Clinical value of different anti-D immunoglobulin strategies for preventing Rh hemolytic disease of the fetus and newborn: A network meta-analysis.

BACKGROUND: Several anti-D immunoglobulin strategies exist for preventing Rh hemolytic disease of the fetus and newborn. This study systematically assessed the clinical value of those therapeutic strategies.
METHODS: The Web of Science, PubMed, EMBASE, China National Knowledge Infrastructure (CNKI) and Wanfang databases were searched for eligible studies that evaluated the value of different anti-D immunoglobulin strategies in preventing maternal anti-D antibody sensitization. Combined odds ratios (ORs) and their 95% confidence intervals (CIs) were calculated. The network meta-analysis was conducted using Stata 14.2 and WinBUGS 1.4.3 software.
RESULTS: Twenty-four original studies involving 64860 patients were included. Among all therapeutic measures, injecting 300 μg anti-D immunoglobulin at 28 and 34 gestational weeks (antenatal 5/E) appeared to be the most effective measure for preventing maternal antibody sensitization (surface under the cumulative ranking curve [SUCRA] = 96.8%), while a single injection at 28 gestational weeks (SUCRA = 89.2%) was the second most effective. Administering no injection or a placebo (SUCRA = 0.0%) was the least effective intervention measure.
CONCLUSION: Among the therapeutic measures, antenatal 5/E appeared to be the best method for reducing the positive incidence of anti-D antibodies in the maternal serum; thus, it may be the most effective treatment for preventing fetal hemolytic disease.

Introduction

Hemolytic disease of the fetus and newborn (HDFN) can lead to fetal hemolytic anemia, jaundice, intellectual retardation, premature birth, abortion and stillbirth. HDFN is an important cause of neonatal morbidity and death [1-3]. To reduce the incidence of HDFN and mortality among fetuses and neonates, anti-D immunoglobulin has been tested in clinical trials in the United Kingdom and United States since the 1960s. Anti-D immunoglobulin has been used to prevent postpartum disease in RhD-negative women and has greatly reduced HDFN-related morbidity as well as fetal and neonatal mortality [4]. The anti-D antibody production rate in the maternal serum after immunization has also decreased significantly from 12–13% to approximately 1.2%. Prenatal prophylaxis with anti-D immunoglobulin in Rh-negative mothers can further reduce anti-D antibody production in maternal sera, which has further reduced the incidence of hemolytic diseases in fetuses and newborns since 1980 [5-11].

However, multiple countries recommend various anti-D immunoglobulin injection schemes, and no consensus has been reached on the use of anti-D immunoglobulin worldwide. Routine prenatal anti-D prophylaxis (RAADP) is recommended in some countries, while postpartum anti-D immunoglobulin injections are still used in other countries. Furthermore, the injection dose differs in some countries due to the lack of available immunoglobulin. Lee et al. suggested that administering low doses of anti-D immunoglobulin (50 μg) provided no benefit [12]. However, excessive doses may increase the risk of allergic reactions and infectious diseases.

Until now, no meta-analysis has been conducted to evaluate the association between anti-D antibody production rates in the maternal serum and various therapeutic strategies regarding anti-D immunoglobulin. We conducted this study to systematically evaluate the preventive effects of anti-D immunoglobulin on HDFN via network meta-analysis based on all related published data.

Material and methods

Search strategy

A comprehensive search strategy was employed to search the PubMed, EMBASE, Web of Science, China National Knowledge Infrastructure (CNKI) and Wanfang databases. The latest search was conducted on 7 July 2019. The following keywords were used in accordance with the search strategy: “RhD-negative” OR “D-negative” OR “Rh(D) Immuno-Globulin” OR “Anti-D Immunoglobulin” OR “Anti-D Antibody” OR “the hemolytic disease of the newborn” OR “haemolytic disease of the newborn” OR “HDFN” et al.

Inclusion and exclusion criteria

The inclusion criteria were as follows: 1) randomized controlled studies on administering anti-D immunoglobulin injections to RhD-negative pregnant women; 2) Rh-positive fetuses in intrauterine pregnancies of Rh-negative pregnant women; 3) reported dose and frequency of anti-D immunoglobulin injections; and 4) reported positive incidence of anti-D antibody in postpartum mothers. Duplications, reviews, case reports, conference abstracts, and studies without useful data were excluded.

Study selection

Two authors (XXH and FQR) screened the abstracts and titles of eligible publications and judged whether to further review the full text independently. We contacted the trial author when full texts were unavailable. Full texts were independently reviewed by XXH and ZD. In the case of any disagreement during the selection process, the decisions were obtained after group discussion. Finally, we used flow chart to show the total number of retrieved references and the number of included and excluded studies.

Data extraction

Two investigators collected data independently in accordance with predesigned tables, which included the name of the first author, publication year, country, sample size, intervention measures, control measures, and anti-D antibody production rate in the maternal serum.

Two researchers independently assessed the quality of all included studies using the Newcastle-Ottawa quality assessment scale (NOS). This method comprised three parameters of quality: selection (score: 0–4), comparability (score: 0–2), and outcome assessment (score: 0–3), with total scores ranging from 0–9. NOS scores >6 were considered to indicate high-quality studies.

Statistical analysis

Stata statistical software, version 14.2 and WinBUGS 1.4.3 were applied to analyze the relationship between anti-D antibody production rates in the maternal serum and various anti-D immunoglobulin injection regimens. The random-effects model with vague priors for multiarm trials was used. The model parameters were estimated using the Markov chain Monte Carlo method of Gibbs sampling. The results are reported as the odds ratio (OR) and standardized mean difference(SMD) with 95% confidence intervals (CIs). To evaluate the inconsistency between direct and indirect effect estimates for the same comparisons, we evaluated each closed loop in the network. In a closed loop, we employed the inconsistency factor (IF) to evaluate heterogeneity among the included studies. Node analysis showed that the direct and indirect comparisons of each node did not differ (P>0.05), and the consistency model was used for convergence. To rank the treatments, we used the surface under the cumulative ranking probabilities (SUCRA). A comparison-adjusted funnel plot was used to assess the presence of small-study effect and publication bias.

Results

Characteristics of eligible studies

Fig 1 shows the literature retrieval procedure. After further discussing and considering the retrieved articles, 24 eligible articles[5,8,10-31] were ultimately identified, and 64860 patients were included in this network meta-analysis, with an average sample size of 2702.5 (range 54–12825). Among those studies, nine intervention-measure dosages for administering anti-D immunoglobulin were analyzed: 250 μg within 28 gestational weeks (antenatal 1/A), 300 μg within 28 gestational weeks (antenatal 2/B), 50 μg within 28 and 34 gestational weeks (antenatal 3/C), 100 μg between 28 and 34 gestational weeks (antenatal 4/D), 300 μg between 28 and 34 gestational weeks (antenatal 5/E), placebo or blank control group (blank/F),100 μg ≤ dosage < 200 μg within 72 h postpartum (postnatal 1/G), 200 μg ≤ dosage < 300 μg within 72 h postpartum (postnatal 2/H), and 300 μg ≤ dosage < 500 μg within 72 h postpartum (postnatal 3/I). All articles were written in good-quality English. summarizes the main characteristics of all included cohort studies. Table 2 has the treatment abbreviations.

Fig 1

Flow diagram of included studies.

Table 2

Treatment abbreviations.

Full name	Abbreviations
Administered 250 μg within 28 gestational weeks	antenatal 1/A
Administered 300 μg within 28 gestational weeks	antenatal 2/B
Administered 50 μg within 28 and 34 gestational weeks	antenatal 3/C
Administered 100 μg between 28 and 34 gestational weeks	antenatal 4/D
Administered 300 μg between 28 and 34 gestational weeks	antenatal 5/E
Placebo or blank control group	blank/F
Administered 100 μg ≤ dosage < 200 μg within 72 h postpartum	postnatal 1/G
Administered 200 μg ≤ dosage < 300 μg within 72 h postpartum	postnatal 2/H
Administered 300 μg ≤ dosage < 500 μg within 72 h postpartum	postnatal 3/I

Network meta-analysis results

Network relationship and inconsistency test. In this network meta-analysis, the association between various anti-D immunoglobulin strategies and their clinical value in HDFN was analyzed for 24 cohort studies comprising 64860 patients and nine treatment measures. Fig 2 shows the network relationship among the different treatment measures. Nodes are proportional to the number of patients included in the corresponding treatments, and edges are weighted according to the number of studies included in the respective comparisons. Nine treatment measures formed three triangles and two quadrilateral closed loops. The inconsistency factor was obtained under the inconsistency model using Gemetc software. Fig 3 shows the inconsistency plot used to identify heterogeneity among studies in the closed loop of this network meta-analysis. Three triangular loops and two quadratic loops are present in the network meta-analysis. The results showed that the inconsistency factor (IF) was 0.11 ≤ 2.13, and the 95% confidence interval (CI) contained 0, suggesting that statistical inconsistency may not exist among these five closed loops (Fig 3). Furthermore, node analysis was used to analyze the differences between direct and indirect comparisons among treatment measures (Table 3). P>0.05 indicates that no statistical inconsistencies were observed, suggesting that a network meta-analysis can be accomplished by directly or indirectly comparing different therapeutic measures. Thus, data on various treatment measures can be converged using consistency models.

Fig 2

Network relationship of the included treatment measures.

Fig 3

Inconsistency test.

Table 3

Node analysis.

Node analysis results
Side	Direct		Indirect		Difference		P>z
	Coef.	Std. Err.	Coef.	Std. Err.	Coef.	Std. Err.
A H	0.863	0.429	-1.012	42.161	1.874	42.163	0.965
B E	-2.123	2.001	5.147	3110.084	-7.269	3110.085	0.998
B H	2.605	1.47	3.023	0.637	-0.417	1.602	0.794
B I	1.852	0.218	1.435	1.587	0.417	1.602	0.794
C F	1.899	0.239	6.653	203.357	-4.754	203.357	0.981
D G	1.441	0.314	1.876	0.905	-0.435	0.958	0.65
D I	0.989	0.297	0.555	0.911	0.435	0.958	0.65
F G	-3.269	0.974	-2.678	0.465	-0.591	1.061	0.578
F H	-1.791	0.631	-3.259	0.998	1.468	1.181	0.214
F I	-3.377	0.237	-2.351	1.068	-1.026	1.102	0.352
G H	-0.773	1.229	1.039	0.724	-1.812	1.426	0.204
G I	-0.339	1.418	-0.556	0.401	0.218	1.473	0.882

Results of the Bayesian network meta-analysis. According to the network of comparisons (Table 4), the antenatal 5/E, antenatal 2/B, antenatal 4/D, antenatal 1/A, postnatal 3/I, postnatal 2/H, and antenatal 3/C regimens significantly reduced the serum anti-D antibody-positive rates compared with that of the blank/F regimen alone (antenatal 5/E vs. blank/F: OR = 0.00, 95% CI = 0.00–0.04; antenatal 2/B vs. blank/F: OR = 0.01, 95% CI = 0.00–0.01; antenatal 4/D vs. blank/F: OR = 0.01, 95% CI = 0.01–0.03; antenatal 1/A vs. blank/F: OR = 0.05, 95% CI = 0.01–0.18; postnatal 3/I vs. blank/F: OR = 0.04, 95% CI = 0.02–0.06, P<0.05; postnatal 2/H vs. blank/F: OR = 0.11, 95% CI = 0.04–0.31; antenatal 3/C vs. blank/F: OR = 0.15, 95% CI = 0.09–0.24; all P<0.05). This indicated that injections of anti-D immunoglobulin, whether before or after delivery, significantly reduced the incidence of maternal serum that was positive for anti-D antibody in Rh-negative mothers with Rh-positive fetuses. Moreover, antenatal 5/E, antenatal 2/B, antenatal 4/D, antenatal 1/A and postnatal 3/I were the most effective treatment measures for reducing the incidence of maternal anti-D antibody positivity (antenatal 5/E vs. antenatal 2/B: OR = 0.12, 95% CI = 0.00–6.05; antenatal 2/B vs. antenatal 4/D: OR = 0.41, 95% CI = 0.20–0.82, P<0.05; antenatal 4/D vs. postnatal 3/I: OR = 0.39, 95% CI = 0.22–0.67, P<0.05; postnatal 3/I vs. antenatal 1/A: OR = 0.78, 95% CI = 0.20–3.08, P>0.05). Similarly, we used a forest plot to represent the network meta-analysis results (Fig 4). All immunization schemes were significantly more effective than was the blank control scheme (P<0.05).

Table 4

Network meta-analysis result.

postnatal 3/I	3.04	1.72 (0.81,3.66)	27.71 (17.66,43.50)	0.02 (0.00,0.98)	0.39 (0.22,0.67)	4.15	0.16 (0.10,0.24)	1.28 (0.32,5.08)
	(1.02,9.03)					(2.17,7.94)
0.33 * (0.11,0.98)	postnatal 2/H	0.56 (0.17,1.92)	9.11 (3.20,25.92)	0.01 (0.00,0.37)	0.13 (0.04,0.41)	1.36	0.05 (0.02,0.16)	0.42 (0.18,0.98)
						(0.43,4.29)
0.58 (0.27,1.24)	1.77	postnatal 1/G	16.14 (7.00,37.22)	0.01 (0.00,0.61)	0.23 (0.13,0.40)	2.42	0.09 (0.04,0.22)	0.75 (0.17,3.30)
	(0.52,6.02)					(0.93,6.29)
0.04 * (0.02,0.06)	0.11*	0.06 * (0.03,0.14)	Blank/F	0.00 (0.00,0.04)	0.01 (0.01,0.03)	0.15	0.01 (0.00,0.01)	0.05 (0.01,0.18)
	(0.04,0.31)					(0.09,0.24)
52.84 * (1.02,2730.18)	160.72 * (2.70,9562.99)	90.71 * (1.64,5031.73)	1464.44 * (27.65,77566.59)	antenatal 5/E	20.48 (0.38,1099.57)	219.16 (4.03,11931.17)	8.35 (0.17,421.95)	67.86 (1.05,4398.56)
2.58 * (1.48,4.48)	7.85 * (2.44,25.26)	4.43 * (2.48,7.92)	71.49 * (35.95,142.18)	0.05 (0.00,2.62)	antenatal 4/D	10.70 (4.66,24.57)	0.41 (0.20,0.82)	3.31 (0.78,13.98)
0.24 * (0.13,0.46)	0.73	0.41 (0.16,1.08)	6.68 *(4.19,10.67)	0.00 * (0.00,0.25)	0.09 * (0.04,0.21)	antenatal 3/C	0.04 (0.02,0.08)	0.31 (0.07,1.28)
	(0.23,2.31)
6.33 * (4.15,9.65)	19.24 * (6.12,60.49)	10.86 * (4.58,25.73)	175.29 * (94.99,323.46)	0.12 (0.00,6.05)	2.45 * (1.22,4.91)	26.23 * (12.14,56.70)	antenatal 2/B	8.12 (1.96,33.64)
0.78 (0.20,3.08)	2.37*	1.34 (0.30,5.89)	21.58 * (5.64,82.53)	0.01 * (0.00,0.96)	0.30 (0.07,1.27)	3.23	0.12 (0.03,0.51)	antenatal 1/A
	(1.02,5.49)					(0.78,13.37)

* indicates a significant difference in the data (P<0.05).

Fig 4

Forest plot of the network meta-analysis.

Rank probability. Injecting 300 μg of anti-D immunoglobulin at 28 and 34 gestational weeks (antenatal 5/E) was the most effective treatment (surface under the cumulative ranking curve [SUCRA] = 96.8%; Fig 5), and administering 300 μg within 28 gestational weeks (antenatal 2/B) was the second most effective (SUCRA = 89.2%). Administering no injection or a placebo was the least effective (SUCRA = 0.0%).

Fig 5

SUCRA for preventing maternal antibody sensitization.

Assessment of publication bias and small-sample effect detection. Fig 6 shows the comparison-correction funnel plots of the included comparison. The funnel diagram is basically symmetric, and the regression line is less tilted; therefore, this study may have a small sample effect and slight publication bias.

Fig 6

Correction funnel plot.

Discussion

In 2012, the National Institute of Health and Clinical Optimization (NICE) proposed that preventing maternal antibody sensitization via routine prenatal anti-D prophylaxis (RAADP) is a cost-effective method (http://www.nice.org.uk/). The British Committee for Standards in Haematology (BCSH) published the latest guidelines in 2014, recommending the use of anti-D immunoglobulin to prevent haemolytic disease of the fetus and newborn [32]. These guidelines recommend that RAADP be performed in RhD-negative pregnant women in their third trimester of pregnancy. RAADP includes the following regimens: a single dose of 300 μg (1500 IU) anti-D immunoglobulin between 28 and 30 gestational weeks or a two-dose regimen of 100 μg (500 IU) anti-D immunoglobulin at 28 and 34 gestational weeks. A Kleihauer-Betke test should be performed after delivery to estimate whether fetomaternal hemorrhaging exceeded 4 ml, then another 100ug(500 IU) should be administered within 72 hours of delivery. In 2017, the American College of Obstetricians and Gynecologists (ACOG)[33] recommends prophylactic anti-D immune globulin to unsensitized Rh D-negative women at 28 weeks of gestation. After birth, if the baby is Rh D positive, these mothers should receive anti-D immune globulin within 72 hours of birth.

However, an observational study noted that compliance with the single injection regimen was better than that with the two-injection regimen[34]. A single injection may also reduce costs. No evidence exists to assess the efficacy of these therapeutic strategies[32].

Therefore, we conducted a network meta-analysis comparing multiple treatment measures. The results revealed that the antenatal 5/E, antenatal 2/B, antenatal 4/D, antenatal 1/A, postnatal 3/I, postnatal 2/H and antenatal 3/C regimens significantly reduced serum anti-D antibody positive rates compared with that of the blank/F regimen alone, indicating that anti-D immunoglobulin immunotherapy, whether administered before or after delivery, significantly reduced the incidence of maternal serum anti-D antibody positivity in Rh-negative mothers with Rh-positive fetuses. Moreover, antenatal 5, antenatal 2, antenatal 4, antenatal 1 and postnatal 3 were the most effective treatment measures for reducing the incidence of maternal anti-D antibody positivity. Therapeutic regimens antenatal 5 and antenatal 2 were likely the most effective regimens for preventing hemolytic diseases in fetuses and newborns.

The SUCRA for preventing maternal antibody sensitization indicated that the 300-μg anti-D immunoglobulin injection at 28 and 34 gestational weeks (antenatal 5/E) was likely the most effective regimen (SUCRA = 96.8%), and administering 300 μg within 28 gestational weeks (antenatal 2/B) was likely the second most effective (SUCRA = 89.2%). Administering no injection or a placebo was the least effective regimen (SUCRA = 0.0%). The anti-D immunoglobulin mechanism of action, which is closely related to the drug duration and dose, may explain these results. Anti-D immunoglobulin is extracted from the serum and used to prevent neonatal hemolysis.

RhD-positive red blood cells (containing the D antigen) from the fetus stimulate antibody production in RhD-negative mothers. During pregnancy and delivery of the first RhD-positive fetus to RhD-negative mothers, the red blood cells of the RhD-positive fetuses enter the RhD-negative mothers and stimulate the mothers to produce IgG anti-D antibodies. When an RhD-negative mother later carries an RhD-positive fetus, the antibodies in the maternal serum enter the fetal blood circulation via the placental barrier and can cause neonatal hemolysis.

However, during the pregnancy with the first RhD-positive fetus, or within 72 hours after delivery, RhD-negative mothers can be intramuscularly injected with 300 μg anti-D immunoglobulin, which can bind to the D antigen leaked into the mother's serum and desensitize it, thus blocking anti-D antibody production in the mother's serum. Anti-D immunoglobulin had no significant preventive effect on mothers who had already produced anti-D antibodies.

Twenty-five micrograms (125 IU) of anti-D immunoglobulin can typically protect against a fetal-maternal hemorrhage (FMH) of approximately 1–2 ml of blood. Therefore, 100 μg (500 IU) of anti-D antibody can prevent an FMH of approximately 8 ml, and 300 μg can prevent an FMH of approximately 30 ml. An FMH of greater than 30 ml is uncommon [35]. However, pharmacokinetic studies have shown that anti-RhD levels vary among patients, and some may have insufficient anti-RhD levels during childbirth [36]. A single injection of 300 μg anti-D immunoglobulin maintained a high immunopreventive effect for approximately 12 weeks. Bowman et al. [37]suggested that women who failed to give birth within 12 weeks after receiving the prenatal doses should receive a second dose of anti-D immunoglobulin to maintain the immunopreventive effect.

Routine prenatal prophylaxis with anti-D immunoglobulin is unlikely to benefit the current pregnancy or improve pregnancy outcomes, but it can reduce the anti-D antibody production during subsequent pregnancies. Chilcott et al.[38]noted that routine anti-D immunoglobulin injections should prevent future hemolytic diseases in infants. In many countries, including the United Kingdom and Australia, the guidelines recommend routine universal prenatal anti-D immunoglobulin prevention (http://www.ranzcog.edu.au/ and http://www.rcog.org.uk/). The incidence of D-negative individuals varies among ethnic groups, with the highest being in Basques (30% -35%), followed by North American and European Caucasians (15%) [38]. In China, RhD-negative individuals constitute approximately 0.3% of the population [39]. Routine use of anti-D immunoglobulin is the main method of decreasing the erythrocyte alloimmunity ratio.

Conclusions

Several limitations must be considered when interpreting the results of this meta-analysis. First, the literature included in this study spanned a long time period, and the titer or quality of anti-D immunoglobulin varies over time, which may affect the outcome. Second, the recruited participants were all from western countries, and no studies could be found regarding Asians and anti-D immunoglobulin. This might limit the application of our conclusions, and research on other races should be conducted.

In conclusion, this study showed that the current first-line recommendation is two 300-μg prenatal immunizations at 28 and 34 gestational weeks. If the anti-D immunoglobulin supply is inadequate, the second alternative should be a single 300-μg prenatal immunization at 28 gestational weeks.

PRISMA NMA checklist of Items to include when reporting a systematic review involving a network meta-analysis.

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The raw data of all included studies.

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28 Jan 2020

PONE-D-19-24055

Clinical value of different anti-D immunoglobulin strategies for preventing Rh hemolytic disease of the fetus and newborn: A network meta-analysis

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Reviewer #1: The authors investigated the various anti-d immunoglobulin strategies used for prevention of Rh hemolytic disease of the fetus and newborn using a network meta-analysis. Of the nine therapeutic measures studied, they found that giving 300 µg anti-D immunoglobulin at 28 and 34 weeks of gestation was the most effective measure for preventing maternal antibody sensitization, followed by a single injection at 28 weeks of gestation. This is interesting work clearly written.

Reviewer’s comments;

1. Page 3 (Results, last sentence): Suggest adding - Table 2 has the treatment abbreviations.

2. Page 5 (Table 2): This table was incorrectly labeled as Table 3. Please correct.

3. Page 9 (Discussion, middle of first paragraph): Suggest adding 100 µg to the sentence that states that …then another 500 IU should be administered…

4. Page 9 (Discussion, first paragraph): ACOG is the American College of Obstetricians and Gynecologists. Please correct.

5. Page 9 (Discussion, first paragraph): The sentence …(ACOG) recommended a single injection of 300 µg anti-D immunoglobulin at 28 weeks of pregnancy with an RhD–positive baby, then another injection of 300 µg again after birth… should be corrected as it is confusing as written. ACOG recommends prophylactic anti-D immune globulin to unsensitized Rh D-negative women at 28 weeks of gestation. After birth, if the baby is Rh D positive, these mothers should receive anti-D immune globulin within 72 hours of birth.

6. Page 10 (Discussion, top paragraph, first line): The area in parenthesis after …anti-D antibody positive rates compared with that of the blank/F regimen alone… can be deleted as it already appears in the Results section.

7. Page 10 (Discussion, top paragraph, line 10): Similarly as in #6 above, the area in parenthesis after …incidence of maternal anti-D positivity…can be deleted.

8. Page 11 (Discussion, next to last paragraph, last four lines): Please clarify what you mean by …but few studies have focused on the first fetus born to RhD-negative mothers. Supply references of the few studies. Also, please reference the last sentence …Some studies have also found that using anti-D immunoglobulin may cause hemolysis in fetuses during pregnancy.

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1 Feb 2020

Dear reviewers:

I am very grateful to your comments for the manuscript. According with your advice, we amended the relevant part in manuscript. Some of your questions were answered below.

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The authors’ answer: the manuscript has been revised as PLOS ONE's style requirements

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The authors’ answer: The new Figure 2 has been repalced.

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The authors’ answer: The tables has been inclued in the manuscript.There is no supplementary tables.

4. We note you have included two different tables in your manuscript labelled as Table 3; 'Table 3 Treatment abbreviations' and 'Table 3 Network meta-analysis result'. Please review these and ensure that you refer to all tables in the text of your manuscript so that they can be separately identified; if accepted, production will need this reference to link the reader to each Table.

The authors’ answer: Table label has been modified .Table 3 Treatment abbreviations has been replaced by Table 2 Treatment abbreviations.

Review Comments to the Author

Reviewer #1: The authors investigated the various anti-d immunoglobulin strategies used for prevention of Rh hemolytic disease of the fetus and newborn using a network meta-analysis. Of the nine therapeutic measures studied, they found that giving 300 µg anti-D immunoglobulin at 28 and 34 weeks of gestation was the most effective measure for preventing maternal antibody sensitization, followed by a single injection at 28 weeks of gestation. This is interesting work clearly written.

Reviewer’s comments;

1. Page 3 (Results, last sentence): Suggest adding - Table 2 has the treatment abbreviations.

The authors’ answer: The sentence has been added.

2. Page 5 (Table 2): This table was incorrectly labeled as Table 3. Please correct.

The authors’ answer: The Table label has been corrected.

3. Page 9 (Discussion, middle of first paragraph): Suggest adding 100 µg to the sentence that states that …then another 500 IU should be administered…

The authors’ answer: The “100 µg” has been added in the sentence.

4. Page 9 (Discussion, first paragraph): ACOG is the American College of Obstetricians and Gynecologists. Please correct.

The authors’ answer: “American Association of Obstetricians and Gynecologists” has been replaced by “American College of Obstetricians and Gynecologists”.

5. Page 9 (Discussion, first paragraph): The sentence …(ACOG) recommended a single injection of 300 µg anti-D immunoglobulin at 28 weeks of pregnancy with an RhD–positive baby, then another injection of 300 µg again after birth… should be corrected as it is confusing as written. ACOG recommends prophylactic anti-D immune globulin to unsensitized Rh D-negative women at 28 weeks of gestation. After birth, if the baby is Rh D positive, these mothers should receive anti-D immune globulin within 72 hours of birth.

The authors’ answer: The confusing sentence has been revised.

6. Page 10 (Discussion, top paragraph, first line): The area in parenthesis after …anti-D antibody positive rates compared with that of the blank/F regimen alone… can be deleted as it already appears in the Results section.

The authors’ answer: The area in parenthesis has been deleted.

7. Page 10 (Discussion, top paragraph, line 10): Similarly as in #6 above, the area in parenthesis after …incidence of maternal anti-D positivity…can be deleted.

The authors’ answer: The area in parenthesis has been deleted.

8. Page 11 (Discussion, next to last paragraph, last four lines): Please clarify what you mean by …but few studies have focused on the first fetus born to RhD-negative mothers. Supply references of the few studies. Also, please reference the last sentence …Some studies have also found that using anti-D immunoglobulin may cause hemolysis in fetuses during pregnancy.

The authors’ answer: Confused sentences have been deleted.

--------------------------------------------------------------------

We acknowledge the reviewer’s comments and suggestions very much, which are valuable in improving the quality of our manuscript.

Sincerely yours,

Xiaohui Xie1;Yi Zhang2

1.Department of Obstetrics and Gynecology，the First People's Hospital of Neijiang,Neijiang 641000,Sichuan Province, P. R. China2．Department of General Surgery，the First People's Hospital of Neijiang,Neijiang 641000,Sichuan Province, P. R. China

January 30, 2020

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

13 Feb 2020

PONE-D-19-24055R1

Clinical value of different anti-D immunoglobulin strategies for preventing Rh hemolytic disease of the fetus and newborn: A network meta-analysis

PLOS ONE

Dear Mr Zhang,

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. There are two additional comments from the reviewer that must be addressed. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Academic Editor

PLOS ONE

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: (No Response)

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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

**********

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

Reviewer #1: 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

**********

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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

**********

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 investigators have addressed previous comments and have made changes to the manuscript. However, I have two additional comments:

1. Results of the Bayesian network meta-analysis (lines 6-7): Based on Table 4, the results for postnatal 3/I vs blank/F should be OR=0.04, 95% CI=0.02-0.06. Please clarify.

2. Figure 5: The labeling of the various curves should be reevaluated. For example, antenatal 5/E had a SUCRA of 96.8%, however the Figure has antenatal 1 with that SUCRA. Antenatal 2/B had a SUCRA of 89.2%, however the Figure has antenatal 3 with that SUCRA.

**********

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Reviewer #1: No

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18 Feb 2020

Reviewer #1: The investigators have addressed previous comments and have made changes to the manuscript. However, I have two additional comments:

1. Results of the Bayesian network meta-analysis (lines 6-7): Based on Table 4, the results for postnatal 3/I vs blank/F should be OR=0.04, 95% CI=0.02-0.06. Please clarify.

Answer: the results for postnatal 3/I vs blank/F has been revised.

2. Figure 5: The labeling of the various curves should be reevaluated. For example, antenatal 5/E had a SUCRA of 96.8%, however the Figure has antenatal 1 with that SUCRA. Antenatal 2/B had a SUCRA of 89.2%, however the Figure has antenatal 3 with that SUCRA.

Answer:The labeling of the various curves has been reevaluated.The Figure 5 has been replaced.

Supporting Information files and PRISMA checklist are uploaded.please check

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

21 Feb 2020

Clinical value of different anti-D immunoglobulin strategies for preventing Rh hemolytic disease of the fetus and newborn: A network meta-analysis

PONE-D-19-24055R2

Dear Dr. Zhang,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Frank T. Spradley

Academic Editor

PLOS ONE

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

**********

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

**********

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

Reviewer #1: 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

**********

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

**********

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: (No Response)

**********

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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

26 Feb 2020

PONE-D-19-24055R2

Clinical value of different anti-D immunoglobulin strategies for preventing Rh hemolytic disease of the fetus and newborn: A network meta-analysis

Dear Dr. Zhang:

I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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.

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With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Frank T. Spradley

Academic Editor

PLOS ONE

Table 1

Main characteristics of all included studies.

First author	Year	Country	Sample size	I/C	Intervention	Control	Multivariate analysis	NOS
Ascari WQ	1968	America	1280	781/499	postnatal 3/I	blank/F	YES	7
Ascari WQ	1969	America	2876	1834/1042	postnatal 3/I	blank/F	YES	8
Bryant EC	1969	America	355	191/164	postnatal 3/I	blank/F	NO	8
Jennings ER	1968	Canada	493	258/235	postnatal 3/I	blank/F	NO	7
Pollack W	1968	America	1286	787/499	postnatal 3/I	blank/F	NO	8
Robertson JG	1969	Scotland	212	100/112	postnatal 3/I	blank/F	NO	7
Stenchever MA	1971	America	54	26/28	postnatal 3/I	blank/F	NO	7
White CA	1970	America	5438	3784/1654	postnatal 3/I	blank/F	NO	8
Dudok D	1968	Holland	662	333/329	postnatal 3/I	blank/F	NO	9
Clake CA	1968	England	197	95/102	postnatal 3/I	blank/F	NO	9
Buchanan DI	1969	Canada	430	223/207	postnatal2/H	postantal 1/G	NO	9
Chown B	1969	Canada	2216	358/500;858/500	postnatal1/G; postantal3/I	blank/F	NO	8
John GC	1969	Canada	202	65/42;53/42	postnatal1/G; postantal3/I	blank/F	NO	9
Tovey LA	1983	England	9178	3875/5303	antenatal 4/D	postantal 1/G	NO	7
Huchet J	1987	France	1189	599/590	antenatal 4/D	postantal 1/G	YES	8
Bowam JM	1987	Canada	12836	9303/3533	antenatal 2/B	postantal 3/I	NO	6
Trolle B	1989	Denmark	700	354/346	antenatal 2/B	postantal 2/H	NO	8
Mayne S	1997	England	2851	1425/1426	antenatal 4/D	postantal 3/I	NO	9
Mackenzie IZ	1999	England	6466	3320/3146	antenatal 4/D	postantal 3/I	NO	9
Mackenzie IZ	2004	England	491	248/243	antenatal 2/B	postantal 3/I	NO	9
Lee D	1995	England	1180	648/532	antenatal 3/C	blank/F	YES	8
Bowam JM	1978	Canada	2361	2109/252	antenatal 5/E	antenatal 2/B	NO	7
Bowam JM	1978	Canada	2612	1598/1014	antenatal 2/B	postantal 3/I	NO	7
Hermann M	1984	Sweden	9295	4895/4400	antenatal 1/A	postantal 2/H	NO	7