Donor vs. Donor or Recipient vs. Recipient? A Singular Case of Post Hematopoietic Stem Cell Transplantation Autoimmune Hemolytic Anemia in a Pediatric Patient

Hematopoietic Stem Cell Transplantation (HSCT) is a curative treatment for certain blood disorders, but can lead to complications such as Autoimmune Cytopenia (AIC), particularly Autoimmune Hemolytic Anemia (AIHA). AIHA after HSCT often involves donor-derived antibodies attacking donor red blood cells, but this case report describes a unique case in a 7-year-old male with beta-thalassemia major. Following HSCT, he developed AIHA nine months after transplantation from an unrelated donor. Despite initially successful engraftment, chimerism analysis revealed a decline in donor cells coincident with the onset of AIHA. The patient had severe hemolysis, a positive Direct Coombs Test (DAT) for IgG and C3d, and several antibodies including anti-e, anti-c, and anti-D. Treatment with cyclosporine and prednisone initially stabilized his condition, but discontinuation resulted in relapse, worsening chimerism, and increased antibody titers. Analysis confirmed the predominance of recipient immune cells, suggesting that AIHA was driven by the recipient's immune response against his own red blood cells. This case emphasizes the complexity of immune reconstitution after HSCT and highlights a recipient-versus-recipient mechanism in post-transplant AIHA.

AIC: Autoimmune Cytopenias; AIHA: Autoimmune Haemolytic Anaemia; DAT: Direct Antiglobulin Test; HB: Hemoglobin; HSCT: Hematopoietic Stem-Cell Transplantation; IAT: Indirect Antiglobulin Test; LDH: Lactate Dehydrogenase; MUD: Matched Unrelated Donor; RBCs: Red Blood Cells; WAIHA: Warm Autoimmune Hemolytic Anemia

Hematopoietic Stem Cell Transplantation (HSCT) is a potentially curative treatment for both malignant and certain non-malignant blood disorders. Autoimmune Cytopenia (AIC) is a rare but potentially serious complication of transplantation, especially in pediatric patients. The most common form of post-transplant AIC is Autoimmune Hemolytic Anemia (AIHA), with an estimated incidence of 2.4% to 6% of patients undergoing HSCT [1]. AIHA typically manifests 5-10 months after HSCT, usually as Warm Autoimmune Hemolytic Anemia (WAIHA) [2,3]. Although the mechanisms underlying the development of post-HSCT AIHA are not fully understood, poor immune reconstitution leading to loss of self-tolerance appears to be a contributing factor. Most reported cases of AIHA occur in the context of complete donor chimerism, suggesting that the autoantibodies are derived from donor plasma cells against donor Red Blood Cells (RBCs), defining a "donor versus donor" scenario [4-6]. Here, we describe a specific case of post-HSCT AIHA in a pediatric patient in which we hypothesized a recipient immune response against his own RBCs.

This is a case of a 7-year-old male patient diagnosed with beta-thalassemia major with blood group O RhD positive (CCDee kk). In July 2019, he underwent HSCT from an Unrelated Donor (MUD) with blood group O RhD negative (ccdee kk).

Pre-HSCT immunohematology evaluations and subsequent evaluations five months post-HSCT showed negative results for both the Direct Coombs Test (DAT) and the Indirect Antiglobulin Test (IAT). Molecular chimerism analysis confirmed complete engraftment of the graft with 98% donor cells, resulting in an O RhD negative blood group.

Over the next three months, repeat chimerism assessments showed a progressive decrease in the percentage of donor cells from 98% to 84%.

Nine months after HSCT, the patient was hospitalized for a first episode of post-HSCT AIHA. Initial laboratory results showed: Hemoglobin (Hb) of 4.4 g/dL, Lactate Dehydrogenase (LDH) of 1049 U/L and total bilirubin of 2.4 mg/dL. DAT was positive for IgG1+C3d. Two antibodies were eluted from the red cells: a pan reactive antibody and an anti-e antibody, both confirmed in the serum by papain enzyme. In addition, anti-C and anti-D antibodies were detected in the serum with titers of 1:32 and 1:64, respectively. The anti-D antibody, initially masked by other antibodies, was identified by serum adsorption with O RhD positive red cells. A repeat chimerism assay showed 84% donor cells.

Over the next six months, the patient was treated with cyclosporine and prednisone, resulting in progressive improvement in hemolysis indices (Hb 14.3 g/dL, reticulocytes 3.4%, LDH 260 U/L) and a significant reduction in antibody titers, leading to complete negativity of the IAT. However, the DAT remained positive with IgG specificity.

Following the observation of IAT negativity, clinicians discontinued pharmacologic therapy. Five months after cessation of therapy, during which time parameters remained stable, new serum antibodies appeared, with titers increasing up to 1:128. The DAT was positive for IgG1+IgM+C3d. This was associated with further deterioration of chimerism, showing 52% donor cells and 48% recipient cells.

The following year, the patient was monitored in their home country and remained stable with favorable hemolysis indices. However, in 2023, the patient was readmitted to our center due to a severe relapse of AIHA, which was associated with a drastic reduction in chimerism, reaching 37% donor cells and 63% recipient cells. The antibody titer was 1:64 with DAT positive for IgG1+IgG3. Repeat elution confirmed the presence of antibodies detected in the initial study. Hemolysis indices were Hb 10.1 g/dL, reticulocytes 22.9%, total bilirubin 2.58 mg/dL, and LDH 400 U/L.

Chimerism analysis of sorted cell populations confirmed a predominance of recipient cells: CD3+ 63%, CD19+ 53%, CD56+ 60%, and CD14+ 51%. Additionally, post-transplant immunologic reconstitution analysis showed a decrease in regulatory T cells and an increase in B lymphocytes. This was associated with hemolytic crises, increasing antibody titers, and worsening chimerism favoring recipient cell predominance.

AIHA is a disease characterized by autoimmune destruction of RBCs. It can occur either as a primary disease or as a secondary complication of various conditions, including infection, malignancy, transplantation, and autoimmune disease. In the context of transplantation, AIHA is of particular interest as it typically develops 5-10 months post-transplantation, often in WAIHA with IgG antibodies reactive at 37°C [3].

In the case discussed, the patient developed warm AIHA with IgG antibodies reactive at 37°C exactly 9 months after HSCT. The complexity of post-transplant AIHA arises from the delicate balance between the reconstituted donor immune system and the still-present but depleted recipient immune system [7]. Regulatory T cells (Tregs) are critical in this context, as they suppress autoreactive T and B cells and prevent abnormal immune responses [8-10]. Post-transplant immunologic analysis of the patient showed a decrease in Tregs and an increase in B lymphocytes, which was associated with the development of AIHA.

A key aspect of the case is the blood groups of the recipient (O pos CCDee) and the donor (O neg ccdee). After transplantation, the patient developed a pan reactive antibody that masked anti-e, anti-C, and anti-D antibodies. In the differential diagnosis between autoantibodies and alloantibodies, chimerism analysis was fundamental (Table 1). Previous studies have suggested that AIHA occurs when more than 95% of the cells are donor-derived, supporting a donor-versus-donor mechanism in which the donor's autoreactive B lymphocytes attack the donor's red blood cells [4].

However, in this particular case, AIHA coincided with a decrease in the percentage of donor cells and an increase in the percentage of recipient cells. This change led to a worsening of the anemia and an increase in the antibody titer, suggesting that the recipient's lymphocytes were responsible for the production of autoantibodies. Another severe relapse of AIHA confirmed this trend, with a decrease in donor cells and an increase in recipient cells. Chimerism analysis of sorted cell populations showed a predominance of recipient immune cells, with high percentages of T lymphocytes, B lymphocytes, NK cells, and monocytes.

These data support the hypothesis that, in this specific case, AIHA is mediated by recipient cells, suggesting a recipient-versus-recipient mechanism. The identified antibodies (anti-e, anti-C, anti-D) can therefore be classified as autoantibodies produced by the recipient's immune cells in response to post-transplant immune dysfunction (Table 2).

The authors declare no conflict of interest.

This work was supported by the Italian Ministry of Health with Current Research funds.

AB, CD, and EC collected the data. AB and PB wrote the manuscript. CD, FL, OP, and MV critically reviewed the manuscript for important intellectual content. All the authors gave their approval to the submitted version of the manuscript.

Considering the retrospective nature of the analysis, the current study did not require the approval of the local ethics committee according to current legislation. Data were retrospectively analyzed in line with personal data protection policies.

1. Greenmyer JR, Ariagno S, Ali A, Pence L, O'Shea M, Greenmyer LA, Khan S, Kuhn A, Martin C, Ferdjallah A, Kohorst M. Autoimmune cytopenias following pediatric hematopoietic cell transplant. Bone Marrow Transplant. 2024 Jan;59(1):117-120. doi: 10.1038/s41409-023-02116-y. Epub 2023 Oct 4. PMID: 37794111.
2. González-Vicent M, Sanz J, Fuster JL, Cid J, de Heredia CD, Morillo D, Fernández JM, Pascual A, Badell I, Serrano D, Fox L, de la Serna J, Benito A, Couselo JM, Molina B, Díaz MÁ, Sanz MÁ. Autoimmune hemolytic anemia (AIHA) following allogeneic hematopoietic stem cell transplantation (HSCT): A retrospective analysis and a proposal of treatment on behalf of the Grupo Español De Trasplante de Medula Osea en Niños (GETMON) and the Grupo Español de Trasplante Hematopoyetico (GETH). Transfus Med Rev. 2018 Mar 3:S0887-7963(17)30164-5. doi: 10.1016/j.tmrv.2018.02.005. Epub ahead of print. PMID: 29573905.
3. Gabelli M, Ademokun C, Cooper N, Amrolia PI. Pathogenesis, risk factors and therapeutic options for autoimmune haemolytic anaemia in the post-transplant setting. Br J Haematol. 2022 Jan;196(1):45-62. doi: 10.1111/bjh.17648. Epub 2021 Jun 30. PMID: 34195990.
4. Wang M, Wang W, Abeywardane A, Adikarama M, McLornan D, Raj K, de Lavallade H, Devereux S, Mufti GJ, Pagliuca A, Potter VT, Mijovic A. Autoimmune hemolytic anemia after allogeneic hematopoietic stem cell transplantation: analysis of 533 adult patients who underwent transplantation at King's College Hospital. Biol Blood Marrow Transplant. 2015 Jan;21(1):60-6. doi: 10.1016/j.bbmt.2014.09.009. Epub 2014 Sep 28. PMID: 25262883.
5. Szanto CL, Langenhorst J, de Koning C, Nierkens S, Bierings M, Huitema ADR, Lindemans CA, Boelens JJ. Predictors for Autoimmune Cytopenias after Allogeneic Hematopoietic Cell Transplantation in Children. Biol Blood Marrow Transplant. 2020 Jan;26(1):114-122. doi: 10.1016/j.bbmt.2019.07.022. Epub 2019 Jul 22. PMID: 31344451.
6. Koo J, Giller RH, Quinones R, McKinney CM, Verneris MR, Knight-Perry J. Autoimmune cytopenias following allogeneic hematopoietic stem cell transplant in pediatric patients: Response to therapy and late effects. Pediatr Blood Cancer. 2020 Sep;67(9):e28591. doi: 10.1002/pbc.28591. Epub 2020 Jul 13. PMID: 32658382.
7. Ogonek J, Kralj Juric M, Ghimire S, Varanasi PR, Holler E, Greinix H, Weissinger E. Immune Reconstitution after Allogeneic Hematopoietic Stem Cell Transplantation. Front Immunol. 2016 Nov 17;7:507. doi: 10.3389/fimmu.2016.00507. PMID: 27909435; PMCID: PMC5112259.
8. Li Z, Rubinstein SM, Thota R, Savani M, Brissot E, Shaw BE, Majhail NS, Mohty M, Savani BN. Immune-Mediated Complications after Hematopoietic Stem Cell Transplantation. Biol Blood Marrow Transplant. 2016 Aug;22(8):1368-1375. doi: 10.1016/j.bbmt.2016.04.005. Epub 2016 Apr 14. PMID: 27095688.
9. Michniacki TF, Ebens CL, Choi SW. Immune-Mediated Cytopenias After Hematopoietic Cell Transplantation: Pathophysiology, Clinical Manifestations, Diagnosis, and Treatment Strategies. Curr Oncol Rep. 2019 Aug 15;21(10):87. doi: 10.1007/s11912-019-0838-7. PMID: 31414187.
10. Michalak SS, Olewicz-Gawlik A, Rupa-Matysek J, Wolny-Rokicka E, Nowakowska E, Gil L. Autoimmune hemolytic anemia: current knowledge and perspectives. Immun Ageing. 2020 Nov 20;17(1):38. doi: 10.1186/s12979-020-00208-7. PMID: 33292368; PMCID: PMC7677104.