Hemolytic Disease of the Fetus and Newborn: Understanding the Testing Needed to Confirm
the Identity of the Causative Antibody
First, we would like to thank the authors for the thought-provoking questions, which
are relevant to the case report. The authors have asked the question about how the
diagnosis of anti-M alloimmunization was made in the neonate.
Mother's plasma and breast milk had shown anti-M antibodies (the process of detection
and quantification has been described in detail below). There was fetal anemia and
signs of early hydrops on fetal scans prior to delivery. At birth, the neonate had
presented with hemolytic hyperbilirubinemia. M antigen typing of the newborn was done
and showed a positive reaction. Both mother and newborn had the same blood group,
A positive, which eliminates other causes of neonatal anemia and jaundice such as
ABO or Rhesus incompatibility.
The authors have further raised queries about the methodology involved in the detection
and quantification of anti-M antibodies in maternal plasma and breast milk. We did
not mention these in the original case report, considering the readership would be
mostly obstetricians and fetal medicine specialists, and the key message we want to
convey through this case report is that anti-M alloimmunization is a reality and that
there is a phenomenon of ethnic susceptibility, hence it is much more common in Asians
than Caucasians.[1] The methodology of anti-M detection and quantification are described below.
Breast Milk Collection
Breast milk samples were collected after sufficient feeding was established. There
was no protocol available for the collection of breast milk. We, therefore, decided
to collect samples within 2 to 5 days of establishment of feed to maintain uniformity
in the study. Samples were collected before giving the next feed. At least 1 mL of
breast milk (∼ 16 drops) was collected from every mother in a sterile container without
anticoagulant. Fresh uncentrifuged breast milk was used for testing.[2]
Antibody Screen and Identification
Antibody Screen and Identification
Red cell antibody screen and indirect Coombs test (ICT)/indirect antiglobulin test
(IAT) were performed using column agglutination technique (LISS/Coombs IDCard, DiaMed
Gmbh, Bio-Rad Laboratories, Switzerland). Fifty microliters of screen cells and 25
μL of mother's plasma/breast milk were incubated at 37°C for 15 minutes and then centrifuged
for 10 minutes in the DiaMed card centrifuge. ICT was performed using 3 to 5% suspension
of saline-washed pooled phenotyped “O” group cells and patient plasma along with negative
and positive controls. Positive control for IAT was 1 in 10 dilutions of immunoglobulin
G anti-D, while negative control was AB group plasma. Identification of antibody,
if present, was performed using 11-cell identification panel (ID-DiaPanel, DiaMed
Gmbh, Bio-Rad Laboratories, Switzerland) in the Coombs phase.
Titers of Anti-M Red Cell Alloantibodies
Titers of Anti-M Red Cell Alloantibodies
Titers were performed for anti-M red cell alloantibodies if identified using the column
agglutination technique. Titers were performed in the saline (NaCl, Enzyme test and
Cold agglutinins DiaMed Gmbh, Bio-Rad Laboratories, Switzerland) and Coombs (LISS/Coombs
IDCard, DiaMed Gmbh, Bio-Rad Laboratories, Switzerland) phase. Breast milk was titrated
in the same way as the plasma. For the Coombs phase titer, the plasma and milk were
treated with dithiothreitol (Sigma Aldrich Co., St. Louis, MO, USA) for inactivation
of immunoglobulin M antibodies. The titer was determined by testing the serial twofold
(2-1024) dilution of the plasma/breast milk against selected red cells. One hundred
microliter of plasma/breast milk was added to a tube containing 100 μL saline labeled
as 1:2. Further, 100 μL was transferred from the tube 1:2 to 1:4 and continued through
the remaining tubes that contained 100 μL saline each. Twenty-five microliters from
each of these dilutions were added to the correspondingly labeled columns in the card.
Fifty microliters of 0.8% red cells (M antigen positive O red cells) were added to
the columns and titers were reported as the column with 1+ agglutination.
