Blood Bank questions

2015_Oostendrop_Whenbloodtransfusionmedicinebecomescomplicatedduetointerferencebymonoclonalantibodytherapy 2019_Werle_OvercomingDarabyadditionofdaraFABfragmentstopatientplasma 2019_Ye_RiskofRBCalloimmunizationinMMpatientstreatedbyDara 2015_Chapuy_Resolvingthedarainterferencewithbloodcompatibilitytesting

 Focus on the articles by Chapuy, Oostendrop, Hosokawa, Werle and Ye. Because they are all on the same topic, make sure to scan the articles to find similarities and make your reading easier. and answer those questions.  

1) What is daratumumab and what is its action during treatment?.     

2) What are the disadvantages of the daratumumab treatment in blood bank tests? OPTIONAL: In other tests in the clinical lab? 

N E W M E T H O D S A N D A P P R O A C H E S

When blood transfusion medicine becomes complicated due to

interference by monoclonal antibody therapy

Marlies Oostendorp,1 Jeroen J. Lammerts van Bueren,2 Parul Doshi,3 Imran Khan,3

Tahamtan Ahmadi,3 Paul W.H.I. Parren,2,4 Wouter W. van Solinge,1 and Karen M.K. De Vooght1

BACKGROUND: Monoclonal antibodies (MoAbs) are

increasingly integrated in the standard of care. The

notion that therapeutic MoAbs can interfere with clinical

laboratory tests is an emerging concern that requires

immediate recognition and the development of

appropriate solutions. Here, we describe that treatment

of multiple myeloma patients with daratumumab, a novel

anti-CD38 MoAb, resulted in false-positive indirect

antiglobulin tests (IATs) for all patients for 2 to 6 months

after infusion. This precluded the correct identification of

irregular blood group antibodies for patients requiring

blood transfusion.

STUDY DESIGN AND METHODS: The IAT was

performed using three- and 11-donor-cell panels.

Interference of daratumumab and three other anti-CD38

MoAbs was studied using fresh-frozen plasma spiked

with different MoAb concentrations. Additionally it was

tested whether two potentially neutralizing agents, anti-

idiotype antibody and recombinant soluble CD38

(sCD38) extracellular domain, were able to inhibit the

interference.

RESULTS: The CD38 MoAbs caused agglutination in

the IAT in a dose-dependent manner. Addition of an

excess of anti-idiotype antibodies or sCD38 protein to the

test abrogated CD38 MoAb interference and successfully

restored irregular antibody screening and identification.

DISCUSSION: CD38 MoAb therapy causes false-

positive results in the IAT. The reliability of the test could

be restored by adding a neutralizing agent against the

CD38 MoAb to the patient’s plasma. This study

emphasizes that during drug development, targeted

therapeutics should be investigated for potential

interference with laboratory tests. Clinical laboratories

should be informed when patients receive MoAb

treatments and matched laboratory tests to prevent

interference should be employed.

D
rug interference is a well-known phenomenon

in laboratory medicine,
1

but can be different

for each drug and each analytical method. For

many drugs, interference with laboratory tests

is unknown and is often discovered by chance, for exam-

ple, when unexpected laboratory results are found which

cannot be explained by the patient’s condition.

Monoclonal antibodies (MoAbs) represent a novel

class of therapeutics, which are increasingly used in a

variety of pathologic conditions, including solid tumors,

leukemia, infections, and cardiovascular and inflamma-

tory diseases.2 An important advantage of MoAbs is their

specific targeting. Since many laboratory tests are also

based on specific antibody–antigen interactions, possible

MoAb interference in laboratory medicine is considered

an increasing problem. For example, several MoAbs (sil-

tuximab, rituximab, infliximab, cetuximab, trastuzumab,

bevacizumab, adalimumab, and ofatumumab) were previ-

ously shown to generate false-positive results in serum

protein and immunofixation electrophoresis, tests that are

ABBREVIATIONS: MM 5 multiple myeloma; sCD38 5

soluble CD38; VSB 5 veronal saline buffer.

From the
1
Department of Clinical Chemistry and Haematology,

University Medical Center Utrecht, and
2
Genmab, Utrecht,

The Netherlands; the 3Janssen R&D LLC, Spring House

(Ambler), Pennsylvania; and the Department of

Immunohematology and Blood Transfusion,
4
Leiden University

Medical Center, Leiden, The Netherlands.

This study was funded by Genmab.

Address reprint requests to: Karen M.K. de Vooght, Depart-

ment of Clinical Chemistry and Haematology, University Medi-

cal Center Utrecht, P.O. Box 85500, 3508 GA, Utrecht, The

Netherlands; e-mail: k.devooght@umcutrecht.nl.

Received for publication August 9, 2014; revision received

March 25, 2015; and accepted April 5, 2015.

doi:10.1111/trf.13150

VC 2015 AABB

TRANSFUSION 2015;55;1555–1562

Volume 55, June 2015 TRANSFUSION 1555

used for diagnosis and follow-up of patients with multiple

myeloma (MM) or Waldenstr€om’s macroglobulinemia.3,4

In a Phase I and II trial with daratumumab, a novel

IgG1j anti-CD38 MoAb which effectively targets and kills

human MM cells,5-7 we observed an unexpected interfer-

ence in routine laboratory tests used in blood transfusion

medicine. All patients receiving daratumumab showed

false-positive indirect antiglobulin tests (IATs), used for

the detection of irregular blood group antibodies.

Although this might only appear to be a clinical laboratory

problem, the interference seriously complicated the selec-

tion of suitable blood products for transfusion for these

patients and was therefore further investigated. Solutions

to prevent MoAb interference were investigated and impli-

cations for patient safety are discussed.

MATERIALS AND METHODS

Additional methods descriptions are provided in the Sup-

porting Information, available in the online version of this

paper.

Study characteristics

MM patients (single center n 5 11, male/female 5 7/4,

age 58 6 9 years) were enrolled in a Phase I and II safety

and dose escalation study with daratumumab (HuMax-

CD38, Genmab A/S, Copenhagen, Denmark; Clinical Trial

Identifier NCT00574288, http://clinicaltrials.gov/show/

NCT00574288). The clinical trial was approved by the

institutional ethics committee and written informed con-

sent was obtained from all patients.

Patients received a low dose of daratumumab 1 day

before the first full dose of 8 to 16 mg/kg. Three weeks

after the first full dose, patients received another dose.

The next day, 8 to 16 mg/kg daratumumab was given in a

weekly interval for 8 weeks. Peak daratumumab concen-

trations in serum were more than 100 mg/mL for all
patients (range, 110-438 mg/mL). As the multicenter trial is
still ongoing, follow-up times for patients regarding the

data on required blood transfusions are variable.

(In)direct antiglobulin testing

To investigate the ability of daratumumab to induce in

vitro red blood cell (RBC) agglutination, fresh-frozen

plasma (FFP) was spiked with 0.01, 0.1, 1.0, and 10.0 mg/
mL daratumumab. IAT was subsequently performed in

the low-ionic-strength solution (LISS) gel column aggluti-

nation technique with anti-IgG present in the gel matrix,

using a three-cell Surgiscreen panel or an 11-cell Resolve

C panel, both containing 0.8% donor RBC suspensions (all

reagents from Ortho Clinical Diagnostics, Raritan, NJ). As

a control, the IAT was repeated with RBCs from MM

patients not receiving daratumumab. For the direct anti-

globulin test (DAT), a 0.8% suspension of the patient’s

own RBCs was made in LISS diluent (Bio-Rad, Hercules,

CA). This was subsequently tested in the LISS/Coombs gel

column technique (Bio-Rad), containing polyspecific anti-

IgG and anti-C3d within the gel matrix. Autocontrol

experiments were performed in the LISS gel agglutination

column technique, by mixing a 0.8% suspension of the

patient’s own RBCs with the patient’s plasma. All aggluti-

nation strengths were graded from 0 to 41 (0 5 no aggluti-

nation; 0.51 5 very weak agglutination; 11 5 weak

agglutination; 21 5 agglutination; 31 5 strong agglutina-

tion; 41 5 very strong agglutination).

Antibody elution from RBCs

Antibodies were recovered from RBCs

by acid elution

using an elution kit (Gamma Elu Kit II, Immucor Inc.,

Norcross, GA) according to the manufacturer’s instruc-

tions. In brief, RBCs were washed four times with wash

buffer as provided by the manufacturer. Next, washed

RBCs were incubated with the eluate solution for approxi-

mately 30 seconds at room temperature. After centrifuga-

tion and correction of the pH to 6.4 to 7.6, the obtained

eluate was used in an IAT as described earlier.

Agglutination with other CD38 antibodies

To test whether in vitro RBC agglutination is a class-

specific issue, three other CD38 antibodies were pro-

duced: Clones 38SB19, MOR03087, and Ab79. Clones

38SB19 and MOR03087 represent surrogates for the anti-

CD38 SAR650984 (humanized) and MOR202 (human),

respectively. Clone Ab79 is a human anti-CD38 in preclini-

cal development. The heavy- and light-chain sequences of

38SB19, MOR03087, and Ab79 were obtained from patent

applications WO 2008/047242, WO 2012/041800, and WO

2012/092612, respectively, and cloned into mammalian

expression plasmids containing human j and c1 constant
regions. The antibodies were generated by transient trans-

fection in HEK293 cells as described by Vink and col-

leagues.
8

IAT was performed using FFP spiked with 0.01,

0.1, 1.0, and 10.0 mg/mL antibodies, as described above.

Preventing MoAb interference using anti-idiotype

antibodies and soluble CD38

The prevention of anti-CD38 MoAb interference was stud-

ied by repeating the indirect antiglobulin experiment using

FFP spiked with 10.0 mg/mL daratumumab and adding a
neutralizing daratumumab anti-idiotype antibody (see

below) at five and 10 times the daratumumab concentra-

tion. The anti-idiotype antibody was also tested using

plasma of MM patients participating in the current trial

(i.e., daratumumab present in vivo and not added in vitro).

The performance of the anti-idiotype antibody was

further investigated by spiking plasma of a patient

with known irregular antibodies (anti-E and anti-K) with

10 mg/mL daratumumab or the combination of

OOSTENDORP ET AL.

1556 TRANSFUSION Volume 55, June 2015

http://clinicaltrials.gov/show/NCT00574288

http://clinicaltrials.gov/show/NCT00574288

daratumumab and a five- or 10-fold excess of anti-

idiotype antibody. Antibody identification experiments

were subsequently performed using an 11-cell screening

panel.

Recombinant soluble CD38 (sCD38) was investigated

as another potential solution to prevent MoAb interfer-

ence (see below). To this extent, sCD38 was added to

plasma spiked with 10 mg/mL daratumumab or 38SB19 in
10- and 20-fold higher concentrations (concentration dif-

ference with anti-idiotype antibody due to the mono- and

bivalent binding capacity of sCD38 and anti-idiotype,

respectively). Hereafter, standard IATs were performed

using a 3-cell screening panel. The effect of sCD38 was

subsequently evaluated using plasma of a patient with

known anti-K spiked with daratumumab.

Generation of the daratumumab anti-idiotype

antibody

Anti-idiotype antibodies against daratumumab were

generated by BioGenes (Berlin, Germany). Briefly, 8-

week-old female BALB/C mice (Charles River Laborato-

ries, Sulzfeld, Germany) were immunized with daratu-

mumab. After isolation of mouse splenocytes and fusion

with SP2/0 mouse myeloma cells (DSMZ, Braunschweig,

Germany), the resulting hybridomas were tested for

binding to daratumumab by an enzyme-linked immuno-

sorbent assay (ELISA). Binding to the human MoAb

HuMab-KLH, a human IgG1 antibody directed against

mariculture keyhole limpet hemocyanin (KLH), was

used in the ELISA for negative selection.
9

Positive clones

were selected and stable antibody-producing clones

were generated by two rounds of limiting dilution clon-

ing. The generated anti-daratumumab clones were

tested for their potential to block daratumumab binding

to CD38-expressing cells. Anti-idiotype Clone 5-3-9 of

the mouse IgG1j subclass was selected for its potency

to block the interaction between daratumumab and

CD38.

Cloning, expression, and purification of sCD38

The sCD38 was generated by transient transfection in

HEK293 cells as described by de Weers and colleagues.5 A

construct similar to the previously described pEE13.4-

HACD38 was made synthetically and was fully codon

optimized (GeneArt, Regensburg, Germany), replacing the

HA tag encoding part by a His tag (HHHHHH) encoding

part. The construct was cloned in pEE13.4 and named

pEE13.4HisCD38. Plasmid DNA was transiently trans-

fected in HEK293F cells using 293fectin (both Invitrogen,

Carlsbad, CA). Proteins were purified from culture super-

natant by chromatography (BD Talon, BD Biosciences,

Palo Alto, CA), and their appropriate molecular weights

were confirmed by sodium dodecyl sulfate-

polyacrylamide gel electrophoresis.

Antibody-induced complement-dependent

cytotoxicity of human RBCs

The assay was performed with whole blood from three

healthy donors collected in heparin tubes. The number of

RBCs was determined after counting in the presence of try-

pan blue, after which the RBCs were washed with RPMI

1640. Finally, the RBCs were diluted to 1 3 10
8

cells/mL in

veronal saline buffer (VSB11, Lonza, Basel, Switzerland).

Test antibodies were diluted 23 in VSB11, added to RBCs,

and incubated for 30 minutes at 48C. After being washed

twice with VSB11, cells were resuspended in VSB11 and

active or inactivated normal human serum (inactivated for

30 min at 608C) was added. As a control for 100% lysis, water

was added to the RBCs. Cells were incubated for 1 hour at

378C. Subsequently, free hemoglobin (Hb) was measured in

the supernatant using a Hb assay kit (Abnova, Taipei City,

Taiwan) according to the manufacturer’s instructions.

RESULTS

Daratumumab infusion causes positive irregular

antibody screening results

Regular blood group serologic testing (i.e., irregular anti-

body screening) is standard of care for all hematologic

patients in the University Medical Center Utrecht, even

when there is no direct clinical need for transfusion. This

is a precautionary measure to allow quick delivery of RBCs

if requested and is performed using direct and IATs. Before

daratumumab treatment, plasma of all patients showed

negative direct and IATs. However, after MoAb infusion,

positive results with generally 21 reactions strengths were

found for the IAT for all patients, suggesting interference

of daratumumab in the antiglobulin test (see Fig. S1, avail-

able as Supporting Information in the online version of

this paper, for a graphical representation on the mecha-

nism of the MoAb interference). Results remained positive

for 2 to 6 months after the last daratumumab infusion and

were not only observed in the gel column technique, but

also in the tube technique using albumin or polyethylene

glycol (not shown). Interestingly the DAT was negative

after infusion for all patients (n 5 11, multiple tests per

patient), indicating that there are no IgGs bound to the

RBCs of daratumumab-treated patients. In addition, the

IAT autocontrol, which tests the agglutination of the

patients’ plasma with their own RBCs, was also negative

for all patients. This implies that daratumumab present in

the patient’s plasma does not induce agglutination of the

patient’s own RBCs in the IAT. These data were confirmed

by the observation that acid eluates prepared from

daratumumab-treated patients’ RBCs did not show any

agglutination with the patients’ own RBCs as well as donor

RBCs (n 5 11). Taken together, these results suggest a

rapid in vivo clearance of a small RBC fraction to which

daratumumab is bound. This is supported by a minor, but

TRANSFUSION COMPLICATED DUE TO MoAb THERAPY

Volume 55, June 2015 TRANSFUSION 1557

clinically nonsignificant, decrease in Hb levels after infu-

sion and an increase in reticulocyte count (Fig. 1).

The ability of daratumumab to indirectly induce RBC

agglutination in vitro was further investigated by repeating

the IAT using FFP spiked with increasing doses of daratu-

mumab. As shown in Table 1, daratumumab induced RBC

agglutination in a dose-dependent manner. No differences

were found in agglutination patterns when using RBCs

from untreated MM patients (Table 1).

False-positive IATs are class-specific for anti-CD38

MoAbs

IAT was repeated with plasma spiked with the humanized

anti-CD38 MoAb 38SB19 and human anti-CD38 MoAbs

MOR03087 and Ab79. Comparable dose-dependent agglu-

tination patterns were found as described for daratumu-

mab (Table 1), which were of the same magnitude for

38SB19 and somewhat weaker for MOR03087 and Ab79

(Table S1, available as Supporting Information in the

online version of this paper). This indicates that the false

positive IAT is not unique for daratumumab, but is a

class-specific problem for anti-CD38.

Daratumumab can be recovered from donor RBCs

by acid elution

Daratumumab was recovered from donor RBCs incubated

with daratumumab-spiked plasma using acid elution. The

eluate contained sufficient daratumumab to again induce

weak agglutination of donor RBCs in the IAT (Table S2,

available as Supporting Information in the online version

of this paper). The mean daratumumab concentration in

the eluate was 86.8 6 36.7 ng/mL, as measured using an

ELISA. This indicates that daratumumab binding to RBCs

is indeed the cause of RBC agglutination in the IAT.

CD38 shows no age-dependent expression pattern

We investigated whether expression of CD38 depends on

RBC age. Therefore, RBCs were separated into five age

fractions using discontinuous Percoll gradient centrifuga-

tion. All fractions were subsequently used for IAT with

daratumumab-spiked plasma. No differences were

observed in agglutination patterns between the RBC frac-

tions (Table S3, available as Supporting Information in the

Fig. 1. Infusion of daratumumab (dashed vertical lines)

resulted in a small, clinically nonsignificant decrease in Hb

levels (A) and a compensatory rise in reticulocyte count (B).

Adverse events encountered with daratumumab infusion did

not include anemia or hemolysis and patients did not

require blood transfusion.

TABLE 1. RBC agglutination patterns of plasma supplemented with daratumumab (top) and another CD38 antibody
38SB19 (bottom) in the IAT using a three-cell RBC screening panel or RBCs from untreated MM patients

Cell MM patient

Anti-CD38 1 2 3 1 2

Daratumumab (mg/mL)
0.00 – – – – –
0.01 – – – ND ND
0.1 0.51 0.51 0.51 ND ND
1.0 11 11 11 11 21
10 21 21 21 11 21

38SB19 (mg/mL)
0.00 – – – ND ND
0.01 – – – ND ND
0.1 0.51 0.51 0.51 ND ND
1.0 11 11 11 ND ND
10 11 11 11 ND ND

ND 5 not determined.

OOSTENDORP ET AL.

1558 TRANSFUSION Volume 55, June 2015

online version of this paper), although the reticulocyte

fraction could not be clearly evaluated due to the absence

of significant amounts of reticulocytes in healthy adults.

Nevertheless, CD38 expression on RBCs does not appear

to be restricted to certain cellular ages.

Flow cytometry analysis revealed a limited level

of staining by daratumumab of CD38 molecules on

reticulocytes and RBCs (see Fig. S3, available as Support-

ing Information in the online version of this paper), which

corresponds to previously published results.10 It is likely

that CD38 is present on all RBCs, albeit at a different den-

sity per cell. Consequently, only a small number of RBCs

has sufficient CD38 density to allow relevant levels of dar-

atumumab binding, resulting in in vivo clearance or in

vitro interference in the IAT.

Daratumumab-induced RBC depletion is not

caused by complement-mediated lysis

After daratumumab infusion, patients showed a Hb

decrease of approximately 1.6 g/dL (Fig. 1A). In vitro

experiments did not show daratumumab-induced com-

plement-mediated lysis (Fig. 2), suggesting that

complement-mediated lysis is not involved in the clear-

ance of daratumumab-loaded RBCs. We therefore specu-

late that the small daratumumab-loaded RBC fraction

disappears from the circulation by Fc-receptor–mediated

clearance in the spleen.
11

Blocking the interference of CD38 MoAbs in the

IAT

We investigated whether a specific daratumumab anti-

idiotype antibody was able to abrogate daratumumab-

mediated RBC agglutination in the IAT. RBC agglutination

induced by plasma spiked with 10 mg/mL daratumumab
was completely blocked using daratumumab anti-

idiotype antibodies at five- and tenfold excess concentra-

tions (Table 2). The anti-idiotype antibody was also tested

using plasma from MM patients who were treated with

daratumumab. Addition of anti-idiotype antibodies in the

laboratory assay prevented agglutination in the IAT (Table

2). In Fig. S2, available as Supporting Information in the

online version of this paper, a graphical representation of

Fig. 2. Daratumumab-mediated complement-dependent

cytotoxicity (CDC) was evaluated in three different donors.

No significant Hb release was observed when RBCs were

incubated with daratumumab in the presence of 10% active

normal human serum (NHS), indicating that daratumumab

does not induce complement mediated-lysis of RBCs. Anti-P

was used as positive control for CDC lysis. Water was added

to the RBCs as a control for 100% lysis. Results are expressed

as mean 6 SD, n 5 3. (w) Active NHS; (�) inactivated NHS.

TABLE 2. False-positive irregular antibody screening results can be effectively blocked using the daratumumab
anti-idiotype antibody at a five- or 10-fold excess concentration (top). The anti-idiotype antibody also successfully

diminishes positive reactions caused by daratumumab present in plasma of a daratumumab-treated patient (in vivo
daratumumab concentration > 200 mg/mL; middle). sCD38 extracellular domain protein (sCD38) efficiently prevents

the interference of both daratumumab and 38SB19 (bottom)

Cell 1 Cell 2 Cell 3

Plasma 1 10 mg/mL dara 11 11 11
Plasma 1 10 mg/mL dara 1 53 anti-idiotype – – –
Plasma 1 10 mg/mL dara 1 103 anti-idiotype – – –
Plasma 1 10 mg/mL dara, corrected for dilution 11 11 11

Dara patient plasma (>200 mg/mL dara) 21 21 21
Dara patient plasma 1 53 anti-idiotype – – –

Plasma 1 10 mg/mL dara 11 11 11
Plasma 1 10 mg/mL dara 1 103 sCD38 – – –
Plasma 1 10 mg/mL dara 1 203 sCD38 – – –
Plasma 1 10 mg/mL dara, corrected for dilution 11 11 11
Plasma 1 10 mg/mL 38SB19 11 11 11
Plasma 1 10 mg/mL 38SB19 1 103 sCD38 – – –
Plasma 1 10 mg/mL 38SB19 1 203 sCD38 – – –
Plasma 1 10 mg/mL 38SB19, corrected for dilution 11 11 11

TRANSFUSION COMPLICATED DUE TO MoAb THERAPY

Volume 55, June 2015 TRANSFUSION 1559

the mechanism by which anti-idiotype antibodies prevent

daratumumab-induced RBC agglutination is provided.

Next, the performance of the anti-idiotype antibody

was tested using daratumumab-spiked plasma from a

randomly selected subject with known anti-E and anti-K

antibodies. As expected, daratumumab caused agglutina-

tion of all RBC suspensions of the 11-cell identification

panel and the donor’s own RBCs (Table 3). Adding the

anti-idiotype antibody in a fivefold excess concentration

resulted in the original agglutination pattern (i.e., without

daratumumab), typical for the presence of anti-E and

anti-K (Table 3). This indicates that the anti-idiotype anti-

body does not interfere with the binding of clinically rele-

vant irregular antibodies and allows correct irregular

antibody identification.

As an alternative to a daratumumab-specific anti-

idiotype antibody, sCD38 extracellular domain protein

(sCD38) was tested as a generic solution to prevent inter-

ference by anti-CD38 MoAbs. As shown in Table 2, sCD38

can be successfully applied to block interference by dara-

tumumab as well as 38SB19. sCD38 also allowed correct

identification of known irregular antibodies in plasma

spiked with daratumumab (not shown). sCD38 therefore

provides a generic solution to prevent false-positive indi-

rect antiglobulin results caused by anti-CD38 MoAbs.

DISCUSSION

Present findings

MoAbs are a rapidly expanding class of drugs with

increasing clinical applications. The possible interference

of such therapeutics in laboratory testing, however, is

often poorly investigated. Here, we describe that infusion

of the monoclonal anti-CD38 daratumumab causes a

false-positive result in the IAT used in blood transfusion

medicine. We found that a small fraction of RBCs express

a low level of CD38 molecules per cell, which appears

unrelated to RBC age. In all patients, daratumumab infu-

sion resulted in a mild and temporal decrease in Hb,

accompanied by an increase in reticulocyte count, with-

out resulting in clinically relevant anemia (Fig. 1). We

speculate that this decrease in Hb is likely not due to

complement-mediated lysis (Fig. 2), but due to Fc-

receptor–mediated clearance in the spleen.
11

It was fur-

thermore observed that anti-CD38 MoAb interference in

the IAT is not specific for daratumumab, as comparable

dose-dependent interference was also observed for three

additional anti-CD38.

Clinical perspective

The use of daratumumab leads to in vitro RBC agglutina-

tion and thereby to false-positive results in the IAT, which

is used to detect irregular antibodies. Although this may

appear only a clinical laboratory problem, there are

important consequences for blood transfusion medicine,

as the presence of irregular antibodies to clinically rele-

vant blood groups cannot be ruled out using the standard

tests. This concern should be recognized when patients

require a blood transfusion. In Phase I and II trials in

which 10 of 78 MM patients treated with daratumumab

worldwide required transfusion, no major transfusion-

related events were observed. It should be noted that

these transfusions were not directly related to the small

Hb decrease caused by daratumumab, but were due to

the underlying hematologic malignancy or a completely

unrelated condition or therapy (e.g., hip replacement sur-

gery). All patients were required to undergo blood typing

before being treated with daratumumab. In addition,

potential mitigation strategies are under development,

that can be implemented across blood banks globally to

prevent any potential blood transfusion problems, two of

which (i.e., the anti-idiotype antibody and sCD38) are

described in the present work.

Mitigation strategies

Different scenarios on how to cope with MoAb interference

can be envisioned depending on the clinical condition of

the patient. During acute, life-threatening situations non–

cross-matched blood group O D– RBCs can be transfused

as this product is suitable for any combination of the ABO

and D blood types. This strategy, that doesn’t take the

potential presence of alloantibodies into account, is identi-

cal for patients not receiving MoAb therapy. In elective sit-

uations, extensive typing and matching for clinically

TABLE 3. RBC agglutination patterns of an 11-cell identification panel with plasma from a patient with known
irregular antibodies against blood groups E and K and spiked with daratumumab. Cells 3 and 6 of the identification
panel were E1 and Cells 2 and 7 were K1. Adding a fivefold excess daratumumab anti-idiotype antibody recovers

the original agglutination pattern and allows correct identification of the known irregular antibodies

Cell

1 2 3 4 5 6 7 8 9 10 11 Autocontrol

Plasma – 31 31 – – 21 31 – – – – –
Plasma 1 dara 11 31 31 0.51 11 31 31 11 11 11 21 21
Plasma 1 dara 1

anti-idiotype
– 31 31 – – 21 31 – – – – –

OOSTENDORP ET AL.

1560 TRANSFUSION Volume 55, June 2015

relevant blood group antigens (i.e., D, C, c, E, and e and

Kell, Kidd, Duffy, and MNS antigens) can be performed.

Although this strategy prevents mismatching for the most

common blood groups and also prevents development of

irregular antibodies against these blood groups, it has sev-

eral disadvantages. First, it is very time-consuming. Sec-

ond, only a limited number of matching donors will be

available, likely resulting in shortage of compatible blood

products if the blood loss is too extensive. Third, and most

importantly, the presence of other irregular antibodies still

cannot be excluded due the positive cross-matching results

caused by the anti-CD38 MoAb. Although posttransfusion

alloimmunization occurs in only 2% to 3% of the general

population,12 the incidence increases to approximately 9%

in patients with hematologic malignancies.13 Alloantibod-

ies can be directed against any of over 300 different blood

groups and can cause (delayed) hemolytic transfusion

reactions if they remain undetected. This risk can be easily

avoided if the MoAb interference is blocked during labora-

tory testing.

We developed two solutions to overcome the interfer-

ence of anti-CD38 MoAbs in the IAT. First, a specific neu-

tralizing anti-idiotype antibody, a reagent that is usually

generated during drug development programs, can be

employed and, second, the recombinant sCD38 extracel-

lular domain, which could provide a generic solution to

attenuate the interference of anti-CD38. Both approaches

resulted in abrogation of the interference of anti-CD38 in

the IAT, without interfering with irregular antibody detec-

tion. It is therefore recommended that national reference

laboratories for blood transfusion medicine are provided

with these reagents, to allow safe and timely blood trans-

fusion for patients receiving anti-CD38 MoAb therapy. In

addition, patients may carry a blood transfusion card indi-

cating that they receive anti-CD38 MoAb therapy.

MoAb interference in other clinical laboratory tests

The described interference with blood group serologic

testing represents an example of the type of interactions

that may occur between biologics present in the patient’s

serum and specific tests in the clinical laboratory. Such

interference may occur more often with the increasing

use of MoAb therapies and may apply to a wide variety of

laboratory tests, as also previously demonstrated for sev-

eral MoAbs in serum protein and immunofixation electro-

phoresis.3 Specifically, potential interference with

laboratory tests that are based on selective antibody–anti-

gen interactions, similar to the ones targeted by the thera-

peutic molecule, should be investigated during drug

development. A close interaction between the researchers,

clinicians, and clinical laboratory experts is therefore

critical.

In conclusion, the potential interference of MoAb

therapeutics with laboratory tests is considered an

increasing problem due to their increasing clinical use. As

described here, interference of anti-CD38 MoAbs in the

IAT may delay or even prevent the selection of suitable

blood products for transfusion. Proper solutions, like the

two solutions presented in this work, should therefore be

developed in concordance with the development of the

MoAb therapeutic, to allow for convenient and correct

laboratory testing and to ensure patient safety. As these

solutions are to be used for patient follow-up during

MoAb treatment, we suggest an extension of the definition

of companion diagnostics, to also include these specific

reagents which prevent inference of MoAbs in laboratory

testing. Finally, as the type of interference of the therapeu-

tic with clinical laboratory testing is not always predict-

able, it is advised that clinical laboratories should always

be informed when patients are treated with biologics.

ACKNOWLEDGMENTS

The authors thank Gerdien Walbeek and Brigitte van Oirschot

(Laboratory of Clinical Chemistry and Haematology, UMC

Utrecht, The Netherlands) for performing the blood group sero-

logic testing and the Percoll age separation of RBCs, respectively.

MO, JJLvB, PWHIP, WWvS, and KMKdV were responsible for writ-

ing the manuscript; literature search; figure design; study design;

data collection, analysis, and interpretation; and final approval of

manuscript. PD, IK, and TA were responsible for writing the

manuscript, data interpretation, and final approval of

manuscript.

CONFLICT OF INTEREST

MO, WWvS, and KMKdV have disclosed no conflicts of interests.

JJLvB and PWHIP are employees of Genmab, own Genmab war-

rants and/or stock, and are listed as inventors on daratumumab

patent applications owned by Genmab. Genmab funded this

study. PD, IK, and TA are employees of Janssen. Janssen has an

exclusive worldwide daratumumab license and development

agreement from Genmab.

REFERENCES

1. Young D. Effects of drugs on clinical laboratory tests. 5th ed.

Washington, DC: AACC Press; 2000.

2. Ruuls SR, Lammerts van Bueren JJ, van de Winkel JG, et al.

Novel human antibody therapeutics: the age of the Umabs.

Biotechnol J 2008;3:1157-71.

3. Genzen JR, Kawaguchi KR, Furman RR. Detection of a

monoclonal antibody therapy (ofatumumab) by serum pro-

tein and immunofixation electrophoresis. Br J Haematol

2011;155:123-5.

4. McCudden CR, Voorhees PM, Hainsworth SA, et al. Interfer-

ence of monoclonal antibody therapies with serum protein

electrophoresis tests. Clin Chem 2010;56:1897-9.

TRANSFUSION COMPLICATED DUE TO MoAb THERAPY

Volume 55, June 2015 TRANSFUSION 1561

5. de Weers M, Tai YT, van der Veer MS, et al. Daratumumab, a

novel therapeutic human CD38 monoclonal antibody, indu-

ces killing of multiple myeloma and other hematological

tumors. J Immunol 2011;186:1840-8.

6. van der Veer MS, de Weers M, van Kessel B, et al. The thera-

peutic human CD38 antibody daratumumab improves the

anti-myeloma effect of newly emerging multi-drug thera-

pies. Blood Cancer J 2011;1:e41.

7. van der Veer MS, de Weers M, van Kessel B, et al. Towards

effective immunotherapy of myeloma: enhanced elimination

of myeloma cells by combination of lenalidomide with the

human CD38 monoclonal antibody daratumumab. Haema-

tologica 2011;96:284-90.

8. Vink T, Oudshoorn-Dickmann M, Roza M, et al. A simple,

robust and highly efficient transient expression system for

producing antibodies. Methods 2014;65:5-10.

9. Lammerts van Bueren JJ, Bleeker WK, Bøgh HO, et al. Effect

of target dynamics on pharmacokinetics of a novel thera-

peutic antibody against the epidermal growth factor recep-

tor: implications for the mechanisms of action. Cancer Res

2006;66:7630-8.

10. Albeniz I, Demir O, T€urker-Sener L, et al. Erythrocyte CD38

as a prognostic marker in cancer. Hematology 2007;12:409-

14.

11. Frank MM, Schreiber AD, Atkinson JP, et al. NIH conference.

Pathophysiology of immune hemolytic anemia. Ann Intern

Med 1977;87:210-22.

12. Heddle NM, Soutar RL, O’Hoski PL, et al. A prospective

study to determine the frequency and clinical significance of

alloimmunization post-transfusion. Br J Haematol 1995;91:

1000-5.

13. 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 at the publisher’s website:

Table S1. RBC agglutination patterns of fresh frozen

plasma spiked with two other anti-CD38 antibodies

MOR03087 (top) and Ab79 (bottom) in the indirect anti-

globulin test using a 3-cell RBC screening panel.

Table S2. Agglutination strengths of RBC eluates pre-

pared from donor RBCs incubated with daratumumab-

spiked plasma using a 3-cell screening panel. The aver-

age daratumumab concentration in the eluate was

86.8 6 36.7 ng/mL, as measured using an ELISA. No

human IgG was detected in acid eluate control samples

of RBCs that were not incubated with daratumumab.

Table S3. Agglutination patterns of age-separated RBCs

from three healthy donors with daratumumab-spiked

plasma. For Fraction 1, agglutination patterns could not

be determined due to a very low yield, likely caused by

the absence of significant amounts of reticulocytes in

healthy donors.

Fig. S1. Daratumumab (DARA) in the patient’s serum

binds to the test RBCs. After adding the anti-IgG rea-

gent, RBC agglutination is observed, thereby generating

a false positive result. The presence of irregular antibod-

ies is masked by the presence of DARA.

Fig. S2. Daratumumab (DARA) specific anti-idiotype

antibodies are added to the patient’s serum and bind to

daratumumab. If the patient has no irregular antibod-

ies, the anti-idiotype blocks the binding of DARA to

RBCs and no agglutination is observed in the indirect

antiglobulin test. However, if the patient does have

irregular antibodies, the anti-idiotype also specifically

binds DARA and RBC agglutination is solely due to the

presence of irregular blood group antibodies. Inhibition

of DARA binding to RBCs was also obtained using a

sCD38 protein.

Fig. S3. A) Dot-plot of double staining, showing the

erythrocyte population in green (CD235a-FITC1/CD71-

APC-) and the reticulocyte population in red (CD235a-

FITC1/CD71-APC1). B) Histograms showing the PE-

fluorescence for reticulocytes and erythrocytes. Results

indicate that red blood cells express low levels of CD38.

OOSTENDORP ET AL.

1562 TRANSFUSION Volume 55, June 2015

Research Article

Transfus Med Hemother 2019;46:423–430

Daratumumab Interference in Pretransfusion
Testing Is Overcome by Addition of
Daratumumab Fab Fragments to Patients’ Plasma

Egon Werle Josefin Ziebart Eleonora Wasmund Kristin Eske-Pogodda

Institute of Laboratory Diagnostics, Microbiology, and Transfusion Medicine, Dietrich-Bonhoeffer-Klinikum,
Neubrandenburg, Germany

Received: August 24, 2018
Accepted: November 23, 2018
Published online: March 12, 2019

Prof. Dr. med. Egon Werle
Dietrich-Bonhoeffer-Klinkum
Salvador-Allende-Strasse 30
DE–17036 Neubrandenburg (Germany)
E-Mail WerleE @ dbknb.de

© 2019 S. Karger AG, Base

l

E-Mail karger@karger.com
www.karger.com/tmh

DOI: 10.1159/000495773

Keywords
Daratumumab · Pretransfusion testing · CD38 ·
Fab fragments · Interference

Abstract
Background: Daratumumab (DARA), an IgG1κ human
monoclonal anti-CD38 antibody, is used for the treatment
of refractory myeloma for example. Binding of DARA to
CD38 on red blood cells (RBCs), however, leads to panag-
glutination in indirect antiglobulin testing and possibly
masks clinically relevant alloantibodies. Dithiothreitol elim-
inates panreactivity by destroying CD38 but has the draw-
back of modifying certain blood group antigens and, there-
by, impairs the detection of alloantibodies. Methods: DARA
was digested for 16 h at 37 ° C using immobilized papain in
a spin column, centrifuged, and washed, and the DARA-Fab
fragments in pooled flow-throughs were stored at –20 ° C.
DARA-Fab and test cells (ID-DiaCell I-II-III or ID-DiaPanel;
BioRad) were incubated with human plasma spiked with
DARA (plasma concentration up to 1,000 mg/L) or plasma
from patients under DARA therapy at 37 ° C for 15 min.
Thereafter, ID-Cards LISS/Coombs were used. Results: Im-
munofixation electrophoresis showed complete fragmen-
tation of DARA into Fc and Fab fragments by papain prote-
olysis. DARA-Fab efficiently prevented RBC agglutination by
patients’ plasma and by plasma spiked with DARA. More-
over, DARA-Fab did not interfere with the detection of allo-
antibodies. Conclusion: We present a quite easy, reproduc-
ible, and cost-effective method for DARA-Fab fragment

preparation. Blocking CD38 epitopes with DARA-Fab easily
overcomes DARA interference in pretransfusion testing
without affecting alloantibody detection.

© 2019 S. Karger AG, Basel

Introduction

Daratumumab (DARA) is a human monoclonal IgG1κ
antibody used for the treatment of multiple myelomas
and other hematological tumors increasing in frequency
[1]. The antibody binds to CD38 which is expressed on
lymphoid and myeloid cells [2, 3]. Therapeutic monoclo-
nal antibodies may disturb laboratory diagnostics. DARA
interferes, for example, with myeloma cell detection by
flow cytometry [4] and disturbs the detection and quan-
titation of monoclonal proteins by immunofixation elec-
trophoresis (IFE). The latter problem could be solved by
use of a specific anti-DARA antibody [5]. Patients treated
with DARA often develop anemia with progressing dis-
ease or following chemotherapy [1]. Since CD38 is also
expressed at low levels on red blood cells (RBCs), DARA
causes positive reactions in indirect antiglobulin tests
(IATs), e.g., antibody detection (screening) tests, anti-
body identification panels, and antihuman immuno-
globulin crossmatches. Typically, there is no reactivity
of patients’ plasma with patients’ RBCs in antihuman
immunoglobulin (autocontrol) since CD38 expression is
downregulated during treatment, thereby preventing he-
molysis in vivo [6]. To overcome interference of DARA

Werle/Ziebart/Wasmund/Eske-PogoddaTransfus Med Hemother 2019;46:423–430424
DOI: 10.1159/000495773

in alloantibody screening, treatment of test cells with di-
thiothreitol (DTT) is widely used in serological laborato-
ries [7]. DTT denaturates CD38 on the cell surface by re-
ducing disulfide bonds. However, DTT also destroys or
modifies some other blood group antigens, e.g., KEL, DO,
JMH, LU, IN, and YT [8], which results in impaired sen-
sitivity to detect alloantibodies against these blood group
antigens. There are several recommendations on how to
deal with this problem, such as extending RBC phenotyp-
ing to ≥3 months after any recent blood transfusion or
genotyping prior to DARA treatment [9]. Only recently,
a modified method for DTT treatment to reduce the he-
molysis up to 28 or even 33 days of storage was published
[10, 11]. Use of papain-treated cell panels also exhibited
severe limitations [12]. An alternative approach is block-
ing the binding site of DARA with soluble CD38 peptides
[13], which, however, is too expensive for routine testing.
Blocking of the antigen binding site of DARA by incuba-
tion of patients’ plasma with anti-idiotypic antibodies
may also be intriguing; however, these antibodies are not
commercially available [14]. In addition, umbilical cord
RBCs as screening cells are not typically available in a rou-
tine transfusion laboratory, and, furthermore, cord cells
may have altered expression of some antigens [9]. In a
recently published case report, a commercially available
product was used to prevent DARA interference in IAT
[15]. In summary, pretreatment of RBCs with DTT is still
performed in most laboratories as recommended by
Chapuy et al. [7] in 2015, and it is widely used despite the
above-mentioned drawbacks until today.

In summer 2017, we started experiments and devel-
oped a novel and simple method to overcome DARA in-
terference by blocking the CD38 epitopes on RBCs by
DARA-Fab fragments (DARA-Fab) which were gener-
ated by papain proteolysis of DARA antibodies. This
method was validated since autumn 2017 in our labora-
tory in parallel to the DTT treatment. Simultaneous incu-
bation of patients’ plasma and DARA-Fab with screening
cells prevented panagglutination in pretransfusion test-
ing without affecting the detectability of all alloantibodies
tested. This procedure is cost-effective and very suitable
for routine serological testing.

Materials and Methods

Effect of DARA on IAT
The agglutination potential of DARA in IAT was demonstrated

by incubation of 50 µL 0.8% ID-DiaCell I-II-III screening cells
(BioRad, Hercules, CA, USA) with 25 µL standard human plasma
(SHP; Siemens, Erlangen, Germany) spiked with DARA (Dar-
zalex®; Janssen, Beerse, Belgium). DARA plasma concentrations
(ranging from 200 mg/L to 1.5 µg/L) were prepared by serial dilu-
tion of 200 mg/L DARA in SHP. IAT was performed using ID-
Cards LISS/Coombs (BioRad) containing polyspecific anti-IgG
and anti-C3d antibodies within the gel matrix. The ID-Cards were

incubated at 37  ° C for 15 min in an ID-incubator 37 SI (BioRad)
and afterwards centrifuged in an ID-centrifuge 12 SII (BioRad) for
10 min at 1,030 rpm. Agglutination of RBCs was evaluated by as-
signing the numbers 0 (no agglutination), 0.5+ (very weak agglu-
tination), 1+ (weak agglutination), 2+ (moderate agglutination),
3+ (strong agglutination), and 4+ (very strong agglutination).

IgG Subclass Measurement before and after DARA
Administration
IgG1, IgG2, IgG3, and IgG4 levels before and 1 day after

Darzalex® administration were measured in plasma samples with
latex-enhanced immunonephelometry (BN ProSpec analyzer, N
AS IgG1/2/3/4 reagents; Siemens Healthcare Diagnostics GmbH,
Eschborn, Germany).

DARA-Fab Preparation
Fab fragments were generated from Darzalex® using the

PierceTM Fab preparation kit (No. 44985; Thermo Scientific,
Waltham, MA, USA) according to manufacturer’s instructions
with minor modifications. Briefly, 250 µL immobilized papain solu-
tion were placed into a 0.8-mL spin column, and buffer was dis-
carded by centrifugation at 4,125 g for 2 min. Digestion buffer was
prepared directly before use by dissolving 3.5 mg cysteine HCl in
Fab digestion buffer. Resin was washed with 0.5 mL digestion buffer
by centrifugation of columns at 4,125 g for 2 min. Subsequently, 100
µL of DARA (2 mg) and 400 µL digestion buffer were mixed and
added to the spin column. Antibodies were digested for 16 h at
37   ° C by constantly inverting the recapped spin column. DARA-
Fab were extracted by centrifugation at 4,125 g for 2 min. Resin was
washed with 100 µL phosphate-buffered saline (PBS; pH 7.2), and
pooled flow-throughs were stored at –20   ° C. Digestion was con-
trolled by IFE using a Hydrasys 2 scan with the Hydragel 4 IF kit
(Sebia, Évry, France). Native DARA (20 g/L) diluted 1: 20 and un-
diluted DARA-Fab solution were mixed 1: 2 with Hydragel IF dilu-
ent, and 10 µL of the dilution were applied to the gel per track. For
immunofixation, anti-γ heavy-chain and anti-κ light-chain antisera
were used for both native and digested DARA, and staining was
done with IF acid violet (Sebia). DARA-Fab purification with the
NAbTM Protein A Plus Spin Column of the PierceTM Fab preparation
kit was done once but omitted thereafter as discussed later on.

DARA-Fab Testing
To test the efficiency of DARA-Fab to mask CD38 on RBCs, 15

µL of DARA-Fab and 50 µL of 0.8% ID-DiaCell I-II-III screening
cells or the ID-DiaPanel (a set of 11 panel cells) for antibody iden-
tification (BioRad) were incubated simultaneously with 25 µL SHP
spiked with DARA (concentrations of 100, 250, 500, or 1,000 mg/L
in SHP) or with 25 µL of plasma from DARA-treated patients (n =
8, age: 71 ± 7 years). Incubation was done in glass tubes in a 37  ° C
water bath for 15 min. Cells were agitated several times to prevent
pelleting of erythrocytes and to facilitate the binding DARA-Fab
to CD38 epitopes. A whole cell suspension was transferred to the
microcolumn of an ID-Card LISS/Coombs and centrifuged for 10
min at 1,030 rpm. Agglutination of RBCs was evaluated by assign-
ing the numbers 0–4+ as described above. In case of incomplete
inhibition of DARA binding to test cells, the DARA-Fab volume
was increased to 30 µL.

Adding 30 µL DARA-Fab significantly reduces the DARA con-
centration and may contribute to the negative results. Therefore,
we also tested a modified pipetting scheme: 50 µL 1.4% RBCs, 40
µL SHP, 5 µL DARA (3.5 g/L), and 30 µL DARA-Fab. This mixture
results in nearly identical relative RBC and DARA concentrations
in a final volume of 125 µL as recommended in the validated in-
structions from BioRad (25 µL patients’ plasma and 50 µL 0.8%
RBC solution).

Daratumumab Fab Fragment Preparation
for Pretransfusion Testing

425Transfus Med Hemother 2019;46:423–430
DOI: 10.1159/000495773

Flow Cytometry
Flow cytometry was performed with ID-DiaPanel cells in order

to be able to refer the extent of the reactivity of DARA-spiked SHP
in IAT to the expression of CD38 on the cell surface of RBCs. Test
cells (106/tube) were stained in PBS containing 2.5% fetal calf se-
rum in a total volume of 50 µL using mouse antihuman CD38
phycoerythrin-Texas red (ECD, A99022; Beckman Coulter, Brea,
CA, USA). As isotype control, cells were incubated with mouse
IgG1 ECD (A07797; Beckman Coulter) in a separate tube. Incuba-
tion with antibodies was performed at room temperature for 30
min protected from light. Afterwards, cells were washed twice and
resuspended in 200 µL BD CellWash. Measurement of a total
of 1 × 105 cells was performed with a NaviosTM 10-color flow cy-
tometer (Beckman Coulter). Evaluation of flow-cytometric data
was performed with Kaluza® software (version 1.5a; Beckman
Coulter). The differences between the median fluorescence inten-
sity (MFI) of anti-CD38-labeled RBCs and the MFI of the isotype
control were calculated and compared to the reaction strength of
DARA-spiked SHP in IAT in these panel cells.

CD38 density has been described for CD8+ lymphocytes [16]
but not for RBCs. Therefore, we stained CD38 on RBCs and on
CD8+ lymphocytes of healthy patients (n = 6; age 41 ± 13 years) to
estimate the CD38 density on RBCs by calculating the anti-CD38
MFI (CD8+) quotient and the anti-CD38 MFI (RBC) quotient and
referring the quotient to the number of CD38 on CD8+ lympho-
cytes described in the literature. Whole blood (200 µL) was incu-
bated with mouse antihuman CD8 FITC (A07756; Beckman Coul-
ter) and mouse antihuman CD38 ECD or mouse IgG1 ECD at
room temperature for 30 min protected from light. Cells were
washed twice and resuspended in 200 µL BD CellWash. For analy-
sis of CD38 on RBCs, 10 µL of the cell suspension were diluted 1:
20 in CellWash. The residual volume was treated with lysing solu-
tion (VersaLyse, A09777; Beckman Coulter) for 10 min at room
temperature, washed once, and resuspended in 200 µL CellWash
for measurement of CD8+ cells.

Irregular Antibody Screening
Fifty microliters of ID-DiaCell I-II-III cells and ID-DiaPanel

(BioRad) cells were incubated with 25 µL plasma from patients
with an irregular alloantibody (anti-E, anti-K, anti-c, anti-D, anti-
M, and anti-Fy[a]). Moreover, these native plasma samples were
tested in parallel after spiking with DARA (20 g/L) to give a final
DARA concentration of 500 mg/L in the patients’ plasma. Finally,
the patients’ plasma samples, spiked with DARA, were incubated
with DARA-Fab (15 µL). Thereby, the ability of DARA-Fab to pre-
vent panreactivity without altering the reaction pattern and reac-
tion strength of the alloantibodies was evaluated.

In addition, we tested the possible influence of DARA-Fab on
the sensitivity to detect alloantibodies with commercially available
test sera using the Data-Cyte® Plus panel (Grifols, Frankfurt, Ger-
many).

For these experiments, we used a modified pipetting scheme to
exclude that the dilution of RBCs and alloantibodies by the addi-
tion of an increased volume of DARA-Fab (15 or 30 µL) may influ-
ence the detection limit of alloantibodies. Adjustments were draft-
ed to ensure a concentration of plasma which may contain alloan-
tibodies and a concentration of RBC in the final volume identical
to the original pipetting scheme. This was realized by an increased
final volume.

We used 50 µL Grifols panel cells for antibody identification
adjusted to 1.4%, 40 µL test serum, 5 µL DARA (3.5 g/L) diluted in
SHP (equal to 500 mg/L DARA concentration in patients’ plasma),
and 15 µL DARA-Fab. SHP was added to give a final volume of 125
µL. In a few cases, we had to add 30 µL DARA-Fab in a final volume
of 125 µL (without SHP). We tested the reaction pattern and

strength of the test sera with or without addition of DARA and
with or without addition of DARA-Fab.

Test sera were diluted with SHP in order to give a reactivity
strength of about 2+. The following antibodies were used (distrib-
utor, lot number, sell-by date, dilution): anti-Jk(a) (SD Nostik,
1Ja043, 05-2019, 1: 8), anti-Jk(b) (Biolith, 504k, 10-2019, 1: 3), anti-
Fy(a) (Optima, T04616, 03-2019, 1: 30), anti-Fy(b) (SD Nostik,
2Fb004, 07-2019, 1: 30), anti-S (Optima, P26316, 06-2019, 1: 30),
anti-s (Optima, F06441, 06-2019, 1: 30), anti-Le(a) (Optima,
S06771, 06-2019, 1: 8), anti-Lu(a) (Biolith, 507S, 12-2018, 1: 5), an-
ti-Kp(a) (SD Nostik, 2Ra026, 07-2019, 1: 10), anti-C (Biolith,
302kMS24, 05-2018, 1: 1,000), anti-e (Optima, H93641, MS-16/
-21/-63, 1: 500), and anti-C(w) (Biolith, 204K, 01-2019, 1: 5).

Results

Measuring the IgG1 serum concentration by nephe-
lometry 1 day after administration of 16 mg/kg Darzalex
resulted in a relative increase of about 200–400 mg/L IgG1
as expected [17].

SHP, spiked with 200 mg/L DARA, was serially diluted
to ascertain the minimum DARA concentration neces-
sary to induce agglutination of erythrocytes in IAT
(Fig.  1). Up to 97.7 µg/L DARA induced a 2+ reaction.
Diminution of the reaction was first seen with 48.8 µg/L.
Complete loss of RBC agglutination was shown at a
DARA concentration of 6.1 µg/L.

Papain proteolysis of 2 mg DARA for 16 h at 37  ° C un-
der continuous inverting was very effective. IFE of native
and papain-digested DARA antibodies shows complete
fragmentation of DARA into Fc and Fab fragments de-
tected by anti-γ heavy-chain and anti-κ light-chain anti-
serum, respectively (Fig. 2).

Fig. 1. Serial dilution of 200 mg/L DARA in standard human
plasma demonstrates capability of DARA to agglutinate screening
cells in indirect antiglobulin testing down to a concentration of
12.2 µg/L.
Fig. 2. Immunofixation electrophoresis of native and papain-di-
gested DARA. The gel shows complete digestion of the antibodies
resulting in the fragmentation into the Fc region detected by anti-γ
heavy-chain antiserum (G) and DARA-Fab fragments detected by
anti-κ light-chain antiserum (K).

1 2

Werle/Ziebart/Wasmund/Eske-PogoddaTransfus Med Hemother 2019;46:423–430426
DOI: 10.1159/000495773

The protocol allowed the reproducible generation of
DARA-Fab fragments, ready to use for incubation with
test cells without further purification steps. According to
our experience, elimination of Fc fragments by absorp-
tion to protein A should not have a measurable effect on
DARA-Fab results in IAT. DARA-Fab activity was main-
tained after storage at –20   ° C for several months. Spin
columns containing immobilized papain can be stored in
PBS at 4  ° C for several weeks without decreasing efficien-
cy of Fab generation when columns are washed with
digestion buffer prior to new digestion. The complete
DARA fragmentation was verified after each DARA-Fab
production by IFE.

Efficiency of CD38 masking by DARA-Fab fragments
was first tested exemplarily in a few screening cells incu-

bated with SHP spiked with DARA obtaining the follow-
ing final concentrations: 100, 250, 500, and 1,000 mg/L
(Fig. 3); 15 µL DARA-Fab were sufficient to prevent bind-
ing of DARA to these cells and RBC agglutination at all
DARA concentrations.

Furthermore, all 3 ID-DiaCell I-II-III screening cells
were incubated with the plasma of the 8 patients receiving
DARA therapy. Simultaneous incubation of cells with 15
µL Fab resulted in overriding of DARA-induced panag-
glutination. Representative results of 3 patients are shown
in Figure 4.

A set of 11 ID-DiaPanel cells for antibody identifica-
tion (BioRad) was incubated with SHP spiked with DARA
(500 mg/L) with or without simultaneous addition of 15
or 30 µL DARA-Fab and, thereafter, applied to ID-Cards

10
0

m
g/

l

25
0

m
g/
l

50
0

m
g/
l

w/o Fab

2+ 2+ 2+

10
0
m
g/
l
25
0
m
g/
l
50
0
m
g/
l

+ Fab

0 00

10
00

m
g/

l
10
00
m
g/
l

+ Fab w/o Fab

0 2+

Fig. 3. Incubation of screening cells with
100, 250, 500, and 1,000 mg/L DARA in
standard human plasma resulted in a
strong agglutination of erythrocytes. Si-
multaneous incubation of screening cells
with 15 µL DARA-Fab with DARA inhib-
ited the agglutination reaction efficiently.
w/o, without.

Fig. 4. Plasma of myeloma patients treated
with DARA resulted in agglutination of
screening cells (I, II, III). Simultaneous
incubation of screening cells with 15 µL
DARA-Fab inhibited the agglutination re-
action efficiently. In patients 1, 2, and 3,
blood was drawn 7, 0, and 2 weeks after the
last DARA administration and after 15, 13,
and 1 full DARA administrations, respec-
tively.

Daratumumab Fab Fragment Preparation
for Pretransfusion Testing

427Transfus Med Hemother 2019;46:423–430
DOI: 10.1159/000495773

LISS/Coombs and centrifuged (Fig. 5). Two panel cells (3
and 8) did not react with DARA. Very low expression of
CD38 on these cells was confirmed by flow cytometry
(MFI 22 ± 25). ID-DiaPanel cells 4–6, 10, and 11 showed
1+ or 2+ reactions, which could be prevented by addition
of 15 µL DARA-Fab solution. The agglutination of the
ID-DiaPanel cells 1, 2, 7, and 9, which all exhibited 2+
reactions, were strongly reduced showing only very slight
RBC agglutination (0.5+ reaction or below). For these
cells, the experiment was repeated using 30 µL DARA-
Fab solution resulting in complete prevention of aggluti-
nation. Despite different reactivity of identification cells
to DARA, flow cytometry showed similar MFI for cells
exhibiting 1+ and 2+ reactions (103 ± 20 and 105 ± 10,
respectively).

To estimate CD38 density on RBCs, we stained CD38
on RBCs and on CD8+ cells in blood samples of healthy
individuals and compared the MFI of anti-CD38 on
both cell types. CD8+ cells showed an anti-CD38 MFI of
1,643 ± 969 while RBCs exhibited an anti-CD38 MFI of
139 ± 31. Therefore, we hypothesized that the CD38 den-
sity on RBSs is about 10 times lower than on CD8+ cells.

Incubation of test cells with DARA-Fab fragments pre-
vented panagglutination of screening cells and did not in-
terfere with the detection of alloantibodies or result in
false-positive results in negative screening cells. This is
shown with patients’ plasma containing anti-K and anti-E
antibodies which were additionally spiked to give a final
plasma concentration of 500 mg/L DARA (Fig.  6). The
same was true for patients’ plasma containing alloanti-

Fig. 5. Comparison of the CD38 expression and the respective isotype control on ID-DiaPanel cells shows low
CD38 expression with varying density on RBCs (a). Indirect antiglobulin test of ID-DiaPanel cells incubated with
500 mg/L DARA in standard human plasma (w/o Fab) and panel cells incubated simultaneously with 500 mg/L
DARA and 15 or 30 µL DARA-Fab (b). DARA-Fab prevents DARA-induced agglutination of erythrocytes in a
dose-dependent manner.

Werle/Ziebart/Wasmund/Eske-PogoddaTransfus Med Hemother 2019;46:423–430428
DOI: 10.1159/000495773

bodies directed against the blood group antigens c, D, M,
and Fy(a) (data not shown).

We further tested alloantibody detection using a mod-
ified pipetting scheme to guarantee that the detection
sensitivity for alloantibodies is not at all impaired by the
addition of DARA-Fab and/or DARA spike solution.
The detection limit of alloantibodies is expected to be
unchanged because the concentration of plasma which
may contain alloantibodies and the RBC concentration
are identical despite an increased final volume. We found
no significant differences in the reaction strength of al-
loantibodies when we compared the results without or
with the simultaneous addition of DARA spike solution
and DARA-Fab. In detail, DARA-Fab did not reduce the
reaction strength of these test sera: Jk(a), Jk(b), Fy(a),
Fy(b), S, s, Le(a), Lu(a), Kp(a), C, e, and C(w). We also
compared this modified pipetting scheme with the
“standard” scheme (50 µL RBC 0.8%, 25 µL plasma ±
DARA, ± 15 µL DARA-Fab) and also found no signifi-
cant effect of plasma dilution on the reaction strength as
shown in Figure 6.

Discussion

DARA, a monoclonal IgG1 type κ antibody directed
against CD38, successfully depletes CD38-expressing
myeloma cells. RBCs physiologically express low levels of
CD38, which result in panagglutination of RBCs in sero-
logical testing and, thereby, complicate alloantibody de-
tection and compatibility testing [17]. Erythrocytes in
subjects receiving DARA treatment are supposed to be
protected from hemolysis by downregulation of CD38
which manifests in a negative direct antiglobulin testing
and a negative autocontrol in these patients. A slight de-
crease in hemoglobin levels, however, has been suggested
[1, 13].

The DARA-Fab preparation described in the present
paper is quite easy and requires no special equipment.
Purification, i.e., elimination of Fc fragments by use of
protein A, is not necessary because Fc fragments do not
interfere with DARA and DARA-Fab binding to RBCs
during the incubation. Moreover, when adding the mix-
ture of RBCs, DARA, and DARA-Fab onto the microcol-
umns of the gel card, the low amount of Fc fragments as
compared to the patients’ plasma IgG concentration is
not expected to disturb the detection of RBC-bound an-
tibodies by antihuman immunoglobulin.

We decided to use papain proteolysis resulting in the
cleavage of DARA into 1 Fc fragment and 2 Fab frag-
ments as opposed to pepsin treatment which results in the
generation of 1 Fc and 1 F(ab)2 fragment. Papain diges-
tion was preferred because 2 Fab fragments should mask
more CD38 antigens than 1 F(ab)2 fragment. In addition,
we supposed that the smaller Fab molecules would better
bind to CD38 than the larger F(ab)2 fragment. Moreover,
antibodies against light chains in the ID-Card LISS/
Coombs we use in routine diagnostics may bind better to
F(ab)2 fragments than to Fab fragments, thereby imitat-
ing the presence of an alloantibody. We suspected that
Fab fragments would be less prone to this unfavorable
interference.

Flow cytometry was used to analyze whether there
might be an individual-specific percentage of RBCs with
higher expression of CD38 prone to antibody-mediated
degradation and which also might be responsible for the
positive IAT. We wanted to exclude that young RBCs, for
example, might express high levels of CD38 while CD38
is downregulated in older RBCs. However, the scatter
plots demonstrated a homogenous expression of CD38
on RBCs of a certain patient or certain donor of test cells.
In addition, these investigations demonstrated different
CD38 densities on the surface of RBCs between different
patients’ RBCs or ID panel cells.

In a few panel cells, CD38 density was below the detec-
tion limit of flow cytometry, and this was associated with
a missing reaction in IAT. Cells with weakly positive IAT

Fig. 6. Reactivity of screening cells with plasma from patients with
an anti-E or anti-K alloantibody without or spiked with 500 mg/L
DARA. Lower figures show screening cells incubated with 15 µL
DARA-Fab and patient’s plasma containing an anti-E or anti-K
alloantibody and 500 mg/L DARA. DARA-Fab incubation does
not interfere with recognition of alloantibodies.

Daratumumab Fab Fragment Preparation
for Pretransfusion Testing

429Transfus Med Hemother 2019;46:423–430
DOI: 10.1159/000495773

showed also a low MFI in flow cytometry arguing for a very
low antigen density. An MFI of about 100 was associated
with a reaction strength of 2+. The mean number of CD38
molecules per CD8+ T lymphocyte in blood was reported
to be about 2,000 in healthy subjects [16]. Considering the
comparison of MFI values between CD8+ lymphocytes
and erythrocytes in our analyses, we estimated the CD38
density to be about 200 molecules per RBC. To sum up,
flow cytometry experiments showed a homogenous ex-
pression of CD38 with varying density on patients’ and
antibody identification cells, and it showed a correlation of
CD38 density to the reactivity of test cells in IAT.

Elimination half-life of DARA averages at 110 ± 42 h
after the first full dose and 587 ± 487 h in case of admin-
istration of 16 mg/kg DARA after the seventh last infu-
sion [17]. Therefore, panreactivity can persist up to sev-
eral weeks after the last DARA application depending on
the doses administered, antibody adsorption on blood
cells expressing CD38, absorption in the tissue, and the
number of blood transfusions a patient has received dur-
ing or after DARA therapy. Persistence of DARA anti-
bodies was demonstrated with plasma from a patient that
induced strong 2+ reactions in screening cells 7 weeks
after the last DARA administration (Fig. 4).

Serial dilution of DARA in SHP revealed agglutination
of RBCs when a concentration as low as 12.2 µg/L DARA
was added to screening cells. Strong 2+ reactions were
visible for concentrations ≥97.7 µg/L. No agglutination
of RBCs was observed at a DARA concentration of 6.1
µg/L (Fig. 1). This is in line with the findings of Oosten-
dorp et al. [13] who reported no agglutination when ap-
plying DARA at a concentration ≤10 µg/L. These data
and further pharmacodynamic data [17] explain the per-
sistence of DARA interference several weeks after drug
administration (Fig. 4).

We started Fab testing using plasma concentrations of
100, 250, and 500 mg/L DARA since 214 and 575 mg/L
are described as mean predose serum concentrations at
the end of weekly dosing after administration of 8 and 16
mg/kg DARA, respectively [17]. Our nephelometric mea-
surements directly before DARA application (16 mg/kg)
and on the next day showed an exclusive increase in IgG1
concentration ranging from 200 to 400 mg/L. However,
higher peak serum concentrations of 426–993 mg/L can
occur directly after administration. Therefore, we addi-
tionally tested DARA-Fab with plasma containing a final
concentration of 1,000 mg/L DARA. As expected from
prior dilution series, all 4 concentrations resulted in sim-
ilar 2+ reactions without treatment and could be abol-
ished completely when cells were simultaneously incu-
bated with DARA-Fab fragments in IAT. Most experi-
ments presented here were performed using 500 mg/L to
show DARA-Fab efficiency in case of high mean predose
DARA plasma concentrations.

Prevention of RBC agglutination by DARA-Fab
(Fig.  3–6) also demonstrates that DARA-Fab fragments
which displace DARA from CD38 in a competitive way
do not lead to RBC agglutination by interaction with an-
tihuman globulin in the microcolumn gel matrix. The ID-
Card LISS/Coombs contain polyspecific IgG antisera
which means that antibodies against κ and λ light chains
should be present in the microcolumn gel and might lead
to RBC agglutination by binding to DARA-Fab. Howev-
er, the anti-light chain activity in IAT was shown to be too
low to cause a false-positive RBC agglutination (Fig. 3).

The main disadvantage of DTT treatment of test
cells, which is the current standard procedure in case of
DARA-induced panagglutination, is the destruction or
modification of other blood group antigens. Therefore,
alloantibodies against these blood groups might be over-
looked. In contrast, DARA-Fab treatment does not
interfere with antibody screening or identification as
demonstrated with plasma from patients containing anti-
E and anti-K alloantibodies (Fig.  6). Spiking of plasma
with DARA induced panagglutination of screening cells,
which could be prevented by the addition of DARA-Fab
to the incubation tube.

Agglutination of RBCs was visible down to a very low
antibody concentration of 12.2 µg/L DARA in SHP on
ID-Cards LISS/Coombs because the sensitivity of ID-
Cards LISS/Coombs is very high. Only 100–500 bound
IgG molecules per cell are sufficient to induce an aggluti-
nation in IAT.

Using 50 µL of 0.8% erythrocyte solution (0.08 ×
1012/L) and 25 µL plasma, spiked with 12.2 µg/L DARA
(molecular weight 148 kDa), about 300 DARA antibodies
per RBC are incubated enabling visible agglutination. In
healthy individuals, the expression of about 2,000 CD38
molecules per CD8+ T lymphocyte has been described
[16]. On RBCs, CD38 is expressed at much lower levels.
Our experiments using flow cytometry showed a factor of
about 10 so that one could assume a number of about 200
CD38 molecules per RBC. We digested 2 mg DARA col-
lecting the DARA-Fab in 600 µL buffer. Assuming a
DARA-Fab yield of > 50%, we use DARA-Fab in 4- to
8-fold excess to the number of DARA antibodies when
adding 15 µL DARA-Fab to erythrocytes and 25 µL 500
mg/L DARA. Regarding the excess of DARA antibodies
(1.3 × 107 antibodies/cell) and DARA-Fab fragments
(> 5 × 107 DARA-Fab/cell) used, presumably all CD38
molecules will bind one of these molecules with similar
binding affinities in a competitive manner. If < 100 CD38 antigens per cell are bound by DARA, the ID-Card LISS/ Coombs system will not be able to detect these few bound antibodies as mentioned above. Therefore, 15 µL are suf- ficient to prevent agglutination in most cases. In case of persisting low agglutination reactions (< 1+), increasing the ratio of DARA-Fab fragments to DARA antibodies by

Werle/Ziebart/Wasmund/Eske-PogoddaTransfus Med Hemother 2019;46:423–430430
DOI: 10.1159/000495773

increasing DARA-Fab volume to 30 µL helps to override
DARA-induced agglutination and enables identification
of even a weak alloantibody reaction. In addition to these
considerations, we used a modified pipetting scheme
which considers the higher dilution of test cells and
DARA antibodies by an increased DARA-Fab volume.
However, also these experiments with various alloanti-
bodies showed no detectable impairment of the detection
strength of alloantibodies by DARA-Fab. The competi-
tive mode of action of DARA-Fab is in accordance with
these findings.

Monoclonal antibodies will be of increasing impor-
tance for therapy in oncoming years, and it might be that
these antibodies under development also may interact
with routine blood compatibility testing. Murphy et al.
[18] strongly recommended that one may pay attention
to a possible effect of new therapeutics on serological test-

ing during drug development or phase 1 studies. In case
of monoclonal antibodies with reactivity against RBCs,
the procedure proposed in the present paper might also
be appropriate.

In summary, this investigation describes a cost-effi-
cient and easy-to-use method for Fab preparation. More-
over, the study demonstrates that Fab fragments may
override complications in antibody screening and identi-
fication by therapeutic monoclonal antibodies reacting
with RBCs in pretransfusion testing without any negative
effect on alloantibody detection.

Disclosure Statement

The authors declare that they have no competing interests.

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Vox Sanguinis (2020) 115,

207

–212

ORIGINAL PAPER © 2019 International Society of Blood TransfusionDOI: 10.1111/vox.12864

Risk of RBC alloimmunization in multiple myeloma patients
treated by Daratumumab
Zhan Ye, Laurie A. Wolf, Daniel Mettman & Fred V. Plapp
University of Kansas Medical Center, Kansas City, Kansas, USA

Received: 29 March 2019,
revised 11 October 2019,
accepted 21 October 2019,
published online 14 November 2019

Background Daratumumab (DARA) is a human monoclonal antibody for the
treatment of multiple myeloma (MM). DARA binds to CD38 on RBCs and inter-
feres with detection of RBC alloantibodies. The objective of this study was to
evaluate the risk of RBC alloimmunization in MM patients treated with DARA.

Materials and methods A retrospective study of the complete serological profile
and transfusion history of 45 MM patients received transfusion and treated with
DARA from July 2015 to December 2018 was undertaken. All cases with positive
Ab screens were treated with DTT to identify RBC alloantibodies.

RBC

transfusion

history was monitored between the first DARA dose to the last or extending to
the first negative Ab screen after the last DARA dose if the Ab screen was ever
positive. Forty-six MM patients received transfusion but not DARA were studied
as control group.

Results Totally 184 Ab screens were done on 45 patients transfused with ABO-
Rh compatible RBCs, phenotypically matched units or both. None of them
showed detectable alloantibodies after DTT treatment. The duration of Ab screen-
ing positivity varied markedly, ranging from 25 days to 5 months after the last
dose. Two of 46 patients in the control group had preexisting alloantibodies but
no new alloantibodies were detected during study period.

Conclusions Our results indicate that the risk of forming new RBC alloantibodies
after transfusion in MM patients treated with current regimens is very low and
no DARA-associated difference in the alloimmunization risk. No significant dif-
ference in alloimmunization is detected between ABO-Rh compatible and pheno-
typically matched transfusion.

Key words: RBC antigens and antibodies, Serological testing, Transfusion medi-
cine, Immunohaematology.

Introduction

Daratumumab (DARA) is a human immunoglobulin (Ig) G1

monoclonal antibody (Ab) that recognizes highly expressed

CD38 on multiple myeloma (MM) cells [1]. In November

2015, the US Food and Drug Administration (FDA)

approved DARA as monotherapy for relapsed/refractory

MM patients who had already received three previous treat-

ments. One year later, DARA received additional FDA

approval as combination therapy with lenalidomide or

bortezomib and dexamethasone for MM patients who had

received at least one prior therapy [2–4].

DARA in patients’ plasma also binds weakly expressed

CD38 on reagent human red blood cells (RBC). Although

it does not affect ABO/Rh typing [5], DARA causes posi-

tivity of indirect antiglobulin tests (IAT) performed at

37°C. During phase I and II trials of DARA, plasma of all
DARA-treated patients demonstrated weak (1+) panreac-
tivity with all RBC panels using anti-human globulin (gel,

tube and solid phase), for up to 6 months [5,6].

Many methods have been developed to negate

DARA

interference including destruction of CD38 on reagent

RBC by dithiothreitol (DTT) or proteolytic enzymes such

as trypsin and papain [5,7]; masking CD38 with F(ab’)2

fragments of DARA [8]; using CD38-negative reagent

Correspondence: Zhan Ye, Department of Pathology and Laboratory
Medicine, University of Kansas Medical Center, 4000 Cambridge Street,
Kansas City, Kansas 66160, USA.
E-mail: zye2@kumc.edu

207

https://orcid.org/0000-0003-2378-3813

https://orcid.org/0000-0003-2378-3813

https://orcid.org/0000-0003-2378-3813

mailto:

RBC such as cord RBC [9]; and neutralizing DARA in

patient plasma with anti-DARA idiotype antibody or

recombinant human soluble CD38 [5,6]. DTT denaturation

of CD38 on reagent RBC by disruption of disulphide

bonds in its extracellular domain is the most widely

adopted method to negate DARA interference worldwide

[10]. The other methods are unlikely to replace DTT

because of their higher cost, lack of availability or thor-

ough validation, or destruction of multiple clinically sig-

nificant antigens (Ag) on reagent RBC.

DTT denatures Ag from nine blood group systems

(Dombrock, Indian, John Milton Hagen, Kell, Knops,

Landsteiner-Wiener, Lutheran, Raph, Cartwright) and

interferes with detection of their corresponding Abs in

patients’ plasma [6]. Most of these Abs are rarely encoun-

tered, with the exception of anti-Kell Ab, which is clini-

cally significant. For this reason, Kell-negative units are

provided unless the recipient is Kell-positive. Hosokawa

et al. recently demonstrated that a lower concentration of

DARA (0�01 mol/L) preserved detection of anti-Kell while
negating DARA interference [11].

Risk of alloimmunization and potential Ab-mediated

haemolysis could be significantly reduced through pheno-

typically or genotypically matching the most common

clinically significant RBC Ags such as Rh, Kell, Kidd,

Duffy and MNS. DARA-treated patients transfused with

phenotypically or genotypically matched RBC have not

experienced Ab-mediated haemolysis or alloimmunization

[12,13]. To ensure accuracy, RBC phenotype must be

determined prior to the initiation of DARA therapy and in

the absence of a positive DAT and transfusion in the prior

3 months. Genotyping, which is usually performed by ref-

erence laboratories, incurs extra expense and prolonged

turnaround time [12]. Cushing et al. report that the

annual cost of transfusion per DARA-treated patient is

almost doubled by using universal genotyping, compared

to a DTT-based algorithm with selective genotyping [14].

Furthermore, the availability of phenotypically or geno-

typically matched units may be limited.

Our hospital transfusion service received its first

DARA-treated patient specimens in July 2015, prior to

DARA’s FDA approval. Since then, 145 patients have

been treated with DARA through December 2018. To our

knowledge, almost all published studies regarding DARA

interference in pre-transfusion testing have focused on

resolution of DARA-induced panreactivity. While one

study has reported the incidence of RBC alloimmunization

in a relatively small number of DARA-treated patients,

direct correlation between transfusion and alloimmuniza-

tion was not investigated [14]. Therefore, a retrospective

study of the serological profile and transfusion history of

all MM patients treated with DARA in our hospital was

undertaken. The aim of this study was to evaluate the risk

of RBC alloimmunization in DARA-treated patients after

transfusion with either ABO-Rh compatible or phenotypi-

cally matched RBCs.

Material and method

Patients

A total of 145 MM patients were treated at the University

of Kansas Hospital (TUKH) with DARA (Darzalex; Jans-

sen-Cilag Pty Ltd) from July 2015 to December 2018.

TUKH followed the indications and protocol of DARA

treatment described in the FDA Darzalex prescribing

information [15]. The patients’ history of DARA adminis-

tration including initiation date, end date, RBC transfu-

sion, stem cell transplant history and blood bank

serological results (ABO-Rh typing, Ab screen and identi-

fication) were collected. To compare alloimmunization

rate, the same information was also collected for MM

patients treated at TUKH from January 2013 to June 2015

before DARA was available. A total of 328 patients were

managed by chemotherapy and stem cell transplant only.

Study period

The study period of DARA group always started with the

first dose of DARA. However, the end-point varied based

on RBC Ab screen results, which included the following

two conditions: (1) 6 months after the last DARA dose if

patients’ Ab screen remained negative or (2) the first neg-

ative Ab screen after the last DARA dose if the Ab screen

had ever been positive. If patients passed away prior to

these end-points, their expiration dates became the end-

point. The study period of the non-DARA group started

from their first TUKH visit to the last one between Jan-

uary 2013 and June 2015.

Blood typing and antibody screen

Patient blood typing and a two-red cell Ab screen were

performed utilizing tube tests and gel column agglutina-

tion technology, respectively. (ORTHOTM ID-Micro Typing

System gel column technology, Ortho Clinical Diagnos-

tics, Raritan, NJ with Panoscreen, Immucor, Norcross,

GA.)

Ab identification was performed on all patients with a

positive Ab screen in tube tests with low-ionic-strength

saline (LISS) and panel red cells (Panocell-10 or -20,

Immucor, Norcross, GA). Direct antiglobulin testing (DAT)

was performed on all samples with a positive autocontrol.

All DATs were performed with polyspecific antiglobulin

reagent and subsequently tested with monospecific anti-

IgG and anti-C3b and anti-C3d reagents, if positive. Acid

© 2019 International Society of Blood Transfusion
Vox Sanguinis (2020) 115, 207–212

208 Z. Ye et al.

eluates were prepared from patient samples if not per-

formed in the preceding six months or if the strength of

the reactivity increased.

All cases with positive Ab screens in the DARA Transfu-

sion Group were tested with 0�2 M DTT-treated RBCs to
identify RBC alloantibodies either in our hospital or a refer-

ence laboratory. DTT treatment method was validated in

our hospital on 1 July 2017. A detailed method of DTT

treatment of RBCs is described in AABB Technical Manual

and a previous publication [5,16]. Quality control was per-

formed using untreated and DTT-treated cells tested with

anti-Kpb to verify the denaturation of Kell system Ags.

Subsequent Ab identification included testing the reactive

plasma with DTT-treated RBCs in tube tests with LISS.

RBC transfusion

Red blood cells transfusion history was monitored

throughout the study period for both DARA and non-

DARA groups. There were two types of transfused RBC

units according to the degree of RBC Ag match: (1) ABO-

Rh compatible and (2) ABO compatible plus phenotypi-

cally matched for Rh, Kell, Duffy, Kidd and Ss.

The electronic crossmatch was performed on non-

DARA group when no historical or current alloantibody

was detected. A serologic crossmatch was performed in

tube tests, including immediate spin and antiglobulin

phases of testing with LISS, on DARA group and any

patients with historical or detectable alloantibodies.

Donor units selected for non-DARA group were ABO-

Rh compatible and negative for corresponding Ags if they

had any historical or detectable alloantibodies. Prior to

validation of DTT method in our hospital, all patients in

the DARA group received ABO compatible plus phenotyp-

ically matched RBCs. After that, they all received ABO-Rh

compatible units negative for K Ag and corresponding

Ags if an alloantibody was detected.

Statistical analysis

The Fisher’s exact test was conducted by Software R [17].

Odds ratio with 95% confidence intervals (CI) was obtained

through adding 1 s to all elements in the contingency table

to estimate the variance due to the 0 incidence in all groups

[18]. Significance level was set at 0�05.

Results

Patients

From May 2015 to December 2018, a total of 145 patients

were treated with DARA at TUKH. Twelve patients were

excluded from analysis: 10 patients had missing records

of initiation or end date of DARA treatment, 1 patient

expired 10 days after the initiation of DARA without an

Ab screen being performed and 1 patient only

received

one treatment and then switched to elotuzumab due to

intolerance of DARA. Among 133 patients with complete

records, 45 patients were transfused with RBC (DARA

Transfusion Group) during the study period while 88

patients were not.

A total of 328 MM patients were treated at TUKH from

January 2013 to June 2015, among whom 46 patients

received RBC transfusion (Non-DARA Transfusion Group).

The demographics of both groups are summarized in

Table 1.

Blood transfusion

Two hundred and forty-six units of RBCs were transfused

to 45 patients in the DARA Transfusion Group: 32

patients only received ABO-Rh compatible RBCs, 1

patient only received ABO compatible plus

phenotypically

matched RBCs and 12 were transfused with both ABO-Rh

compatible and ABO compatible plus phenotypically

matched RBCs. All 46 patients in the Non-DARA Transfu-

sion Group received 284 units of

ABO-Rh compatible

RBCs without phenotypical match. Two patients with RBC

alloantibodies in this group received ABO-Rh compatible

plus Ag-negative RBCs. The number of patients and RBC

units transfused is listed in Table 2.

Antibody screen and identification

The results of Ab screen and identification of both the

DARA Transfusion Group and Non-

DARA Transfusion

Group are summarized in Table 3.

Two of 46 patients in the

Non-DARA Transfusion

Group had a positive Ab screen (anti-Jka and anti-K) at

the beginning of study period. Both antibodies were gen-

erated after transfusion prior to treatment at our hospital.

The Abs persisted after transfusion of ABO compatible

plus Ag-negative RBCs during the study interval. No new

alloantibodies were detected during the study period.

Table 1 Patient demographics

Number of
patients Age

Gender ratio
(female/male)

Percentage
of death

DARA
transfusion

group

45 65�1 – 10�6 21/24 27% (12/45)

Non-DARA

transfusion
group

46 59 – 10�1 24/22 20% (9/46)

© 2019 International Society of Blood Transfusion
Vox Sanguinis (2020) 115, 207–212

RBC alloimmunization and Daratumumab 209

In the DARA Transfusion Group, none of 45 patients

had a positive Ab screen prior to the first DARA treat-

ment. Forty-two of the 45 patients developed positive Ab

screens during DARA treatment and 3 patients’ Ab

screens remained negative. The reason for the negative

Ab screens in these 3 patients was not determined but it

was noted that all of them passed away within 3 weeks

after the last DARA dose.

The duration of Ab screen positivity varied markedly,

ranging from 25 days to 5 months after the last DARA

dose. Altogether, 184 Ab screens were performed after the

initiation of DARA on these patients and none of them

had detectable alloantibodies after DTT treatment. Eight

patients in the DARA Transfusion Group had an addi-

tional 55 Ab screens performed after the study period (ad-

ditional 1–9 months) and none of them were positive.

As described in Table 2, patients in the DARA Transfu-

sion Group received either ABO-Rh compatible RBCs,

ABO compatible plus phenotypically matched units or

both. They also received an additional 41 units of ABO-

Rh compatible RBC following the study period. None of

these patients developed alloantibodies, even after pro-

longed transfusion, regardless of which RBC selection

strategy was chosen.

Alloimmunization risk comparison

None of the 45 patients in the DARA Transfusion Group

and 46 patients in the Non-DARA Transfusion Group who

received ABO-Rh compatible units developed alloantibod-

ies. Fisher’s exact test showed no significant difference in

the risk of developing alloantibody between the DARA

Transfusion Group and Non-DARA Transfusion Group.

Odds ratio (95% CI) was 1�04 (0�01, 83�65), P = 1.
In the DARA Transfusion Group, 181 ABO-Rh compati-

ble units and 65 ABO compatible plus phenotypically

matched units were transfused. No alloantibody was

detected post-transfusion with either type of blood. There

was no significant difference in the risk of developing

alloantibody between the ABO-Rh compatible units and

ABO compatible plus phenotypically matched units. Odds

ratio (95% CI) was 0�37 (0�004, 29�05), P = 0�47.

Discussion

Decreased risk of RBC alloimmunization has been

reported in patients with immunosuppression. Extensive

studies of Rh-D-negative patients with hematopoietic pro-

genitor cell transplantation, solid organ transplantation

and HIV infection did not detect any anti-D alloimmu-

nization after transfusion of Rh-D-positive RBCs [19–21].

Another study reported that the frequency of anti-D for-

mation was only 20% in hospitalized patients [22], while

it was more than 80% in immunocompetent individuals.

Therefore, the low alloimmunization rate of non-DARA

Table 2 Profile of transfused RBCs

DARA transfusion
group

Non-DARA
transfusion group

ABO-Rh compatible

RBCs only

109 units (32 patients) 284 units (All 46

patients)

2 patients with

positive Ab screen

received

Ag-negative RBCs: 1

unit of Jka-negative

and 2 units of

Kell-negative

ABO

compatible plus

phenotypically

matched RBCs only

3 units (1 patient)

None

Both ABO-Rh

compatible and ABO

compatible plus
phenotypically

matched RBCs

72 units of ABO-Rh

compatible RBCs, 62

units of ABO

compatible plus
phenotypically

matched RBCs (12

patients)
None

Table 3 Ab screen and Ab identification

Ab screen before first DARA dose

Ab screen during study period

New alloantibody detected

Total
Ab screen
performed

Positive Ab
screen

Negative Ab
screen

DARA Transfusion

Group (45 patients)

Negative 184 180 (42 patients) 4 (3 patients) None (after DTT treatment)

Non-DARA Transfusion

Group (46 patients)

Two patients had positive Ab

screen from previous transfusion

(anti-Jka and anti-K)

301 5 (2 patients) 296 (44 patients) None (anti-Jka and anti-K

persisted in those two patients)

© 2019 International Society of Blood Transfusion
Vox Sanguinis (2020) 115, 207–212

210 Z. Ye et al.

Transfusion Group is expected. Although no new alloanti-

bodies were detected during study period, the preexisting

anti-Jka and anti-K were generated after transfusion dur-

ing chemotherapy.

Our study showed no significant difference in alloim-

munization risk between the DARA Transfusion Group

and Non-DARA Transfusion Group, probably due to the

low alloimmunization rate of MM patients receiving

chemotherapy. However, two patients in the non-DARA

group developed alloantibodies before the study period

but no alloantibodies were detected in the DARA group,

even after multiple transfusions. Although we did not

detect a difference of red cell alloimmunization in the

DARA group, others have reported RBC antibody suppres-

sion using DARA. Schuetz et al. recently reported that

DARA was effective in treating paediatric patients with

autoimmune haemolytic anaemia post-hematopoietic stem

cell transplantation [23]. Chapuy et al. also reported a

case of delayed red cell engraftment caused by persis-

tently high titre anti-donor anti-A which was successfully

treated by DARA [24].

Our study had two major limitations. First, sample size

of the DARA Transfusion Group included only 45

patients. Despite the wide usage of DARA since its FDA

approval in 2015, the absolute number of patients

remains small and the majority of them do not require

transfusion. A longer period of data collection may be

necessary to increase statistical power. Second, our study

was done in a single hospital but RBC alloimmunization

rates may vary significantly among different institutions.

Cushing et al. reported a RBC immunization rate of

26�4% (24 of 91 patients) in their DARA-treated group
[14] but we did not detect any new RBC antibody forma-

tion. This difference may result from variation in disease

stages, MM management strategy and RBC immunization

rates in different ethnic groups. A multicenter study

should be undertaken to more accurately assess the

immunization rate of those patients.

Currently, there is no consensus regarding pre-transfu-

sion testing for patients being treated with DARA. Two

similar algorithms have been published recently [12,25].

Both recommended transfusion of uncrossmatched O or

ABO compatible RBCs when patients needed emergent

transfusion. For routine transfusions, pre-transfusion

workups of DARA panreactivity included DTT treatment

of reagent RBC and selection of Kell-negative RBC. If the

DTT method was not available, then phenotypically or

genotypically matched RBCs were selected for transfusion.

When neither DTT treatment nor phenotyping/genotyping

was available, then patients’ samples were sent to a

reference laboratory. To avoid delay, Lancman et al. sug-

gested obtaining patients’ phenotype or genotype prior to

the initiation of DARA therapy [12].

Considering the low incidence of RBC alloimmuniza-

tion in this patient population and lacking apparent bene-

fit of phenotypically matched transfusion, the pre-

transfusion workup of DARA patients could be simplified.

The following protocol is recommended based on our cur-

rent findings: (1) maintain a current database of patients

being treated with DARA; (2) perform Ab screen and

identification prior to the initiation of DARA therapy; (3)

use DTT treatment for patients demonstrating panreactiv-

ity during and after DARA treatment if they have a newly

detected or historical alloantibodies prior to the initiation

of DARA; (4) consider eliminating DTT treatment if pan-

reactivity of Ab screen and identification tests is present

during and within two weeks after the last dose of DARA

treatment on patients without current or historical alloan-

tibodies before the initiation of DARA; and (5) forgo phe-

notyping or genotyping at any time during and after

DARA treatment.

A 2-week interval was chosen because DARA interfer-

ence disappeared as early as 25 days after the last dose.

Regular Ab screen and identification with DTT treatment

should be resumed after this window.

Besides DARA, several other anti-CD38 monoclonal

Abs, such as isatuximab (chimeric IgG kappa Ab),

MOR202 (human IgG1 lambda Ab) and TAK079 (human

IgG1 Ab), are currently in different phases of clinical tri-

als [26]. Panreactivity in pre-transfusion tests has also

been found with anti-CD38 monoclonal Abs other than

DARA [6]. A new human monoclonal IgG4 Ab, anti-CD47

(Hu5F9-G4), is also in clinical trials for the treatment of

haematologic and solid malignancies. Plasma from

patients on anti-CD47 shows panreactivity in all phases

of pre-transfusion testing (IS, room temperature, 37 C,

and IAT with or without enhancement), including ABO

reverse typing. Multiple RBC alloadsorptions and/or mon-

oclonal gamma-clone anti-IgG were the only interven-

tions that eliminated panreactivity [27].

The introduction of more and more therapeutic mono-

clonal Abs will continue to challenge transfusion practice.

Transfusion medicine specialists need to be aware of

potential interference by these new drugs. Extensive test-

ing for compatibility is necessary to prevent haemolytic

transfusion reactions. However, determining the risk of

RBC alloimmunization in patients treated with these novel

therapies may help to eliminate some time-consuming

tests. Experience with the management of DARA-induced

panreactivity should facilitate these evaluations.

© 2019 International Society of Blood Transfusion
Vox Sanguinis (2020) 115, 207–212

RBC alloimmunization and Daratumumab 211

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N E W M E T H O D S A N D A P P R O A C H E S

Resolving the daratumumab interference with blood

compatibility testing

Claudia I. Chapuy,1 Rachel T. Nicholson,1 Maria D. Aguad,1 Bjoern Chapuy,2 Jacob P. Laubach,2

Paul G. Richardson,2 Parul Doshi,3 and Richard M. Kaufman1

BACKGROUND: Daratumumab (DARA), a promising

novel therapy for multiple myeloma, is an IgG1j

monoclonal antibody that recognizes CD38 on myeloma

cells. During routine compatibility testing, we observed

that the plasma of five of five DARA-treated patients

demonstrated a positive antibody screen and

panreactivity on red blood cell (RBC) panel testing. We

hypothesized that the observed panreactivity reflected

DARA binding to CD38 on reagent RBCs, and we

investigated methods to prevent this binding.

STUDY DESIGN AND METHODS: DARA binding to

CD381 or CD38– HL60 cells was assessed by flow

cytometry. To remove cell surface CD38, cells were

incubated with dithiothreitol (DTT) or trypsin. Soluble

CD38 or anti-DARA was used to neutralize DARA in

solution. Routine blood bank serologic methods were

used to test samples from DARA-treated patients and

normal plasma samples spiked with DARA and/or

alloantibodies.

RESULTS: Normal plasma samples spiked with DARA

(0.1-10 mg/mL) and incubated with reagent RBCs
recapitulated the interference observed with samples

from DARA-treated patients. Flow cytometry experiments

confirmed DARA binding to CD381 HL60 cells, but not to

CD38– controls. DTT treatment of CD381 HL60

cells

reduced DARA binding by 92% by denaturing cell surface

CD38. Treating DARA-containing plasma with soluble

CD38 or anti-DARA idiotype also inhibited DARA binding.

CONCLUSION: DARA causes panreactivity in vitro by

binding to CD38 on reagent RBCs. Treating reagent

RBCs with DTT is a robust method to negate the DARA

interference, enabling the safe provision of blood to

DARA-treated patients. Because DTT denatures Kell

antigens, K– units are provided to these patients.

D
aratumumab (DARA) is a promising novel

therapy for multiple myeloma (MM). DARA is

an IgG1j human monoclonal antibody
(MoAb) that specifically targets human CD38,

which is highly expressed on myeloma cells. In preclinical

studies, DARA was highly cytotoxic to tumor cells via mul-

tiple mechanisms, including complement-dependent

cytotoxicity, antibody-dependent cellular cytotoxicity, and

apoptosis.1 In the first-in-human Phase I and II clinical

trial, DARA showed significant anti-MM activity as mono-

therapy in heavily treated patients with relapsed or refrac-

tory disease.2,3 Phase III trials of DARA are currently

getting under way in the United States and across several

countries internationally.

ABBREVIATIONS: DARA 5 daratumumab; GFP 5 green

fluorescent protein; MM 5 multiple myeloma.

From the 1Blood Bank, Department of Pathology, Brigham and

Women’s Hospital, and the
2
Department of Medical Oncology,

Dana-Farber Cancer Institute, Boston, Massachusetts; and the
3Janssen R&D, Spring House, Pennsylvania

Daratumumab and anti-DARA were provided by Janssen, Inc.

Address correspondence to: Richard M. Kaufman, MD, Blood

Bank, Amory 260, 75 Francis Street, Brigham and Women’s

Hospital, Boston, MA 02115; e-mail: rmkaufman@partners.org

The copyright line for this article was changed on 13 April

2015 after original online publication.

This is an open access article under the terms of the Crea-

tive Commons Attribution-NonCommercial License, which per-

mits use, distribution and reproduction in any medium,

provided the original work is properly cited and is not used for

commercial purposes.

Received for publication September 12, 2014; revision

received January 27, 2015; and accepted January 29, 2015.

doi:10.1111/trf.13069

VC 2015 The Authors Transfusion published by Wiley Peri-

odicals, Inc. on behalf of AABB

TRANSFUSION 2015;55;1545–1554

Volume 55, June 2015 TRANSFUSION 1545

On routine screening in the blood bank, we observed

that five of five patients who had received DARA in a

Phase I and II clinical trial had positive antibody screens.

The plasma of these patients was panreactive in routine

serologic tests, preventing the blood bank from providing

cross-match–compatible red blood cell (RBC) units.

Adsorptions using ZZAP-treated or untreated RBCs failed

to remove the interference.

The expression of CD38 on human RBCs has been

demonstrated previously.
4-7

For example, Albeniz and

coworkers7 performed Western blot analyses of CD38 on

human RBCs of cancer patients and healthy controls.

While they found increased CD38 expression on RBC

membranes of cancer patients, a signal confirming weak

expression on normal RBCs was also detected.7 We

hypothesized that the panreactivity observed in the blood

bank was caused by direct binding of DARA to endoge-

nous CD38 on reagent RBCs. Previous investigators

reported that CD38 is sensitive to denaturation by the

reducing agent dithiothreitol (DTT) and that enzymatic

digestion with trypsin can cleave CD38 from the cell sur-

face.
8,9

We explored methods to negate the DARA inter-

ference by removing RBC surface CD38 or by neutralizing

DARA in solution.

MATERIALS AND METHODS

Patient samples

The Brigham and Women’s Hospital blood bank received

whole blood (EDTA) samples collected as part of routine

clinical care from adult patients with refractory MM

receiving DARA. All of these patients were enrolled in a

Phase I and II clinical trial conducted at Dana-Farber

Cancer Institute (Protocol NCT00574288, Dana-Farber

Cancer Institute Protocol 10-429). Baseline (DARA-free)

samples were tested in all subjects (n 5 11). Subse-

quently, DARA-containing samples were tested in the

subset of enrolled subjects who required blood transfu-

sion (n 5 5). The samples were tested in the blood bank

using routine serologic methods, including solid-phase

(TANGO optimo automated blood bank system, Bio-

Rad, Hercules, CA) and tube testing (polyethylene glycol

[PEG], low-ionic-strength saline [LISS], or no enhance-

ment).10 Serologic tests performed on patient samples

were as follows: ABO/Rh type, antibody screen, RBC

panel, direct antiglobulin test (DAT), and antihuman

globulin (AHG) cross-match. All DATs were done with

monospecific testing for IgG and C3. Eluates were pre-

pared from patient samples with a positive DAT using

acid elution. Patient samples that were panreactive in

routine tests were further tested using DTT-treated rea-

gent RBCs (below). Institutional review board approval

was obtained to study methods to prevent DARA binding

on these patient samples.

Serologic testing of DARA-spiked plasma samples

or DARA-treated patient samples

Normal (DARA-free) plasma samples with or without RBC

alloantibodies were spiked with increasing concentrations

of DARA (0.1-10 mg/mL; provided by Janssen R&D, Spring
House, PA). These samples were analyzed using routine

blood bank serologic testing with solid phase (TANGO

optimo, Bio-Rad) and tube testing using PEG, LISS, or no

enhancement.10 Agglutination was graded per routine (0

[no agglutination], M1 [macroscopically positive (weak)],

11, 21, 31, 41). Microscopic examinations were per-

formed only on DATs. For all serologic studies, 3% to 5%

cell suspensions of reagent RBCs (Bio-Rad, Biotestcell 1,

Ref. 816014100, Batch ID 959) in phosphate-buffered

saline (PBS), pH 7.3, were used. RBC panels used for anti-

body identification were from different manufacturers:

Bio-Rad, Immucor (Norcross, GA), Ortho Clinical Diag-

nostics (Raritan, NJ), and Medion Diagnostics (Miami,

FL). Cells were chosen depending on their specific antigen

expression profile. Panels included five to 12 cell lines.

RBC alloantibody identification required obtaining a posi-

tive agglutination reaction on three cells with the antigen

present (rule-in) and a negative agglutination reaction on

three cells with the antigen absent (rule-out). To demon-

strate removal of CD38 from reagent RBCs, RBCs were left

untreated, treated with 0.2 mol/L DTT (Sigma, St Louis,

MO; detailed protocol below), or treated with 1% trypsin

(incubated at 37�C for 30 min)11 and tested against DARA-

spiked plasma samples or plasma from DARA-treated

patients. For neutralization studies, DARA-spiked plasma

samples were incubated at room temperature for 15

minutes with recombinant human soluble CD38 (R&D

Systems [Minneapolis, MN], Cat. No. 2404-AC; final con-

centration, 0.05-5 mg/mL) or mouse anti-DARA idiotype
(Janssen; final concentration, 5 mg/mL; reported by Oos-
tendorp et al., submitted for publication). A mouse anti-

human antibody (mouse IgG1j, MOPC-21, Sigma-Aldrich,
St Louis, MO) was used as an isotype control for the anti-

idiotype. Eluates were prepared from DARA-treated RBCs

using acid elution.10

DTT treatment of reagent RBCs

A detailed method for DTT treatment of RBCs is

described in the AABB Technical Manual.10 Briefly,

0.2 mol/L DTT was prepared by diluting 1 g of DTT in

32 mL of PBS, pH 8.0. K1, E1 control RBCs were used to

verify that DTT treatment had denatured the K antigen

while preserving the E antigen. Reagent and control RBCs

(100 mL of a 3%-5% suspension) were washed four times
with PBS, pH 7.3, before adding 400 mL of 0.2 mol/L DTT
to each tube. The RBCs were incubated at 37�C for 30

minutes with periodic mixing by inversion (three to four

times during incubation). The RBCs were washed four

times with PBS, pH 7.3, and used for subsequent testing.

CHAPUY ET AL.

1546 TRANSFUSION Volume 55, June 2015

Cell culture

Human HL60 cells were propagated in RPMI supple-

mented with 10 mmol/L HEPES buffer, 2 mmol/L L-gluta-

mine, 50 U/mL penicillin, 50 U/mL streptomycin, and

10% heat-inactivated fetal bovine serum (FBS; all from

Life Technologies, Grand Island, NY).

Generation of CD381 HL60 cells and CD38–, green

fluorescent protein–positive control HL60 cells

HL60 cells were transduced with human CD38 or green

fluorescent protein (GFP), the latter serving as a negative

control for DARA binding. The cDNA of human CD38 was

obtained in pDONR221 (HsCD00045212) from the DF/

HCC DNA Resource Core (http://plasmid.med.harvard.

edu/PLASMID/Home.jsp). Sanger sequencing was used to

confirm that the plasmid contained the full-length open

reading frame of human CD38. The human CD38

sequence was cloned into pMSCV-puro using a standard

Gateway LR reaction according to the manufacturer’s

directions (Life Technologies). Generation of the control

vector pMSCV-puro-GFP, packaging of retroviral particles,

and infection were performed as previously described.
12

Two days after transduction, HL60 cells were selected for

Fig. 1. Generation of a one-tube HL60 cell model system to study DARA binding. (A) Flow cytometric assessment of CD38 expres-

sion on patient RBCs was performed after gating on GlyA1 RBCs using an anti-CD38 FITC–conjugated antibody (black) or an

isotype control (gray). (B) Flow cytometric assessment of CD38 surface expression in stably transduced HL60-CD38 cells (black)

compared to nontransduced HL60 cells (gray). Human CD38 was detected using mouse anti-CD38 directly conjugated to allo-

phycocyanin. (C) Flow cytometric assessment of GFP expression in stably transduced HL60-GFP cells (black) compared to non-

transduced HL60 cells (gray). (D) For DARA-binding studies, HL60-CD38 cells and control HL60-GFP cells (2.5 3 105 cells each)

were incubated with increasing concentrations of DARA. DARA binding was assessed by flow cytometry using an anti-human

antibody labeled with PE. Separate gating on GFP1 cells (HL60-GFP) and GFP– cells (HL60-CD38) allowed the assessment of

DARA binding to each cell type in one tube. (E) Quantification of dose-dependent binding of DARA to HL60-CD38 cells, meas-

ured by flow cytometry (black; y-axis reflects the geometric mean) and compared to isotype control (gray). Plasma from a

DARA-treated patient (Patient 3, Table 1) also showed significant binding to HL60-CD38 cells. (F) Flow cytometric assessment of

DARA binding to HL60-CD38 (black) compared to HL60-GFP cells (gray). All data show a representative example of at least three

independent experi

ments. Error bars indicate SD.

DARATUMUMAB BLOOD BANK INTERFERENCE

Volume 55, June 2015 TRANSFUSION 1547

http://plasmid.med.harvard.edu/PLASMID/Home.jsp

http://plasmid.med.harvard.edu/PLASMID/Home.jsp

72 hours with 1 mg/mL puromycin (Sigma-Aldrich). Trans-
duction efficiency of HL60-GFP and HL60-CD38 cells was

assessed by flow cytometry (Fig. 1).

Detection of CD38 on RBCs and on transduced

HL60

cells by flow cytometry

Reagent RBCs (Ortho Clinical Diagnostics) were incubated

for 30 minutes with anti-CD235A-phycoerythrin (PE; anti-

GlyA, eBioscience [San Diego, CA], 12-9987-82, 1:1000)

and either a monoclonal mouse anti-CD38-fluorescein

isothiocyanate (FITC; BD Biosciences [Sparks, MD], Clone

HIT2) or a monoclonal mouse anti-human FITC-labeled

IgG1j isotype control (BD, Clone 15H6). Enforced expres-
sion of human CD38 on HL60 cells after transduction was

detected using a monoclonal mouse anti-CD38 directly

conjugated to allophycocyanin (BD, 560980). This was

done as follows: 5 3 10
5

cells were washed twice with

PBS, resuspended in 10 mL of antibody, and 90 mL of PBS
and incubated for 30 minutes at room temperature in the

dark. Per sample, 20,000 events were recorded. All flow

cytometry data were acquired on a cell analyzer (BD LSR

Fortessa, BD Biosciences). Data analysis and graphics

generation were performed with computer software

(FlowJo, V10.0.6 for MacOS, Tree Star, Ashland, OR).

Detection of DARA binding to transduced HL60

cells by flow cytometry

For DARA-binding studies, a mixture of HL60-CD38 and

HL60-GFP cells (2.5 3 105 each) was added to the same

tube and incubated with increasing concentrations of

DARA (0.1-1 mg/mL) for 1 hour at room temperature. An
IgG isotype antibody (SouthernBiotech [Birmingham, AL];

human IgG1 kappa-UNLB, Cat. No. 0151K-01) in equal

concentrations to DARA was used as a negative control.

Detection of DARA or control antibody binding was

assayed using a PE-labeled goat anti-human IgG (South-

ernBiotech, Cat. No. 2040-09, final concentration, 0.1 mg/
106 cells) incubated with HL60-CD38 and HL60-GFP cells

for 30 minutes at room temperature in a final volume of

100 mL. Separate gating on HL60-CD38 and HL60-GFP

cells was performed to assess specific DARA binding to

each cell population.

Negation of DARA binding to transduced HL60

cells

To remove cell surface CD38, mixtures of HL60-CD38 and

HL60-GFP cells (2.5 3 105 each) were incubated for 30

minutes at 37�C with DTT (0.1-10 mmol/L) or trypsin

(1%-2%) before incubation with DARA. The DTT concen-

tration was optimized for HL60 cells. Cell death was

observed when HL60 cells were treated with the standard

DTT concentration used to treat RBCs (0.2 mol/L). After

DTT incubation, cells were washed twice with PBS; after

trypsin incubation cells were washed once with growth

medium to inactivate trypsin and then twice with PBS.

The subsequent incubation with DARA and detection of

DARA with PE-labeled goat anti-human IgG followed the

same steps as described. For neutralization studies,

DARA-containing plasma (final concentration, 0.5 mg/mL)
was incubated for 15 minutes with either anti-DARA idio-

type (final concentration, 5 mg/ml) or a control mouse
anti-human antibody (mouse IgG1j, MOPC-21, Sigma-
Aldrich) at room temperature. Likewise, increasing con-

centrations (0.05-5 mg/mL) of recombinant soluble human
CD38 (R&D Systems) or identical concentrations of bovine

serum albumin (BSA, Sigma-Aldrich) were incubated with

DARA before mixing with HL60 cells for 15 minutes at

room temperature.

Statistical analysis

Comparison of groups was performed with a two-sided

unpaired t-test using computer software (GraphPad

Prism, Version 6.0c for Mac, GraphPad Software, La Jolla,

CA; www.graphpad.com).

RESULTS

DARA causes plasma panreactivity in vitro

On routine screening in the blood bank, five of five

patients with MM receiving DARA were observed to have

a positive antibody screen and panreactive plasma in RBC

TABLE 1. Summary of blood bank testing of DARA-treated patients

Patient
(number of
samples tested) Sex

Age
(years)

DARA dose/
week (mg/kg)

Period from
last DARA dose to
BB screen (days)

Result of antibody
screen and RBC panel DAT

Result of RBC
panel using DTT-

treated cells

1 (3) Male 48 8 0-7 Pan reactive Positive Negative
2 (3) Male 68 8 7 Pan reactive Positive Negative
3 (10) Female 44 8 6-13 Pan reactive Negative Negative
4 (1) Female 66 16 0 Pan reactive Positive Negative
5 (1) Male 59 16 0 Pan reactive Not done Negative

BB 5 blood bank.

CHAPUY ET AL.

1548 TRANSFUSION Volume 55, June 2015

http://www.graphpad.com

Fig. 2. Evaluation of methods to prevent DARA binding to CD38 on transduced HL60 cells. (A) DARA (0.5 mg/mL) binds selectively

to HL60-CD38 cells (black) and not control HL60-GFP cells (gray). Treating the cells with increasing concentrations of DTT (0-10

mg/mL) resulted in a dose-dependent reduction in DARA binding. (B) Quantification of reduced DARA binding after pretreating

HL60-CD38 cells with increasing concentrations of DTT or (C) trypsin compared to isotype control binding (gray). (D, E) Incuba-

tion of DARA-spiked plasma (0.5 mg/mL) with soluble CD38 (D) or anti-DARA idiotype (E) caused reduced DARA binding to

HL60-CD38 cells, while negative controls using BSA (D) or a mouse anti-human IgG (mah control; E) showed no effect on DARA

binding. (F) Quantitative assessment of DARA adsorption by HL60 CD381 cells. DARA-spiked plasma (0.5 mg/mL) was incubated

with HL60 CD381 cells or HL60 CD38– cells. After incubation, the plasma supernatants were assayed for residual DARA using

flow cytometry. Significant adsorption of DARA was detected after incubation with the higher dose (5 3 106 cells), but not the

lower dose (1 3 106 cells) of CD381 adsorbing cells. All data show a representative example of at least three independent experi-

ments. Error bars indicate SD.
DARATUMUMAB BLOOD BANK INTERFERENCE

Volume 55, June 2015 TRANSFUSION 1549

panel testing (Table 1). All samples were initially screened

using a solid-phase method (TANGO optimo); confirma-

tory testing was performed by tube testing with PEG

enhancement. Patients requiring transfusion on multiple

occasions had several longitudinal samples sent to the

blood bank. In total, the blood bank observed the pan-

reactivity in 18 of 18 samples from these patients. The

majority of the patients (3/5) had a positive DAT (IgG

only) and positive autocontrol. None of the five DARA-

treated patients showed signs of hemolysis. Typically,

Fig. 3. Eluate experiments: DARA binds specifically to RBC CD38. (A) Untreated or DTT-treated RBCs were incubated with DARA

and then washed. Eluates were then prepared by the acid elution technique.9 The eluates were added to a mixture of HL60-

CD38 cells and control HL60-GFP cells, and DARA binding was assessed by flow cytometry. (B, left panel) An eluate of untreated

RBCs contained recovered DARA that bound to HL60-CD38 cells (black) but not to HL60-GFP cells (gray). (Right panel) An elu-

ate of DTT-treated RBCs did not contain CD38-binding activity (i.e., did not contain recovered DARA). (C, left panel) An RBC elu-

ate prepared from a DAT-positive DARA-treated patient (Patient 4, Table 1) bound to HL60-CD38 cells (black) but not to HL60-

GFP control cells (gray). (Right panel) Incubating the patient eluate with anti-DARA idiotype reduced binding to HL60-CD38

cells. (D) Quantification of flow cytometry studies of the patient eluate. Incubating the patient eluate with anti-DARA idiotype

reduced binding to HL60-CD38 cells, while incubating with a mouse anti-human IgG control antibody did not have a substantial

effect. All data show a representative example of at least three independent experiments. Error bars indicate SD.

CHAPUY ET AL.

1550 TRANSFUSION Volume 55, June 2015

reaction strengths of the DARA-treated patient samples

were graded as weakly positive (M1 [macroscopically

positive] to 11) in both solid-phase and tube. When heter-

ologous adsorption studies were performed, the panreac-

tivity persisted after three passes using untreated RBCs.

ABO/Rh typing of patient RBCs was unaffected.

To verify that DARA was causing the observed agglu-

tination reactions, we first confirmed by flow cytometry

that CD38 is weakly expressed on human RBCs, as

reported previously
4-7

(Fig. 1A). Next, we spiked normal

plasma samples with increasing concentrations of DARA

(0.1-10 mg/mL). When these samples were incubated
with reagent RBCs, panreactivity was observed at anti-

human globulin phase using no enhancement, PEG, and

LISS, at all concentrations tested. Reaction strengths

were graded as M1 (0.1-0.5 mg/mL) or 11 (1.0-10 mg/
mL). Six cycles of heterologous adsorptions10 using

untreated RBCs failed to eliminate the interference.

DATs performed on reagent RBCs incubated with

DARA-spiked plasma were positive (IgG only) at all

concentrations tested (0.1-10 mg/mL), with strengths
varying between microscopically positive for concentra-

tions of 0.1-0.25 mg/mL and 11 for all higher
concentrations.

Negating the DARA interference in an HL60 cell

model system

To study methods of eliminating the DARA interference in

the blood bank, we established a model system in HL60

cells (Figs. 1B-1D). The use of transfected HL60 cells to

study CD38 function was reported previously.8 Here, HL60

cells were stably transduced with either CD38 (Fig. 1B) or

GFP, which served as a CD38– control (Fig. 1C). Flow

cytometry confirmed specific, dose-dependent binding of

DARA in spiked plasma to CD381 HL60 cells but not to

CD38– controls (Figs. 1E and 1F).

Using this system, we evaluated methods to remove

CD38 antigen from the cell surface. Incubating CD381

HL60 cells with 10 mmol/L DTT reduced DARA binding

by 92% (p < 0.001, Figs. 2A and 2B). Treating CD381 HL60

cells with 2% trypsin reduced DARA binding by 40%

(p < 0.001, Fig. 2C).

In addition to CD38 antigen removal, we attempted

to inhibit DARA binding to CD381 HL60 cells by neu-

tralizing DARA in plasma. Soluble CD38 added to

DARA-spiked plasma reduced DARA binding to CD381

HL60 cells in a dose-dependent manner (Fig. 2D). Simi-

larly, addition of a neutralizing mouse anti-DARA idio-

type antibody decreased DARA binding by 95%. A

nonspecific control antibody had no effect on DARA

binding (Fig. 2E).

We also investigated using CD381 HL60 cells as

DARA-adsorbing cells (Fig. 2F). DARA-spiked plasma was

incubated with either HL60 CD381 cells or HL60 CD38–

control cells. The adsorbed plasma was then assayed for

the presence of residual DARA by flow cytometry. While

plasma adsorbed with 1 3 10
6

HL60 CD381 cells con-

tained high levels of residual DARA, plasma adsorbed with

5 3 106 HL60 CD381 cells contained significantly reduced

levels of residual DARA (p < 0.001). Adsorbing plasma

with HL60 CD38– control cells did not significantly reduce

the level of DARA detected.

DARA is present in eluates of untreated, but not

DTT-treated, RBCs

Untreated or DTT-treated RBCs were incubated with

DARA and then washed (Fig. 3A, Steps 1 and 2). Eluates

were then generated from untreated or DTT-treated RBCs

and used for flow studies on HL60 CD381 cells and

CD38– control cells (Fig. 3A, Steps 3-5). Eluates prepared

from the untreated RBCs contained specific IgG binding

to CD381 HL60 cells, while eluates prepared from DTT-

treated RBCs contained no detectable IgG binding to

CD381 HL60 cells (Figs. 3A and 3B). These eluates were

further serologically tested against a minipanel of five

RBC lines. No reactivity was observed in the eluate of

DTT-treated RBCs, but the eluate of untreated RBCs was

TABLE 2. Representative serology results: identification of anti-E in the presence of DARA*

Screening cell Plasma Alloantibody Antibody screen result Panel cells Panel result

Cell 1 No DARA – 0 Untreated No reactivity
Cell 2 0
Cell 1 No DARA Anti-E 0 Untreated Anti-E
Cell 2 11
Cell 1 1 DARA – 11 Untreated Panreactivity
Cell 2 11
Cell 1 1 DARA Anti-E 11 Untreated Panreactivity
Cell 2 11
Cell 1 1 DTT 1 DARA – 0 DTT-treated No reactivity
Cell 2 1 DTT 0
Cell 1 1 DTT 1 DARA Anti-E 0 DTT-treated Anti-E
Cell 2 1 DTT 11

* Screening Cell 1 5 phenotype R1R1, DCe; Screening Cell 2 5 phenotype R2R2, DcE.

DARATUMUMAB BLOOD BANK INTERFERENCE

Volume 55, June 2015 TRANSFUSION 1551

panreactive. These results are consistent with DTT dena-

turing RBC CD38 epitopes, preventing DARA binding.

An RBC eluate was prepared from a DARA-treated

patient sample (Patient 4, Table 1). The eluate was pan-

reactive on RBC panels. Flow cytometry confirmed that

the eluate contained binding activity to CD381 HL60 cells

but not to CD38– controls (Fig. 3C). Binding to CD381

HL60 cells was specifically inhibited by the addition of

anti-DARA idiotype (Figs. 3C and 3D), confirming the

presence of DARA in the patient sample.

Negating the DARA interference with blood bank

tests

We performed a series of experiments on DARA-spiked

plasma samples (1.0 mg/mL) using routine blood bank
serologic assays. Treating reagent RBCs with DTT or

trypsin eliminated the panreactivity with these samples.

This allowed identification of underlying clinically sig-

nificant alloantibodies (anti-E, anti-Fya, anti-Jka, or anti-

s) in the presence of DARA. Representative blood bank

serology results for identifying anti-E are shown in

Table 2. Similar results were obtained by neutralizing

DARA-spiked samples with either anti-DARA idiotype

or soluble hCD38 (data not shown).

Adding anti-DARA idiotype to the plasma of DARA-

treated patients specifically eliminated positive antibody

screen reactions (Table 3), confirming that the positive

antibody screens seen in the clinical samples were directly

caused by DARA. Finally, using DTT-treated reagent RBCs,

the DARA interference was completely eliminated from

the plasma of all five DARA-treated patients and all 18

patient samples, allowing the safe release of blood prod-

ucts for these patients.

DISCUSSION

The use of anti-CD38 is a promising treatment for patients

with MM.
2,3

A problem with DARA is that it interferes with

blood compatibility testing, complicating the safe release

of blood products. Here, we show that direct binding of

DARA to endogenous CD38 on RBCs causes the panreac-

tivity observed in antibody screens and other pretransfu-

sion tests. Several lines of evidence support this

conclusion. First, a fluorescently labeled anti-CD38 was

shown by flow cytometry to bind directly to reagent RBCs.

Second, normal plasma samples spiked with DARA and

incubated with reagent RBCs recapitulated the interfer-

ence observed in the blood bank with samples from

DARA-treated patients. Third, an eluate prepared from the

RBCs of a DARA-treated patient bound only to CD381

cells and not to CD38– control cells. Fourth, an eluate pre-

pared from untreated RBCs that had been incubated with

DARA likewise bound only to CD381 cells and not to

CD38– control cells (i.e., contained recovered DARA).

Finally, when a specific anti-DARA neutralizing antibody

was added to DARA-treated patient samples or DARA-

spiked samples, RBC agglutination reactions were pre-

vented. Surface expression of RBC CD38 appears to be rel-

atively low, potentially explaining the weak agglutination

reactions seen in vitro. Despite binding to RBCs, DARA did

not cause significant hemolysis in the five treated patients.

Multiple rounds of adsorption with untreated or

ZZAP-treated RBCs failed to remove the panreactivity

from the plasma of DARA-treated patients. ZZAP con-

tains DTT, so ZZAP-treated RBCs are predicted to have

denatured CD38 surface antigen that would fail to bind

DARA. In contrast, we speculate that adsorptions using

untreated RBCs failed to remove the panreactivity from

DARA-treated patient samples due to low expression of

intact CD38 antigen on the adsorbing RBCs. The low

expression of CD38 on RBCs is reflected in the flow

cytometry results shown in Fig. 1A. Experiments using

transduced CD381 HL60 cells as adsorbing cells (Fig.

2F) demonstrated detectable removal of DARA from

spiked plasma only when a high number of adsorbing

cells was used. RBCs appear to express considerably

less surface CD38 than the transduced CD381 HL60

cells; thus we would expect RBCs to function relatively

poorly as DARA-adsorbing cells.

We evaluated a number of potential methods to neg-

ate the DARA interference in the blood bank. The extrac-

ellular domain of human CD38 contains six disulfide

bonds that are critical to the protein structure.
13

Previous

TABLE 3. Neutralization of DARA in patient plasma samples

Plasma sample DARA dose
Days from last
DARA infusion

Initial antibody
screen result

(Cells 1 and 2) Neutralization

Antibody screen result
(Cells 1 and 2)

after neutralization

Normal plasma spiked
with DARA

1 mg/mL NA Positive Anti-DARA idiotype (10 mg/mL) Negative
Isotype control (10 mg/mL) Positive

DARA-treated Patient 3 8 mg/kg/week 7 Positive Anti-DARA idiotype (100 mg/mL) Negative
Isotype control (100 mg/mL) Positive

DARA-treated Patient 5 16 mg/kg/week 0 Positive Anti-DARA idiotype (100 mg/mL) Negative
Isotype control (100 mg/mL) Positive

CHAPUY ET AL.

1552 TRANSFUSION Volume 55, June 2015

investigators reported that the enzymatic activity of CD38

was highly sensitive to reducing agents such as DTT
14

and 2-mercaptoethanol.
9

Berthelier and colleagues
8

reported that treating CD381 HL60 cells with DTT

decreased the binding of specific MoAbs to CD38 by

denaturing the protein. Additionally, trypsin was reported

to cleave the ectodomain of CD38 from the cell mem-

brane.
8

These findings provided the basis for our hypoth-

esis that DTT or trypsin could prevent DARA binding by

disrupting the extracellular domain of CD38 on RBCs.

Using the HL60 model system, we confirmed that DTT is

highly effective in denaturing CD38 and preventing DARA

binding. CD38 binding activity was completely absent

from an eluate prepared from DTT-treated RBCs incu-

bated with DARA. We further showed using samples from

DARA-treated patients that DTT pretreatment of RBCs

eliminated the DARA interference with blood bank tests.

Trypsin pretreatment of HL60 cells and of RBCs was also

successful in reducing DARA binding, but was less effi-

cient than DTT treatment.

Another approach to prevent DARA binding was

neutralization of free DARA in plasma by adding solu-

ble CD38 or an anti-DARA idiotype. Both methods

were highly effective in preventing DARA binding, and

DARA neutralization in solution is simpler to perform

than DTT treatment of RBCs. Disadvantages of these

neutralization methods, however, are higher costs and

a lack of widespread availability of the reagents. Large

quantities of soluble CD38 would be needed to treat

clinical samples from DARA-treated patients. [Correc-

tion added on 21-April-2015, after first online publica-

tion: “DTT-treated” changed to “DARA-treated” in the

preceding sentence] In contrast, DTT is very inexpen-

sive and is already used by blood banks.10

A potential drawback of DTT treatment is the dis-

ruption of a limited number of blood group antigens

(Table 4). The sensitivity of virtually all clinically signifi-

cant RBC antigens to DTT or trypsin has previously been

defined.
10

In routine clinical practice, anti-K is the only

commonly encountered, clinically significant antibody to

a DTT-sensitive RBC antigen. In practice, this issue is

readily addressed by providing K– units to DARA-treated

patients. More than 90% of all donated RBC units are K–

.
15

It is possible that using the DTT method to evaluate

a DARA-treated patient could result in a potentially sig-

nificant RBC alloantibody (e.g., anti-k, anti-Yta) being

missed, but this would be a very rare event. Here, we

have shown that the DTT method allows for the detec-

tion of alloantibodies in the presence of DARA from

those blood group systems—aside from Kell—that

account for the majority of clinically significant hemo-

lytic reactions: Rh, Duffy, Kidd, and MNS.

In addition to DARA, many antibody-based cancer

therapies are in various stages of development that might

similarly interfere with routine blood bank tests. In the

future, the DTT-based method described here may be use-

ful to eliminate novel in vitro interferences in the blood

bank, in those cases where the involved RBC antigen con-

tains extracellular disulfide bonds.

In conclusion, we showed that DARA potently inter-

feres with routine blood bank serologic tests by directly

binding to CD38 on RBCs. DTT treatment of reagent RBCs

is a robust method to negate the DARA interference,

allowing the safe provision of RBC units to DARA-treated

patients. Before patients are started on DARA, RBC anti-

gen phenotyping or genotyping is recommended.

ACKNOWLEDGMENTS

The authors thank William Savage and Leslie Silberstein for their

reviews of the manuscript, and the blood bank technologists at

Brigham and Women’s Hospital for their help with developing

and validating the DTT method. We also thank Barbara Bierer for

making this study possible.

CONFLICT OF INTEREST

PD is a full-time employee of Janssen. All other authors have dis-

closed no conflicts of interest.

REFERENCES

1. de Weers M, Tai YT, van der Veer MS, et al. Daratumumab, a

novel therapeutic human CD38 monoclonal antibody,

TABLE 4. DTT-sensitive blood group systems*

Blood group system name ISBT symbol Transfusion reaction potential

Dombrock DO Immediate/delayed, mild to severe
Indian IN Very rare, decreased cell survival with IN1
John Milton Hagen JMH Delayed (rare)
Kell KEL Immediate/delayed, mild to severe
Knops KN No
Landsteiner-Wiener LW Delayed, none to mild
Lutheran LU No to moderate
Raph RAPH No to moderate
Cartwright YT Delayed (rare); mild

* Adapted from the Blood Group Antigen Facts Book.
15

DARATUMUMAB BLOOD BANK INTERFERENCE

Volume 55, June 2015 TRANSFUSION 1553

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1554 TRANSFUSION Volume 55, June 2015