SUMMARY  

• Daratumumab binds to CD38 found at low levels on red blood cells (RBCs) and may result in a positive indirect Coombs test (indirect antiglobulin test; IAT). Daratumumab-mediated positive indirect Coombs test may persist for up to 6 months after the last daratumumab infusion. It should be recognized that daratumumab bound to RBCs may mask detection of antibodies to minor antigens in the patient’s serum. The determination of a patient’s ABO and Rh blood type are not impacted.¹
  • Type and screen patients prior to starting daratumumab.¹
  • In the event of a planned transfusion notify blood transfusion centers of this interference IATs. If an emergency transfusion is required, non-cross- matched ABO/RhD-compatible RBCs can be given per local blood bank practices.¹
• Positive IATs in fresh-frozen plasma (FFP) samples and samples from multiple myeloma patients in which daratumumab has been introduced or administered, respectively, have been reported in the literature.^1-3
• Chapuy et al (2015)² and Oostendorp et al (2015)³ reported daratumumab anti-idiotype antibodies and recombinant soluble CD38 (sCD38) blocked all daratumumab interference in the IAT of FFP + daratumumab samples and in plasma samples from multiple myeloma patients.
• Chari et al (2017)⁴ conducted an analysis of RBC transfusions and transfusion-related adverse events (AEs) in the SIRIUS study. Seven patients at the authors’ institutions who had antibody screening after receiving DARZALEX demonstrated agglutination of all RBCs on panel testing.
• Chapuy et al (2015)² published findings that treating RBCs with dithiothreitol (DTT) effectively eliminates daratumumab interference in serologic testing. Treating RBCs with trypsin demonstrated less efficient elimination of interference by daratumumab.
• Chapuy et al (2016)⁵ conducted a multicenter international study to validate the DTT method for use by blood banks worldwide. Using DTT-treated RBCs, 100% of blood bank laboratories sampled worldwide were able to identify a clinically significant antibody initially masked by the presence of daratumumab.
• Additional citations are included in the References section for your information.^6-22

PRODUCT LABELING

• DARZALEX (daratumumab) [Prescribing Information]. Horsham, PA: Janssen Biotech, Inc.; www.janssenlabels.com/package-insert/product-monograph/prescribing-information/DARZALEX-pi.pdf
• DARZALEX FASPRO (daratumumab and hyaluronidase-fihj) [Prescribing Information]. Horsham, PA: Janssen Biotech, Inc.; www.janssenlabels.com/package-insert/product-monograph/prescribing-information/DARZALEX+Faspro-pi.pdf

BACKGROUND

• Daratumumab binds to CD38 found at low levels on RBCs and may result in a positive indirect Coombs test. Daratumumab-mediated positive indirect Coombs test may persist for up to 6 months after the last daratumumab infusion. It should be recognized that daratumumab bound to RBCs may mask detection of antibodies to minor antigens in the patient’s serum. The determination of a patient’s ABO and Rh blood type are not impacted.¹

CLINICAL DATA

Daratumumab Interference with Blood Compatibility Testing

Chapuy et al (2015)² published similar findings from the same phase 1 and 2 studies at their single center (Dana Farber Cancer Institute). In total, investigators observed positive antibody screens in 5 of 5 patients who received daratumumab after routine serological testing. Results from treating RBCs with DTT as a method for blocking daratumumab IAT interference are summarized.

Study Design/Methods

• Normal plasma samples were supplemented with daratumumab 0.01, 0.1, 1.0, and 10.0 µg/mL and analyzed using routine testing with solid phase (TANGO optimo, Bio-Rad) and tube techniques (PEG, LISS, or no enhancement).
  • Agglutination strengths were record as: 0 = no agglutination; 0.5+ = very weak agglutination; 1+ = weak agglutination; 2+ = agglutination; 3+ = strong agglutination; 4+ = very strong agglutination
• To test the effectiveness of treating RBCs with DTT to eliminate daratumumab IAT interference:
  • Washed reagent and control RBCs were treated with 400 µL of 0.2 mol/L DTT, then incubated at 37°C for 30 minutes (periodic inversion completed 3-4 times during incubation), and washed an additional 4 times. Eluates were then prepared from DTT-treated and untreated RBCs to further test reactivity.
  • HL60 cells were stably transduced with either human CD38 (HL60-CD38) or green fluorescent protein as a control (GFP; HL60-GFP). These cells were used to further evaluate methods to remove the CD38 antigen from the cell surface.
• Blocking daratumumab interference with daratumumab anti-idiotype antibody and soluble CD38 were also assessed.

Results

Observed Irregular Antibody Screening in the Phase 1 and 2 Study

• Positive antibody screens (generally +1) were observed in 5 of 5 patients treated with daratumumab who underwent routine screening in the blood bank. In total, 18 samples were collected, and all demonstrated positive results.
  • Positive direct antiglobulin test (DAT; IgG only) and positive auto-control were observed in 3/5 patients.
  • No signs of hemolysis were observed.

Failure of Adsorption to Eliminate Interference

• Panreactivity was observed at all daratumumab concentrations and could not be removed even after 6 cycles of heterologous adsorption.

Blocking Daratumumab Interference with DTT-Treated RBCs

• Following incubation of HL60-CD38 cells with 10 mmol/L DTT, daratumumab binding on the cell surface was reduced by 92% (P<0.001). Treating HL60-CD38 cells with 2% trypsin reduced daratumumab binding by 40% (P<0.001).
• Eluates from untreated and DTT-treated RBCs were tested against a minipanel of 5 RBC lines.
  • No reactivity was observed in the eluate of DTT-treated RBCs (which did not contain daratumumab), and reactivity was observed in the eluate of untreated RBCs.
• In plasma samples with daratumumab 1.0 µg/mL, treating reagent RBCs with DTT and trypsin each resulted in elimination of panreactivity.

False-Positive Indirect Antiglobulin Tests Post DARZALEX Treatment

Oostendorp et al (2015)³ published findings from a phase 1 and 2 safety and dose-escalation study in which 11 multiple myeloma patients at their single center received DARZALEX 8 to 16 mg/kg intravenous infusions on weeks 1, 4, and then weekly until week 8.  Peak daratumumab serum concentrations exceeded 100 µg/mL for all patients (range 110-438 µg/mL).  The investigators observed that all patients who received DARZALEX demonstrated false-positive IATs for 2 to 6 months after the last DARZALEX infusions.

In-Vitro Study Design/Methods

• The ability of daratumumab to produce red blood cell agglutination was assessed using FFP samples supplemented with daratumumab 0.01, 0.1, 1.0, and 10.0 µg/mL.
  • IAT performed using the low-ionic-strength solution (LISS) gel column agglutination technique (anti-IgG present in the gel matrix)
  • IAT also performed with samples from multiple myeloma patients prior to receiving DARZALEX treatment as a control
  • The DAT was performed using the LISS/Coombs gel column technique (Bio-Rad), containing polyspecific anti-IgG and anti-C3d within the gel matrix.
  • Agglutination strength was recorded as: 0 = no agglutination; 0.5+ = very weak agglutination; 1+ = weak agglutination; 2+ = agglutination; 3+ = strong agglutination; 4+ = very strong agglutination
• Agglutination by other CD38 monoclonal antibodies were assessed using similar methods.
• Analyses were conducted with FPP + daratumumab 10.0 µg/mL samples in which daratumumab anti-idiotype antibody or sCD38 were introduced to assess these approaches as potential solutions for blocking daratumumab interference.

Results

Observed Irregular Antibody Screening in the Phase 1 and 2 Study

• Plasma from all patients demonstrated negative IATs and DATs prior to DARZALEX administration.
• After DARZALEX administration, positive IAT results (generally 2+) were observed for 11 patient samples tested and the IAT autocontrol was negative for all patients.
• The positive IAT results persisted for 2-6 months after the last DARZALEX treatment in 8 patients.
• DAT results were negative (indicating a lack of IgGs bound to the RBCs) for all patients tested.

Irregular Antibody Screening In-vitro

• Daratumumab induced RBC agglutination in a dose-dependent manner in a 3-cell RBC panel and in RBCs from untreated multiple myeloma patients. See Table: RBC Agglutination Observed in IAT of Plasma Samples with Daratumumab.
  • Similar RBC agglutination patterns observed with other CD38 monoclonal antibodies.
  • No differences in agglutination patterns observed when RBCs were separated into 5 age fractions.

Blocking daratumumab interference with daratumumab Anti-Idiotype Antibodies

• Daratumumab anti-idiotype antibodies at 5- and 10-fold excess concentrations blocked all daratumumab interference in the IAT of FFP + daratumumab samples and in plasma samples from multiple myeloma patients.
  • In plasma from a patient with known anti-E and anti-K antibodies, the addition of the daratumumab anti-idiotype antibodies resulted in elimination of daratumumab interference and accurate determination of anti-E or anti-K antibodies.
• Addition of sCD38 also blocked daratumumab interference in the IAT.

Analysis of RBC Transfusions and Transfusion-Related Adverse Events in the SIRIUS Study

Chari et al (2017)⁴ conducted an analysis of RBC transfusions and transfusion-related AEs in the SIRIUS study.

Study Design/Methods

• SIRIUS was a phase 2, open-label, international, multicenter study.
• Patients in the study had multiple myeloma and received ≥3 prior lines of therapy, including a proteasome inhibitor and an immunomodulatory drug, or had disease which was refractory to both a proteasome inhibitor and an immunomodulatory drug.
• In part 1 of the study, patients were randomized 1:1 to receive:
  • DARZALEX 16 mg/kg every week for 8 weeks, DARZALEX 16 mg/kg every 2 weeks for 16 weeks, then DARZALEX 16 mg/kg every 4 weeks thereafter (n=16), or,
  • DARZALEX 8 mg/kg every 4 weeks (n=18)
• Response was evaluated and the DARZALEX 16 mg/kg dose was established as the recommended dose for further evaluation in part 2 of the study. An additional 90 patients were enrolled in the DARZALEX 16 mg/kg group.
• Patients were RBC phenotyped by tube methodology prior to receiving their first dose of DARZALEX.
• If the patient’s antibody screen was positive, antibody identification was performed using a panel of reagent RBCs.
• Antibody screens and RBC panels were performed using gel cards.
• If panagglutination reactions were detected on gel cards, additional testing was conducted using LISS.

Results

Transfusions in the Overall Study Population

• As of a clinical cut-off date of January 9, 2015, 49 of 124 (39.5%) patients received 236 transfusions.
  • A total of 47 (37.9%) patients received 147 packed red blood cell transfusions.
  • A total of 17 (13.7%) patients received 89 platelet transfusions.
  • No patient received a whole blood or fresh frozen plasma transfusion.
• Hemolysis was not reported.
• One transfusion-related reaction was observed following platelet transfusion.

Transfusion Experience at Clinical Study Sites

• The experience of 15 patients was described (n=8 from Mount Sinai, n=7 from the Levine Cancer Institute).
  • At Mount Sinai, no transfusion AEs were observed, no new unexpected RBC alloantibodies were identified, and transfusions increased hemoglobin values (median, 1.2 g/dL).
  • At the Levine Cancer Institute, 6 of 7 patients responded to transfusions, with a median hemoglobin change of 1.7 g/dL.
• Blood typing of patients from Mount Sinai is described in Table: Blood Typing of Mount Sinai Patients.
• Patient blood transfusions from both Mount Sinai and the Levine Cancer Institute are described in Table: Blood Transfusions.

Dithiothreitol-Based Method to Resolve the Daratumumab Interference with Blood Compatibility Testing

Chapuy et al (2016)⁵ conducted a multicenter international study to validate the DTT method for use by blood banks worldwide.

Study Design/Methods

• Blood banks received 2 unknown plasma samples:
  • Sample 1 was spiked with daratumumab alone (10 mcg/mL).
  • Sample 2 was spiked with daratumumab plus a clinically significant antibody (anti-D, anti-Fya, or anti-s).
• Sites were instructed to perform an antibody screen using their usual method (tube, gel, or solid phase), then to repeat the antibody screen using DTT-treated RBCs (gel or tube).
  • If the antibody screen remained positive with DTT-treated RBCs (sample 2), sites were asked to identify the unknown antibody using a DTT-treated RBC panel (gel or tube).
• The primary outcome was the proportion of study sites able to identify the unknown antibody in the presence of daratumumab.

Results

• Paired plasma sample unknowns were shipped to 25 study sites. See Table: Results of Multicenter Serologic Analysis of Daratumumab-Spiked Plasma Sample Unknowns.
• For the initial antibody screen (sample 1), 10 sites used tube testing, 9 sites used gel, and 6 sites used solid phase.
• All sites observed daratumumab interference with the antibody screen and at 24 of 25 sites (96%), DTT treatment of screening cells eliminated the positive reactions seen with sample 1.
• All sites were able to correctly identify the unknown antibody using the DTT method: anti-Fya (9 sites), anti-s (10 sites), and anti-D (6 sites).
• Ninety percent of sites (17/19) reported that they planned to use the DTT method to manage clinical samples from daratumumab-treated patients in the future.

Literature Search

A literature search of MEDLINE^®, Embase^®, BIOSIS Previews^®, and Derwent Drug File databases (and/or other resources, including internal/external databases) was conducted on 22 July 2024.