21-Deoxycortisone: A Novel Sensitive and Specific Newborn Screening Marker for Congenital Adrenal Hyperplasia

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript describes a retrospective study to evaluate the performance of 21-deoxycortisone as a novel biomarker for second-tier newborn screening for Congenital Adrenal Hyperplasia (CAH).  The study utilizes stored newborn bloodspot specimens to measure 21-deoxycortisone levels and compares sensitivity and specificity of this approach to the standard algorithm for the program that utilizes 21-deoxycortisol.  Although the abstract contains a number of obvious errors, the remainder of the manuscript is well written, clear, and presents novel data of interest to the community.

 

Specific Comments:

Abstract:

Line 10: “We postulated that 21-deoxycortisone, the 11β-hydroxysteroid dehydrogenase metabolite of 21-deoxycortisone, may also be an accurate bloodspot marker of CAH.” -the second 21-deoxycortisone should be 21-deoxycortisol.

Line 12: 21DE should be defined at first use.

Line 14: 21-deoxycortisol should be 21-deoxycortisone

Line 15: 21-deoxycortisol should be 21-deoxycortisone

Line 18: “TP” should be defined at first use.

Line 19: “TN should be “FN” and this abbreviation for false negative should be defined at first use.

Line 19: “FP” should be defined at first use.

 

Introduction:

Line 53: “Children with classical CAH (SW-CAH and SV-CAH) that are missed by screening may have benefiĴed from early detection and treatment.” – Why is SW-CAH included in this sentence? The manuscript does not report missing any SW-CAH cases in the study period.

Discussion:

Line 220: “Based on our retrospective screening data, screening sensitivity will be improved without any changes to 17OHP immunoassay thresholds as several FN specimens with elevated 21-DF and 21-DE proceeded to second tier LC-MS/MS analysis and would have been detected after LC-MS/MS second tier analysis.” -  The data does not appear to support this statement.  As shown in Table 1, all false negative cases that went to second-tier testing had elevations in both 21DF and 21DE.   All the samples that had elevated 21DE but normal 21DF would fail to be sent for second-tier testing under the current first-tier protocol, so the improved sensitivity of the 21DE analyte would never be realized in a prospective screening scenario and the screening sensitivity is not actually improved.

Table A1:  How was Case #10 detected by screening?  Based on the description of the screening algorithm at the time, this sample with low 21-DF does not appear to meet your screen positive criteria.

Author Response

Please see attached letter with responses to the editors and both reviews. Thank you

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This study describes a second‑tier LC-MSMS method that measures 21‑deoxycortisone (21-DE) as an additional, highly sensitive and specific biomarker for congenital adrenal hyperplasia (CAH).  21-DE was measured retrospectively in 494 samples, including true positive, false negative and false positive specimens. The data from this study suggest that 21-DE, as a NBS marker for (classic) CAH, has higher sensitivity and specificity than 21-deoxycortisol (21-DF).

Comments:

1. Abstract: Please review, there is a typo:

“21-deoxycortisol is a sensitive and specific blood marker for congenital adrenal hyperplasia (CAH). We postulated that 21-deoxycortisone, the 11β-hydroxysteroid dehydrogenase metabolite of 21-deoxycortisone, may also be an accurate bloodspot marker of CAH.”

21-deoxycortisone (21-DE) is formed from 21-deoxycortisol.

2. Abstract: The abbreviation 21-DE is used in the abstract (Line 12), but was not defined at first use (Line 11).

 

2. Abstract, Lines 14 -16:

“The test was considered positive if 21-deoxycortisol was detected. The sensitivity and specificity of 21-deoxycortisol as a marker for classical CAH was calculated and compared to 21-deoxycortisol data from a previous New Zealand study. The method was linear to 1000nmol/L and precision was 7.3-10.3%.... .”

This paragraph is not clear. In this study, was the determination of a positive screen based on retrospective measurement of 21‑DF? (not 21-DE?)

 

4. LC-MS/MS Analysis & Results sections:

Please indicate whether analytical interference was evaluated as part of the validation of the 21‑DE method.

5. Discussion: As noted by the authors in the Discussion section, a limitation of this study is the lack of a long-term stability evaluation for 21-DE, however, inclusion of short‑term stability data (e.g., 1 week to 60 days of storage at room and other temperatures) would strengthen the manuscript.
6. Discussion: “In this study, 21DE was detected in all TP specimens and in all but one FN samples and was not detected in any FP samples.)  

   21‑DE was not detected in one of the identified FN specimens.  Please discuss the possible reasons. (Both 21-DF and 21-DE were < 2 nmol/L in this specimen.  Sample collected in 2006.  Stability issues?

    

    

 

Author Response

Please see attached letter responding to the review and the editorial comment

Author Response File: Author Response.pdf