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IMMUNOHAEMATOLOGY New method for overcoming the

interference produced by
Original article anti-CD38 monoclonal antibodies
in compatibility testing
Emma Castro Izaguirre1, María del Mar Luis-Hidalgo1, Luis Larrea González1,
Cristina Arbona Castaño1

Background - Plasma of patients taking anti-CD38 monoclonal antibodies

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(MoAbs) leads to panagglutination in the indirect antiglobulin test (IAT), that

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Centro de Transfusión
1
can mask clinically significant alloantibodies. Dithiothreitol (DTT) treatment
de la Comunidad Valenciana, of test RBCs is the more widespread method for avoiding this interference.
Valencia, Spain Current DTT 0.2 mol/L method is time consuming and damages several red
blood groups antigens. This study aims to evaluate low concentration DTT

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treatment of RBCs adapted for gel testing.
Materials and methods - Four DTT concentrations (0.01, 0.02, 0.03, and
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0.04 mol/L), and three gel test brands were evaluated on six DARA patient’s
samples. Briefly, the method consists of pipetting 50 μL of 0.8% RBCs on
AHG micro columns, followed by 25 μL of DTT, thoroughly mixing and 15
min incubation at 37 °C. Then, 25 μL of serum/plasma is added to proceed
to IAT. In order to asses the effect of DTT 0.04 mol/L on different blood group
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antigens, serial dilutions of sera containing anti-K, -k, -Kpb, -Lub, -Yta and
anti-JMH antibodies were tested against DTT-RBCs. One sample of a DARA
patient with known alloantibodies as well as samples of two patients
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inoculated with anti-K and anti-Fya were evaluated.


Results - RBCs treatment with DTT 0.04 mol/L for 15 min completely eliminated
anti CD38 panagglutination in all samples studied and worked with different
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reactivity intensities in IAT and gel brands. The new method allowed the detection
of underlying anti-D, anti-E, anti-K and anti-Fya alloantibodies. Titration assays
demonstrated no denaturation of Kell, Lutheran, Cartwright and JMH antigens.
Discussion - The new DTT method adapted for gel testing is efficacious,
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simple and only adds 15 min over regular IAT. Pheno/genotyping before DARA
treatment or transfusion of K negative RBCs may be unnecessary.

Keywords: daratumumab, panagglutination, anti-CD38, DTT, gel test.

INTRODUCTION
Plasma of patients taking an anti-CD38 monoclonal antibody (MoAb) as a treatment
for multiple myeloma (MM) causes panagglutination in the indirect antiglobulin test
Arrived: 8 January 2020
Revision accepted: 23 March 2020
(IAT) that can mask clinically significant alloantibodies. This is an important issue
Correspondence: Emma Castro Izaguirre for patients that need blood transfusions since it produces a delay in releasing red
e-mail: emmacastroizaguirre@gmail.com
blood cells (RBC). With an increasing number of patients being treated with these

Blood Transfus 2020; 18: 290-4 DOI 10.2450/2020.0004-20


© SIMTIPRO Srl
290 All rights reserved - For personal use only
No other use without premission
New method for eliminating DARA interference

types of drugs, it is important to find the best way to avoid 0.2 mol/L DTT-treated RBCs. For the study group, four
this interference. different concentrations of DTT (0.01, 0.02, 0.03 and
CD38 is a glycoprotein found on the cellular surface of 0.04 mol/L) and three incubation times of RBCs with DTT at
many tissues, as well as haematopoietic and immune 37 °C (15, 30 and 40 min) were assayed. All sera/plasma samples
system cells. It is also weakly expressed on normal RBC were also tested using three different brands of gel test cards.
membranes. Anti-CD38 MoAbs bind cross-linked to Dithiothreitol treatment of red blood cells
test RBCs and cause panagglutination in IAT1. This pan One gram of DTT (Fisher Scientific, Merelbeke, Belgium)
reactivity can not be removed by regular adsorption/ was diluted in 32 mL of phosphate-buffered saline (PBS)
elution techniques1. There are several theoretical pH 8.0, to obtain DTT 0.2 mol/L. Aliquots of 2 mL were
approaches for eliminating the interference in IAT1-7, but stocked at −20 °C until needed. Blood plasma/serum
up till now, the majority of transfusion services have used samples were submitted to standard gel IAT with 0.2 mol/L
the Dithiothreitol (DTT) method. DTT-treated RBCs according to the method described in

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Dithiothreitol, a reducing compound, denatures CD38 by the AABB Technical Manual9. Brief ly, this involves mixing

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cleaving the disulphide bonds1. As a consequence, DTT one volume of PBS washed RBCs with four volumes of
damages CD38 proteins but also other RBC antigens, 0.2 mol/L DTT at pH 8.0, followed by incubation at 37 °C
resulting in the failure to detect antibodies against for 30-45 min. RBCs and DTT solution are manually mixed
clinically relevant blood group systems that include: Kell, 4-5 times during incubation. After DTT treatment, RBCs

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Lutheran, JMH, LW, Cromer, Indian, Knops, Dombrock, are washed four times with PBS at pH 7.0, and packed.
Cartwright, and Raph8. DTT-treated RBCs are then diluted to 3-5% or 0.8% with
PBS, before undergoing IAT. All samples included in this
A detailed method for the treatment of RBCs with DTT
PR
is described in the American Association of Blood Banks study were tested in parallel following the new method.
(AABB) Technical Manual9. Around two hours are needed to The new method consists of treating test RBCs with
complete pre-transfusion testing with DTT-treated RBCs. low DTT concentrations directly in gel columns. RBCs
Given this, we can conclude that, although DTT is along with DTT were incubated for 15-40 min at 37 °C.
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effective, it is time-consuming and may miss some RBCs are not washed with PBS after DTT treatment.
important alloantibodies. Plasma/serum is added to micro-columns immediately
Recently, Hosokawa et al.4 made an important contribution after DTT treatment to start the IAT.
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to the field. By reducing the concentration of DTT from Indirect antiglobulin test methods
0.2 mol/L to 0.01mol/L, and employing an IAT tube technique Serum/plasma samples were screened for the presence
with an automated cell washing centrifuge, they reduced of irregular antibodies using commercial RBC panels of
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the time needed to complete the antibody screening and 3-4 cells: ID-Diacell I-II-II (BioRad, DiaMed GmbH,
cross-match to around 60 min. In addition, this approach Cressier FR, Switzerland), Serascan Diana 4 (Diagnostic
also reduces the denaturation of K antigen. Grifols S.A., Barcelona, Spain), and Across Gel® Cell
Worldwide, most blood transfusion services rely on gel Screen 4 (DiaPro, Istanbul, Turkey). For auto-control
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testing and no longer use tube techniques or cell washing and cross-match RBC preparation, the corresponding
centrifuges in their routine procedures. Therefore, this LISS were used: ID Diluent 2 (BioRad, DiaMed GmbH),
study aims to evaluate a DTT technique to mitigate the DG Gel Sol (Diagnostic Grifols S.A), and Across LISS Gel
panagglutination produced by anti-CD38 MoAbs therapy, (DiaPro). Anti_Human Globulin (AHG) gel test cards of
adapted for gel-based IAT. three different brands were used: Liss/Coombs ID Cards
(BioRad, DiaMed GmbH), Anti-Human Globulin DG Gel
MATERIALS AND METHODS 8 Anti-IgG (Rabbit) (Diagnostic Grifols S.A.), and Across
Blood samples of six patients under daratumumab Gel® AHG IgG+C3d (DiaPro).
(DARA) therapy, submitted to our laboratory for Low DTT concentrations (0.01, 0.02, 0.03 and 0.04 mol/L)
compatibility workup, were included in the study. All were prepared by diluting DTT 0.2 mol/L stock solution
samples were tested according to our routine IAT with with PBS at pH 7.0.

Blood Transfus 2020; 18: 290-4 DOI 10.2450/2020.0004-20


All rights reserved - For personal use only 291
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Castro Izaguirre E et al

New dithiothreitol method procedure DTT concentration working for every gel test brand and a
The new method consists of pipetting 50 μL of 0.8% test 15-min incubation time. As far as the efficacy of different
RBCs to every AHG card micro-column, followed by the DTT concentrations is concerned, samples tested with
addition of 25 μL of low concentration DTT (0.01, 0.02, BioRad showed elimination of the interference with
0.03 or 0.04 mol/L) directly to the micro-column top. As DTT 0.02 mol/L and 15-min incubation. Conversely, it
a dilution factor control, in one micro-column we put was necessary to increase DTT concentration up to
25 μL of PBS instead of DTT. After carefully mixing RBCs 0.04 mol/L and 15-min incubation for the complete
and DTT or PBS five times with the pipette tip, AHG cards elimination of pan reactivity of samples tested with
are incubated at 37 °C for 15-30 min. Finally, 25 μL of Grifols or Across AHG gel test.
plasma/serum is added, mixed thoroughly five times, The combination of RBC treatment with DTT 0.04 mol/L
and incubated for 15 min at 37 °C, followed by standard directly on AHG gel cards and 15-min incubation at 37 °C
centrifugation and reading. Agglutination grading from was capable of eliminating the DARA interference of all

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1+ to 4+ was used10. patient’s samples, regardless of the gel test brand used and

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In order to assess the impact of this technique on degree of initial panagglutination.
DTT-sensitive blood groups, different concentrations of Detection of underlying alloantibodies
DTT were added to every IAT run, and three different One sample of a DARA patient with anti-D and anti-E
antisera (anti-k, anti Kpb and anti-Lub) were assayed in alloantibodies, previously detected with the reference

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parallel to patients’ sera. DTT 0.2 mol/L method, was assayed with the new method.
Moreover, serial dilutions of commercial anti-k, anti-Kpb, Screening IAT showed pan reactivity with all RBCs, but
anti Lub and patients’ sera with anti-K, anti-Yta, and two different intensities were observed: 4+ with cells I and
PR
anti-JMH alloantibodies were tested against RBCs treated II and 2+ with cell III (ID-Diacell panel) or III and IV (Grifols
with the highest DTT concentration assayed in this study panel). Reactivity with cell III of BioRad and III and IV of
(0.04 mL/L). Grifols disappeared after treatment with DTT 0.04 mol/L
In order to demonstrate the ability of the new method and 15-min incubation. Remaining reactivity with cells I
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to detect underlying alloantibodies, a serum sample of a and II revealed the presence of underlying alloantibodies.
patient on DARA therapy containing alloantibodies was Antibody identification workup with an 11-RBC panel treated
included. Two more samples of patients under DARA with the new DTT method revealed the presence of two
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treatment were inoculated with one specimen containing alloantibodies of anti-D and anti-E specificities.
high-titre anti-K plus anti-Fya. Two DARA patients’ Two additional samples of patients with anti-CD38
samples were also inoculated with a serum specimen interference, inoculated with anti-K and anti-Fya serum at
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containing high-titre anti-K. Brief ly, one volume of serum a ratio of 2 volumes of DARA patient’s sera to one volume
with known alloantibodies was added to two volumes of of anti-K and anti-Fya serum were tested. Using DTT
DARA patient’s sera, and pan reactivity was demonstrated 0.04 mol/L and 15-min incubation, both alloantibodies
before submitting them for DTT testing. were correctly identified. Two more samples of
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patients on DARA therapy were inoculated with serum


RESULTS containing high-titre anti-K. Samples were tested using
Efficacy of different dithiothreitol concentrations in 0.04 mol/L DTT and a 15-min incubation scheme and,
reducing daratumumab interference again, alloanti-K was correctly identified.
There was a clear difference between gel test brands and Effect of dithiothreitol 0.04 mol/L on different blood
panagglutination intensity of reaction. For every serum group antigens
sample, the degree of reactivity was higher with Grifols or All low DTT concentrations tested (from 0.01 mol/L to
Accross AHG gel test, compared to BioRad. 0.04 mol/L) preserved the antigens of different blood
Combinations of DTT concentrations from 0.01 mol/L groups’ systems known to be susceptible to high DTT
to 0.04 mol/L, and incubation times with RBCs at 37 °C, (0.2 mol/L) concentrations, as shown in every run using
from 15 to 40 min were assayed. The objective was to find a anti-k, anti-Kpb, and anti-Lub polyclonal antisera.

Blood Transfus 2020; 18: 290-4 DOI 10.2450/2020.0004-20


292 All rights reserved - For personal use only
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New method for eliminating DARA interference

Serial double dilutions of polyclonal anti-k, anti-Kpb and DTT method prevents detection and identification
anti-Lub antisera, as well as three patients’ sera containing of alloantibodies of several blood group systems.
anti-Yta, anti-JMH and anti-K alloantibodies, were DTT 0.2 mol/L also damages RBC membranes, producing
submitted to IAT with RBCs treated with 0.04 mol/L for visible haemolysis, and causing some RBC stroma to
15 min at 37 °C. In parallel, the same antisera/sera dilutions be trapped on top of gel columns, giving a false positive
were tested with untreated RBCs with the addition of image.
25 μL PBS to maintain the same RBC dilution factor. The new method uses lower DTT concentrations so it
These tests showed that anti-k, anti-Kpb, anti-Lub, anti-K, does not cause as much damage to RBC membranes, thus
anti-Yta, and anti-JMH titres with DTT-treated RBCs preventing false positive images and haemolysis. Another
did not vary significantly compared to untreated RBCs, advantage is the elimination of washing RBCs before and
confirming that the new DTT method preserves clinically after DTT treatment, which significantly reduces the TAT.
significant RBC group antigens. Even an alloantibody Other methods have been proposed for eliminating the

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with a titre as low as 1 (anti-JMH) was detected with interference1-7, such as anti-idiotype antibodies and CD38

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DTT-treated RBCs (Table I). soluble antigens; however, these are still not commercially
available2.
Recent research has been directed to the development
Table I - Titration of several antibodies against red blood cells
untreated and treated with dithiothreitol 0.04mol/L of DARA Fab fragments6,7 that block CD38 epitopes on

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Antibody Titre without DTT Titre after DTT the RBC membranes, overcoming DARA interference.
Anti-K 32 16 DARAex (Imusyn; patent-pending) is a new technology
based on DARA Fab fragments that mask CD38 antigens in
Anti-k 16 16
PR
Anti-Kp b
16 16 RBCs. It is a very promising approach that also preserves
Anti-Lub 8 8 the integrity of red blood group antigens. However, the
Anti-Yta 64 64 major drawback of this method is the price, which makes
Anti-JMH it unaffordable for most blood transfusion services.
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1 1
DTT: dithiothreitol. Furthermore, DARAex pre-incubation of RBCs before IAT
is about 30 min, while our method only needs 15 min. In
addition, the usefulness of DARAex is probably restricted
Time-to-test results
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to patients taking DARA, and it is not known whether it


The turnaround time (TAT) of the new method for an
works in patients treated with Isatuximab, MOR202,
antibody screening with four cells was 45 min compared
or other anti-CD38 MoAbs. On the other hand, our low
to the 120 min needed for the current DTT 0.2 mol/L
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concentration DTT method works with every anti-CD38


technique; this is 75 min quicker. Therefore, the new
MoAb because it is based on direct CD38 glycoprotein
method takes only 15 min more than a regular IAT.
denaturation and, therefore, is not affected by different
DISCUSSION MoAb binding affinities. Furthermore, the new method
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This study demonstrates that the elimination of anti-CD38 described here, is safe, fast and inexpensive.
interference can be perfectly accomplished with lower By using this new DTT method, clinically significant blood
DTT concentrations (0.04 mol/L), while preserving Kell group antigenicity will be preserved and complementary
and other blood groups’ antigens and reducing the TAT preventive strategies, such as full phenotyping/genotyping
of pre-transfusion compatibility tests. From an economic before daratumumab treatment, or the use of K negative
point of view, this method significantly reduces the costs RBCs10, may not be necessary.
of the reagents and of laboratory technical staff. The Osaka Hospital group4 was the first to describe the
One of the major problems for hospital transfusion benefits of reducing DTT concentration for RBC treatment
services in performing compatibility tests for patients to overcome DARA interference. Although their DTT
on anti-CD38 MoAbs therapy, is the long TAT of the 0.01 mol/L method significantly reduces TAT compared
current DTT 0.2 mol/L method. Furthermore, the current to regular DTT 0.2 mol/L treatment, it is not useful for

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All rights reserved - For personal use only 293
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Castro Izaguirre E et al

hospitals working exclusively with gel test technology. REFERENCES


In Spain, many transfusion services no longer have tube 1. Chapuy CI, Nicholson RT, Aguad MD, et al. Resolving the daratumumab
interference with blood compatibility testing. Transfusion
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transfusion medicine becomes complicated due to interference by
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patients. 3. Chapuy CI, Nicholson RT, Aguad MD, et al. DARA-DTT Study Group for
the BEST Collaborative International validation of a dithiothreitol
Compared to our method, the TAT of the Osaka method (DTT)-based method to resolve the daratumumab interference with
is 15 min longer. Moreover, our study demonstrates blood compatibility testing. Transfusion 2016; 56: 2964-72.
4. Hosokawa M, Kashiwagi H, Nakayama K, et al. Distinct effects of
that not only K antigen is unaffected by DTT 0.04 mol/L daratumumab on indirect and direct antiglobulina test: a new method
treatment, but also k, Kpb, Lub, Yta, and JMH antigens are employing 0.01 mol/L dithiothreitol for negating the daratumumab
interference with preserving K antigenicity (Osaka method). Transfusion
not denatured. 2018; 58: 3003-13.
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daratumumab interference in transfusion test. N Engl J Med

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test the integrity of other red blood cell antigens because 2018; 379: 90-1.

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interference in pretransfusion testing is overcome by addition of
-Indian and -Raph specificities. Another limitation is that Daratumumab Fab fragments to patient’s plasma. Transfus Med
Hemother 2019;46:423-30.
incubation of DTT 0.04 mol/L along with patient’s sera,
7. Karen N, Chinoca Ziza,Tainá A, et al. A blockage monoclonal antibody
where the final DTT concentration reaches 0.01 mol/L, protocol as an alternative strategy to avoid anti-CD38 interference in

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8. Reid ME, Lomas-Francis C, Olsson ML. The blood groups antigen facts
but this is only a theoretical inconvenience as long as book. 3rd ed. Amsterdam: Elsvier/AP, 2012
compatibility testing is performed in IAT to look for 9. Fung MK, Grossman BJ, Hillyer CD et al. AABB Technical Manual. 18th ed.
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clinically significant, IgG, alloantibodies. 10. Rumsey DH, Cieselski DJ. New protocols in serologic testing: a review
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CONCLUSIONS 2000;16: 131-7.
11. Anani WQ, Marchan MG, Bensing KM, et al. Practical approaches and costs
The new method described here fulfils the needs of for provisioning safe transfusions during anti-CD38 therapy. Transfusion
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many transfusion services in eliminating the anti-CD38 2017; 57:1470-9.

interference while maintaining the ability to detect the


vast majority of blood group alloantibodies. Following
on from the Osaka method, we would like to name the
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method described in this article as the Valencia method.


ACKNOWLEDGEMENTS
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The Authors would like to acknowledge Mrs. Amparo


Bernardez, Mrs. Nuria Martinez and Mrs. Isabel Terrón
for their technical expertise in performing all the tests.
AUTHORSHIP CONTRIBUTIONS
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EC developed the study concept and design, conducted


the study, and also drafted the manuscript. dML and
LL contributed to the revision and correction of the
manuscript. CA is responsible for the critical revision of
the final manuscript.

The Authors declare no conf lict of interests.

Blood Transfus 2020; 18: 290-4 DOI 10.2450/2020.0004-20


294 All rights reserved - For personal use only
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