side-scatter height (SSC-H) parameter on a bi-exponential scale. Based on the SSC-H signals from the reference beads, the following size ranges were established: 10⁵ to 1,000 nm and 10⁶ to 10,000 nm. The EVEPs were considered to fall within the size range ≤ 1000 nm (10⁵) and 10⁴, although events above this range were occasionally detected. This calibration strategy confirmed that the EVEPs analysed in this study predominantly correspond to microvesicles and large vesicles.

To estimate the size range of EVEPs, a calibration was performed using different The authors repeatedly refer to vesicle quantification; however, no such quantification has actually been performed.

 

Comments 2:

The authors repeatedly refer to vesicle quantification; however, no such quantification has actually been performed

 

Response 2

We thank the reviewer for the comment; as the wording we used was incorrect, certain lines in the text have been amended to improve clarity. The following has been added to lines 31 and 32 of the text: showed a higher percentage of marker-positive events by flow cytometry; Line 259: Comparison of the percentage of tetraspanin-positive EVEPs between patients with B-ALL and Line 253-256: expression appears to show a slight tendency to be higher in PB EVEPs from patients with B-ALL compared to the healthy group, without being close to showing a significant difference. The above result suggests that the three markers evaluated do not constitute a differential marker of malignancy by themselves; Lines 280 and 281: average percentage of marker-positive

 

Comments 3:

Cell budding is a multifactorial process; it may just as well result from treatments rather than tumor activity. These vesicles could also originate from transfusions, from immune activation potentially induced by vaccination, or from another condition such as a simple infection.

Response 3

Line 507-512: was added: It is important to bear in mind that the release of EVs is a multifactorial process that may be influenced not only by tumour activity, but also by other clinical and physiological conditions, such as infections and inflammation. In this study, all PB and BM samples were obtained at the time of diagnosis, prior to the start of anti-leukaemia therapy, which allows us to interpret the observed EVEP profiles as representative of a baseline disease state.

Comments 4:

Regarding the presence of CD3⁺CD19⁺ vesicles, this does not in any way demonstrate that they originate from leukemic cells. One would at least expect associated cellular phenotyping performed at the same time.

 

Response 4

We agree with this comment. We added supplementary materials figure S3

With regard to the reviewer’s comment on CD3+CD19+ vesicles, we acknowledge that our study does not experimentally demonstrate that these vesicles originate from leukaemic cells or that they result from vesicle fusion. However, we would like to clarify the rationale behind our proposal of these possibilities. We propose two non-mutually exclusive mechanisms: (1) direct secretion from leukaemic cells with an aberrant dual phenotype, or (2) post-secretion fusion between CD19+and CD3+ vesicles.*

In our analysis, we observed that, in bone marrow samples, patients with B-ALL expressed only CD19+ (but not CD3+), patients with B-ALL expressed only CD3+ (but not CD19+), whilst CD3⁺CD19+ double-positive events were identified exclusively in extracellular vesicle-enriched populations (EVEPs) from peripheral blood, mainly from patients with B-ALL but not in control samples lacking EVEPs. *Furthermore, a recent study by Lozano-Andrés et al. (2023) demonstrated that lipoproteins can form physical associations with extracellular vesicles, which could influence their detection and composition. This finding supports the possibility that vesicle-vesicle or vesicle-lipoprotein interactions occur in the peripheral blood circulation rather than in the bone marrow, which could contribute to the generation of double-positive events.

 

Comments 5:

In my opinion, the microscopy does not provide reliable information. The issue of aggregate analysis also arises: how can one be certain that these aggregates are not generated by the sample preparation protocol? No data from healthy donors are provided

 

Response 5

 

In response to the comment, three CTRL (healthy) samples were included (we added figure 4B) to demonstrate that the labelling of CD3+CD19+ EVEPs is specific and does not appear to be a result of the mounting protocol. It is important to note that this finding arises from flow cytometry observations, which can be seen in question 4. The ImageJ2 EVAnalyzer software is capable of detecting labelling on a vesicle-by-vesicle basis, which we used to confirm our observations via confocal microscopy of obtaining some EVs (microvesicles) with dual CD3+CD19+ labelling. Precipitation by centrifugation facilitated interaction between the EVs, and upon disruption, there was a consistent tendency for a greater qualitative quantity of aggregates to remain in those samples derived from patients with B-ALL.

 

Comments 6:

 

A substantial revision of the manuscript is needed, and a short communication format might be more appropriate. Including a larger number of patients would likely provide more robust statistical data. At present, these data appear too preliminary for publication.

 

Response 6

Thank you for your comment; we have decided to forward your submission to Short Communications.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by Carmona-Zamudio MA, et al describes an exploratory study with an aim to characterize extracellular vesicle–enriched populations (EVEPs) obtained from peripheral blood and bone marrow of adult patients with B cell acute lymphoblastic leukemia (B-ALL) and to compare them with the clinical immunophenotype (CIP). The latter is further specified among the objectives to describe the presence of double-positive CD3⁺CD19⁺ extracellular vesicles (EVs) that seems to be ‘the finding’ of the study. The presentation is detailed and logical. The discussion is well-taken. The main limitation is the number of few patients (n=8) and no deeper exploration to the development of the double-positive CD3+CD19+ EVs.

Major comments

1. The Abstract would benefit to include the number of patients and samples that were studied in triplicates (information identified first on line 151, subsection 2.3)? The chapter ‘Discussion’ is to include evaluation of the benefits and drawbacks when triplicates were included the analysis process.

2. The chapter ‘Introduction’ would benefit of additional information about the known biological function reflecting various cell membrane markers (CD markers) of interest for this study (eg. CD38, CD63, CD81, etc). This theoretical information would support underlying expectations and the presentation of the used CD-markers (chapter ‘Material and methods’). Furthermore, the authors’ reasoning of the results in the chapter ‘Discussion’ would benefit to highlight the aspect that the choice of the CD markers will reflect what is possible to identify and how the authors did choose the used CD markers with the theoretical expectations. Also, for a reader less familiar of the biology reflected in various markers, the manuscript would be easier to study. It’s highly supported that as now presented there is information of various size of EVs and also about the double-positive CD3⁺CD19⁺ ones.

3. There is one unclear aspect of time relationship between patients’ diagnosis of their B-ALL and time point for sampling in peripheral blood (PB) and bone marrow (BM) for analyses in this study. Please, complement with this information. The chapter ‘Discussion’ is proposed to evaluate the influence of this time gap. If the aim is to evaluate the potential of EVEPs in diagnostics of B-ALL, then it’s also of interest to discuss at least on the theoretical level how the findings of EVEPs would change due to changes of leukemic cells in BM and further in PB over time and due to given anti-leukemic therapy?

4. The information of predefined heterogenous population of EVs of interest belongs the chapter ‘Material and methods’ (subsection 2.2) (lines 87-91). If the authors want to give some information related to the MISEV2023 already in the chapter ‘Introduction’, it may be presented more on common level. Please, revise.

5. In the subsection 2.1. there is information of one patient with T-ALL but who was excluded from the study (line 100). This needs to be specified more in details: was the per protocol study population defined as nine patients with B-ALL and thus, the T-ALL patient was a protocol failure?

6. In the subsection 3.1. ‘Clinical information’, the authors are asked to add more detailed information of which material (peripheral blood white blood cells, plasma; bone marrow, etc) were used to diagnose patients’ leukemia type (lines 187-188).

7. The authors’ conclusion of CD3+CD19+ vesicles suggesting an existence of a leukemic niche would benefit from a presentation of more clear hypothetic background also here in the conclusion. Although the finding is evaluated in the chapter ‘Discussion’, presentation of a hypothesis to be studied further, i.e. existence of cells expressing both T and B cell lineage that are involved in the evolution of leukemic cells in a specific bone marrow niche and ultimately developing to B-ALL lineage. Based on the chapter ‘Introduction’, this is ‘the finding’ of the study the authors want to highlight by including the conclusions? It’s not unusual that ‘stressed readers’ only read ‘Abstract’ and ‘Conclusion’, why more detailed conclusion is suggested.                                                      
8. The manuscript’s scientific value would be higher if the authors could include any data of explorative, for example in vitro studies concerning the development of the double-positive CD3+CD19+ EVs.

Minor comments

1. The text body includes very long sentences that may lead to misunderstanding and in any case slows down reading. Please, check sentences and divide them to 2 to 3 separate ones (for example on lines 47-51, 68-73 and 73-82, i.e. over 5-10 lines). Also, to revise to two separate sentences on lines 124-126, please.

 

2. The English writing of the following sentence needs several corrections as following: “…in the isolation process by of EVs populations, [31, 32], their Menck K and Parolini I et al could provide a greater substantial number of markers of interest, including cell surface markers, in addition to showing intervesicular fusion phenomena that could occur in vivo [331 – 36]. (lines 58-61)

Comments:

1. the form genetive is created by ‘of’; alternatively, ‘… EVs populations’ isolation process Menck K…’ .
2. In general, one doesn’t refer to a given references (here used ‘their’ referring to references 31 and

• The form ‘greater’ requires a comparison to what something is compared to; for example, ‘greater than …’ Here it’s understood not needing any comparison.

1. All references can be given in the end.

 

3. Abbreviations are to be explained when they are used for the first time, and don’t need to be repeated later on. Especially, in the chapter ‘Discussion’ irregular and unnecessary use of abbreviations is observed. Also, in general, it’s not supported to start a sentence by an abbreviation but the word is written out. Please, check these two elements (eg. ‘MISEV2023’, on line 88).
4. Please, revise the writing to express one statement, for example ‘suggests’ since the results are base very few patient samples (line 292).
5. The writing of the reference ‘31’ in the following sentence is to be revised: ‘….reported strategies^[31].’ (line 379).
6. The writing in the sentence: ‘Others researchers have been reporterd existence of CD3+CD19+ cells…’ needs a revision; please, see above deleted ‘s’, deleted ‘been’, the right tempus for ‘reported’ and added ‘existence of’ as well as ‘+’ for CD19. Please, check if the intended meaning is the right one (line 472).

Comments on the Quality of English Language

Please, see my 'Minor comments'

Author Response

Reviewer 2

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files. We appreciate your comments, which we have taken on account, and would like to provide the following information:

We would like to thank you for your valuable comments and suggestions; below are our responses

Comments 1:

The Abstract would benefit to include the number of patients and samples that were studied in triplicates (information identified first on line 151, subsection 2.3)? The chapter ‘Discussion’ is to include evaluation of the benefits and drawbacks when triplicates were included the analysis process.

 

Response 1

We have included the information from the abstract. Line 26-28: 12 individuals were initially recruited (8 with B-ALL, 1 with T-ALL, and 3 healthy controls). The study focused on the 8 B-ALL patients and 3 controls, whilst the T-ALL sample was used as a specificity control

We have amended line 424-431: All validation experiments were performed in triplicate to improve reproducibility, optimize selection strategies and ensure consistency in the detection of EVEPs. However, despite these technical advantages, the biological variability inherent in human samples represents a significant limitation. As in clinical diagnostic settings, EVEPs profiles can vary significantly between individuals due to underlying biological and pathological heterogeneity. Therefore, whilst triplicate measurements enhance technical reliability, they do not fully account for inter-sample variability, underscoring the need for larger cohorts to validate and generalize these findings.

 

Comments 2:

The chapter ‘Introduction’ would benefit of additional information about the known biological function reflecting various cell membrane markers (CD markers) of interest for this study (eg. CD38, CD63, CD81, etc). This theoretical information would support underlying expectations and the presentation of the used CD-markers (chapter ‘Material and methods’). Furthermore, the authors’ reasoning of the results in the chapter ‘Discussion’ would benefit to highlight the aspect that the choice of the CD markers will reflect what is possible to identify and how the authors did choose the used CD markers with the theoretical expectations. Also, for a reader less familiar of the biology reflected in various markers, the manuscript would be easier to study. It’s highly supported that as now presented there is information of various size of EVs and also about the double-positive CD3⁺CD19⁺ ones.

Response 2

 

Following the comment, the following paragraphs were added to the text.

Lines: 66–76                   

There are currently several methods for characterizing EVs; one of these involves surface proteins, also known as CDs, which have played a crucial role in this characterization, primarily through the use of flow cytometry. This has enabled the identification of tetraspanins such as CD9, CD63 and CD81, which are widely recognized as canonical markers of EVs [8, 37, 38]. Furthermore, lineage-specific markers, such as CD19 and CD10 for B cells and CD3 for T cells, are essential for immunophenotypic characterization [39].

Markers such as CD34 and CD20 further contribute to the identification of cellular differentiation and maturation states. Therefore, the selection of these markers allows not only the identification of EVs, but also the exploration of their potential cellular origin and biological relevance in leukemic contexts [40, 41]

 

Comments 3:

 

There is one unclear aspect of time relationship between patients’ diagnosis of their B-ALL and time point for sampling in peripheral blood (PB) and bone marrow (BM) for analyses in this study. Please, complement with this information. The chapter ‘Discussion’ is proposed to evaluate the influence of this time gap. If the aim is to evaluate the potential of EVEPs in diagnostics of B-ALL, then it’s also of interest to discuss at least on the theoretical level how the findings of EVEPs would change due to changes of leukemic cells in BM and further in PB over time and due to given anti-leukemic therapy?

 

Response 3

We have been placed on line: 507-523

It is important to note that the release of EVs is a multifactorial process that may be influenced not only by tumour activity, but also by other clinical and physiological conditions, such as infections and inflammation [27, 52, 66]. In this study, all PB and BM samples were obtained at the time of diagnosis, prior to the initiation of antileukemic therapy, which enables us to interpret the observed EVEPs profiles as representative of a baseline disease state.

However, although the patients were diagnosed de novo, detailed clinical information on concomitant conditions was not systematically available. Therefore, we cannot rule out the possibility that factors other than leukaemia may have contributed to the observed EVEPs profiles.

Furthermore, it has previously been reported that both the concentration and phenotypic composition of EVs can change over time as a result of disease progression or therapeutic intervention [65, 75, 76].

In this context, the EVEPs profiles described here should be interpreted as a single cross-sectional representation of the disease at the time of diagnosis; further longitudinal studies will be required to assess their dynamic behavior and their potential utility in monitoring disease progression and response to treatment.

 

Comments 4:

The information of predefined heterogenous population of EVs of interest belongs the chapter ‘Material and methods’ (subsection 2.2) (lines 87-91). If the authors want to give some information related to the MISEV2023 already in the chapter ‘Introduction’, it may be presented more on common level. Please, revise.

Response 4

The information was presented in lines 100 and 103,

Section 2.1 contains information on a patient with T-cell ALL who was, however, excluded from the study (line 100). This needs to be specified in more detail: was the study population defined ‘by protocol’ as nine patients with B-ALL, and was the patient with T-ALL therefore considered a case of non-compliance with the protocol?

In line:26 In the abstract we clarify sample patient with T-cell ALL

The text in line 113-116 has been reorganized: A total of twelve individuals were enrolled in this study. The primary analysis included eight adult patients with B-ALL and three healthy controls (volunteer donors matched for age and sex). One patient with T-ALL serving instead as a control to verify the specificity of the assay (Supplementary Materials, Figure S1).

In subsection 3.1, ‘Clinical information’, the authors are requested to provide more detailed information on which material (peripheral blood white cells, plasma, bone marrow, etc.) was used to diagnose the type of leukaemia in the patients (lines 204–207).

Line 204-207:

Diagnostic analyses were performed primarily using BM or BP, from which whole blood was obtained; leukocytes were subsequently isolated from the leukocyte layer.

 

 

Comments 5:

The authors’ conclusion of CD3+CD19+ vesicles suggesting an existence of a leukemic niche would benefit from a presentation of more clear hypothetic background also here in the conclusion. Although the finding is evaluated in the chapter ‘Discussion’, presentation of a hypothesis to be studied further, i.e. existence of cells expressing both T and B cell lineage that are involved in the evolution of leukemic cells in a specific bone marrow niche and ultimately developing to B-ALL lineage. Based on the chapter ‘Introduction’, this is ‘the finding’ of the study the authors want to highlight by including the conclusions? It’s not unusual that ‘stressed readers’ only read ‘Abstract’ and ‘Conclusion’, why more detailed conclusion is suggested

 

Response 5

Line 533-535

Following the reviewer’s suggestion, the conclusion has been amended as follows: This can be supported by comparison with the CIP obtained. On the other hand, the occasional identification of CD3+CD19+ cells could be associated with leukaemic processes in peripheral blood.

The scientific value of the manuscript would be enhanced if the authors could include data from exploratory studies, such as in vitro studies, concerning the development of double-positive CD3+CD19+ EVs.

We appreciate the reviewer’s comment and acknowledge that in vitro studies would be necessary to accurately demonstrate the presence of CD3⁺CD19⁺ EVs, as well as to elucidate the mechanisms underlying their formation. However, at present we are unable to carry out such experiments due to limitations in resources, time and the availability of experimental infrastructure.

Nevertheless, we have considered conducting future experiments aimed at: (1) assessing vesicular fusion, (2) inhibiting vesicular fusion using specific drugs, and (3) analysing the interaction of B and T lymphoid lineages separately. These studies will enable us to address the questions raised by the reviewer more rigorously and will strengthen the biological basis of our findings.

 

Comments 6:

 

The text body includes very long sentences that may lead to misunderstanding and in any case slows down reading. Please, check sentences and divide them to 2 to 3 separate ones (for example on lines 47-51, 68-73 and 73-82, i.e. over 5-10 lines). Also, to revise to two separate sentences on lines 124-126, please. Modifications in lines 47-53, 67-82.

 

The English writing of the following sentence needs several corrections as following: “…in the isolation process by of EVs populations, [31, 32], their Menck K and Parolini I et al could provide a greater substantial number of markers of interest, including cell surface markers, in addition to showing intervesicular fusion phenomena that could occur in vivo [331 – 36]. (lines 58-61). Modifications in lines 59-61.

Comments:

the form genetive is created by ‘of’; alternatively, ‘… EVs populations’ isolation process Menck K…’ .

In general, one doesn’t refer to a given references (here used ‘their’ referring to references 31 and The form ‘greater’ requires a comparison to what something is compared to; for example, ‘greater than …’ Here it’s understood not needing any comparison.

All references can be given in the end.

Abbreviations are to be explained when they are used for the first time, and don’t need to be repeated later on. Especially, in the chapter ‘Discussion’ irregular and unnecessary use of abbreviations is observed. Also, in general, it’s not supported to start a sentence by an abbreviation but the word is written out. Please, check these two elements (eg. ‘MISEV2023’, on line 88).

Please, revise the writing to express one statement, for example ‘suggests’ since the results are base very few patient samples (line 292).

The writing of the reference ‘31’ in the following sentence is to be revised: ‘….reported strategies[31].’ (line 379).

The writing in the sentence: ‘Others researchers have been reporterd existence of CD3+CD19+ cells…’ needs a revision; please, see above deleted ‘s’, deleted ‘been’, the right tempus for ‘reported’ and added ‘existence of’ as well as ‘+’ for CD19. Please, check if the intended meaning is the right one (line 472).

 

Response 6

In response to the reviewer’s suggestions, the text will be reviewed and corrected for spelling and grammar

Reviewer 3 Report

Comments and Suggestions for Authors

The disease setting is not well chosen for the study as there is no need for a liquid biopsy.

In terms of lymphoid markers that would make more sense with lymphomas.

The 'invasiveness' of the method is the same as of a robust and validated BM or PB immunoprophilling by FACS. It would have to be done from urine or saliva to be less invasive.

I think this could be treted as technical testing that could be moved to more suited setting.

 

Author Response

Reviewer 3

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files. We appreciate your comments, which we have taken on account, and would like to provide the following information:

We would like to thank you for your valuable comments and suggestions; below are our responses

 

Comments 1:

 

The disease setting is not well chosen for the study as there is no need for a liquid biopsy. In terms of lymphoid markers that would make more sense with lymphomas. The 'invasiveness' of the method is the same as of a robust and validated BM or PB immunoprophilling by FACS. It would have to be done from urine or saliva to be less invasive. I think this could be treted as technical testing that could be moved to more suited setting.

Response 1

While we agree that flow cytometry-based immunophenotyping of bone marrow or peripheral blood remains the current gold standard for leukemia, we respectfully suggest that this approach reflects optimal diagnostic conditions that are not always achievable in routine clinical practice.

In many cases, bone marrow samples may be of limited volume, diluted, and may even clot or be insufficient for multiple subsequent analyses. In this context, our approach does not aim to replace established diagnostic methods but rather to complement them by leveraging plasma-derived extracellular vesicles (EVs) as an additional and underexplored source of tumor-associated information.

Regarding concerns about invasiveness, we clarify that our methodology requires no additional procedures beyond standard blood collection. Instead, it seeks to maximize the analytical value of plasma, which is often already obtained in clinical workflows. Extracellular vesicles (EVs) offer a stable and enriched source of biomarkers that can expand the diagnostic potential of a single sample. Furthermore, in many healthcare settings, especially in low- and middle-income countries, access to flow cytometry is not always immediate. Samples often need to be transported to specialized centers, which can lead to delays of more than 48 to 72 hours. These delays can compromise cell viability and affect immunophenotypic analyses. In contrast, plasma samples can be cryopreserved and transported more easily, with less impact on their integrity, supporting their usefulness in decentralized or resource-limited settings.

Overall, we propose that EV-based analysis be considered a complementary and exploratory strategy, particularly valuable in scenarios where conventional approaches have logistical or technical limitations.

Finally, we agree with the reviewer that lymphomas represent a highly relevant and promising context for the future application of this methodology, and we appreciate this valuable suggestion.

Reviewer 4 Report

Comments and Suggestions for Authors

Lines 25, 48, 105, 106:The abbreviation PB is already explained in line 22.

Lines: 58, 59: It is better to avoid this type of wording when describing biological processes, as 100% purity cannot be assumed in such studies.

Line 235: This comparison should be interpreted with caution, as control EVEPs were derived from peripheral blood, whereas patient EVEPs were obtained from bone marrow.

Figure 3A: Check the gating strategy for CD34+ cells

Please check the explanation of abbreviations throughout the manuscript, as they are repeated multiple times.

The number of patients is unclear. You state that eight patients were included, but one was excluded due to T-ALL, which suggests that only seven samples should be analyzed. However, later you refer to eight samples—please clarify.

In line 379, the reference number should not remain in superscript.

Comments on the Quality of English Language

The manuscript would benefit from improved English.

Author Response

Reviewer 4

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files. We appreciate your comments, which we have taken on account, and would like to provide the following information:

We would like to thank you for your valuable comments and suggestions; below are our responses

 

Comments 1:

The abbreviation PB is already explained

Response 1

in lines 22 and 50.

Comments 2:

Lines: 58, 59: It is better to avoid this type of wording when describing biological processes, as 100% purity cannot be assumed in such studies.

Response 2:

The text was rewritten and ended up on lines 57-60

Although methodologies exist that seek to purify extracellular vesicles, biological processes still interfere with selectivity, so absolute purity cannot be achieved in the isolation of EV populations [31, 32],

Comments 3:

Line 235: This comparison should be interpreted with caution, as control EVEPs were derived from peripheral blood, whereas patient EVEPs were obtained from bone marrow.

Response 3:

In patients with B-ALL, both bone marrow and peripheral blood samples were processed; on the other hand, in controls, only peripheral blood samples were taken. Although comparisons were made between PB vs. PB and BM vs. PB, it is correct that the interpretation should be done with caution depending on the type of comparison and especially considering the dispersion of the data and the n of the study.

 

 

 

 

Comments 4:

The number of patients is unclear. You state that eight patients were included, but one was excluded due to T-ALL, which suggests that only seven samples should be analyzed. However, later you refer to eight samples—please clarify.

Response 4:

Line 113-116 the text was reorganized: A total of twelve individuals were enrolled in this study. The primary analysis included eight adult patients with B-ALL and three healthy controls (volunteer donors matched for age and sex). One patient with T-ALL serving instead as a control to verify the specificity of the assay (Supplementary Materials, Figure S1).

 

Comments 5:

Figure 3A: Check the gating strategy for CD34+ cells

Response 5:

It was decided not to perform a gatin analysis based on CD34 derived from the swarm effect; we could not be sure of the passage of unique events

Comments 6:

Please check the explanation of abbreviations throughout the manuscript, as they are repeated multiple times.

Response 6:

Resolved

Comments 7:

In line 379, the reference number should not remain in superscript.

Resolved

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I would like to thank the authors for this revised version. This version is clearer and better suited to the style of short articles. The results are still very preliminary and descriptive, but the authors clearly state the limitations of their study.

Reviewer 3 Report

Comments and Suggestions for Authors

As in the first review.