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*Corresponding author: E-mail: shayanthebest911@gmail.com; 
 
Cite as: Ashfaq, Muhammad Shayan, and Muhammad Hasan. 2025. “Anti-N Antibody Reacting at 37°C: An Unusual Reaction 
in Antibody Screening in a 20-Year-Old Male Liver Donor”. Asian Journal of Immunology 8 (1):82-85. 
https://doi.org/10.9734/aji/2025/v8i1162. 
 

 
 

Asian Journal of Immunology 
 
Volume 8, Issue 1, Page 82-85, 2025; Article no.AJI.135018 
 

 
 

 

 

Anti-N Antibody Reacting at 37°C: An 
Unusual Reaction in Antibody 

Screening in a 20-Year-Old Male Liver 
Donor 

 
Muhammad Shayan Ashfaq a* and Muhammad Hasan a 

 
a Department of Pathology and Laboratory Medicine, Section of Hematology & Transfusion Medicine, 

Aga Khan University Hospital, Karachi, Pakistan. 
 

Authors’ contributions  
 

This work was carried out in collaboration between both authors. Authors MSA and MH contributed to 
the design, drafting and critical revision of the manuscript. Both authors read and approved the final 

manuscript. 
 

Article Information 
 

DOI: https://doi.org/10.9734/aji/2025/v8i1162  
 

Open Peer Review History: 
This journal follows the Advanced Open Peer Review policy. Identity of the Reviewers, Editor(s) and additional Reviewers,  

peer review comments, different versions of the manuscript, comments of the editors, etc are available here: 
https://pr.sdiarticle5.com/review-history/135018  

 
 

Received: 23/02/2025 
Published: 29/04/2025 

 
 

ABSTRACT 
 

Anti-N antibodies usually possess cold-reactive properties which make them clinically insignificant 
and are mostly classified as naturally occurring IgM antibodies. These antibodies sometimes trigger 
reactions at body temperature (37°C) or in the anti-human globulin phase resulting in serious 
medical outcomes such as delayed hemolytic transfusion reactions or hemolytic disease of the 
newborn. This case shows an extraordinary naturally occurring anti-N antibody which was detected 
in a 20-year-old male liver donor during standard antibody testing because of its unexpected 
reactivity at 37°C. The absence of any previous blood transfusions or medication use in the patient 
made this case stand out as particularly intriguing. The antibody identification was successful 

Case Report 

https://doi.org/10.9734/aji/2025/v8i1162
https://pr.sdiarticle5.com/review-history/135018


 
 
 
 

Ashfaq and Hasan; Asian J. Immunol., vol. 8, no. 1, pp. 82-85, 2025; Article no.AJI.135018 
 
 

 
83 

 

following the 'pre-warm' technique implementation while antigen phenotyping validated it as an IgG-
type anti-N antibody. This medical case demonstrates the critical importance of recognizing 
unusual antibody responses during blood transfusions and organ transplants because uncommon 
reactions may lead to serious consequences. 

 

 
Keywords: Anti-N antibody; MNS blood group system; transfusion medicine; serological testing; 

delayed hemolytic transfusion reactions. 
 

1. INTRODUCTION 
 

The MNS blood group system, discovered by 
Landsteiner and Levine in 1927, was the second 
to be identified after the ABO system. Among the 
antibodies in the MNS system, anti-M is a 
common "naturally occurring" antibody (Perrault, 
1973). The S antigen was identified in 1947 by 
Walsh and Montgomery after the development of 
the antiglobulin test (Harmening, 2018). Most 
anti-M antibodies are cold-reactive and do not 
activate complement or react with enzyme-
treated RBCs (Thakral et al., 2010). They are 
rarely associated with hemolytic transfusion 
reactions (Sancho et al., 1998). 
 
Anti-N antibodies are less common than anti-M 
and are also typically naturally occurring, cold-
reactive IgM or IgG agglutinins that do not 
activate complement or react with enzyme-
treated RBCs (Perrault, 1973; Taj et al., 2025). 
They are clinically insignificant unless reactive at 
37°C and have been linked to rare cases of mild 
hemolytic disease of the fetus and newborn 
(HDFN) (Ballas et al., 1985; Wiebe et al., 2024). 
Potent anti-N antibodies are more frequently 
found in individuals of African descent with a 
specific RBC phenotype (M+ N- S- s-) due to the 
absence of the N antigen (Harmening, 2018; 
Katagiri et al., 2024). Immune anti-N antibodies 
are extremely rare (Klein & Anstee, 2013). We 
report a case of naturally occurring anti-N that 
reacts at 37°C, identified during routine antibody 
identification testing (Kumawat et al., 2015). 
 

2. CASE REPORT 
 
A 20-year-old guy from Karachi, who’s healthy 
and doesn't have any other medical issues, 
decided to donate a liver to his dad. His father 
had been struggling with chronic liver disease 
because of a hepatitis B infection, so they set up 
a liver transplant at Dow University of Health 
Sciences. This donor had never had a blood 
transfusion or taken any medication. For the 
usual pre-transplant checks, they sent a test 
request to Aga Khan University Hospital to 
identify any antibodies. The first blood tests 

came back showing a positive auto-control, 
which means his red blood cells reacted with his 
own serum at room temperature. But when they 
did a Direct Antiglobulin Test (DAT) with anti-IgG 
+ C3d, it came back negative, ruling out any 
autoantibodies. They then checked three 
different antibody screening panels (ID-Diacell I-
II-III, Biorad) and found positive reactions in all of 
them (2+, 3+, and 3+). They also noticed that the 
auto-control was positive right from the spin 
phase. So, they decided to run the antibody 
screening again using a method where they 
warmed everything up, and surprise! They got 
positive results across all three panels. To dig 
deeper, they used the Papain treatment method 
(ID-Diacell Papain Kit), which surprisingly turned 
up a negative result for the red cell antibody 
screening. But when they went ahead with the 
antibody identification using the warm technique, 
they found out he had an anti-N antibody using 
this 11-cell identification panel (ID-Diacell, 
Biorad). The reaction was pretty strong, 3+, with 
homozygous N+ N+ cells (Panels 4, 10, and 11) 
and negative with heterozygous M+ N+ cells 
(Panels 1, 3, 7, and 8), plus also negative with N-
negative cells (Panels 2, 5, 6, and 9). They also 
checked other antigens for N, S, s, and M and 
found that he was M-, N-, S-, and s-. They 
treated his plasma with dithiothreitol, which 
confirmed there was an IgG-type anti-N antibody 
present. The antibody titer was 1:2. This report 
emphasizes a naturally occurring anti-N antibody 
that reacts at 37°C, which could actually be 
pretty important even though the donor hadn’t 
had any blood transfusions before. It really brings 
home the need to check for these naturally 
happening antibodies during routine blood tests 
because their reactions at body temperature can 
affect transfusion practices. 
 

3. DISCUSSION 
 
Anti-N antibodies belong to the MNS blood group 
system and are usually naturally occurring and 
mainly cold-reactive IgM antibodies. These 
antibodies are often clinically insignificant unless 
they decide to react at 37°C or during the anti-
human globulin (AHG) phase of testing. In this 



 
 
 
 

Ashfaq and Hasan; Asian J. Immunol., vol. 8, no. 1, pp. 82-85, 2025; Article no.AJI.135018 
 
 

 
84 

 

case, we found the anti-N antibody in a 20-year-
old male liver donor who had a serological profile 
showing an uncommon blood group discrepancy. 
While anti-N antibodies are generally more of a 
cold-reactive type and don’t usually bind 
complement, if they do react at 37°C, that’s when 
we start to worry about their clinical significance. 
 
Normally, anti-N antibodies don’t cause major 
issues, as they are linked to non-pathological 
clinical outcomes. They typically don't lead 
to hemolytic transfusion reactions 
(HTRs) or hemolytic disease of the fetus and 
newborn (HDFN) unless we see them react at 
body temperature (37°C), like in this instance 
(Perrault, 1973). A transfusion reaction is more 
likely if these antibodies show strong reactivity at 
37°C, but that's pretty rare. When it happens, it 
can cause delayed hemolytic reactions, resulting 
in some transfusion-associated complications. 
Besides, while HDFN can be a concern in cases 
of maternal-fetal blood group incompatibility, 
instances of HDFN linked to anti-N are super 
rare (Harmening, 2018). 
 
Interestingly, the IgM class of anti-N antibodies 
usually reacts in colder conditions and doesn’t 
typically bind to complement or react with 
enzyme-treated red blood cells (RBCs). This is 
kind of similar to anti-M antibodies, which show 
the same cold-reactive behavior and limited 
clinical relevance, unless they react at body 
temperature (Thakral et al., 2010). However, in 
our case, the antibody was behaving unusually 
by reacting at 37°C, reminding us to stay on our 
toes about atypical antibody behaviors in blood 
donors, especially when we’re talking 
about organ transplant recipients who might 
need careful crossmatching and serologic 
evaluations. 
 
The phenotyping results were pretty noteworthy 
since the donor's red cell antigen profile showed 
the absence of the M, N, S, and s antigens, 
which are usually part of testing in the MNS 
blood group system. Plus, after treating 
with Dithiothreitol (DTT), we confirmed that the 
anti-N antibody was of the IgG nature, hinting 
that it could have some clinical significance in 
certain transfusion scenarios (Sancho et al., 
1998). 
 
Finding an anti-N antibody in a healthy individual 
with no prior blood transfusions is pretty unusual, 
given that naturally occurring antibodies in the 
MNS system are typically IgM types and don’t 
usually trigger immune responses. The titer of 

1:2 here suggests the anti-N antibody could 
definitely be clinically important at 37°C 
(Kumawat et al., 2015). This just goes to show 
how important it is to do a thorough antibody 
screening for blood donors; even naturally 
occurring antibodies might need our attention in 
a clinical setting. 
 

4. CONCLUSION 
 
In conclusion, this case really emphasizes the 
importance of rare blood group 
antibodies in organ 
transplantation and transfusion medicine. It’s a 
reminder for clinicians and lab staff to keep an 
eye out for unusual antibody profiles, especially 
when there are discrepancies in routine 
serological testing. We need more studies and 
awareness about these antibodies to 
avoid adverse reactions in transfusions 
and organ transplant procedures. 
 

DISCLAIMER (ARTIFICIAL INTELLIGENCE) 
 
Author(s) hereby declare that NO generative AI 
technologies such as Large Language Models 
(ChatGPT, COPILOT, etc.) and text-to-image 
generators have been used during the writing or 
editing of this manuscript.  
 

CONSENT  
 
As per international standards or university 
standards, patient(s) written consent has been 
collected and preserved by the author(s). 
 

ETHICAL APPROVAL 
 
It is not applicable. 
 

COMPETING INTERESTS 
 

Authors have declared that no competing 
interests exist. 
 

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