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Maternal serum and cord blood anti-A and Anti-B antibody levels as a predictor of significant hyperbilirubinemia in newborns


1 Department of Paediatrics and Neonatology, Pondicherry Institute of Medical Sciences, Puducherry, India
2 Department of Pathology and Transfusion Medicine and Obstetrics and Gynaecology, Pondicherry Institute of Medical Sciences, Puducherry, India
3 Department of 2Obstetrics and Gynaecology, Pondicherry Institute of Medical Sciences, Puducherry, India

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Date of Submission16-Dec-2021
Date of Decision01-Jun-2022
Date of Acceptance26-Jun-2022
Date of Web Publication12-Dec-2022
 

   Abstract 

BACKGROUND: Hemolytic disease of the newborn (HDN) was more common due to Rh incompatibility. Its prevalence has decreased due to introduction of Immunoglobulin G (IgG) prophylaxis against RhD antigen. HDN due to ABO incompatibility has become more common.
AIM: The aim of this study was to study the predictive value of umbilical cord serum and maternal serum anti-A and anti-B antibody titer for the occurrence of significant hyperbilirubinemia in newborns.
SETTINGS AND DESIGN: This was a prospective cohort study which included 139 “O” blood group mothers and their offspring with A or B blood groups.
MATERIALS AND METHODS: We analyzed the IgG anti-A, and anti-B antibody titers from cord blood and maternal serum and correlated them with features of significant hyperbilirubinemia. Positive predictive value (PPV), negative predictive value (NPV), sensitivity and specificity were calculated.
RESULTS: Twenty-eight newborns out of 139 (20 %) developed significant hyperbilirubinemia and required phototherapy. One newborn required immunoglobulin infusion. Out of these 28 newborns' maternal serum, 22 (79%) newborns' maternal serum had IgG antibody titer of ≥128. Cord blood serum IgG antibody titer was 1:2 in all newborns, which was not significant. The direct Coombs test was positive in six (4%) newborns with maternal IgG antibody titer ≥128.
CONCLUSION: Maternal IgG antibody titer was ≥1:128 and can predict significant hyperbilirubinemia in newborns with a sensitivity of 53.5%, specificity of 98.2%, PPV of 88%, NPV of 89%, and P value (P > 0.001).

Keywords: Direct Coombs test, hemolytic disease of the newborn, indirect Coombs test, total serum bilirubin


How to cite this URL:
Toshniwal PU, Krishnan L, Kingsley S, Kurvila S, Manikandan M. Maternal serum and cord blood anti-A and Anti-B antibody levels as a predictor of significant hyperbilirubinemia in newborns. Asian J Transfus Sci [Epub ahead of print] [cited 2023 Jan 28]. Available from: https://www.ajts.org/preprintarticle.asp?id=363222



   Introduction Top


ABO incompatibility, Rh incompatibility, and G6PD deficiency are major causes contributing to significant neonatal hyperbilirubinemia. After the introduction of Rh immunoglobulin G (IgG) prophylaxis, Rh hemolytic disease has dramatically declined. Hence, ABO incompatibility is now the most common cause of hyperbilirubinemia in newborns.[1]

ABO incompatibility was first reported by Halbrecht (1944) and Rosenfield (1955). Thereafter, many studies have been conducted to determine the appropriate laboratory test to confirm ABO incompatibility.[2] The prevalence of ABO hemolytic disease is 15%–25% of all maternal and fetal pairs. However, ABO hemolytic disease of the newborn (HDN) is confined to approximately 1% of group “O” women who have antenatal high titer IgG antibodies. HDN occurs exclusively in newborns of blood group “A” and “B” born to mothers of blood group “O.”[3] The incidence of ABO HDFN is reported as 0.33%–2.2%.[1] ABO HDFN occurs almost exclusively in neonates of blood group “A” or “B” who are born to group “O” mothers,[2] because IgG anti-A or anti-B occurs commonly in group “O” than in group “A” or “B.”[2] Anti-A and anti-B antibodies in group “O” individuals predominantly tend to be IgG type and, therefore, can cross the placenta and cause Hemolytic disease of foetus and Newborn.[2]

Jaundice due to ABO incompatibility is often present as a benign condition, not requiring any intervention, but some newborns may be severely affected.[4] ABO HDN tends to be relatively mild in nature, because red blood cells (RBCs) of the newborn have not matured to have adult levels of “A” and “B” antigens. However, the strength of newborn ABO blood group antigens can vary, and therefore, the degree of hemolysis and the severity of HDN can be unpredictable.[5]

Close monitoring of these newborns is required to prevent the rise of total serum bilirubin (TSB) ≥20 mg/dl, which can lead to acute bilirubin encephalopathy, kernicterus, and sensorineural hearing loss. The American Academy of Pediatrics (AAP) 2004 guidelines recommend predischarge reassessment of newborns to look for the development of hyperbilirubinemia. Severe hyperbilirubinemia is a major cause of readmission of babies to hospital. Hence, identifying the cause of hyperbilirubinemia is very important to prevent complications.[6] This study aims to predict significant hyperbilirubinemia in newborns from maternal and cord blood anti-A and anti-B antibody titers. Hence, the newborn at risk of significant hyperbilirubinemia can be closely monitored and treated.


   Materials and Methods Top


This is a prospective cohort study conducted at the Department of Pediatrics, Pondicherry Institute of Medical Sciences, Puducherry, India. The study aimed to predict the occurrence of significant hyperbilirubinemia by analyzing the maternal and cord blood IgG antibody titer of blood group “O” mothers delivered with a newborn of blood group “A” or “B.” This study was done from November 2016 to April 2018 and is approved by the Institute Review Board and Ethics Committee.

The inclusion criteria were babies born with blood group “A” or “B” to an “O” blood group mother with a gestational age ≥34 weeks and weight ≥2000 g.

The exclusion criteria include all the newborns with nonhemolytic cause of jaundice and all mothers with A, B, or AB blood group, Rh incompatibility, and mothers with positive indirect Coombs test (ICT).

One hundred and thirty-nine “O” blood group mothers and their offspring with either “A” or B blood groups were included in this study.

Collection of samples

Maternal blood

All eligible mothers with “O” blood group were confirmed in the hospital information system, and 4 ml of clotted blood was drawn from the mother at the time of labor and sent to blood bank for maternal IgG anti-A and B titers. This sample was centrifuged at 3000 rpm/min for 3 min and serum was separated and stored at −70°C until the offspring's blood group was confirmed.

Cord blood

Immediately after the birth, umbilical cord blood samples (2-ml ethylenediaminetetraacetic acid and 4-ml clotted) were collected from the babies born to mothers of blood group “O” for blood grouping and typing, direct Coombs test (DCT), peripheral smear for spherocytes, hemoglobin level, reticulocyte count, and red cell distribution width (RDW). Clotted blood was sent to blood bank for IgG antibody titers. All the samples were labeled with name and hospital number at the collection site.

All babies were continuously checked for the development of significant hyperbilirubinemia clinically and doing TSB at the 48th h after delivery.

Procedures

The blood group of the babies was identified by ABD forward (Tulip Gel Matrix) card, DCT by gel card (IgG + anti-C3d) according to blood bank standard operating protocol. ICT was performed in multigravida mothers to exclude the other irregular blood group antibodies. Complete blood count was analyzed by Horiba automated analyzer, peripheral smear by Leishman stain, and reticulocyte count with supravital stain.

Antibody titer

The titer of an antibody was determined by testing the serial two-fold dilution of the serum against selected red cells.

The IgG antibody titration was done in maternal serum treated with 0.01 M dithiothreitol (DTT). Corresponding 3%–5% saline suspended pooled red cells were used for titration . The 0.01 M DTT was prepared by dissolving 0.154 g of DTT (Sigma-Aldrich, St. Louis, MO, USA), in 100 mL of PBS , pH 7.3. Grading of the reaction was done according to the American Association of Blood Bank guidelines.[7] The titer is the reciprocal of the highest serum dilution at which macroscopic agglutination is observed.

Babies were followed up for the development of clinical jaundice soon after birth. All the babies were followed up for hyperbilirubinemia at 24 h by transcutaneous bilirubinometer and clinical icterus and at the 48th h by TSB.

Criteria for phototherapy, intravenous immunoglobulin, and exchange transfusion in newborn

In this study, we have taken TSB at the 48th h as per hospital protocol, hence all newborns and their TSB values at the 48th h were taken for comparing with cord blood and maternal serum IgG antibody titers for predicting hyperbilirubinemia.

Hyperbilirubinemia is defined as TSB level that exceeds the hour-specific threshold value of phototherapy, according to the guidelines presented by the AAP. We have taken the TSB value at the 48th h and plotted it in the nomogram given by AAP. This nomogram has three zones to consider hyperbilirubinemia in newborns based on their risk factors. The first one is for low-risk newborns or no risk factor, the second one is for intermediate risk, and the third is for those with high risk. Phototherapy should be started to the newborn with hemolytic jaundice in high-risk zone and when the cutoff is ≥11 mg/dl. The TSB cutoff for intravenous immunoglobulin (IVIG) and exchange transfusion is 16 mg/dl at the 48th h of life as per AAP nomogram for exchange transfusion.[8] The requirement of phototherapy increased with increasing titers. This suggested that the more the maternal titers, the greater is the risk of development of significant hyperbilirubinemia and required phototherapy.

Sensitivity, specificity, positive predictive value, and negative predictive value

To find out the anti-A and anti-B antibody titers as predictors of the occurrence of clinical significant jaundice, a calculation was made on the sensitivity, specificity, positive predictive value (PPV), and negative predictive value at the cutoff point of 1/16,1/32,1/64,1/128,1/256, and 1/512 and plotted on the receiver operating characteristic curve (ROC). Sensitivity, specificity, PPV, and negative predictive value were calculated with antibody titers and compared with those newborns who received phototherapy with the help of 2 × 2 table.

Statistical analysis

Assuming a prevalence of 15%, for a sensitivity of 90%, precision of 5%, and confidence level of 95%, 139 “O” group mothers and their A or B group babies require to be studied. The data were analyzed using the IBM SPSS Statistics for Windows, version 21.0, 2018 (IBM Corp, Armonk, New York, USA).

Parameters including sensitivity, specificity negative, PPV, and negative predictive value of maternal Coombs titers were calculated with the significant hyperbilirubinemia. Pearson Chi-square test was used to compare the antibody titers with Hb, TSB, DCT, and reticulocyte percentage. Chi-square test was used to compare the phototherapy with titers. P < 0.05 was considered statistically significant.


   Results Top


One hundred and thirty-nine newborns were included in this study. Of the 139 newborns, 81 (58%) were males and 58 (42%) were females and 79 (57%) were “B” group and the remaining 60 (43%) were “A” blood group [Table 1].
Table 1: Demography of the study population

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Among the 139 newborn babies, 65 (47%) had hemoglobin of <15 gm%, 15 (11%) had elevated reticulocyte counts, 6 (4%) had positive DCT, peripheral smear showed spherocytes in 5 (3.5%) newborns, and 28 (20%) developed significant hyperbilirubinemia [Table 2].
Table 2: Laboratory parameters

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Among the 28 newborns with significant hyperbilirubinemia, 16 (57%) were “B” blood group and 12 (43%) were “A” blood group [Table 3], 9 (32%) had low hemoglobin levels, 6 (21%) had positive DCT, 7 (54%) had high reticulocyte percentage, and 5 (18%) newborns' peripheral smear showed spherocytes [Table 2].
Table 3: Distribution of blood group based on significant hyperbilirubinemia

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In the cord blood, IgG antibody titer was 1:2 in all the samples. In the maternal serum sample, 62 (45%) had more than 1:128 titer and 77 (55%) had <1:128 titer. Antibody titers were compared with offspring who developed significant hyperbilirubinemia, 22 (78.5%) babies developed significant hyperbilirubinemia with the titer more than 1:128, and 6 (21.5%) babies developed significant hyperbilirubinemia with the titer <1:128 [Table 4].
Table 4: Distribution of maternal and cord blood IgG antibody titer

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The ROC curve [Figure 1] was drawn by a nonparametric method using SPSS software, and area under curve (AUC) was 0.802. This curve and corresponding AUC showed that among the 139 maternal sera, IgG titer ≥1:128 newborns developed significant hyperbilirubinemia.
Figure 1: ROC for IgG antibody titer in predicting development of significant neonatal hyperbilirubinemia

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Among the 28 neonates identified with significant hyperbilirubinemia, 22 (79%) received phototherapy with titer ≥1:128 and 6 (21%) developed titer ≤1:128 [Table 5].
Table 5: Influence of maternal anti-A and anti-B antibody titer on phototherapy

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The sensitivity, specificity,positive predictive value (PPV), and negative predictive value (NPV) were calculated for maternal IgG antibody titers in predicting significant neonatal hyperbilirubinemia. In the 1:128 titer, sensitivity (78.5%), specificity (64%), PPV (35.4%), NPV (92.2%), odds ratio [OR] = 6.5, 95% confidence interval [CI] = 2.4–17.3, and P = 0.0002 were deduced [Table 5].

Hemolytic features were compared between maternal IgG antibody titer ≥1:128 and ≤1:128 with the laboratory parameters. The TSB at the 48th h of life was statistically significant (P < 0.001) with a mean of 10.55 mg/dl and hemoglobin was also statistically significant (P < 0.001) with a mean of 14.75 g/dl. Whereas reticulocyte count percentage (P = 0.289), RDW (P = 0.317), and mean corpuscular hemoglobin concentration (P = 0.283) values were not statistically significant. Newborns with positive DCT and the presence of spherocytes in the peripheral smear had titer ≥1:128 [Table 6].
Table 6: Maternal IgG antibody titer and blood parameters

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   Discussion Top


ABO incompatibility-induced hemolytic anemia is more common and less severe. In the “O” blood group mother, IgG antibodies are more than the mothers with blood group “A” or “B.” Severity of the ABO HDN is due to the strength of IgG anti-A and anti-B antibodies titer. The higher the antibody transfer transplacentally, the higher the destruction of newborns' red cells. In general, 15%–25% of all maternal/fetal pairs are ABO incompatible, but ABO HDN is roughly confined to 1% of such group “O” women who have antenatal high titer IgG antibodies.[9]

To predict the occurrence of clinically significant jaundice in ABO-incompatible pregnancies, it is important to know the cutoff point of the maternal anti-A and anti-B antibody titers. In our study, we tried to predict the occurrence of significant hyperbilirubinemia in neonates of A and B groups born to “O” group mothers based on cord blood and maternal IgG antibody titers.

Titration values can provide information on the relative amount of anti-A and anti-B antibodies present in serum, and this information is especially important in cases of ABO HDFN and ABO-incompatible bone marrow and solid organ transplantations.[10] Titration of ABO antibodies is usually performed with serial two-fold dilutions of serum with selected red cells.

In our study, 139 newborn babies were included. There were 60 (43%) A group neonates and 79 (57%) “B” group neonates. The number of neonates who had significant hyperbilirubinemia was 28 (20%). All these neonates were treated with phototherapy and one baby additionally required IVIG. None of the neonates required exchange transfusion.

In our study, 16/28 (57%) “B” blood group neonates developed significant hyperbilirubinemia and 12/28 (43%) were “A” blood group. Usha et al. in India found that A blood group neonates developed severe hyperbilirubinemia compared to B blood group neonates.[3] However, in our study, B blood group neonates were more than the A blood group. One of the reasons for this could be, our study included B blood group (79) babies more than that of A (60) blood group. However, blood groups were not statistically significant (P = 0.534) in babies with significant hyperbilirubinemia.

In our study, cord blood IgG antibody titer was 1:2 in all 139 neonates. Hemachandrika and Gunasingh in India found that IgG antibody levels in cord blood of group A and B blood neonates born to “O” were <1:32.[4] Ukita et al. from Japan found that seven (5%) neonates had significant jaundice and their cord IgG antibody titer was 1:4–1:64, whereas the maternal IgG antibody titer of these seven (5%) neonates was 1:1024–1:8092.[11]

In our study, 62 (44.6%) maternal sera showed IgG antibody titer ≥128 and 17 (12.2%) had IgG titer of 1:256. Bao et al. from China found that IgG antibody titer ≥64 is associated with hemolytic disease in a newborn.[12]

Among the 28 babies who had significant hyperbilirubinemia, 22 had ≥1:128 titer. The newborns with maternal IgG antibody titer ≥1:128 and <1:128 showed statistically significant (P < 0.05) differences for hemoglobin, TSB at 48 hours ,spherocytes and DCT positivity. This indicates that, if the maternal serum is more than 1:128, there is a chance of hemolytic anemia. Hence, titer ≥1:128 is considered to be a predictor of significant hemolysis in this study.

DCT is an important test to differentiate immune and nonimmune hemolytic anemia. In our study, among the six DCT-positive newborns, four (66.66%) developed significant hyperbilirubinemia and their IgG antibody titers were 1:256 and two babies were DCT positive but did not develop ABO HDN with the titer of 1:128. This was also seen by Ukita et al.'s study, where they found that among the 43 DCT-positive newborns, only 4 newborns developed hyperbilirubinemia. The authors found that DCT-positive newborns had IgG1 or IgG2 antibodies bound to RBC. If a small amount of antibodies are bound to the neonatal RBC, DCT might become positive, but neonates may not develop ABO HDN, whereas a large amount of antibodies bound to RBC will result in DCT positive and develop ABO HDN as well. Neonates with DCT negative with ABO HDN have IgG3 antibody bound to their RBC.[11]

Management of ABO HDN is usually successful with only the phototherapy provided by modern equipment. In our study, among the 28 newborns who developed significant hyperbilirubinemia, all the newborns received phototherapy. However, one newborn received phototherapy and IVIG with the titer of 1:256. Bakkeheim et al. noticed that invasive interventions such as IVIG and exchange transfusion increased with antibody titer ≥512 with a sensitivity of 90% and specificity of 72% for invasive treatment.[6] We also noticed that with titer ≥1:256, there is an increase in the requirement of phototherapy in a newborn. Kadri from Indonesia found that IgG antibody titer ≥1:256 in maternal serum is associated with the development of significant hyperbilirubinemia with a PPV of 79.27% and NPV of 17.57%.[11]

For the IgG antibody of 1:128 in maternal serum, sensitivity of 78.5%, specificity of 64%, PPV of 35.4%, NPV of 92.2%, OR of 6.5, and 95% CI of 2.4–17.3 with P < 0.005 were deduced from our study. We conclude that the predictive value for significant hyperbilirubinemia in maternal serum can be taken as an IgG antibody titer ≥1:128.

Requirement of phototherapy increased with increasing titers. This suggested that the more the maternal titers, the greater is the risk of development of significant hyperbilirubinemia and required phototherapy.


   Conclusions Top


In our study, we found that IgG antibody titers in the “O” blood group maternal serum ≥ 128 is a good predictor for the development of significant neonatal hyperbilirubinemia in ABO HDN with features of hemolysis. IgG antibody titer levels of A and B blood group newborns delivered by O group mother's cord blood are not predictive for the development of significant hyperbilirubinemia in ABO HDN. The method adopted is simple and does not require any special equipment or reagents. It can be routinely done in any blood center.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Han P, Kiruba R, Ong R, Joseph R, Tan KL, Wong HB. Haematolytic disease due to ABO incompatibility: Incidence and value of screening in an Asian population. Aust Paediatr J 1988;24:35-8.  Back to cited text no. 1
    
2.
Esan AJ. Hemolytic disorders of the newborn, current methods of diagnosis and treatment: A review study. Hematol Blood Transfus Disord 2016;3:1-18.  Back to cited text no. 2
    
3.
Usha KK, Sulochana PV. Detection of high risk pregnancies with relation to ABO haemolytic disease of newborn. Indian J Pediatr 1998;65:863-5.  Back to cited text no. 3
    
4.
Hemachandrika C, Gunasingh TS. Correlation between the levels of anti- A/B IgM/IgG antibodies and cord blood bilirubin levels in haemolytic disease of the new-born. Indian J Basic and Appl Med Res 2016;6:596-601.  Back to cited text no. 4
    
5.
Yaseen H, Khalaf M, Rashid N, Darwich M. Does prophylactic phototherapy prevent hyperbilirubinemia in neonates with ABO incompatibility and positive Coombs' test? J Perinatol 2005;25:590-4.  Back to cited text no. 5
    
6.
Bakkeheim E, Bergerud U, Schmidt-Melbye AC, Akkök CA, Liestøl K, Fugelseth D, et al. Maternal IgG anti-A and anti-B titres predict outcome in ABO-incompatibility in the neonate. Acta Paediatr 2009;98:1896-901.  Back to cited text no. 6
    
7.
Roback JD, Combs MR, Grossman BJ, Hillyer CD, editors. Technical Manual. 16th ed. Bethesda: American Association of Blood Banks; 2008.  Back to cited text no. 7
    
8.
American Academy of Pediatrics Subcommittee on Hyperbilirubinemia. Management of hyperbilirubinemia in the newborn infant 35 or more weeks of gestation. Pediatrics 2004;114:297-316.  Back to cited text no. 8
    
9.
Kadri N. The role of maternal anti-A and anti-B antibody titers in predicting ABO hemolytic disease of the newborn. Med J Indones 1998;7:79-83.  Back to cited text no. 9
    
10.
Bao R, Wang S, Yang Q. Clinical study of the relationship between prenatal antibody titer and hemolytic disease of newborn. Int J Clin Exp Med 2017;10:1637-42.  Back to cited text no. 10
    
11.
Ukita M, Takahashi A, Nunotani T, Kihana T, Watanabe S, Yamada N. IgG subclasses of anti-A and anti-B antibodies bound to the cord red cells in ABO incompatible pregnancies. Vox Sang 1989;56:181-6.  Back to cited text no. 11
    
12.
Sungnoon D, Viroje C, Sasijit V, Kleebsbai S. Association of maternal ABO IgG antibodies with neonatal jaundice due to ABO incompatibility at Siriraj Hospital. J Hematol Transfus Med 2016;26:2559.  Back to cited text no. 12
    

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Correspondence Address:
Simon Kingsley,
No 83, Vaigai Street, Vasantham Nagar, Puducherry - 605 110
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ajts.ajts_182_21



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