Severe haemolytic disease of a newborn with variant D mimicking blocked-D phenomenon

Abstract

Anti-D is still the most common antibody causing severe haemolytic disease of the fetus and newborn (HDFN). In a mother with a very high titer of anti-D, antibodies can coat and block the D antigens on the red blood cells of the newborn. This blocking phenomenon prevents agglutination of the D-positive red cells with the IgM anti-D typing reagent, giving false negative results. Here, we report the case of a newborn with variant D phenotype and severe HDFN, which mimicked the blocked-D phenomenon, which, at the first instance, confused both the treating clinicians and the transfusion service personnel.

Background

Rh blood group is one of the most complex blood group systems. Of the 64 different antigens of the Rh system, it is the D antigen which is routinely typed and has the highest clinical value.¹ Individuals who lack the D antigen may develop anti-D on exposure to a minimal amount (as low as 0.01 mL) of Rh D+ allogenic red cells. Generally, the D antigen test in the laboratory yields a clear result; however, there exists a grey area described as D variant or serological weak D antigen.²

There are several theories that explain weak, partial or variant D phenotypes. Daniel et al defined the D variants as ‘Weak D antigens have all D epitopes but with weak expression while partial D antigens lack one or more D epitopes. Individuals with partial D antigens can make anti-D; those with weak D antigens cannot.’³ The molecular basis for D variants is either the amino acid substitution in the Rh D protein or the product of genetic recombination between Rh and RHCE genes.³ Rh D typing of the mother and later administration of RhIG (Rh Immune globulin) is an important aspect of antenatal care. However, anti-D is still one of the common antibodies causing immune-mediated haemolytic disease of the fetus and newborn (HDFN). The blocking phenomenon prevents agglutination of the D-positive red cells with the IgM anti-D typing reagent, giving false negative results. Here, we report a case of a newborn with variant D phenotype and severe HDFN which mimicked the blocked-D phenomenon, which, at the first instance confused both the treating clinicians and transfusion service personnel.

Case presentation

A male baby was born to a 30-year old woman (para-2, intrauterine death-1, live-1) at 37 weeks of gestation at a peripheral hospital. The couple were of Dravidian ethnicity and the biological father was same on both the occasions. Her first pregnancy had an adverse outcome as intrauterine death due severe pre-eclampsia at 36 weeks of gestation. Her blood group was reported as ‘A Negative’. The blood group of the stillborn based on the autopsy was ‘O Positive’ and the mother received anti-D RHIG following the birth of the first child. There was no history of transfusion in the past and during this pregnancy also. Her antenatal period for the second pregnancy (ie, current pregnancy) was also uneventful. Antenatal antibody screening was not performed in the primary healthcare unit.

The newborn had pale skin, poor feeding, fast heart rate and rapid breathing at rest at birth. Subsequent laboratory findings indicated HDFN which includes hyperbilirubinemia (total bilirubin 25 mg/dL, direct bilirubin 0.9 mg/dL and indirect bilirubin 24 mg/dL) with anaemia (haemoglobin 50 g/L) and the peripheral smear showed polychromasia and anisocytosis with nucleated RBCs within 24 hours of birth. The baby was typed as O Rh (D) negative with a positive direct antiglobulin test (DAT). ABO incompatibility (maternal group A/neonate group O) was ruled out. Other non-immune mediated causes for HDFN were ruled out (ie, alpha thalassemia major, hereditary spherocytosis, glucose-6-phosphate dehydrogenase (G6PD) deficiency, pyruvate kinase deficiency and the UGT1A1 gene promoter associated with Gilbert syndrome). In view of the rising hyperbilirubinemia, the baby received IvIg (500 mg/kg over 4 hours) and leukoreduced, irradiated O RhD negative packed red blood cell top up transfusion at the referral hospital as per NICE 2016 guidelines on management of jaundice in newborn babies under 28 days.^{4 5} Our laboratory received samples of the father, mother and newborn for further immunohaematolgical workup to establish the reason for severe immune-mediated destruction of red cell in a newborn where there was no documented Rh D or ABO incompatibility. All the samples of the newborn was obtained prior to start of any treatment in the newborn.

Investigations

Immunohaematology workup

We performed blood grouping by the column agglutination technique using a fully automated blood grouping equipment (Ortho Auto-Vue Innova System, Buckinghamshire, England). The mother’s sample was typed ‘A Rh D Negative’. Serum grouping showed unexpected agglutination (grade 4+) with pooled reagent red cells of ‘A’ and ‘O’ blood groups. The indirect antiglobulin test was positive. The presence of anti-D red cell alloantibody along with anti-Le^b antibody was confirmed using commercially available antibody screening and antibody identification red cell panels (BioRad ID, Micro Typing System, Switzerland). To avoid interference of anti-D, Rh D Negative ‘A’ and ‘O’ group pooled reagent red cells were used to resolve ABO discrepancy. Maternal red cells were found to have r’r (dCe/dce) and Le (a-, b-) phenotype. The IgM and IgG anti-D titers were 2 and 512, respectively using R1R1 cells. IgG titer was performed using the dithiothreitol-treated maternal serum. The father’s sample was typed as O Rh (D)+, and further as R1R1or R1r’ (DCe/DCe or DCe/dCe) phenotype.

The newborn was typed as ‘O’ for the blood group. However, mixed field reaction was noted on typing for D antigen (majority of the cells were settled at the bottom of the gel column with few agglutinates) as shown in figure 1. A DAT using polyclonal antihuman globulin reagent gave a strong positive (+4) reaction. Antibodies from RBCs were eluted by heat for further testing. Anti-D antibody was identified in the eluate and serum of the newborn. We suspected blocked-D phenomenon leading to the weaker or mixed field reaction while typing for Rh D antigen on the newborn’s sample. Repeating the blood group of the newborn on follow-up helped us to resolve the problem. All immunohaematological techniques were performed as per the departmental standard operating procedure (SOP) (prepared in accordance with AABB methods).⁶

Figure 1.

The newborn’s blood group at birth.

Differential diagnosis

Blocked-D phenomenon, serological weak D in the baby, alpha thalassemia major, hereditary spherocytosis, G6PD deficiency and ABO incompatibility.

Outcome and follow-up

On follow-up, the baby’s blood group was typed as ‘O Rh (D)−’ in third month of life as shown in figure 2. DAT was repeated and found to be negative. The Rh phenotyping of the baby was ‘ce’. The weak D testing showed a positive (+2) reaction performed by the gel column technology using IgG anti-D as shown in figure 3. Partial Rh D typing performed using commercially available cell panels (BioRad ID, Micro Typing System) showed D variant type III. Hence, we reported the case as variant D phenotype in a newborn baby with features of HDFN.

Figure 2.

The newborn’s blood group after 3 months.

Figure 3.

Weak D testing showing a positive result (arrow mark indicates agglutinations).

Discussion

HDFN is characterised by the increased rate of red blood cell (RBC) destruction. The principle clues which suggest haemolytic anaemia include increased number of reticulocytes and/or circulating nucleated RBCs, unconjugated hyperbilirubinemia, a positive direct antiglobulin test and characteristic changes in red cells in blood films.^{7 8} Based on aetiology, haemolysis in newborn can be immune or non-immune mediated. Non-immune mediated causes include alpha thalassemia major, hereditary spherocytosis and G6PD deficiency.⁷ In the current case, non-immune causes were ruled out as the newborn was born to Rh D Negative mother with Rh D incompatibility in parents and DAT of the baby was positive. Hence, it was concluded that it was a case of HDFN due to Rh incompatibity.

Despite antenatal and postnatal RhIg prophylaxis, 1–3 in 1000 Rh D Negative women develop anti-D.^{9 10} The lack of uniform and universal anti-D immunoprophylaxis is the reason for this higher rate of alloimmnunisation in India.^11–13 However, in developed countries, even with immunoprophylaxis, anti-D is still a leading offender, as shown by Gottvall et al (60%) and Howard et al (42%).^{14 15} There are several reasons why anti-D still causes HDFN, including D antigen typing errors in pregnant women and newborns, under-recognised sensitisation during pregnancy and insufficient dose of RhIg and immunisation secondary to blood transfusion.^{16 17} Administration of RhIg to Rh D Negative pregnant women is the cornerstone of the prevention of alloimmunisation to D antigen and the subsequent prevention of HDFN. It is a very safe and effective therapy. Revised guidelines of British Committee For Standards In Haematology (BCSH) in 2014 summarise ‘administration of anti-D Ig, following potentially sensitising events, as soon as possible and always within 72 hours of the sensitising event’.¹⁸ This regimen is recommended by obstetric professional societies and serves as the standard of care in many countries.^18–20 In spite of the established guideline by regulatory bodies like Federation of Obstetric and Gynaecological Societies of India, the use of anti-D prophylaxis in all enlisted sensitising events such as abortions, ectopic pregnancy and medical termination of pregnancy (MTP) is as low as 10%–20% owing the cost factor.²¹ This case highlights the importance of anti-D immunoprophylaxis which needs to implemented across the country.

Serological problems on Rh D typing pose a major challenge to the immunohaematologist. Blocked-D is one such phenomenon.^22–25 Sulochana et al described a case of Rh D Negative blood group in a newborn baby with severe HDFN born to a ‘B Rh D Negative ’ second gravida. The baby was grouped as ‘B Rh D Negative’ by direct grouping; however, anti D was recovered from elution, and post elution, red D antigen was detected on the red cells of the baby.²⁴ Verma et al also described a similar case in which elution studies with chloroquine diphosphate confirmed the presence of the D antigen.²⁵ Roziers et al and Katharia et al showed that, between the chloroquine diphosphate and glycine EDTA methods of elution, the glycine-HCl/EDTA method was more effective in reducing the strength of the reaction of DAT.^{26 27} However, in the present case, we could not achieve complete elution of the antibodies by the glycine-HCl/EDTA method and failed to obtain DAT negative cells from the newborn’s RBCs. Hence, we decided to repeat the blood grouping of the newborn at 3–4 months of life to confirm the RhD status. On repeating the Rh typing at third month of life, the baby was typed as Rh D Negative with the Rh phenotype of ‘ce’ at the saline phase. Serological weak D status was confirmed by testing the red cells with IgG anti-D by IAT.

The unique feature of this case is that it was a D-variant (type – III) on RBC of newborn was the target of polyspecific anti-D produced by the mother’s. The mother’s anti-D reacted against all types of D epitopes and not specifically with the newborn’s D epitope as depicted from the mother’s serum reaction with other D positive cells. Though the variant D phenotype can trigger alloantibody production, we could not establish it in the present case. The patient had a history of prior sensitising events (previous pregnancy) which could have been the cause of alloimmunisation in this case.

The alloimmunisation potential of variant D is not very clear. DEL variants, weak D type 4.2, 11, 15, 21 and 57 can stimulate anti-D production. DVa stimulated the production of anti-D in the D-woman during her first pregnancy and resulted in mild HDFN of her second baby who also appeared to have the variant D antigen.²⁸ The mother had received anti-D immunoglobulin after the first pregnancy. DIII red cells are known to react with all monoclonal ant-D, yet must lack at least one D epitope as some individuals with DIII phenotype make anti-D.²⁹ A weak D type 3 fetus appears to have stimulated anti-D production in the mother with Rh ‘D’-Negative ‘C’ Negative phenotype.^{30 31} Though weak D testing is not recommended for the patients, it should be routinely done for the fetuses or newborns of Rh D Negative mothers. Sandler et al have proposed an algorithm for identifying serological weak D and to determine the candidacy for RhIG and Rh D types for transfusions.³² They recommend Rh genotyping whenever a discordant Rh D typing results are noted on serology. Owing to the limitation of resources, Rh genotyping of the newborn was not possible.

The present case illustrates the complexity of Rh D typing in neonates. It is not advisable to report R D status in cases with positive DAT. The variant D in newborns with positive DAT can mimic the blocked-D phenomenon.

Learning points.

• One should be careful while interpreting the blood group in patients with strong DAT positive results

• The blocked-D phenomenon can mimic the Rh ‘D’Negative blood group in newborns with severe haemolytic disease with Positive DAT. d

• Variant D phenotype may pose a problem in interpreting and reporting the Rh ‘D’ blood group.