Front. Pediatr. Frontiers in Pediatrics Front. Pediatr. 2296-2360 Frontiers Media S.A. 10.3389/fped.2024.1475623 Pediatrics Systematic Review Direct antiglobulin test for the prediction of neonatal hyperbilirubinemia needing an intervention: a systematic review and diagnostic test accuracy meta-analysis Kumar KrishnegowdaVijay 1 RamaswamyViraraghavan Vadakkencherry 2 AbiramalathaThangaraj 3 4 BandyopadhyayTapas 5 SAbdul Kareem Pullattayil 6 Kannan LoganathanPrakash 7 8 9 * † Department of Neonatology, Institute of Medical Sciences and SUM Hospital, Bhubaneswar, Orissa, India Department of Neonatology, Ankura Hospital for Women and Children, Hyderabad, Telangana, India Department of Neonatology, Kovai Medical Center and Hospital (KMCH), Coimbatore, Tamil Nadu, India KMCH Research Foundation, Coimbatore, Tamil Nadu, India Department of Neonatology, ABVIMS and Dr. RML Hospital, New Delhi, India Health Sciences Librarian, Queen’s University, Kingston, ON, Canada Neonatal Unit, James Cook University Hospital, Middlesbrough, United Kingdom Clinical Academic Office, Faculty of Medical Sciences, Newcastle University, Newcastle, United Kingdom Department of Physics, University of Durham, Durham, United Kingdom

Edited by: Francesco Pegoraro, University of Florence, Italy

Reviewed by: Tudor Lucian Pop, University of Medicine and Pharmacy Iuliu Hatieganu, Romania

Ramesh Vidavalur, Cayuga Medical Center, United States

 Correspondence: Prakash Kannan Loganathan pkannanloganathan@nhs.net

ORCID Prakash Kannan Loganathan orcid.org/0000-0003-3717-8569

 28012025 2024 121475623 04082024 27122024 © 2025 Kumar Krishnegowda, Ramaswamy, Abiramalatha, Bandyopadhyay, S and Kannan Loganathan. 2025Kumar Krishnegowda, Ramaswamy, Abiramalatha, Bandyopadhyay, S and Kannan Loganathan

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 Importance

The direct antiglobulin test (DAT) is commonly used as a screening test for predicting significant neonatal hyperbilirubinemia requiring intervention. However, evidence for this approach is limited.

Objective

The aim of this study was to evaluate the diagnostic utility of DAT in predicting the need for phototherapy and double volume exchange transfusion (DVET) in neonates with ABO and Rhesus (Rh) incompatibility conditions.

Methods

MEDLINE, Embase, CENTRAL, CINAHL, and Web of Science were searched from inception until 1 February 2024. Randomized controlled trials (RCTs) and non-RCTs were eligible for inclusion. Two reviewers screened the titles and abstracts blinded to each other. A Bayesian bivariate random-effects model was employed for the diagnostic test accuracy meta-analyses. Risk of bias was assessed using Quality Assessment for Studies of Diagnostic Accuracy 2 and certainty of evidence (CoE) was adjudged according to the Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) guidelines.

Results

In total, 53 studies were included in the systematic review and 28 were synthesized in the meta-analysis. For the need for phototherapy outcome, the pooled sensitivity [95% credible interval (CrI)] and specificity (95% CrI) of DAT in ABO incompatibility (18 studies, n = 10,110) were 56.1% (44.5%–67.8%) and 83.6% (71.6%–90.8%). For Rh incompatibility (three studies, n = 491), the sensitivity and specificity were 40.4% (12.2%–81.7%) and 89.9% (72.7%–94.6%). The CoE was predominantly low. For the need for DVET outcome, the pooled sensitivity and specificity of DAT in ABO incompatibility (three studies, n = 2,652) were 83.6% (35.8%–99.6%) and 74.5% (40.3%–92.7%). For Rh incompatibility (two studies, n = 240), the sensitivity and specificity were 80.3% (34.2%–97.3%) and 68.0% (25.3%–92.1%). The CoE was predominantly very low.

Conclusion

In ABO and Rh incompatibility, DAT probably has moderate specificity and low sensitivity for predicting the need for phototherapy. For DVET, though DAT is possibly a better predictor due to its acceptable sensitivity, the predictive interval was wide. Thus, we do not suggest the routine use of DAT screening to predict the need for phototherapy and DVET. However, it may be used as a second-tier investigation for risk stratification of high-risk neonates.

Systematic Review Registration

https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022297785, PROSPERO (CRD42022297785).

 neonatal jaundice Coombs test meta-analysis newborn exchange transfusion section-at-acceptanceNeonatology Introduction

Neonatal hyperbilirubinemia (NNH) is a common diagnosis in neonates, requiring treatment in the initial postnatal days (1). While the majority of these neonates needing treatment are managed with phototherapy, a small proportion may require additional therapies such as intravenous immunoglobulin (IVIG) and double volume exchange transfusion (DVET). Despite the availability of effective interventions, the risk of serious adverse outcomes including kernicterus, cerebral palsy, hearing loss, and even mortality remains a significant concern (2). These adverse outcomes could be prevented by the timely identification and treatment of neonates at risk of developing severe NNH.

Evidence-based guidelines have recommended the use of various screening tools for early identification and treatment of NNH (1, 3). Despite the incorporation of these tools into clinical practice, instances of serious adverse outcomes secondary to NNH have been reported. This is often attributed to factors such as challenges in identifying high-risk neonates, limitations of the screening tools, and adaptation of policies such as early discharge from healthcare facilities. As a result, regional guidelines suggest risk stratification based on gestational age, bilirubin levels, and the presence of various other morbidities such as sepsis, asphyxia, and ABO, Rhesus (Rh), and other blood group incompatibilities (1, 4, 5). The inclusion of additional tests such as the direct antiglobulin test (DAT) has also been recommended (1, 5).

DAT is commonly performed in suspected cases of hemolytic disease of the newborn due to ABO or Rh incompatibility (6). However, the current clinical guideline does not recommend routine testing of umbilical cord blood for DAT in ABO and Rh incompatibility (5). As the evidence for the same is contentious, many centers continue to use routine DAT testing as a screening test to identify at-risk neonates (6–10).

Thus, we conducted a systematic review and diagnostic test accuracy (DTA) meta-analyses with an aim to evaluate the diagnostic utility of DAT in predicting NNH requiring treatment in neonates with ABO and Rh incompatibility conditions.

 Methods

The protocol was registered with PROSPERO (CRD42022297785) (11), and the reporting is in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses of Diagnostic Test Accuracy Studies (PRISMA-DTA) (12).

Study eligibility

Randomized controlled trials (RCTs), non-RCTs, and conference abstracts were eligible for inclusion. Studies that evaluated the diagnostic performance of DAT in predicting the need for treatment of NNH in various mother-neonate blood group pairs, including but not exclusively ABO and Rh incompatibility, were also eligible for inclusion. Case reports, traditional reviews, and systematic reviews were excluded. There were no language restrictions.

 Patient population

We included studies conducted in different healthcare settings in late preterm and term neonates. Studies reporting on preterm neonates of <34 weeks' gestation, NNH secondary to enzyme deficiency, sepsis, liver disorders, and those who had received fetal therapy were excluded.

 Index test

A DAT was performed on umbilical cord blood or during the first 14 days of postnatal life. We included studies that had used the two commonly used methods of DAT measurement (gel or tube agglutination methods). For the generation of the 2 × 2 table, we considered a weakly positive DAT as a positive test.

 Reference standard

A serum bilirubin test was performed in the first 14 days of postnatal life.

 Target condition

We considered the following outcomes: the need for phototherapy, DVET, and IVIG therapy in the first 14 days, irrespective of the treatment thresholds recommended by different guidelines. We did not include outcome parameters such as clinical jaundice and significant hyperbilirubinemia not requiring intervention.

 Information sources

We conducted a comprehensive search of MEDLINE, Embase, CENTRAL, CINAHL, and Web of Science from inception till 1 February 2024. (AS, VK) (Supplementary Table 1). In addition, we searched the bibliographies of included studies and review articles to identify potentially eligible studies for inclusion.

 Study selection and data extraction

All the study titles and abstracts were screened independently in duplicates by two authors (TA, TB) using an online software platform (Rayyan QCRI, Doha) (13). Data extraction was independently performed by the two investigators (VR, VK) using a preformed data extraction form, and disagreements were resolved by consensus. We recorded data items, including blood group, timing of DAT measurement, outcomes, and test accuracy measures [true positives (TPs), true negatives (TNs), false positives (FPs), and false negatives (FNs)].

 Study quality assessment

The risk of bias and applicability concerns of the included studies were assessed using the Quality Assessment for Studies of Diagnostic Accuracy 2 tool (QUADAS-2) (14). Two authors (VK, PK) independently assessed the quality of the studies.

 Data synthesis

Data synthesis was conducted in accordance with the Cochrane Handbook for Systematic Reviews of Diagnostic Test Accuracy (15). We estimated individual sensitivities and specificities, along with their 95% confidence intervals using DTA measures. These findings are presented using forest plots and the summary receiver operating characteristic (SROC) space. In addition, we used a Bayesian bivariate random-effects approach to estimate median pooled sensitivity and specificity, along with 95% credible intervals (CrI). Bayesian analysis was performed using METABayesDTA version 1.4 and R software version 4.3.1 (R Foundation for Statistical Computing, Vienna, Austria) using the “meta4diag' and “meta” packages (16, 17). Vague priors were used. A Stan sampler with two chains, 500 warm-up iterations, and 1,500 total iterations was utilized. Model fit was assessed by visualizing the correlation residual plot and the frequency table probability residual plot. Model convergence was confirmed by examining the trace and density plots.

A meta-analysis was performed for each outcome: the need for phototherapy, the need for DVET, and the requirement of IVIG therapy. Within each outcome, the DTA measures were estimated for specific maternal and neonatal blood group combinations. For the DTA meta-analysis, we included only studies with ABO or Rh incompatibility, as these are the two commonly encountered clinical scenarios. Studies that evaluated DAT in blood group scenarios other than ABO or Rh incompatibility were included in the narrative review. ABO or Rh blood group incompatibilities were categorized into ABO incompatibility [Mother blood group (MBG): O; Rh positive and baby blood group (BBG): A or B irrespective of Rh status], Rh incompatibility (MBG: A, or B or AB or O with Rh negative and BBG: A or B or AB or O with Rh positive), or ABO/Rh incompatibility (MBG: O with Rh positive and BBG: A or B with Rh positive or negative OR MBG: A, B, AB, O with Rh negative and BBG: A or B or AB or O with Rh positive).

Subgroup and sensitivity analysis

Heterogeneity was explored using subgroup analysis of the covariates, DAT testing methods and treatment thresholds, as per the guidelines. We performed a post-hoc sensitivity analysis by excluding studies with applicability concerns in the patient selection domain. For publication bias, we examined the regression coefficient line for plot asymmetry. A funnel plot of the log diagnostic odds ratio against 1/(effective sample size)1/2 was generated. For assessing the certainty of the evidence, we used GRADEPro, following GRADE guidelines for DTA meta-analysis for each target condition and blood group combination (18).

 Results Study selection

After the removal of duplicates, 1,409 titles and abstracts were screened. Of these, 73 were selected for full-text review. Among them, 51 were included in the systemic review. Of the included studies, 28 were synthesized in a DTA meta-analysis, and 23 studies were included in the narrative review (Figure 1). The excluded studies with valid reasons for their exclusion are listed in Supplementary Table 2.

PRISMA flow chart of the literature search.

 Characteristics of included studies

A total of 28 studies (n = 20,935) (10, 19–44) were included in the meta-analysis, with 20 studies (10, 19, 26, 28, 30, 32–44) (n = 11,385), 3 studies (10, 22, 29) (n = 491), and 9 studies (10, 20, 21, 23–25, 27, 31) (n = 9,561) assessed DAT in the ABO, Rh and ABO/Rh incompatibility scenarios, respectively. Only two studies (23, 25) included late preterm along with term neonates, while the others only included term neonates. Furthermore, 16 studies (20, 22, 24, 25, 28–33, 36, 39–42, 45) did not specify the method of DAT assessment, 7 studies (10, 19, 23, 27, 35, 37, 38) utilized the gel method, and 5 studies (21, 26, 34, 43, 44) used the column agglutination method. Most DAT assessments were performed with cord blood samples (10, 19, 20, 22–31, 33–37, 40–44), while two studies (21, 38) performed the assessments during the initial postnatal days. Of the included studies, 28 studies (10, 19–44) (n = 20,162) reported the outcome measure of the need for phototherapy, 7 studies (19, 22, 27, 29, 31, 32, 44) (n = 3,338) were on DVET, and 2 studies (19, 45) (n = 1,005) were on IVIG therapy. The characteristics of the included studies synthesized in the meta-analysis are provided in Table 1, and those in the narrative review are summarized in Appendix I in the Supplementary Material.

Characteristics of included studies in the meta-analysis.

No Author, study design, country Sample size Gestation (mean/median) Blood group scenario Index test (DAT method and timing) Reference standard (Bilirubin/Tcb/clinical examination and timing) Target condition (Treatment threshold)
1 Secil, 2024RetrospectiveTurkey 820 38.8 (1.3) wks ABOa Method: gel, Timing: cord Tcb followed by Bilirubin till 96 h Phototherapy, Threshold: AAP
2 Daunov, 2024RetrospectiveUnited States 579 39 (38–40) wks ABOa Method: NS, Timing: cord Bilirubin IVIG, Threshold: AAP
3 Novoselac, 2023RetrospectiveCroatia 182 39 (39–40) wks ABO/Rh negativec Method: column agglutination, Timing: cord (Rh negative) and clinical jaundice (ABO) Bilirubin (clinical jaundice) Phototherapy, Threshold: AAP
4 Omran, 2023RetrospectiveSaudi Arabia 611 39 wks ABOa Method: NS, Timing: cord Tcb followed by Bilirubin Phototherapy, Threshold: AAP
5 Gabbay, 2023RetrospectiveUnited States 542 39.4 (36–41.7) wks ABO/Rh negativec Method: NS, Timing: cord Bilirubin or Tcb at 12 and 24 h Phototherapy, Threshold: AAP
6 Duete, 2022RetrospectiveBrazil 8 NS Rh negativeb Method: NS, Timing; cord Bilirubin Phototherapy/DVET, Threshold: NS
7 Chowdhary, 2022RetrospectiveIndia 426 38.4 (37.5–39.2) wks ABOa Method: gel, Timing: cord Bilirubin at 24 h (DAT positive) and clinical jaundice Phototherapy/DVET/IVIG, Threshold: AAP
8 Kardum, 2020RetrospectiveCroatia 1360 39 (38–40) wks ABO/Rh negativec Method: NS, Timing: cord Bilirubin or Tcb within 2 days Phototherapy, Threshold: NICE
9 Alkhater, 2021RetrospectiveSaudi Arabia 251 38 (1.4) wks ABO/Rh negative Method: gel, Timing: cord Bilirubin [Clinical jaundice, positive DAT, hospital protocol (IDM and SGA)] Phototherapy, Threshold: NICE
10 Mehta, 2021RetrospectiveUnited States 158 Late preterm and term ABO/Rh negativec Method: gel, Timing: cord Bilirubin before discharge Phototherapy, Threshold: AAP
11 Fei, 2020RetrospectiveUnited States 100 39.1 (1.4) wks ABOa Method: column agglutination (Fei_A); gel (Fei_B)Timing: cord Bilirubin Phototherapy, Threshold: AAP
12 Ligsay, 2020RetrospectiveUnited States 6,622 Late preterm and term ABO/Rh negativec Method: NS, Timing: cord Bilirubin Phototherapy, Threshold: AAP
13 Shin, 2019RetrospectiveSouth Korea 303 38 wks ABO/Rh negativec Method: gel, Timing: cord Bilirubin Phototherapy/DVET, Threshold: AAP
14 Altuntas, 2019ProspectiveTurkey 83 38.9 (1.2) wks ABOa Method: NS, Timing: cord Bilirubin at 6, 24 h, and until 15 days Phototherapy, Threshold: AAP
15 Zonneveld, 2017ProspectiveSuriname 232 38 wks Rh negativeb Method: NS, Timing: cord Bilirubin Phototherapy, Threshold: NICE
16 Schutzman, 2010Retrospective 700 39 (0.48) wks ABOa Method: NS, Timing: cord Bilirubin (cord sample followed every 8 h in positive DAT) Phototherapy, Threshold: AAP
17 Tatopoulos 2010RetrospectiveUnited States 257 Term ABOa Method: gel, Timing: day 1 Bilirubin Phototherapy/DVET, Threshold: AAP
18 Bakkeheim, 2009ProspectiveNorway 98 39.8 (39.3–40.3) wks ABOa Method: gel, Timing: cord Bilirubin (clinical jaundice) Phototherapy/DVET/IVIG, Threshold: Norwegian
19 Sarici, 2002ProspectiveTurkey 136 39.2 (1.1) wks ABOa Method: NS, Timing: NS Bilirubin Phototherapy, Threshold: AAP
20 Geelkerken, 1999RetrospectiveNetherlands 143 NS ABO/Rh negativec Method: NS, Timing: cord Bilirubin Phototherapy/DVET, Threshold: NS
21 Dudin, 1993RetrospectivePalestinian 1,530 38 wks ABOa Method: NS, Timing: NS Bilirubin within the first week Phototherapy/DVET, Threshold: NS
22 Diane, 1993RetrospectiveUnited States 143 39 (1.3) wks ABOa Method: NS, Timing: cord Bilirubin Phototherapy, Threshold: Cockington
23 Comos, 1991RetrospectiveSpain 1,933 37 wks ABOa Method: NS, Timing: cord Bilirubin in first 2 days Phototherapy, Threshold: Cockington
24 Brouwers, 1988ProspectiveNetherlands 200 40 wks ABOa Method: NS, Timing: cord Bilirubin Phototherapy, Threshold: NS
25 Meberg, 1998ProspectiveNorway 2,335 Term ABOa Method: NS, Timing: cord Tcb followed by Bilirubin Phototherapy, Threshold: Hillingdon hospital bilirubin chart
26 Han, 1988ProspectiveSingapore 251 NS ABOa Method: column agglutination, Timing: cord Bilirubin at 24 and 48 h Phototherapy, Threshold: NS
27 Whyte, 1981ProspectiveScotland 142 Term ABOa Method: column agglutination, Timing: cord Bilirubin Phototherapy, Threshold: NS
28 Peevy, 1978RetrospectiveUnited States 696 NS ABOa Method: column agglutination, Timing: cord Bilirubin DVET, Threshold: NS

Wks, weeks; DAT, direct antiglobin test; TcB, transcutaneous bilirubin; AAP, American Academy of Pediatrics; NS, not specified; IVIG, intravenous immunoglobulin; DVET, double volume exchange transfusion; Rh, Rhesus; NICE, National Institute for Health and Care Excellence; IDM, infant of diabetic mother; SGA, small for gestational age.

ABO: Mother blood group—O and Rhesus positive; and neonate blood group—A or B and Rhesus positive or negative.

Rhesus negative: Mother blood group—A, B, AB, or O and Rhesus negative; and neonate blood group—A, B, AB, or O and Rhesus positive.

ABO/Rhesus negative: Mother blood group—O and Rhesus positive; and neonate blood group—A or B and Rhesus positive or negative OR Mother blood group—A, B, AB, or O and Rhesus negative; and neonate blood group—A, B, AB, or O and Rhesus positive.

 Risk of bias and applicability

Overall, the studies were adjudged to have a moderate risk of bias (Table 2). The common reason to adjudge studies as having a high risk of bias was in the domain of reference standard, as the DAT result was not blinded to the clinicians. For patient selection, high risk was attributed to the non-consecutive inclusion of neonates. Applicability concerns in the patient selection domain were related to the selective inclusion of neonates by prior testing. The proportion of studies assessed to have an overall risk of bias of “low,” “high,” or “unclear” is presented in Figure 2.

QUADAS-2 assessment of the studies for risk of bias and applicability concerns.

Risk of Bias Applicability concerns
Study rob_PS rob_IT rob_RS rob_FT ac_PS ac_IT ac_RS
1 Secil, 2024 High Low High Low Low Low Low
2 Daunov, 2024 Low Low High Low Low Low Low
3 Gabbay, 2023 Low Low High Low Low Low Low
4 Gabbay, 2023 Low Low High Low Low Low Low
5 Novoselac, 2023 High Low High Unclear High Low Low
6 Omran, 2023 High Low High High Low Low Low
7 Chowdhary, 2022 Low Low High Low Low Low Low
8 Duete, 2022 High Low High Unclear High Low Low
9 Alkhater, 2021 Low Low High Unclear Low Low Low
10 Mehta, 2021 Low Low High Low Low Low Low
11 Kardum, 2021 Low Low Low Low Low Low Low
12 Ligsay, 2020 Low Low High Low Low Low Low
13 Fei, 2020 High Low High Low Low Low Low
14 Fei, 2020 High Low High Low Low Low Low
15 Shin, 2019 High Low High Unclear High Low Low
16 Altuntas, 2019 Low Low High Unclear Low Low Low
17 Zonneveld, 2017 High Low Low Low Low Low Low
18 Schutzman, 2010 Low Low High Unclear Low Low Low
19 Tatopoulos, 2010 Low Low High Low Low Low Low
20 Bakkeheim, 2009 Low Low High Low High Low Low
21 Sarici, 2002 Low Low High Low Low Low Low
22 Geelkerken, 1999 High Low Low High Low Low Low
23 Meberg, 1998 Low Low High Low Low Low Low
24 Dudin, 1993 Low Low High Unclear Low Low Unclear
25 Diane, 1993 Low Low High Low Low Low Low
26 Comos, 1991 Low Low Low Low Low Low Low
27 Han, 1988 Low Low High Low Low Low Low
28 Brouwers, 1988 High Low High Low Low Low Low
29 Whyte, 1981 High Low High Low Low Low Low
30 Peevy, 1978 High Low Low Unclear Low Low Low

Rob, risk of bias; PS, patient selection; IT, index test; RS, reference standard; FT, flow and timing; ac, applicability concern.

Proportions of studies with low, high, or unclear risk of bias and applicability concerns as per QUADAS-2.

 Main results

We have summarized the study-specific sensitivity and specificity of all the included studies for the target conditions: the need for phototherapy (Supplementary Figures 1, 2), DVET (Supplementary Figures 3, 4), and IVIG therapy (Supplementary Figures 5, 6), employing forest plots and the SROC curves.

For the primary target condition, the need for phototherapy, a total of 28 studies (n = 20,162) (10, 19–44) were included. In the meta-analysis of studies evaluating DAT in ABO incompatibility (18 studies; n = 10,110) (10, 19, 26, 28, 30, 32–43), the posterior median sensitivity and specificity were 56.1% (95% CrI: 44.5%–67.8%) and 83.6% (95% CrI: 71.6%–90.8%), respectively. For the Rh isoimmunization scenario (three studies; n = 491) (10, 22, 29), the pooled sensitivity and specificity were 40.4% (95% CrI: 12.2%–81.7%) and 89.9% (95% CrI: 72.7%–94.6%), respectively. Studies that had evaluated DAT in ABO/Rh incompatibility (nine studies; n = 9,561) (10, 20, 21, 23–25, 27, 31) yielded a pooled sensitivity and specificity of 35.8% (95% CrI: 19.6%–57.0%) and 82.5% (95% CrI: 61.9%–93.1%), respectively (Figures 3, 4, Table 3).

(A) Forest plot depicting the sensitivity of studies utilizing the DAT as an index test to predict the need for phototherapy in ABO, Rh, and ABO/Rh incompatibility scenarios. (B) Forest plot depicting the specificity of studies utilizing the DAT as an index test to predict the need for phototherapy in ABO, Rh, and ABO/Rh incompatibility scenarios.

(A) SROC plot of studies utilizing the DAT as an index test to predict the need for phototherapy in ABO incompatibility scenarios. (B) SROC plot of studies utilizing the DAT as an index test to predict the need for phototherapy in Rh incompatibility scenarios. (C) SROC plot of studies utilizing the DAT as an index test to predict the need for phototherapy in ABO/Rh incompatibility scenarios.

Main analysis of studies based on target condition and blood group settings.

Studies/participants Blood group scenario Sensitivity (95% CrI) Specificity (95% CrI) LR+ (95% CrI) LR− (95% CrI) DOR (95% CrI)
Phototherapy
 1 18 studies; n = 10,110 ABO incompatibilitya 0.561 (0.445–0.678) 0.836 (0.716–0.908) 3.40 (2.08–5.72) 0.526 (0.399–0.668) 6.513 (3.36–12.20)
 2 Three studies; n = 491 Rh incompatibilityb 0.404 (0.122–0.817) 0.899 (0.727–0.946) 3.60 (0.919–10.56) 0.67 (0.207–1.013) 5.61 (0.90–42.32)
 3 Nine studies; n = 9,561 ABO/Rh incompatibilityc 0.358 (0.196–0.570) 0.825 (0.619–0.931) 2.055 (1.08–4.0) 0.782 (0.591–0.964) 2.687 (1.129–5.81)
Exchange transfusion
 1 Three studies; n = 2,652 ABO incompatibilitya 0.836 (0.358–0.996) 0.745 (0.403–0.927) 2.917 (1.42–5.89) 0.290 (0.08–0.76) 10.21 (1.89–58.12)
 2 Two studies; n = 240 Rh incompatibilityb 0.803 (0.342–0.973) 0.680 (0.253–0.921) 2.32 (0.70–9.40) 0.305 (0.04–1.43) 8.96 (0.47–108.94)
 3 Two studies; n = 446 ABO/Rh incompatibilityc 0.501 (0.139–0.855) 0.890 (0.670–0.949) 4.26 (1.07–11.93) 0.574 (0.16–0.98) 7.64 (1.09–56.05)

CrI, credible interval; LR+, positive likelihood ratio; LR−, negative likelihood ratio; DOR, diagnostic odds ratio; n, sample size; Rh, Rhesus.

ABO incompatibility: Mother blood group—O and Rh positive; and neonate blood group—A or B and Rh positive or negative.

Rh incompatibility: Mother blood group—A, B, AB, or O and Rh negative; and neonate blood group—A, B, AB, or O and Rh positive.

ABO/Rh incompatibility: Mother blood group—O and Rh positive; and neonate blood group—A or B and Rh positive or negative OR Mother blood group—A, B, AB, or O and Rh negative; and neonate blood group—A, B, AB, or O and Rh positive.

For the target condition, the need for DVET, a total of seven studies (n = 3,338) (19, 22, 27, 29, 31, 32, 44) were included. In ABO incompatibility settings (three studies; n = 2,652) (19, 32, 44), the pooled sensitivity and specificity were 83.6% (95% CrI: 35.8%–99.6%) and 74.5% (95% CrI: 40.3%–92.7%). In Rh incompatibility (two studies; 240 infants) (22, 29), the pooled sensitivity and specificity were 80.3% (95% CrI: 34.2%–97.3%) and 68.0% (95% CrI: 25.3%–92.1%). Whereas for ABO/Rhesus incompatibility (two studies; n = 446) (27, 31), the pooled sensitivity and specificity were 50.1% (95% CrI: 13.9%–85.5%) and 89.0% (95% CrI: 67.0%–94.9%) (Table 3 and Supplementary Figure 7).

For the target condition, the need for IVIG (two studies; n = 1,005) (19, 45), we could not perform a meta-analysis as only a limited number of studies on each blood group scenario had reported on this outcome.

Subgroup analysis Treatment thresholds

Subgroup analysis was conducted for the need for phototherapy for the studies which used the American Academy of Pediatrics (AAP) threshold chart in the ABO (nine studies, n = 3,231) (19, 26, 28, 30, 35, 36, 38, 39) and ABO/Rh incompatibility (six studies, n = 7,807) (20, 21, 23, 25, 27) scenarios. Whereas subgroup analyses were performed for studies using National Institute for Health and Care Excellence (NICE) threshold charts in the Rh (two studies, n = 483) (10, 29) and ABO/Rh (two studies; n = 1,611) (10, 24) incompatibility scenarios. There were no significant differences in the subgroup analyses except for an improvement in sensitivity when using AAP charts for the need for phototherapy in ABO incompatibility scenarios. The sensitivity and specificity were 80.3% (95% CrI: 34.2%–97.3%) and 68.0% (95% CrI: 25.3%–92.1%), respectively (Supplementary Table 3).

 DAT method

The subgroup analysis based on the method used for DAT measurement (gel vs. tube method) showed no significant difference for the outcomes of phototherapy and DVET in any of the blood group scenarios (Supplementary Table 3).

 <italic>Post-hoc</italic> sensitivity analysis

We also performed a post-hoc sensitivity analysis by excluding studies with applicability concerns in patient selection (21, 22, 27, 37). For the need for phototherapy, we found no differences from the primary analysis for ABO incompatibility (17 studies, n = 10,012) (10, 19, 26, 28, 30, 32–36, 38–43), Rh incompatibility (two studies, n = 483) (10, 29), and ABO/Rh incompatibility (seven studies, n = 9,076) (10, 20, 23–25, 31) (Supplementary Table 4).

 Certainty of evidence

The certainty of evidence for the outcome measure, the need for phototherapy, was very low for the pooled sensitivity in ABO, Rh, and ABO/Rh incompatibility. This was primarily due to inconsistency, followed by the risk of bias and imprecision. In contrast, the certainty of evidence for specificity varied between low to moderate in ABO, Rh, and ABO/Rh incompatibility, mainly due to the risk of bias and inconsistency. For the outcome measure, the need for DVET, the certainty of evidence for sensitivity was very low. For specificity, it remained very low to low across ABO, Rh, and ABO/Rh incompatibility scenarios. Downrating the certainty of evidence was done mainly due to the risk of bias and inconsistency (Table 4).

GRADE assessment for the certainty of evidence for the effect estimates of the sensitivity and specificity of DAT in predicting the need for phototherapy and DVET.

Target condition Blood group scenario DTA measure No of studies (n) Factors that may decrease certainty of evidence Rating up the evidence for the ose–responsegradient, plausible confounding, and large effect Test accuracy CoE
Pooled point estimate Risk of bias Indirectness Inconsistency Imprecision Publication bias
Phototherapy ABO incompatibilitya Sensitivity 18 studies 10,110 neonates 0.56 (0.45–0.68) Seriousb Not serious Very seriousc Seriousd None None ⊕○○○Very low
Specificity 18 studies 10,110 neonates 0.84 (0.72–0.91) Seriousb Not serious Seriouse Not serious None None ⊕⊕○○Low
Rh incompatibilityf Sensitivity Three studies 491 neonates 0.40 (0.12–0.82) Seriousg Not serious Very serioush Very seriousi None None ⊕○○○Very low
Specificity Three studies 491 neonates 0.90 (0.73–0.95) Seriousg Not serious Not seriousj Not serious None None ⊕⊕⊕○Moderate
ABO/Rh incompatibilityk Sensitivity Nine studies 9,561 neonates 0.35 (0.19–0.57) Seriousl Not serious Seriousm Seriousn None None ⊕○○○Very low
Specificity Nine studies 9,561 neonates 0.82 (0.62–0.93) Seriousl Not serious Not seriouso Not serious None None ⊕⊕⊕○Moderate
DVET ABO incompatibilitya Sensitivity Three studies 2,652 neonates 0.84 (0.36–1.00) Seriousp Not serious Seriousq Seriousr None None ⊕○○○Very low
Specificity Three studies 2,652 neonates 0.74 (0.40–0.93) Seriousp Not serious Not seriouss Serioust None None ⊕⊕○○Low
Rh incompatibilityf Sensitivity Two studies 240 neonates 0.80 (0.34–0.97) Very seriousu Not serious Not seriousv Seriousw None None ⊕○○○Very low
Specificity Two studies 240 neonates 0.68 (0.25–0.92) Very seriousu Not serious Seriousx Seriousy None None ⊕○○○Very low
ABO/Rh incompatibilityk Sensitivity Two studies 446 neonates 0.50 (0.14–0.85) Very seriousz Not serious Not seriousaa Seriousab None None ⊕○○○Very low
Specificity Two studies 446 neonates 0.89 (0.67–0.95) Very seriousz Not serious Not seriousac Not serious None None ⊕⊕○○Low

CrI, credible interval; n, sample size; DVET, Double volume exchange transfusion; Rh, Rhesus.

ABO incompatibility: Mother blood group—O and Rh positive; and neonate blood group—A or B and Rh positive or negative.

As assessed by QUADAS-2, in the patient selection domain, 6 out of 18 studies had a high risk of bias, and in the reference standard domain, 17 out of 18 studies had a high risk of bias. We did not downgrade further, as blinding to the DAT results might not have had an impact if the guideline threshold had been used.

For individual studies, the sensitivity varied markedly from 14% to 92%. We downgraded by two levels as we could not perform meta-regression or subgroup analysis for all covariates.

We downgraded by one level due to the wide CrIs related to the true positives and false negatives.

For individual studies, the specificity varied from 46% to 98%. We downgraded by one level as we could not perform meta-regression or subgroup analysis for all covariates.

Rh incompatibility: Mother blood group—A, B, AB, or O and Rh negative; and neonate blood group—A, B, AB, or O and Rh positive.

As assessed by QUADAS-2, in the patient selection domain, two out of three studies had a high risk of bias, and in the reference standard domain, two out of three studies had a high risk of bias. We did not downgrade further, as blinding to the DAT results might not have had an impact if the guideline threshold had been used.

For individual studies, the sensitivity varied markedly from 8% to 100%. We downgraded by two levels as we could not perform a meta-regression or subgroup analysis for all covariates.

We downgraded by two levels due to the wide CrIs related to the true positives and false negatives.

We did not downgrade as the specificity varied between 88% and 93%.

ABO/Rh incompatibility: Mother blood group—O and Rh positive; and neonate blood group—A or B and Rh positive or negative OR Mother blood group—A, B, AB, or O and Rh negative; and neonate blood group—A, B, AB, or O and Rh positive.

As assessed by QUADAS-2, in the patient selection domain, three out of nine studies had a high risk of bias, and in the reference standard domain, seven out of nine studies had a high risk of bias. We did not downgrade further, as blinding to the DAT results might not have had an impact if the guideline threshold had been used.

For individual studies, sensitivity varied markedly from 12% to 94%. We downgraded by one level as we could not perform a meta-regression or subgroup analysis for all covariates.

We downgraded by one level due to the wide CrIs related to the true positives and false negatives.

For individual studies, the specificity varied markedly from 6% to 99%. We did not downgrade as the majority of studies (9 out of 10) had a range from 75% to 99%.

As assessed by QUADAS-2, in the patient selection domain, one out of three studies had a high risk of bias, and in the reference standard domain, two out of three studies had a high risk of bias. We did not downgrade further, as blinding to the DAT results might not have had an impact if the guideline threshold had been used.

For individual studies, the sensitivity varied from 66% to 100%. We downgraded by one level as we could not perform a meta-regression or subgroup analysis for all covariates.

We downgraded by one level due to the wide CrIs related to the true positives and false negatives.

For individual studies, the specificity varied markedly from 64% to 85%. We did not downgrade.

We downgraded by one level due to the wide CrIs related to the true negatives and false positives.

As assessed by QUADAS-2, in the patient selection domain, two out of two studies had a high risk of bias, and in the reference standard domain, one out of two studies had a high risk of bias. We downgraded by two levels.

Both studies had a sensitivity of 100%, so we did not downgrade.

We downgraded by one level due to the wide CrIs related to the true positives and false negatives.

Specificity ranged from 58% to 92%. We downgraded by one level.

We downgraded by one level due to the wide CrIs related to the true negatives and false positives.

As assessed by QUADAS-2, in the patient selection domain, two out of two studies had a high risk of bias, and in the reference standard domain, one out of one study had a high risk of bias. We downgraded by two levels.

The sensitivity was 50% in both studies.

We downgraded by one level due to the wide CrIs related to the true positives and false negatives.

The specificity was 88% in one study and 92% in the other. We did not downgrade.

 Publication bias

For the outcome measures, the need for phototherapy and DVET, we did not detect publication bias as Deek's plot for asymmetry was not statistically significant (Supplementary Figures 8, 9).

 Discussion

In our systematic review and DTA meta-analysis, we found that the DAT had low sensitivity but moderate specificity in predicting the need for phototherapy in ABO and Rh incompatibility scenarios. The evidence certainty for sensitivity ranged from very low to low, while for specificity, it was low to moderate. For the outcome measure, the need for DVET, the DAT showed varied sensitivity and specificity across ABO and Rh incompatibility scenarios, with the evidence certainty ranging from very low to low for both sensitivity and specificity.

The sensitivity of DAT for predicting the need for phototherapy was 56.1% in ABO incompatibility, 40.4% in Rh incompatibility, and 35.8% in studies that had evaluated either ABO or Rh incompatibility pairs, with only modest changes in specificity in the aforementioned groups, which was moderate. In addition, the predictive interval for Rh and ABO/Rh incompatibility was as low as 14%, indicating that the DAT is a poor predictor of the need for phototherapy in these scenarios. The poor predictive ability of DAT in Rh and ABO/Rh incompatibility could likely be attributed to a false positive rate of approximately 15% due to the routine use of maternal anti-D immunoglobulin in Rh-negative mothers and the passive transfer of these immunoglobulins to infants (46). The specificity of DAT for predicting the need for phototherapy was 83.6%, 89.9%, and 82.5% in ABO, Rh, and ABO/Rh incompatibility, respectively. However, not accounting for other causes of NNH, such as G6PD deficiency and extravasation, likely could have impacted the specificity of DAT. In contrast, the sensitivity and specificity of the DAT for predicting the need for DVET varied markedly across different blood group incompatibility scenarios. For ABO and Rh incompatibility, the sensitivity was above 80%, while the specificity was relatively lower.

To illustrate the practical implications of our study findings, we could consider 1,000 neonates with ABO incompatibility with a 20% probability [based on Gabbay et al. 2023 (20)] of them needing phototherapy for NNH. In this scenario, there would be 243 positive test results. However, 131 (41%) neonates would be falsely diagnosed as having ongoing hemolysis (false positive), whereas, among the 757 negative DAT results, 88 (12%) would be incorrect diagnoses (false negative). This high rate of false positives could lead to delayed discharge, additional investigations, an increased burden on the healthcare system, and heightened parental anxiety. On the other side, false negatives pose a significant risk, as high-risk neonates might be discharged earlier and followed up less frequently, placing the infants at risk of significant hyperbilirubinemia. In addition, the cost implications are considerable. Nevertheless, the moderate specificity of DAT for ABO incompatibility would allow 669 (88%) out of the 757 cases with negative results to have the correct diagnosis (true negatives). This improved specificity may facilitate early discharge and reduce costs.

The studies we included had a moderate risk of bias, especially in the reference standard domain. This was due to the non-blinding of the DAT test results to clinicians, which could significantly influence the need for an intervention. In addition, there was uncertainty in the flow and timing domain due to the poor reporting of the included studies.

Our review has crucial clinical implications, as it is the first comprehensive review addressing the utility of DAT in the management of neonates with blood group incompatibility. Previously, conflicting evidence on the utility of DAT led to varied recommendations by academic bodies. The AAP recommends a routine umbilical cord blood DAT for all mothers with a history of antenatal antibody screen being positive or unknown or with an Rh-negative blood group (1). The NICE advises against using routine umbilical cord blood DATs to predict significant NNH (4). As a DAT has a low sensitivity in most situations, a DAT may not be used as a screening tool for predicting the need for NNH treatment. Our findings imply that a negative DAT result in an incompatibility scenario can reasonably identify neonates who are less likely to require treatment for NNH and may be restricted to at-risk neonates to assess their risk stratification. Moreover, the sensitivity and specificity of a DAT in the prediction of DVET varied markedly with a wide predictive interval and very low to low evidence certainty. Therefore, we do not suggest the use of the DAT as a screening test for predicting severe hyperbilirubinemia requiring DVET.

Despite including several studies, the certainty of evidence remained very low to low for sensitivity and low to moderate for specificity for the need for phototherapy outcome measure, implying the need for future studies with improved study designs.

Strengths and limitations

To the best of our knowledge, this is the first systematic review to assess the predictive ability of DAT for determining the need for an intervention in NNH. We conducted a thorough literature search and performed clinically relevant analyses based on various blood group combinations, including various subgroup and post-hoc sensitivity analyses. We adhered to the standards recommended by the Cochrane Screening and Diagnostic Tests Methods group and followed the PRISMA reporting guidelines. We assessed the certainty of evidence as guided by the GRADE working group. However, our study had some limitations. Initially, we planned to include all blood group combinations in the meta-analyses, but we restricted the analysis to clinically relevant incompatibility scenarios. We were unable to do many of the pre-planned subgroup analyses such as based on gestational age and DAT strength due to the non-availability of data. We could not evaluate the effect of the updated AAP guidelines on phototherapy and DVET on the sensitivity and specificity of DAT, as none of the included studies evaluated these thresholds. Finally, we did not account for ethnic differences or consider other causes of significant hyperbilirubinemia.

 Conclusions

In ABO and Rh incompatibility, DAT probably has moderate specificity but low sensitivity for predicting the need for phototherapy. For the need for DVET, DAT is possibly a poor predictor as the sensitivity and specificity varied markedly across blood groups with wide predictive intervals. Thus, we do not suggest the use of DAT as a screening test for predicting hyperbilirubinemia requiring either phototherapy or DVET.

 Data availability statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

 Author contributions

VK: Data curation, Formal Analysis, Methodology, Writing – original draft, Writing – review & editing. VR: Conceptualization, Data curation, Formal Analysis, Methodology, Writing – original draft, Writing – review & editing. TA: Conceptualization, Data curation, Methodology, Writing – original draft, Writing – review & editing. TB: Conceptualization, Writing – original draft, Writing – review & editing. AS: Writing – original draft, Writing – review & editing. PK: Conceptualization, Formal Analysis, Methodology, Supervision, Writing – original draft, Writing – review & editing.

 Funding

The author(s) declare that no financial support was received for the research, authorship, and/or publication of this article.

 Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

 Publisher's note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

 Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fped.2024.1475623/full#supplementary-material

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