Daratumumab is an IgG1-k monoclonal antibody that binds selectively to CD38, a glycoprotein expressed on human plasma cells [1]. Daratumumab has efficacy in improving organ functions and hematologic responses [2-4]. On the other hand, daratumumab can induce unexpected panagglutination in blood antibody identification tests [5,6]. Despite this, the interference provoked by daratumumab in cell lineages, such as lymphocytes, has not been documented extensively.

Immunoglobulin light-chain (AL) amyloidosis is a fetal form of systemic amyloidosis resulting from the clonal expansion of CD38＋ plasma cells [4]. The heart is the second most commonly damaged organ, followed by the kidney [7]. Cardiac amyloidosis (CA) is usually associated with an unfavorable prognosis, necessitating heart transplantation for a significant number of patients [8]. The treatment of AL involves using therapies derived from multiple myeloma, specifically targeting plasma cells. Until recently, there was no established standard treatment with recognized approval [9].

Allosensitization is an important factor for heart transplantation, and the presence of anti-human leukocyte antigens (HLA) antibodies increases the risk of antibody-mediated rejection or graft failure [10,11]. Flow-cytometric crossmatch (FCMXM) is used widely in conjunction with complement-dependent lymphocytotoxicity crossmatch (CDCXM) to detect anti- HLA antibodies in histocompatibility laboratories [12]. On the other hand, there are reports showing that certain monoclonal antibodies (e.g., rituximab) can cause false positive FCMXM results [13,14]. Recently, there have been reports of daratumumab-induced false- positive results in FCMXM in patients undergoing kidney transplantation [15].

This paper presents the case of daratumumab-induced false-positive FCMXM in a CA patient who was enlisted on the heart transplantation waitlist, consequently delaying organ matching for the transplant. In addition, this study examined the CD38 expression levels on various cell types in peripheral blood and the altering patterns of these cells under daratumumab-treated conditions, dithiothreitol (DTT), or pronase, which is commonly used in B cell FCMXM to prevent false-positive results. Finally, a straightforward solution to mitigate interference by treating donor cells with DTT is proposed.

A 47-year-old male was diagnosed with CA in May 2016 based on a comprehensive assessment of the clinical manifestations, chest computed tomography (CT), and heat biopsy (Fig. 1). Despite the treatment with bortezomib or lenalidomide, CA progressed to heart failure, and daratumumab was initiated in May 2022. The patient was listed for heart transplantation (HT) and did not have detectable HLA antibodies in his immune profile. For seven months, 900 mg of Daratumumab was administered monthly, and in December 2022, the first deceased heart was matched. In CDCXM, the T and B cells showed negative results, but T cell FCMXM showed a positive result with 150 MCS and 3.8 FR, but a negative result in B cell FCMXM. The HT was delayed because of the positive result of T cell FCMXM. One month later, a second deceased heart was matched. CDCXM showed negative results in both T and B cells, but FCMXM showed a positive result in T cells (MCS 88, FR 2.2) and B cells (MCS 177, FR 4.9). The donor cells were treated with 0.05 M of DTT, and FCMXM was repeated. The FCMXM results were negative in T and B cells. Consequently, the patient received the second matched deceased heart transplant without complications.

Fig. 1. Clinical course of daratumu-mab-induced false positive flow cytometry crossmatch in a CA patient. The T cell flow cytometry cutoff was set with a median channel shift (MCS) of 40, and a fluorescence ratio (FR) of 2.0. For B cell flow cytometry, the cutoff values were established at MCS 100 and FR 2.0.
Abbreviations: CDCXM, complement-dependent lymphocytotoxicity cross-match; FCMXM, flow-cytometric crossmatch; DTT, dithiothreitol.

1.Study subjects

In the spiking tests, 15 crossmatching pairs were incorporated, all exhibiting negative results in CDCXM and FCMXM. Three samples from healthy donors were gathered randomly, and the CD38 expression levels were measured. Concerning the sera from daratumumab-treated CA patients, the study assessed four individuals diagnosed with CA and treated with daratumumab, with the most recent medication administered within one year.

2.Flow cytometry crossmatch

Negatively selected T and B cells from peripheral blood leukocytes were incubated with serum and CD3 or CD19 monoclonal antibodies for 15 minutes at room temperature and washed with PBS. FITC-conjugated anti-human IgG (Jackson Immunoresearch, Baltimore Pike, PA, USA) was used as a secondary antibody. The cells were acquired and analyzed using a BD FACSLyric flow cytometer (BD, Franklin Lakes, New Jersey, USA) and FACSuite software (ver 1.5). Flow cytometry cutoff medical channel shift (MCS) and fluorescence ratio (FR) were set. The T and B cell FCMXM cutoffs of MCS were 40 and 100, respectively, and the FR was 2.0 in both T and B cells. In B cell FCMXM, the B cells were treated with 500 μL of pronase in a water bath at 37℃ for 15 minutes.

3.Daratumumab spiking and DTT treatment of lymphocytes

After inducing false positive FCMXM with daratumumab spiking (500 μg/mL), the cells were treated with 0.1 M of DTT and incubated for 10 minutes at room temperature. After the DTT treatment, FCMXM was repeated. Two higher daratumumab concentrations were tested to induce false positivity (data not shown), but 500 μg/mL was sufficient.

4.Immunophenotyping of peripheral blood lymphocytes

The CD38 expression levels on T, B, and NK cells were analyzed in the specimens obtained from three health controls. The evaluation was performed under three conditions: neat (untreated), DTT-treated, and pronase-treated. FITC-conjugated anti-human CD38 (clone GR7A4) was used for cell staining. The results were then analyzed using Kaluza Analysis Software (version 2.2).

1.Effect of daratumumab spiking and resolution of false positivity by DTT treatment

Table 1 lists the false positive FCMXM results induced by the daratumumab spiking experiments. Among the 15 pairs with negative results in CDCXM and FCMXM, 13 pairs showed false positive results only in T cell FCMXM. Their median MCS and FR were 68 (range, 41∼108) and 1.9 (1.4∼2.6), respectively. Two pairs showed negative results in T cell FCMXM, with a median MCS of 33 (28∼38) and a median FR of 1.35 (1.3∼1.4). No induced false positive FCMXM results were detected in B cell FCMXM, with a median MCS of 4 (0∼15) and median FR of 1.0 (0.8∼1.1). Flow cytometry was re-conducted after a 0.1 M DTT treatment, and all 15 pairs showed negative results in T cell FCMXM.

Table 1

False positive results by daratumumab spiking induced and resolved results by a DTT treatment in flow cytometry crossmatch

	Positive	Negative	Total
Daratumumab (500 ug/mL) spiking on recipient sera
T cells (N)	13	2	15
Median MCS (range)	68 (41∼108)	33 (28∼38)	64 (28∼108)
Median FR (range)	1.9 (1.4∼2.6)	1.35 (1.3∼1.4)	1.8 (1.3∼2.6)
B cells (N)	0	15	15
Median MCS (range)	-	4 (0∼15)	4 (0∼15)
Median FR (range)	-	1 (0.8∼1.1)	1 (0.8∼1.1)
Post-DTT (0.1 M, 10 minutes) treatment on donor lymphocytes
T cells (N)	0	15	15
Median MCS (range)	-	6 (0∼23)	6 (0∼23)
Median FR (range)	-	1.1 (0.8∼1.2)	1.1 (0.8∼1.2)

Abbreviations: DTT, dithiothreitol; MCS, median channel shift; FR, fluorescence ratio; N, number of cases.

2.HLA crossmatching in daratumumab-treated patients’ sera with DTT treatment

Table 2 lists the crossmatch results of the sera from four CA patients treated with daratumumab. The serum samples were obtained from individuals with varying time intervals since their latest daratumumab treatment. Case 1 was a 60-year-old female whose serum was collected one week after the most recent daratumumab treatment. Cases 2 and 3 were two male patients whose sera were collected four weeks after the latest daratumumab treatment. Case 4 was a 64-year-old male whose serum was obtained nine months after the most recent daratumumab treat-ment. Among them, three sera (cases 1, 2, and 3) showed positive results in T cell FCMXM with the random donor’s lymphocytes. The MCS values for cases 1, 2, and 3 were 73.0, 87.0, and 68.0, respectively. Case 4 tested negative in T cell FCMXM. Every four sera showed negative in B cell FCMXM. Flow cytometry was re-conducted after the DTT treatment on the donor’s lymphocytes. All cases tested negative in T and B cell FCMXM. The MCS values of cases 1, 2, and 3 were 14, 10, and 11, respectively.

Table 2

False positive crossmatch results in cardiac amyloidosis patients with the daratumumab treatment and the resolved results by the DTT treatment

Case	Age	Sex	Weight (kg)	Dara (mg)	Last treatment of Daratumumab	PRA	FCMXM						FCMXM with DTT (0.1 M, 10 minutes)
							T cell	MCS	FR	B cell	MCS	FR	T cell	MCS	FR	B cell	MCS	FR
1	60	F	74.0	1,000	One week ago	Neg	Pos	73.0	1.9	Neg	3.0	1.0	Neg	14	1.1	Neg	0.0	1.0
2	77	M	54.0	800	Four weeks ago	Neg	Pos	87.0	2.2	Neg	8.0	1.1	Neg	10	1.1	Neg	0.0	1.0
3	56	M	70.1	1,000	Four weeks ago	Neg	Pos	68.0	1.9	Neg	0.0	1.0	Neg	11	1.1	Neg	0.0	1.0
4	64	M	64.6	1,000	Nine months ago	Neg	Neg	0.0	1	Neg	0.0	1.0	Neg	0	1.1	Neg	0.0	0.9

Abbreviations: Dara, daratumumab; PRA, panel reactive antibody; FCMXM, flow cytometry crossmatch; DTT, dithiothreitol; F, female; M, male; Pos, positive; Neg, negative.

3.CD38 expression on peripheral blood lymphocytes and the effect of the DTT treatment

CD38 expression patterns in various cell types were measured under various conditions (Fig. 2). In T cells, including the CD8＋ and CD4＋ populations, the mean positive percentage of the CD38＋ cell population was 27.8% (10.4∼26.9%) under the natural condition (neat). In contrast, the mean CD38＋ expression level in B and NK cells was 70.0% (52.8∼ 82.3%) and 87.1% (83.8∼89.0%), respectively. Under the pronase treatment conditions, the CD38 expression level on CD8＋ T cells did not change significantly, but CD38＋ cells were not observed in the CD4＋ T cells. Regarding the change in CD38 expression on B cells and NK cells, there was no significant difference in the expression level. After the DTT treatment, the CD38 expression level decrea-sed substantially in all cell types. The mean decreased percentages in T, B, and NK cells were 24.6%, 57.1%, and 50.7%, respectively.

Fig. 2. CD38 expression levels in the peripheral blood T, B, and NK cells in neat, pronase (500 μg) treated at 37℃ for 15 minutes and DTT (0.1 M) treated at room temperature for 10 minutes conditions.

This paper reports the case of daratumumab-induced false positive FCMXM in a patient awaiting HT. Moreover, daratumumab-induced false positivity was demonstrated with a spiking test, and a simple solution by treating donor lymphocytes with DTT was proposed. DTT is used routinely in most clinical histocompatibility laboratories to determine if a positive crossmatch is due to an IgM or IgG class antibody [16].

Regarding the DTT concentration used to address induced false positivity, the concentration recommended by Ho et al. [15] (0.05 M) was adopted for the HT patient. This concentration, constituting half of the laboratory’s standard setting, effectively alleviated the false positivity observed in FCMXM. In the present study, however, 0.1 M DTT was chosen. The DTT treatment mitigated the false positive results of FCMXM.

In the daratumumab spiking tests, a daratumumab concentration of 500 μg/mL was chosen based on the pharmacokinetic data of daratumumab, and half of the peak level of the full saturation status was selected. The recommended dosing regimen is a 16 mg/kg injection weekly for the first eight weeks, then every two weeks for 16 weeks, and every four weeks thereafter. At the saturation level, the peak daratumumab concentration in the peripheral blood reached 1,000 μg/mL [17].

Regarding the crossmatch test with daratumumab-treated CA patients’ sera, the fourth patient’s serum treated with daratumumab nine months earlier did not show a false positive in FCMXM. Daratumumab-induced false-positive results were reported in indirect antiglobulin tests in transfusion medicine, and the daratumumab-induced false-positive effect could last up to six months [18]. Only the fourth CA patient’s serum with a daratumumab treatment did not show a false positive FCMXM, which is substantiated by a previous study.

In the spiking test and crossmatching with sera from CA patients treated with daratumumab, false positives were observed exclusively in T cell FCMXM. The target of daratumumab, CD38, is a 46-kilodalton (kDa) type II transmembrane glycoprotein. The glycoprotein also exists in a soluble form of 39 kDa in biological fluids [19,20]. It is present in hematopoietic cells and can be expressed in various cells, including regulatory T cells, regulatory B cells, and myeloid-derived suppressor cells [21]. CD38 is crucial in B cell activation, differentiation, and maturation [22]. Although the precise quantification of CD38 expression in peripheral T and B cells remains unknown, the spiking tests suggest that the CD38 expression level is potentially higher in T cells than in B cells.

The hypothesis was tested by calculating the CD38 positive cell population by flow cytometry from three healthy individuals. Flow cytometry showed that the CD38 expression level was higher in B cells and NK cells than in T (CD8＋ and CD4＋) cells (Fig. 2). This finding contrasts with the experiment and case results, but the reason for this discrepancy is unclear.

Under the pronase treatment condition, no notable changes were observed in the levels of CD38 expression in CD8＋ T cells, B cells, and NK cells. On the other hand, the CD38-expressing CD4＋ cell population was not observed after pronase treatment. A pronase treatment of lymphocytes is used widely to reduce nonspecific immunoglobulin binding in FCMXM, especially in B cell crossmatch [23]. This assay was conducted to assess the impact of pronase on CD38 expression in B cells, but no significant changes were observed. The unexpected disappearance of CD38 expression in CD4＋ T cells following the pronase treatment was noted but was not further explored because it was beyond the scope of the current study.

The DTT or pronase treatment tests revealed a more than 50% decrease in CD38 expression on B and NK cells, while only a 14.8% decrease was observed on T cells. DTT is used widely in CDCXM to remove the effects of IgM-type antibody reactivity [24]. Fortunately, a previous study suggested that HLA antigens could be intact under the DTT treatment [15].

According to previous studies in transfusion medicine, daratumumab induced pan reactivity on red blood cells (RBC) panel testing, and DTT could inhibit the interference of daratumumab [25]. On the other hand, the DTT treatment could also eliminate the Kell antigen, a significant antigen on the RBC surface [26]. Moreover, the anti-CD47 monoclonal antibody (ALX148), one of the immune checkpoint inhibitors, also could exhibit interference in pretransfusion testing; it could be resolved with all adsorption methods [27].

Based on the measurements of the CD38 expression level under various conditions, the DTT treatment is a simple and safe method to resolve the interference of daratumumab on FCMXM. CD38 is expressed in various cell types, including RBC and other lymphocytes. With the continuous development of monoclonal antibody therapeutics, interference with antibody therapeutics in tests with cellular compone-nts is expected to become more common [28]. Obtaining precise patient information and medication history and identifying the cause of false positives will become increasingly crucial to determining appropriate resolutions.

daratumumab was a direct cause of a false-positive FCMXM, an issue effectively addressed by treating the cells with DTT. Patients treated with daratumumab, including those with CA awaiting HT, may be susceptible to daratumumab-associated interference in FCMXM and its subsequent consequences.

Therefore, verifying the information regarding the patient’s medical condition and the use of immunotherapeutic agents, such as daratumumab, is essential when conducting tests involving cellular components because daratumumab can influence various types of cells expressing CD38.

배경: CD38을 표적으로 하는 항-CD38 단클론성 항체인 daratumumab은 관련된 질환의 치료에 광범위하게 사용되고 있다. 특히, daratumumab을 사용하는 환자의 수혈 전 검사에서 검사용 적혈구와 비특이적 반응을 보여 판독에 주의를 기울여야 하는 것이 잘 알려져 있다. 본 연구에서는 in-vitro 및 ex-vivo 실험에서 daratumumab에 의한 위양성 간섭현상 및 DTT를 사용한 간섭현상 해결에 대한 실험적인 근거를 제시하고자 하였다.

방법: Daratumumab으로 치료받은 4명의 심근 아밀로이드증(Cardiac amyloidosis, CA) 환자와 3명의 건강인을 포함한 15개의 교차시험군을 대상으로 하였다. 유세포 교차시험은 환자 혈청 또는 daratumumab이 주입된 혈청을 사용하였으며, 음성 선택된 T 및 B 세포로 수행되었다. 혈청에 500 μg/mL의 daratumumab을 주입한 후 10분 동안 T 및 B 세포에 DTT를 처리하였다. 전향적 유세포 교차시험은 daratumumab으로 치료받은 CA 환자의 혈청으로 수행되었다. T, B 및 NK 세포의 CD38 발현 수준은 림프구의 DTT 또는 pronase 처리 전후에 측정하여 비교하였다.

결과: 500 μg/mL의 daratumumab 주입은 T 세포 유세포 교차시험에서 위양성 간섭효과를 유발하였다. 특히 0.1 M DTT의 투여는 유세포 교차 시험에서 유발된 위양성을 효과적으로 제거하였다. 또한 DTT는 T, B 및 NK 세포에서 CD38 발현양을 감소시켰다.

결론: 본 연구에서는 유세포 교차시험을 통해 일반적인 치료용량에서 daratumumab에 의한 간섭현상이 발생할 수 있음을 확인했으며, DTT 처리에 의해 효과적으로 해결할 수 있음을 실험적으로 입증하였다. Daratumumab은 CD38을 발현하는 여러 종류의 세포에 영향을 미칠 수 있으므로 세포를 이용한 검사를 시행하는 경우 환자의 질환명 및 daratumumab과 같은 면역 치료제의 사용에 대한 정보를 확인하는 것이 매우 중요하다.

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Samsung Medical Center IRB (IRB no. 2023-04-123-001).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.