Analytical Validation of GFR_NMR: A Blood-Based Multiple Biomarker Assay for Accurate Estimation of Glomerular Filtration Rate

Round 1

Reviewer 1 Report

The chronical kidney decease (CKD) is a widespread ailment. The monitoring of the kidney function is critical for a diagnosis of it. Thus the topic of the present paper is timely and important. The manuscript presents an account of a solid research devoted to the analytical performance evaluation of the glomerular filtration rate by means of nuclear magnetic resonance (GFR_NMR). As a result, several important results have been achieved:
- Excellent analytical precision and accuracy is anticipated in clinical practice;
- No analytical interference was observed for the majority of the relevant substances tested;
- The presence of substances, including additive solutions and therapeutic agents, will have little impact in clinical practice on the results of the GFR_NMR equation;
- GFR_NMR testing after overnight fasting should be considered in diabetes management;
- Naproxen > 0.39 mmol/L interfered with GFR_NMR, whereas other common analgetic drugs such as ibuprofen, acetylsalicylic acid, and acetaminophen did not interfere with GFR_NMR testing;
- In patients with or without CKD receiving active ribavirin treatment for hepatitis C, GFR_NMR should be replaced by alternative test methods to minimize the risk of missing test results with GFR_NMR;
- An excellent analytical performance of the GFR_NMR assay was demonstrated.

The presentation of the results and their discussion are logic and professional, the manuscript is carefully and clearly written.

Thus I recommend the manuscript for publication in Diagnostics as is.

Author Response

RESPONSE: We are very grateful for his kind response to our manuscript. We would like to thank the reviewer for his time and effort during the peer-review.

Author Response File: Author Response.pdf

Reviewer 2 Report

Analytical validation of GFRNMR: A blood-based multiple biomarker assay for accurate estimation of glomerular filtration rate

Summary: The authors present a very nice validation study of a new method to estimate the glomerular filtration rate.

Strengths: All validation tests are well described and repeated sufficiently to confirm the results. The inclusion of possible confounding chemicals was very nicely done.

Weaknesses: None noted.

Specific comments:

1. Page 3, lines 102-104. Can these two sentences be combined? I had to read them about 3 times before I caught the minor wording change between “calibrator” and “control”.
2. Starting on page 4 and used throughout. The authors use the word “volunteer”, which suggests that blood was collected specifically for this work. In that case, IRB approval would be required. If the blood was not collected specifically for this work, but rather some random serum sample was used for these tests, that should be clarified. Similarly, the use of “donor” in Table 3 and line 494 also suggests that an IRB approval would be needed.
3. Page 5, line 251. Since the “LoD Variant Approach…” is in quotes, is there a reference for this?
4. Page 7 line 304, minor English correction. “lied” should either be a simple “was” or it should be “lay”. I prefer the simple “was”.
5. Page 14, lines 497-499. I do not understand what the authors are trying to say here. Please rephrase.
6. A general comment/observation. Not being familiar with the pseudo-Voight fitting method, how does it handle overlapping peaks? It seems to me that with myo-inositol having resonances at 3.5 and 3.6, valine having resonances at 3.6, and glucose having resonances also at 3.5, if the method doesn’t handle overlapping peaks well, that could explain the issue with high glucose concentration. In that case, does the method already fit glucose? If not, would the GFRNMR method be improved by including glucose in the fit? Also, the reference for the pseudo-Voight fitting doesn’t point to a method paper describing the fitting, but rather to another paper that just uses it. A reference describing the fitting itself would be helpful. It appears to be just a way to fit the individual peaks, but I was unable to find anything about how it handles overlap.

Author Response

1. Page 3, lines 102-104. Can these two sentences be combined? I had to read them about 3 times before I caught the minor wording change between “calibrator” and “control”.

RESPONSE: We agree with the reviewer and revised the manuscript accordingly. Now we state: “Calibrator and quality control samples were prepared by filling 600 µL of AXINON serum calibrator or control 2.0, respectively, into a 5 mm NMR tube and capping with a barcoded cap for identification.”

2. Starting on page 4 and used throughout. The authors use the word “volunteer”, which suggests that blood was collected specifically for this work. In that case, IRB approval would be required. If the blood was not collected specifically for this work, but rather some random serum sample was used for these tests, that should be clarified. Similarly, the use of “donor” in Table 3 and line 494 also suggests that an IRB approval would be needed.

RESPONSE: We apologize for the confusion. The study used anonymized data (no personal reference can be established) and consequently was not subject to ethical consultation by the responsible ethics committee of the Bavarian Medical Association. All donating subjects gave written informed consent to use residual serum for research according to the Declaration of Helsinki (revised version of the 64th WMA General Assembly, Fortaleza, Brazil, October 2013), and following the standards of ICH-GCP E6 (R2). According to the reviewer’s comment, we now use the term “donor” throughout the entire manuscript.

3. Page 5, line 251. Since the “LoD Variant Approach…” is in quotes, is there a reference for this?

RESPONSE: We now provide EP17-A2 as reference and have revised the section as follows: “In case Cochran’s C test failed, the “LoD Variant Approach: Nonparametric Analysis” was used (trial and error experiment design according to EP17-A2).

4. Page 7 line 304, minor English correction. “lied” should either be a simple “was” or it should be “lay”. I prefer the simple “was”.

RESPONSE: Corrected accordingly.

5. Page 14, lines 497-499. I do not understand what the authors are trying to say here. Please rephrase.

RESPONSE: We revised the sentence as follows: ”The expansion of the interference experiments by evaluating in-vivo samples of patients with a high likelihood for the presence of certain interfering substances, as done by others [23], would have complemented our approach.”

6. A general comment/observation. Not being familiar with the pseudo-Voight fitting method, how does it handle overlapping peaks? It seems to me that with myo-inositol having resonances at 3.5 and 3.6, valine having resonances at 3.6, and glucose having resonances also at 3.5, if the method doesn’t handle overlapping peaks well, that could explain the issue with high glucose concentration. In that case, does the method already fit glucose? If not, would the GFRNMR method be improved by including glucose in the fit? Also, the reference for the pseudo-Voight fitting doesn’t point to a method paper describing the fitting, but rather to another paper that just uses it. A reference describing the fitting itself would be helpful. It appears to be just a way to fit the individual peaks, but I was unable to find anything about how it handles overlap.

RESPONSE: We thank the reviewer for pointing this out. In direct response to the comment, we allowed ourselves to add a section with an additional reference to explain our readers how overlapping signals were handled by simultaneous fitting. The sections reads now like this: “Metabolite quantification used curve-fitted pseudo-Voigt profiles [30]. In case of severe or frequent overlapping signals derived from interfering substances in the targeted spectral region, simultaneous fitting for the metabolite and the interfering substance(s) were performed [31] [PMID: 32987827].”

Author Response File: Author Response.pdf