Bone marrow failure (BMF) syndromes are a heterogeneous group of benign hematological conditions with common clinical features including reduced bone marrow cellularity and peripheral blood cytopenias. Acquired aplastic anemia (AA) is caused by T helper(Th)1-mediated immune responses and cytotoxic CD8+ T cell-mediated autologous immune attacks against hematopoietic stem and progenitor cells (HSPCs). Interferon-γ (IFNγ), tumor necrosis factor-α, and Fas-ligand are historically linked to AA pathogenesis because they drive Th1 and cytotoxic T cell-mediated responses and can directly induce HSPC apoptosis and differentiation block. The use of omics technologies has amplified the amount of data at the single-cell level, and knowledge on AA, and new scenarios, have been opened on \"old\" point of view. In this review, we summarize the current state-of-art of the pathogenic role of IFNγ in AA from initial findings to novel evidence, such as the involvement of the HIF-1α pathway, and how this knowledge can be translated in clinical practice.

Previous studies have verified the dysfunction of mesenchymal stem cells (MSCs) for immunoregulation in acquired aplastic anemia (AA) patients. Exosomes derived from MSCs can partially substitute MSCs acting as immune regulator. Dysfunction of exosomes (Exos) derived from AA-MSC (AA-Exos) may play a key role in immunologic dissonance.
In this study, CD3 + T cells were collected and cocultured with AA-Exos and exosomes derived from HD-MSC (HD-Exos). The proliferation, differentiation and activation of CD3 + T cells were detected to compare the immunosuppressive effects between AA-Exos and HD-Exos. An immune-mediated murine model of AA was structured to compare the therapeutic effect of AA-Exos and HD-Exos. Furthermore, total RNA including miRNA from exosomes we purified and total RNA of CD3 + T cells were extracted for RNA-seq in order to construct the miRNA-mRNA network for interactions and functional analysis.
AA-Exos had impaired inhibition effects on CD3 + T cells in terms of cell proliferation, activation and differentiation compared with exosomes from HD-Exos. HD-Exos showed a more effective rescue of AA mice compared to AA-Exos. Importantly, we found some differentially expressed miRNA involved in immune response, such as miR-199, miR-128 and miR-486. The Gene Ontology analysis of differentially expressed genes (DEGs) revealed involvement of various cellular processes, such as lymphocyte chemotaxis, lymphocyte migration and response to interferon-gamma. The Kyoto Encyclopedia of Genes and Genomes analysis illustrated upregulation of critical pathways associated with T cell function after coculturing with AA-Exos compared with HD-Exos, such as graft-versus-host disease, Th17 cell differentiation and JAK-STAT signaling pathway. A miRNA-mRNA network was established to visualize the interaction between them.
In summary, AA-Exos had impaired immunosuppressive effect on T cells, less ability to rescue AA mice and differently expressed miRNA profile, which might partly account for the pathogenesis of AA as well as provide a new target of AA treatment.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired X-linked, clonal hematopoietic stem cell disease. Patients with PNH may complain of vague symptomatology that contributes to the challenge of its diagnosis. This is especially true in the clinical context of a coinciding hematologic disorder. Aplastic anemia (AA) is an additional immune-mediated illness that results in the destruction of hematopoietic precursors and pancytopenia. The authors encourage screening for PNH clones in patients initially diagnosed with AA, treating underlying hematologic disease to prevent clonal expansion, and further research to investigate the effectiveness of eculizumab in an unusual \"classical\" PNH secondary to AA with hypercellular bone marrow.

Acquired aplastic anemia (AA) is a bone marrow failure disorder characterized by pancytopenia, and immunosuppressive therapy (IST) is the optional first-line management. Several studies identified the influencing factors on IST response; however, there are still a considerable number of patients suffering from poor prognoses. In this study, we enrolled 61 AA patients aged ≤ 40 years old, and whole-exome sequencing (WES) found unexpected high FANC heterozygous germline mutations (28/61, 45.9%). Patients with FANC mutations have a significantly lower absolute reticulocyte count and CD34+ % in the bone marrow and also lower 3-, 6-, and 9-month IST response than that without mutation, which were 0% vs. 25% (P = 0.017), 26.3% vs. 42.1% (P = 0.495), and 29.4% vs. 72.2% (P = 0.011), especially in anti-thymocyte globulin combined with the cyclosporin A (ATG + CsA) group, which were 0% vs.33.4% (P = 0.143), 25% vs.83.3% (P = 0.103), and 25% vs. 100% (P = 0.003), respectively. The event-free survival in the FANCwt group was also better than that in the FANCmut group (P = 0.016) and also showed in patients who received ATG + CsA treatment (P = 0.045). In addition, all the adverse effects of FANC germline mutation were not significant in stem cell-transplanted group. Our result indicated that the WES-based detection of FANC heterozygous germline mutations may have a great meaning in predicting IST response of acquired AA. This study was registered at chictr.org.cn (# ChiCTR2100054992).

Differential diagnosis of pancytopenia with bone marrow (BM) hypoplasia represented by aplastic anemia (AA) is often challenging for physicians, because no laboratory tests have been established, until recently, to distinguish immune-mediated BM failure, which includes acquired AA (aAA) and a subset of low-risk myelodysplastic syndrome (MDS), from non-immune BM failure, which is primarily caused by genetic abnormalities in hematopoietic stem cells (HSCs). HSCs of healthy individuals often undergo somatic mutations, and some acquire phenotypic changes that allow them to escape immune attack against themselves. Once an immune attack against HSCs occurs, HSCs that undergo somatic mutations survive the immune attack and continue to produce their progenies with the same genetic or phenotypic changes. The presence of mature blood cells derived from mutated HSCs in the peripheral blood serves as evidence of the immune-mediated destruction of HSCs. Glycosylphosphatidylinositol-anchored protein-deficient (GPI[-]) blood cells and HLA class I allele-lacking (HLA[-]) leukocytes are two major aberrant cell types that represent the immune mechanism underlying BM failure. This review focuses on the importance of identifying immune mechanisms using laboratory markers, including GPI(-) cells and HLA(-) leukocytes, in the management of BM failure.

BACKGROUND:  Aplastic anemia (AA) is characterized by pancytopenia and hypocellular marrow in the absence of an abnormal infiltrate or increase in reticulin fibrosis. The diagnosis of AA is challenging at times due to decreased cellularity and overlapping morphological features with other bone marrow failure syndromes. Hepatitis-associated aplastic anemia (HAAA) is a rare variant in which patients typically present with jaundice and hepatitis followed by pancytopenia almost within 6 months. Post-hepatitis AA accounts for approximately 1-5% of cases, and invariably such cases are negative for the known hepatitis virus as well. There is limited literature available to understand the correlation of AA with hepatitis with none reported at the national level in our region. As AA is relatively more prevalent in Southeast Asia as compared to the western world and hepatitis is a prevalent disease in our population, the main purpose of this study was to assess the hepatic profile and determine the association of hepatitis in AA at the time of diagnosis.
METHODS:  A cross-sectional study was carried out at the National Institute of Blood Disease and Bone Marrow Transplantation, Karachi, from November 2019 to December 2020 after the informed consent from patients. The study included all treatment-naïve patients of acquired AA with no prior history of taking steroids, immunosuppressive treatment, or chemoradiation therapy. Liver function tests, complete blood count, prothrombin time (PT), and activated partial thromboplastin time were performed, along with viral profiles (HAV, Hep B, Hep C, and HIV). SPSS version 23 (IBM Corp., Armonk, NY) was used for statistical analysis. Mean and standard deviations were computed for quantitative variables while percentages and frequencies were reported for qualitative variables. T-test was used to observe the main difference between groups and a p-value <0.05 was considered to be significant.
RESULTS:  Out of a total of 351 patients, 29 (8.2%) patients with AA tested positive for viral hepatitis. Hepatitis A was the most prevalent hepatitis (4.0%), followed by hepatitis C (3.7%). The comparison of platelet counts in patients with and without hepatitis was reported to be of statistical significance (p-value < 0.05). A significant statistical difference (p-value < 0.0001) was found in platelet count and PT in patients of AA with and without hepatitis.
CONCLUSIONS:  Overall, this study revealed that <10% of patients of AA had a positive screening for hepatitis A, B, and C and low platelet count, and PT was statistically significant when compared between the patients with and without hepatitis. Hepatitis being prevalent in our part of the world might have an important causal association with AA. Patients with AA should be screened for liver functions and viral hepatitis at the time of diagnosis. In addition to hepatitis A, B, and C and HIV, other causes of hepatitis should also be screened such as parvovirus B19, human herpes virus 16, and adenovirus which are not included in routine diagnostic viral testing panel.

Cyclosporine (CsA) is a component of the first-line treatment for acquired aplastic anemia (acquired AA) in pediatric patients. This study aimed to develop a population pharmacokinetic (PK) model of CsA in Chinese pediatric patients with acquired AA to inform individual dosage regimens. A total of 681 CsA whole blood concentrations and laboratory data of 157 pediatric patients with acquired AA were retrospectively collected from two hospitals in Shanghai. A nonlinear mixed-effect model approach was used to build the population PK model. Potential covariate effects of age, body weight, and biochemical measurements (renal and liver functions) on CsA PK disposition were evaluated. Model fit was assessed using the basic goodness of fit and a visual predictive check. The CsA concentration data were accurately described using a two-compartment disposition model with first-order absorption and elimination. Body weight value was implemented as a fixed allometric function on all clearance and volume of distribution parameters. Total bilirubin level was identified as a significant covariate on apparent clearance (CL/F), with a 1.07% reduction per 1 nmol/L rise in total bilirubin level. The final estimates for CL/F and central volume (Vc/F) were 29.1 L/h and 325 L, respectively, for a typical 28 kg child. Other covariates (e.g., gender, age, albumin, hemoglobin, hematocrit, serum creatinine, and concomitant medication) did not significantly affect the PK properties of CsA. This population PK model, along with a maximum a posteriori Bayesian approach, could estimate individual PK parameters in pediatric patients with acquired AA to conduct individual CsA therapy.

Aplastic anemia (AA) and myelodysplastic syndrome (MDS) are clonal diseases with hemopoietic stem cell (HSC) abnormalities,which are sometimes difficult to be distinguished from each other.The development of molecular detection techniques has facilitated our understanding of the molecular pathogenesis of the two diseases.This article reviewed the somatic mutation (SM) and cytogenetic changes of AA,and analyzed their molecular relationship with MDS and their roles in disease transformation.The most common somatic change in AA is the loss of PIGA and HLA alleles,which,along with trisomy 8 and del(13q),is related to the immune pathogenesis of AA.Among the 5 most common mutations in AA,PIGA and BCOR/BCORL1 mutations are related to a good prognosis,while DNMT3A and ASXL1 mutations are likely associated with clonal evolution and a poor prognosis.The risk factors for secondary MDS after AA include SM and cytogenetic changes such as del(7q) associated with poor prognosis,prolonged disease duration after diagnosis,onset age of AA,and leukocyte telomere attrition.Although role of SM in disease progression remains unclear because of its dynamic change and unknown significance,prognostic assessment based on the monitoring of SM and clinical features may guide the treatment.

Secondary poor graft function (sPGF) increases the risk of life-threatening complications after hematopoietic stem cell transplantation (HSCT). The incidence, clinical outcomes, and risk factors of sPGF have not been elucidated in haploidentical (haplo-) HSCT for acquired aplastic anemia (AA) patients. We retrospectively reviewed 423 consecutive AA patients who underwent haplo-HSCT between January 2006 and December 2020 and report a 3-year cumulative incidence of 4.62% (95% confidence interval [CI]: 3.92%-10.23%) of sPGF. While no primary PGF occurred. The median time to sPGF was 121 days (range 30-626 days) after transplantation. To clarify the risk factors for sPGF, 17 sPGF cases and 382 without PGF were further analyzed. Compared to patients without PGF, the 2-year overall survival was significantly poorer for sPGF patients (67.7% vs 90.8%, p =.002). Twelve sPGF patients were alive until the last follow-up, and 7 achieved transfusion independency. The multivariable analyses revealed that later neutrophil engraftment (OR 2.819, p=.049) and a history of refractory cytomegalovirus viremia (OR=7.038, p=.002) post-transplantation were associated with sPGF. There was weak evidence that a history of grade 3-4 acute graft-versus-host disease increased the risk of sPGF (p=.063). We advocated better post-transplantation strategies to balance the risk of immunosuppression and viral reactivation for haplo-HSCT in AA patients.

Acquired aplastic anemia (aAA) is an autoimmune disease, characterized by infiltration of T lymphocytes in the bone marrow with destruction of hematopoietic stem cells by the effector cells. Interferon-gamma (IFN-γ) and perforin are important mediators of cell destruction. In this flow cytometry-based study, we have investigated the percentage of intracellular IFN-γ+ and perforin+ CD5+ T cells in peripheral blood of newly diagnosed aAA patients before and after immunosuppressive therapy (IST). Patients were categorized as per standard disease severity and response to IST. The median percentage of IFN-γ+ and perforin+ CD5+ T cells was higher in untreated patients compared to healthy controls. The percentage of these cells was also increased in untreated severe and very severe aplastic anemia when compared with non-severe aplastic anemia patients. In patients before and after IST the median percentage of T cells producing IFN-γ and perforin was elevated in non-responders as compared to partial plus complete responders. The higher percentage of IFN-γ+ and perforin+ CD5+ T cells may be useful as an early diagnostic marker for aberrant activation of immune system and predict poor response to IST in aAA patients, who will benefit from alternative therapy.