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The human erythrocyte embodies various antigens on its surface. Any discrepancy in ABO blood typing due to the variants or weaker subtypes often leads to immunohematology [IH] work-up challenges. The major subtypes of antigen A are A1 and A2, wherein only the A1 subgroup reacts with anti-A1 lectin (Dolichos biflorus). Erythrocytes reacting with anti-A and Anti-A1 are typed as A1, while those reacting exclusively with anti-A alone are called A2.[1] However, A1 and A2 are not just the only subtypes of antigen A; there are other subtypes too, depending on the agglutination with anti-A. The variants such as A3, A3B, Aend, and Ax show agglutination with anti-A while subtypes, namely, Am, Ay, and Ael cells, do not. The genetic variations in the ABO locus lead to the polymorphism of certain genes. Therefore, to differentiate the phenotypes, serological techniques like an agglutination of cells using anti-A, anti-B, anti-A, B, and anti-H, serum grouping of ABO antibodies, the presence of anti-A1 and the adsorption elution experiments with polyclonal anti-A from B and O typed individuals all have been used in the IH laboratories worldwide. Furthermore, the weaker variants of antigen A and B can be accidentally speculated as group O and if, somehow, this variant is administered to an O typed recipient; it will decrease the chance of survival of red cells and can be detrimental to the recipient both immunologically as well as clinically. Therefore, it is crucial to identify these subtypes accurately to avoid their mistyping. We report our experience of an AMFA B Rh (D) positive blood type in an Indian male term newborn presenting with immune-mediated neonatal hyperbilirubinemia [NNH] due to Rh (D) iso-immunization with his mother, who was typed as A1B Rh (D) negative. The red blood cell (RBC) typing of ABO and Rh (D) antigens was performed using reagents (Tulip Diagnostics (P) Ltd, Goa). The conventional test tube [CTT] agglutination test was used for both cell and serum grouping using these monoclonal antisera. The IH work-up included the direct anti-globulin test (DAT) and indirect anti-globulin test (IAT), and antibody detection using poly-specific (anti-IgG,-C3d) column agglutination test (; Ortho Clinical Diagnostics, New Jersey, USA). The adsorption and elution method was used as further IH work-up to confirm the status of A antigen on the newborn's erythrocytes following serological methods as described in the American Association of blood bank standards.[2] Physically, the term newborn was appropriate for gestational age [AGA] and presented with pallor and deep icterus up until his soles. He was active, and alert with good spontaneous movements and normal reflexes. The bodyweight of the newborn was 2.5 kg. Despite his respiratory and cardiovascular findings within the normal specified limits, he had moderate hepatomegaly on abdominal examination. The risk factors for neonatal sepsis and gross congenital anomalies were absent. His total serum bilirubin [TSB] was 481.7 μ mol/L at 16 h after birth (in newborns, the TSB would normally range from 34.0 to 110 μmol/L within 24 h of birth). He was transferred to the neonatal intensive care unit for the early management of his rising TSB concentration. Unlike his mother's blood type, the term newborn's blood subtype showed mixed field agglutination [MFA] with anti-A monoclonal anti-sera and positive agglutination reactions with anti-B and anti-Rh (D) monoclonal anti-sera (grade 4 + each) in both CTT and CAT methods respectively [Table 1] and [Figure 1]a and [Figure 1]b. The control used for cell grouping was valid. In CTT, the monoclonal anti-A antisera showed a weak positive reaction with A tube against the CAT method, wherein an MFA was seen. The final group of the newborn was interpreted as AMFA B Rh (D) positive. The DAT of the newborn was positive [Grade 4+] [Figure 1]c. The adsorption and elution method showed a positive agglutination (grade 2+) with A antigen along with negative 8th saline wash [Figure 1]d. The final clinical diagnosis of NNH with a term AGA newborn due to Rh (D) incompatibility was arrived at. The neonate became anemic on the 3rd day of life, with a hemoglobin level of 7.4 g/dL. The peripheral blood smear showed spherocytes with moderate anisopoikiolocytosis consistent with the ongoing in-vivo hemolysis. The critical rise of his TSB concentration within 24 h of birth warranted a justifiable bedside clinical decision to perform a double volume exchange transfusion [DVET] with phototherapy. For DVET, we selected an O Rh (D) negative, leukocyte-reduced packed red blood cell [PRBC] unit in citrate phosphate dextrose adenine (CPDA-1) anticoagulant preservative solution, stored up to five days after collection and cross-matched with the mother's serum. In addition, the selected compatible PRBC unit was reconstituted with the thawed AB plasma to adjust the volume and hematocrit of the reconstituted whole blood to 410 mL and 45%, respectively. In addition, on the 3rd day post DVET, a top-up transfusion of 25 mL cross-matched compatible and leukoreduced O Rh (D) negative PRBC was transfused to the newborn. There was a gradual reduction in his serum concentration of TSB.
Table 1: Showing ABO and Rh (D) blood typing of Mother and Newborn performed by $CAT method
Figure 1: (a) The mother's blood type was A1B Rh (D) negative (b) Her 3-day old term newborn's blood type was AMFA B Rh (D) Positive (c) The newborn's DAT was positive with 4+ grade and valid control (d) On performing adsorption & elution, the eluate showed 2+ grade of reaction with type A erythrocytes and a valid 8th saline wash in the respective control wellsThe A subgroups can often result in an ABO typing discrepancy.[3] Amongst these A subgroups, the serological phenomenon called MFA is commonly seen, with the A3 phenotype, which is rare but a known rare subgroup of A, occurring with a frequency of <1:1000.[4] To prove the presence of a weak variant of A antigen on the DAT positive erythrocytes, the adsorption elution method was used.[2] It was then finally interpreted as a probable A variant. Further, all the problems related to the sample, such as fibrin residues in the red cell suspension, reagents, and technical errors, were ruled out. Just like our report, Svensson et al.,[5] in their A variant/A3 phenotyped sample, found the classical MFA of both agglutinating and non-agglutinating cells confirmed on nuclear magnetic resonance. Furthermore, there is a possibility that the baby may, eventually, produce anti-A1 antibodies as he grows up, thereupon adding clinical significance for its future. It's a well-known fact that ABO antigens are poorly developed at birth. We are aware that the poorly developed antigen expression may give rise to MFA as well. In fact, this may resolve spontaneously as the child grows up. However, if persistent, family study for the molecular characterization of the A variant would be required. With appropriate management, the newborn showed optimal developmental milestones, consistent improvement in his body weight, and a normal liver size on his follow-up visit. He was finally discharged on the 28th day of birth from our hospital. In the discharge summary, we advised that the blood group should be repeated after six months of age for the confirmation of the ABO status. To conclude, caution must be, exercised when MFA is noted in routine blood typing using CAT. Thus, vigilant reporting of the newborn's blood type from a hospital-based blood transfusion laboratory while dealing with subgroups[6] will help ensure safer transfusion practices amongst the neonates.
Informed consent
As per our policy, informed consent is obtained from all the recipients before blood transfusion in accordance with our hospital's blood transfusion policy.
Ethical Approval
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional ethics committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
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Conflicts of interest
There are no conflicts of interest.
References 
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CheckDOI: 10.4103/ijpm.ijpm_136_22
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