Immunohematology Case Studies

2019 – Multiple common antibodies and an

antibody to a high-prevalence antigen in a
patient with a transplanted bone marrow

Mirela Raos
Head of Transfusion Medicine Division
Clinical Department of Transfusion Medicine and
Transplantation Biology
University Clinical Hospital Zagreb, Croatia
[email protected]
Clinical History

• 41-year-old female Caucasian with Myelodysplastic

syndrome (MDS-RAEB-1) was admitted to the hospital
for allogeneic unrelated bone marrow transplantation
(BMT)
• Two red cell units are immediately ordered (Hgb=60
g/L)
Clinical History

• She had two pregnancies

• During the last delivery she received red cell
transfusions
• A month ago she received platelets
• There were no chemotherapeutic regimens before
priming
Serologic History

• Indirect antiglobulin test (IAT) was performed on two

occasions last year and was negative (the last one was
done a month ago when she received platelets)
Current Sample Presentation Data

ABO/D: O D positive
Antibody Screen Method: Indirect Antiglobulin Test (IAT)
using Column Agglutination Technology (CAT)
polyspecific (Biovue, Ortho Clinical Diagnostics)
Antibody Screen Results: Positive
Antibody Identification Method: IAT using CAT-
Polyspecific and Neutral (Biovue, Ortho Clinical
Diagnostics) and IAT in tube - IgG
Antibody Identification Preliminary Results: likely anti-E
and anti-Cw in IAT with untreated and papain-treated
cells, but additional alloantibody is suspected, the
autocontrol is negative
Antibody identification panel
CAT

D C c E e Cw K k Fya Fyb Jka Jkb Lea Leb P1 M N S s IAT Enz

1 + 0 + 0 0 + 0 + 0 + + + 0 + + 0 + 0 + 3+ 4+
2 0 + 0 0 + 0 0 + 0 0 + + 0 0 + + + 0 0 w 1+
3 0 + + 0 + 0 0 + 0 + 0 + 0 + + + + + + 1+ 0
4 0 0 + + + 0 0 + 0 w + 0 0 + + + 0 + + 2+ 4+
5 0 0 + + 0 0 0 0 0 + 0 + 0 + + + + + + 3+ 4+
6 0 0 + 0 + 0 + + + + + + 0 + + + + + 0 1+ 1+
7 0 0 + 0 + 0 + + 0 + + 0 0 + + + 0 0 + w 1+
8 0 0 + 0 + 0 0 + + 0 + + + 0 + 0 + 0 + 1+ 1+
9 0 0 + 0 + 0 0 + + 0 + + + 0 0 + 0 + 0 1+ 1+
10 0 0 + 0 + 0 0 + 0 0 + 0 0 0 + 0 + + 0 1+ 1+
11 0 0 + 0 + 0 0 + + 0 0 + 0 + 0 + + + + 1+ 0
AC 0 NT

AC (autocontrol): negative
 Antibody identification panel
Tube test

D C c E e Cw K k Fya Fyb Jka Jkb Lea Leb P1 M N S s RT 37C IgG

1 + 0 + 0 0 + 0 + 0 + + + 0 + + 0 + 0 + 0 2+ 2+
2 0 + 0 0 + 0 0 + 0 0 + + 0 0 + + + 0 0 0 0 0
3 0 + + 0 + 0 0 + 0 + 0 + 0 + + + + + + 0 0 w
4 0 0 + + + 0 0 + 0 w + 0 0 + + + 0 + + 0 1+ 1+
5 0 0 + + 0 0 0 0 0 + 0 + 0 + + + + + + 0 3+ 3+
6 0 0 + 0 + 0 + + + + + + 0 + + + + + 0 0 0 1+
7 0 0 + 0 + 0 + + 0 + + 0 0 + + + 0 0 + 0 0 0
8 0 0 + 0 + 0 0 + + 0 + + + 0 + 0 + 0 + 0 0 1+
9 0 0 + 0 + 0 0 + + 0 + + + 0 0 + 0 + 0 0 0 w
10 0 0 + 0 + 0 0 + 0 0 + 0 0 0 + 0 + + 0 0 0 1+
11 0 0 + 0 + 0 0 + + 0 0 + 0 + 0 + + + + 0 0 0
AC 0 0 0

AC (autocontrol) negative
RT = room temperature
Challenge with the Current
Presentation

• There are probably anti-E and anti-Cw alloantibodies in

the patient’s plasma
• Multiple alloantibodies are reactive with all panel cells
in IAT with untreated cells and with some enzyme-
treated cells, and the autocontrol is negative
• An initial review of results would suggest that an
additional alloantibody to a high-prevalence antigen, or
more likely multiple alloantibodies, besides anti-E and
anti-Cw alloantibodies, are present in the patient’s
plasma
Interim Antibody Identification
Possible Answers and Next Steps

• Reactivity appears to be an alloantibody to a high

frequency antigen, or more likely multiple alloantibodies,
besides anti-E and anti-Cw alloantibodies, present in the
patient’s plasma
• An inconsistency of reactivity with all cells in the panel
was shown (the reaction with enzyme-treated cell was
not enhanced and with some cells it was negative), and
the autocontrol was negative
• Further testing is needed for a conclusion, particularly
phenotyping and testing with pheno-matched RBCs
Further Work

• Phenotyping

RBC ABO Rh Kell Kidd Lewis MNS Duffy P1

CcD.ee,
Patient O K-k+ Jk(a+b-) Le(a-b+) M+N+S-s+ Fy(a+b-) P1+
Cw neg
Updated Clinical Information

• Due to urgency, the patient immediately received 1 O D

positive E-, Cw-, K- and Jk(b-), (Fy(b+), S+) red cell unit
that was compatible in the tube test, but was positive in
CAT (XM in IAT)

• Pre-transfusion Hgb=60 g/L, DAT negative

• Post-transfusion Hgb=71 g/L, DAT positive

DAT (CAT): anti-IgG (1+), -IgA, -IgM, C3c, -C3d neg

Eluate: non-specific antibody
Further Work

• Testing with pheno-matched RBCs and papain-treated

and 0.2 M dithiothreitol (DTT) treated RBCs, as well as
with cord RBCs and autologous RBCs in CAT
• Reaction was positive with 1 unit of pheno-matched O
D positive E-, Cw-, K-, Jk(b-), Fy(b-), S- RBCs in IAT (2+)
• After treating the RBCs with papain, the reaction was
negative, while after treating RBCs with 0.2 M DTT, it
remained positive (1+)
• Antibody reacted in IAT (1+) with cord O D positive E-,
Cw- RBCs
Further Testing Results and
Interpretations

Adsorption and elution

• RBC phenotypes
- E+, K-, Jk(b-), Fy(b+), S+
- E+, K-, Jk(b+), Fy(b-), S+
- E+, K-, Jk(b+), Fy(b+), S-
• Anti-K, -Jkb, -Fyb and -S were excluded from the
patient’s plasma by adsorption and elution studies
Anti-HLA screening
• Anti-HLA screening (ELISA) detected anti-HLA class I
and II antibodies in the patient’s serum
Updated Clinical Information

Bone marrow transplantation

• The patient underwent allogenic unrelated BMT

• Prior to BMT, she received myeloablative conditioning
therapy according to Flu/Bu4/ATG protocol
• Flu/Bu4/ATG protocol consisted of 5 days of fludarabine
(total dosage of 250 mg iv.), 4 days of busulphane (total
dosage of 792 mg iv.) and 2 days of antithymocyte
globulin (total dosage of 300 mg iv.)
• Before BMT, one plasmapheresis was done due to
major ABO incompatibility (titer anti-A IgM 32, IgG 64)
Updated Clinical Information

Transfusion support

• Prior to BMT, she received two more O D positive E-,

Cw-, K- and Fy(b-), Jk(b+), S+ red cell units that were
compatible with the tube test, but positive in CAT (XM in
IAT)

• Pre-transfusion Hgb=66 g/L, DAT positive

• Post-transfusion Hgb=67 g/L, DAT positive

DAT (CAT): anti-IgG (2+), -C3d (2+)

Eluate: non-specific antibody
Further Work

Donor
ABO/D: A D positive
IAT: negative

RBC ABO Rh Kell Kidd Lewis MNS Duffy P1

ccD.ee,
Donor A K-k+ Jk(a+b+) Le(a-b+) M+N-S+s+ Fy(a-b+) P1+
Cw neg
CcD.ee,
Patient O K-k+ Jk(a+b-) Le(a-b+) M+N+S-s+ Fy(a+b-) P1+
Cw neg
Further Work

• A blood sample (pre-transfusion and post-transfusion

sample) was urgently sent to the International Blood
Group Reference Laboratory (IBGRL), Bristol
• After transfusion, additional testing was done in our
laboratory (please see tables on next two slides)
 Antibody identification panel
CAT

D C c E e Cw K k Fya Fyb Jka Jkb Lea Leb P1 M N S s IAT Enz El

1 + 0 + 0 0 + 0 + 0 + + + 0 + + 0 + 0 + 4+ 4+ 1+
2 0 + 0 0 + 0 0 + 0 0 + + 0 0 + + + 0 0 2+ 2+ 1+
3 0 + + 0 + 0 0 + 0 + 0 + 0 + + + + + + 2+ 2+ 1+
4 0 0 + + + 0 0 + 0 w + 0 0 + + + 0 + + 4+ 4+ 1+
5 0 0 + + 0 0 0 0 0 + 0 + 0 + + + + + + 4+ 4+ 1+
6 0 0 + 0 + 0 + + + + + + 0 + + + + + 0 2+ 2+ 1+
7 0 0 + 0 + 0 + + 0 + + 0 0 + + + 0 0 + 2+ 0 0
8 0 0 + 0 + 0 0 + + 0 + + + 0 + 0 + 0 + 2+ 2+ 1+
9 0 0 + 0 + 0 0 + + 0 + + + 0 0 + 0 + 0 2+ 2+ 1+
10 0 0 + 0 + 0 0 + 0 0 + 0 0 0 + 0 + + 0 2+ 0 2+
11 0 0 + 0 + 0 0 + + 0 0 + 0 + 0 + + + + 2+ 2+ 1+
AC 1+

AC (autocontrol): positive (mixed field appearance was not noted)

DAT (CAT): anti-IgG (2+), -C3d (2+), -IgA, -IgM, -C3c negative
El (eluate): unidentified antibody
 Antibody identification panel
Tube technology

D C c E e Cw K k Fya Fyb Jka Jkb Lea Leb P1 M N S s RT 37C IgG

1 + 0 + 0 0 + 0 + 0 + + + 0 + + 0 + 0 + 0 2+ 3+
2 0 + 0 0 + 0 0 + 0 0 + + 0 0 + + + 0 0 0 0 3+
3 0 + + 0 + 0 0 + 0 + 0 + 0 + + + + + + 0 0 2+
4 0 0 + + + 0 0 + 0 w + 0 0 + + + 0 + + 3+ 4+ 3+
5 0 0 + + 0 0 0 0 0 + 0 + 0 + + + + + + 3+ 4+ 3+
6 0 0 + 0 + 0 + + + + + + 0 + + + + + 0 0 0 2+
7 0 0 + 0 + 0 + + 0 + + 0 0 + + + 0 0 + 0 0 1+
8 0 0 + 0 + 0 0 + + 0 + + + 0 + 0 + 0 + 0 0 2+
9 0 0 + 0 + 0 0 + + 0 + + + 0 0 + 0 + 0 0 w 2+
10 0 0 + 0 + 0 0 + 0 0 + 0 0 0 + 0 + + 0 0 0 1+
11 0 0 + 0 + 0 0 + + 0 0 + 0 + 0 + + + + 0 w 2+
AC 0 0 0

AC (autocontrol) negative
RT = room temperature
Further Testing Results and
Interpretations

From our repeated testing

• Reactions were stronger after transfusion

• DAT became positive and a non-specific antibody was
detected in the eluate
• An anti-E and anti-Cw antibody, as well as an antibody
to a high-prevalence antigen were present in the
patient’s plasma
• Investigation stopped there as we sent a sample for an
urgent investigation to IBGRL, Bristol, and we did not
repeat adsorption and elution studies
Further Testing Results and
Interpretations

From testing at IBGRL

• In the Preliminary Report, the presence of anti-E and
anti-Cw was confirmed in the patient’s plasma, an
antibody to a high-prevalence antigen of anti-Yta
specificity was found and an anti-Jkb antibody was
suspected, but further testing was needed.
• The patient’s cells (from the pre-transfusion sample)
were Yt(a-b+)
• An anti-Jkb antibody was afterwards confirmed in the
patient’s plasma
• Four examples of Yt(a-), E-, Cw-, Jk(b-) cells were
compatible with the patient’s plasma and no additional
antibodies were detected
Updated Clinical Information

• The patient received eight more red cell units (E-, Cw-,
Jk(b-) before she was discharged from the hospital
• She was serologically monitored during transfusion
support (please see the table on the next slide)
• By means of PCR-STR monitoring for chimerism, she
was determined to be a 100% donor
• 3 months after BMT, only anti-Cw and -E specificity
(only with the sensitive enzyme technique) were
identified in the patient’s plasma, and anti-Jkb and –Yta
were below the sensitivity of the tests
Updated Clinical Information

• Red cell units transfused and laboratory findings prior

and after all red cell transfused (table).
RBC unit transfused
/Transfusion event 1. 2. 3. 4. 5. 6. 7. 8.

Number of RBC units transfused 1 2 1 2 2 1 1 1

XM (IAT in CAT) pos pos pos pos pos pos neg neg

Hgb prior transfusion (g/L) 60 66 45 49 63 65 69 79

DAT prior transfusion neg pos pos pos pos pos NT NT

Hgb after transfusion (g/L) 71 67 56 76 74 78 86 86

DAT after transfusion pos pos pos pos pos pos NT NT

Total Bilirubin (µmol/L) 46 39 23 NT 10 12 20 27

LDH (U/L) 290 229 364 NT 189 188 268 437

NT = not tested
Conclusions

• The patient received allogenic unrelated BMT for the

MDS and she immediately needed transfusion support
• Anti-Cw and anti-E alloantibodies were identified in the
patient’s plasma
• A suspected antibody of the anti-Yta specificity to a
high-prevalence antigen was confirmed at IBGRL, Bristol
• Another antibody, of anti-Jkb specificity, was suspected
and confirmed to be present in the patient’s plasma at
IBGRL, Bristol
• She was transfused with incompatible red cell units
(Yt(a+) in emergency, with no ill effects
Summary of Case Challenges

• A patient with MDS was admitted to the hospital for an allogeneic

unrelated BMT and red cell units were immediately ordered
• Before transfusion, anti-E, anti-Cw and an antibody to a high-
prevalence antigen (later found to be anti-Yta) were identified in the
patient’s plasma
• During the immunosuppressive myeloablative conditioning therapy
prior to BMT, the patient developed an anti-Jkb as a subsequent
immune response to transfused red cell units
• She received incompatible red cell units and had a good
hematological response
• After BMT, the patient was determined to be a 100% donor
• She received the last red cell unit on day +29 after BMT and was soon
dismissed from hospital
• Three months after BMT, anti-Jkb and -Yta were not detectable
Lessons Learned by the Case

• A patient with MDS developed multiple common antibodies (anti-E,

anti-Cw) and an antibody to a high-prevalence antigen (anti-Yta) after
16 units of platelets were transfused a month ago, when the IAT was
negative. During immunosuppressive myeloablative conditioning
therapy for allogenic unrelated BMT, she developed another antibody
of anti-Jkb specificity after a transfusion of two Jk(b+) red cell units.
All antibodies were developed despite the patient’s diagnosis and
immunosuppressive therapy
• In a patient with an antibody to a high-prevalence antigen, an
underlying anti-Jkb was very difficult to detect. As samples were
urgently sent to IBGRL in Bristol, adsorption and elution studies to
detect newly developed antibodies after transfusion were not done.
An anti-Jkb was detected afterwards in Bristol. From our panel
results, it can be seen that reactions with two Jk(b-) RBCs were
negative in the enzyme which could lead us to suspect an anti-Jkb
Lessons Learned by the Case

• Compatible red cell units were not available, so incompatible blood

was transfused (Yt(a+), E-, Cw- Jk(b-)) with no evidence of
decreased red cell survival. Clinical significance of anti-Yta is
variable, and some have been implicated in an immediate HTR.
Therefore, after this case, we set up a Monocyte Monolayer Assay
(MMA) to be able to predict clinical significance of unknown
antibodies in the future
• In this case, the transplant donor was Jk(a-b+) and one must be
aware that the patient’s anti-Jkb and anti-Yta could also cause acute
or delayed hemolysis of the donor’s Jk(b+) RBCs from the BMT and
contribute to morbidity and mortality, but this was not the case, as
the patient normally recovered hematopoiesis
ISBT Terminology of the YT Blood
Group System

• The anti-Yta to a high frequency antigen was first found

in 1956
• The antithetical antibody, anti-Ytb, detects an antigen
on red cells of about 8% of white people and was found
eight years later
• YT, the Cartwright system, now includes 5 antigens; an
inherited Yt(a-b-) phenotype has not been found.
• Cartwright is the 11th human blood group system
recognized by ISBT (ISBT 011)
• The Cartwright blood group antigens are encoded by
the ACHE gene on chromosome 7q22, which produces
acetylcholinesterase (AchE)
ISBT Terminology of the Yt Blood
Group System

• Two single nucleotide changes in ACHE are

associated with Yta/Ytb polymorphism: 1057C>A in exon
2 encodes His353Asn and 1432C>T, a silent mutation in
exon 3 in the codon for Pro477
• Yta is not affected by trypsin, but is destroyed by α-
chymotrypsin treatment of red cells. Papain and ficin
may also destroy the antigen, but this appears to
depend on the anti-Yta used. Yta and Ytb are sensitive to
disulphide bond reducing agents
• YT antigens are present on red cells from cord blood
samples, but the strength of Yta on cord cells is weaker
than that on the red cells of adults
Brief Review of the Blood Group
Antibody
• Anti-Yta and -Ytb are stimulated by pregnancy or
transfusion, neither is ‘naturally occuring’
• YT antibodies are mostly IgG and require an
antiglobulin test to agglutinate red cells
• Some anti-Yta bind the complement, others do not
• YT antibodies do not cause HDFN
• Anti-Yta has been implicated in an immediate HTR
• Many patients with anti-Yta have received multiple
transfusion of Yt(a+) red cells with no ill effects
• For transfusion purposes, each sample of anti-Yta must
be accessed independently. For the strong examples,
Yt(a-) rare units is recommended. An MMA may also be
proposed.
References

1. Daniels G. Human Blood Groups, 3rd ed, Oxford, UK:

Blackwell Publishing 2013
2. Reid M, Lomas-Francis C, Olsson M. The Blood Group
Antigen FactsBook, 3rd ed, Academic Press, Inc. San
Diego, California, USA 2012
2. Franchini M, Gandini G, Aprili G. Non-ABO red blood
cell alloantibodies following allogenic hematopoietic stem
cell transplantation. Bone Marrow Transplantation
2004;33:1169-1172