The role of carbon nanoparticle in lymph node detection and parathyroid gland protection during thyroidectomy for non-anaplastic thyroid carcinoma- a meta-analysis.

OBJECTIVE: To assess the efficiency of the carbon nanoparticles (CNs) in lymph node identification and parathyroid gland (PG) protection during thyroidectomy for non-anaplastic thyroid carcinoma (N-ATC).
METHODS: A systematic literature search for relevant literatures published up to December 2018 in PubMed, EMBASE, Web of Science and Cochrane Library was performed. Both English and Chinese literatures were retrieved and analyzed. Randomized controlled trials or nonrandomized controlled trials comparing the use of CNs with the use of methylene blue or a blank control in patients undergoing thyroidectomy for N-ATC were enrolled in this study. The primary outcomes included the number of lymph nodes harvested, the rate of lymph nodes involved, and the rates of accidental parathyroidectomy, hypoparathyroidism, and hypocalcemia. Weighted mean differences (WMDs), odds ratios (ORs) and risk differences (RDs) were calculated for the dichotomous outcome variables. Between study heterogeneity was tested using the Q tests and the I2 statistics. All analyses were performed using Review Manager (version 5.3.5).
RESULTS: 25 studies comprising 3266 patients were included in this analysis. The total number of lymph nodes harvested in the CNs groups was significantly higher than that in the control groups (WMD, 2.36; 95% CI, 1.40 to 3.32; P <0.01). Administrating CNs was associated with a lower incidence of accidental PG removal (OR = 0.28, 95% CI = 0.21 to 0.37, P<0.01) and lower rates of both postoperative transient hypoparathyroidism (OR = 0.46, 95% CI = 0.33 to 0.64, P <0.01) and transient hypocalcemia (OR = 0.46, 95% CI = 0.33 to 0.65, P <0.01). No significant difference was found concerning lymph node metastatic rates between CNs group and control group. Subgroup analysis indicated that the application of CNs in reoperation thyroidectomy reduced both the rate of transient hypoparathyroidism (OR = 0.21, 95% CI = 0.06 to 0.75, P = 0.02) and the possibility of accidental PGs removal (OR = 0.21, 95% CI = 0.07 to 0.62, P = 0.004, P<0.05).
CONCLUSIONS: The application of CNs in thyroidectomy for N-ATC results in higher number of lymph node harvested and better PG protection during both initial and reoperation thyroidectomy.

Introduction

Thyroid cancer is one of the most common types of n class="Disease">cancer in the world, the incidence of which increased dramatically in the recent two decades [1]. More than 95 percent of the thyroid carcinomas were non-anaplastic thyroid carcinoma (N-ATC), including papillary thyroid carcinoma, follicular thyroid carcinoma, and medullary thyroid carcinoma [1]. Total thyroidectomy combined with levelⅡ-Ⅵ neck dissection for clinically N1b cases or central compartmental dissection (CND) for cN0 cases is widely advocated as one of the standard treatment protocols for N-ATC, whereas lobectomy is only accepted for T1 cases without high risk factors [2-4]. However, it’s a challenge for surgeons to perform a total thyroidectomy with or without CND due to the potential risk of postoperative transient hypocalcemia/hypoparathyroidism (incidence rate 20%-60%) or permanent ones (incidence rate 2%-7%) based on the previous reports and even higher for those who undergo a reoperation [5, 6].

Carbon nanoparticles (CNs) have been successfully attempted for lymph node dissection in breast carcinoma, gastric carcinoma, and a few other malignancies [7, 8]. CNs have a mean diameter of 150 nm larger than the size of the capillary endothelial cell gap (20–50 nm), and thus cannot enter the blood vessels. However, they can penetrate the lymphatic capillary endothelial cell gap (120–500 nm) and may be phagocytized by macrophages. Thus, these CNs specifically accumulate in the lymph nodes, staining them black and easily been identified with naked eyes [9, 10]. However, the parathyroid glands (PG) remains unstained due to their possible lack of lymphatics. This so called “negative development” not only increases the number of lymph nodes harvested during compartmental dissection, but also facilitates the surgeons’ distinguishing of the PGs from the lymph nodes and decreases the possibility of unintentional removal of PGs [11-13]. The pattern of CNs metabolism is the hypothetic basis that CNs can be used as a tracer to detect the sentinel nodes.

In the past decade, CNs had been successfully attempted as a negative developer to protect parathyroid gland (PG) during initial thyroidectomy [14]. Although a couple of meta-analyses evaluating the value of CNs in initial thyroidectomy had been published, the data need to be updated [15, 16]. Furthermore, there are still doubts about the efficiency of CNs in reoperation thyroidectomy due to the hypothesis that the lymphatic capillaries may be destroyed during initial surgery [17, 18]. Therefore, we performed a meta-analysis with more comprehensive and updated studies to summarize the role of CNs in lymph node detection and PG protection during both initial and reoperation thyroidectomy.

Materials and methods

Search strategy

Two authors conducted independently a search for relevant literatures up to December 2018 in PubMed, En class="Chemical">MBASE, Web of Science, and Cochrane Library. The following medical search headings were used: ("thyroid gland"[MeSH Terms] OR ("thyroid"[All Fields] AND "gland"[All Fields]) OR "thyroid gland"[All Fields] OR "thyroid"[All Fields] OR "thyroid (usp)"[MeSH Terms] OR ("thyroid"[All Fields] AND "(usp)"[All Fields]) OR "thyroid (usp)"[All Fields]) and ("carbon"[MeSH Terms] OR "carbon"[All Fields]). We manually searched the references of eligible studies and ClinicalTrials.gov to ensure identification of relevant published and unpublished studies. Chance-corrected agreement (i.e. kappa) in the screening stages and the eventual final set of included studies was calculated.

Inclusion criteria

Studies included in the meta-analysis need to fulfill the following criteria: (1) human n class="Chemical">N-ATC confirmed by pathology; (2) patients underwent thyroidectomy/lobectomy and/or neck dissection; (3) studies designed to compare the use of CNs with the use of methylene blue (MB) or with blank control; (4) studies on human beings; (5) full text available in English or Chinese.

Exclusion criteria

Studies were excluded if they (1) had a sample size less than 15; (2) included pregnancy or adolescent (aged<16); (3) included patients with benign and n class="Disease">malignant thyroid diseases, and complete data of the malignancies were unavailable. Exclusion criteria (2) was set to reduce the publication bias because most of the studies reported only the cases in adult or adolescents over 16 years old. When two or more studies were reported by the same authors and/or institution, either the most recent study or the higher quality study was included in the analysis to excluded the possible duplicate cases.

Data extraction

Two reviewers (SW Xu and ZF Li) independently performed the first-stage screening of titles and abstracts based on the inclusion criteria. In the second-stage screening, the two reviewers retrieved and reviewed the possible relevant articles in full text to confirm the included articles, and then they recorded the following data independently: first author, publication year, sample size, description of study population (age, sex), study design (RCT or NRCT), surgical procedure (Cn class="Chemical">Ns injection dose, points and waiting time), lymph nodes details (number of harvested and involved LN), parathyroid protection outcome. Outcome of parathyroid protection was evaluated based on the following parameters: number of the PGs identified and/or incidentally removed and patient numbers with transient or permanent postoperative hypoparathyroidism/hypocalcemia. Both hypoparathyroidism and hypocalcemia were accepted as parameters to judge parathyroid function impair, and the threshold values in each report were adopted. When hypoparathyroidism or hypocalcemia persisted for 6 months or more, it was defined as permanent, otherwise it was defined as transient. Any discrepancies were resolved by discussion or referred to the corresponding author (HW Peng).

Quality assessment

Concerning the quality of study design, the RCT was assessed according to the Jadad Scoring system, which consists of 3 items: randomization (0–2 points), blinding (0–2 points), and descriptions of the withdrawals and dropouts (0 or 1 point). The total possible score was 5 points. The Newcastle-Ottawa Scale was used for n class="Chemical">NRCT.

Statistical analysis

All analyses in the current meta-analysis were performed using RevMan 5.3.5 (free software downloaded from http://www.cochrane.org). The results are presented as weighted mean differences (WMDs) and Odds ratios (ORs) with a 95% confidence interval (CI). A P value< 0.05 was considered statistically significant, except where otherwise specified. Moreover, study heterogeneities were quantified using the Q-test and the I statistic. When P> 0.1and I< 50%, a fixed-effect model was used; otherwise, a random-effects model was applied. Possible publication bias was tested by Begg’s funnel plot.

Results

Study selection and description

Fig 1 details the study flowchart of the initial search and the subsequent selection of relevant articles. The initial search retuned 4488 studies. In the first-stage screening, 4202 irrelevant references and 230 duplicates were excluded. The remaining 56 studies with full text were further evaluated in the second-stage screening, 31 studies of which were excluded due to the reasons listed in the diagram. Finally, 25 studies fulfilled the inclusion criteria for the meta-analysis [9–13, 18–37]. Begg’s test for 25 studies present in Fig 2 and the Kappa Coefficient was 0.68 (95% CI:0.66–0.70). Table 1 summarizes all the individual studies and their characteristics. Of these 25 studies, 13 were RCT, 12 were NRCT. A total of 3266 n class="Species">patients were included in this meta-analysis, of which 1496 were included in the CN group, 1770 in the Control group (1496 in blank control and 181 in MB control). 3248 patients had PTC; 12 had FTC, and 6 had MTC. The two groups had no significant difference in terms of age (MD, -0.43; 95% CI, -1.35–1.26, P = 0.95) and sex (OR, 1.05; 95% CI, 0.87–1.25, P = 0.63). There were 3096 initial surgeries and 170 reoperations in this analysis.

Fig 1

Study flow diagram.

Fig 2

The funnel plots of the publication bias calculated from the Begg's test.

Table 1

Characteristics of the articles included in the meta-analysis.

Study	Study period	Group	Patients (N)	Age	M	F	Etiology
Bai, Y.C (2013)	Jun 2010 to Mar 2012	Experimental	48	46.3±9.2	9	39	48PTC
		Control	73	N	7	33	73PTC
Chaojie (2016)	Feb2011 to Feb 2014	Experimental	64	N	11	53	64PTC
		Control	52	N	9	43	52PTC
Chen, W (2014)	Jan2013 to Dec 2013	Experimental	36	38.23±10.67	5	31	36PTC
		Control	36	34.64±8.75	8	28	36PTC
Deng, W (2014)	July 2011 to Dec 2013	Experimental	18	N	4	14	18PTC
		Control	33	N	12	21	33PTC
Fu, H (2017)	Oct 2015 to May 2016	Experimental	75	45.41±10.2	21	54	75PTC
		Control	73	45.32±9.74	21	52	7PTC
Gao, B (2015)	Jan 2012 to Dec 2014	Experimental	27	49.4±2.5	2	25	27PTC
		Control	27	52.5±1.8	2	25	27PTC
Gao, Q (2014)	Jan2010 to Oct 2012	Experimental	50	42±1.28	12	38	50PTC
		Control	50	N	9	41	50PTC
Gu, J (2015)	Jun 2012 and Aug 2014	Experimental	50	46.98±9.027	10	40	47PTC+1FTC+2MTC
		Control	50	47.76±13.91	6	44	48PTC+1FTC+1MTC
Hao, R. T (2012)	Jan2008 to Dec 2009	Experimental	100	41	14	86	100PTC
		Control	100	44	11	89	100PTC
Liu, F (2017)	Apr 2016 and Feb 2017	Experimental	48	51.98 ± 3.1	N	N	48PTC
		Control	48	51.98 ± 3.1	N	N	48PTC
Liu, Y (2018)	Feb 2013 to May 2015	Experimental	45	46.17 ±10.20	17	28	45PTC
		Control	47	45.39±12.03	12	35	47PTC
Long, M (2017)	Jan2012 to May 2013	Experimental	42	44.5 ± 9.6	9	33	42PTC
		Control	46	43.8 ± 10.3	11	35	46PTC
Shen, H (2014)	Mar 2012 to Jul 2013	Experimental	45	N	N	N	42PTC+3FTC
		Control	64	N	N	N	57PTC+6FTC+1MTC
Shi, C (2016)	Jan2014 and Feb 2015	Experimental	52	45.2±5.8	6	46	52PTC
		Control	45	42 ±4.3	6	39	45PTC
Su, A. P (2016)	Apr 2013 to Mar 2015	Experimental	195	43.38 ± 11.65	57	138	55PTC
		Control	181	45.67 ± 13.50	55	126	181PTC
Tian, W (2014)	Apr 2012 to Oct 2013	Experimental	50	36.4±2.5	5	45	50PTC
		Control	50	44.5±5.8	11	39	46PTC+4FTC
Wang, B (2015)	Mar 2013 to Mar 2014	Experimental	28	30.25 ± 6.04	1	27	28PTC
		Control	27	29.44 ± 6.27	2	25	27PTC
Wang, B (2016)	Jan2013 to Jan2014	Experimental	90	44.36 ± 11.48	25	65	90PTC
		Control	141	44.09 ± 12.41	37	104	141PTC
Wang, X. L (2009)	NM	Experimental	18	44	10	8	17PTC+1MTC
		Control	18	40	7	11	16PTC+1FTC+1MTC
Xu, X. F (2017)	Sep 2013 to Aug 2014	Experimental	57	45.37±10.71	5	52	57PTC
		Control	57	42.68±14.43	4	53	57PTC
Xue, S (2018)	Jan2010 to Dec 2012	Experimental	106	44.88 ± 7.78	20	86	106PTC
		Control	300	44.35 ± 10.28	66	234	300PTC
Yu, W (2016).	Aug 2012 to Jun 2013	Experimental	41	41.6±17.1	8	33	41PTC
		Control	41	41.7±18.9	11	30	41PTC
Yu, WB (2016)	Jan2012 to Jun 2013	Experimental	70	44.5 ± 17.4	14	56	70PTC
		Control	70	45.5 ± 19.0	17	53	70PTC
Zhu H (2017)	Jan 2013 to Feb 2015	Experimental	60	60.4±7.18	24	36	60PTC
		Control	60	62.5±7.65	25	35	60PTC
Zhu, Y (2016)	Apr 2010 to Apr 2011	Experimental	81	46.75±12.09	14	67	81PTC
		Control	81	44.31± 10.37	16	65	81PTC

M/F = male/female; CN = carbon nanoparticles; PTC = papillary thyroid cancer; FTC = follicular thyroid cancer; MTC = medullary thyroid cancer

The quality assessment details for the RCTs and NRCTs are presented in Tables 1 and 2 in S1 File.

Surgical procedure

The CNs were provided by Chongqing LUMMY Pharmaceutical Co., Ltd. In most of the studies, the Cn class="Chemical">Ns were injected underneath the fibrous thyroid capsules at two or three points around the tumor, 0.1–0.2ml for each point, with 5-10mins wait before thyroidectomy (Table 3 in S1 File).

Lymph node removal

Analysis of the number of harvested lymph nodes were possible in 21 studies. A random-effects model was applied in this analysis (P<0.01, I = 97%). WMD analysis showed that the total nun class="Chemical">mber of harvested lymph nodes in the CN group was significantly higher than that in the control groups (WMD, 2.36; 95% CI, 1.40 to 3.32; P<0.01, Fig 3). The rate of LN black-stained varied between 73.3% and 95.3%. Eighteen out of the 21 studies provided adequate information for the analysis of LN metastatic rate (= number of tumors involved LN/ number of harvested LNs). A random-effects model was applied to assess heterogeneity (P<0.01, I = 96%). No difference was found between the CNs group and the Control group regarding LN metastatic rate (OR = 1.07, 95% CI = 0.75 to 1.51, P = 0.71, Fig 4).

Fig 3

Forest plots of the total number of lymph nodes harvested in groups.

(Experimental = Carbon nanoparticle group, Control = Blank or methylene blue group, Total = total number of LN harvested, Mean = the average number of LN harvested in each patient, SD = Standard deviation).

Fig 4

Forest plots of the total metastatic rate of lymph nodes harvested in groups.

(Experimental = Carbon nanoparticle group, Control = Blank or methylene blue group, Total = total number of LN, Events = the number of LN with metastasis).

Parathyroid gland protection

A fixed-effects model was used to analyze the data (P = 0.89, I = 0%). After analyzing the accidental removal of the PGs using 23 eligible studies, we found that the possibility of accidental PGs removal was decreased by 30% in CNs group compared with control group (OR = 0.28, 95% CI = 0.21 to 0.37, P<0.01, Fig 5).

Fig 5

Forest plots of accidental parathyroid removal rate in groups.

(Experimental = Carbon nanoparticle group, Control = Blank or methylene blue group, Total = The number of patient, Events = the number of parathyroid glands removed accidentally).

The overall postoperative transient hypoparathyroidism data were available in 16 studies. A random-effects model was used, P<0.04, I = 42%), and subgroup analysis of transient n class="Disease">hypocalcemia was possible 15 studies (A random-effects model was applied, P<0.08, I = 36%). Data analysis showed that the application of CN decreased the rate of both postoperative transient hypoparathyroidism and transient hypocalcemia equally by 46% (OR = 0.46, 95% CI = 0.33 to 0.64, P<0.01, Table 2, Fig 1 in S2 File; OR = 0.46, 95% CI = 0.33 to 0.65, P <0.01, Table 2, Fig 2 in S2 File). A random-effects model was applied to our data (P = 0.44, I = 0%), and 7 studies had a followed-up time of at least 6 months, thus the data of permanent hypocalcemia were analyzed. No significant difference concerning permanent hypocalcemia was found between the CNs group and control group (OR = 0.55, 95% CI = 0.09 to 3.43, P = 0.52, Table 2, Fig 3 in S2 File).

Table 2

Summary for subgroup analysis in the meta-analysis.

Subgroup analysis	Studies	Experiment (n)	Control (n)	OR	I2(%)	P
The number of LN harvested	21	1323	1572	(WMD)2.36	97	<0.01
The metastatic rate of LN	18	1208	1469	1.07	96	0.71
Accidental parathyroid gland removal	23	1326	1418	0.3	0	<0.01
Transient hypoparathyroidism.	16	1017	1275	0.46	42	<0.01
Transient hypocalcemia	15	881	1147	0.46	36	<0.01
Permanent hypocalcemia	7	590	840	0.50	0	0.45
Reoperation transient hypoparathyroidism	2	91	79	0.21	25	0.02
Reoperation accidental PGs removal	2	91	79	0.21	0	0.004

CNs application during reoperation

Two out the 25 included studies focused on reoperation thyroidectomy. Subgroup analysis of the data using a random-effects model demonstrated that the use of CNs significantly reduced the postoperative transient n class="Disease">hypoparathyroidism rate by 21% (OR = 0.21, 95% CI = 0.06 to 0.75, P = 0.02, Table 2, Fig 4 in S2 File), and the possibility of accidental PGs removal was decreased by 21% (OR = 0.21, 95% CI = 0.07 to 0.62 P = 0.004, Table 2, Fig 5 in S2 File).

Discussion

Protection of the PGs and the recurrent laryngeal nerves are important techniques related to thyroidectomy, particularly total thyroidectomy con class="Chemical">mbined with CND or reoperation thyroidectomy. Anatomically, the location of PGs, particularly the lower two, are variable, which results in a high risk of being accidentally removed during surgery [38]. Removal of all PGs inevitably leads to transient or permanent hypoparathyroidism and hypocalcemia, which has an adverse impact on the quality of life of the patients. Exposure and identification of the PGs intraoperatively facilitates preservation of the PG function. In addition, PTC has a trend of involving youngers and the 5-year survival rate is reported up to 98% [1]. As a result, omission of micro-metastases of the central compartmental lymph nodes may lead to reoperation, which may subsequently increase the incidence of complications and mortality. Therefore, effective techniques are urgently needed to improve the thoroughness of CND while preserving the PGs, particularly for reoperation.

CNs, approved by China Food and Drug Administration, have been efficiently attempted to assist lymph node dissection for n class="Disease">gastric cancer, breast cancer and other malignancies [7, 8]. In the recent years, CNs are used as a novel and safe surgical technique to trace the regional lymph nodes and localize the PG during thyroidectomy [23]. So far, no toxic side effects have been reported in humans. However, when the CNs were injected improperly and they spread out of the thyroid capsule, they may stain the surgical field, which makes surgical procedure more difficult [23]. In most of the included studies, the dose of CNs is 0.1 to 0.2 ml per point and 2–3 points around tumor within the thyroid capsule were recommended. After being properly injected, the CNs enter the lymphatic capillaries and stain lymph nodes in 10-15min, rather than PGs and the recurrent laryngeal nerve [10, 13, 18–20, 27, 32–37]. It is hypothesized that this technique facilitates intraoperative identification of LNs and improves thoroughness of LN dissection for thyroid carcinoma.

The current meta-analysis demonstrates that the total number of lymph node harvested in the Cn class="Chemical">N group was approximately 2.36 more than that in the control groups. But the ratio of metastatic lymph nodes has no significant difference between the two groups. The results were similar to previous studies. This denotes that CNs play a key role in accurate identification of the lymph nodes, but they cannot improve the detection of metastatic lymph nodes. The possible explanation is that CNs facilitate identification of the tiny LNs which are black dyed by both surgeons and pathologists. However, CNs have no tumor trophism and they dye both the normal LNs and the metastatic LNs black without preference [26, 35].

Hypoparathyroidism is a common complication after thyroid surgery, especially after reoperation.

It is a disorder characterized by hypocalcemia, low or inappropriately normal intact n class="Gene">parathyroid hormone (PTH) levels, and often hyperphosphatemia, which may be transient when it recovers within a few weeks or one month after thyroid surgery, or permanent when hypoparathyroidism persists for at least six months postoperatively. The incidence of transient hypoparathyroidism had been reported to be 20% to 60%, while that of permanent hypoparathyroidism was 0% to 7% [5, 6]. Our study revealed that application of CNs during thyroidectomy with or without CND reduces the incidence of accidental PG removal by approximately 30% and reduces the incidence of both postoperative transient hypoparathyroidism and transient hypocalcemia equally by 46%. However, there were still doubts about whether this technique leads to protection of long-term parathyroid function. In our subgroup analysis of permanent hypocalcemia using 7 studies who had a followed-up period of at least 6 months, no significant better PG function outcome was found in CNs group. We hypothesize that CNs facilitate identification of the PGs and lead to better protection of the glands’ structures and blood supply. However, the benefit still cannot reach a statistic significance base on the data of these 7 studies yet.

It is assumed that lymphatic tracer cannot accurately drains to the sentinel nodes, the first lymphatic station from the primary cancer in case of reoperation, because the local structures could possibly be destroyed in the initial surgery. Whether previous surgeries disturb the role of CNs in black-staining the lymph nodes and negative developing the PGs during thyroidectomy remains unknown. To the best of our knowledge, no previous meta-analysis concerned the outcome of the application of CNs for reoperation thyroidectomy has been reported so far. Our systemic review retrieved only 2 studies who focused on CNs application for reoperation thyroidectomy. These reports demonstrated CNs technique not only have a benefit of PGs identification but also decrease the incidence of postoperative transient hypoparathyroidism by 20% [26, 35]. We believe although the disturbance of the drainage routes of the CNs leads to failure of sentinel node localization, it does not impact their drainage to the regional LNs, which facilitates identification of the black-stained LN and negative developing of the PGs.

The current meta-analysis has some limitations and the results should be interpreted with caution. First, all the studies including in this meta-analysis were conduct only in China and the subjects were all Chinese. Second, long-term PG function outcome is difficult to figure out because most of the studies had a follow-up period of less than 6 months. Third, average Jadad Score and Newcastle-Ottawa score were only 3 and 7.4 respectively, which indicate the study design was of moderate quality. Thus, further research is needed to verify the conclusions of the current study.

Conclusions

This is the meta-analysis so far, firstly focused on the application of CNs during both initial thyroidectomy and reoperation thyroidectomy for n class="Chemical">N-ATC. This meta-analysis demonstrates that the application of CN for thyroidectomy improve the lymph node detection and PG protection not only for initial surgery but also reoperation.

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18 Nov 2019

PONE-D-19-26418

The Role of Carbon Nanoparticle in Lymph Node Detection and Parathyroid Gland Protection during Thyroidectomy- a Meta Analysis

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In addition, authors should note that their Figures 4-7 demonstrate that accidental parathyroid removal & hypo-parathyroidism is worse with CN (experimental) than with control. This is the opposite of what the authors report in the results and discussion and conclusion. Please explain. it is either a mistake in conclusion or a mistake in the way the treatment assignment variable direction is coded.

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Reviewer #1: The authors have performed a systematic review with meta-analysis of the use of carbon nanoparticles in thyroid surgery. There are several areas where this manuscript could be improved for clarity throughout. I would suggest re-review for English language. There are several grammatical errors and typos within the manuscript. Additionally, much of the methods and results are not clear in the manuscript and require additional description in the manuscript. With additional clarity of these issues, this could be an interesting paper.

Methods

1. Please clarity the diagnosis included in the paper. Thyroid carcinoma is a varied and diverse group of diseases, ranging from anaplastic to papillary, which in the recent ATA guidelines has been downgraded in many cases. It needs to be clear which disease you are examining in the review.

2. Why did you only include studies that include single medical team/surgeon? This seems like an odd inclusion criteria.

3. For your exclusion criteria, what did you include for "incomplete data"? You provide to description of what is considered complete data in your inclusion criteria. Do patient need to have follow up? Data on calcium levels? Of note, many of the included papers do not have simple data, like the average age. Why were these papers included while others were not?

4. In the abstract is it stated that only RCTs or non randomized CT are included however in the methods, it appears that retrospective trials were included. Can you please clarify this contradiction?

Results

1. Please note the results in the figure diagrams are unclear. I cannot confirm the number of studies included or excluded and for which reasons based on the formatting errors.

2. The actual patient population and surgeries performed in the respective studies in unclear - how many were reoperations? How many were original surgeries? This is important data for the reader to be able to see and assess

3. Examining the results and figure, there is a high degree of variability in the actual number of studies included. I can't seem to find a single figure that includes all 25 included papers. Many of the sub analyses have small sample sizes (2 papers), which is quite different that the 25 papers reported in the results. Each sub analysis should be well described in the methods and results section, with a clearly states nun class="Chemical">mber or papers included and sample size for that analysis. Without this, the results of your paper are presented in a misleading way, implying there is more power to you analysis than their actual is

Figures and tables

1. I would suggest editing these figures and tables for consistent text format and readability. The content is cut off in several of the images. Additionally, there are far too many figures and tables, with scattered data throughout that is not easier for the reader to assess and interpret for themselves.

**********

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

[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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

15 Jan 2020

Dear reviewers,

Thank you for your review of our manuscript: PONE-D-19-26418 - “The Role of n class="Chemical">Carbon Nanoparticle in Lymph Node Detection and Parathyroid Gland Protection during Thyroidectomy- a Meta Analysis”. We really appreciate the reviewers’ valuable comments. We are resubmitting a revised manuscript, per their suggestions. We describe below the changes we have made as well as our response to the reviewer’s comments. The main changes were highlighted in red.

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

Response:

The manuscript has been revised according to PLOS ONE's style requirements.

2. Please provide the full search strategy and full search terms used for at least one database used in the supplementary information. In addition, please provide additional details regarding the assessment of publication bias in your systematic review and meta-analysis. Specifically, please provide the funnel plots calculated from the Begg's test and/or the regression plots from the Egger's test.

Response:

The full search strategy and full search terms used has been listed in the Method section: page 4, line 69-72 . In addition, the funnel plots calculated from the Begg's test has been added in the text: page 7, line 125-126.

3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

Response:

Yes, I will provide the relevant information if needed.

Reviewers’ comments:

1. In addition, authors should note that their Figures 4-7 demonstrate that accidental parathyroid removal & hypo-parathyroidism is worse with CN (experimental) than with control. This is the opposite of what the authors report in the results and discussion and conclusion. Please explain. it is either a mistake in conclusion or a mistake in the way the treatment assignment variable direction is coded.

Response:

Thanks for your careful review. I made a mistake in the way the treatment assignment variable direction is coded, which have been corrected in the revised manuscript.(Modified at line 149, line 154, and line 163)

2. Also, authors should report the chance-corrected agreement (i.e. kappa) in the screening stages and the eventual final set of included studies.

Response:

The chance-corrected agreement has been added and inserted in page 6, line 117.

3. Please clarity the diagnosis included in the paper. Thyroid carcinoma is a varied and diverse group of diseases, ranging from anaplastic to papillary, which in the recent ATA guidelines has been downgraded in many cases. It needs to be clear which disease you are examining in the review.

Response:

Papillary thyroid carcinoma, follicular thyroid carcinoma, and medullary thyroid carcinoma comprising the subjects of the 25 studies included in the current meta-analysis. It’s impossible to distinguish them base on the data available. I combined these 3 pathologic types and termed as Non-anaplastic thyroid carcinoma, and make revision in the text where appropriate. The details were inserted in page 6, line 119-121.

4. Why did you only include studies that include single medical team/surgeon? This seems like an odd inclusion criteria.

Response:

I delete the criteria in manuscript. ( Modified in page 4, line 78-79.)

5. For your exclusion criteria, what did you include for "incomplete data"? You provide to description of what is considered complete data in your inclusion criteria. Do patients need to have follow up? Data on calcium levels? Of note, many of the included papers do not have simple data, like the average age. Why were these papers included while others were not?

Response:

Thanks for your critical review. “Incomplete data” is vague and inaccurate, so I deleted this exclusion criteria. (Modified in page 4, line 82-83.)

In addition, only 7 studies had a follow-up time of at least 6 months and they are eligible for the subgroup analysis of postoperative permanent hypocalcemia/hypoparathyroidism. I mentioned this in page 14, line 173.

6. In the abstract is it stated that only RCTs or non randomized CT are included however in the methods, it appears that retrospective trials were included. Can you please clarify this contradiction?

Response:

All the studies included in the meta-analsis were RCTs or Non-RCTs, and there was no retrospective trials included. I have corrected in the appropriate part of the text: page 5, line 98-101.

7. Please note the results in the figure diagrams are unclear. I cannot confirm the number of studies included or excluded and for which reasons based on the formatting errors.

Response:

The figures have been corrected and inserted in the appropriate place in the text.

8. The actual patient population and surgeries performed in the respective studies is unclear - how many were reoperations? How many were original surgeries? This is important data for the reader to be able to see and assess

Response:

I added the mentioned data in page 6, line 122-123.

9. Examining the results and figure, there is a high degree of variability in the actual number of studies included. I can't seem to find a single figure that includes all 25 included papers. Many of the sub analyses have small sample sizes (2 papers), which is quite different that the 25 papers reported in the results. Each sub analysis should be well described in the methods and results section, with a clearly states nun class="Chemical">mber or papers included and sample size for that analysis. Without this, the results of your paper are presented in a misleading way, implying there is more power to your analysis than their actual is

Response:

Thanks for your comment. In fact, studies included had different objectives and provided only their associate data. Therefore, there was no analysis including 25 studies. In addition, I add the data of number or papers included and sample size for each subgroup analysis according to your advice. (Modified at 112-185 )

10. I would suggest editing these figures and tables for consistent text format and readability. The content is cut off in several of the images. Additionally, there are far too many figures and tables, with scattered data throughout that is not easier for the reader to assess and interpret for themselves.

Answer: Thank you for your kindly advice. I summarized the result of each subgroup analysis in Table 2 for more intuitive.

We look forward to your acceptance.

Best regards,

Peng Hanwei.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

15 May 2020

PONE-D-19-26418R1

The Role of Carbon Nanoparticle in Lymph Node Detection and Parathyroid Gland Protection during Thyroidectomy for Non-Anaplastic Thyroid Carcinoma- a Meta-Analysis

PLOS ONE

Dear Prof. Peng,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS On class="Chemical">NE’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.

We would appreciate receiving your revised manuscript by Jun 29 2020 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Biniam Kidane

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

-Fig 1 is cut off. It needs to be fixed please.

-line 141-142 and Fig 3 are discordant: "WMD analysis showed that the total number of harvested lymph nodes in the Cn class="Chemical">N group was significantly higher than that in the control groups" however Fig 3 shows the opposite. Please address this.

-Supplemental figures 1 and 2 also show the opposite of your conclusions; they show that control performs better whereas your conclusions state that experimental (CN) performs better.

-Supplemental figure 3 and line 179-183: this is inappropriate use of fixed effects modeling. With only 2 studies dealing with heterogenous populations and also including comparisons with near-zero values, there is an unacceptably high risk of unstable estimates with fixed effects modeling. This should be done with random effects.

[Note: HTML markup is below. Please do not edit.]

[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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

14 Jun 2020

Dear reviewers,

Thank you for your review of our manuscript: PONE-D-19-26418 - “The Role of n class="Chemical">Carbon Nanoparticle in Lymph Node Detection and Parathyroid Gland Protection during Thyroidectomy- a Meta Analysis”. We really appreciate the reviewers’ valuable comments. We are resubmitting a revised manuscript, per their suggestions. We describe below the changes we have made as well as our responses to the reviewer’s comments. The main changes were highlighted in red.

1. Fig 1 is cut off. It needs to be fixed please.

Response:

We have fixed the Fig 1 in the manuscript. (Modified at line 124-125 )

2. Line 141-142 and Fig 3 are discordant: "WMD analysis showed that the total number of harvested lymph nodes in the Cn class="Chemical">N group was significantly higher than that in the control groups" however Fig 3 shows the opposite. Please address this.

Response：

Thanks for your sharp review. We made a mistake in the way the treatment assignment variable direction is coded, which have been corrected in the revised manuscript. (Fig3, Modified at line 147)

3. Supplemental figures 1 and 2 also show the opposite of your conclusions; they show that control performs better whereas your conclusions state that experimental (CN) performs better.

Response：

We are so sorry that we made the same mistakes in our first revision in the Supplemental fig 1 and fig 2. We have revised these data and check throughout the manuscript to confirm that there is no similar mistakes remained.

4. Supplemental figure 3 and line 179-183: this is inappropriate use of fixed effects modeling. With only 2 studies dealing with heterogenous populations and also including comparisons with near-zero values, there is an unacceptably high risk of unstable estimates with fixed effects modeling. This should be done with random effects.

Response：

Thank you for your kindly advise. Random effects have been used in Supplemental figure 3, figure 4, figure5 and the related text in the manuscript have been revised. (See line 32-33, 170, 173-174, 177-180, Table2)

Thank you for your consideration of our revised manuscript. We look forward to the re-review of our manuscript and are prepared to make additional revisions as required.

Yours Faithfully,

Hanwei Peng

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

20 Aug 2020

PONE-D-19-26418R2

The Role of Carbon Nanoparticle in Lymph Node Detection and Parathyroid Gland Protection during Thyroidectomy for Non-Anaplastic Thyroid Carcinoma- a Meta-Analysis

PLOS ONE

Dear Dr. Peng,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS On class="Chemical">NE’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.

There are still some issues raised by the reviewers and some additional edits that would aid the manuscript, especially improving the description of the CN technique in a more detailed manner.

Please submit your revised manuscript by Oct 04 2020 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 n class="Chemical">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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Claudio Andaloro

Academic Editor

PLOS ONE

[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 #2: (No Response)

Reviewer #3: (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 #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: I Don't Know

Reviewer #3: I Don't Know

**********

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 #2: Yes

Reviewer #3: 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 unan class="Chemical">mbiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: No

Reviewer #3: 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 #2: Major comments:

How exactly does using CN help identify parathyroid glands? Is it just that the parathyroid glands are not mistaken for lymph nodes since the lymph nodes would turn black? Are parathyroid glands commonly excised because they are thought to be lymph nodes or is it simply because they get stuck to the capsule? Please clarify specifically how the Cn class="Chemical">N would help identify parathyroid glands.

If the CN helps identify more lymph nodes but does NOT help identify more diseased lymph nodes, is it worth utilizing? What would be the benefit of identifying and excising more benign lymph nodes?

Minor comments:

Please include the pseudo confidence interval as part of the legend of Figure 2

There are a number of punctuation and grammatical errors throughout the manuscript, please review carefully and correct these

Reviewer #3: Introduction: Please clear the matter of the high percentages that you described as post surgical incidence of hypocalcemia and hypoparathyroidm: they are valid only in transient cases. n class="Disease">Permanent hypocalcemia or hypoparathyroidism are described in the literature usually in less than 2% of cases.

Please describe the CN technique more detailed.

Material: Please explain the reason for with you did consider only adolescents over 16 yers old and adults in your study.

Please define more clearly " Included patients with benign thyroid disease unable to be separated from N-ATC.

Results: Please describe the definition of transient respective;;y permanent hypocalcamie/hypoparathyroidism. Some studies use the PTH value less than 10 pg/mL, others less than 7 pg/mL. Some do state the permanent condition six months after the surgery, other use a different time interval. Please do comments which threshold and time interval were considered in the evaluated studies.

Discussions: Till this section there was do specific talk about transient versus permanent hypoparathyroidism. Also the number pf references used, when comparing the current results with the previous results is small and insuficient.

**********

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 #2: No

Reviewer #3: 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.

15 Sep 2020

Dear reviewers,

Thank you for your review of our manuscript: PONE-D-19-26418 - “The Role of n class="Chemical">Carbon Nanoparticle in Lymph Node Detection and Parathyroid Gland Protection during Thyroidectomy- a Meta Analysis”. We really appreciate the reviewers’ valuable comments. We are resubmitting a revised manuscript, per their suggestions. We describe below the changes we have made as well as our responses to the reviewer’s comments. The main changes were highlighted in red.

Q1: How exactly does using CN help identify parathyroid glands? Is it just that the parathyroid glands are not mistaken for lymph nodes since the lymph nodes would turn black? Are parathyroid glands commonly excised because they are thought to be lymph nodes or is it simply because they get stuck to the capsule? Please clarify specifically how the Cn class="Chemical">N would help identify parathyroid glands.

Response: CN technique was described in detail in introdunction. (Modified at line 54-59)

Q2: Please include the pseudo confidence interval as part of the legend of Figure 2

Response: We describe the 95% confidence interval in results. (Modified at line 127)

Q3: There are a number of punctuation and grammatical errors throughout the manuscript, please review carefully and correct these

Response: Thanks for your sharp review. We made a mistake in punctuation and grammatical errors, which have been corrected in the revised manuscript. (Modified at line 108, 115-117, 138, 153, 160, 174, 184, 254)

Q4: Please clear the matter of the high percentages that you described as post surgical incidence of hypocalcemia and hypoparathyroidism: they are valid only in transient cases. Permanent hypocalcemia or hypoparathyroidism are described in the literature usually in less than 2% of cases.

Response: A more accurate expression was modified as your kindly advise. (Modified at line 46-47)

Q5: Please describe the CN technique more detailed.

Response: The same as Q1.

Q6: Please explain the reason for with you did consider only adolescents over 16 years old and adults in your study.

Response: To reduce the publication bias because most of the studies included only report the cases in adult or adolescents over 16 years old. (Modified at line 87-89)

Q7: Please define more clearly " Included patients with benign thyroid disease unable to be separated from N-ATC.

Response: Thank you for your kindly advise. We have modified it more clearly in exclusion criteria. (Modified at line 86-87)

Q8: Please describe the definition of transient respectively permanent hypocalcamie/hypoparathyroidism. Some studies use the PTH value less than 10 pg/mL, others less than 7 pg/mL. Some do state the permanent condition six months after the surgery, other use a different time interval. Please do comments which threshold and time interval were considered in the evaluated studies.

Response: We describe the definition of hypoparathyroidism and hypocalcemia in Discussion. (Modified at line 224-228)

Actually, most studies included in meta-analysis report the cases of hypoparathyroidism and hypocalcemia, but not the value of PTH in detail.

Thank you for your consideration of our revised manuscript. We look forward to the re-review of our manuscript and are prepared to make additional revisions as required.

Yours Faithfully,

Hanwei Peng

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

30 Sep 2020

PONE-D-19-26418R3

The Role of Carbon Nanoparticle in Lymph Node Detection and Parathyroid Gland Protection during Thyroidectomy for Non-Anaplastic Thyroid Carcinoma- a Meta-Analysis

PLOS ONE

Dear Dr. Peng,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS On class="Chemical">NE’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.

Dear Authors, You did not answer some of previous points raised by reviewer. Please pay attention to this matter. Moreover, a reviewer raised the following question "If the CN helps identify more lymph nodes but does n class="Chemical">NOT help identify more diseased lymph nodes, is it worth utilizing?  What would be the benefit of identifying and excising more benign lymph nodes?" It requires an explanation

the question about hypoparathyroidism definition is still not addressed,

Please submit your revised manuscript by Nov 14 2020 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 n class="Chemical">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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Claudio Andaloro

Academic Editor

PLOS ONE

[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 #3: (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 #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: 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 #3: 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 unan class="Chemical">mbiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: 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 #3: The question about hypoparathyroidism definition is still not addressed. Row 224-228 from discussions o not contain informations about n class="Disease">hypoparathyroidism and hypocalcemia - transient versus permanent. This does not play a major role in the manuscript results but does really influence the motivation and the importance of the technique described in the study.

**********

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 #3: 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.

3 Oct 2020

Dear reviewers,

Thank you for your prompt re-review of our manuscript: PONE-D-19-26418 - “The Role of Carbon Nanoparticle in Lymph Node Detection and Parathyroid Gland Protection during Thyroidectomy- a Meta Analysis”. After discussing the comments raised by the reviewer, we made changes per the reviewers’ comments. We are now resubmitting a revised manuscript to correct the errors together with this cover letter for further evaluation.

Q1: Hypoparathyroidism definition is still not addressed.

Response: We addressed the hypoparathyroidism definition as well as diagnostic criteria in the current study in the manuscript. (See line 104-110)

Q2: If the CN helps identify more lymph nodes but does NOT help identify more diseased lymph nodes, is it worth utilizing? What would be the benefit of identifying and excising more benign lymph nodes?

Response: Thanks for your question. This is really a crucial point that readers may concern. In fact, in the results section, we stated " WMD analysis showed that the total number of harvested lymph nodes in the Cn class="Chemical">N group was significantly higher than that in the control groups (WMD, 2.36; 95% CI, 1.40 to 3.32; P＜0.01, Fig 3).” (see line 157-159) and “No difference was found between the CNs group and the Control group regarding LN metastatic rate (OR = 1.07, 95% CI = 0.75 to 1.51, P=0.71, Fig 4).” (See line 163-164). These denoted that the numbers of both diseased lymph nodes and benign lymph nodes were higher in the CN group than that in the control group. Therefore, we advocate that this technique is worth utilizing. We revised a sentence in the discussion section (line 229-231) and added a sentence in this paragraph (line 234-237) to express our points of view.

Thank you for your consideration of our re-revised manuscript. We look forward to further review of our manuscript and are prepared to make additional revisions as required.

Yours Faithfully,

Hanwei Peng

Submitted filename: Response to Reviewers.docx

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7 Oct 2020

The Role of Carbon Nanoparticle in Lymph Node Detection and Parathyroid Gland Protection during Thyroidectomy for Non-Anaplastic Thyroid Carcinoma- a Meta-Analysis

PONE-D-19-26418R4

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15 Oct 2020

PONE-D-19-26418R4

The Role of Carbon Nanoparticle in Lymph Node Detection and Parathyroid Gland Protection during Thyroidectomy for Non-Anaplastic Thyroid Carcinoma- a Meta-Analysis

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