Exeley Inc.

Daniels, G.

IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019 1
Immuno_35_1_04     •     March 11, 2019 8:39 AM
This update of the Augustine (AUG) blood group system (Daniels 
G. The Augustine blood group system, 48 years in the making. 
Immunohematology 2016;32:100–3) describes two antigens 
that have been added to the Augustine system (International 
Society of Blood Transfusion system 36), bringing the number of 
antigens in the system to four. Further information on the clinical 
significance of Augustine system antibodies and the function of 
the Augustine glycoprotein, equilibrative nucleoside transporter 
1, is presented. Immunohematology 2019;35:1–2.
Key Words: Augustine, SLC29A1, adenosine transport, 
erythropoiesis, ENT1
New Augustine Antigens
Since the publication of the original review,1 two new 
antigens have been added to the Augustine (AUG) blood group 
system: AUG3 and AUG4 (Table 1).
An antibody to a low-prevalence antigen (AUG3) caused 
severe hemolytic disease of the fetus and newborn (HDFN) 
requiring two exchange and four top-up transfusions. Targeted 
exome sequencing for blood group genes revealed that the baby 
was heterozygous for a novel variant in the Augustine gene, 
SLC29A1, encoding p.Thr387Pro in the fifth extracellular loop 
of equilibrative nucleoside transporter 1 (ENT1). The baby’s 
brother, father, two paternal aunts, and grandmother were all 
heterozygous for the variant allele, and their red blood cells 
(RBCs) were all AUG:3.2
An antibody to a high-prevalence antigen (AUG4) in 
a white woman, who had been pregnant and transfused, 
was first found in 1995. Recent whole exome sequencing 
revealed homozygosity for a missense mutation in SLC29A1 
encoding p.Asn81Ser. The variant was present in 0.1 percent 
of individuals in the Exome Aggregation Consortium but was 
never found in a homozygous state. The AUG:–4 RBCs of the 
propositus were AUG:1,2, confirming that ENT1 is present, 
but flow cytometric analysis revealed an approximately 
30 percent reduction in surface ENT1 relative to that on 
AUG:1,2,4 (common phenotype) RBCs. The antibody of the 
propositus reacted with AUG:1,2 and AUG:1,–2 RBCs, but not 
with AUG:–1,–2 (null phenotype) RBCs.3
Clinical Significance of Augustine System 
Antibodies
An acute hemolytic transfusion reaction caused by anti-
AUG2 (anti-Ata)4 and severe HDFN caused by anti-AUG32 
confirm previous reports that Augustine system antibodies 
have the potential to be dangerously hemolytic.
Functional Aspects of the Augustine Glycoprotein
Analysis of mature RBCs and developing erythroid 
cells from the three previously reported AUG:–1,–2 (null 
phenotype) siblings revealed macrocytosis 
and abnormal morphologies together with 
a decreased level of proliferation during 
erythropoiesis and delayed erythroblast 
maturation. This finding suggests a role 
of ENT1-mediated adenosine transport in 
erythroid differentiation.5
An update on the Augustine blood group 
system
G. Daniels
Blood Group revie w
Table 1. Antigens of the Augustine system
Antigen Molecular basis
Number Name Prevalence Nucleotides Exon Amino acids Reference
AUG1 High c.589+1G>C 6 Slice site 1
AUG2 Ata High c.1171G>A 12 Glu391Lys 1
AUG3 ATML Low c.1159A>C 12 p.Thr387Pro 2
AUG4 ATAM High c.242A>G 3 p.Asn81Ser 3
2 IMMUNOHEMATOLOGY, Volume 35, Number 1, 2019
Immuno_35_1_04     •     March 11, 2019 8:39 AM
G. Daniels
References
 1. Daniels G. The Augustine blood group system, 48 years in the 
making. Immunohematology 2016;32:100–3.
 2. Millard GM, McGowan EC, Wilson B, et al. A proposed new 
low-frequency antigen in the Augustine blood group system 
associated with a severe case of haemolytic disease of the fetus 
and newborn. Transfusion 2018;58:1320–2.
 3. Vrignaud C, Mikdar M, Raneri A, et al. Characterization of a 
novel high-prevalence red blood cell antigen in the Augustine 
blood group system (abstract). Vox Sang 2018;113(Suppl 
1):64–5.
 4. Raval JS, Harm SK, Wagner B, Triulzi DJ, Yazer MH. 
Acute hemolytic transfusion reaction attributed to anti-Ata. 
Immunohematology 2016;32:140–2.
 5. Mikdar M, Le Van Kim C, Kinet S, et al. Lack of the nucleoside 
transporter ENT1 in individuals with the rare Augustine-null 
blood group is associated with an abnormal erythropoiesis and 
microcytosis (abstract). Vox Sang 2018;113(Suppl 1):64.
Geoffrey Daniels, PhD, retired, previously Head of Diagnostics, 
International Blood Group Reference Laboratory, Bristol, UK, geoff.
daniels.bris@outlook.com.
The editorial staff of Immunohematology welcomes manuscripts 
pertaining to blood group serology and molecular genetics for 
consideration for publication. We are especially interested in 
review articles, case reports, papers on platelet and white cell 
serology, scientific articles covering original investigations or new 
blood group alleles, papers on molecular testing, and papers on 
new methods for use in the blood bank. To obtain instructions for 
submitting scientific articles, case reports, and review articles, 
see Instructions for Authors in every issue of Immunohematology 
or e-mail a request to immuno@redcross.org. Include fax and 
phone numbers and e-mail address with all manuscripts 
and correspondence. E-mail all manuscripts to immuno@
redcross.org.
Manuscripts


An update on the Augustine blood group system

Castilho, L.

An update on the MNS blood group system

Howard, P.J.

Guerra, L.

Kuttner, D.K.

George, M.R.

IMMUNOHEMATOLOGY, Volume 35, Number 4, 2019 159
The antigens associated with the MNS blood group system (ISBT 
002) are located on glycophorin A (GPA) and glycophorin B (GPB). 
The most frequently encountered antibodies to antigens in this 
system by a transfusion medicine service are those directed 
against M, N, S, and s. Individuals lacking GPA typically have 
red blood cells that lack M, N, and Ena, whereas those lacking 
both GPA and GPB lack M, N, and Ena as well as S, s, and U. 
Such individuals may develop a rare antibody, anti-Ena, directed 
against determinants on GPA. This antibody is capable of causing 
hemolytic transfusion reactions and hemolytic disease of the 
fetus and newborn. This case report describes a pregnant woman 
found to have anti-Ena. Molecular testing supported an Mk 
phenotype that was found in several members of her immediate 
family. Immunohematology 2019;35:159–161.
Key Words: Ena, antibody, prenatal, MNS
Introduction
The MNS blood group system (ISBT 002) consists of red 
blood cell (RBC) antigens located on glycophorin A (GPA) and 
glycophorin B (GPB). The GYPA and GYPB genes encoding these 
glycophorins are located on the long arm of chromosome 4.1,2 
The most frequently encountered antibodies to antigens in this 
system by a transfusion medicine service are those directed 
against M, N, S, and s. In rare cases, individuals may lack GPA 
or both GPA and GPB on their RBCs because of gene deletion, 
and such individuals may develop a rare antibody, anti-Ena, 
to determinants located on GPA. Anti-Ena does not target a 
precise antigen on GPA but rather is an “umbrella” term for 
antibodies directed at external regions of GPA made by rare 
people who lack all or part of GPA. Three broad categories of 
anti-Ena have been defined according to the effect of protease 
enzymes on the antigenic determinants that each detects, 
namely anti-EnaTS, -EnaFS, and -EnaFR.3 The results from 
a previous study by Tanner and Anstee4 showed that RBCs 
lacking GPA, the major sialoglycoprotein on the RBC surface, 
lack Ena. This deficiency reduces the overall zeta potential of 
an intact RBC resulting in RBCs with a higher propensity to 
agglutinate than RBCs expressing GPA.4 Antibodies against 
Ena have been associated with hemolytic transfusion reactions 
and hemolytic disease of the fetus and newborn (HDFN).5 
We present a case of the rare Mk phenotype with allo-anti-
Ena in a maternal prenatal sample. We discuss the clinical 
ramifications of this finding.
Case Report
A 28-year-old Caucasian woman, never transfused, 
primigravida at approximately 23 weeks’ gestation, presented 
for routine prenatal care at her local community hospital. A 
routine type and antibody detection test was performed and 
found to be positive, and her sample was flagged for further 
testing by the Immunohematology Reference Laboratory 
(IRL) at Miller-Keystone Blood Center (Bethlehem, PA). The 
IRL identified anti-Ena by indirect antiglobulin testing. The 
presence of this antibody is worrisome for both mother and 
fetus because anti-Ena of an IgG isotype has been implicated 
in both acute and delayed hemolytic transfusion reactions 
and HDFN.3 The patient was referred to the Maternal Fetal 
Medicine department at Penn State Hershey Medical Center 
(Hershey, PA). 
The initial antibody detection test performed at Hershey 
Medical Center demonstrated weak positive reactivity with all 
three reagent screening RBCs and with 11 of 11 panel RBCs at 
the antiglobulin phase using a solid-phase red cell adherence 
assay. The direct antiglobulin test was negative. Further 
testing by our IRL revealed that the patient’s RBCs typed 
M–N–S–s– with licensed RBC antisera (Immucor, Norcross, 
GA). This finding prompted further testing of the patient’s 
plasma against a U– and a U+var reagent RBC, both of which 
were reactive. This reactivity was not detected when the RBCs 
were treated with ficin. 
These results, together with the patient’s non-African 
ethnicity, allowed the IRL to rule out anti-U and anti-Wrb. 
Classically, it was expected that both of these antibodies would 
remain reactive with ficin-treated reagent RBCs. Furthermore, 
the testing of the patient’s plasma aginast ficin-treated reagent 
RBCs in low-ionic-strength solution (tube method) resulted in 
no agglutination. These findings are consistent with a subtype 
of Ena that is sensitive to ficin treatment, historically known 
Clinical approach after identification of a rare 
anti-Ena in a prenatal sample
P.J. Howard, L. Guerra, D.K. Kuttner, and M.R. George
Case report
160 IMMUNOHEMATOLOGY, Volume 35, Number 4, 2019
P.J. Howard et al.
as EnaFS. However, because of the lack of MkMk or En(a–) 
reagent RBCs available for testing against the patient’s plasma, 
these findings were not confirmed. 
IgG titers were then performed without enhancement 
media at 37°C for 60 minutes. The initial titer was 1, and 1 
month later, the titer was 2; these results are within procedural 
variation and the titer difference was considered insignificant 
by the IRL. The sample was referred to the National Molecular 
Laboratory at the American Red Cross (Philadelphia, PA) 
for performance of RBC genotyping by PreciseType HEA 
Molecular BeadChip (Immucor). This molecular platform 
interrogates targets to predict expression of M, N, S, s, and 
U. The patient sample failed to amplify for all the assays that 
interrogate GYPA and GYPB targets. RBCs from the presumed 
father of the fetus demonstrated an M+N–S+s+ phenotype by 
serological and molecular methods from the same laboratories 
as the patient. The patient’s molecular and serologic findings 
from testing of the patient’s sample were consistent with an 
Mk phenotype, which is a null phenotype for antigens on both 
GPA and GPB, encoded by homozygosity for GYP*01N. No 
typing of the patient’s RBCs with antisera against other high-
prevalence antigens in the MNS system was performed.
Given the exceedingly rare occurrence of En(a–) 
individuals (<0.1%), clinical encounters with alloantibodies to 
Ena are limited. If transfusions were required for the mother 
or fetus, our options were autologous donations, directed 
donations from family members who match the patient’s 
phenotype, or via rare blood donor registries. Fortunately, this 
patient came from a large family, most of whom were tested 
both serologically and molecularly. A blood group genotyping 
platform (ID CORE XT; Grifols, Barcelona, Spain) that also 
interrogates GYPA and GYPB targets to predict the common 
antigens in the MNS system was used to analyze samples from 
the family members at Grifols laboratory. Four siblings were 
predicted to express the same Mk phenotype as the patient. 
These results were confirmed, also by Grifols laboratory, by 
allele-specific polymerase chain reaction (PCR) and agarose 
gel electrophoresis, which showed an absence of products 
for GYPA exons 2–7; GYPB exons 1–2 and 4–6; and GYPB 
pseudo-exon 3 (data not shown). A pedigree demonstrates 
the likely inheritance pattern of the GYPA and GYPB genes 
and the MNS antigens (Fig. 1). Given the rarity in the general 
population and prevalence within this family, we can postulate 
that Mk phenotype inheritance is due to consanguinity among 
the parents in the pedigree, although an inquiry to confirm 
this was not pursued. 
The patient’s antibody titers remained unchanged 
throughout her pregnancy (minimum 1; maximum 2), with 
low reactivity in a mononuclear monolayer assay (MMA) 
performed by Grifols laboratory. The MMA is used to predict 
an antibody’s ability to promote macrophagocytic adherence 
and phagocytosis upon exposure to a cognate antigen with or 
without a source of complement.
The consensus in this case was to manage this as a high-
risk pregnancy with frequent intensive monitoring, including 
antibody titers. At term, the patient underwent induction of 
labor and successfully delivered a healthy baby boy by vaginal 
route. The delivery was without event, no transfusions were 
necessary antepartum or postpartum, and a neonatal sample 
revealed no bound RBC antibodies (i.e., negative direct 
antiglobulin test) or evidence of hemolysis.
Fig. 1 This pedigree describes the inheritance of the GYPA and GYPB alleles and expression of these antigens within the MNS blood group 
system. Arrow notes the predicted phenotype of the proposita.
MS/Mk
M+S+N–s–
Haplotypes:          MS: GYPA*M – GYPB*S          Ns: GYPA*N – GYPB*s          Mk: GYPA*deletion – GYPB*deletion
Mk/Ns
M–S–N+s+
MS/Ns
M+S+N+s+
MS/Ns
M+S+N+s+
Mk/Mk
M–S–N–s–
Mk/Mk
M–S–N–s–
Mk/Mk
M–S–N–s–
Mk/Mk
M–S–N–s–
Mk/Mk
M–S–N–s–
MS/Mk
M+S+N–s–
MS/Mk
M+S+N–s–
IMMUNOHEMATOLOGY, Volume 35, Number 4, 2019 161
Anti-Ena in a prenatal sample
Discussion
Because Ena is a high-prevalence antigen, there are 
few case reports of anti-Ena. This rarity affords the medical 
community limited data about the potential clinical sequelae 
of this antibody, which is concerning in a pregnant woman. 
The identification of the antibody resulted in this case being 
monitored as a high-risk pregnancy, based on previous 
case reports.6 Providing this patient with rare En(a–) RBCs 
(or MkMk RBCs) via national or international blood donor 
registries would have been exceedingly difficult. Donors in a 
registry do not guarantee that RBC units of a desired phenotype 
will be available when needed. Autologous donation was also 
considered for the purposes of antepartum or postpartum 
use as well as future use, if needed, in the form of frozen 
RBCs. The allogenic donor registry and autologous donation 
approaches were discussed and understood by the patient. 
These approaches were unnecessary, however, because the 
patient had many compatible family members available and 
willing to donate blood by directed allogenic donations.
The Mk phenotype within this family is due to deletions 
involving exons 2–7 of the GYPA and exons/pseudo-exon 1–6 
of the GYPB genes rather than a mutation in both the GYPA 
and GYPB genes.7 This genetic alteration results in the absence 
of GPA, GPB, and the constitutive antigens of the MNS blood 
group system. We surmise that our patient was exposed to the 
Ena present on the GPA of her baby’s RBCs in utero, or perhaps 
during an undetected miscarriage, which led to a subsequent 
alloimmunization. However, given the infrequency in which 
fetal antigens contact the maternal immune system and the 
low titer of the maternal antibody, there is a possibility that 
the anti-Ena may have been non-RBC related (“naturally-
occurring”).
In conclusion, this case report describes the identification 
of a rare anti-Ena in a prenatal patient and the subsequent 
testing of family members in preparation for the delivery. The 
clinical finding of a rare antibody for which there are limited 
data requires leveraging every resource available to predict its 
behavior and provide safe blood products to patients who may 
require transfusion.
Acknowledgments
The authors are indebted to Melissa Groholtosky, BS, 
BB(ASCP); Joseph Kneebone, MT(ASCP); Patty Brenner, BS, 
MT; and Julianna Corchado, BS, MLT, at Miller-Keystone 
Blood Center (Bethlehem, PA) and the laboratory personnel at 
the American Red Cross (Penn-Jersey Region, Philadelphia, 
PA) in the diagnostic workup of this case. 
References
 1. Heathcote DJ, Carroll TE, Flower R. Sixty years of antibodies 
to MNS system hybrid glycophorins: what have we learned? 
Transfus Med Rev 2011;25:111–24.
 2. Siebert PD, Fukuda M. Human glycophorin A and B are 
encoded by separate, single copy genes coordinately regulated 
by a tumor-promoting phorbol ester. J Biol Chem 1986;261: 
12433–6.
 3. Issitt PD, Anstee DJ. The MN blood group system. In: Applied 
blood group serology. Miami, FL: Montgomery Scientific 
Publications, 1985:501–7. 
 4. Tanner MJ, Anstee DJ. The membrane change in En(a–) 
human erythrocytes. absence of the major erythrocyte 
sialoglycoprotein. Biochem J 1976;153:271–7.
 5. Walker PS, Bergren MO, Busch MP, Carmody AM, Perkins HA. 
Finnish En(a-) propositus with anti-EnaFS and anti-EnaFR: 
in vitro and in vivo characteristics. Vox Sang 1987;52:103–6.
 6. Al-Jada NAH. A Jordanian family with three sisters apparently 
homozygous for Mk and evidence for clinical significance 
of antibodies produced by MkMk individuals. Transfusion 
2016;57:376–8.
 7. Rahuel C, London J, Vignal A, et al. Alteration of the genes for 
glycophorin A and B in glycophorin-A-deficient individuals. 
Eur J Biochem 1998;177:605–14.
Philip Howard, MD, MLS (ASCP) (corresponding author), Medical 
Director, Mid-Atlantic Region American Red Cross, Biomedical 
Services, 611 West Brambleton Avenue, Norfolk, VA 23510, 
Philip.Howard2@redcross.org; Lauro Guerra, MLS(ASCP)SBB, 
Laboratory Manager, Immunohematology Center, Grifols Diagnostic 
Solutions, San Marcos, TX; Donald K. Kuttner, DO, Medical Director, 
Miller Keystone Blood Center, Bethlehem, PA; and Melissa R. George, 
DO, FCAP, FASCP, Medical Director, Transfusion Medicine; Program 
Director, Pathology Residency; and Associate Director, Transfusion 
Medicine Fellowship, Penn State Health Milton S. Hershey Medical 
Center, Hershey, PA.


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