Abstract
Introduction: DEL is known to be one of the weakest D variants, which can be detected by the adsorption-elution technique or by molecular study. Currently, in Thailand, we do not routinely test for DEL variants serologically or genetically among serologic RhD-negative blood donors. Case Presentation: We reported 2 cases of alloimmunization after transfused with Rh DEL, RHD*DEL1 allele, in the Thai population. The first case was a 73-year-old male with anemia who presented with post-cardiac arrest and septic shock. The patient was group B, RhD-negative, and was transfused with RhD-negative red blood cells (RBCs). Antibody screening and identification found that the patient developed anti-D and anti-Mia during the admission course. The second case was a 38-year-old woman with pseudomyxoma peritonei who developed anti-D after receiving four units of RhD-negative RBCs during cytoreductive surgery with hyperthermic intraperitoneal chemotherapy. Both patients did not receive anti-D immunoglobulin and had no previous history of anti-D detection. We retrospectively investigated and found two units of RHD*DEL1 among the RBCs transfused to these patients. Discussion: Previous reports of several cases of anti-D alloimmunization in RhD-negative recipients transfused by RHD*DEL1, an Asian-type DEL, are limited only to East Asia. We first identified 2 patients with anti-D alloimmunization after receiving the RHD*DEL1 RBCs in the Thai population. This raises concern about Rh DEL screening among D-negative Thai blood donors and whether to remove DEL units from the D-negative inventory to improve patient safety.
Introduction
The DEL phenotype is defined as an extremely weak expression of the D antigen on red blood cells. Serologically, DEL is detectable only by adsorption and elution assay [1, 2]. There is no agglutination in the conventional indirect antiglobulin test on D typing; therefore, most individuals with DEL are classified as D-negative. Among the 45 reported alleles of DEL phenotype [3], the most frequently reported mutation in East Asia is RHD*DEL1 (c.1227G>A) or Asian-type DEL. This variant represents approximately 17–30% of the negative phenotype D in countries of East Asia [4]. The prevalence of RhD-negative in the Thai population is 0.3% [5] with previously reported DEL phenotypes and RHD*DEL1 of 37.7% and 33.6% among RhD-negative blood donors [6]. However, this is the first report of anti-D alloimmunization in two recipients in Southeast Asia.
Case Report
The first case was a 73-year-old man who presented with cardiac arrest at the emergency department due to acute kidney injury and metabolic acidosis. Return of spontaneous circulation was achieved after cardiopulmonary resuscitation, and the patient was admitted to the medicine unit. During an approximately 2-month hospital stay, the patient developed pneumonia with severe septic shock and an unknown cause of anemia (hemoglobin 7.0–8.3 g/dL) without an obvious bleeding site. A total of 10 units of group B, RhD-negative red blood cells (RBCs) were transfused on different occasions during his admission. To our surprise, 6 weeks after admission, the patient developed anti-D and anti-Mia antibodies, as demonstrated in online supplementary Figure S1 (for all online suppl. material, see https://doi.org/10.1159/000533625). The anti-Mia reacted from the room temperature phase to the 37°C phase, but not the indirect antiglobulin phase. It is difficult to know if this antibody was naturally occurring or alloimmunization due to a previous transfusion. We performed the Mia antigen typing from the patient’s cells and found it to be Mi(a-). Unfortunately, information on the RhCE phenotype of the patient was not available, and he passed recently after anti-D was detected.
All units transfused into this patient were retrospectively investigated: nine of the ten donors had genotypically negative RhD using the multiplex sequence-specific primer (PCR-SSP) of RHD at intron 4, exon 7, and exon 10 [7]. The only unit positive for this multiplex PCR was then further tested for the RHD*DEL1 allele using the PCR-SSP technique and was found to be positive for this variant (shown in online suppl. Fig. S2). The PCR-SSP method we utilized involved amplifying the extracted genomic DNA using the forward and reverse primers, along with the PCR conditions described in the previous literature [8]. In brief, the thermocycler was set as follows: denaturation at 94°C for 5 min, then 35 cycles of 30 s at 94°C, 40 s at 68°C, and 30 s at 72°C. The obtained 348 bp PCR products indicate the RHD*DEL1 (c.1227G>A). The 629 bp growth hormone gene was used as an internal control.
The donor had the D−C+c+E−e+ phenotype. The duration from transfusion of the DEL unit (November 8, 2021) to anti-D detection (December 16, 2021) was 38 days; during this period, periodic antibody screening was performed and remained negative, including the test on December 2, 2021 (24 days after transfusion). The patient did not receive platelet products or Rh immunoglobulin during this admission. His last transfusion before this event was 5 years ago at a provincial hospital. We then retested the affected donor on the next visit and found similar genetic results. We coated donor RBCs with anti-D and conducted flow cytometry to quantify the D antigen [9]. Our findings revealed 22 D antigen sites on the donor RBCs, which confirmed the donor’s DEL status.
The second case was a 38-year-old woman who developed anti-D after receiving four units of RBCs in a cytoreductive surgery with subtotal colectomy and hyperthermic intraperitoneal chemotherapy (HIPEC) procedure to treat pseudomyxoma peritonei. She had two RhD-positive children (more than 10 years ago) with an unknown history of Rh immunoglobulin prophylaxis in both pregnancies, and her last transfusion was 6 months earlier during a surgery. She was referred to our institute for further management. Her blood group typing was group A, D−C+c+E−e+, K−, and negative antibody screening. Two months earlier, the patient had undergone cytoreductive surgery with HIPEC and had received four units of group A, RhD-negative RBCs. After the follow-up imaging study pointed out the remaining tumors, the surgeons decided to set up another operation for tumor removal and requested six units of group A, RhD-negative RBCs. This time, 35 days after receiving RBC transfusions, her type and screen testing were positive for anti-D with the titer of 1:1,024. The patient did not receive any blood transfusions or Rh immunoglobulin after the first procedure. We reviewed the four negative RhD units transfused into this patient and detected one DEL unit. The donor serological test showed DEL by the adsorption and elution method with the D−C+c+E−e+ phenotype. The genotyping study of this donor demonstrated positive for D with the RHD*DEL1 allele by PCR-SSP. Quantification of the D antigen by flow cytometry showed 32 D antigen sites on donor RBCs.
Discussion
The prevalence of DEL in RhD-negative blood donors in the Asian population is relatively high compared to Caucasians [4, 10]. However, routine serologic methods applied in clinical tests would type DEL as D-negative and would be transfused into D-negative recipients. The alloimmunization of anti-D from DEL donors in Asia has been reported previously [11‒16], as summarized in Table 1, particularly by the Asian DEL allele. This allele was identified to have a complete RhD-positive epitope pattern [17, 18]. Anti-D is one of the clinically significant antibodies that can cause major hemolytic transfusion reactions and severe hemolytic disease of the fetus and newborn. In our cases, 2 RhD-negative patients were transfused with DEL, RHD*DEL1 allele, and developed the D antibody. Focusing on the first case, the duration between alloimmunization was between 24 and 38 days after DEL blood transfusion. This would postulate that this patient developed a primary immune response caused by donors of the DEL phenotype with the RHD*DEL1 allele. While for the second case, we do not have data on antibody testing in between to know the exact onset of anti-D alloimmunization. However, the very high anti-D titer on day 35 would suggest that this could be due to a secondary immune response rather than a primary response. Previous reports of anti-D alloimmunization from Asian-type DEL were mostly secondary immunizations, except for one Chinese recipient who reported having a primary immune response indicated by the onset of antibody development on day 22 [14]. We also show the scanty D antigen sites on both donor RBCs by flow cytometry. Therefore, this study supports that DEL can cause primary anti-D alloimmunization despite the small amount of D antigens on the RBC. This is the first study of the Southeast Asian population of anti-D alloimmunization from donors with DEL phenotype. There is only a limitated study on DEL in this region. The serological study in Myanmar showed a prevalence of 15.8% of the DEL phenotype among RhD-negative blood donors [19]. Therefore, further studies of DEL in the Southeast Asia area are warranted. A recent study by Nuchnoi and colleagues has suggested a cost-effective method for Asian-type DEL screening in Thai blood donors [20]. The current study would support the benefit of this practice, removing the DEL units from the D-negative inventory to improve patient safety. Whereas patients and pregnant women with Asian-type DEL should, probably, be treated as RhD-positive individuals without concern for D alloimmunization [21].
Summary of previous reports of anti-D alloimmunization in Asia by DEL red blood cells
| Case | Recipient | Age, years | Sex | Blood group | DEL exposure, units | DEL allele | Alloimmunization | Prior transfusion/pregnancy | Reference |
|---|---|---|---|---|---|---|---|---|---|
| 1 | Japanese | 67 | F | B, D− | 2 from 59 | RHD*DEL1 | Secondary | D+ transfusion | Yasuda et al. [11] (2005) |
| 40 years | |||||||||
| 2 | Taiwanese | 64 | M | D− | 2 from 6 | n/a | n/a | n/a | Chen et al. [12] (2006) |
| 3 | Taiwanese | 73 | M | AB, D− | 4 from 6 | n/a | Secondary? (day 6) | n/a | Chen et al. [12] (2006) |
| 4 | Korean | 68 | M | O, D− | 1 from 4 | RHD*DEL1 | Secondary? (day 9) | None | Kim et al. [13] (2009) |
| 5 | Chinese | 33 | F | B, D− | 1 | RHD*DEL1 | Secondary | Pregnancy | Shao et al. [14] (2012) |
| 6 | Chinese | 45 | M | D− | 1 from 2 | RHD*DEL1 | Secondary | D+ transfusion | Shao et al. [14] (2012) |
| 20 years | |||||||||
| 7 | Chinese | 68 | M | D− | 2 | RHD*DEL1 | Primary (day 22) | none | Shao et al. [14] (2012) |
| 8 | Russian (Korean donor) | 64 | M | A, D− | 2 | RHD*DEL1 | Primary? (day 5–7) | none | Yang et al. [15] (2015) |
| 9 | Chinese | 44 | F | D− | 1 from 4 | RHD*DEL1 | Secondary (day 4) | Pregnancy | Wen et al. [16] (2022) |
| 10 | Thai | 73 | M | B, D− | 1 from 10 | RHD*DEL1 | Primary (day 24–38) | None | This study |
| 11 | Thai | 38 | F | A, D− | 1 from 4 | RHD*DEL1 | Secondary? (day 35) | Pregnancy | This study |
| Case | Recipient | Age, years | Sex | Blood group | DEL exposure, units | DEL allele | Alloimmunization | Prior transfusion/pregnancy | Reference |
|---|---|---|---|---|---|---|---|---|---|
| 1 | Japanese | 67 | F | B, D− | 2 from 59 | RHD*DEL1 | Secondary | D+ transfusion | Yasuda et al. [11] (2005) |
| 40 years | |||||||||
| 2 | Taiwanese | 64 | M | D− | 2 from 6 | n/a | n/a | n/a | Chen et al. [12] (2006) |
| 3 | Taiwanese | 73 | M | AB, D− | 4 from 6 | n/a | Secondary? (day 6) | n/a | Chen et al. [12] (2006) |
| 4 | Korean | 68 | M | O, D− | 1 from 4 | RHD*DEL1 | Secondary? (day 9) | None | Kim et al. [13] (2009) |
| 5 | Chinese | 33 | F | B, D− | 1 | RHD*DEL1 | Secondary | Pregnancy | Shao et al. [14] (2012) |
| 6 | Chinese | 45 | M | D− | 1 from 2 | RHD*DEL1 | Secondary | D+ transfusion | Shao et al. [14] (2012) |
| 20 years | |||||||||
| 7 | Chinese | 68 | M | D− | 2 | RHD*DEL1 | Primary (day 22) | none | Shao et al. [14] (2012) |
| 8 | Russian (Korean donor) | 64 | M | A, D− | 2 | RHD*DEL1 | Primary? (day 5–7) | none | Yang et al. [15] (2015) |
| 9 | Chinese | 44 | F | D− | 1 from 4 | RHD*DEL1 | Secondary (day 4) | Pregnancy | Wen et al. [16] (2022) |
| 10 | Thai | 73 | M | B, D− | 1 from 10 | RHD*DEL1 | Primary (day 24–38) | None | This study |
| 11 | Thai | 38 | F | A, D− | 1 from 4 | RHD*DEL1 | Secondary? (day 35) | Pregnancy | This study |
n/a, no available data; ?, did not have sufficient information to interpret.
Statement of Ethics
Patients or their legal representatives have provided their written informed consent to gain access to and use of confidential and personal medical information used to write the manuscript and publish the manuscript in a scientific journal. No samplings or tests have been conducted for this article or manuscript. This study protocol was reviewed and approved by the Siriraj Institutional Review Board (SIRB), approval number [Si 262/2023].
Conflict of Interest Statement
The authors have no conflicts of interest to declare.
Funding Sources
There are no funding sources for the production of the data used or the writing of the manuscript.
Author Contributions
Kanyapon Suksard and Thongbai Rungroung were contributed to the serological testing of the samples. Komon Luangtrakool and Pradermchai Saetam performed the molecular assays. Sutthisak Chamsai performed flow cytometry for quantifying antigen site. Janejira Kittivorapart wrote the manuscript. Janejira Kittivorapart, Kulvara Kittsares, and Parichart Permpikul designed the experiments and critically examined the manuscript.
Data Availability Statement
All the data generated or analyzed during this study are included in this article and its online supplementary material. Further inquiries can be directed to the corresponding author.
References
