The Prevalence of Titanium Dioxide Particles in Synovial Fluid Samples Drops after European Union Ban

Round 1

Reviewer 1 Report

The authors present a brief report  about the analysis of crystals in human synovial fluids before and after the TiO2 European ban. The identification of crystal in SF is done by Raman analysis and white light polarization. Raman analysis is adequate to characterize crystals. The number of samples analysed is high. The authors present clearly the limitations of the study. This study is interesting for the scientific community.

But there is some questions and comments to be addressed before publication. These following comments are made to improve the actual version of the manuscript.

Questions:

Are all TiO2 crystal  birefringent ? Do the authors found TiO2 crystal non birefringent ?

Minor modifications:

Abstract and discussion:  considering all the limitations and potential bias of the study, the authors should moderate their statement. In the abstract, a sentence should be added to moderate the results or present limitations. In the first sentence of the discussion: “We here present a clear decrease in prevalence…”. The word “clear” should be removed. Even if the level of significance is p<0.01, there is numerous cofounding factors taht could induce this level of significance. Moderated sentences need to be used.  

Section 2.2 test methods:

The authors need to specify the objective of the microscope and the numerical aperture.

It is not clear the statement “1s/pixel”. What do the authors refer when they talk about pixel? Is it the pixel of an image or the CCD or another item? Please be more explicit.

Could the authors indicate the approximate number of spectra acquired per sample ? To give an idea for the readership.

Author Response

Dear reviewer,

Thank you for taking your time reviewing our manuscript and providing us with your feedback. We have read your comments carefully and adapted our manuscript based on your ideas. Please find our response to your queries below. 

Queries:

The authors present a brief report  about the analysis of crystals in human synovial fluids before and after the TiO2 European ban. The identification of crystal in SF is done by Raman analysis and white light polarization. Raman analysis is adequate to characterize crystals. The number of samples analysed is high. The authors present clearly the limitations of the study. This study is interesting for the scientific community.

Thank you for reading our manuscript and your comments.

But there is some questions and comments to be addressed before publication. These following comments are made to improve the actual version of the manuscript.

Questions:

Are all TiO2 crystal  birefringent ? Do the authors found TiO2 crystal non birefringent ?

Both anatase and rutile are positively birefringent. We have added a small text to the introduction for clarification (with reference):

We retrieved the samples by arthrocentesis, subsequently using polarized light microscopy to locate birefringent material (TiO₂ is birefringent [17]), followed by a characterization of particles with Raman spectroscopy.

Minor modifications:

Abstract and discussion:  considering all the limitations and potential bias of the study, the authors should moderate their statement. In the abstract, a sentence should be added to moderate the results or present limitations. In the first sentence of the discussion: “We here present a clear decrease in prevalence…”. The word “clear” should be removed. Even if the level of significance is p<0.01, there is numerous cofounding factors taht could induce this level of significance. Moderated sentences need to be used.  

We have added the following sentence to the abstract: These results are hinting towards a possible relationship between the EU-ban and the identified decrease in prevalence. And we have removed the word clear in the first sentence of the discussion.

Section 2.2 test methods:

The authors need to specify the objective of the microscope and the numerical aperture.

The text now reads: The sample was then placed under the Hybriscan iRPolM integrated Raman polarized light microscope (Hybriscan Technologies B.V., Nijkerk, the Netherlands), which operates a 20x 0.8NA objective.

It is not clear the statement “1s/pixel”. What do the authors refer when they talk about pixel? Is it the pixel of an image or the CCD or another item? Please be more explicit.

In this case pixel refers to the Raman spectrum. We performed a hyperspectral analysis in which we measured several spectra of the objects. Each pixel contains a full Raman spectrum. We have clarified this by adding (Raman spectrum) after the word pixel in this sentence.

Could the authors indicate the approximate number of spectra acquired per sample ? To give an idea for the readership.

We refer to Raman spectra with the word pixel (as it is a hyperspectral image). We have measured approximately 60 to 120 pixels per object (as mentioned in the manuscript).

Reviewer 2 Report

This study investigated the prevalence of Titanium Dioxide (TiO2) particles in synovial fluid during and after a ban of the particle. The prevalence of TiO2 decreased during and after the ban, showing how regulation directly impacts prevalence. Overall, the study read well, with an uplifting finding and conclusion.

I have some questions and comments that hopefully can help improve the manuscript.

Abstract

l. 10. Please add that particles are studied during the ban and afterward.

l. 14. The methods section says up to 40 microliters. Please write whichever is correct.

Introduction

l. 47. Many abbreviations. Please write all out in full: NLPR, THP, LPS, IL, and TNF. 

Methods

l. 61. "Consecutive" Does this mean some patients provided more than one sample?

l.66. Microscopy is a skill that needs training and is individual (PMID: 27837341). Please add what training the rheumatologist performing the microscopy had. 

l. 76 "Up to 40 mikroliters". If not all were 40 microliters, consider reporting the mean (SD) analyzed amount in the results section. 

l. 79. I have yet to work with the Hybridscan - is calibration necessary? If so, please describe. 

l. 79. Birefringence detection is enhanced if the microscope has a compensator. Did the microscope have a compensator? If so, please add. If not, consider adding it as a limitation depending on the strength of TiO2 birefringences.

l. 80. Who located the birefringent material?

l. 85. Please add a reference for comparison spectras. Also, when was a spectra considered confirmative - visual plot inspection by experts? Or distinct peaks? Margin error for peaks?

Results

l. 104. Please add the number of patients. Also, the text says 302 samples were analysed, but the figure text to figure 2 says that 265 samples were analysed; why do these numbers differ?  

l. 108. Given the size of the particles, I agree with the authors that they are most likely congregated particles. This comment is not a result and should be moved to the discussion. 

Figure 1. Consider adding peak values on the graph so others can use the spectras for reference more easily. 

Table 1. In the TiO2 positive group, the prevalence is highest in the clinical diagnosis "other." Please add how many positives each group had (psoriatic arthritis, bacterial, and causa ignota).

Figure 2. Why does the 95% CI stop around Dec 22? 

Discussion

l. 159 The limited resolution of optical microscopes is indeed a challenge. The introduction mentions that most identified TiO2 particles are less than <100nm. The small size and the small analysed amount of synovial fluid impose a risk of false negatives. I suggest adding this explicitly to the discussion. 

Given the size of TiO2 particles, a different analysis approach might have been more appropriate. Was this considered? Why was polarized light microscopy selected?  

The discussion does not mention the strengths of the study. Is this deliberate? I want to compliment the authors for doing the blank sample examination. This analysis increases confidence that there is a small-to-no amount of false positives. Consider mentioning this analysis as a strength.      

Author Response

Dear reviewer,

Thank you for taking your time reviewing our manuscript and providing us with your feedback. We have read your comments carefully and adapted our manuscript based on your ideas. Please find our response to your queries below. 

Queries:

This study investigated the prevalence of Titanium Dioxide (TiO2) particles in synovial fluid during and after a ban of the particle. The prevalence of TiO2 decreased during and after the ban, showing how regulation directly impacts prevalence. Overall, the study read well, with an uplifting finding and conclusion.

Thank you for reading our manuscript and your comments.

I have some questions and comments that hopefully can help improve the manuscript. 

Abstract

Please add that particles are studied during the ban andafterward.

The abstract now reads: The prevalence of titanium dioxide nanoparticles in synovial fluid samples is studied during and after the transitional period

The methods section says up to 40 microliters. Please write whichever is correct.

The abstract now reads: From each sample, up to 40 (mu)l of fluid was analysed with Raman Spectroscopy for the presence of titanium dioxide particles. 

Introduction

Many abbreviations. Please write all out in full: NLPR, THP, LPS, IL, and TNF. 

The text now reads: Like MSU crystals [15], TiO2 nanoparticles can mediate inflammation through the NLRP-3 (NOD-, LRR- and pyrin domain containing protein 3) inflammasome. Co-stimulation of THP-1 macrophages with LPS (lipopolysaccharides) and TiO2 nanoparticles led to an in-crease in secretion of pro-inflammatory cytokines interleukin 1β, interleukin 6, and TNF-α (tumour necrosis factor) [16]. This mechanism is an argument for a potential role of TiO2 in the induction or progression of inflammatory joint disease.

THP-1, however, is the name of a cell line and not an abbreviation. 

Methods

"Consecutive" Does this mean some patients provided more than one sample?

We consider every patient sample as a new patient. We expect that from a small minority of patients more than one sample was collected, but arthrocentesis is usually only performed at the initial visit and the overwhelming majority of patients is only punctured once.

Microscopy is a skill that needs training and is individual (PMID: 27837341). Please add what training the rheumatologist performing the microscopy had. 

We acknowledge the difficulty behind microscopy (author TLJ was also an author on the mentioned publication). TLJ performed the microscopy analysis and has over 20 years of experience as a specialist. The manuscript now reads: All samples have been reviewed by a rheumatologist (>20 years of experience) with polarized light microscopy for the presence of pathological crystals, such as monosodium urate for gout.

"Up to 40 mikroliters". If not all were 40 microliters, consider reporting the mean (SD) analyzed amount in the results section. 

That would definitely have been an interesting addition, however, we are unable to retrospectively retrieve data on the exact amount of microliters used.

I have yet to work with the Hybridscan - is calibration necessary? If so, please describe. 

The following text is added to the method: Before each sample, the Raman spectroscope is calibrated with (sequentially) CCD camera offset measurements, CCD camera dark current measurement, a Neon calibration standard, a PMMA calibration standard, a NIST intensity calibration measurement and a background (air) measurement.

Birefringence detection is enhanced if the microscope has a compensator. Did the microscope have a compensator? If so, please add. If not, consider adding it as a limitation depending on the strength of TiO2 birefringences.

When using ordinary polarized light microscopy (as in routine clinical practice), the compensator indeed is necessary to distinguish between for example MSU and CPP crystals. In our opinion, the compensator however only enhances the identification and not the detection of the analysis. The identification is performed with Raman spectroscopy, the detection is based on the compensator. We respectfully disagree that this is a limitation in our method, as the specificity of Raman spectroscopy is almost 100%.

Who located the birefringent material?

Author TN located the birefringent material. This is added to the manuscript, which now reads: Birefringent objects were located manually (TN), which were then scanned with the integrated Raman spectroscope

Please add a reference for comparison spectras. Also, when was a spectra considered confirmative - visual plot inspection by experts? Or distinct peaks? Margin error for peaks?

We added a reference for the spectra. The spectra were examined with visual plot inspection. The method is altered and now reads: The spectra were compared using visual plot examination with earlier reported spectra of anatase and rutile [18].

Results

Please add the number of patients. Also, the text says 302 samples were analysed, but the figure text to figure 2 says that 265 samples were analysed; why do these numbers differ?  

We initially analyzed 265 samples for a conference abstract but later had the opportunity to include some extra patient samples. The 265 next to the figure is an error and it should indeed be 302 samples, thank you for noticing. We have altered the number next to the figure.

Given the size of the particles, I agree with the authors that they are most likely congregated particles. This comment is not a result and should be moved to the discussion. 

Thank you for noticing this error. We have altered the results to read: The average size of the particles was 4.4 (SD ±1.4) (mu)m [text removed].

The discussion now reads: Due to the large average size of the particles, we suspect that the identified particles were most probably congregates of clustered nanoparticles.

Figure 1. Consider adding peak values on the graph so others can use the spectras for reference more easily. 

We have added peak values to the graphs.

Table 1. In the TiO2 positive group, the prevalence is highest in the clinical diagnosis "other." Please add how many positives each group had (psoriatic arthritis, bacterial, and causa ignota).

We have added these values and recalculated the P-value.

Figure 2. Why does the 95% CI stop around Dec 22? 

The 95% CI is based on the 3-month MOVAG filter which was used to fit an exponential decreasing trend to. After Dec 2022, there are no samples positive for titanium dioxide within this window, therefore the proportion (and the 95% CI) reaches zero (quite drastically indeed).

Discussion

159 The limited resolution of optical microscopes is indeed a challenge. The introduction mentions that most identified TiO2 particles are less than <100nm. The small size and the small analysed amount of synovial fluid impose a risk of false negatives. I suggest adding this explicitly to the discussion. 

Thank you for this comment. We have added this to the limitations, which now read: Due to the large average size of the particles, we suspect that the identified particles were most probably congregates of clustered nanoparticles. Individual particles are often <100 nm and chances are these could be missed in our analysis.

Given the size of TiO2 particles, a different analysis approach might have been more appropriate. Was this considered? Why was polarized light microscopy selected?  

This report describes a coincidental finding of a large patient study to the additional value of Raman spectroscopy for the diagnosis of gout and pseudogout. We acknowledge that other modalities might also have been useful, but for example scanning electron microscopy (which is more appropriate given the size of the particles) is not suited for the analysis of such a large cohort.

The discussion does not mention the strengths of the study. Is this deliberate? I want to compliment the authors for doing the blank sample examination. This analysis increases confidence that there is a small-to-no amount of false positives. Consider mentioning this analysis as a strength.      

Thank you for this compliment. We have added the following paragraph to the discussion:

Strong points of this study include the inclusion of blank samples, which increases the confidence that there is a small chance for false positives. Furthermore, a large number of patients were included and there was no selection of patient samples, as we analyzed all available samples from our outpatient clinic.