|Year : 2019 | Volume
| Issue : 1 | Page : 114-116
Blocked D in RhD hemolytic disease of fetus and newborn
Department of Transfusion Medicine, Kovai Medical Center and Hospital, Coimbatore, Tamil Nadu, India
|Date of Web Publication||22-Apr-2019|
Dr. Rajeswari Subramaniyan
Department of Transfusion Medicine, Kovai Medical Center and Hospital, Coimbatore, Tamil Nadu
Source of Support: None, Conflict of Interest: None
A 3-day-old term neonate was admitted to our hospital for the evaluation of neonatal jaundice. The neonate was born to an RhD-negative multiparous mother who had never received anti-D prophylaxis. The neonate's direct antiglobulin test was 4+. Blood group was B, and Rh D typing was negative using tube technique and positive (2+) using gel card. The mother had anti-D, and anti-D titers were 1:1024. After heat elution, RhD typing of the neonate was positive using tube (2+) and gel technique (4+). The eluate was shown to have anti-D. As the red cells of the neonate were saturated with maternal anti-D, commercial monoclonal anti-D could not bind to D antigen which resulted in false-negative D typing in the neonate. Such blocking phenomenon is rare. The discrepancy was identified while using two different techniques (tube and gel) which aided in early diagnosis and tailored an appropriate treatment. The neonate improved after the initiation of a combination of intravenous immunoglobulin and phototherapy.
Keywords: Agglutination, blocked D, hemolytic disease of fetus and newborn, maternal anti-D
|How to cite this article:|
Subramaniyan R. Blocked D in RhD hemolytic disease of fetus and newborn. Glob J Transfus Med 2019;4:114-6
| Introduction|| |
The incidence of RhD hemolytic disease of fetus and newborn (HDFN) has declined from 16% to 0.1% worldwide, thanks to the introduction of postnatal and routine antenatal anti-D immunoprophylaxis. D antigen is highly immunogenic. Next to ABO antibodies, anti-D is the most common antibody implicated in the HDFN. The production of D antigen starts as early as 5 weeks of gestation and completes before birth. The disease spectrum ranges from isolated positive direct antiglobulin test (DAT) at birth with no evidence of red cell destruction to occasional intrauterine death. Anti-D is the most common cause of severe HDFN. The critical titer for anti-D is 1:16 at the anti-human globulin phase., D antigen sites of RhD fetal red cells are saturated with anti-D in alloimmunized pregnancy with high anti-D titers. Rarely, the bound anti-D prevents the binding of commercial Rh typing agent (monoclonal IgM anti-D), thereby resulting in false-negative RhD typing (blocked D phenomenon). Blocking of the antigen sites by potent monoclonal antibodies resulting in false-negative antigen typing is an uncommon phenomenon. False-negative K typing has been observed with high titer maternal anti-K (anti-K titer >1:256). Similarly, Lee et al. reported a case of false-negative Fy a typing wherein murine monoclonal anti-Fy a (MIMA-19) was blocked by maternal anti-Fy a antibodies (anti-Fy a titer >1:256). Hereby, we present one such case, wherein maternal anti-D blocked the binding of monoclonal anti-D resulting in a false-negative D typing in the neonate.
| Case Report|| |
A 3-day-old term neonate was admitted to our hospital for the evaluation of neonatal jaundice. The mother was multiparous (P2 L1A1) with a male child alive and healthy. She had not received the anti-D prophylaxis before. Laboratory parameters of the neonate at the time of admission were as follows: hemoglobin (Hb): 15.6 g/dL (normal range: 15.0–24.6 g/dL), total bilirubin: 24.16 mg/dL (normal range: 0–1 mg/dL); indirect bilirubin (IB): 22.0 mg/dL (normal range: 0–0.75 mg/dL); and corrected reticulocyte count: 8.2% (normal range: 2%–6%). We received a request for blood grouping and DAT of the neonate.
| Materials and Methods|| |
Blood grouping and RhD typing were performed using conventional tube technique (CTT) using monoclonal antisera (IgM anti-D: Clone P3 × 61, Tulip diagnostics, Goa, India) and gel technique (IgM anti-D clone LHM 59/20 (LDM3) +175–2, Biorad, Cressier, Switzerland; IgM anti-D clone P3 × 61+TH-28, Tulip diagnostics, Goa, India). An appropriate negative control (Erybank Rh-hr Control, Tulip Diagnostics, Goa, India) was used for D typing. Monospecific gel card for IgG and C3d (DC Screening I, Biorad, Cressier, Switzerland) was used to determine DAT. Red cell antibody screening and identification were performed in LISS-Coombs gel card using commercial 3-cell panel (Diacel I-II-III, Biorad, Cressier, Switzerland) and 11-cell panel (Diapanel, Biorad, Cressier, Switzerland), respectively. Antibody titers were tested using CTT. Rh-K phenotyping was carried out using gel card (DiaClon Rh-Subgroups + K, Biorad, Cressier, Switzerland).
| Results|| |
Blood group of the neonate was B, and RhD typing was inconsistent. CTT showed a negative reaction with anti-D. RhD typing was repeated using gel card (Biorad and Tulip diagnostics). The gel card of Tulip diagnostics yielded negative results while the gel card of Biorad showed positive (2+) reaction with anti-D. Control column was negative. Neonate's DAT was positive (4+) for IgG only.
Blood grouping and RhD typing of the mother was B RhD negative. The mother was found to have anti-D using red cell antibody screening and identification. Anti-D titer in the mother's serum was 1:1024. Anti-D was identified in the neonate's serum as well. Since the results of RhD typing in the neonate were discordant using different techniques coupled with high maternal anti-D titer and strong DAT of neonate's red cells, blocked D phenomenon was suspected.
To confirm this, the neonate's red cells were subjected to heat elution at 56°C for 10 min as described in AABB technical manual, 17th edition. RhD typing of the eluted red cells was repeated using CTT and gel card as mentioned above. Using CTT, the grade of reaction with anti-D enhanced to 2+. Using gel techniques (both Biorad and Tulip), the grade of reaction with anti-D increased to 4+, thereby confirming the presence of D antigen. Red cell antibody identification of the eluate showed the presence of anti-D. Rh-K phenotyping of the mother was rr (dce/dce) and that of the neonate was R1r (DCe/dce). The neonate was started on phototherapy and high-dose (0.5 g/kg body weight over a period of 2 h) intravenous immunoglobulin. Subsequently, serum IB declined to 12.8 mg/dL. On day 8, the neonate was transfused with B RhD negative, Rh-K matched packed red cells in view of anemia (Hb: 7.3g/dL). On day 12, the neonate recovered and discharged with a serum IB level of 2.1 mg/dL and a hematocrit of 48%.
| Discussion|| |
Blocked phenomenon should be suspected with high maternal antibody titers, strong DAT, and absence of the cognate antigen on fetal/neonatal red cells tested using saline-reacting antisera. Wiener demonstrated the blocking phenomenon in vitro for the first time. They proposed that false-negative D typing (blocked D) could be due to prozone phenomenon. It is nothing but a zone of antibody excess, given the D antigen sites are saturated with anti-D. In the index case, the mother had high titers of anti-D (1:1024). The D antigen sites of the neonatal red cells were completely coated by the potent maternal anti-D, leaving no antigen site for commercial anti-D. This resulted in false-negative D typing. RhD typing was accurately determined as RhD positive only after heat elution(anti-D was eluted from the neonatal red cells). Very few reports of blocked antigen resulting in false-negative typing are available in the literature [Table 1]. Monoclonal antibodies can be used for red cell antigen phenotyping even in the setting of a positive DAT due to their high specificity. Nevertheless, monoclonal antisera are not without disadvantages. Potentiator (like bovine serum albumin) is one of the ingredients of these antisera to enhance the antigen-antibody reaction. Red cells coated with IgG anti-D antibodies can agglutinate in the presence of these potentiators. Hence, it is strongly emphasized to include Rh control along with the D typing. Wang et al. observed positive control results using gel card due to the inclusion of macromolecular potentiators in the gel columns. RhD typing cannot be ascertained in such a scenario. The discrepancy in RhD typing results observed in the index case could probably be explained by the sensitive technique (gel technique) and distinct clones used for the preparation of monoclonal antisera. It is therefore advisable to use a sensitive technique in addition to the CTT for the determination of the blood grouping and RhD typing.
|Table 1: Clinical and laboratory features of blocked D cases reported in the literature|
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| Conclusion|| |
Commercial monoclonal antisera to blood group antigens could give rise to false-negative results when typing antibody-coated fetal or neonatal red cells. Although diagnostically challenging, a systematic approach utilizing multiple techniques solves the blood group discrepancy in such cases.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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