Persistence of Monosodium Urate Crystals and Calcium Pyrophosphate Crystals in Synovial Fluid Samples After Two Weeks of Storage at 4 °C and −20 °C: A Longitudinal Analysis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The purpose of this manuscript is to investigate the persistence of MSU and CPP crystals in synovial fluid when they are not immediately examined. This issue has been addressed in several publications, all of which are reviewed in the Introduction, with results similar to those presented in this manuscript: MSU crystals can be identified some time after sampling, but CPP crystals appear to degrade rapidly. These results, although not original, are important, particularly for clinicians who do not work in an environment where synovial fluid analysis is routinely performed.

In any case, the authors should explain how this study contributes to previous research.

The methodology of this study needs to be described in more detail. What was the interobserver reproducibility of synovial fluid analysis? Were the measurements taken simultaneously? If there was a discrepancy between the two readers, how long did it take to organise a second reading?
The method used to calculate the number of crystals should also be reported: on how many fields was it calculated? What was the definition of "decreased number of MSU/CPP crystals"?

Figure 1 should also include the number of samples included/analysed.

In the discussion, the authors suggest that EDTA protects leukocytes from cell lysis. Were they able to confirm this hypothesis by counting the number of cells before and after storage?

Author Response

Comment #1.1
The purpose of this manuscript is to investigate the persistence of MSU and CPP crystals in synovial fluid
when they are not immediately examined. This issue has been addressed in several publications, all of
which are reviewed in the Introduction, with results similar to those presented in this manuscript: MSU
crystals can be identified sometime after sampling, but CPP crystals appear to degrade rapidly. These
results, although not original, are important, particularly for clinicians who do not work in an
environment where synovial fluid analysis is routinely performed.
In any case, the authors should explain how this study contributes to previous research.
Response:
Our study supports the existing understanding that MSU crystals tend to persist in synovial fluid longer
than CPP crystals. Previous research shows substantial heterogeneity in storage conditions, with storage
times ranging from 24 hours to 24 weeks, temperatures ranging from room temperature to -80 °C, and
the use of various preservatives. The protocol used in our study aligns with practices in most local
settings: storage at 4 °C and 20 °C in EDTA-coated containers, with a typical wait time of approximately
1–2 weeks. We have added this point to the Discussion section (line 266-269).
Comment #1.2
The methodology of this study needs to be described in more detail. What was the interobserver
reproducibility of synovial fluid analysis? Were the measurements taken simultaneously? If there was a
discrepancy between the two readers, how long did it take to organise a second reading?
Response:
We have added the following details to the Methods section of the manuscript regarding SF examination
(line 108-131):
Synovial fluid examination was conducted on the same day that samples were obtained from the
hospital laboratory. The specimens were prepared by one of the co-investigators (JS) as soon as possible
after collection. Two examiners (KJ and NW) performed crystal identification sequentially using the
same microscope and slide. First, KJ examined the slide and recorded the presence or absence of
crystals. Subsequently, NW conducted an independent examination, also noting the presence or
absence of crystals and performing a crystal count from 10 high-power fields following a pre-set clock-
wise pattern. KJ and NW were blinded to each other’s findings. For SF without crystals, at least 10 high-
power fields must be surveyed before the absence of crystal could be reported. The examination
typically required no more than 15 minutes for KJ and no more than 30 minutes for NW (due to the
additional crystal count).
All follow-up examinations after 2-week storage followed the same protocol as the baseline
examination. Frozen samples were thawed for 20 minutes before being mounted on a slide for crystal
analysis. Both examiners received only the prepared slides and were therefore blinded to the storage
condition of the samples.
In cases of inconsistent findings (i.e., one positive and one negative result), a second examination was
planned to be performed on the same day within one hour of the initial readings to minimize sample
deterioration. However, no discrepancies between the two examiners were observed during the study.
While we did not formally calculate interobserver agreement, we were reassured by both examiners’
extensive experience and the high consistency of their findings throughout the study.
Comment #1.3
The method used to calculate the number of crystals should also be reported: on how many fields was it
calculated? What was the definition of "decreased number of MSU/CPP crystals"?
Response:
If present, the crystal count was performed by one of the co-investigators (NW) by counting crystals in
10 high-power fields. This protocol is consistent with a previous study by Tausche AK et al. (doi:
10.1097/RHU.0b013e31829cde53). The presence of crystals and the corresponding crystal count results
have been added as supplementary material (Table S1 of the supplementary documents).
Furthermore, we have presented the mean crystal count compared the mean crystal count between
baseline and at 2 weeks for both 4°C and -20°C storage using paired t-test (Table 1). These additional
analyses have been added to the Methods section. (line 149-150)
The term “decreased number of MSU/CPP crystals” in Table 2 was defined as any sample in which the
post-storage crystal count was lower than the baseline count. This includes both samples in which
crystals became undetectable after storage and those in which crystals remained identifiable but were
reduced in number. These details have been added to the Methods section (line 138-141).
Comment #1.4
Figure 1 should also include the number of samples included/analysed.
Response:
A total of 49 samples were included, and all underwent the same analysis procedure. Following the
baseline analysis, each sample was divided into two aliquots: one stored at 4°C and the other at –20°C.
After 2 weeks, all samples were re-analyzed. We chose not to include the sample number directly in the
figure itself, as doing so would have been repetitive (all steps involved the same 49 samples). Instead,
we added the sample number to the figure caption to maintain clarity and ensure the figure remains
easy to follow. The caption for Figure 1 has been revised accordingly.
Comment #1.5
In the discussion, the authors suggest that EDTA protects leukocytes from cell lysis. Were they able to
confirm this hypothesis by counting the number of cells before and after storage?
Response:
We agree that changes in WBC count could support our hypothesis that EDTA helps prevent cell lysis
and contributes to crystal preservation. However, as the primary objective of our study was to
investigate crystal persistence, WBC counts were not performed. We explored the possibility of
accessing automated WBC count data from specimens analyzed by the hospital’s central laboratory.
Unfortunately, this was not feasible, as follow-up WBC counts were not available, and accessing the data
would require patient identifiers - violating our study protocol, which mandates the use of anonymized
SF samples only. We have included this limitation in the Discussion section of the manuscript (line 260-
262).

Reviewer 2 Report

Comments and Suggestions for Authors

The authors performed repeated SF analysis to see the persistence of crystals two weeks after aspiration. Raw numbers show good persistence with MSU and a drop for CPP. From my perspective, it adds little to existing evidence. In addition, the results may be hampered by the number of samples, especially for CPP, and the lack of statistical analyses that discard random findings. Moreover, incorporating non-crystal fluids provides minimal contribution to the study's objective (crystal preservation for analysis).

I have addressed some comments and issues:

Intro

Line 42: The reference here demonstrates that a short training makes observers capable of identifying crystals reliably. Accordingly, the authors should rewrite their statement.

Line 69: In the Intro, the authors discussed prior literature regarding storing synovial fluid samples, first focusing on heparin-containing tubes and later introducing the option of EDTA tubes. The study by Pastor et al (PMID  32007932) used EDTA and found less CPP identification over time with it, which should be mentioned.

Comment: There are a couple of papers suggesting the value of adding DMSO to preserve crystals (see doi:10.1177/00045632221076349 and 10.37349/emd.2023.00002), please revise and comment.

Methods

Line 85: The study design is longitudinal, as two observations were performed over two different time points.

Line 91: The authors excluded fluids of less than 5mL, while a minimal amount is sufficient to perform polarized light microscopy. I see this approach as unnecessary.

Line 95: I guess the authors refer to 400x (40x objective plus 10x binoculars).

Lines 96-98: Since the analysts did not work simultaneously, an examining protocol (i.e., number of fields to revise until a sample is coined as “non-crystal”) would be highly recommended. Please explain.

Line 115: More statistical insight is needed. The authors must perform 95% CI estimations and before-after analyses, considering the type of crystal and storing temperature as covariates.

Results

Line 132: The authors should give the crystal counts at 2 weeks (for both +4ºc and -20ºC).

Comment: The number of samples is probably too low for the objective. Was any cell count at baseline and follow-up performed?

Discussion

Lines 147 and 205: The authors stated that “their study demonstrated…”. Numbers suggest it, but statistical comparisons are needed to reach that conclusion.

Comment:  I suggest the authors discuss how cell clotting and debris would also explain the decline in identifying CPP crystals, particularly as this crystal is often better detected by shape than by birefringence (contrary to MSU).

Author Response

Comment #2.1
The authors performed repeated SF analysis to see the persistence of crystals two weeks after
aspiration. Raw numbers show good persistence with MSU and a drop for CPP. From my perspective, it
adds little to existing evidence. In addition, the results may be hampered by the number of samples,
especially for CPP, and the lack of statistical analyses that discard random findings. Moreover,
incorporating non-crystal fluids provides minimal contribution to the study's objective (crystal
preservation for analysis).
I have addressed some comments and issues:
Intro
Line 42: The reference here demonstrates that a short training makes observers capable of identifying
crystals reliably. Accordingly, the authors should rewrite their statement.
Response:
The statement has been revised to the following: ‘Specific training in crystal identification by
experienced examiners has been shown to improve the accuracy and reliability of crystal detection in
synovial fluid.’ (line 42-44)
Comment #2.2
Line 69: In the Intro, the authors discussed prior literature regarding storing synovial fluid samples, first
focusing on heparin-containing tubes and later introducing the option of EDTA tubes. The study by
Pastor et al (PMID 32007932) used EDTA and found less CPP identification over time with it, which
should be mentioned.
Response:
We have added the following statements regarding the study by Pastor et al (doi:
10.3899/jrheum.190468) to the introduction section: In a study by Pastor et al., synovial fluid samples
were stored at 20°C and 4°C using either heparin or ethylenediaminetetraacetic acid (EDTA) as
preservatives. MSU crystals remained detectable in over 90% of samples after 7 days, while the
detection rate of CPP crystals declined to 67% after 7 days of storage at 20°C with EDTA as preservative.
(line 56-61)
Comment #2.3
Comment: There are a couple of papers suggesting the value of adding DMSO to preserve crystals (see
doi:10.1177/00045632221076349 and 10.37349/emd.2023.00002), please revise and comment.
Response:
Thank you for the recommendation. We have included the study by Pérez-Ruiz et al.
(doi:10.37349/emd.2023.00002) in the Introduction section of the revised manuscript. This study
suggested that the addition of DMSO to synovial fluid samples helped prevent cell lysis and preserved
high agreement in crystal identification after storage at –20°C for 3 months. The following statement has
been added: “For frozen synovial fluid, DMSO appeared to help prevent cell lysis and maintained high
crystal identification agreement after 3-month storage. However, DMSO is not commonly available in
our local health facilities.” (line 61-63)
We did not include the study by Pavic et al. (doi: 10.1177/00045632221076349) in our manuscript, as it
focused on the preservation of cellular morphology in synovial fluid with DMSO, rather than on the
persistence of crystals.
Comment #2.4
Methods
Line 85: The study design is longitudinal, as two observations were performed over two different time
points.
Response:
The manuscript title and the description of the study design in the Methods section (line 92) has been
revised to reflect the longitudinal design.
Comment #2.5
Line 91: The authors excluded fluids of less than 5mL, while a minimal amount is sufficient to perform
polarized light microscopy. I see this approach as unnecessary.
Response:
We agree that this exclusion criterion may have been unnecessarily strict and could have introduced
selection bias, as higher-volume samples (i.e., more inflammatory) were more likely to be included.
However, we believe this limitation does not significantly impact our primary objective, which was to
analyze changes in crystal presence over time within the same samples, rather than to compare findings
between different samples. We have acknowledged this limitation in the Discussion section of the
revised manuscript (line 255-260).
Comment #2.6
Line 95: I guess the authors refer to 400x (40x objective plus 10x binoculars).
Response:
The texts have been corrected to 400x. (line 113)
Comment #2.7
Lines 96-98: Since the analysts did not work simultaneously, an examining protocol (i.e., number of
fields to revise until a sample is coined as “non-crystal”) would be highly recommended. Please explain.
Response:
SF examination was conducted on the same day that samples were obtained. The specimens were
prepared by one of the co-investigators (JS). Two examiners performed crystal identification
sequentially using the same microscope and slide (examination by KJ followed by NW). NW also
performed a crystal count from 10 high-power fields following a pre-set clock-wise pattern. KJ and NW
were blinded to each other’s findings. For SF without crystals, at least 10 high-power fields must be
surveyed before the absence of crystal could be reported. A full description of the examination protocol
has been added to the Methods section of the manuscript. (line 109-123)
Comment #2.8
Line 115: More statistical insight is needed. The authors must perform 95% CI estimations and before-
after analyses, considering the type of crystal and storing temperature as covariates.
Response:
We performed McNemar’s test to compare the proportion of samples with detectable crystals at
baseline to those after 2-week storage at 4°C and –20°C. When the expected values in any cell of the 2x2
contingency table were too low, a binomial sign test was used instead. Similar comparisons were
conducted separately for the proportion of samples with detectable MSU crystals and CPP crystals. A p-
value of 0.05 or higher was interpreted as indicating no significant difference in the proportion of
detectable crystals between baseline and after storage, whereas a p-value below 0.05 indicated a
statistically significant difference. Table 1 has been revised to include these additional analyses.
Furthermore, we constructed a multivariable logistic regression model to explore factors associated with
the persistence of crystals after 2-week storage. The model included all samples that were crystal-
positive at baseline (20 stored at 4°C and 20 stored at –20°C). The dependent variable was the presence
of detectable crystals at 2 weeks. Covariates included storage temperature (4°C vs –20°C), crystal type
(MSU vs CPP), and baseline crystal count. For each covariate, we reported the regression coefficient,
odds ratio, 95% confidence interval, and p-value. A p-value < 0.05 was considered indicative of a
statistically significant association with crystal persistence at 2 weeks. The results of this analysis are
presented in Table S2 of the supplementary material.
The corresponding texts in the Methods section (line 135-158) and the Results section (line 188-191)
have been revised to reflect these changes.
Comment #2.9
Results
Line 132: The authors should give the crystal counts at 2 weeks (for both +4ºc and -20ºC).
Response:
Crystal count results has been included as supplementary material (Table S1 of the supplementary
documents). The details on how the crystal count was performed was included in the Methods section
of the manuscript. (line 114-117)
Comment #2.10
Comment: The number of samples is probably too low for the objective. Was any cell count at baseline
and follow-up performed?
Response:
We agree that the low sample number was one of the limitations of the current study. AS such, we have
added how we estimated the sample size in the Methods section. (line 100-107) The limitation has been
discussed in the Discussion section. (line 262-265)
As also mentioned by the other reviewer, we recognized that changes in WBC count could support our
hypothesis that EDTA helps prevent cell lysis and contributes to crystal preservation. However, as the
primary objective of our study was to investigate crystal persistence, WBC counts were not performed.
We explored the possibility of accessing automated WBC count data from specimens analyzed by the
hospital’s central laboratory. Unfortunately, this was not feasible, as follow-up WBC counts were not
available, and accessing the data would require access to patient identification data – a violation of our
study protocol. We have included this limitation in the Discussion section of the manuscript. (line 260-
262)
Comment #2.11
Discussion
Lines 147 and 205: The authors stated that “their study demonstrated…”. Numbers suggest it, but
statistical comparisons are needed to reach that conclusion.
Response:
We have performed McNemar’s test to compare the proportion of samples with detectable crystals at
baseline to those after 2-week storage at 4°C and –20°C (Table 1). The detection of CPP crystals
appeared to decline to a greater extent than MSU crystals after 2-week storage at both 4°C and –20°C;
however, the differences in proportions between baseline and 2 weeks did not reach statistical
significance in both types of crystals.
We have also performed logistic regression analysis to explore the links between the persistence of
crystals at 2 weeks and a number of covariates (baseline crystal count, storage temperature and type of
crystal). The results of the regression analysis can be found in Table S2 in the supplementary material.
We have revised the interpretation of the findings to more accurately reflect the results of this analysis.
The revised statement has been included in the Discussion section (line 266-269).
Comment #2.12
Comment: I suggest the authors discuss how cell clotting and debris would also explain the decline in
identifying CPP crystals, particularly as this crystal is often better detected by shape than by
birefringence (contrary to MSU).
Response:
We agree that cell clotting and debris formation could have contributed to the decline in CPP crystal
detection, given that CPP crystals are often identified based on their characteristic morphology rather
than strong birefringence. We have added this point into our Discussion section (line 217-220).

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have appropriately addressed my remarks.

I have no further comments.

Author Response

We are grateful for your insights and suggestions.

Reviewer 2 Report

Comments and Suggestions for Authors

Thanks for the effort in addressing my previous comments and submitting an improved manuscript. I have some additional comments:

• Incorporating statistical analyses enhances the soundness of the findings. Regarding sample size estimation, you predicted 100% persistence at -20ºC but only 50% at +4ºC. May you provide the source for that hypothesis (particularly, as having several papers published on the topic)? In addition, the final sample size was close but did not meet the estimations (49 samples, 20 with crystals). Please comment in the Discussion.
• Thanks for providing the observation protocol, which helps us understand the methods followed. However, I have difficulties understanding how the crystal count was managed, given that there were two observers (and a third in conflicting cases). Please explain.
• A sample was considered as having no crystals after examining 10 fields. In the study by Bernal et al (DOI 10.1002/acr.24874), most crystals were found in <10 fields, but 10% required more than 10 fields to be identified. I may agree that it probably has little impact on your study. Still, it probably merits a comment, especially as the number of fields needed to be scanned to rule out crystals has received scant attention in the literature. 
• Thanks for providing crystal counts. It is to me a bit surprising that the MSU crystal count showed even an increase over 2 weeks, contrary to a prior study that proved no new MSU crystal formation in stored fluids (Tausche, J Clin Rheumatol 2013). Observer bias? A comment on that is needed.
• I see Figure 2 is a bit blurred, needs to be improved.

Author Response

Thank you for your comments. Our responses and the list of amendments are included below. Please
note that the revised text added in this second round of revision is highlighted in blue, while text
highlighted in yellow corresponds to changes made during the first round of revision.
Reviewers’ comments
Comment#1
Incorporating statistical analyses enhances the soundness of the findings. Regarding sample size
estimation, you predicted 100% persistence at -20ºC but only 50% at +4ºC. May you provide the source
for that hypothesis (particularly, as having several papers published on the topic)? In addition, the final
sample size was close but did not meet the estimations (49 samples, 20 with crystals). Please comment
in the Discussion.
Response:
We estimated the percentage of crystal persistence based on studies by Kerolus et al. and Pastor et al.
At the more extreme end, Kerolus et al. reported 100% persistence of MSU crystals after 8 weeks of
storage at 4°C, while CPP crystals became undetectable over the same period. In the study by Pastor et
al., 77% of CPP crystals and 100% of MSU crystals remained detectable after 1 week of storage at 4°C.
Based on these findings, we predicted that, overall, approximately half of the crystals would remain
detectable after 2 weeks at 4°C, while all or nearly all crystals would persist in samples stored at –20°C.
The final number of included samples (49) fell slightly short of the calculated sample size of 50. This
shortfall was due to logistical limitations in our study protocol. Based on prior communication with the
central laboratory, we anticipated that approximately 30 synovial fluid samples would be available each
month. Accordingly, we scheduled data collection over a 2-month period (February–March 2024).
Our study protocol required immediate examination of synovial fluid samples upon arrival at the central
hospital laboratory. This necessitated that both examiners (KJ, NW) and the study coordinator (JS)
remain on-site to perform crystal analysis on the same day. All investigators coordinated their schedules
to be present during the study period. In total, we received 57 samples, of which 49 were included in the
final analysis. Unfortunately, some eligible samples were not processed due to the unavailability of one
or more investigators at the time of sample arrival.
We have revised the Discussion section to ensure that the sample size issue is clearly explained (line
271-275). References to support sample size estimation has also been included (line 102, reference 9,
11).
Comment#2
Thanks for providing the observation protocol, which helps us understand the methods followed.
However, I have difficulties understanding how the crystal count was managed, given that there were
two observers (and a third in conflicting cases). Please explain.
Response:
The presence of crystals (yes or no) and the type of crystals (MSU or CPP) were assessed independently
by two examiners (KJ and NW). In cases of disagreement (e.g., one examiner reported the presence of
crystals while the other reported their absence), the study coordinator (JS) arranged a second
examination by the same two investigators (KJ and NW) within one hour from the initial analysis to
reach a consensus.
Crystal count was performed by a single investigator (NW), who counted the number of crystals (if
present) in 10 HPF following a clockwise pattern.
We have revised the Methods section to ensure that this procedure is clearly described (lines 116–124).
Comment#3
A sample was considered as having no crystals after examining 10 fields. In the study by Bernal et al (DOI
10.1002/acr.24874), most crystals were found in <10 fields, but 10% required more than 10 fields to be
identified. I may agree that it probably has little impact on your study. Still, it probably merits a
comment, especially as the number of fields needed to be scanned to rule out crystals has received
scant attention in the literature.
Response:
Thank you for raising this point. In our study, a sample was considered negative if no crystals were
identified in at least 10 HPF. Notably, under this protocol, no inconsistent findings were reported
between the two examiners. This may provide some support for the adequacy of the 10 HPF threshold
in distinguishing crystal-positive from crystal-negative samples. Nonetheless, our study was not
primarily designed to prove or disprove this hypothesis, so further research is needed to confirm its
validity.
We have added a discussion of this point into our Discussion section. (line 258-263)
Comment#4
Thanks for providing crystal counts. It is to me a bit surprising that the MSU crystal count showed even
an increase over 2 weeks, contrary to a prior study that proved no new MSU crystal formation in stored
fluids (Tausche, J Clin Rheumatol 2013). Observer bias? A comment on that is needed.
Response:
We observed that synovial fluid samples appeared to contain a greater amount of amorphous debris
and cellular clumps after 2 weeks of storage. During follow-up crystal examinations, many fields
contained debris clumps with multiple MSU crystals embedded within. We suspect that the storage and
thawing process may have led to some cell lysis, resulting in the formation of dense debris clusters that
concentrated pre-existing crystals. This may have contributed to the apparent increase in crystal count.
However, we are unable to confirm whether our impression of increased debris clustering and
decreased WBCs is factual, as WBC counts were not performed. We also acknowledge that the observed
increase in crystal count could potentially be attributed to observer bias. Nonetheless, we do not believe
this observation reflects new crystal formation.
We have included these points into the Discussion section (line 214-217)
Comment#5
I see Figure 2 is a bit blurred, needs to be improved.
Response:
A new figure with improved resolution has been prepared.