Abstract
Many authors have reported poor prognostic value of anti-D antibody titer in the setting of Hemolytic Disease of Fetus and Newborn (HDFN). According to literature, HDFN cases with IgG1 and IgG3 have more severity compared to IgG2 and IgG4.Therefore, we planned this study to evaluate the prevalence and prognostic value of IgG subtypes in the setting of Rh HDFN. This was a retrospective study performed at a tertiary care center in north India from October 2015 to November 2017. Women with anti-D antibody were included in the study and categorized on the basis of presence of specific IgG subtype. “DAT IgG1/IgG3 ID” card (BIO-RAD) was used for determining the subclass of IgG. Various clinical, laboratory & interventional parameters were used to categorize fetal outcome in severe and non-severe cases. Perinatal outcome was then compared between women with different IgG subclass profile. Subclass distribution among 80 alloimmunized women was 26.2% for IgG1, 15% for IgG3, 46.2% for IgG1 + IgG3 and the rest had neither IgG1 nor IgG3. Severity of HDFN was significantly higher when IgG1 &/or IgG3 were present alone or in combination, compared to cases with absence of IgG1 or IgG3 (p value < 0.05). Risk of severe HDFN was significantly higher in the presence of IgG1 &/or IgG3 and the severity was highest when both IgG1 and IgG3 were present. We recommend that IgG subclass determination should be included in a multi—parameter protocol for more accurate prediction HDFN severity to ensure timely referral and intervention.
Keywords: Haemolytic disease of fetus and newborn, IgG subclass, Column agglutination technology, Maternal alloimmunization, Fetal outcome
Introduction
Haemolytic disease of the fetus and newborn (HDFN) is a clinical condition characterised by development of IgG antibodies in the mother against fetal red cell antigens, which cause hemolysis in the fetus by crossing the placenta. Red cells sensitized with maternal IgG are destroyed by macrophages in the fetal spleen. Anti-D antibody, targeted against RhD antigen, is the most common antibody responsible for severe HDFN [1, 2]. Most Rh antibodies are IgG in nature including anti-D.
Fetal outcome in case of maternal alloimmunization against RhD antigen depends on various factors such as antibody specificity, titer, IgG subclass and antigen density on fetal red cell surface. Although there is a decline in cases of HDFN due to availability of anti-D prophylaxis in recent times, this does not obliviate the need of non-invasive tests for early and accurate prediction of fetal outcome [3]. Several studies have demonstrated poor correlation between anti-D titer and fetal outcome [4, 5]. In fact, not only the titer of antibodies but other characteristics are also important in determining fetal outcome i.e. IgG subclass and Gm allotypes [6, 7]. Reports available on IgG subclass composition of maternal anti-D and its potential to cause HDFN are contradictory [8–10]. Therefore, this study was planned to evaluate the prevalence and prognostic value of IgG subclasses in RhD sensitized antenatal women.
Materials and Methods
Sanjay Gandhi Post-Graduate Institute of Medical Sciences (SGPGIMS), Lucknow, India is a tertiary care hospital providing advanced care to high risk pregnancy cases. Approximately 100-120 intrauterine transfusions (IUTs) are performed every year on alloimmunized antenatal women by obstetric unit in collaboration with the department of Transfusion Medicine. This was a retrospective analysis of antenatal alloimmunized women from October 2015 to November 2017.
All antenatal women attending outpatient obstetric services were screened for the presence of irregular red cell antibodies. Out of these, most of the antenatal women were referred from other centers in view of bad obstetric history. Majority of them were found to be RhD negative.
Antenatal women with anti-D antibody were included in the study. Presence of other factors that can adversely affect fetal outcome, such as red cell antibody in addition to anti-D and comorbidities, was considered as exclusion criteria. Women with recent anti-D prophylaxis and RhD antigen negative fetus/neonates were also excluded. Data collected included maternal obstetric history, maternal antibody identification and titration, antigen typing of father, mother and fetus (before first IUT), fetal direct antiglobulin test (DAT), and various clinical/laboratory/interventional parameters of fetal or neonatal outcome. Institutional ethical clearance was obtained for the study.
In this study, antenatal women with anti-D were categorised into 4 subgroups on the basis of IgG subclass: (1) IgG1 only, (2) IgG3 only, (3) IgG1 + IgG3 and (4) neither IgG1 nor IgG3.
HDFN Case Definition and Categorization of Fetal Outcome
Each pregnancy sensitized with anti-D antibody was followed until its final outcome was known. HDFN was diagnosed following BCSH guidelines (BCSH 2016) in the fetus and newborns [11]. Fetal outcome was determined and categorized on the basis of combination of clinical and lab parameters as well as the intervention required. For the purpose of the study, fetal outcome was classified into three categories namely unaffected, mild-moderate and severe HDFN (Table 1).
Table 1.
Categorization of fetal outcome
| Category | DATb | Evidence of hemolysisc | Cord Hb (g-dl−1) | Intervention required (IUT, ET, simple transfusion and phototherapy) |
|---|---|---|---|---|
| Unaffecteda | Negative | No | > 12 | No |
| Mild-Moderatea | Positive | Yes | 10–12 | Yes but limited to phototherapy &/or simple transfusion |
| Severe | Positive | Yes | < 10 | Intra uterine/exchange transfusion/stillbirth/Hydrops |
aUnaffected + Mild-Moderate cases = Non-severe cases
bDirect Antiglobulin Test
cIncludes both clinical (Pallor, icterus, hepatospleenomegaly etc.) and laboratory (Peripheral blood smear, Increased reticulocyte count, Microspherocytosis, Indirect hyperbilirubinemia) evidence of hemolysis
Identification of Antibody Specificity
Screening followed by identification of irregular red cell antibodies was done using column agglutination technology (CAT) in indirect antiglobulin phase. A commercially available three-cell antigen panel (ID Diacell I, II, III; BIO-RAD, California, USA) and an extended 11-cell panel (ID DiaPanel, 11 cell panel, BIO-RAD, California, USA) were used for the screening and identification of antibody respectively according to the manufacturers’ instructions.
Determination of IgG Subclasses
Maternal serum samples with only anti-D antibody were further processed for IgG subtyping in indirect antiglobulin phase using CAT. Commercially available gel based subtyping cards (ID-Card “DAT IgG1/IgG”, BIO-RAD, California, USA) were used for this purpose. Fifty μl of the pooled ORhD positive red cell suspension (1%) followed by 25 μl serum of the patient were added into the microtubes containing monoclonal anti-IgG1 & anti-IgG3 in 2 different dilutions. Then ID-Card was incubated for 15 min at 37 °C followed by centrifugation for 10 min. Results were interpreted as per the manufacturer’s instructions. Microtubes giving positive reaction with the lower dilution (high concentration of IgG subclass) or with both, lower and higher dilutions were considered as positive for that particular IgG subclass.
In the study, we solely focused our analysis on the presence or absence of various IgG subtypes in maternal sera and its effect on fetal outcome. Although ID-Card used for subclass determination also provides relative concentration of IgG subtypes in the form of dilutions, this information was not used for analysis.
Statistical Analysis
Impact of IgG subclass on severity of HDFN was evaluated by estimation of odds ratio between severely affected and non-severely affected cases. Non-severely affected cases included unaffected cases as well as cases with mild to moderate HDFN.
Chi square test or Fisher Exact test were used for estimation of significance of association. Two sided P value of equal to or less than 0.05 was considered significant. Odds ratio was presented with 95% confidence intervals (CIs). All statistical analyses were performed using SPSS Statistics version 18 (IBM, Armonk, NY, USA).
Results
A total of 490 antenatal women were screened for the presence of red cell alloantibodies. All women had more than one pregnancy (range, 2–8). Antibody screen was positive in 151 (30.8%) cases and anti-D was the most common antibody, found in all the cases. Incidentally, all screen positive women were RhD negative and none of the RhD positive mothers demonstrated red cell alloimmunization.
Distribution of screen positive cases is given in Fig. 1. Out of the total 151 screen positive cases, 125 (82.7%) women had anti-D alone while rest 26 (17.3%) had at least one alloantibody in combination with anti D. A total of 71 women were excluded from the study due to various reasons including 26 patients with at least one antibody in addition to anti-D. For the final analysis, 80 patients with anti-D alone were selected. About 88% of the cases had either IgG1 &/or IgG3 while 12%, though positive for anti-D antibody, were negative for both of these subclasses. IgG1 and IgG3 were found to be present alone in 21 (26.2%) and 12 (15%) patients respectively. A combination of IgG1 and IgG3 was present in 37 (46.2%) women.
Fig. 1.
Summary of RhD sensitized antenatal cases with distribution of IgG subclass
Median anti-D titer was variable among women with different IgG subclass profiles. Patients with IgG1 usually had higher overall anti-D titre compared to other categories (IgG3 and negative for both IgG1 and IgG3).
Table 2 shows correlation of IgG subclass with fetal outcome. The odds of developing severe HDFN for women with IgG1 and IgG3 in combination were 3.6 times the odds for women with IgG1 or IgG3 alone (95% CI 1.3–9.8). Highly significant difference in the severity of the disease (OR = 2.6, 95% CI 1.4–14.2) was also observed between women negative for both IgG1 and IgG3, compared to those with either IgG1 or IgG3 alone. We could not find any significant difference in the severity of HDFN between women with IgG1 and IgG3 alone (p = 0.591).
Table 2.
Correlation of IgG subclass with fetal outcome
| IgG subclass (N) | Median anti-D titer (range) |
Severe (N, %) |
Non-severe (N, %) |
OR (95% CI) |
p value |
|---|---|---|---|---|---|
|
IgG1 (n = 21) |
128 (8–1024) |
09 (42.8%) |
12 (57.2%) |
1.5 (0.3–6.5) |
0.591 |
|
IgG3 (n = 12) |
32 (4–256) |
04 (33.3%) |
08 (66.7%) |
||
|
IgG1 or IgG3 (n = 33) |
64 (4–1024) |
13 (39.4%) |
20 (60.6%) |
3.6 (1.3–9.8) |
0.009 |
|
IgG1 + IgG3 (n = 37) |
64 (16–512) |
26 (70.2%) |
11 (29.8%) |
||
|
IgG1 or IgG3 (n = 33) |
64 (4–1024) |
13 (39.4%) |
20 (60.6%) |
2.6 (1.4–14.2) |
0.023 |
|
Neither IgG1 nor IgG3 (n = 10) |
32 (8–256) |
02 (20.0%) |
08 (80.0%) |
Discussion
Various factors affect the severity of HDFN in a RhD sensitized antenatal women. Many researchers had highlighted the prognostic role of IgG subtypes in the setting of Rh HDFN [6, 12]. Rh titre has its own limitations in predicting fetal outcome [4], therefore the prognostic significance of IgG subtypes was evaluated here in this study.
Table 3 summarises the prevalence of IgG subclasses in various studies. It is generally accepted that anti-D antibodies are predominantly IgGl and IgG3; rarely IgG2 and IgG4 [10, 13]. However, many authors have demonstrated the presence of IgG2 &/or IgG4 in RhD sensitized women [12, 14]. Our data is consistent with Choudhuri et al. [15] who reported that both IgG1 and IgG3 subclasses might be absent even with high overall anti-D titers. The prevalence of combined IgG1 and IgG3 was approximately double that of IgG1 alone in this study. Our data are in concordance with Pollock et al.: who observed combined IgG1 and IgG3 as the predominant subclass in their study, while Iyer et al. reported IgG1 alone as most common IgG subclass category [10, 14].
Table 3.
IgG subclass distribution in various studies and its correlation with fetal outcome
| Author | Subclass prevalence (%) | Correlation of pregnancy outcome with IgG subclass |
|---|---|---|
| Emilija [18] | IgG1-35.5, IgG3-6.6, IgG1 + IgG3-57.7 | No |
| Pollock and Bowman [10] | IgG1-32.6, IgG3-3.06, IgG1 + IgG3-64.2 |
Yes (IgG1 + IgG3 > IgG1 > IgG3) |
| Iyer et al. [14] |
IgG1-48.5, IgG3-12.1, IgG1 + IgG3-21.5 IgG2 &/or IgG4-6.5 Other-11.2 |
Yes (IgG1 > IgG1 + IgG3 > IgG3) |
| Choudhuri et al. [15] | IgG1-20, IgG3-3.5, IgG1 + IgG3-24.7, Neither IgG1 nor IgG3- 51.7 |
Yes (IgG + IgG3 > IgG1 or IgG3 > neither IgG1 nor IgG3) |
| Parinaud et al. [6] | IgG1-29.4, IgG3-28.2, IgG1 + IgG3-42.4 | IgG1 > IgG1 + IgG3 > IgG3 |
| Present study | IgG1-26.2, IgG3-15, IgG1 + IgG3-46.2, Neither IgG1 nor IgG3-12.5 |
Yes (IgG1 + IgG3 > IgG1 or IgG3 > neither IgG1 nor IgG3) |
The destruction of red cells sensitized with anti-D is primarily carried out by mononuclear-macrophage system. IgG coated erythrocytes bind to the Fc receptor on the macrophage and are subsequently destroyed by phagocytosis [16].
Various studies have shown contradictory results with regard to an association between the IgG subtype and severity of Rh HDFN. We observed significantly increased risk of severe HDFN when either of IgG1 or IgG3 was present. The risk of severe HDFN was further increased many folds when both IgG1 and IgG3 were present together. This reflects that both IgG1 and IgG3 play critical role in the pathogenesis of HDFN. Our data is in concordance with Pollock et al. and Choudhuri et al., who found that the combined presence of IgGl and IgG3 leads to very poor fetal outcome [10, 15]. In present study, severely affected cases were 70.2% when the maternal anti-D antibody had a combination of IgGl and IgG3. Opposite to our findings, mild to moderate disease was reported by Parinaud et al. when these subtypes existed together [6].
We also found that the severity of the disease was not significantly different between women with IgG1 and IgG3 alone even after difference in overall anti-D titer (128 and 32 for IgG1 and IgG3 respectively). This could be explained by greater affinity of IgG3 antibodies to bind Fc receptors on macrophages than that of IgGl antibodies. For effective interaction between sensitized red cells and monocytes, 4–5 times more IgG1 molecules are required than IgG3 [17].
In our study, 8 out of 10 mothers with neither of IgG1 and IgG3 delivered non-severely affected infants while 2 mothers, both with a anti-D titer of 256, delivered severely affected infants. It seems that significantly high levels of anti-D antibodies always lead to severe HDFN irrespective of the IgG subclass.
There were several limitations in this study. We could not demonstrate the presence or absence of IgG2/4; rather, cases with neither of IgG1 and IgG3 were used for making comparisons. Quantification of IgG subclass was not done. Impact of IgG subclass on fetal outcome was not evaluated at individual anti-D titer category due to less number of cases in each category titer.
Conclusion
The presence of IgG1 and IgG3 impacted the severity of HDFN significantly, with the risk of severe HDFN increasing 2.6 fold when IgG1 or IgG3 was present in isolation, to a further 3.6 times when both were present. By early identification of IgG subclass one can plan timely intervention to prevent the dreaded outcomes of HDFN. Most transfusion centres are familiar with easy-to-use column agglutination technique (CAT). The study results suggest that IgG subclass determination should be included in a multi—parameter protocol for more accurate prediction HDFN severity. This multi-parameter approach can lead to better assessment of the expected intensity of HDFN, in combination with the titer and specificity of the antibodies.
Author’s Contributions
All the authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Bharat Singh, Rajendra Chaudhary, Rahul Katharia and Mandakini Pradhan. The first draft of the manuscript was written by Bharat Singh and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Funding
No funding was required.
Availability of Data and Material
The authors declare that they have all the data and material to support their published claims.
Compliance with Ethical Standards
Conflicts of interest
The authors declare that they have no conflicts of interest.
Ethical Approval
This was a retrospective study conducted on already available data of a student’s thesis. The requisite approval was taken from the institute ethics committee.
Consent to Participate
This was a retrospective study based on a student’s thesis data. Informed consent was taken from the participants during data collection for thesis purpose.
Consent for Publication
Each author has approved the final version of the submitted manuscript and the submission to ‘INDIAN JOURNAL OF HEMATOLOGY AND BLOOD TRANSFUSION’.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
References
- 1.Suresh B, Sreedhar Babu KV, Arun R, Jothibai DS, Bharathi T. Prevalence of “unexpected antibodies” in the antenatal women attending the Government Maternity Hospital, Tirupati. J Clin Sci Res. 2015;4:22–302. doi: 10.15380/2277-5706.JCSR.13.034. [DOI] [Google Scholar]
- 2.Pahuja S, Gupta SK, Pujani M, Jain M. The prevalence of irregular erythrocyte antibodies among antenatal women in Delhi. Blood Transfus. 2011;9:388–393. doi: 10.2450/2011.0050-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Chavez GF, Mulinare J, Edmonds LD. Epidemiology of Rh haemolytic disease of the newborn in the United States. J Am Med Assoc. 1991;265:3270–3274. doi: 10.1001/jama.1991.03460240066029. [DOI] [PubMed] [Google Scholar]
- 4.Gupte SC, Bhatia HM. Correlation of Rh(D) antibody titer and obstetric history with severity of Rh haemolytic disease of the newborn. Indian J Hematol. 1985;3:76–80. [Google Scholar]
- 5.Morley G, Gibson M, Eltringham D. Use of discriminant analysis in relating maternal anti-D levels to the severity of haemolytic disease of the newborn. Vox Sang. 1977;32:90–98. doi: 10.1111/j.1423-0410.1977.tb00611.x. [DOI] [PubMed] [Google Scholar]
- 6.Parinaud J, Blanc M, Grandjean A. IgG subclasses and Gm allotypes of anti-D antibodies during pregnancy: correlation with the gravity of the fetal disease. Am J Obstet Gynec. 1985;151(1111–1):115. doi: 10.1016/0002-9378(85)90393-x. [DOI] [PubMed] [Google Scholar]
- 7.Schanfield M, Schoeppner S, Stevens J (1980) New approaches to detecting clinically significant antibodies in the laboratory. Immunobiology of the erythrocyte, pp 305–323 [PubMed]
- 8.Shaw DR, Conley ME, Knox FJ. Direct quantitation of IgG subclasses 1, 2 and 3 bound to red cells by Rh(D) antibodies. Transfusion. 1988;28:127–131. doi: 10.1046/j.1537-2995.1988.28288179015.x. [DOI] [PubMed] [Google Scholar]
- 9.Hadley AG, Kumpel BM, Leader KA. Correlation of serological, quantitative and cell-mediated functional assays of alloantibodies with the severity of hemolyric disease of the newborn. Br J Hematol. 1991;77:221–228. doi: 10.1111/j.1365-2141.1991.tb07981.x. [DOI] [PubMed] [Google Scholar]
- 10.Pollock JM, Bowman JM. Anti-Rh(D) IgG subclasses and severity of Rh haemolytic disease of the newborn. Vox Sang. 1990;59:176–179. doi: 10.1111/j.1423-0410.1990.tb00854.x. [DOI] [PubMed] [Google Scholar]
- 11.White J, Qureshi H, Massey E, Needs M, Byrne G, Daniels G, Allard S. Guideline for blood grouping and red cell antibody testing in pregnancy. Transfus Med. 2016;26:246–263. doi: 10.1111/tme.12299. [DOI] [PubMed] [Google Scholar]
- 12.Zupanska B, Brojer E, Richard Y, Lenkiewicz B, Seyfried H, Howell P. Serological and immunological characteristics of maternal anti-Rh D antibodies in predicting severity of hemolytic disease of the newborn. Vox Sang. 1989;56:247–253. doi: 10.1111/j.1423-0410.1989.tb02037.x. [DOI] [PubMed] [Google Scholar]
- 13.Lambin P, Debbia M, Puillandre P. IgG1 and IgG3 anti-D in maternal serum and on the RBCs of infants suffering from HDN: relationship with the severity of the disease. Transfusion. 2002;42:1537–1546. doi: 10.1046/j.1537-2995.2002.00239.x. [DOI] [PubMed] [Google Scholar]
- 14.Iyer YS, Kulkarni SV, Gupte SC. Distribution of IgG subtypes in maternal anti-D sera and their prognostic value in Rh haemolytic disease of the new-born. Acta Hematol. 1992;88:78–81. doi: 10.1159/000204656. [DOI] [PubMed] [Google Scholar]
- 15.Choudhuri J, Chacko MP, Kuruvilla AK, Jose R, Mathews J, Regi A, Daniel D. Clinical impact and prevalence of IgG1 and IgG3 subclasses in antenatal alloimmunized women: experience from a tertiary medical centre in South India. ISBT Sci Ser. 2016;11:58–61. doi: 10.1111/voxs.12260. [DOI] [Google Scholar]
- 16.Engelfriet CP (1981) Immune destruction of red cells. In: 34th Annual Meeting of the American Association of Blood Banks, Washington p 93
- 17.Zupanska B, Thomson EE, Mary AH. Fc receptors for IgG, and IgG3 on human mononuclear cells: an evaluation with known levels of erythrocyte bound IgG. Vox Sang. 1986;50:97–103. doi: 10.1111/j.1423-0410.1986.tb04854.x. [DOI] [PubMed] [Google Scholar]
- 18.Velkova E. Correlation between the amount of anti-D antibodies and IgG Subclass with severity of Hemolytic Disease of Fetus and Newborn. Maced J Med Sci. 2015;3(2):293. doi: 10.3889/oamjms.2015.058. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The authors declare that they have all the data and material to support their published claims.