Prevalence and Comparative Clinical Profile of Major versus Minor Blood Group Incompatibility in Neonates with Pathological Unconjugated Hyperbilirubinemia: A Prospective Tertiary-Center Study

Background: Hemolytic disease of the newborn remains a leading cause of pathological hyperbilirubinemia, yet the comparative burden of minor blood group incompatibility — often overlooked in favor of ABO and RhD — remains poorly characterized in Indian cohorts. We sought to compare the prevalence and clinical profiles of ABO, RhD, and minor blood group incompatibilities among neonates with hemolytic hyperbilirubinemia at a North Indian tertiary-care center.
Methods: In this prospective observational study, 135 neonates with identifiable hemolytic etiologies were enrolled from January 2020 to September 2021. ABO and RhD status were determined by standard tube technique; minor blood group antibodies were screened using a 3-cell panel and confirmed with an 11-cell panel. Continuous variables were compared using the Kruskal-Wallis test (non-normally distributed data) with Dunn's post-hoc test and Bonferroni correction; categorical variables with small expected cell counts were analyzed using Fisher's exact test (Monte Carlo simulation). A binary logistic regression model was constructed to identify predictors of severe hyperbilirubinemia (total serum bilirubin ≥17 mg/dL), with multicollinearity assessed by variance inflation factor (VIF) and model fit by the Hosmer-Lemeshow test.
Results: ABO incompatibility was the most common hemolytic cause (45.9%, n=62), followed by RhD (32.6%, n=44) and minor blood group incompatibility (21.5%, n=29). While no statistically significant differences were detected in baseline bilirubin levels across the three groups (Kruskal-Wallis H = 0.962, p = 0.618), the overall phototherapy duration differed significantly (H = 6.785, p = 0.034). However, pairwise post-hoc comparisons did not survive Bonferroni correction (all adjusted p > 0.05). Exchange transfusion and direct Coombs test (DCT) positivity rates differed significantly across groups (Fisher's exact test, p = 0.006 and p = 0.002, respectively), with all exchange transfusions confined to the RhD group. No significant differences were observed between major (ABO + RhD combined) and minor incompatibility for any outcome. In the logistic regression model (after removing gestational age due to multicollinearity, VIF = 31.9), birth weight emerged as an independent predictor of severe hyperbilirubinemia (adjusted OR 1.001, 95% CI 1.000–1.003, p = 0.011), while male gender showed an unexpected apparent protective effect (adjusted OR 0.300, 95% CI 0.101–0.894, p = 0.031) that contradicts established literature and likely reflects chance variation in this small sample. The model demonstrated adequate fit (Hosmer-Lemeshow p = 0.688) but was limited by 5.0 events per variable.
Conclusion: ABO incompatibility remains the dominant hemolytic etiology in our setting, but minor blood group incompatibility accounted for over one-fifth of cases and produced severe hyperbilirubinemia rates comparable to RhD. The statistically significant differences in treatment intensity (exchange transfusion, DCT positivity) support the need for systematic antibody screening in neonates with pathological jaundice. The comparable severity of minor and major incompatibility challenges the assumption that minor blood group mismatches are invariably mild.

ABO incompatibility; RhD incompatibility; minor blood group incompatibility; neonatal hyperbilirubinemia

1. Bhutani VK, Zipursky A, Blencowe H, et al. Neonatal hyperbilirubinemia and rhesus disease of the newborn: incidence and impairment estimates for 2010 at regional and global levels. Pediatr Res. 2013;74(Suppl 1):86-100. [Google Scholar] [Crossref]

2. Moise KJ. Management of rhesus alloimmunization in pregnancy. Obstet Gynecol. 2008;112(1):164-176. [Google Scholar] [Crossref]

3. Zipursky A, Bhutani VK. Neonatal hyperbilirubinemia: an evidence-based approach to care. BMJ. 2020;369:m1004. [Google Scholar] [Crossref]

4. American Academy of Pediatrics Subcommittee on Hyperbilirubinemia. Management of hyperbilirubinemia in the newborn infant 35 or more weeks of gestation. Pediatrics. 2004;114(1):297-316. [Google Scholar] [Crossref]

5. Karagol BS, Zenciroglu A, Okumus N, et al. Hemolytic disease of the newborn caused by irregular blood subgroup incompatibilities: report of 106 cases. Am J Perinatol. 2012;29(6):449-454. [Google Scholar] [Crossref]

6. Bose B, Chakraborty S, Chakraborty S, Chaudhuri S. ABO hemolytic disease of the newborn: a hospital-based prospective study. Indian J Pediatr. 2018;85(5):367-371. [Google Scholar] [Crossref]

7. Yasar B, Caksen H, Abuhandan M, et al. Minor blood group incompatibility as a cause of neonatal hyperbilirubinemia: a single-center experience. J Matern Fetal Neonatal Med. 2021;34(14):2254-2260. [Google Scholar] [Crossref]

8. Smits-Wintjens VEHJ, Walther FJ, Lopriore E. Rhesus haemolytic disease of the newborn: postnatal management, associated morbidity and long-term outcome. Semin Fetal Neonatal Med. 2008;13(4):265-271. [Google Scholar] [Crossref]

9. Jauniaux RM, Rodeck CH. Rhesus alloimmunization: the obstetrician's view. BMJ. 1995;311(7005):663-664. [Google Scholar] [Crossref]

10. Moran P, Robson SC, Reid M, Lindow SW, et al. Antenatal screening for haemolytic disease of the fetus and newborn due to other red cell antibodies: a population-based study. BJOG. 2018;125(13):1687-1696. [Google Scholar] [Crossref]

11. Okwundu CI, Nagpal S, Musekiwa A, Bhakta N. Phototherapy for neonatal jaundice: an overview of systematic reviews. Cochrane Database Syst Rev. 2020;(12):CD012207. [Google Scholar] [Crossref]

12. Kaplan M, Muraca M, Hammerman C, et al. Imbalance between production and conjugation of bilirubin: a fundamental concept in the mechanism of neonatal jaundice. Pediatrics. 2002;110(4):e47. [Google Scholar] [Crossref]

13. Christensen RD, Yaish HM, Lemons RS. Neonatal hemolytic jaundice: morphologic features of erythrocytes that will help you diagnose the underlying condition. Neonatology. 2014;105(4):243-249. [Google Scholar] [Crossref]

14. Huang MJ, Kua KE, Teng HC, et al. Risk factors for severe hyperbilirubinemia in neonates. Pediatr Res. 2004;56(5):682-689. [Google Scholar] [Crossref]

15. Vandborg PK, Hansen BM, Greisen G, et al. Dose-response relationship of phototherapy for hyperbilirubinemia. Pediatrics. 2012;130(2):e352-e357. [Google Scholar] [Crossref]

16. Kaplan M, Bromiker R, Hammerman C, et al. Post-phototherapy neonatal bilirubin rebound: a potential cause of significant hyperbilirubinaemia. Arch Dis Child Fetal Neonatal Ed. 2006;91(2):F120-F123. [Google Scholar] [Crossref]

17. Yueh MF, Chen S, Nguyen N, et al. Developmental, genetic, dietary, and xenobiotic influences on neonatal hyperbilirubinemia. Mol Pharmacol. 2017;91(4):297-304. [Google Scholar] [Crossref]

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