Review Reports - The Single Antigen Luminex Bead Assay for the Definition of HLA-Specific Antibodies Revisited: Improved Reactivity by Incubation at 37 Degrees Celsius

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study explores the impact of incubation temperature on Luminex-based HLA antibody detection, comparing standard ambient conditions to physiologic temperature (37°C). By assessing 42 serum samples using single antigen bead assays for HLA class I and II, the authors observed increased mean fluorescence intensity (MFI) at 37°C and noted that nonspecific reactivities, often against irrelevant HLA antigens, sometimes shifted from positive to negative. The findings suggest that performing the assay at 37°C may provide a more accurate reflection of a patient's in vivo humoral immune status, with potential implications for organ transplantation assessment.

Comments:

In abstract, the findings suggest increased MFI and improved specificity at 37°C, but lack numerical or statistical data to support these claims.
The observed shift from positive to negative in some sera due to nonspecific reactivity is clinically interesting, but the absence of outcome correlation (e.g., rejection episodes) weakens the translational impact.
The conclusion is cautiously stated (“might affect transplantation”) but should be more assertive or suggest next steps (e.g., validation in a clinical cohort).
Why objectives, methods, results, conclusions are separate from abstract.
The introduction provides a thorough historical background, effectively tracing the development of HLA antibody detection from CDC assays to current Luminex-based platforms. However, it would benefit from a clearer and more concise structure, as it presently includes redundancies and tangential information.
There is an overemphasis on cryoagglutinins and IgM antibodies, which are acknowledged as clinically irrelevant; this discussion could be shortened or more tightly integrated with the point about nonspecific reactivities in Luminex testing.
Statements like “elevation in temperature substantially influences antibody binding” are made without citing adequate recent evidence; these claims would be more convincing if backed by quantitative data or references to recent immunological studies.
The mention of previous findings (e.g., Battle et al.) about prozone effects and denatured antibodies is relevant and supports the need for assay refinement. However, the connection to the current study’s specific aims should be stated more clearly.
The sentence structure at times is awkward or grammatically incorrect, making the narrative difficult to follow. For example, “serum sample of a proband/patient will incubated” should be corrected to “will be incubated.”
The authors state they do not aim to find the assay’s maximum efficiency but to simulate physiological conditions. This distinction is important but should be articulated earlier to set realistic expectations for the reader.
While the link between normothermic organ transport and the rationale for 37°C incubation is logical, the manuscript should briefly discuss whether temperature-induced changes in MFI values would alter clinical decision-making (e.g., virtual crossmatch outcomes or unacceptable antigen listing).
The inclusion of both HLA class I and class II antibodies is appropriate and enhances the comprehensiveness of the analysis; however, it would be helpful to clarify if these 42 sera (19 class I, 23 class II) came from the same or different patient samples.
It is unclear whether any replicates (technical or biological) were included in the temperature comparison, and no mention is made of how variability between replicates (if any) was handled.
It is unclear whether any statistical analyses were conducted to quantify the frequency or magnitude of MFI shifts across the cohort. Even basic descriptive statistics (e.g., number or percentage of samples with shifts) would provide a more robust summary.
While individual case examples (Figures 2–8) are well described, the reliance on qualitative language such as “clearer results” or “more accurate interpretation” weakens the scientific rigor. These statements should be supported by numeric MFI values and standardized thresholds.
The discussion lacks detailed statistical interpretation or frequency-based summarization of results. Even qualitative data synthesis (e.g., % of sera affected) would strengthen the argument.
The discussion would benefit from deeper integration with prior literature. Several references are cited, but specific comparisons to findings from similar temperature-altered or sensitivity-focused HLA testing studies are missing.
The explanation of “false-positive” reactivity to HLA-A80 is insightful, but the discussion does not provide a generalizable framework for identifying and managing such artifacts across different alleles or patient populations.
The conclusion is brief but aligns with the study’s aim: normothermic serum incubation could enhance the physiological relevance of HLA antibody testing and improve transplant immunological assessments. However, a stronger final statement proposing validation in larger cohorts or clinical correlation would be more impactful.

Author Response

We thank the reviewer for the valuable and important comments and suggestions. Below we answer to the suggestions and remarks in a point-to-point manner. The changes were made via track change but were at the end unreadable and therefore we had zo accept the change and present them here. Sorry for that.

Comments:

In abstract, the findings suggest increased MFI and improved specificity at 37°C but lack numerical or statistical data to support these claims.
Answer: We introduced statistical analysis in the abstract for specific beads. Furthermore, we add numerical data in the results e.g. page 8 to page 12. We additionally included Figures 2 a, b and Tables 3 and 4 with numerical and % data where appropriate.
The observed shift from positive to negative in some sera due to nonspecific reactivity is clinically interesting, but the absence of outcome correlation (e.g., rejection episodes) weakens the translational impact.
Answer: The study presented here is observational, and we did not yet compare any of the outcomes of the transplantation. This will be the scope for a follow-up study in which not only the number of samples tested will be higher but also especially the correlation to any of the outcomes will be analyzed. In the near future we aim for a study including several centers.
The conclusion is cautiously stated (“might affect transplantation”) but should be more assertive or suggest next steps (e.g., validation in a clinical cohort).
Answer: Thank you for the remark. We elaborate on this point in the abstract, discussion and conclusion. The proposed method must be validated using a larger cohort, with longer observation time for graft survival or surrogate markers.
Why objectives, methods, results, conclusions are separate from abstract.
Answer: Thanks it is now corrected; our mistake
The introduction provides a thorough historical background, effectively tracing the development of HLA antibody detection from CDC assays to current Luminex-based platforms. However, it would benefit from a clearer and more concise structure, as it presently includes redundancies and tangential information.
Answer: done

There is an overemphasis on cryoagglutinins and IgM antibodies, which are acknowledged as clinically irrelevant; this discussion could be shortened or more tightly integrated with the point about nonspecific reactivities in Luminex testing.
Answer: done

Statements like “elevation in temperature substantially influences antibody binding” are made without citing adequate recent evidence; these claims would be more convincing if backed by quantitative data or references to recent immunological studies.

Answer: Done so far literature available

The mention of previous findings (e.g., Battle et al.) about prozone effects and denatured antibodies is relevant and supports the need for assay refinement. However, the connection to the current study’s specific aims should be stated more clearly.

Answer: Done we also presented the citation of Schnaidt, Transplantation of 2011, who elaborated of the topic.

The sentence structure at times is awkward or grammatically incorrect, making the narrative difficult to follow. For example, “serum sample of a proband/patient will incubated” should be corrected to “will be incubated.”

Answer: Corrected

The authors state they do not aim to find the assay’s maximum efficiency but to simulate physiological conditions. This distinction is important but should be articulated earlier to set realistic expectations for the reader.

Answer: Done in the introduction

While the link between normothermic organ transport and the rationale for 37°C incubation is logical, the manuscript should briefly discuss whether temperature-induced changes in MFI values would alter clinical decision-making (e.g., virtual crossmatch outcomes or unacceptable antigen listing).

Answer: We elaborated on the topic in the discussion.

The inclusion of both HLA class I and class II antibodies is appropriate and enhances the comprehensiveness of the analysis; however, it would be helpful to clarify if these 42 sera (19 class I, 23 class II) came from the same or different patient samples.

Answer: The selection of the sera is depicted in M&M.

It is unclear whether any replicates (technical or biological) were included in the temperature comparison, and no mention is made of how variability between replicates (if any) was handled.

Answer: Unfortunately, we did not do any replicates. This will be part of the following study. We, however, measured some samples in two different machines. All assays were performed by the same person.

It is unclear whether any statistical analyses were conducted to quantify the frequency or magnitude of MFI shifts across the cohort. Even basic descriptive statistics (e.g., number or percentage of samples with shifts) would provide a more robust summary.

Answer; Done; Figures 2a and 2b, S1, S2, and Tables 3 and 4 and 2x2 tables for specific beads. All reported in the main text in the part results and in the abstract. Pages 6, 7 and supplement.

While individual case examples (Figures 2–8) are well described, the reliance on qualitative language such as “clearer results” or “more accurate interpretation” weakens the scientific rigor. These statements should be supported by numeric MFI values and standardized thresholds.

Answer: Done, MFI values are added in results pages 8-12

The discussion lacks detailed statistical interpretation or frequency-based summarization of results. Even qualitative data synthesis (e.g., % of sera affected) would strengthen the argument.

Answer: Done

The discussion would benefit from deeper integration with prior literature. Several references are cited, but specific comparisons to findings from similar temperature-altered or sensitivity-focused HLA testing studies are missing.

Answer: Done so far available

The explanation of “false-positive” reactivity to HLA-A80 is insightful, but the discussion does not provide a generalizable framework for identifying and managing such artifacts across different alleles or patient populations.

Answer: Discussed, clarified in Page 14, 15

The conclusion is brief but aligns with the study’s aim: normothermic serum incubation could enhance the physiological relevance of HLA antibody testing and improve transplant immunological assessments. However, a stronger final statement proposing validation in larger cohorts or clinical correlation would be more impactful.

Answer: done

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presented for review titled “The single antigen Luminex bead assay for the definition of HLA-specific antibodies revisited: HLA-specific antibodies like it warm" is an important investigation into the impact of incubation temperature (21°C vs. 37°C) on the detection of HLA-specific antibodies using the Luminex single antigen bead assay.

The subject matter of this article is important as it provides preliminary data that could impact clinical practice and policy in HLA laboratories.

The systematic analysis of the topics raised by the Authors has been presented in a clear and coherent manner. This manuscript is well-organized and appropriately references relevant literature. The language of the work is understandable and easy to read. The manuscript is generally well written and clear.

While the manuscript addresses an important topic, I have several minor and moderate suggestions that could strengthen the work.

The introduction is a bit long and includes some repeated or background information that isn’t directly needed. I suggest shortening it by about one-third and focusing more on why studying temperature effects is important. Some of the extra technical details could be moved to the supplement.
The study used a small number of patients and samples. Authors should try to explain why this number of samples was chosen, or to include a power calculation to show if the study had enough data to support its conclusions. Also they should include basic statistical comparisons (e.g. paired statistical tests, p-values) to determine whether the observed shifts in MFI values between temperatures are statistically significant.
Authors should explain how the 30 patients were selected. Were they chosen randomly, or based on specific clinical features?
The authors list different changes in MFI results (like going from positive to negative), but they don’t clearly explain what these changes mean for patients. Authors should discuss potential mechanisms and what does it means for transplant safety or rejection risk.
Authors should clarify whether the same bead lots were used throughout the study and whether any steps (besides having the same individual perform the assays) were taken to control for inter-day variation.
The term “clinically irrelevant” isn’t clearly explained. Do the authors mean non-donor-specific antibodies, IgM, or something else? Authors should define it and explain why those antibodies are considered not important for transplant outcomes.
In my opinion, the title "HLA-specific antibodies like it warm" is too informal for a scientific paper. I suggest that the authors consider revising the title to more professionally reflect the focus of the study.

Author Response

The subject matter of this article is important as it provides preliminary data that could impact clinical practice and policy in HLA laboratories.

While the manuscript addresses an important topic, I have several minor and moderate suggestions that could strengthen the work.

We thank the reviewer for the suggestions. We tried to address the questions and remarks point by point.

The introduction is a bit long and includes some repeated or background information that isn’t directly needed. I suggest shortening it by about one-third and focusing more on why studying temperature effects is important. Some of the extra technical details could be moved to the supplement.

Response: The introduction is now focused and shortened. For the sake of reproducibility, we retained technical details in M&M.

2. The study used a small number of patients and samples. Authors should try to explain why this number of samples was chosen, or to include a power calculation to show if the study had enough data to support its conclusions. Also they should include basic statistical comparisons (e.g. paired statistical tests, p-values) to determine whether the observed shifts in MFI values between temperatures are statistically significant.

Response: We introduced p-values in the abstract and the main text so far possible. Since number of patients is admittedly low, we give the MFI values and, where appropriate, % changes e.g. pages 6 and 7.

3. Authors should explain how the 30 patients were selected. Were they chosen randomly, or based on specific clinical features?

Response: Done in M&M, page 3

4. The authors list different changes in MFI results (like going from positive to negative), but they don’t clearly explain what these changes mean for patients. Authors should discuss potential mechanisms and what does it means for transplant safety or rejection risk.

Response: We elaborate on this in the discussion. Since we did not present any clinical data, we cannot report on rejection episodes etc. This will be part of a follow-up study with a larger cohort.

5. Authors should clarify whether the same bead lots were used throughout the study and whether any steps (besides having the same individual perform the assays) were taken to control for inter-day variation.

Response: Done in M&M There was one person who performed the testing.

6. The term “clinically irrelevant” isn’t clearly explained. Do the authors mean non-donor-specific antibodies, IgM, or something else? Authors should define it and explain why those antibodies are considered not important for transplant outcomes.

Response: Agree we elaborate on that as far as possible. Explained in the introduction.

7. In my opinion, the title "HLA-specific antibodies like it warm" is too informal for a scientific paper. I suggest that the authors consider revising the title to more professionally reflect the focus of the study.

Response: Agree and changed.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors have sincerely amended the manuscript.

Author Response

Comment 1: Authors have sincerely amended the manuscript.

Response 1: We thank the reviewer for the time and efforts to make the ms better.

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for the detailed responses and revisions, which have significantly improved the manuscript. I am fully satisfied with the changes. For full transparency, my only remaining suggestion is to briefly state the rationale for not performing a power calculation (e.g., limited sample availability and the hypothesis-generating design of this pilot study).

Author Response

Comments 1: I am fully satisfied with the changes. For full transparency, my only remaining suggestion is to briefly state the rationale for not performing a power calculation (e.g., limited sample availability and the hypothesis-generating design of this pilot study).

Respense 1: We thank the reviewer for the time and efforts invested to make our ms. better. The remark is very important and is taken well into consideration for our future steps. Our explorative rertospective testing was a try out. We observed deviations in all directions. The nature of the differences observed will be analyzed on a bigger cohort using samples from external proficiency testing exercises because of the availability of a good control and involving several cooperating centers reducing the costs. The results will be made available as soon as analyzed. A before hand power calculation for the data shown here was not possible because we had first to see what is going on with the increase of temperature in the Luminex assay.