Patients receiving an ABO-incompatible hematopoietic stem cell transplantation (SCT) are at an increased risk for immune-mediated hematological complications including immediate and delayed hemolysis, late red blood cell engraftment, and pure red cell aplasia [1, 2]. Much research effort has been invested in the unraveling of the immunological mechanisms underling these complications and approaches to prevent them . Only minimal attention has been paid to the fact that in some SCT patients, even after years, a persistent patient A- and/or B-antigen is detected in the clinical laboratory, despite 100% white cell donor chimerism. The impact for the patient can be substantial: fear that the transplantation was not successful, concern of relapse, and other anxieties influence the quality of life. Little is known about the possible causes of this phenomenon, making appropriate counseling and reassurance of patients by the clinician difficult. In this letter, we describe two cases and a short review on the putative causes of persistent blood group antigens after SCT.
The first patient is a 24-year-old Dutch male, who received an allogeneic T-cell-depleted myeloablative stem cell transplantation (SCT) from an unrelated donor in January 2008, for treatment of pre-B-acute lymphoblastic leukemia (ALL). Conditioning for this SCT was performed using anti-thymocyte globulin, cyclophosphamide, and total body irradiation. The patient's original blood group was A Rh D positive, the donor's blood group was B Rh D positive. Donor chimerism was 100% in T- and non-T-cell fraction (NK cells) 29 days after SCT. Blood group typing 3 months after SCT showed a decrease in blood group A cells (2+ reaction strength, on a scale of 0 to 4+), and strong agglutination of B cells (4+), taken into account the transfusion of seven units of erythrocytes. Results of blood group typing (Ortho AutoVue Innova, Ortho-Clinical Diagnostics, Beerse, Belgium) 6 months after SCT until now, showed a persistent A-antigen (1+ reaction), despite a 100% white cell donor chimerism. The patient did not show any signs of hemolysis and his hemoglobin was normal. Each visit to the hematology outpatient clinic, he asked his hematologist for his blood group typing results. Although the presence of a persistent blood group antigen as such is not harmful, it affected the patient's serenity. His main worry was that he did not get rid of all original stem cells and might be prone to relapse of his disease.
The second patient is a 21-year-old Dutch male, who received an allogeneic T-cell-depleted myeloablative SCT from an unrelated donor in March 2008, for treatment of Fanconi Anemia. Conditioning for this SCT was performed using anti-thymocyte globulin, cyclophosphamide, fludarabine, and busulfan. The patient's original blood group was A Rh D positive, the donor's blood group was O Rh D positive. Donor chimerism was 100% in T- and non-T-cell fraction 15 days after SCT. Blood group typing 3 weeks after SCT showed a decrease in blood group A cells (reaction strength, 0.5+), taken into account the transfusion of two units of erythrocytes. From then on, the results of blood group typing showed a persistent A-antigen (reaction, 0.5+), despite a 100% white cell donor chimerism.
The major goal of allogeneic SCT for hematological malignancies is complete eradication of malignant cells together with the development of complete donor chimerism. With the use of increasingly sensitive techniques, mixed chimerism, defined as a mixture of both donor and recipient-derived hematopoietic cells, is frequently observed after allogeneic SCT, especially in patients who receive T-cell-depleted grafts . Residual autologous hematopoiesis may represent residual normal cells belonging to one or more specific subsets that survived the conditioning regimen and that are tolerated by the transplanted donor immune system. However, these cells may also originate from the malignant clone and represent residual disease restricted by an active donor-derived immune system [4, 5].
Traditionally white cell chimerism (PCR on microsatellites) is used as a marker for SCT donor engraftment. In most cases of malignant disease, both doctor and patient are satisfied when 100% donor engraftment is achieved. Another putative marker for successful transplantation is, in case of a blood group incompatible allogeneic SCT, the obtainment of donor ABO blood group. The value of this marker to the patient is often under-appreciated by the physician. Patients often ask for their blood group typing results. The two patients described in this report asked for their results every time they visited their hematologist at the outpatient clinic. They considered the persistence of A-antigen as an indication of engraftment failure, despite 100% white cell donor chimerism.
Studies on mixed chimerism after allogeneic SCT have predominantly focused on the B- and T-lymphocyte population , but not on distinct myeloid cell lineages such as nucleated erythroid precursors and megakaryocytes . Schaap et al.  demonstrated a great individual difference in hematopoietic cells involved in this mixed chimerism after allogeneic bone marrow transplantation. Manfroi et al.  showed, by studying an enriched erythroblastic precursor population, that mixed chimerism in a 21-year-old man with acute lymphoblastic leukemia who received an allogeneic unrelated BMT, was owing to a persistent clone of recipient's red blood cell (RBC) precursors. The sensitivity of white cell chimerism analysis depends on the methodology that is used. However, sensitivity of the current real-time PCR techniques exceeds that of routine serologic blood typing assays to detect minor cell populations. In our patients, the persistent A-antigen could be detected easily (without use of microscopy). In contrast, the more sensitive white blood cell chimerism assay showed 0% patient chimerism. Additional blood group typing in our patients (Rh, Kell, Duffy, and Kidd blood group systems), almost 2 years after SCT, did not show a mixed field reaction (visible by eye or microscope) in any of the identified blood group antigens. This suggests the absence of mixed chimerism in erythroid precursors. Although a significant difference in white blood cell and RBC donor chimerism was not detected in our patients, we cannot rule out that a difference between white cell and red cell donor chimerism is the explanation for a persistent A-antigen.
Besides a difference in white and red cell chimerism, the persistent A-antigen in our patients could be owing to a partly transformation of donor B erythrocytes to AB erythrocytes. There is hardly any literature about the ABO modification of erythrocytes outside the traditional blood cell producing tissues. In blood, A-, B-, and H-antigens are, in addition to erythrocytes, found in the plasma of ABH secretors and nonsecretors although greater quantities are present in the former . Wichmann et al. , Garratty et al. , and Arndt et al.  were the first to describe the uptake of soluble A-antigen onto transplanted O RBCs. As described by Garratty et al. , a blood group A1 leukemia patient who had received a group O SCT was typed as group O 1 month post-transplant. Four months later, the patient's RBCs were reacting weakly with some anti-A and anti-A,B in tube and gel methods. By use of flow cytometry, in which the transplant patient's fixed RBCs were incubated with anti-A or anti-A,B followed by fluorescein isothiocyanate anti-Ig, a small increase in A-antigen on all RBCs was shown. A minor population of A cells could not be detected in the patient's sample and the patient was not having a relapse. The finding of full RBC donor chimerism (at least by serological techniques) for non-ABO antigens in our two patients supports the hypothesis that adsorption of A-antigen could result in the persistence of A-antigen after SCT.
Another hypothetical and harmless cause for the transformation of donor B erythrocytes to AB erythrocytes is the presence of a highly active tissue A-transferase that is able to convert remaining H-antigen on circulating B cells, resulting in RBCs expressing both A- and B-antigens. Comenzo et al.  reported that following transplantation of a liver from a group AB donor, a proportion of the red cells of a group O child became transiently group AB, as did group O transfused red cells. The authors hypothesize that the patient's anomalous ABO phenotype was caused by hepatic transferase activity.
Despite the abovementioned acquired types of an aberrant A-antigen, the phenomenon of an additional weak A- or B-antigen has also been described in healthy individuals. A- and B-specified transferases are very similar and have overlapping functions. Thus, under the appropriate conditions, the A1-transferase can synthesize group B determinants  and the B-transferase has the potential to synthesize blood group A-active structures, using the same donor and acceptor substrates as the A-transferase [15, 16]. Several laboratories observed that highly potent monoclonal anti-A reagents capable of agglutinating Ax red cells also weakly agglutinated some group B cells. This led to the recognition that the phenomenon described above, the ability of B-transferase to produce A determinants in vitro, may also occur in vivo [17, 18]. Red cells are described as B(A) when, despite coming from a B subject who lacks the A gene, they are weakly agglutinated by potent anti-A monoclonals. Although less likely, the donor of the here-described patient A, typed elsewhere as blood group B, could have had the B(A) phenotype.
In general, the observation of persistence of patient ABO antigens, despite 100% donor chimerism, after allogeneic SCT, worries the patient more than the doctor. In our clinical practice, we encountered five patients with a persistent patient blood group antigen after allogeneic SCT in the last 2 years. In the literature, no data on incidence and clinical consequences of this persistent ABO antigen can be found. None of our patients showed any signs of engraftment failure or relapse of the disease, with a follow-up time ranging from 1 to 8 years. Except for engraftment failure itself, all causes of a persistent ABO antigen, as described in this report, are benign of origin. Although a persistent patient ABO antigen, despite 100% donor chimerism, has no clinical relevance, the psychological implications, like the patient's anxiety of engraftment failure and relapse, can, however, be significant. It is important to inform the patient about the putative causes of this phenomenon and their often benign character.