ORIGINAL RESEARCH
A blockage monoclonal antibody protocol as an alternative strategy
to avoid anti-CD38 interference in immunohematological testing
Karen N. Chinoca Ziza,1† Tainá A. Paiva,1† Sabrina R. Mota,1 Marcia Regina Dezan ,2
Luciana Cayres Schmidt,3 Denise Menezes Brunetta ,4 Gustavo Ricci,1 Fernando Valadares Basques,3
Fernando Barroso-Duarte,4 Vanderson Rocha,1,2,5 Alfredo Mendrone-Junior,2 and
Carla Luana Dinardo 1,2
BACKGROUND: As CD38 is expressed on red blood
cells (RBCs), the plasma of patients on daratumumab
(DARA) reacts with the panel cells of pretransfusion
tests, masking underlying alloantibodies. The
treatment of RBCs with dithiothreitol (DTT) is the
most disseminated method to overcome DARA effect
on immunohematological tests, but it hampers the
identification of potentially harmful antibodies. Our
goal was to validate a new strategy, the blockage
monoclonal antibody protocol (BMAP), to mitigate the
DARA interference on RBCs using anti-CD38 and
antihuman globulin.
METHODS: Samples of patients receiving DARA
were included in the study. Sera were tested using
both DTT- and BMAP-treated RBCs, which comprised
three steps: 1) titration of monoclonal anti-CD38, 2)
treatment of RBCs obtained from donors with anti-
CD38, and 3) blockage of anti-CD38–adsorbed RBCs
with antihuman globulin.
RESULTS: Twenty patients were included in the study.
Donor RBCs were treated with anti-CD38 and
successfully blocked with antihuman globulin. In
19 patients, DARA-mediated agglutination was eliminated
using both DTT- and BMAP-treated RBCs. In one patient,
agglutination persisted when tested against the BMAP-
treated RBCs, and alloantibodies were identified. Patient
samples were mixed with commercial anti-D, -C, −e, -K,
−Jka, -Kpb and tested against antigen-positive BMAP-
treated RBCs, resulting in detection of these antibodies.
CONCLUSION: This study validated a new strategy
to minimize the interference of DARA on immuno-
hematological tests. The protocol preserves the
integrity of RBC antigens, permitting the detection of
antibodies from all blood group systems. The BMAP
has potential use in other situations where specific
antibodies may interfere with pretransfusion screening.
M
ultiple myeloma (MM) is a plasma cell neo-
plasm with a chronic and incurable course
requiring successive treatments that prolong
and improve the patient’s quality of life. In
the past decade, the therapeutic landscape of MM has evolved
dramatically with the validation of proteasome inhibitors and
immunomodulatory agents.1 Despite the improvement, the
disease typically relapses and new classes of drugs are needed.2
Monoclonal antibodies represent a novel class of therapeutics
in MM, targeting different antigens on plasma cells.3,4 Of these
targets, anti-CD38 antibodies are in advanced stages of clinical
development, with the approval of daratumumab (DARA) for
the treatment of MM patients in November 2015 by the US
Food and Drug Administration.5–7
The use of DARA and other anti-CD38 therapies leads
to interference with routine immunohematological tests,
ABBREVIATIONS: AHG = antihuman globulin; BMAP = blockage
monoclonal antibody protocol; DARA = daratumumab;
DAT = direct antiglobulin test; DTT = dithiothreitol; IATs = indirect
antiglobulin tests; MM = multiple myeloma.
From the 1Discipline of Hematology Transfusion and Cell Therapy,
University of São Paulo School of Medicine (FMUSP), São Paulo,
Brazil; 2Fundação Pró-Sangue Hemocentro de São Paulo, São
Paulo, Brazil; 3Fundação Hemominas, Belo Horizonte, Minas
Gerais, Brazil; 4HEMOCE – Hematology and Hemotherapy Centre,
Fortaleza, Ceará, Brazil; and the 5Churchill Hospital, NHSBT,
Oxford University, Oxford, United Kingdom.
Address reprint requests to: Carla Luana Dinardo, Fundação
Pró-Sangue Hemocentro de São Paulo, Av. Dr. Enéas de Carvalho
Aguiar, 155, 1 floor, Cerqueira César-São Paulo- SP, Brazil,
05403-000. e-mail: caludinardo@gmail.com.
†
Contributed equally
Received for publication January 29, 2018; revision received
December 19, 2018, and accepted January 3, 2019.
doi:10.1111/trf.15202
© 2019 AABB
TRANSFUSION 2019;9999;1–9
TRANSFUSION 1
CHINOCA ZIZA ET AL.
representing a new challenge to blood banks worldwide.8–16
CD38 is weakly expressed on normal human red blood cells
(RBCs) and consequently anti-CD38 monoclonal antibodies
can bind directly to CD38 expressed on RBCs, causing pan-
reactivity in vitro.9,17 Although DARA does not interfere with
ABO/D typing, the plasma of DARA-treated patients con-
sistently causes positive agglutination reactions in indirect
antiglobulin tests (IATs), including antibody screening, anti-
body identification panels, and IAT crossmatches.8,9 Aggluti-
nation due to DARA occurs in tests using normal saline,
low-ionic-strength saline, and polyethylene glycol.10 The
false-positive IAT can persist for 2 to 6 months after ending
the treatment with DARA, and it has the potential to mask
the detection of RBC irregular antibodies in the patient’s
serum, compromising the transfusion’s safety.8 Dealing with
the interference leads to time-consuming pretransfusion
assays. Appropriate solutions to negate the effect of anti-
CD38 monoclonal antibodies on RBCs are important to rule
out the presence of clinically significant alloantibodies and
minimize the delay in issuing a blood product.
Since DARA was approved for clinical use, many litera-
ture reports have been dedicated to describing methods to
overcome its interference with immunohematological pre-
transfusion tests.8–18 The currently described strategies in the
literature are dithiothreitol (DTT)-treated RBCs, trypsin-treated
RBCs, cord cell antibody screen, soluble anti-CD38, anti-CD38
idiotype, and phenotype and genotype matching.8–18 Pub-
lished reports suggest that the most commonly used method
is DTT-treated RBCs.9–13 Although the DTT technique is inex-
pensive and fairly easy to apply, the primary limitation is the
damaging of RBC antigens other than CD38, which results in
failure to detect antibodies against clinically relevant blood
group systems: Kell, Dombrock, Yt, and Lutheran.9,10 Missing
antibodies against these blood group antigens may lead to
clinically significant hemolytic reactions.19 All the mitiga-
tion methods already reported have their own advantages
and disadvantages, and currently there is no standard-of-
care technique.
There is a classical protocol in immunohematology
designed to minimize the interference of autoantibodies
when attempting to phenotype RBCs using antihuman
globulin (AHG).20 The use of AHG blocks the autoanti-
bodies that are bound to the RBC membrane by covering
their Fc portion and leaving the other RBC antigens free
to be targeted on the phenotyping assay.20 This strategy
does not damage the RBC structure, letting the antibodies
bind to the antigens on the erythrocyte surface. We postu-
lated that this protocol could be adapted to block CD38
sites on RBCs (after being saturated with monoclonal anti-
CD38), preventing them from being targeted in the IAT by
the anti-CD38 in the patient’s serum.
Our aim is to describe and validate a new strategy to
mitigate the DARA interference in pretransfusion tests. As a
fast, safe, and low-cost method is needed, we propose in
this study a blockage monoclonal antibody protocol (BMAP)
2 TRANSFUSION
using anti-CD38 and AHG, with the benefit of keeping intact
the RBC structure, overcoming the major disadvantage of
the commonly used DTT method.
MATERIALS AND METHODS
Patient samples
Whole blood samples (ethylenediaminetetraacetic acid and
serum tubes) that were collected as part of routine clinical
care of adult patients with MM receiving DARA from the
University of São Paulo Medical School, Hematology and
Hemotherapy Center of Ceará and Fundação HEMOMINAS
were included in the study. Samples were forwarded to the
clinical immunohematology laboratory of the hospital of the
University of São Paulo Medical School and to the immuno-
hematology laboratory of the Hematology and Hemotherapy
Center of Ceará to perform the routine serologic tests
(ABO/Rh typing, antibody screening, antibody identifica-
tion, direct antiglobulin test [DAT] and auto-control) in a
gel column technique (Bio-Rad). Patient samples that were
panreactive in the antibody screening were further tested
using DTT-treated RBCs and RBCs treated with the pro-
posed BMAP, as described below. To ensure the reproduc-
ibility of the BMAP, the protocol was performed by four
laboratory technicians of different immunohematology cen-
ters involved in the study.
Institutional review board approval was obtained to
study methods to prevent DARA binding on these patient
samples. The study was conducted in accordance with the
ethical principles of the Declaration of Helsinki.
DTT treatment of RBCs
A detailed method for DTT (Sigma) treatment of RBCs is
described in the AABB Technical Manual.21 Briefly, 0.2 mol/L
of DTT was prepared by diluting 1 g of DTT in 32 mL of
phosphate-buffered saline (pH 8). Kpb + control RBCs were
used to verify that DTT treatment had effectively denatured
Kell blood group system antigens. RBCs were washed three
times with saline, before adding 400 μL of DTT (0.2 mol/L) to
each tube (100 μL of RBCs +400 μL of DTT). They were then
incubated at 37
C for 30 minutes with periodic mixing by
inversion (three to four times during incubation). Finally,
RBCs were washed three times with saline and used for anti-
body screening.
Blockage monoclonal antibody protocol
The validation of the BMAP was performed in three steps.
Figure 1 outlines the study workflow.
Step I: Anti-CD38 titration
The anti-CD38 used in this study was the residual content of
DARA bottles (20 mg/mL; Janssen R&D), after its total infusion
in the patients (generously provided by Fernando Barroso-
Duarte and Fundação HEMOMINAS). A small amount (10 μL)
 BMAP TO AVOID DARA LABORATORIAL INTERFERENCE

Fig. 1. Schematization of the BMAP with anti-CD38 and AHG. (A) Donor’s RBCs adsorption with anti-CD38. (B) Blocking step with AHG
binding to the Fc portion of anti-CD38. (C) IAT performed with the BMAP-treated RBCs. (D) Test result: On the left side, anti-CD38 of patient’s
serum does not cause agglutination reaction, because there is no free CD38 on RBCs surface. On the right side, the presence of alloantibodies
in patient’s serum cause agglutination on the IAT allowing their identification. [Color figure can be viewed at wileyonlinelibrary.com]

of anti-CD38 was suspended in saline at 1/1000 concentration
and stored at −20
C. For the experiments, an aliquot of this solu-
tion was thawed and used for titration. As there is an interindivi-
dual variability in the amount of CD38 on erythroid membrane
(Appendix S1, available as supporting information in the online
version of this paper), anti-CD38 titration was performed using
all donor RBCs that would be blocked to compose the antibody
screening panel to be used in this study. Only RBCs stored for
less than 7 days were used in the study, as this was the maximum
storage length standardized in our reference laboratory.

TRANSFUSION 3
CHINOCA ZIZA ET AL.
The anti-CD38 suspension (1:1,000) was titrated to
identify the drug concentration resulting in 1+ agglutination
with selected blood donor RBCs. First, anti-CD38 suspen-
sion was subjected to serial dilution with normal saline,
from titer 1 to 256. Nine tubes were used for the dilution
process: 100 μL of anti-CD38 were added to the first and to
the second tube (titer 1 and 2, respectively). Next, 100 μL of
normal saline was added to the tubes, starting from the sec-
ond one. Then, 100 μL of the anti-CD38 suspension of the
second tube (titer 2) were transferred to the third tube and
so on, until the last tube (titer 256).
After the dilution process, donor RBCs (O type) with the
following phenotype were selected: 1) Phenotype I: ccDee;K–k
+,Kp(a-b+);Fy(a-b-); Jk(a + b+); M + N + S-s+; P1-, Le(a + b-);
Dia-, Lu(a-b+); 2) Phenotype II: ccddee; K–k+, Kp(a-b+);
Fy(a + b+); Jk(a + b-); M-N + S + s-; P1+, Le(a-b-); Dia-; and
3) Phenotype III: CcDee; K + k+; Fy(a + b+); Jk(a-b+); M + N+
S + s-; Le(a-b+), Dia+. A suspension was prepared by diluting
10 μL of donor RBCs in 1 mL of low-ionic-strength solution
(Biorad). Then, 50 μL of 1% donor RBCs suspension and 25 μL
of each titer of anti-CD38 were added to the micro columns of
monospecific (anti-IgG) cards (Biorad), which were incubated
for 15 minutes at 37
C and centrifuged for 10 minutes, as
specified by the manufacturer. Agglutination was graded from
0 to 4+, as specified elsewhere.21 Anti-CD38 titer resulting in 1+
was selected for the next step. If more than one dilution
resulted in 1+ agglutination, then the more concentrated titer
should be tested first.
Step II: Treatment of RBCs with anti-CD38
Before starting the protocol, DAT was performed with all
selected donor RBCs (Phenotypes I, II, and III) to certify the
absence of autoantibodies and/or complement fractions on
erythroid membrane. Then, 300 μL of donor RBCs (undiluted)
were added to 300 μl of anti-CD38 (titer determined in the
previous step), incubated for 15 minutes at 37
C, and washed
three times with 1 mL of normal saline, discarding the super-
natant. This treatment was performed with the three selected
RBC units. DAT was repeated after the adsorption to confirm
the linkage of anti-CD38 antibodies to RBCs with 1+. If DAT
agglutination strength after adsorption was 1+, the blockage
could be done. If 2+ or more, then more diluted anti-CD38
should be tested before blocking the RBCs.
In the course of BMAP validation, the adsorption of
RBCs with undiluted DARA (20 mg/mL) for blockage was
tested but failed. Details about these initial experiments are
given in Appendix S1.
Step III: Blockage of anti-CD38–treated RBCs
with AHG
The blockage of anti-CD38-treated RBCs was performed
with monospecific anti-human IgG (Fc specific) produced
in rabbits - AHG (Fresenius HemoCare, São Paulo, Brazil),
in the proportion of 950uL of AHG and 50uL of RBCs cov-
ered with anti-CD38 (undiluted). It was performed with the
4 TRANSFUSION
tube technique, with 20-minute incubation at room temper-
ature, followed by three cycles of washing with 1 mL of nor-
mal saline, discarding the supernatant.20 All previously
treated donor RBCs were blocked with AHG. Then, 10 μL of
each BMAP-treated RBC unit were diluted in 1 mL of low-
ionic-strength saline and this suspension was used in subse-
quent tests. DAT was performed using the postblockage
RBCs to confirm that there was no anti-CD38 (Fc fraction)
uncovered by AHG on erythroid membrane.
After the three steps of the BMAP were completed,
antibody screening was performed, testing 25 μL of patients’
sera with 50 μL of BMAP-treated RBCs (1% suspension) in
both monospecific (anti-IgG) and polyspecific (anti-IgG and
anti-C3d) cards (Biorad). Patients’ samples were tested at
three different times of the study to ensure reproducibility
and reliability of the antibody screening using BMAP-treated
RBCs. Also, four different laboratory technicians performed
the protocol in parallel, using the same samples.
To evaluate whether the BMAP was capable of accurately
detecting antibodies, 10 μL of commercial antisera of different
specificities—anti-D, -C, -K, −Jkb, -S, -Kpb (Lorne Laborato-
ries)—were added to 200 μL of saline, and 10 μL of this solu-
tion was added to 500 μL of the sera of three samples of
nonalloimmunized patients using DARA. Antibody screening
was then performed using RBCs treated with the BMAP.
To prove that the most important erythrocyte antigens
are not affected by the BMAP, the expression of C, c, E, e; K;
Fya, Fyb; S, s; and Jka and Jkb in BMAP-treated RBCs was
tested by phenotyping in the gel method according to the
manufacturer’s instruction (Biorad).
Stability of BMAP-treated RBCs
The stability of BMAP-treated RBCs stored at 2 to 6
C was
evaluated after 12 hours, 24 hours, 36 hours, 7 days, 21 days,
and 28 days after the treatment with anti-CD38 and AHG,
using the sera of nonalloimmunized patients using DARA to
perform the antibody screening.
Cost analysis
The approximate costs of the BMAP were calculated based
on the value of the supplies and of labor per patient sample.
The costs were obtained from the amount paid by Hospital
das Clínicas of University of São Paulo School of Medicine
to the manufacturers and included all supplies: gloves,
pipette tips, AHG, test tubes, gel cards, and saline. Values
may vary according to the buyer.
RESULTS
Patients’ samples
Twenty patients with MM using DARA who required routine
serologic tests were included in the study from March to
September 2017. Eleven patients were from Hospital das
Clínicas/University of São Paulo, seven were from Fundação

HEMOMINAS, and two from the Hematology and Hemother-
apy Center of Ceará. No information was provided about dos-
age and duration of DARA treatment, but the goal of the study
was to develop a method to remove the interference of this
drug irrespective of the postinfusion interval. All included
patients (n = 20) presented with positive antibody screening
and panagglutination with all RBCs of the identification panel.
Consequently, all samples were tested with both DTT- and
BMAP-treated RBCs.
RBC blockage with anti-CD38 and AHG
Titration of the anti-CD38 suspension (1/1,000) in monospe-
cific (IgG-only) cards was the first step for validating the BMAP.
Anti-CD38 titers selected for this study varied from 16 to
128, suggesting the existence of an interindividual variability of
CD38 antigens sites on the erythroid membrane, which was
further explored in additional experiments (Appendix S1).
The second step was to treat selected RBCs with anti-CD38.
RBCs obtained from three different blood donors were selected,
all exhibiting negative DAT before the beginning of the protocol.
After the adsorption of anti-CD38, DAT was again performed
and resulted positive (1+) in all treated RBCs. In the third step,
AHG was added to anti-CD38–treated RBCs and, after that, DAT
resulted negative. Figure 2 exhibits DAT results in all steps of the
protocol: negative with untreated RBCs, positive after adsorption
with anti-CD38, and negative after blockage with AHG.
Antibody screening using BMAP-treated RBCs
Antibody screening on all included samples included both
BMAP- and DTT-treated RBCs. The results of antibody
screening using treated and nontreated (BMAP- and DTT-
treated) RBCs are described in Table S2, available as support-
ing information in the online version of this paper. In 19 of
20 patients, DARA-mediated agglutination was eliminated
when sera were tested against both DTT- and BMAP-treated
RBCs on monospecific cards (IgG only). In 1 of 20 patients, it
was observed that DTT treatment reduced the agglutination
Fig. 2. Results of direct antiglobulin test (DAT) using untreated, anti-CD38–adsorbed and BMAP-treated RBCs of different phenotypes.
(A) Negative DAT of untreated RBCs showing that there are no antibodies bound to the RBCs surface. (B) Positive DAT after anti-CD38
adsorption. (C) Negative DAT after blocking RBCs with AHG. [Color figure can be viewed at wileyonlinelibrary.com]
BMAP TO AVOID DARA LABORATORIAL INTERFERENCE
strength in RBCs of Phenotypes I and III (from 2+ to 1+) and
removed the agglutination in RBCs of Phenotype II. When
the same sample was tested against BMAP-treated RBCs, the
agglutination strength in RBCs of Phenotypes I and III was
increased (from 2+ to 3+) and negative in RBCs of Phenotype
II, suggesting the presence of antibody-directed antigens pre-
sent only in RBCs of Phenotypes I and III. In the patient’s
immunohematology records before the start of DARA treat-
ment, irregular antibodies had already been identified.
Antibody identification confirmed the following specificities:
anti-D, -C, −G, and −Lea. The increase in agglutination
strength observed when antibodies were present in the
patients’ sera may be due to AHG transfer from CD38 to
nearby IgG antibodies.
One-half of the included samples were stored at 2 to
6
C up to 7 days and tested three times by different labora-
tory technicians using BMAP-treated RBCs, and the results
were reproducible. Anti-CD38 titration was repeated in
every experiment for each batch of RBCs selected for block-
age. Even though no significant changes in the selected
titers were observed throughout the experiments, the stabil-
ity of the frozen anti-CD38 suspension was not evaluated in
this study, and consequently, the recommendation is to per-
form the titration before initiating any RBC BMAP.
As described in the Methods section, antibody screen-
ing was performed using both BMAP- and DTT-treated
RBCs in monospecific (IgG only) and polyspecific (anti-IgG
and anti-C3d) gel column cards. It was observed that when
sera containing DARA were tested against BMAP-treated
RBCs in polyspecific cards, there was a significant decrease
in the strength of DARA-mediated agglutination, but it did
not disappear. When the IAT was performed using BMAP-
treated RBCs in the monospecific cards, DARA interference
was removed (Table 1).
Commercial anti-D, -C, -K-, −Jkb, -S, and -Kpb were added
to the sera of three study patients and antibody screening was
repeated using DTT- and BMAP-treated RBCs (Table 2). Anti-
D, -C, −Jkb, and -S were accurately detected by both methods.
TRANSFUSION 5
CHINOCA ZIZA ET AL.

TABLE 1. Strength of RBC agglutination on polyspecific and monospecific LISS cards when testing sera of DARA-
treated patients against BMAP-treated RBCs
RBCs RBCs
I II III I II III
Polyspecific card - LISS/antiglobulin test 2+ 2+ 2+ ! Using BMAP RBCs w w w
Monospecific card - anti-IgG w / 1+ w / 1+ w / 1+ ! Using BMAP RBCs 0 0 0
BMAP = blockage monoclonal antibody protocol; DARA = daratumumab; LISS = low-ionic-strength saline

Completely negative reactions were obtained when sera treated
with anti-K and anti-Kpb were tested against DTT-treated RBCs.
In contrast, agglutination with antigen-positive RBCs was
observed when using BMAP-treated RBCs, accurately indicating
the presence of antibodies. Results comparing the efficacy of
BMAP and DTT strategies in the presence or absence of anti-
Kpb are shown in Fig. 3.
Phenotyping for C, c, E, e; K; Fya, Fyb; S, s; and Jka and
Jkb was performed with blood donors’ BMAP-treated RBCs.
Satisfactory results were observed, showing that antigens
were still detectable and that no false-positive agglutination
took place (not shown).
Stability of BMAP-treated RBCs
The stability of BMAP-treated RBCs stored at 2 to 6
C was
tested five times after preparation: at 12 hours, 24 hours,
36 hours, 7 days, 21 days, and 28 days. Satisfactory results,
meaning a negative antibody screening, were obtained only
in the 12- and 24-hour storage length (Table 3).
Cost analysis
The calculated costs associated with the BMAP, including
supplies and labor, are described in Table 4.
DISCUSSION
This study validated and proved the efficacy of the BMAP
using AHG and monoclonal anti-CD38 to mitigate the inter-
ference of DARA on immunohematological tests. The pro-
posed serologic technique proved to be accurate in detecting
underlying irregular antibodies against diverse blood group
systems, including Kell, demonstrating that the protocol does
not modify the erythroid membrane or damage any RBC
antigen. It was tested with 20 samples of patients using DARA
and removed the drug interference in all cases. It was also
shown that enough anti-CD38 to perform the BMAP could be
obtained from bottles of DARA after its total infusion in
patients, reducing the costs associated with the method and
foreseeing its application to other monoclonal drugs that tar-
get CD38 or other RBC antigens, depending on the amount
expressed on the erythroid membrane.
The main advantage of the studied BMAP is overcom-
ing the major drawback of the most currently applied
method to eliminate DARA interference on pretransfusion
tests: DTT treatment of RBCs, which hampers the evaluation
of antibodies directed against antigens of the potentially
clinically relevant blood group systems Kell, Dombrock, Yt,
and Lutheran. During oncologic therapy, patients may
develop antibodies directed against antigens of these other
systems, which were not included in the prospective
antigen-matched transfusion program.22 These antibodies
may be associated with significant delayed posttransfusion
hemolytic reactions. Also, if they are targeted to high-
frequency antigens (i.e., k, Jsb, Kpb, Hy, Gya), differentiation
with anti-CD38 interference is impossible using the DTT
strategy, compromising transfusion safety. The use of soluble
CD38 and anti-CD38 idiotype are alternatives to the BMAP
that, similarly, do not compromise the evaluation of any
blood group system antigen. However, these methods have
restricted applicability in daily practice because of the lim-
ited availability and high cost.8,13,18
In the immunohematological investigation, the most appro-
priate scenario is that in which the BMAP is complementary to

TABLE 2. Antibody screening using BMAP-treated RBCs and samples of patients using DARA treated with different
commercial antibodies
Untreated RBCs BMAP-treated RBCs
RBCs Anti-D Anti-C Anti-K Anti-Jkb Anti-S Anti-D Anti-C Anti-K Anti-Jkb Anti-S
I 2+ 1+ 1+ 2+ 2+ 2+ 0 0 1+ 0
II 1+ 1+ 1+ w+ 2+ 0 0 0 0 1+
III 2+ 4+ 2+ 2+ 2+ 2+ 4+ 2+ 2+ 1+
Phenotype I: ccDee;K–k+,Kp(a-b+);Fy(a-b-); Jk(a + b+); M + N + S-s+; P1-, Le(a + b-); Dia-, Lu(a-b+).
Phenotype II: ccddee; K–k+, Kp(a-b+); Fy(a + b+); Jk(a + b-); M-N + S + s+; P1+, Le(a-b-); Dia-.
Phenotype III: CcDee; K + k+; Kp(a-b+); Fy(a + b+); Jk(a-b+); M + N + S + s-; P1-; Le(a-b+); Dia+.
BMAP = blockage monoclonal antibody protocol; DARA = daratumumab.

6 TRANSFUSION
 BMAP TO AVOID DARA LABORATORIAL INTERFERENCE

Fig. 3. Results of antibody screening of DARA-treated patients using BMAP-RBCs and DTT-treated RBCs. (A) Patient’s serum with DARA
agglutinates all three untreated RBCs. Agglutination was eliminated using both BMAP-treated RBCs and DTT-treated RBCs. (B) Patient’s
serum with DARA + anti-Kpb agglutinates untreated RBCs and BMAP-treated RBCs. Agglutination was eliminated in DTT-treated RBCs.
[Color figure can be viewed at wileyonlinelibrary.com]

DTT. As there is a possible transfer of AHG to nearby IgG alloan-
tibodies when the BMAP is applied to samples containing irregu-
lar antibodies, methods such as adsorption and elution can be
hampered. Therefore, DTT should be the method of choice for
antibody identification, while the BMAP with selected donor
RBCs should be used in parallel to reveal irregular antibodies
directed to antigens that might have been damaged by the DTT
treatment.
As a suggested workflow, RBCs from two randomly
selected donor units and from commercial antibody screen-
ing panels might undergo daily BMAP and DTT treatment,
respectively. The specific phenotype of the donor cells does
not need to be known, as selection of two samples should
ensure that at least one would carry Kell, Dombrock, Yt,
and Lutheran antigens of high prevalence. The DTT-treated
commercial antibody screening cells would be used to
detect alloantibodies to common RBC antigens except for
anti-K. The BMAP-treated donor samples would allow the
detection of alloantibodies to antigens of high prevalence
that had been damaged by DTT treatment of the commer-
cial antibody-screening cells. RBCs should be replaced once
a week or every time hemolysis is detected and, in this cir-
cumstance, anti-CD38 titer should be reassessed. Daily, the
selected donor RBCs should undergo BMAP treatment, and
the commercial antibody screening cells should be treated
with DTT (or less frequently depending on the facility-specific
protocols). Unless the selection of RBCs for the BMAP includes
a K+ sample, donor units for transfusion should be K-. This
workflow is described in Table 5.
To help blood banks, AABB developed a bulletin with
recommendations regarding DARA interference with sero-
logic testing.15 To prevent delays in the pretransfusion pro-
cess, it was suggested that hospitals should inform the
transfusion service when patients start receiving the anti-
CD38 monoclonal antibody. Also, it is suggested that RBC
phenotyping and genotyping and antibody screening should

TABLE 3. Stability of BMAP-treated RBCs stored at 2 to 6
C

BMAP-treated RBCs Untreated RBCs
Donor RBCs Immediate 12 h 24 h 36 h Immediate 12 h 24 h 36 h
I 0 0 0 1+ 1+ 1+ 1+ 1+
II 0 0 0 1+ 1+ 1+ 1+ 1+
III 0 0 0 1+ 1+ 1+ 1+ 1+
BMAP = blockage monoclonal antibody protocol.

TRANSFUSION 7
CHINOCA ZIZA ET AL.
TABLE 4. BMAP cost analysis
BMAP Costs
Titration supplies US$2.53
BMAP supplies US$0.11
Antibody screening supplies US$1.98
Labor US$21.63
BMAP = blockage monoclonal antibody protocol.
be performed before the initiation of DARA treatment. How-
ever, the information if the patient is using DARA usually
lacks in the blood requests. The most common situation
involves the transfusion laboratory suspecting of DARA inter-
ference based on the results of pretransfusion tests in a patient
diagnosed with MM and having to confirm that with the medi-
cal team. In this scenario, if extended phenotype was not per-
formed before, even the selection of antigen-matched RBC
units would be hampered and the performance of IAT cross-
match with DTT-treated erythrocytes would be error prone.
On the other hand, the BMAP could be applied in this situa-
tion to remove anti-CD38 interference with both antibody
screening and IAT crossmatch, decreasing the risks of incom-
patible transfusions and, consequently, increasing transfusion
safety.
Several monoclonal antibody therapies are in advanced
stages of development, and similarly to DARA, they might
interfere with routine blood bank tests. For future applica-
tion, the BMAP with AHG possibly can be used to overcome
the panreactivity in vitro for other monoclonal drugs target-
ing RBC antigens, enhancing the importance of the devel-
oped technique. However, the applicability of the BMAP to
mitigate the interference of other monoclonal drugs on the
immunohematological tests would depend on the antigen
density of the target antigen, as the risk of spontaneous
agglutination is higher than with CD38, which is present
at a low antigen density on the erythroid membrane.
The fact that the BMAP has some drawbacks is impor-
tant to highlight. The most relevant ones are the technical
difficulty of the method, the unavailability of anti-CD38 for
some health institutions, and the need to titrate anti-CD38
TABLE 5. Suggested workflow for the implementation
of the BMAP in the routine of an immunohematology
reference laboratory
1×/week
• Selection of RBCs from two blood donors
• Storage at 4  2
C
• Anti-CD38 titration
1×/day
• Visual inspection of stored RBCs
• BMAP treatment of selected donor RBCs
• DTT treatment of commercial antibody screening RBCs*
* May be required less frequently if the facility-specific protocol
allows for storage and use of DTT-treated RBC for longer than
24 hours.
BMAP = blockage monoclonal antibody protocol; DTT = dithiothreitol.
8 TRANSFUSION
before performing the tests and having to strictly select the
dilution, which results in low strength of RBC agglutination
(1+), as more heavily coated RBCs were associated with
false-positive results. These factors can make it difficult to
implement the BMAP in a busy transfusion service or in a
reference laboratory where multiple DARA patients may be
tested every day. Another drawback is that the BMAP was
validated for fresh (<7 days of collection) noncommercial
RBCs. In this study, we selected samples from donors whose
phenotype composition was suitable for composing a
screening panel. As such, the service must have a certain
number of phenotyped donors to gather those samples.
None of the patients tested with the BMAP in this study
had the immunohematological tests compromised due to
panagglutination, and the values of monoclonal immuno-
globulin varied from 0.3 to 7.7 g/dL. However, higher levels
of gamma globulin can affect pretransfusional testing and,
potentially, the BMAP. Finally, in the present study, the left-
overs of DARA were used for performing the laboratory tests
after patients’ consent, similarly to what is usually done to
investigate drug-induced autoimmune hemolysis. Each ser-
vice must consult the local ethics committee and the hospi-
tal regiment before using this source to perform the BMAP.
In conclusion, the BMAP using anti-CD38 and AHG is
capable of neutralizing the effect of DARA on routine immu-
nohematological tests. It represents an inexpensive, accu-
rate, and simple method that preserves the integrity of
RBCs, allowing the identification of clinically significant
alloantibodies. In the future, the BMAP can be applied to
minimize the interference on pretransfusion tests of any
monoclonal drug that incidentally targets erythroid lineage,
with no need to acquire different specific reagents.
CONFLICT OF INTEREST
The authors have disclosed no conflicts of interest.
REFERENCES
1. Moreau P, Touzeau C. Multiple myeloma: from front-line to
relapsed therapies. Am Soc Clin Oncol Educ Book 2015;35:
e504-11.
2. Kumar SK, Lee JH, Lahuerta JJ, et al. Risk of progression and
survival in multiple myeloma relapsing after therapy with
IMiDs and bortezomib: a multicenter international myeloma
working group study. Leukemia 2012;26:149-57.
3. Touzeau C, Moreau P, Dumontet C. Monoclonal antibody ther-
apy in multiple myeloma. Leukemia 2017;31:1039-47.
4. van de Donk NWCJ, Moreau P, Plesner T, et al. Clinical efficacy
and management of monoclonal antibodies targeting CD38
and SLAMF7 in multiple myeloma. Blood 2016;127:681-95.
5. Lokhorst HM, Plesner T, Laubach JP, et al. Targeting CD38
with daratumumab monotherapy in multiple myeloma. N Engl
J Med 2015;373:1207-19.

6. Darzalex package insert [Internet]. Horsham (PA): Janssen Bio-
tech, Inc.; 2015 [cited 2016 Apr 01]. Available from: http://
www.accessdata.fda.gov/drugsatfda_docs/label/2015/
761036s000lbl.pdf.
7. McKeage K. Daratumumab: first global approval. Drugs 2016;
76:275-81.
8. Oostendorp M, Lammerts van Bueren JJ, Doshi P, et al. When
blood transfusion medicine becomes complicated due to inter-
ference by monoclonal antibody therapy. Transfusion 2015;55:
1555-62.
9. Chapuy CI, Nicholson RT, Aguad MD, et al. Resolving the dara-
tumumab interference with blood compatibility testing. Trans-
fusion 2015;55:1545-54.
10. Chapuy CI, Nicholson RT, Aguad MD, et al. International vali-
dation of a dithiothreitol (DTT)-based method to resolve the
daratumumab interference with blood compatibility testing.
Transfusion 2016;56:2964-72.
11. Moreau P, van deDonk NWCJ, Miguel JS, et al. Practical con-
siderations for the use of daratumumab, a novel CD38 mono-
clonal antibody, in myeloma. Drugs 2016;76:853-67.
12. Anani WQ, Duffer K, Kaufman RN, et al. How do I work up
pretransfusion samples containing anti-CD38? Transfusion
2017;57:1337-42.
13. Anani WQ, Marchan MG, Bensing KM, et al. Practical
approaches and costs for provisioning safe transfusions during
anti-CD38 therapy. Transfusion 2017;57:1470-9.
14. Hannon JL, Clarke G. Transfusion management of patients
receiving daratumumab therapy for advanced plasma cell mye-
loma. Transfusion 2015;55:2770.
15. Mitigating the anti-CD38 interference with serologic testing
[Internet]. AABB Association Bulletin #16–02. Bethesda (MD):
BMAP TO AVOID DARA LABORATORIAL INTERFERENCE
AABB; 2016 [cited 2016 May 29]. Available from: https://www.
aabb.
org/programs/publications/bulletins/Documents/ab16-02.pdf.
16. DeVooght KM, Oostendorp M, van Solinge WW. Dealing with
anti-CD38 (daratumumab) interference in blood compatibility
testing. Transfusion 2016;56:778-9.
17. Zocchi E, Franco L, Guida L, et al. A single protein immunolog-
ically identified as CD38 displays NAD+ glycohydrolase, ADP-
ribosyl cyclase and cyclic ADP-ribose hydrolase activities at the
outer surface of human erythrocytes. Biochem Biophys Res
Commun 1993;196:1459-65.
18. Schmidt AE, Kirkley S, Patel N, et al. An alternative method to
dithiothreitol treatment for antibody screening in patients
receiving daratumumab. Transfusion 2015;55:2292-3.
19. Reid M, Lomas-Francis C, Olsson ML. The blood group antigen
factsbook. 3rd ed. Waltham (MA): Academic Press; 2012.
20. Sererat TS, Veidt D, Dutched A. A quick and simple method
for phenotyping IgG-sensitized red blood cells. Immunohema-
tology 2000;16:154-6.
21. Fung MK, Grossman BJ, Hillyer CD, et al., editors. Technical
manual. 18th ed. Bethesda (MD): AABB; 2014.
22. Schonewille H, Haak HL, van Zijl AM. Alloimmunization after
blood transfusion in patients with hematologic and oncologic
diseases. Transfusion 1999;39:763-71.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the
online version of this article.
Appendix S1. Supporting Information.
TRANSFUSION 9