Clinical value of combination detection of direct antiglobulin test and serum albumin globulin ratio in severe hyperbilirubinemia caused by ABO hemolytic disease of the newborn

Abstract Background To explore of a combination of antiglobulin test(DAT) and albumin globulin ratio(AGR) could predict the severity of ABO hemolytic disease of the newborn(ABO-HDN). Methods The measurement of DAT, AGR and combination detection of DAT and AGR was done to predict severe ABO-HDN hyperbilirubinemia in 270 full-term infants based on whether the infants received transfusions of blood components. The infants were divided into three groups according to the results of DAT and ARG and compared the differences of phototherapy day and hospitalization day of the three groups. Results Of the 270 cases enrolled in this study, 69 infants were DAT positive. Peak total bilirubin, AGR, and positive DAT were independently associated with the need for blood components transfusion. ROC curve analysis for blood components transfusion showed that DAT cutoff value >± with a sensitivity of 39.4% and a specificity of 83.9%, AGR cutoff value <2.05 with a sensitivity of 54.1% and a specificity of 85.7%, and combination detection of DAT and ARG with a sensitivity of 62.1% and a specificity of 91.2%. The AUCs for DAT, AGR, and combination detection of DAT and AGR were .621, .740, and .750 respectively. The phototherapy day and hospitalization day were significantly longer in group of AGR <2.05 and DAT >± than that of a group of AGR <2.05 and group of DAT >±. Conclusions DAT and ARG could be early predictors for the severity ABO-HDN hyperbilirubinemia and combination detection of DAT and AGR could further increase its predictive value.


Introduction
Hemolytic disease of the newborn (HDN) is alloimmunization hemolysis caused by maternal-fetal blood group incompatibility [1].The clinical manifestations of HDN include jaundice, anemia, extramedullary hematopoiesis, hydrops fetalis even neonatal death [2].ABO-HDN has emerged as the leading cause of HDN.Unlike Rh-HDN, ABO-HDN is usually a problem of the neonate rather than the fetus [3].Previous metaanalysis concerning the incidence of ABO incompatibility in pregnancies showed that about 15% to 25% of pregnancies can have ABO incompatibility and 20% develop HDN [4].ABO-HDN refers to a milder disease usually treated successfully with phototherapy.However, previous reports [5,6] have described that severe ABO-HDN cases should be treated with RBC transfusion and exchange transfusion therapy.Moreover, our latest research [7] enrolled 123 infants with severe hyperbilirubinemia in Wuhan and demonstrated that ABO-HDN is the leading etiology of severe neonatal hyperbilirubinemia which should be treated with exchange transfusion therapy.
Although ABO-HDN is a common clinical situation, there are many unresolved questions including clinical course and predictive factors.Early and rapid identification of neonates with a high risk of severe ABO-HDN is critical for pediatricians to reduce neonatal morbidity and will result in better patient management [8,9].Meanwhile, the predictive value of laboratory tests for detection of the hemolysis and its severity is still controversial, which leads to that there are no unified criteria or guidelines for the diagnosis and management of ABO-HDN.Among these immunohematological laboratory tests, the direct antiglobulin test (DAT) is a classic experiment to detect antibodies or complements bound directly to the patient's RBCs.DAT is regarded as the cornerstone in the diagnosis of HDN [10,11], however, the incidence of a positive DAT in ABO-HDN ranges widely [12,13] because of the low concentration of IgG anti-A or anti-B [14,15], the different uses of DAT techniques and population examined [16].Meanwhile, the DAT itself may be affected by many factors including anti-RBC IgA, low affinity antibody, antihuman globulin activity and centrifugation technique, our previous research which was concerning the interference in DAT showed that rheumatoid factor can lead to both false decreases and false increases in DAT [17].Therefore, DAT could not support the diagnosis of severe hemolysis alone, and combined detection of additional identification factors is required to diagnose the severity of ABO-HDN.
Albumin globulin ratio(AGR), calculated as albumin/globulin, is actually a reflection of all non-albumin proteins.AGR has been reported to be a novel prognosticator of many diseases including cancer, polyangiitis, and heart failure [18,19].
In plasma, bilirubin binds to albumin to further metabolism, which may lead to the potential decrease of albumin concentration and the decline of AGR level.To our knowledge, no study has investigated the clinical value of the association of AGR with the severity of ABO-HDN.Therefore, in the study, our aim is to explore whether the combination detection of AGR and DAT would predict the severity of ABO-HDN.
This study was performed in line with the principles of the Declaration of Helsinki.

Study subjects
From June 2020-December 2021, 1280 infants with neonatal hyperbilirubinemia underwent combination detection of DAT, free antibody test, and elution test in Wuhan Children's Hospital (Wuhan Maternal and Child Healthcare Hospital), Tongji Medical College, Huazhong University of Science & Technology were selected.Exclusion criteria: (1) with HDN due to non-A/B RBC alloantibodies, (2) gestational age less than 37 weeks, (3) with other causes may lead to hyperbilirubinemia, including sepsis, intracranial hemorrhage, G6PD deficiency, (4) discharge required by guardians of neonates who did not meet the clinical cure standard, (5) received operations during hospitalization, (6) with history for treatment in other hospitals, (7)

Free antibody test
The free antibody test is used for the detection of IgG alloantibodies in neonatal plasma.The procedure is as follows: 50 lL of neonatal plasma and 50 lL of the reagent cells are added to the microcolumn gel card.
Next, incubation at 37 C for 15 min.Finally, centrifugation with g-force 128.1 ± 1.0g for 9 m.

Elution test
The principle of the elution test refers to the release of the IgG alloantibodies adsorbed on the RBC membrane to the elution liquid by chemical or physical methods.The procedure is as follows: 1 Volume of three-time washed RBC is mixed evenly with equal volume of saline.Then, centrifuge immediately after incubate at 56 C for 15 min.Take out the upper liquid, which is the elution liquid.100 lL elution liquid and 50 lL of the reagent cells are added to microcolumn gel card.Next, incubation at 37 C for 15 min.Finally, centrifugation with g-force 128.1 ± 1.0g for 9 m.

Result interpretation
Positive free antibody test (elution test): when the antibody in neonatal plasma (elution liquid) is directed against the ABO blood group.
ABO-HDN: the infants with positive elution test.

Data collection
The basic conditions of all infants have been collected from an electronic medical record system anonymously, including age of hospitalization, sex, gestational age, delivery mode, feeding mode, age of jaundice, age of peak total bilirubin, birth weight, body weight at hospitalization, days of phototherapy, days of hospitalization, as well as the results of laboratory tests during hospitalization, including ABO blood type, peak total bilirubin, HGB, RBC, AGR and DAT at the same time, the general data of the mothers were recorded, including age, gestational day, pregnancy and delivery history.

Outcome measures
Phototherapy is the first line treatment of neonatal hyperbilirubinemia in China and blood components only be used when phototherapy is ineffective.Therefore, we define the hyperbilirubinemia requiring blood components transfusions as severe hyperbilirubinemia.The primary outcome was the infants received blood components transfusions, including IVIG, albumin, RBC and exchange transfusion therapy during hospitalization.The secondary outcomes included the days of phototherapy and the length of hospitalization.

Statistical analysis
The measurement data were tested by Kolmogorov-Smirnov test or Shapiro-Wilk test to see if they were of normal distribution.Normally distributed data were expressed by mean ± standard deviation (x ± s) and data that were not normally distributed were presented as median and 25th-75th centiles.T-test or Mann-Whitney test was used to compare the differences of measurement data between groups with and without blood components.Chi-square test was used to compare the differences of count data between groups with and without blood components.Next, the variables with p < .05were entered in a univariate binary logistic regression analysis continuously to identify the predictors of blood components transfusion.Next, the predictors with p < .1 were entered in a multivariate binary logistic regression analysis together to identify the ultimate predictors of blood components transfusion.
A receiver operating characteristic (ROC) curve was made to compare the difference of prediction value of AGR, DAT, and combination detection of AGR and DAT and the result is presented as the area under the curve (AUC).To find the cutoff value for AGR and DAT as predictors for blood components transfusion in ABO-HDN infants, the Youden index was calculated.The Youden index is presented as the fetal AGR and DAT levels with corresponding sensitivity and specificity.
A Kruskal Wallis test was performed to compare differences of phototherapy day and hospitalization day among group of AGR < 2.05, group of DAT >± and group of AGR <2.05 and DAT >±.The difference of P value less than .05was considered to be significant.These analyses were performed using the SPSS 20.0 statistical software (IBM, Chicago, Illinois, USA).The figures were performed using the GraphPad Prism 8.0 software (San Diego, California, USA).

Results
During the study period, 458 infants were confirmed as HDN and admitted to Wuhan Children's Hospital (Wuhan Maternal and Child Healthcare Hospital), Tongji Medical College, Huazhong University of Science & Technology.Of which 455 cases were diagnosed as ABO-HDN during the study period and thus were eligible for this study.A total of 185 cases were excluded, as 46 cases were discharge required by guardians of neonates, 43 cases were excluded because the gestational age was less than 37 weeks, 52 cases with other causes of hyperbilirubinemia, including 40 cases of sepsis and 12 cases of intracranial hemorrhage, respectively, 21cases received operations during hospitalization, 19 infants has the history for treatment in other hospitals and 4 infants with severe congenital malformation.The derivation of the study population is shown in Figure 1.The baseline characteristics of the study group are presented in Table 1.Finally, 270 cases were enrolled in this study, of which 109 infants received blood components infusion.Among them, 80 infants received one kind of blood components, 29 infants received more than one kind of blood components, respectively and the number of infants who received one time, two times, three times or more than three times of blood components infusions were 65, 29, 13 and 2, respectively.Details showed in Table 2.
An ROC curve was plotted and the AUCs for DAT and AGR were .621and .740,respectively.The Youden index was calculated at .398 with a cutoff AGR of <2.05 with a sensitivity of 54.1% and a specificity of 85.7%, the Youden index was calculated at .233 with a cutoff DAT level of >± with a sensitivity of 39.4% and a specificity of 83.9%.An ROC curve was plotted and the AUC for combination detection of DAT and ARG was .750with a sensitivity of 62.1% and a specificity of 91.2% (Figure 2).Phototherapy days for group of AGR <2.05, group of DAT > ± and group of AGR <2.05 and DAT > ± were 6.0(5.0,7.0), 6.0(5.0,7.0), and 8.0(6.0,9.0),respectively.Hospitalization days for group of AGR <2.05, group of DAT > ± and group of AGR <2.05 and DAT > ± were 7.0(6.0,8.0), 7.0(6.0,8.0), and 9.0(7.0,10.0), respectively.There are significant statistical difference phototherapy day of and hospitalization day among three groups (Figure 3)..001 a a Compared with DAT negative.b Although the p value of DAT ± was .197, the p value of DAT was .015by the univariate binary logistic regression analysis and DAT ± was one classification of DAT intensity levels.Therefore, DAT ± was entered in the multivariate binary logistic regression analysis.

Discussion & conclusions
In present study, we prove that DAT > ± and AGR <2.05 were possible predictors for severe ABO-HDN hyperbilirubinemia and combination detection of DAT and ARG can improve the predictive value.
In severe ABO-HDN cases, transfusions of blood components were required usually to reduce jaundice, prevent and treat anemia, however, blood components transfusion may lead to transfusion transmitted diseases, anaphylaxis, hypersensitivity, thrombosis, electrolyte disturbance and renal failure as well as high hospitalization cost.
The incidence of positive DAT in ABO-HDN ranges greatly due to the different DAT techniques and population examined, in this study, the incidence of positive DAT is 25.6% (69/270).a 2-year retrospective study with a large sample size of ABOincompatible neonates of black ethnicity showed that 270 of 1537 cases are DAT (with tube method) positive with the positive rate of 17.6% [12].Valsami S et al. retrospectively reviewed routinely performed DAT by microcolumn gel method of all infants born between January 2011 and December 2012 in Greece showed that, among 481 ABO incompatible infants, 64 cases are confirmed as DAT positive (positive rate is 13.3%) [13].The retrospective analysis enrolled 289 infants concerning the laboratory data and serological test results in ABO-HDN from central China showed that 115 cases were DAT (by microcolumn gel method) positive.The positive rate is 39.8% [20].The incidence of positive DAT is inconsistent from that of previous reports.The different race and DAT techniques may be the one reason of the distinction, and the study subject in our study was confirmed ABO-HDN infants, could be the another reason.Therefore, the low and varied positive rate of DAT in ABO-HDN illustrated that DAT alone might be a poor positive predictor of ABO-HDN.
Moreover, we investigated the clinical value of DAT in severe hyperbilirubinemia in the present study.Although ABO-HDN refers to be the major reason of positive DAT, whether DAT could be a prediction factor for severe hyperbilirubinemia is still controversial.Some studies reported that the positive DAT has only a poor predictive value for severe hyperbilirubinemia because of only approximate 23% cases of ABO-HDN with positive DAT will continue to have significant hyperbilirubinemia [21,22].However, our study shows that infants with positive DAT in with blood components group were significantly higher than that in without blood components group (p ¼ .000).Therefore, positive DAT refers to a prediction factor for severe ABO-HDN hyperbilirubinemia.Mehta R's study [23] enrolled 901neonates with gestational age >34 weeks and birth weight >2000 g showed that the risk for hyperbilirubinemia requiring phototherapy in the DAT positive infants is significantly higher than that in the neonates with negative DAT (OR 6.78, 95% CI 2.38-19.33).Previous studies demonstrated that ABO-HDN with a positive DAT is considered a major risk factor for the development of severe hyperbilirubinemia and neurotoxicity [24,25] as well.The combination detection [16] of DAT and detection factors including the blood cell indices, pre-discharge total bilirubin level, cord serum albumin and cord bilirubin/albumin ratio [26] would be more beneficial than that of DAT alone for predicting the severity of ABO-HDN.Among them, cord serum albumin and cord bilirubin/albumin ratio have been well researched.
In plasma, the bilirubin albumin complex formed to transport bilirubin to the liver for further metabolism.On one hand, bilirubin binding to albumin increases the water solubility of bilirubin and improves the plasma capacity of transporting bilirubin.On the other hand, it limits the free permeability of bilirubin to various cell membranes and avoids toxic effect of bilirubin on tissues and cells [27,28].Therefore, low serum albumin level decreases bilirubin clearance and thus increases significant hyperbilirubinemia.Previous studies [29,30] found that term infants cases with low cord albumin <2.8 g/dl developed more significant hyperbilirubinemia requiring phototherapy and exchange transfusion.Khairy MA and colleagues [29] showed that neonates with cord bilirubin/albumin ratio <.61 were at risk of developing significant hyperbilirubinemia needing interventions at the same time.However, both cord serum albumin level and cord bilirubin/albumin ratio are considered to reflect ability of albumin to bind bilirubin.AGR we assessed in the current study reflect the infant level of albumin remaining after binding bilirubin, resulting in that high level of bilirubin may lead to decline of albumin level.Moreover, since this is the first analysis assessing the clinical value of AGR in severe ABO-HDN hyperbilirubinemia to the best of our knowledge, we plotted an ROC curve analysis and showed that AGR cutoff value <2.05 had a good predictive value with a sensitivity of 54.1% and a specificity of 85.7%.Meanwhile, we also demonstrated that combination detection of DAT and ARG had a better predictive value than that of respective detection of DAT and ARG in prediction of severe ABO-HDN hyperbilirubinemia (AUC for combination detection .750VS AUC for DAT .621and AUC for AGR .740).Finally, after dividing the infants into three groups according to the results of DAT and ARG and comparing phototherapy day and hospitalization day of the three groups, we found that group of AGR <2.05 and DAT > ± had significant longer phototherapy day and hospitalization day than that of group of AGR <2.05, group of DAT > ± respectively, prompting that the condition of infants with AGR < 2.05 and DAT > ± is more severe and meaning more hospital costs.With lack of studies done on ARG as a prediction factor of severe ABO-HDN hyperbilirubinemia, this work opens the window for further studies to be performed in this field and we are aware that larger scale trials including Multi ethnic researches and preterm neonates are needed.
To summarize, DAT and AGR can be considered possible predictors for severe ABO-HDN hyperbilirubinemia in term neonates.Infants with either DAT > ± or AGR <2.05 were at risk of developing significant ABO-HDN hyperbilirubinemia.Combination detection of DAT and ARG can improve the predictive value.We recommend that pediatricians pay close attention to the results of DAT and AGR when judging the severity of ABO-HDN hyperbilirubinemia, particularly the term infants with DAT > ± and AGR < 2.05.

Figure 1 .
Figure 1.Flowchart of the study population.

Figure 2 .
Figure 2. ROC Curves of DAT, AGR and combination detection of DAT and ARG.

Table 1 .
Baseline characteristics of study population.

Table 2 .
Distribution of blood components.

Table 3 .
Predictors for blood components transfusion in infants with ABO-HDN hyperbilirubinemia.