Anti-N antibody reacting at 37°C: An unusual occurrence interfering with routine testing: Two interesting cases.

Most anti-N antibodies are naturally occurring, IgM antibodies, and not active above 25°C and are not clinically significant but IgG anti- N has also been described. Immune anti-N resulting from multiple transfusions does occur & has been implicated as the cause of hemolytic transfusion reactions and mild hemolytic disease of fetus and newborn. Anti- N reacting at room temperature can be a cause for ABO blood group discrepancy.

Introduction

MNS blood group system was the second to be discovered by Landsteiner and Levine in 1927 after ABO blood group system. Among antibodies of MNS blood group system, anti-M is a relatively common “naturally occurring” antibody.[1] Anti-N is relatively rare compared with anti-M;[12] in one series of 86,000 patients, only two examples of anti-N were found (Croucher, personal communication).[3] It is very rare for anti-N to be formed as an immune antibody,[3] only two such cases have been reported in literature[3] by Callender and Race[4] and Ballas et al.[5] Perrault found only eight cases of anti-N in 45,000 blood samples against M− N+ (or M+ N+) cells in the auto analyzer, using a low ionic-strength polybrene method.[1] Most anti-N antibodies are naturally occurring IgM antibodies. These are usually not active above 25° C and not considered clinically significant.[6] The anti- N of IgG type has also been described in literature.[1] Immune anti-N resulting from multiple transfusions do occur, usually in people of African origin with M+ N− S− s− U− red cells.[5] Anti-N has been implicated as the cause of hemolytic transfusion reactions (HTRs)[5] and mild hemolytic disease of the fetus and newborn also.[7] Here, we report two cases (one donor and one patient) of naturally occurring anti-N reacting at 37°C.

Case Reports

Case 1

A 60-year-old patient was admitted in neurosurgery ward of our institute to be operated for parietal space occupying lesion with no history of previous transfusion. Blood sample received for four units of packed red cells using conventional tube technique showed preliminary blood group as A Rh D positive, but in the reverse grouping the patient's serum was reacting with all the three pooled A, B and O reagent red cells (4+ agglutination in tube) with negative autocontrol. Three cell screening panel (Diacell, Biorad, 1785, Cressier s/Morat, Switzerland) and 11 cell identification panel using conventional tube technique (Diapanel, Biorad, 1785, Cressier s/Morat, Switzerland) showed the presence of anti-N specificity. Antibody was reacting with similar maximum strength of reaction (4+) after 1 h water bath incubation at 37°C. Dithiothreitol (DTT) treatment of serum showed IgM type of immunoglobulin. Enzyme treatment of red cell could not be performed on patient sample. The anti-N titer was 1:32 at room temperature (doubling dilution). Patient's MNS phenotype was M+ N− S− s+. The red cells which were N− were compatible with patient serum both in the saline phase and the AHG phase. Reverse grouping with N− pooled ABO reagent red cells also resolved patient's blood group discrepancy.

Case 2

The blood group of a 24-year-old female repeat whole blood donor who donated for 5th time was reported as blood group discrepancy as her serum was reacting with pooled reagent O cells. Preliminary blood group of donor using conventional tube technique was O Rh D positive but in the reverse grouping there was a reaction with all three reagent red cells with autocontrol negative. Anti-H lectin with donor red cell was 4+ using conventional tube technique. Three cell screening panel (Diacell, Biorad, 1785, Cressier s/Morat, Switzerland) and 11 cell identification panel (Diapanel, Biorad, 1785, Cressier s/Morat, Switzerland) showed the presence of anti-N specificity using conventional tube technique. The antibody was reactive at room temperature and also at 37°C. DTT treatment of serum showed IgM type of immunoglobulin. Donor's MNS phenotype was M+ N− S− s+. Reverse grouping with N− pooled ABO reagent red cell resolved the patient's the blood group discrepancy. The previous blood grouping results were not traceable, but there was no history of blood transfusion and pregnancy in blood donor, so antibody can be considered as naturally occurring.

Discussion

Anti-N is usually not active at 37°C. It can generally be ignored in transfusion practice and if the room temperature incubation is eliminated from compatibility testing and screening for antibodies, antibody will usually not be detected.[8] The anti-N antibody in both of our cases was IgM type, having a wide thermal amplitude reacting at 37°C and capable of causing HTR. ABO discrepancy due to cold alloantibodies (e.g., anti-M) or autoantibodies (e.g., anti-I) reacting with reverse grouping cells, leading to unexpected positive reactions have been described in the literature.[9] In both the cases, anti-N was detected at an earlier stage as interfering with reverse grouping.