The relationship between maternal anti-A\-B Titers and the severity of positive direct antiglobulin test ABO-Hemolytic disease of the newborn in a syrian population

Mohamad A Mahfood; Suzanne Alshemali; Mohamad Ayman AWAMA

doi:10.4103/joah.joah_101_22

ORIGINAL ARTICLE

Year : 2023 | Volume : 14 | Issue : 2 | Page : 108-114

The relationship between maternal anti-A\-B Titers and the severity of positive direct antiglobulin test ABO-Hemolytic disease of the newborn in a syrian population

Mohamad A Mahfood¹, Suzanne Alshemali², Mohamad Ayman AWAMA¹
¹ Department of Biochemistry and Microbiology, Faculty of Pharmacy, Tishreen University, Latakia, Syria
² Department of Laboratory Medicine, Faculty of Medicine, Tishreen University, Latakia, Syria

Date of Submission	15-Nov-2022
Date of Decision	18-Jan-2023
Date of Acceptance	30-Mar-2023
Date of Web Publication	27-Jul-2023

Correspondence Address:
Dr. Mohamad A Mahfood
Department of Biochemistry and Microbiology, Faculty of Pharmacy, Tishreen University, Latakia
Syria

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/joah.joah_101_22

Abstract

AIMS: The aim of this study was to investigate the relationship between maternal anti-A\-B titers and the values of hemoglobin and indirect bilirubin in ABO-hemolytic disease of the newborn (ABO-HDN) afflicted newborns and to develop a cutoff of total and immunoglobulin G (IgG) antibodies for treatment needs in neonates in Latakia.
STUDY DESIGN: A case–control study of 40 positive direct antiglobulin test (DAT) neonates who were ABO incompatible with their mothers and 20 healthy DAT-negative ABO incompatible neonates. Total and IgG anti-A\-B titers were compared between the two groups, and their correlation with newborns' hemoglobin and indirect bilirubin and the need for treatment were evaluated.
RESULTS: Both the maternal anti-A\-B total and IgG titers in the ABO-HDN group were significantly higher than in the control group (P < 0001). A significant correlation between maternal IgG titers and indirect bilirubin was found, but not between total antibody titers and indirect bilirubin. A negative correlation between total and IgG titers and hemoglobin was also documented. The IgG cutoff value for ABO-HDN incidence and the need for treatment was 128 with sensitivity and specificity of 92.5% and 95%, and the total antibodies cutoff value was 64, with a sensitivity and specificity of 62.5% and 90%, respectively.
CONCLUSION: Maternal total and IgG anti-A\-B can be an important prediction for the occurrence of ABO-HDN and evaluation of the neonates' hyperbilirubinemia after birth. Therefore, the routine detection of the titer of these antibodies and especially in mothers who have a high antibodies titer and a record of a previous jaundiced neonate can be an additional step in the ABO-HDN assessment of neonate.

Keywords: ABO mismatch, ABO-hemolytic disease of fetus and neonates, hyperbilirubinemia, total and immunoglobulin G anti-A\B

How to cite this article:
Mahfood MA, Alshemali S, AWAMA MA. The relationship between maternal anti-A\-B Titers and the severity of positive direct antiglobulin test ABO-Hemolytic disease of the newborn in a syrian population. J Appl Hematol 2023;14:108-14

How to cite this URL:
Mahfood MA, Alshemali S, AWAMA MA. The relationship between maternal anti-A\-B Titers and the severity of positive direct antiglobulin test ABO-Hemolytic disease of the newborn in a syrian population. J Appl Hematol [serial online] 2023 [cited 2023 Sep 27];14:108-14. Available from: https://www.jahjournal.org/text.asp?2023/14/2/108/382406

Introduction

Hemolytic disease of fetus and neonates (HDFN) is a clinical condition caused by the shortened survival of the newborn's red blood cells (RBCs) due to maternal antibodies.^[1] After the advent of prophylaxis with anti-D in 1969 which led to a remarkable reduction of Rh D alloimmunization, ABO incompatibility has become the main cause of HDFN in newborns.^[2] The occurrence of ABO incompatibility ranged from 15% to 25% of pregnancies, and ABO-hemolytic disease of the newborn (ABO-HDN) incidence after birth from 1% to 4% is related to the race of the population.^[3],[4]

The clinical manifestations of ABO-HDN include neonatal anemia, ranging between mild mostly and severe,^[5] early onset of hyperbilirubinemia that can develop into kernicterus in severe cases,^[6],[7] and rarely spontaneous abortion of the fetus.^[8]

Immunoglobulin G (IgG) is considered the only antibody capable of crossing the placenta. Hence, it is the only class of antibodies implicated in HDN pathogenesis.^[9],[10] This makes it reasonable to investigate the titer of IgG Anti-A/-B maternal antibodies after delivery, but there is a difference of opinions about the correlation between the severity of neonate hemolysis and the titer of these antibodies in the mother's serum.^[11]

Owa et al. reported in their studies in Nigeria that maternal antibodies titer of 64 was a cutoff for both treatment and exchange transfusion.^[12] Other studies suggested a titer of 512 as a cutoff for cases needing treatment.^[3],[13] Kadri found that newborns to mothers who had a titer >256 are at risk to develop jaundice 18 times higher than those with titer <256.^[14] On the other hand, several studies found no correlation between maternal antibody Anti-A\-B titers and the probability of ABO-HDN incidence.^[15]

In this study, our aim was to evaluate the correlation between the maternal anti-A\-B titers and the occurrence of ABO-HDN and its severity in the newborns, as expressed by the hemoglobin and bilirubin values and the probability of hemolytic disease in neonates in Syria.

Participants

This study was conducted on 60 cases of newborns with ABO incompatibility (neonates with A or B blood group to O mother) in Obstetrics and Pediatric Hospital and Tishreen University Hospital from March 1 to December 31, 2021. All mothers included in this study were negative for irregular antibodies as determined by antibody screening tests.

Cases were distributed into two groups: The ABO-HDN group (n = 40) included newborns with positive direct antiglobulin test (DAT) and developed anemia and\or jaundice and the control group (n = 20) included DAT-negative healthy newborns to ABO incompatible mothers.

Exclusion criteria included (1) Newborns with hemolysis by the incompatibility of other blood groups (Rh-D, E, etc.) and other RBCs disorders (thalassemia, G6PD deficiency, etc.), (2) the presence of accompanying infections or C-reactive protein >5 mg\dl, and (3) gestational age (GA) <35 weeks.

Ethics

The ethics committee of the Faculty of Pharmacy reviewed and approved the study, approve number 510. The mothers were asked to participate, and the blood samples were collected after obtaining informed consent.

Blood samples

Venous neonatal blood was collected into ethylenediaminetetraacetic acid (EDTA) tubes for DAT test and hematological profiling. If DAT was positive, 5 ml of venous maternal blood was collected into an EDTA tube for the antibody screening test and titration of the total and IgG antibody titer.

Materials and Methods

Direct antiglobulin test

Coombs AntiHuman Globulin Gel cards (Biorex Diagnostics, Antrim Technology Park, UK) were used. About 0.8% suspension of newborn erythrocytes in saline was prepared, then, 50 μl of the suspension was added to the chamber of the card and centrifuged at 2000 rpm for 10 min, and the reaction was graded as specified by the manufacturer's instruction.

Maternal total anti-A\-B titer

Immediate spin (IS) tube method was used for total antibody titration. 3%–5% saline-suspended group A or group B RBCs were added to tubes containing similar volumes of saline's serial dilutions of the plasma sample. After incubation for 5 min and centrifugation for 20 s at 3000 rpm, the result was determined macroscopically, and if negative, microscopically.^[16]

Maternal immunoglobulin G Anti-A\-B titer

One hundred μl of mother's plasma was added to 100 μl of 2-mercaptoethanol (2-ME) 0.1% (Sigma-Aldrich-USA M3148) and incubated for 45 min at 37°C with occasional shaking. The IS tube method and column agglutination technique (CAT) were used for IgG antibody titration. For IS method, after making doubling dilutions of 100 μl treated serum, 50 μl of 3%–5% saline-suspended group A or group B RBCs was added. The results were determined as previously described in the IS method for total antibody titration.

For the Indirect antiglobulin test (IAT) method, 0.8% suspensions of A and B blood groups from healthy blood donors were prepared. A volume of 25 μl of 2-ME-treated maternal plasma was then added and mixed with 50 μl of the corresponding suspension in the card chamber and incubated at 37°C for 15 min and the result was documented after centrifugation.^[16]

Statistical analysis

IBM ® SPSS ® Vol.24, Chicago, USA was used for data analysis, Shapiro–Wilk test was used for the normality test, and the normal distribution data were analyzed by the independent sample t-test. Mann–Whitney U-test, Chi-square, and Fisher's exact probability were used for nonnormal distribution data and the Wilcoxon rank sum test for comparison between groups. Spearman analysis was used to detect the correlation between two categorical factors, the receiver operating characteristic (ROC) curve for the diagnostic efficiency, and P ≤ 0.05 was considered statistically significant.

Results

[Table 1] represents the clinical data of mothers and newborns in both ABO-HDN and control groups. There were no significant differences between the two groups in terms of gravidity, GA of the mother, or sex, birth weight, and blood group distribution of the newborn (P > 0.05), but a family history of previous ABO-HDN was significantly higher in the ABO-HDN group (P < 0.01).

Table 1: Clinical data comparison between the ABO-hemolytic disease of the newborn and control groups

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The maternal total Anti-A and Anti-B titers were both significantly higher in the ABO-HDN group in comparison with the control group using IS method for titration, 64 (8–512) versus 8 (8–64) with P < 0.0001, as shown in [Figure 1].

Figure 1: Comparison of maternal total antibody titer using IS between the ABO-HDN group and the control group. ABO-HDN = ABO-Hemolytic disease of the newborn, IS = Immediate spin

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Our data shows that 25 (62.5%) newborns in the ABO-HDN group had total titer ≥64 while only 2 (10%) newborns in the control group had total titer = 64. [Table 2] shows the titer distribution in both groups using IS.

Table 2: Comparison of total titer between the ABO-hemolytic disease of the newborn group and the control group

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Likewise, the maternal IgG Anti-A\-B was significantly higher in the ABO-HDN group compared to the control group 64 (8–512) versus 8 (8–64), with P < 0.0001, as shown in [Figure 2] and [Figure 3].

Figure 2: Comparison of IgG titer using IAT between the ABO-HDN group and the control group. IgG = Immunoglobulin G, IAT = Indirect antiglobulin test , ABO-HDN = ABO-Hemolytic disease of the newborn

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Figure 3: Comparison of IgG titer using IS between the ABO-HDN group and the control group. IgG = Immunoglobulin G, IS = Immediate spin, ABO-HDN = ABO-Hemolytic disease of the newborn

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Notably, 37 (92.5%) newborns in the ABO-HDN group had IgG titer ≥64, while only 6 newborns in the control group had IgG titer of 64. [Table 3] shows the IgG titer distribution in both groups.

Table 3: Comparison between immunoglobulin G titers using indirect antiglobulin test in the ABO-hemolytic disease of the newborn and the control groups

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Using IS method for IgG titration, there was a significant difference in IgG Anti-A\-B distribution between the groups (P < 0.0001), with 34 (85%) newborns having antibody titer ≥4 in the ABO-HDN group, while only 3 out of 20 newborns in the control group with titer ≥4.

There was a strong negative correlation between maternal total Anti-A\-B titer and newborn' hemoglobin (r = −0.3, P = 0.018), as shown in [Figure 4].

Figure 4: The correlation between total anti-A\-B titer and newborns' hemoglobin

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Moreover, there was a strong negative correlations between maternal IgG anti-A\-B titration and newborn hemoglobin by the two methods were found; with r = −0.35, P = 0.0068 for the CAT method, and r = −0.38, P = 0.0027 for the IS method, as depicted in [Figure 5].

Figure 5: Correlation between IgG titer and newborn hemoglobin using CAT and IS methods. IgG = Immunoglobulin G, CAT = Column agglutination technique

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No correlation was found between the mother's total anti-A\-B titer and newborn total bilirubin (r = 0.15, P = 0.34), as shown in [Figure 6].

Figure 6: Correlation between total antibodies titer and newborns' total bilirubin

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There was a strong positive correlation between maternal IgG Anti-A\-B titer using CAT method and newborns' total bilirubin (r = 0.44, P = 0.00049), but no correlation was found using IS methods (r = 0.14, P = 0.37), as shown in [Figure 7],[Figure 8].

Figure 7: Correlation between IgG titer and newborns' bilirubin using IAT and IS. IgG = Immunoglobulin G, IAT = Indirect antiglobulin test , IS = Immediate spin

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Figure 8: Correlation between total antibodies titer using IS and IgG antibodies using both IAT and IS. IS = Immediate spin, IgG = Immunoglobulin G, IAT = Indirect antiglobulin test

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There was a positive correlation between maternal total anti-A\-B titer and IgG anti-A\-B using the two methods in our study. The correlation was strong in the CAT method (r = 0.31, P = 0.017) and medium in IS method (r = 0.47, P = 0.00017).

In the ABO-HDN group, 30 newborns (75%) received a phototherapy treatment, 4 (10%) received an exchange transfusion and phototherapy, and 6 (15%) neonates developed jaundice without the need for treatment. Using the ROC curve of the diagnostic value of maternal total antibody titer, it indicated that the area under the curve (AUC) of total anti-A\-B titer was 0.839, and the cutoff value for predicting the risk of ABO-HDN and the need for treatment was 64 with a sensitivity of 62.5% and a specificity of 90%, as shown in [Figure 9].

Figure 9: Predicting the risk of ABO-HDN and the need for treatment using maternal total titer. ABO-HDN = ABO-Hemolytic disease of the newborn

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The ROC curve of maternal IgG antibody titer using the CAT method showed that the AUC of IgG titer was 0.989, the cutoff value for predicting the risk of ABO-HDN and need for treatment was 128 with a sensitivity of 92.5% and a specificity of 95%, as shown in [Figure 10].

Figure 10: Predicting the risk of ABO-HDN and the need for treatment using maternal IAT IgG titer. ABO-HDN = ABO-Hemolytic disease of the newborn, IAT = Indirect antiglobulin test, IgG = Immunoglobulin G

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Discussion

The ABO hemolytic disease of neonates is felt the most common reason for neonate hemolytic disease worldwide.^[17] Most cases of ABO-HDN are mild with hyperbilirubinemia, phototherapy often is sufficient, but there were severe cases that needed exchange transfusion. In some countries, ABO-HDN is no less essential than Rh-HDN as a cause for exchange transfusion. In a study in Iceland, among 8 neonates who required exchange transfusion, 5 of these had Rh antibodies and 3 had ABO Blood group incompatibility.^[18]

Our study included 40 DAT-positive ABO incompatible neonates who developed significant jaundice and required treatment and medical care and 20 healthy DAT-negative neonates. Then, total and IgG Anti-A\-B antibodies were carried out and its relationship with neonate's hemoglobin, indirect bilirubin, and their need for therapy. Phototherapy was a satisfactory treatment in 75% of ABO-HDN group neonates. Ten percent of the ABO-HDN group neonates required exchange transfusion, which is considered a clinically significant proportion.

Our study was limited to DAT-positive ABO incompatible neonates, DAT positivity in ABO mismatch cases increased the probability of receiving phototherapy and exchange transfusion. In addition, positive DAT neonates had higher IgG Anti-A\-B in comparison with DAT-negative neonates. In Rahmati et al.'s study, ABO mismatch was the most common reason for DAT positivity in neonates.^[19] In another study, ABO incompatibility was the common cause of DAT positivity in Iceland between 2002 and 2015.^[18] We intend in the following study to investigate the ABO mismatch in DAT-negative neonates and the elution test.

The routine practice of early discharge of newborns leads to a greater rate of hyperbilirubinemia and readmission.^[20] ABO HDN is a major cause of hyperbilirubinemia and identifying susceptible newborns will facilitate early intervention, which can mitigate invasive therapies and the occasional severe sequelae.

Some studies identified the maternal IgG titer of 64 as a cutoff value and neonates with a titer ≥64 are at risk of developing severe HDN.^[12] In our study, all the neonates in the ABO-HDN group had a titer ≥64. This is consistent with the literature. However, in the control group, there were 6 newborns with a titer of 64 and did not develop jaundice, which may be due to many factors such as antibody subtype, placental transfer, and others.^[21]

Our study suggests the titer 128 as a cutoff value for ABO-HDN development and the need for treatment with a sensitivity of 92.5% and specificity of 95%, which differ from Bakkeheim et al. in Norway (cutoff = 512),^[3] Krog et al. in Denmark (cutoff = 256),^[9] and Kadri in Malaysia (cutoff = 256).^[14] These studies had higher cutoff titers.

The titer of maternal total and IgG Anti-A\-B using IS is a simple and common method that can predict the incidence of ABO-HDN and does not need any complex equipment. It can be done in any transfusion center or underdeveloping laboratory. Our result is compatible with Usha and Sulochana,^[22] who found a direct correlation between total and IgG Anti-A\-B and ABO-HDN.

We find that the gender and blood group of the newborn did not affect on the occurrence of ABO-HDN, which is compatible with Firouzi et al.^[23] in Iran, Akgül et al. in Turkey,^[24] and Abbas et al. in Iraq.^[25] However, this differs from Krog et al.^[9] and Kaplan et al.^[26] found a significant relation between ABO-HDN and the newborn blood group. This can be explained by the ethnic difference between the participants in our study and others.^[27]

We find that a pregnant woman with high Anti-A\-B titers who had a previous history of having a jaundiced neonate was at high risk of having another baby with serious ABO-HDN, this outcome is compatible with Rieneck et al.^[28] It proves the importance of monitoring the antibody titer in mothers who have previously given birth to a child with jaundice due to ABO-HDN.

We studied the correlation between maternal total and IgG Anti-A\-B and newborns' hemoglobin, and the correlation was strong. It is speculated that the assay of the maternal total and IgG Anti-A\-B prenatal or perinatal can be a great indicator to predict the probability of ABO-HDN in newborns.

Acknowledgment

The authors thank all the health-care officials and workers at Obstetrics and Pediatric Hospital and Tishreen University Hospital, Department of Pediatrics, for the help with data and sample collection, and for providing the medical care for neonates needs it.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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