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INRA, a new high‑frequency antigen
in the INDIAN (IN023) blood group
system
Sanmukh R. Joshi, Ankita Sheladiya, Kinjal V. Mendapara‑Dobariya
Website:
www.ajts.org

DOI:
Abstract:
10.4103/0973-6247.214337 BACKGROUND: The INDIAN blood group system comprises 4 antigens sensitive to enzymes and
2‑aminoethyl isothiouronium bromide (AET).
AIM: The patient’s antibody was investigated for its specificity to the high‑frequency antigens (HFA)
of this system.
MATERIAL AND METHODS: Low ionic strength solution  (LISS)‑tube/LISS‑indirect antiglobulin
test (IAT) methods were used. The patient’s red blood cells (RBCs) were tested with antisera to
HFA. Her antibody was tested with RBCs lacking the HFA. Furthermore, it was tested with RBCs as
untreated or treated with enzyme or AET. The genetic sequence was studied for mutation in CD44
gene that encodes INDIAN antigens.
RESULTS: The patient was grouped A1B, RhD+, antibody screening test positive, direct antiglobulin
test negative. A negative autocontrol test had suggested to the alloantibody being present. Antibody
had agglutinated RBCs in LISS‑tube at RT and by LISS‑IAT at 37°C. The RBCs of the 11‑cell panel,
those lacking HFA and from 50 random donors, were agglutinated by her antibody indicating its
specificity to the HFA, though the RBCs of Lu (a‑b‑)/In (Lu) type showed a weaker reaction. The
patient’s RBCs were agglutinated by antisera to a number of the enzyme‑sensitive HFA, including
those of INDIAN blood groups. The antibody showed reduced reactivity with the RBCs treated
with papain, chymotrypsin, and AET but resistant to trypsin and dithiothreitol. The patient’s genetic
sequence revealed a novel homozygous mutation 449G>A in exon 5 of CD44.
CONCLUSION: The antibody to enzyme sensitive HFA was tested for serological and molecular
genetics studies and found to be directed to the novel HFA, named as INRA of the INDIAN blood
group system and was assigned a numerical symbol IN: 005 by the International Society of Blood
Transfusion (ISBT).
Keywords:
A new high‑frequency antigen, in the INDIAN blood groups, INRA

Introduction In(b).[3] The INDIAN blood group system was

further expanded in the year 2007 when two
Lok Samarpan Regional
Blood Bank and Research
Center, Surat, Gujarat,
T he INDIAN  (023) blood group system
currently consists of 4 antigens, namely,
In (a), In (b), In3, and In4. Of these, In (a) occurs
more antigens, namely, INFI and INJA were
recognized as part of the system. Both these
antigens were HFA. Of these, the INFI was
India in a low frequency of about 3% in Indians.[1] It detected among the Moroccans, while the
Address for
was less uncommon among the Arabs (11.8%) INJA was found among the individuals who
correspondence: and Iranians  (10.6%) tested. [2] The Salis had their origin in the Indian subcontinent.[4]
Dr. Sanmukh R. Joshi, antibody to a high‑frequency antigen (HFA), These 4 antigens of the INDIAN (023) blood
Lok Samarpan Regional found earlier in a Pakistani patient, was group system are now known by their
Blood Bank and Research
Center,
directed to the antigen that turned out to numerical terms IN: 001, IN: 002, IN: 003, and
Mini Bazar, Varachha be antithetical to In(a), hence was named as IN: 004 assigned by the ISBT.[5]
Road, Surat ‑ 395 006,
This is an open access article distributed under the terms of the
Gujarat, India.
Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License,
E‑mail: sanmukhj@yahoo. which allows others to remix, tweak, and build upon the work non-
How to cite this article: Joshi SR, Sheladiya A,
com commercially, as long as the author is credited and the new creations Mendapara-Dobariya KV. INRA, a new high-frequency
are licensed under the identical terms. antigen in the INDIAN (IN023) blood group system.
Submission: 16‑10‑2016 Asian J Transfus Sci 2017;11:121-3.
Accepted: 23‑12‑2016 For reprints contact: [email protected]

© 2017 Asian Journal of Transfusion Science | Published by Wolters Kluwer - Medknow 121
 Joshi, et al.: INRA, a new high‑frequency antigen in IN023

Antibodies to INDIAN blood group antigens result in was ruled out. In addition, the antibody had reacted
direct agglutination of red blood cells (RBCs) in saline with RBCs of several “null”phenotypes, including
medium but react stronger by the indirect antiglobulin Gerbich‑null, Rh‑null,‑D‑/‑D‑, MkMk, Fy(a‑b‑), Gy(a‑),
test (IAT).[1] The antigens of the system are sensitive to and K0, thus ruling out its specificity to the hitherto
proteolytic enzymes like papain[1] and chemicals like known HFAs. However, the RBCs of the Lu  (a‑b‑)/
2‑aminoethyl isothiouronium bromide  (AET).[6] Such In (Lu) type showed a depressed reactivity with the
properties may provide hints in the identification of antibody. Furthermore, the antibody was not inhibited
the antibodies. We encountered such an antibody to by the soluble proteins of DAF  (chromer) and Yt(a)
HFA that we thought, may belong to the INDIAN blood group antigens so its specificity to those HFAs
blood group system, so we investigated the case from was ruled out. The antibody did not agglutinate RBCs
that angle. if they were treated with enzyme chymotrypsin,
though it reacted with RBCs treated with trypsin or
Materials and Methods dithiothreitol.

The case The patient’s RBCs were typed positive with antisera to a
A 40‑year‑old female had a history of seven pregnancies number of papain‑sensitive and papain-resistant HFAs,
but never received transfusion. She was hospitalized including In(b), In3, IN4, Kn(a), McC(a), Yt(a), U, Vel,
with continuous bleeding p.v. due to the uterine tumor. En(a), Kp(b), Js(b), Wr(b), Ge, CD99. These findings had
In the face of anemia, with the hemoglobin level of corroborated in ruling out the specificity of the patient’s
8 g/dL, patient was planned for transfusion. Her blood alloantibody to these antigens.
specimen was referred to arrange blood units in case
the patient required a transfusion during/after surgery. The genomic analysis revealed the patient to be IN * B (IN
The pretransfusion compatibility tests were initiated. *02) homozygous. In exon 5 of CD44, a novel homozygous
Antibody screening test (AST) was positive, and the cross missense mutation c. 449G>A was revealed, encoding
matching with several blood units was incompatible. p.Arg150His amino acid substitution at the protein
Hysterectomy was performed without blood transfusion. level. This mutation resulted in a lack of expression
The standard serological methods were employed in of a novel HFA to which we have proposed the name
pretransfusion compatibility tests and other serological INRA after the patient and have obtained the ISBT
workups. For molecular genetics study, the DNA was numerical term as IN: 005. The INRA gene sequence has
extracted from the patient’s sample. The exons of the been allocated GenBank accession number KX639826.
hemopoietic isoform of the CD44 gene were amplified Besides this, in exon 2 of the CD44, a novel homozygous
by polymerase chain reaction and sequenced. synonymous (silent) mutation c.255C>T was observed
in a codon for p.His85. However, for the moment, it
Results remains an enigmatic entity.

The patient was grouped as A1B, RhD+ with a positive Discussion

AST. The direct antiglobulin test and the autocontrol
tests were negative, indicating the presence of The antibody to HFAs makes it difficult to find a
alloantibody in her serum. The antibody strongly compatible blood for transfusion. It poses a problem in
agglutinated the RBCs in saline/low ionic strength identification of its serological specificity as well and
solution  (LISS) phase at room temperature and by also gives difficulties in finding the “antigen‑negative”
LISS‑IAT at 37°C. The antibody showed uniform blood unit for transfusion. Sometimes, the properties of
hemagglutination strength on over 50 random samples the corresponding antigen like sensitivity to proteolytic
and 11 commercial RBCs panel. The antibody failed to enzymes or certain chemicals may provide a clue to the
react with the RBCs pretreated with papain enzyme presumptive specificity of the antibody. In the present
and the AET. In accordance with these observations, case, the antibody showed direct agglutination of red cells
the patient’s RBCs were tested and gave positive in saline/LISS medium and that, it did not agglutinate
reaction with anti‑In(b), thus ruling out the specificity the RBCs pretreated with enzyme/AET thus indicating
to this HFA. After initial serological investigations in the specificity to the HFA of the INDIAN blood group
our laboratory, her blood specimen was referred to system.[1,6] As a rule, an alloantibody is developed in a
IBGRL, UK to rule out/rule in the specificity to other person who lacks the corresponding antigen. In other
known antigens of the INDIAN blood group system, words, the specificity of an alloantibody is ruled out, if the
namely, In:3 and In4. As the antibody had reacted with antibody producer is positive for that antigen. In a similar
red cells lacking In3 and In4 antigens, its specificity note, if an antibody has reacted with the RBCs devoid of
against these antigens of the INDIAN blood groups an antigen, the specificity to that antigen is ruled out. This
maneuver in the identification of antibodies may need

122 Asian Journal of Transfusion Science - Volume 11, Issue 2, July-December 2017
 Joshi, et al.: INRA, a new high‑frequency antigen in IN023

an exhaustive rare red cells panel that lacks the HFAs. to confirm our serological findings and to rule out the
Besides, it requires the rare antisera to HFA to be tested. specificity to hitherto known antigens, including those
In the present study, the patient’s red cells were typed of the INDIAN (023) blood group system. In addition,
positive with antisera to the HFAs, thus ruling out the we express our gratitude to Dr. Vanja Crew, PhD, the
specificity of her alloantibody to those antigens. Besides, molecular geneticist at the IBGRL, for testing the patient’s
her antibody had reacted with RBCs lacking the HFAs sample and providing a report on the mutation in the
further ruling out its specificity to hitherto known HFA. hemopoietic isoform of the CD44 giving rise to a lack
of the new HFA. Besides, we acknowledge this team to
The In(a)/In(b) antigens are weakly expressed on red cells
with Lu(a‑b‑)/In(Lu) phenotype.[7,8] The antibody in the obtain the gene access number from GeneBank and the
present case showed depressed reactivity with such RBCs. numerical name from the ISBT on our behalf.
This observation hinted that the antigen defined by the
patient’s antibody may belong to the INDIAN blood group Financial support and sponsorship
system. The genetic sequence studied on hemopoietic Nil.
isoform of the CD44 that carries the INDIAN blood group
antigens,[7] revealed a novel homozygous missense mutation Conflicts of interest
c.449G>A in exon 5, encoding p.Arg150His amino acid There are no conflicts of interest.
substitution at the protein level and that, this mutation
resulted in a lack of expression of a novel HFA in the References
INDIAN (023) blood group system. The name INRA, given
by us to this antigen, was ratified as the novel antigen with 1. Badakere  SS, Joshi  SR, Bhatia  HM, Desai  PK, Giles  CM,
its numerical term as IN005 by the ISBT. Goldsmith KL. Evidence for a new blood group antigen in the
Indian population  (a preliminary report). Indian J Med Res
The CD44 gene is located on chromosome 11 at position 1973;61:563.
p13.[7] The In  (a)/In  (b) polymorphism results from 2. Badakere SS, Parab BB, Bhatia HM. Further observations on the
Ina (Indian) antigen in Indian populations. Vox Sang 1974;26:400‑1.
the single base change in exon 2 of CD44 gene that
3. Giles  CM. Antithetica relationship of anti‑In‑a with the Salis
substitutes one amino acid, from Pro to Arg at position
antibody. Vox Sang 1975;29:73‑6.
46 of the CD44 protein.[9] The common allele, In(b), has
4. Poole  J, Tilley  L, Warke  N, Spring  FA, Overbeeke  MA,
G252 and encodes Arg46 while the rare allele In(a) has van der Mark‑Zoet  JA, et al. Two missense mutations in the
C252 and encodes Pro46. This has resulted from a single CD44 gene encode two new antigens of the Indian blood group
mutation G > C in CD44 at position 252.[9] A lack of system. Transfusion 2007;47:1306‑11. [Erratum in: Transfusion
IN3 and IN4 results from homozygosity for mutations 2007;47:1741].
encoding H85Q and T163K in the CD44 gene.[4] In the 5. International Society of Blood Transfusion Working Party on
present case, the patient lacked a novel HFA (INRA, In5) Red Cell Immunogenetics and Blood Group  Terminology.
Available from: http://www.isbtweb.org/working‑parties/
that has resulted from homozygous mutation 449G>A in
red‑cell‑immunogenetics‑and‑blood‑group‑terminology/. [Last
exon 5 of the CD44 gene encoding an amino acid change accessed on 2016 Oct 08].
Arg150His at the protein level. 6. Joshi SR, Bhatia HM. Effect of 2‑aminoethyl isothiouroniumbromide
on Ina/Inb blood group antigens. Indian J Med Res 1987;85:420‑1.
Conclusion 7. Spring  FA, Dalchau  R, Daniels  GL, Mallinson  G, Judson  PA,
Parsons SF, et al. The Ina and Inb blood group antigens are located
The antibody to the enzyme sensitive hitherto unknown on a glycoprotein of 80,000 MW (the CDw44 glycoprotein)
HFA was investigated. Serological and molecular whose expression is influenced by the In (Lu) gene. Immunology
studies have provided evidence for a novel antigen in the 1988;64:37‑43. Available from: https://www.ncbi.nlm.nih.gov/
pubmed/2454887. [Last accessed on 2016 Oct 08].
INDIAN (023) blood group system. The new HFA named
8. Telen MJ. Lutheran antigens, CD44‑related antigens, and Lutheran
INRA, was assigned the numerical term In: 005 by the ISBT. regulatory genes. Transfus Clin Biol 1995;2:291‑301.
9. Telen  MJ, Udani  M, Washington  MK, Levesque  MC, Lloyd  E,
Acknowledgment Rao N. A blood group‑related polymorphism of CD44 abolishes
Authors are thankful to B. Jones and Nicole Thorton of a hyaluronan‑binding consensus sequence without preventing
the International Blood Group Reference Laboratory, UK hyaluronan binding. J Biol Chem 1996;271:7147‑53.

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