Establishing pregnancy-specific haematological reference intervals in Ghana; a three-center cross-sectional study

Nicodemus Larbi Simpong; Charity Tenu Afefa; Leander Yimpuri; Betty Akum; Afia Safo; Simon-Junior Edziah; David Larbi Simpong; Patrick Adu

doi:10.1371/journal.pone.0274422

Peer Review History

Original SubmissionFebruary 11, 2022
4 Apr 2022 Decision Letter - Orvalho Augusto, Editor PONE-D-22-04279Establishing pregnancy-specific haematological reference intervals in Ghana; a three-center cross-sectional study.PLOS ONE Dear Dr. Adu, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by May 19 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Orvalho Augusto, MD, MPH Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information. 3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized. Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. Additional Editor Comments: This is an important report to improve pregnancy outcomes. The authors make a very compelling argument for building hematologic reference intervals specific to African subpopulations, and in particular for pregnancy. Issues: 1. Please, add line enumeration. 2. As both reviewers point out there is an important amount of typos and orthographic issues to fix. 3. The authors declared that the dataset is fully available. I did not see such data. 4. There are so many abbreviations. Please have a list of abbreviations at the end of the manuscript. 5. The authors removed participants with malaria, and many other important conditions. However, nothing is mentioned about HIV (interestingly hepatitis B is). Why? 6. In the sample size subsection: - the last sentence starting at “The lower and upper reference…” should be removed. That is not part of sample size considerations. 7. The “Reference interval estimation” subsection should be merged with the “Data management and data analyses” 8. Data management and data analyses - Please cite the software used - For Excel, please add as well the version 9. Table 1: - Please a row for overall total 10. Table 2: - Please a row for overall total - For hemoglobin classification, please add a footnote explaining the limits 11. Table 3: - Add a footnote explaining the abbreviations 12. Figure 1: - It is dangerous the mixture boxplots and line plots with presumably errors whiskers (which are in fact percentiles). Please separate these and explain in the footnote how the errors in the line plots are computed 13. Figure 2: - There are no dotted lines. There are dashed lines. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors sought to determine haematological reference intervals for different trimesters of pregnancy among pregnant women which can be used to make clinical decisions. Below are some suggestions to improve the quality of the manuscript. Abstract 1. CLSI should be written out in full since it is not a standard abbreviation 2. Please clarify the version of the guidance document: is C28A2992 referring to C28-A3? 3. It is not clear why Hb >13 stated here and in the discussion should be considered as being high and linked to unfavorable pregnancy outcomes 4. It might be good to limit the number of decimal points for the various parameters as follows: Hb - 1, MCHC - 1, RBC - 2, MCH - 1, MCV - 1 MCH - 1, WBC - 1, and PLT - 0 (in the manuscript) 5. Please indicate in the conclusion the comparison for which the determined reference intervals are lower Keywords 6. the keywords look a bit too long and repetitive, and could be revised to: Haematological; Reference intervals, Intra-pregnancy; Sub-Saharan Africa, pregnancy Introduction 7. CLSI is Clinical and Laboratory Standards Institute, and not Clinical Laboratory Standards Institute 8. Please provide references for statements attributed to CLSI, and also references for previous research in Sub-Saharan Africa which authors indicate showed lower values 9. Please note NCCLS is an older name of CLSI, kindly update to CLSI 10. The last sentence of the introduction sounds like a conclusion and could be revised to explain the aim of the study Methods 11. Please indicate the make and manufacturer of the haematology analyzers used for the analysis 12. Please indicate Quality Assurance/Quality Control measures put in place to ensure reliable results 13. Please give references for the sickling and G6PD tests Results 14. Reference values are supposed to be determined using apparently healthy individuals. Inclusion of the Hepatitis B positive individuals will affect the determined values. Please exclude these individuals from the calculations 15. Presentation of the reference intervals in a tabular form will make it easier for the ready than the figures 16. The minimum of 120 individuals recommended by the CLSI for the determination of reference intervals is to make it possible to calculate 90% confidence intervals around the lower and upper values. The confidence intervals seem to be missing in the manuscript. Discussion, References 17. The discussion is well written but could be updated with more references comparing similar studies. 18. the reference section looks too short. Kindly update Reviewer #2: This manuscript by Adu et al. establishes reference intervals in a cohort of healthy pregnant women across trimesters for key haematology parameters. The authors are congratulated on their work as significant evidence gaps continue to exist in evidence-based cut-offs for test interpretation in pregnancy. However, further clarification and select changes are needed, particularly in the methods section. Specific comments are below: Abstract: - Suggest to remove "Deranged" and replace with "Abnormal" - Suggest to include weeks of gestation for each trimester, space allowing - Methods: - Exclusion criteria: Please clarify whether individuals with acute illness at the time of collection and/or use of prescribed medication (e.g. thyroid supplementation) - Study participants: Please clarify whether iron supplementation was captured during assessment. In addition, was any longitudinal assessment evaluated at the end of the pregnancy? - Reference Interval Determination: Please add confidence intervals for each estimate. CLSI guidelines recommends reporting 90% CIs - Experimental Protocols: There is insufficient information in this section. For every assay, the manufacturer analyzers should be reported as well as their method (i.e. traceability) and performance throughout study duration (i.e. CV for QC levels). In addition, multiple centers were included in this study - were any center-specific differences in methodology evaluated? - Data Analyses: CLSI guideline recommends using the Harris and Boyd method to test for partitions (i.e. gestational-age specific differences). Please clarify why MW and KW tests were selected. In addition, please clarify if outliers were removed and whether normality of data were assessed prior to statistical testing. Results: - Figure 1: Suggest to include these graphs for every parameter in supplemental methods. - Table 1: Please clarify if individuals were excluded on the basis of positive HepB status. - Table 3: Suggest to include RIs in this table as well (or an additional table) rather than in the graphs. In addition, was the differential evaluated? Discussion: - The discussion mainly focuses on Hgb levels and their classification based on WHO standards. It should be highlighted that the WHO standards may not suit the ethnic population of Ghana and it is potentially not a public health problem. If no pregnant individuals or their neonates had serious complications, should this be considered abnormal? Suggest to include a comment on this. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: Yes:** Mary Kathryn Bohn [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0274422.r001
Revision 1
19 May 2022 Author Response Comment Response Page EDITORIAL 1. Please, add line enumeration Line numbers added 2. As both reviewers point out there is an important amount of typos and orthographic issues to fix. The manuscript has been revised to address these 3. The authors declared that the dataset is fully available. I did not see such data. The minimal data set underlying the results described in the manuscript has been uploaded as a Supporting Information file 4. There are so many abbreviations. Please have a list of abbreviations at the end of the manuscript. List of abbreviations included Page 21; Lines 147-155 5. The authors removed participants with malaria, and many other important conditions. However, nothing is mentioned about HIV (interestingly hepatitis B is). Why? Hepatitis B infected participants’ data have been excluded; data has subsequently been re-analysed. All data from HIV positive pregnant women were not included in the initial data collection. Page 6; Line 145 6. In the sample size subsection: - the last sentence starting at “The lower and upper reference…” should be removed. That is not part of sample size considerations. “The lower and upper reference limits were determined non-parametrically using the 2.5th and 97.5th percentiles respectively” has been deleted from the “Sample size” section in the revised manuscript. Page 6; Line 131-136 7. The “Reference interval estimation” subsection should be merged with the “Data management and data analyses” The “reference interval estimation” section has been merged with the “Data management and data analyses section.” Pages 8-9; Line 174 - 190 8. Data management and data analyses - Please cite the software used - For Excel, please add as well the version The Data management and data analyses section of the manuscript has been revised to include “Data was double-entered and checked in a Microsoft Excel 2016 Spreadsheet. For statistical analysis, the Statistical Package for the Social Sciences (SPSS) version 22 (IBM Inc., USA), GraphPad Prism software version 8.0.2 (GraphPad Prism Inc., San Diego, USA) and Stata IC version 16.0 (Stata Corp, College Park, TX, USA) were utilized.” Page 8; Line 174 – 178. 9. Table 1: - Please a row for overall total Row totals have been calculated Page 9; line 197-198 10. Table 2: - Please a row for overall total - For hemoglobin classification, please add a footnote explaining the limits Row totals have been calculated Manuscript has been revised to include a footnote “Anaemia was classified based on the trimester-specific cut-offs set by the World Health Organization; trimester 1 (Hb <11.0 g/dl), trimester 2 (Hb <10.5 g/dl), and trimester 3 (Hb <11.0 g/dl); in accordance with previous meta-analyses report (4), intra-pregnancy Hb >13.0 g/dL was classified as high maternal haemoglobin.” Page 10; line 207-208 Line 209 - 211 11. Table 3: - Add a footnote explaining the abbreviations Footnote defining abbreviations have been added. Page 12; line 249 - 251 12. Figure 1: - It is dangerous the mixture boxplots and line plots with presumably errors whiskers (which are in fact percentiles). Please separate these and explain in the footnote how the errors in the line plots are computed All graphs have been converted to Boxplots; a footnote “(All box plots were constructed with whiskers indicating minimum and maximum data points)” has been added. Page 11; Line 228 – 231. 13. Figure 2: - There are no dotted lines. There are dashed lines. Figure 2 has been converted into a table (table 4) as suggested by both reviewer 1 & 2 Page 13; line 254 - 268 REVIEWER 1 Abstract 1. CLSI should be written out in full since it is not a standard abbreviation Done Page 2; line 29 2. 2. Please clarify the version of the guidance document: is C28A2992 referring to C28-A3? Yes. Please see reference Page 2; line 38 3. 3. It is not clear why Hb >13 stated here and, in the discussion, should be considered as being high and linked to unfavorable pregnancy outcomes This has been clarified under section “full blood count”; this is from a meta-analyses report by Young et al (2019) “Young MF, Oaks BM, Tandon S, Martorell R, Dewey KG, Wendt AS. Maternal hemoglobin concentrations across pregnancy and maternal and child health: a systematic review and meta-analysis. Ann N Y Acad Sci. 2019;1450(1):47-68.” Page 7; Line 163-164 4. 4. It might be good to limit the number of decimal points for the various parameters as follows: Hb - 1, MCHC - 1, RBC - 2, MCH - 1, MCV - 1 MCH - 1, WBC - 1, and PLT - 0 (in the manuscript). All decimals have been limited to 1 in all tables. 5. Please indicate in the conclusion the comparison for which the determined reference intervals are lower This is applicable to all the parameters analysed in the current study. Keywords 6. the keywords look a bit too long and repetitive, and could be revised to: Haematological; Reference intervals, Intra-pregnancy; Sub-Saharan Africa, pregnancy Manuscript revised to reflect suggestions made by reviewer Page 3; line 52-53 Introduction 7. CLSI is Clinical and Laboratory Standards Institute, and not Clinical Laboratory Standards Institute Manuscript revised to read Clinical and Laboratory Standard Institute (CLSI). Page 4; line 83 8. Please provide references for statements attributed to CLSI, and also references for previous research in Sub-Saharan Africa which authors indicate showed lower values Done. 9. Please note NCCLS is an older name of CLSI, kindly update to CLSI Manuscript revised to uniformly read CLSI 10. The last sentence of the introduction sounds like a conclusion and could be revised to explain the aim of the study. The last section of the introduction has been revised to read “Since intra-pregnancy haematological reference intervals have not been explored within the sub-Saharan African region, we aimed to generate empirical baseline data to begin to address this knowledge gap, and stimulate discussion as well as further research that will ultimately lead to improved context-specific intra-pregnancy clinical decision-making process.” Page 5; line 114 - 117 Methods 11. Please indicate the make and manufacturer of the haematology analyzers used for the analysis The section has been revised accordingly to include “5-part Sysmex 20N haematology analyser (Sysmex Corp., Japan)” Page 7; line 160 12. Please indicate Quality Assurance/Quality Control measures put in place to ensure reliable results The “Data was double-entered and checked in a Microsoft Excel 2016 Spreadsheet” in the “Data management and data analyses” has been elaborated by adding “Data entry were at each study site was undertaken independently by two researchers after which they were validated by the research supervisor as a quality control process to ensure that data was accurately captured” to reflect QA procedures employed during data acquisition Page 8; line175 – 178. 13. Please give references for the sickling and G6PD tests Reference provided in the revised manuscript “Barabara J. Bain IB, Michael A Laffan, S. Mitchell Lewis. Dacie and Lewis Practical Haematology: Elsevier Churchill Livingstone; 2011. 650 p.” Results 14. Reference values are supposed to be determined using apparently healthy individuals. Inclusion of the Hepatitis B positive individuals will affect the determined values. Please exclude these individuals from the calculations Data from hepatitis B positive individuals have been excluded and data re-analysed. Page 6; line 145 15. Presentation of the reference intervals in a tabular form will make it easier for the ready than the figures All the reference intervals have been presented in tabular form (see table 4) Page 14 16. The minimum of 120 individuals recommended by the CLSI for the determination of reference intervals is to make it possible to calculate 90% confidence intervals around the lower and upper values. The confidence intervals seem to be missing in the manuscript. Manuscript has been revised to include the 90% confidence intervals for both the lower and upper limits; see table 4. Page 14 Discussion, References 17. The discussion is well written but could be updated with more references comparing similar studies. More references added 18. the reference section looks too short. Kindly update. References added REVIEWER 2 Abstract: 1. Suggest to remove "Deranged" and replace with "Abnormal" - Suggest to include weeks of gestation for each trimester, space allowing Done Page 2; line 29 Page 2; line 35 – 36 Methods 1. Exclusion criteria: Please clarify whether individuals with acute illness at the time of collection and/or use of prescribed medication (e.g. thyroid supplementation) Data from pregnant women with clinically diagnosed acute or chronic diseases were excluded from the study. The only recorded medications the participants were on were the “routine pregnancy drugs” The inclusion criteria section has therefore been revised to include: “All participants were on the following medications: folic acid (5 mg daily), oral iron tablets (200 mg three times daily) and oral multivitamin tables; these medications are generally called routine pregnancy drugs within the Ghana Health Services as they are part of the routine medical care for all pregnant women.” Page 6; line 138 – 141. Study participants: Please clarify whether iron supplementation was captured during assessment. In addition, was any longitudinal assessment evaluated at the end of the pregnancy? Iron supplementation is mandatory for all pregnant women in Ghana as part of routine care. This fact has been included in the No Page 6; line 138 – 141 - Reference Interval Determination: Please add confidence intervals for each estimate. CLSI guidelines recommends reporting 90% CIs Manuscript has been revised to include the 90% confidence intervals for both the lower and upper limits; see table 4. Page 14 Experimental Protocols: There is insufficient information in this section. For every assay, the manufacturer analyzers should be reported as well as their method (i.e. traceability) and performance throughout study duration (i.e. CV for QC levels). In addition, multiple centers were included in this study - were any center-specific differences in methodology evaluated? Each site uses 5-part Sysmex haematology analyser. Since this is a retrospective data that was retrieved from the hospital data management system, we were unable to collect the performance data of the analysers since the data was not prospectively collected. We believe this would have been valid undertaking if the design for the study was a prospective one since this would have allowed us to interrogate the comparability of the laboratory results as well as inter- and intra-laboratory agreement. - Data Analyses: CLSI guideline recommends using the Harris and Boyd method to test for partitions (i.e. gestational-age specific differences). Please clarify why MW and KW tests were selected. The recommendation from Harris and Boyd was employed by comparing the standard deviations of parameters per trimester (table 3). The MW and KW statistical procedures were only employed to test statistical differences between the haematological values. In addition, please clarify if outliers were removed and whether normality of data were assessed prior to statistical testing. The data was explored for outliers using the recommendation from the CLSI prior to analyses. Data was assessed for normality before choosing non-parametric means was selected for the data selection. Results: Figure 1: Suggest to include these graphs for every parameter in supplemental methods. Noted Table 1: Please clarify if individuals were excluded on the basis of positive Hep B status. Data from hepatitis B positive individuals have been excluded from the revised manuscript; see under the revised “Exclusion criteria section”. Page 6; line 145. Table 3: Suggest to include RIs in this table as well (or an additional table) rather than in the graphs. The rational for including table 3 is to provide basis for why a separate or combined reference intervals should be used. The reference intervals are included in table 4. Page 14 In addition, was the differential evaluated? No Discussion: The discussion mainly focuses on Hgb levels and their classification based on WHO standards. It should be highlighted that the WHO standards may not suit the ethnic population of Ghana and it is potentially not a public health problem. If no pregnant individuals or their neonates had serious complications, should this be considered abnormal? Suggest to include a comment on this. We have acknowledged the cross-sectional nature of the study as a key limitation as we could not access pregnancy outcome data. Page 19; line 374 – 386. Attachments Attachment Submitted filename: Responses to reviewer comments - PONE comments.docx https://doi.org/10.1371/journal.pone.0274422.r002
1 Jun 2022 Decision Letter - Orvalho Augusto, Editor PONE-D-22-04279R1Establishing pregnancy-specific haematological reference intervals in Ghana; a three-center cross-sectional study.PLOS ONE Dear Dr. Adu, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Jul 16 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Orvalho Augusto, MD, MPH Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: (No Response) ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In Table 4 (page 14), the 90% confidence interval for the upper limits of the following parameters do not seem technically or clinically possible: HCT (351), MCHC (418 and 223), RDW (447). I would suggest to remove these columns. Reviewer #2: Thank you for submitting this revised version of the manuscript. Most comments have been addressed, but select points still need to be considered. - Both reviewers commented on the availability of quality control data for the hematology system in use. This needs to be included to ensure proper analytical performance was obtained throughout the study (i.e. was internal QC run?) - In my opinion, graphs need to be provided by gestational age. Otherwise, it is very difficult to visualize the data and remark on observed trends. This would be very important to laboratories interpreting these findings. - A reference was added for the >13 cut-off, but there is insufficient rationale to justify its use. Please provide more in-depth rationale and discuss the impact of high HGB on pregnancy outcomes ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: Comment to authors.docx https://doi.org/10.1371/journal.pone.0274422.r003
Revision 2
15 Jun 2022 Author Response RESPONSES TO REVIEWERS’ COMMENTS Establishing pregnancy-specific haematological reference intervals in Ghana; a three-center cross-sectional study (manuscript ID: PONE-D-22-04279) COMMENTS RESPONSE PAGE 1. In Table 4 (page 14), the 90% confidence interval for the upper limits of the following parameters do not seem technically or clinically possible: HCT (351), MCHC (418 and 223), RDW (447). I would suggest to remove these columns. Table 4 has been revised to address this; a footnote “Note: the upper limit of the 90% CI of HCT, MCH, MCHC, and RDW for T2 are not indicated as they were very large to be considered technically feasible” has been added to highlight this change. Page 14, line 281 – 282 2. A reference was added for the >13 cut-off, but there is insufficient rationale to justify its use. Please provide more in-depth rationale and discuss the impact of high HGB on pregnancy outcomes. Manuscript has been revised to include additional information “Maternal haemoglobin and adverse birth outcomes have been shown to follow a U-shaped curve where both low and high gestational haemoglobin are associated with adverse pregnancy outcomes such as preterm, gestational diabetes, stillbirth, pre-eclampsia, and small-for-gestational age (1-3). Therefore, in accordance with previous meta-analyses report (4), intra-pregnancy Hb >13.0 g/dL was classified as high maternal haemoglobin to classify participants with high gestational haemoglobin.” Relevant references have also been cited. Page 8, line 169 – 174 3. Both reviewers commented on the availability of quality control data for the hematology system in use. This needs to be included to ensure proper analytical performance was obtained throughout the study (i.e. was internal QC run?) Per the prevailing protocols in each of the participating laboratory, control samples (low, normal and high) are run daily as quality control measure/check on analyser performance prior to assaying patient sample. Only when the respective haematology analyser passes the quality control tests is the analyser used to assay patient samples. Additionally, each of these daily quality control sample data (low, normal and high) are cumulatively evaluated monthly as a quality assurance measure to identify trends in analyser performance that may require immediate action. Page 7, line 161 - 167 4. - In my opinion, graphs need to be provided by gestational age. Otherwise, it is very difficult to visualize the data and remark on observed trends. This would be very important to laboratories interpreting these findings. This has been addressed in the revised manuscript; in figure 1A, [haemoglobin] is compared per trimester; figure 1C & 1F are specific trimester 2 [Hb] comparison per age category and participants’ place of residence; figure 1D & 1G are specific trimester 3 [Hb] comparison per age category and participants’ place of residence - 1. Dewey KG, Oaks BM. U-shaped curve for risk associated with maternal hemoglobin, iron status, or iron supplementation. Am J Clin Nutr. 2017;106(Suppl 6):1694S-702S. 2. Abeysena C, Jayawardana P, de ASR. Maternal haemoglobin level at booking visit and its effect on adverse pregnancy outcome. Aust N Z J Obstet Gynaecol. 2010;50(5):423-7. 3. Díaz-López A, Ribot B, Basora J, Arija V. High and Low Haemoglobin Levels in Early Pregnancy Are Associated to a Higher Risk of Miscarriage: A Population-Based Cohort Study. Nutrients. 2021;13(5). 4. Young MF, Oaks BM, Tandon S, Martorell R, Dewey KG, Wendt AS. Maternal hemoglobin concentrations across pregnancy and maternal and child health: a systematic review and meta-analysis. Ann N Y Acad Sci. 2019;1450(1):47-68. Attachments Attachment Submitted filename: Responses to reviwer comments.docx https://doi.org/10.1371/journal.pone.0274422.r004
27 Jun 2022 Decision Letter - Orvalho Augusto, Editor PONE-D-22-04279R2Establishing pregnancy-specific haematological reference intervals in Ghana; a three-center cross-sectional study.PLOS ONE Dear Dr. Adu, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Aug 11 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Orvalho Augusto, MD, MPH Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments: This is a very important report and I feel sad to still find issues. Please respond to the reviewer's question. 1. Line 228 - I think the "semester" should be "trimester" 2. For the prevalence of anaemia, please add 95% confidence interval (exact binomial confidence interval. In fact, this could be a separate figure or table. 3. The lines between 226 to 262 fail to describe the results properly. Rather than talking about the medians being compared per different groups, the authors focus only on p-values. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Thank you for submitting a revised manuscript. There are still very abnormal values indicated in Table 4 for the 90% CI for trimester 3 MCHC (223) and trimester 2 TWBC (87). As indicated in the earlier review comment, several of the upper values of the 90% CI are not clinically feasible and recommended the column could be removed. Reviewer #2: (No Response) ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0274422.r005
Revision 3
8 Aug 2022 Author Response Comment Responses Page Editor comment 1. Line 228 - I think the "semester" should be "trimester" Correction effected Page 11; line 230. 2. For the prevalence of anaemia, please add 95% confidence interval (exact binomial confidence interval. In fact, this could be a separate figure or table. Manuscript revised to include the 95% CI (exact binomial confidence interval) “Furthermore, whereas 46.5% of participants had low haemoglobin level and therefore anaemic, 9.3% had higher haemoglobin levels. When participants haemoglobin levels were considered per trimester, 36.4% (95% CI: 0.1719 – 0.5934), 31.6% (95% CI: 0.2487 – 0.3904) and 68.2% (95% CI: 0.5951 – 0.7601) respectively of participants in trimester 1, 2 and 3 were anaemic.” However, since the main import of the manuscript is that using the Caucasian-centric reference intervals (which was the basis for the anaemia prevalence in table 2), may lead to some participants incorrectly classified as anaemic, we are of the opinion that constructing additional table/figure will not be necessary in this instance. Page 10; line 216 - 220 3. The lines between 226 to 262 fail to describe the results properly. Rather than talking about the medians being compared per different groups, the authors focus only on p-values. The description to the figure has been revised to include the respective median to enable the reader better appreciate the data. The revised manuscript thus read “Participants’ haemoglobin levels were compared with the demographic data (figure 1). Overall, the median haemoglobin significantly differed across the trimesters of pregnancy. The median haemoglobin was higher in trimester 1 (11.80 g/dl) but decreased progressively across trimesters 2 (11.00 g/dl) and 3 (10.50 g/dl) [fig 1A: p = 0.0232 (trimester 1 vs trimester 3); p = 0.0005 (trimester 2 vs trimester 3)]. There was a general trend toward higher median haemoglobin levels with advancing age of participants as the respective median haemoglobin levels for 14 – 19 years, 20 – 29 years, 30 – 39 years, and 40 – 49 years were 10.35 g/dl, 10.80 g/dl, 11.00 g/dl, and 11.10 g/dl [fig 1B; p = 0.049 (14 – 19 years vs 30 – 39 years group; p = 0.0278 (14 – 19 years vs 40 – 49 years group)]; a similar trend was also observed when data was explored for trimester 2 (figure 1C) and trimester 3 (figure 1D) although the differences did not reach statistical significance. Also, the median haemoglobin level was significantly higher in participants from Ashanti region (11.80 g/dl) compared to median haemoglobin level of participants from Eastern region (10.50 g/dL) or Upper Est region (10.30 g/dl) [figure 1E: p <0.0001 (Ashanti vs Eastern region); p <0.0001 (Ashanti vs Upper West region)]. Similarly, the median haemoglobin levels in trimester 2 was significantly higher in participants from Ashanti region (11.80 g/dl) compared to participants from Eastern (10.80 g/dl) or Upper West (10.30 g/dl) regions (figure 1F: p = 0.0031 vs Eastern region; p <0.0001 vs Upper West region). Moreover, the median haemoglobin in trimester 3 was higher in participants from Ashanti region (11.65 g/dl) compared to the Upper West (10.50 g/dl) region (figure 1G: p = 0.0240).” Page 12; line 227 – 246. Reviewer 1 There are still very abnormal values indicated in Table 4 for the 90% CI for trimester 3 MCHC (223) and trimester 2 TWBC (87). As indicated in the earlier review comment, several of the upper values of the 90% CI are not clinically feasible and recommended the column could be removed. Corrections effected Page 15; line 284 - 289 Attachments Attachment Submitted filename: Point-by-point responses to reviewer comment 3.docx https://doi.org/10.1371/journal.pone.0274422.r006
30 Aug 2022 Decision Letter - Orvalho Augusto, Editor Establishing pregnancy-specific haematological reference intervals in Ghana; a three-center cross-sectional study. PONE-D-22-04279R3 Dear Dr. Adu, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Orvalho Augusto, MD, MPH Academic Editor PLOS ONE Additional Editor Comments (optional): This is very important work to help establish appropriate clinical guidelines for African pregnant women. I still cannot understand why the authors still test for normality when in the end used non parametric methods. Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The earlier recommendation to remove all the 90% CIs has not rather been replaced with "-" for the abnormally high limits. Keeping some values and putting "-" makes it appear the limit is to infinity. Reviewer #2: The author has addressed all remaining comments by reviewers. Updates to Table 4 and results section are satisfactory. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0274422.r007
Formally Accepted
5 Sep 2022 Acceptance Letter - Orvalho Augusto, Editor PONE-D-22-04279R3 Establishing pregnancy-specific haematological reference intervals in Ghana; a three-center cross-sectional study. Dear Dr. Adu: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Orvalho Augusto Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0274422.r008

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .