RhD blocking phenomenon implicated in an immunohaematological diagnostic dilemma in a case of RhD-haemolytic disease of the foetus

Introduction

Of all red blood cell antigens, RhD is second only to the ABO antigens in importance in blood transfusion. The Rh antigens are fully expressed at the time of birth unlike the weak expression of ABO antigens in neonates¹. Immunisation to D antigen can occur in reaction to less than 0.1 mL of foetal blood, resulting in anti-D alloantibody in the maternal circulation². Haemolytic disease of the foetus and newborn (HDFN) occurs when there is a destruction of foetal/neonatal red cells by IgG antibodies produced by the mother. When Anti-D is the cause of HDFN, the severe anaemia can cause foetal hydrops, tissue hypoxia and even foetal death in utero³. Sensitised pregnant women require close monitoring for early detection of foetal anaemia and to decide whether and, if so, when intrauterine transfusion is required. As a key part of any pre-transfusion testing, the ABO and RhD groups of recipient samples are determined. It has been reported that if a neonate’s red cells are heavily saturated with IgG antibodies, RhD typing with anti-D reagents may give either false negative or false positive results³. We report here the case of blocked RhD in a cord blood sample in a suspected case of RhD-HDFN which was detected in our hospital blood bank laboratory.

Case report

A 32-year old female presented at our hospital for an antenatal check-up in her 20^th week of pregnancy (G_{10 P1+8}). She gave a history of Rh immunoglobulin prophylaxis in her first pregnancy along with a blood transfusion 2 years previously at another hospital. In our laboratory she was found to have “AB Rh(D) negative” blood group together with a positive indirect antihuman globulin test result (4+) by a gel technique (DiaMed, Switzerland). The commercially available antibody screening cells and antibody identification panel cells (DiaMed, Switzerland) confirmed the presence of anti-D red cell alloantibody. The anti-D titre was 256 by the conventional tube technique. Her husband’s ABO blood group was “B Rh(D) positive”. The patient was also phenotyped for other Rh antigens and her Rh phenotype was rr (dce/dce) whereas her husband typed as R1 Rz (DCe/DCE) using commercially available monoclonal antisera.

A cordocentesis procedure (percutaneous umbilical blood sampling) was planned at 20 weeks of gestation because the foetus showed features of hydrops on ultrasound examination in the presence of the high anti-D titre, which was quite suggestive of RhD-HDFN. The cordocentesis corroborated the presence of HDFN as the foetal haemoglobin and haematocrit values were 5.4 g/dL and 13.9%, respectively. As a result, intrauterine transfusion of red blood cells was planned. The foetal samples were sent to our laboratory for immunohaematological workup of HDFN and for preparation of packed red blood cells for intrauterine transfusion.

The foetal red cells were typed for ABO and RhD antigens by the conventional tube technique using commercially available reagents and a gel technique which showed “A RhD negative” blood group. The direct antiglobulin test (DAT) was performed using polyspecific anti-human globulin which gave a strong positive (4+) result. Unable to explain the absence of RhD antigens on foetal red cells, we repeated the RhD phenotyping with both techniques but the results remained unchanged. These immunohaematological results, when reviewed in conjunction with the clinical history including the presence of a high titre of anti-D in the mother and a strong DAT reaction by foetal red cells led to the suspicion of blocked D phenomenon. Two aliquots were, therefore, prepared for dissociating maternal IgG antibodies from DAT-positive foetal red cells. The aliquots were tested by chloroquine diphosphate elution at pH 5.1 according to the recommended method³. DAT performed on chloroquine eluted red cells gave a 2+ reaction (from an initial 4+) using the gel technique. The results of the chloroquine elution method are shown in Table I. After three sequential elutions, foetal red cells could be phenotyped as RhD positive (2+) with anti-D antisera (DiaClon anti-D monoclonal IgG & IgM antibodies) on a gel card (Figure 1). The eluate specificity was found to be anti-D using a three-cell panel on an indirect antiglobulin test (Figure 2). Typing for IgG subclass to establish the class of IgG antibody could not be done because of the paucity of the sample.

Table I.

Results of the immunohaematological work-up on foetal red cells after elution.

Anti-A	Anti-B	Anti-D	DAT	Eluate specificity
4+	0	2+	2+	Anti-D

Figure 1.

Foetal red cell ABO & RhD typing before and after elution.

Figure 2.

Eluate showing anti-D reactivity on 3-cell panel.

The foetus received an intrauterine transfusion of 26 mL of “O RhD negative” red blood cells prepared in accordance with the department’s standard operating procedure. The transfused red cell aliquot was reportedly successful in raising the foetal haemoglobin concentration to 14.1 g/dL and the haematocrit to 41.8%. However, the mother developed an additional antibody (anti-Kidd^b) after the first intrauterine transfusion so the next two intrauterine transfusions were done with RhD and Jk^b negative cross-matched compatible units. The patient delivered a male baby by Caesarean section at another tertiary care hospital where the neonate had icterus at birth so exchange transfusions were planned. Here the neonate was initially phenotyped as “O RhD negative” as the doctors were not then aware that he had previously received intrauterine transfusions with “O RhD negative” red cells. The neonate underwent exchange transfusions with “O RhD negative” blood. Finally, after 6 months, when the baby came for a follow-up at our centre, he was phenotyped as “A RhD positive”.

Discussion

ABO and RhD grouping can be regarded as the most important serological tests performed in a blood bank on pre-transfusion samples. As regard to alloantibodies which have been implicated in HDFN, anti-D is usually associated with the most severe disease. The mere presence of antibody does not, however, indicate that HDFN will occur as only IgG antibodies can cross the placenta. Apart from this, foetal antigens may not include the antigen corresponding to the mother’s antibody. The likelihood of foetal involvement can often be predicted by typing the father’s red cells.

The blocking phenomenon due to maternal anti-D should be suspected in a case of Rh-HDFN if foetal or infant red blood cells give a strongly positive result in a DAT but a negative result on RhD typing with saline reactive anti-D antisera. This phenomenon may lead to a delay in making an antenatal or postnatal diagnosis of RhD-HDFN by routine serological investigation. It is prudent to be alert to the patient’s diagnosis and in such cases, one should immediately repeat the tests using red cells from which antibody has been eluted.

This study also highlights the importance of routine blood grouping and screening for atypical antibodies during antenatal check-ups and the correct interpretation of the results in relation to the clinical history and laboratory findings. In the present study, the foetus showed a false RhD negative result first due to the D antigen blocking phenomenon and was then mistyped again as RhD negative immediately after birth because of previous intrauterine transfusions with RhD negative blood.

The first description of Rh blocking phenomenon was given by Wiener in 1944⁴. A PubMed search revealed only a handful of case reports of blocked D involving neonates^5,6. Sulochana et al.⁵ described a case in which a RhD negative blood group was found repeatedly in a newborn baby with severe HDFN born to a second gravida “B RhD negative” mother. The baby was grouped as “B RhD negative” by direct grouping but, after elution, D antigen was detected. Both mother and baby had high concentrations of anti-D which persisted in the baby even after three exchange transfusions. In this study the authors achieved complete elution (negative DAT); however, eluted cells reacted weakly with IgG anti-D in antihuman globulin phase although blend of IgG-IgM anti-D reagent did not react in direct grouping. Similarly, Moiz et al.⁶ reported blocked D phenomenon in another neonate typed as “A RhD negative” with a strong positive DAT result. Here we have described a case of severe RhD-HDFN in a foetus which presented as RhD negative by anti-D reagent testing but was subsequently determined to be RhD positive by testing eluted red cells with the same anti-D reagent.

Anti-D is the specificity most commonly implicated in this blocking phenomenon; however, the phenomenon is not limited to anti-D as antibodies to other blood group antigens could also possibly give rise to false negative results when phenotyping antibody-coated foetal red blood cells. Two cases of false negative K-typing of foetal cells due to blocking by maternal anti-K antibodies have been reported recently^7,8. In one study, the authors also performed a simulation exercise using anti-K sera from pregnant women with various titres and showed that antibodies with a titre of 256 or greater can block the K antigens on red cells⁷. Thus, one should suspect a blocking phenomenon when a foetus or neonate has a strong DAT and types antigen-negative for the antibody present in the mother.

The discovery of cell-free foetal DNA present in the maternal circulation has provided a potential method for non-invasive assessment of foetal RHD using molecular analysis of a maternal blood sample⁹. Although this was not done in the present case, the use of non-invasive foetal RHD genotyping could have confirmed without any doubt the foetal RhD status in the antenatal period.

In conclusion, when interpreting the results of blood grouping on foetal or neonatal samples from an alloimmunised pregnancy with potent antibody, the blocking phenomenon should be taken into account. A false negative RhD grouping can be seen when maternal IgG antibodies saturate all antigen sites on foetal red cells and leave no antigen sites for the anti-D reagent to attach with. A proper clinical history, including the history of any intrauterine transfusions and results of previous immunohaematological investigations done during the perinatal period, is also very important in order to reach the final diagnosis, as illustrated in this report.