Chimerism-Mediated Tolerance in Intestinal Transplantation.

In this review, the authors outlined concepts and strategies to achieve immune tolerance through inducing hematopoietic chimerism after solid organ transplantation and introduced challenges and opportunities in harnessing two-way alloresponses to improve outcomes after intestinal transplantation (ITx). Next, the authors discussed the dynamics and phenotypes of peripheral blood and intestinal graft T-cell subset chimerism and their association with outcomes. The authors also summarized studies on other types of immune cells after ITx and their potential participation in chimerism-mediated tolerance. The authors further discussed strategies and future directions to promote chimerism-associated tolerance after ITx to overcome rejection and minimize immunosuppression.

The published complete mitochondrial genome of Blue-fronted Redstart (Phoenicurus frontalis) is a chimera and includes DNA from Pink-rumped Rosefinch Carpodacus waltoni eos (Aves: Passeriformes).

The complete mitochondrial genome of Blue-fronted Redstart (Phoenicurus frontalis), GenBank accession number MT360379 (NC_053917), was published by Li and colleages in 2020. Here we show that this mitogenome is actually a chimera containing DNA fragments of both P. frontalis (15,518 bp, 92.5%) and Pink-rumped Rosefinch (Carpodacus waltoni eos, 1258 bp, 7.5%). This mitogenome has been re-used in at least three phylogenies. Our study confirms that mitogenomes are best verified with multiple gene trees, and that any anomalies should be investigated by direct comparison of sequences.

Case report: Persistent hypogammaglobulinemia and mixed chimerism after HLA class-II disparate-hematopoietic stem cell transplant.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for various hematological, immunological and metabolic diseases, replacing the patient's hematopoietic system with donor-derived healthy hematopoietic stem cells. HSCT can be complicated by early and late events related to impaired immunological recovery such as prolonged hypogammaglobulinemia post-HSCT. We present a 16-year-old female patient with sickle-cell disease who underwent HSCT with stem cells from a human leukocyte antigen (HLA) class-II mismatched family donor. While cellular recovery was good post-HSCT, the patient developed mixed chimerism and suffered from cervical lymphadenopathy, recurrent airway infections and cutaneous SLE. She presented with hypogammaglobulinemia and was started on immunoglobulin substitution therapy and antibiotic prophylaxis. B-cell phenotyping showed that she had increased transitional and naïve mature B cells, reduced memory B cells, and diminished marginal zone/natural effector cells. In-depth immunophenotyping and B-cell receptor repertoire sequencing ruled out an intrinsic B-cell defect by expression of activation-induced cytidine deaminase (AID), presence of somatic hypermutations and differentiation into IgG- and IgA-producing plasma cells in vitro. Immunohistochemistry and flow cytometry of lymph node tissue showed a clear block in terminal B-cell differentiation. Chimerism analysis of sorted lymph node populations showed that exclusively patient-derived B cells populated germinal centers, while only a minor fraction of follicular helper T cells was patient-derived. Given this discrepancy, we deduced that the HLA class-II disparity between patient and donor likely hinders terminal B-cell differentiation in the lymph node. This case highlights that studying disturbed cognate T-B interactions in the secondary lymphoid organs can provide unique insights when deciphering prolonged hypogammaglobulinemia post-HSCT.

Body-wide chimerism and mosaicism are predominant causes of naturally occurring ABO discrepancies.

Routine ABO blood group typing of apparently healthy individuals sporadically uncovers unexplained mixed-field reactions. Such blood group discrepancies can either result from a haematopoiesis-confined or body-wide dispersed chimerism or mosaicism. Taking the distinct clinical consequences of these four different possibilities into account, we explored the responsible cause in nine affected individuals. Genotype analyses revealed that more than three-quarters were chimaeras (two same-sex females, four same-sex males, one sex-mismatched male), while two were mosaics. Short tandem repeat analyses of buccal swab, hair root and nail DNA suggested a body-wide involvement in all instances. Moreover, genome-wide array analyses unveiled that in both mosaic cases the causative genetic defect was a unique copy-neutral loss of heterozygosity encompassing the entire long arm of chromosome 9. The practical transfusion- or transplantation-associated consequences of such incidental discoveries are well known and therefore easily manageable. Far less appreciated is the fact that such findings also call attention to potential problems that directly ensue from their specific genetic make-up. In case of chimerism, these are the appearance of seemingly implausible family relationships and pitfalls in forensic testing. In case of mosaicism, they concern with the necessity to delineate innocuous pre-existent or age-related from disease-predisposing and disease-indicating cell clones.

Genomic representativeness and chimerism in large collections of SAGs and MAGs of marine prokaryoplankton.

BACKGROUND: Single amplified genomes (SAGs) and metagenome-assembled genomes (MAGs) are the predominant sources of information about the coding potential of uncultured microbial lineages, but their strengths and limitations remain poorly understood. Here, we performed a direct comparison of two previously published collections of thousands of SAGs and MAGs obtained from the same, global environment. RESULTS: We found that SAGs were less prone to chimerism and more accurately reflected the relative abundance and the pangenome content of microbial lineages inhabiting the epipelagic of the tropical and subtropical ocean, as compared to MAGs. SAGs were also better suited to link genome information with taxa discovered through 16S rRNA amplicon analyses. Meanwhile, MAGs had the advantage of more readily recovering genomes of rare lineages. CONCLUSIONS: Our analyses revealed the relative strengths and weaknesses of the two most commonly used genome recovery approaches in environmental microbiology. These considerations, as well as the need for better tools for genome quality assessment, should be taken into account when designing studies and interpreting data that involve SAGs or MAGs. Video Abstract.

Allogeneic stem cell transplant in primary hemophagocytic lymphohistiocytosis - a single-center experience.

Hemophagocytic Lymphohistiocytosis (HLH) is a rare disorder of immune dysregulation characterized by fever, cytopenias, and splenomegaly. Its primary form poses a therapeutic challenge due to its high fatality when left untreated. We retrospectively analyzed 28 patients who underwent related-donor allogeneic stem cell transplant for primary HLH from 2010 to 2021. Among them were 10 cases of familial HLH, 8 cases of Griscelli syndrome type 2, and 1 case each with PRF1 and STX11 mutations. All the patients underwent transplants with reduced-intensity or myeloablative conditioning and 26 of them achieved neutrophil engraftment at a median of day + 14. The donors were either fully matched (68%) or haploidentical (32%). With a median follow-up of 1 year, overall survival was 68% (n = 19) and disease-free survival was 64.4% (n = 18). OS was better in patients transplanted with a sibling donor (compared to parent donor), who achieved complete donor chimerism, and those transplanted early in the course of the disease (diagnosis to transplant duration less than 6 months).

Prospects and Potential for Chimerism Analysis after Allogeneic Hematopoietic Stem Cell Transplantation.

Chimerism analysis after allogeneic hematopoietic stem cell transplantation serves to confirm engraftment, indicate relapse of hematologic malignancy, and attribute graft failure to either immune rejection or poor graft function. Short tandem repeat PCR (STR-PCR) is the prevailing method, followed by quantitative real-time PCR (qPCR), with detection limits of 1-5% and 0.1%, respectively. Chimerism assays using digital PCR or next-generation sequencing, both of which are more sensitive than STR-PCR, are increasingly used. Stable mixed chimerism is usually not associated with poor outcomes in non-malignant diseases, but recipient chimerism may foretell relapse of hematologic malignancies, so higher detection sensitivity may be beneficial in such cases. Thus, the need for and the type of intervention, e.g., immunosuppression regimen, donor lymphocyte infusion, and/or salvage second transplantation, should be guided by donor chimerism in the context of the feature and/or residual malignant cells of the disease to be treated.

Cell-Based Therapies Induce Tolerance of Vascularized Composite Allotransplants: A Systematic Review.

INTRODUCTION: Vascularized composite allotransplantation (VCA) is the transplantation of multiple tissue types as a solution for devastating injuries. Despite the highly encouraging functional outcomes of VCA, the consequences of long-term immunosuppression remain the main obstacle in its application. In this review, we provide researchers and surgeons with a summary of the latest advances in the field of cell-based therapies for VCA tolerance. METHODS: Four electronic databases were searched: PubMed, Scopus, Cumulative Index to Nursing and Allied Health Literature , and Web of Science. We used the Preferred Reporting Items for Systematic Reviews and Meta-Analysis as the basis of our organization. RESULTS: Hematopoietic stem cells prolonged VCA survival. A combination of immature dendritic cells and tacrolimus was superior to tacrolimus alone. T cell Ig domain and mucin domain modified mature dendritic cells increased VCA tolerance. Bone marrow-derived mesenchymal stem cells prolonged survival of VCAs. A combination of adipose-derived mesenchymal stem cells, cytotoxic T-lymphocyte antigen 4 immunoglobulin, and antilymphocyte serum significantly improved VCA tolerance. Ex-vivo allotransplant perfusion with recipient's bone marrow-derived mesenchymal stem cells increased VCA survival. Recipient's adipose-derived mesenchymal stem cells and systemic immunosuppression prolonged VCA survival more than any of those agents alone. Additionally, a combination of peripheral blood mononuclear cells shortly incubated in mitomycin and cyclosporine significantly improved VCA survival. Finally, a combination of donor recipient chimeric cells, anti-αß-T cell receptor (TCR), and cyclosporine significantly prolonged VCA tolerance. CONCLUSIONS: Evidence from animal studies shows that cell-based therapies can prolong survival of VCAs. However, there remain many obstacles for these therapies, and they require rigorous clinical research given the rarity of the subjects and the complexity of the therapies. The major limitations of cell-based therapies include the need for conditioning with immunosuppressive drugs and radiation, causing significant toxicity. Safety concerns also persist as most research is on animal models. While completely replacing traditional immunosuppression with cell-based methods is unlikely soon, these therapies could reduce the need for high doses of immunosuppressants and improve VCA tolerance.

Chimeric Cell Therapies as a Novel Approach for Duchenne Muscular Dystrophy (DMD) and Muscle Regeneration.

Chimerism-based strategies represent a pioneering concept which has led to groundbreaking advancements in regenerative medicine and transplantation. This new approach offers therapeutic potential for the treatment of various diseases, including inherited disorders. The ongoing studies on chimeric cells prompted the development of Dystrophin-Expressing Chimeric (DEC) cells which were introduced as a potential therapy for Duchenne Muscular Dystrophy (DMD). DMD is a genetic condition that leads to premature death in adolescent boys and remains incurable with current methods. DEC therapy, created via the fusion of human myoblasts derived from normal and DMD-affected donors, has proven to be safe and efficacious when tested in experimental models of DMD after systemic-intraosseous administration. These studies confirmed increased dystrophin expression, which correlated with functional and morphological improvements in DMD-affected muscles, including cardiac, respiratory, and skeletal muscles. Furthermore, the application of DEC therapy in a clinical study confirmed its long-term safety and efficacy in DMD patients. This review summarizes the development of chimeric cell technology tested in preclinical models and clinical studies, highlighting the potential of DEC therapy in muscle regeneration and repair, and introduces chimeric cell-based therapies as a promising, novel approach for muscle regeneration and the treatment of DMD and other neuromuscular disorders.

Donor Lymphocyte Infusion (DLI) post allogeneic stem cell transplant (allo-SCT) in Acute Myeloid Leukemia (AML) and High-Grade Myelodysplastic Syndrome (MDS). A longitudinal retrospective study using peripheral blood (PB) CD34+ and CD3+ donor chimerism (DC) monitoring.

INTRODUCTION: This longitudinal study was based on the outcomes of Donor Lymphocyte Infusion (DLI) for falling peripheral blood (PB) CD34+ and CD3+ donor chimerism (DC). METHODS: From 2012 to 2018, data was collected from the BMT database and electronic medical records (EMR). The primary objective was to compare the indication for DLI based on falling PB CD34+ or CD3+ DC in patients post allo-SCT for AML and MDS and their overall survival (OS). RESULTS: 18/70 patients met the inclusion criteria. Indications for DLI were i) falling PB CD34+ DC ≤ 80 % with morphological relapse, ii) falling PB CD34+ DC ≤ 80 % without morphological relapse and iii) falling PB CD3+ DC ≤ 80 % without falling PB CD34+ DC. Log rank analysis showed falling PB CD34+ DC and morphological relapse had significantly lower OS. Linear regression demonstrated better OS post DLI if there was PB CD34+ and CD3+ chimerism response at 30 days (p = 0.029), GVHD (p = 0.032) and tapering immunosuppression at the time of falling DC (p = 0.042). CONCLUSION: DLI for PB CD34+ DC values ≤ 80 % and morphological relapse had the lowest OS. In this study, full DC was achieved after DLI even with a PB CD3+DC value as low as 13 %, provided the PB CD34+ DC remained > 80 %. Further research is vital in CD34+ DC as a biomarker for disease relapse and loss of engraftment.

Efficacy of Engraftment and Safety of Human Umbilical Di-Chimeric Cell (HUDC) Therapy after Systemic Intraosseous Administration in an Experimental Model.

Cell-based therapies hold promise for novel therapeutic strategies in regenerative medicine. We previously characterized in vitro human umbilical di-chimeric cells (HUDCs) created via the ex vivo fusion of human umbilical cord blood (UCB) cells derived from two unrelated donors. In this in vivo study, we assessed HUDC safety and biodistribution in the NOD SCID mouse model at 90 days following the systemic intraosseous administration of HUDCs. Twelve NOD SCID mice (n = 6/group) received intraosseous injection of donor UCB cells (3.0 × 106) in Group 1, or HUDCs (3.0 × 106) in Group 2, without immunosuppression. Flow cytometry assessed hematopoietic cell surface markers in peripheral blood and the presence of HLA-ABC class I antigens in lymphoid and non-lymphoid organs. HUDC safety was assessed by weekly evaluations, magnetic resonance imaging (MRI), and at autopsy for tumorigenicity. At 90 days after intraosseous cell administration, the comparable expression of HLA-ABC class I antigens in selected organs was found in UCB control and HUDC therapy groups. MRI and autopsy confirmed safety by no signs of tumor growth. This study confirmed HUDC biodistribution to selected lymphoid organs following intraosseous administration, without immunosuppression. These data introduce HUDCs as a novel promising approach for immunomodulation in transplantation.

Analysis of immunogenetics interlaboratory comparisons' success rates. External quality assurance system of the Spanish Society for Immunology GECLID-SEI.

Background: For many years, transplantation outcomes were uncertain and not hopeful, until histocompatibility testing spread. Common criteria for histocompatibility assays and communications' improvement allowed an efficient organ sharing system. The possibility of organ exchanges is closely linked to the importance of interlaboratory comparisons for histocompatibility and immunogenetics methods. The external proficiency testing (EPT) systems are the most powerful quality assurance tools. They help achieve harmonization of analyses, set a standard of performance, and a common interpretation. Methods: The external quality assurance program for diagnostic immunology laboratories (Garantía Externa de Calidad para Laboratorios de Inmunología Diagnóstica, GECLID) program nowadays runs 13 external quality assurance (EQA) histocompatibility and immunogenetics schemes, with the first of them from 2011 to date: serological and molecular: low- and high-resolution human leukocyte antigen (HLA), human platelet antigen (HPA), and killer inhibitory receptor (KIR) typing(HLA-B*27, HLA-B*57:01, and coeliac disease-related HLA), cell-dependent cytotoxicity (CDC) and flow cytometry (FC) crossmatches, anti-HLA and anti-HPA antibodies, and chimerism. Results: A total of 85 laboratories participated in this subprogram in the last 12 years reporting over 1.69 M results: 1.46 M for anti-HLA and anti-HPA antibodies, 203.810 molecular typing data (HLA, HPA, and KIR genes), 2.372 for chimerism analyses, and 39.352 for crossmatches. Based on the European Federation for Immunogenetics (EFI) standards for EPT providers, the mean success rates ranged from 99.2% for molecular typing schemes and antibodies and 94.8% for chimerism, was 96.7% regarding crossmatches, and was 98.9% in serological typing. In 2022, 61.3% of the participating laboratories successfully passed every HLA EQA scheme, although 87.9% annual reports were satisfactory. Most penalties were due to nomenclature errors or misreporting of the risk associated to HLA and disease. Conclusion: This EQA confirms the reliability of HLA and immunogenetics assays in routine care. There is little heterogeneity of results of different assays used by participating laboratories, even when in-house assays are used. Reliability of test results is reasonably granted.

Immune cell profiling in intestinal transplantation.

Since the first published case study of human intestinal transplantation in 1967, there have been significant studies of intestinal transplant immunology in both animal models and humans. An improved understanding of the profiles of different immune cell subsets is critical for understanding their contributions to graft outcomes. While different studies have focused on the contribution of one or a few subsets to intestinal transplant, no study has integrated these data for a comprehensive overview of immune dynamics after intestinal transplant. Here, we provide a systematic review of the literature on different immune subsets and discuss their roles in intestinal transplant outcomes on multiple levels, focusing on chimerism and graft immune reconstitution, clonal alloreactivity, and cell phenotype. In Sections 1, 2 and 3, we lay out a shared framework for understanding intestinal transplant, focusing on the mechanisms of rejection or tolerance in the context of mucosal immunology and illustrate the unique role of the bidirectional graft-versus-host (GvH) and host-versus-graft (HvG) alloresponse. In Sections 4, 5 and 6, we further expand upon these concepts as we discuss the contribution of different cell subsets to intestinal transplant. An improved understanding of intestinal transplantation immunology will bring us closer to maximizing the potential of this important treatment.

New insights in immunomodulation for intestinal transplantation.

Tolerance is the Holy Grail of solid organ transplantation (SOT) and remains its primary challenge since its inception. In this topic, the seminal contributions of Thomas Starzl at Pittsburgh University outlined foundational principles of graft acceptance and tolerance, with chimerism emerging as a pivotal factor. Immunologically, intestinal transplantation (ITx) poses a unique hurdle due to the inherent characteristics and functions of the small bowel, resulting in increased immunogenicity. This necessitates heavy immunosuppression (IS) while IS drugs side effects cause significant morbidity. In addition, current IS therapies fall short of inducing clinical tolerance and their discontinuation has been proven unattainable in most cases. This underscores the unfulfilled need for immunological modulation to safely reduce IS-related burdens. To address this challenge, the Leuven Immunomodulatory Protocol (LIP), introduced in 2000, incorporates various pro-tolerogenic interventions in both the donor to the recipient, with the aim of facilitating graft acceptance and improving outcome. This review seeks to provide an overview of the current understanding of tolerance in ITx and outline recent advances in this domain.

The predictive value of T-cell chimerism for disease relapse after allogeneic hematopoietic stem cell transplantation.

Introduction: Chimerism is closely correlated with disease relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, chimerism rate is dynamic changes, and the sensitivity of different chimerism requires further research. Methods: To investigate the predictive value of distinct chimerism for relapse, we measured bone marrow (BM), peripheral blood (PB), and T-cell (isolated from BM) chimerism in 178 patients after allo-HSCT. Results: Receiver operating characteristic (ROC) curve showed that T-cell chimerism was more suitable to predict relapse after allo-HSCT compared with PB and BM chimerism. The cutoff value of T-cell chimerism for predicting relapse was 99.45%. Leukemia and myelodysplastic syndrome (MDS) relapse patients' T-cell chimerism was a gradual decline from 2 months to 9 months after allo-HSCT. Higher risk of relapse and death within 1 year after allo-HSCT. The T-cell chimerism rates in remission and relapse patients were 99.43% and 94.28% at 3 months after allo-HSCT (P = 0.009), 99.31% and 95.27% at 6 months after allo-HSCT (P = 0.013), and 99.26% and 91.32% at 9 months after allo-HSCT (P = 0.024), respectively. There was a significant difference (P = 0.036) for T-cell chimerism between early relapse (relapse within 9 months after allo-HSCT) and late relapse (relapse after 9 months after allo-HSCT) at 2 months after allo-HSCT. Every 1% increase in T-cell chimerism, the hazard ratio for disease relapse was 0.967 (95% CI: 0.948-0.987, P<0.001). Discussion: We recommend constant monitoring T-cell chimerism at 2, 3, 6, and 9 months after allo-HSCT to predict relapse.

Graft-versus-host disease after autologous stem cell transplantation in a recipient who underwent allogeneic stem cell transplantation 20 years earlier.

A literature review does not provide information about the safety of autologous hematopoietic stem cell transplantation (HSCT) in a recipient who has previously received allogeneic HSCT. We treated a 69-year-old woman with diffuse large B cell lymphoma. The patient received autologous stem cell transplantation (ASCT) in the second complete remission of malignant lymphoma. The patient had undergone allogeneic hematopoietic SCT (allo-HSCT) for chronic myeloid leukemia 20 years ago. Chronic myeloid leukemia had been in complete remission for the previous 20 years. Thus, the patient received autologous and allogenic HSCT 20 years apart. ASCT involves the patient receiving "self" hematopoietic cells from an allogeneic donor. In other words, this is immunologically the second allo-HSCT. The allo-HSCT 20 years ago was undergone by a related healthy brother, a human leukocyte antigen (HLA) 8/8 full matched donor. The conditioning regimen was reduced-intensity consisting of fludarabine and busulfan. The patient did not experience acute or chronic graft-versus-host disease (GVHD) after allo-HSCT. The second transplantation, ASCT was performed to the MEAM conditioning regimen. Engraftment was uneventful, and complete donor chimerism had been achieved even after ASCT. She suffered from an acute gastric mucosal lesion 52 days after ASCT. Pathological finding of gastric mucosa was nonspecific acute gastritis with significant neutrophil infiltration. Sex chromosome analysis of gastric mucosa demonstrated that mucosal cells had XX signals, whereas infiltrating neutrophils had XY signals. We speculated the patient onset of an acute gastric GVHD in this recipient after the second transplantation. This case remarked infiltration of neutrophils triggered GVHD reaction by resetting allogeneic immune reaction after the second transplantation. We describe a rare occurrence of GVHD reaction in a recipient of ASCT following allo-HSCT.

Complete mitochondrial genome MK992912 of Great Knot (Calidris tenuirostris) is a chimera with DNA from Pacific Golden Plover Pluvialis fulva (Aves: Charadriiformes).

A complete mitochondrial genome of Great Knot (Calidris tenuirostris), MK992912, was published by He and colleagues in 2020. Here we show that this mitogenome is actually a chimera containing DNA fragments of both C. tenuirostris (15,567 bp, 92.8%) and Pacific Golden Plover (Pluvialis fulva, 1208 bp, 7.2%). Detecting such errors is possible before publication if each sequenced fragment is separately analyzed phylogenetically before assembling the fragments into a single mitogenome. This mitogenome has been re-used in at least four phylogenies. The error is documented to avoid the perpetuation of erroneous sequence information in the literature.

True Donor Cell Leukemia after Allogeneic Hematopoietic Stem Cell Transplantation: Diagnostic and Therapeutic Considerations-Brief Report.

Donor cell leukemia (DCL) is a rare complication after allogeneic hematopoietic stem cell transplantation (HSCT) accounting for 0.1% of relapses and presenting as secondary leukemia of donor origin. Distinct in phenotype and cytogenetics from the original leukemia, DCL's clinical challenge lies in its late onset. Its origin is affected by donor cell anomalies, transplant environment, and additional mutations. A 43-year-old woman, treated for early stage triple-negative breast cancer, developed mixed-phenotype acute leukemia (MPAL), 12 years later. Following induction chemotherapy, myeloablative conditioning, and allo-HSCT from her fully HLA-matched brother, she exhibited multiple cutaneous relapses of the original leukemia, subsequently evolving into DCL of the bone marrow. Cytogenetic analysis revealed a complex male karyotype in 20 out of 21 metaphases, however, still showing the MPAL phenotype. DCL diagnosis was confirmed by 90.5% XY in FISH analysis and the male karyotype. Declining further intensive chemotherapy including a second allo-HSCT, she was subsequently treated with repeated radiotherapy, palliative systemic therapies, and finally venetoclax and navitoclax but died seven months post-DCL diagnosis. This case underlines DCL's complexity, characterized by unique genetics, further complicating diagnosis. It highlights the need for advanced diagnostic techniques for DCL identification and underscores the urgency for early detection and better prevention and treatment strategies.

Marmosets contain multitudes.

Single-cell RNA sequencing reveals the extent to which marmosets carry genetically distinct cells from their siblings.

Assessment of chimerism by next generation sequencing: A comparison to STR/qPCR methods.

Chimerism analysis is used to evaluate patients after allogeneic hematopoietic stem cell transplant (allo-HSCT) for engraftment and minimal measurable residual disease (MRD) monitoring. A combination of short-tandem repeat (STR) and quantitative polymerase chain reaction (qPCR) was required to achieve both sensitivity and accuracy in the patients with various chimerism statuses. In this study, an insertion/deletion-based multiplex chimerism assay by next generation sequencing (NGS) was evaluated using 5 simulated unrelated donor-recipient combinations from 10 volunteers. Median number of informative markers detected was 8 (range = 5 - 11). The limit of quantitation (LoQ) was determined to be 0.1 % recipient. Assay sample number/batch was 10-20 and total assay time was 19-31 h (manual labor = 2.1 h). Additionally, 50 peripheral blood samples from 5 allo-HSCT recipients (related: N = 4; unrelated: N = 1) were tested by NGS and STR/qPCR. Median number of informative markers detected was 7 (range = 4 - 12). Results from both assays demonstrated a strong correlation (Y = 0.9875X + 0.333; R2 = 0.9852), no significant assay bias (difference mean - 0.08), and 100 % concordant detection of percent recipient increase ≥ 0.1 % (indicator of increased relapse risk). NGS-based chimerism assay can support all allo-HSCT for engraftment and MRD monitoring and simplify clinical laboratory workflow compared to STR/qPCR.