A pregnant women (gravida 2) with a 3-year-old daughter delivered twins (diamniotic dichorionic) at 38 weeks of gestation as normal vaginal delivery. The newborns were normal at the time of birth, but presented with hyperbilirubinemia at 16 h of life. Mother was never transfused previously. Clinically both the newborns were stable hemodynamically with no hepatosplenomegaly, hydrops, hematoma, polycythemia, or twin discordance on examination and were adequately taking breast milk. Blood bank received a requisition for reconstituted whole blood for an exchange transfusion for the twins (T1, T2) as their billirubin [Figure 1] was in exchange zone.
Twins did not require exchange transfusion as phototherapy which was given over 4 days showed a reduction in the bilirubin levels. Both the newborns further showed progressive decrease in hematocrit (HCT) and later developed hepatosplenomegaly. Peripheral smears revealed no evidence for hemolysis, low reticulocyte count (T1-4%, T2-3%). The TORCH titers, serology for parvovirus, sepsis screen were all negative. Quantitative G6PD and thyroid profile were within normal range. Bone marrow examination showed predominantly normoblasts with few cells showing dyserythropoiesis. The other cell lines such as megakaryocytes and cells of lymphoid lineage were normal. Further adenosine deaminase levels were also done to rule out Diamond-Blackfan anemia. The mother and father also had normal beta and alpha thalassemia screen.
Blood sample of the newborn twins as well as the mother were sent to the blood bank. ABO and D blood typing was performed routinely by both manual tube and column agglutination technology (CAT). Direct antiglobulin test (DAT) was done in CAT cards containing polyspecific antihuman globulin (AHG) (anti-IgG and anti-C3d). Screening of irregular antibody and identification was done using surgiscreen and resolve Panel A, ortho-clinical diagnostics (USA) with polyspecific AHG cards. Rh, MNSs and Kell phenotypes were performed in tubes according to manufacturer's instruction by direct agglutination (Rh, K, M, N, S) and by IAT(s). CAT cards (plain and AHG) and rare antisera used for phenotyping were from ortho-clinical diagnostics (USA). Antibody titration was performed on CAT polyspecific cards and by direct agglutination on tubes. The titer was determined by serial two-fold dilutions in saline, tested against selected M+N- red blood cells (RBCs). The reciprocal of the highest dilution that gave a 1+ reaction is referred to as the titer.
Blood group of twins was O RhD positive. DAT was negative for both the twins. Elution of the DAT negative sample was not done.
Blood group of mother was O RhD positive. On antibody screening, mother showed a positive reaction with screening red blood cells, the reaction strength being cell I- (2+); cell II- (4+); cell III- (4+) at both room temperature and AHG phase, indicating presence of an alloantibody in maternal sample of both IgM and IgG type, which was duly confirmed on 1,4-dithiothreitol treatment.
Further antibody identification panel showed antibody specificity to M antigen, reacting at both room temperature and AHG phase. Similar antibody was identified in the serum of both the twins as well, but reacting at AHG phase only (IgG type). Both antibody screen and identification done on the CAT and manual tube showed similar results.
Phenotype of the twins was a heterozygous state of M and N antigen (M+N+). The mother was homozygous for N antigen (M-N+) and father was homozygous for M antigen (M+N-) [Table 1].
The M alloantibody (both IgG and IgM types) showed clear evidence of dosage phenomena with a weaker reaction with the heterozygous cells (M+N+) when compared to a homozygous cell (M+N-). The antibody identification was further enhanced by adding one part (0.1 N) HCl to four parts of the serum before testing (to lower the pH at 6.5). Alloantibody also showed a stronger reaction in albumin than in saline.
The antibody titer (using tube and CAT both) during 1st week of life was 16 (IgG) in twin-1 (T1) and 8 (IgG) in twin-2 (T2). The mother had a titer of 16 (IgG) and 8 (IgM) after the delivery. Anti-M was detectable in the twins until 7th weeks of life. The anti-M titer in the mother were constant at 32 (IgG) and 16 (IgM) until 8 weeks of follow-up postbirth [Figure 2].
Both the newborns initially showed rise in bilirubin, which subsided following phototherapy (started on day 1) on day 4 and did not require exchange transfusion. Twin-1 showed a decrease in hematocrit to 16% at day 6 and was transfused with 40 mL of (M-N+) packed RBCs (PRBC). On day 32, HCT dropped again to 16% but without any rise in bilirubin and both newborns were transfused with M-N+ PRBC. Repeatedly both showed decrease in the red cells, with all rest other cell lines normal, without any feature of hemolysis. On day 49, postbirth both the twins again had low HCT (15%) and were again transfused with (M-N+) PRBC. Twins continued to have anemia until 70 days of life after birth, their hematocrits stabilized their after [Figure 1].