IgM‒C4d Complex Causes the Inaccurate Measurement of Serum Uric Acid Levels via the Uricase Method

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this interesting paper, the authors describe the possible reason for influence of high IgM interference with uric acid (UA) determination in serum - the formation of the IgM-C4d complex. The study seems well-conceived and the conclusions drawn are logical. However, some things remained unclear to me:

1. The methods of determination of UA concentration seem not sufficiently described. How is the concentration determined from ODs at 660 and 800nm (the wavelengths are not even stated in section Measurement of serum UA levels with an automatic biochemical analyzer via the uricase method)? Are the compositions of Reagents 1 and 2 known? If I understand correctly, points 1-9 and 10-27 in measurement are just measurements of the same reaction mixture in time? How long is the time interval between points?
2. In the section Measurement of serum immunoglobulins and complements, I suppose the method is automatized, and certain amount of serum is given to the instrument, which gives out the concentration values? For me it would be interesting to know how they are determined, but if it is not common in this type of paper, I will not insist on addition of details.
3. In Correction test, for the dry slide method, only OD at 670 nm was enough for determination of UA concentration? No subtraction of OD at 800 nm?
4. Regarding data in Table 1, it seems to me that the UA concentrations determined by uricase method and MS differ significantly even for samples in the control group, especially taking into account what the authors say later (line 247) that "the higher limit of the interindividual BV of UA is 8.5" and therefore even corrected uricase test values are not good enough. Can authors comment on that?
5. Why were only samples of Group 2 (samples 9-14) corrected by dry slide or PEG precipitation method (Table 2)? Why not also those of Group 1?
6. In Figure 3, it should be stated what "IP" means, and concentrations of IgM, IgA and IgG should be listed in the same order for all samples, for easier comparison.
7. Western blot results should be better explained - where is the band corresponding to IgM-C4d complex, what is the size of C4d, what is the band of ~45 kDa in panels WB: IgM, are samples no.1-4 in WB the same samples 1-4 from table 1, what is the difference between panels a and b? 

Author Response

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Author Response File: Author Response.doc

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript provides a highly valuable contribution to clinical laboratory medicine by identifying, for the first time, a novel circulating IgM-C4d complex that interferes with the routine uricase method and leads to inaccurate serum uric acid measurements. Furthermore, it rigorously compares alternative analytical strategies and provides a clear clinical recommendation to utilize mass spectrometry for accurate quantification in these specific patient populations
Required Revisions
1. There is a major contradiction in the reported data; the abstract explicitly states that 24 serum samples from 18 patients were collected, whereas the "Patient population" subsection in the Methods claims that only 14 samples from 9 patients were analyzed.

2. The authors note that the alkaline component of the uricase reagent causes the insolubility of the IgM-C4d complex, but they should provide a deeper biochemical explanation as to why this specific alkaline environment triggers the precipitation

3. While the study demonstrates that both the polyethylene glycol precipitation and dry slide based methods still yield unacceptable methodological biases compared to mass spectrometry, the authors should briefly discuss the practical challenges of implementing mass spectrometry as a routine solution in standard clinical laboratories

Author Response

Please see the attachment.

Author Response File: Author Response.doc