Adequate re-establishment of thymopoiesis is critical for long-term immune reconstitution after hematopoietic cell transplantation (HCT), potentially impacting patient survival rates. This study ...aimed to evaluate immune reconstitution in pediatric HCT recipients by quantifying recent thymic emigrants (RTEs), specifically CD3+CD31+CD45RA+ cells.
We conducted a retrospective analysis of 186 pediatric patients transplanted between 2013 and 2020, undergoing their first allogeneic HCT, who were alive in the first 100 days after transplantation with immune recovery evaluation at three time points: day 100, day 180 and day 360 after HCT. We analyzed the distribution of peripheral blood subsets of T, B and natural killer lymphocytes and assessed the impact of underlying disease, HCT type, stem cell source, recipient age, conditioning regimen, graft-versus-host disease (GVHD) occurrence and cytomegalovirus (CMV) reactivation on immune recovery.
At day 100, patients under 10 years exhibited higher RTE CD4+ and CD8+CD31+CD45RA+ counts compared with older patients (5.3 versus 2.2 cells/µL, P = 0.022 and 48 versus 72.8 cells/µL, P = 0.049, respectively). Patients with haploidentical HCT had lower RTE CD4+ counts compared with those with unrelated or related donors (2.4 versus 4.4 versus 7.9 cells/µL, P = 0.024). Administration of rabbit anti-thymocyte globulin negatively impacted RTE CD4+ production (median, 6.5 versus 2.4 cells/µL, P = 0.007). At day 180, the presence of GVHD had a negative influence on RTE production (11.7 versus 56.8 cells/µL, P < 0.001), particularly higher-grade acute GVHD (without, 56.8 cells/µL, grade 1–2, 28.1 cells/µL, grade 3–4, 6.0 cells/µL, P < 0.001). Patients with CMV reactivation had higher CD8+CD31+CD45RA+ compared with those without reactivation (median, 204.6 versus 100.2 cells/µL, P = 0.022). At day 360, no variables significantly affected RTE recovery. Overall survival at 5-year follow-up was 87.7%, with a median of 1170 days (range, 122–3316). Multivariate analysis showed that age >10 years (P = 0.038), negative CMV donor serology (P = 0.0029) and acute GVHD (P = 0.0026) had a negative impact on survival.
This study highlights variations in RTE production based on patient age, donor type and immunosuppression regimen employed.
The minimal residual disease (MRD) status plays a crucial role in the treatment of acute lymphoblastic leukemia (ALL) and is currently used in most therapeutic protocols to guide the appropriate ...therapeutic decision. Therefore, it is imperative that laboratories offer accurate and reliable results through well standardized technical processes by establishing rigorous operating procedures.
Our goal is to propose a monoclonal antibody (MoAb) panel for MRD detection in ALL and provide recommendations intended for flow cytometry laboratories that work on 4-color flow cytometry platforms.
The document includes pre-analytical and analytical procedures, quality control assurance, technical procedures, as well as the information that needs to be included in the reports for clinicians.
Bone marrow-derived stem cells (BMDSCs) play an essential role in organ repair and regeneration. The molecular mechanisms by which hormones control BMDSCs proliferation and differentiation are ...unclear. Our aim in this study was to investigate how a lack of ovarian or/and thyroid hormones affects stem cell number in bone marrow lineage. To examine the effect of thyroid or/and ovarian hormones on the proliferative activity of BMDSCs, we removed the thyroid or/and the ovaries of adult female rats. An absence of ovarian and thyroid hormones was confirmed by Pap staining and Thyroid Stimulating Hormone (TSH) measurement, respectively. To obtain the stem cells from the bone marrow, we punctured the iliac crest, and aspirated and isolated cells by using a density gradient. Specific markers were used by cytometry to identify the different BMDSCs types: endothelial progenitor cells (EPCs), precursor B cells/pro-B cells, and mesenchymal stem cells (MSCs). Interestingly, our results showed that hypothyroidism caused a significant increase in the percentage of EPCs, whereas a lack of ovarian hormones significantly increased the precursor B cells/pro-B cells. Moreover, the removal of both glands led to increased MSCs. In conclusion, both ovarian and thyroid hormones appear to have key and diverse roles in regulating the proliferation of cells populations of the bone marrow.
Flow cytometry has become an increasingly important tool in the clinical laboratory for the diagnosis and monitoring of many hematopoietic neoplasms. This method is ideal for immunophenotypic ...identification of cellular subpopulations in complex samples, such as bone marrow and peripheral blood. In general, 4-color panels appear to be adequate, depending on the assay. In acute leukemias (ALs), it is necessary identify and characterize the population of abnormal cells in order to recognize the compromised lineage and classify leukemia according to the WHO criteria. Although the use of eight- to ten-color immunophenotyping panels is wellestablished, many laboratories do not have access to this technology.
In 2015, the Brazilian Group of Flow Cytometry (Grupo Brasileiro de Citometria de Fluxo, GBCFLUX) proposed antibody panels designed to allow the precise diagnosis and characterization of AL within available resources. As many Brazilian flow cytometry laboratories use four-color immunophenotyping, the GBCFLUX has updated that document, according to current leukemia knowledge and after a forum of discussion and validation of antibody panels.
Recommendations for morphological analysis of bone marrow smears and performing screening panel for lineage (s) identification of AL were maintained from the previous publication. The lineage-oriented proposed panels for B and T cell acute lymphoblastic leukemia (ALL) and for acute myeloid leukemia (AML) were constructed for an appropriate leukemia classification.
Three levels of recommendations (i.e., mandatory, recommended, and optional) were established to enable an accurate diagnosis with some flexibility, considering local laboratory resources and patient-specific needs.
Bernard-Soulier Syndrome (BSS) is an inherited recessive bleeding disorder. In some instances, diagnosis might be restricted to routine blood exams, including bleeding time, prothrombin time (PT), ...and partial thromboplastin time (APTT). Exams such as platelet aggregation, and testing for expression of ristocetin cofactor, or von Willebrand factor may not be commonly performed. This leads to misdiagnosis in a number of patients, which are subsequently treated erroneously. Flow cytometry has been used widely as a tool in the diagnosis of leukemias, lymphomas, and many other immuno-hematological diseases. The purpose of this study was to assess whether flow cytometry could be helpful in the diagnosis of Bernard-Soulier Syndrome in Brazilian patients. For this, we examined a selected group of 15 patients with suspected BSS based on classical diagnosis. We used a panel of antibodies to detect the expression of glycoproteins GPIb , GPIIb, GPIIIa, GPIX, as well as CD9. Abnormalities of GPIb and GPIX were observed in nine of the 15 patients, which included severe reduction of both antigens, of one or the other, or normal levels but weak expression. Strikingly, this abnormality correlated with severely reduced expression of CD9 in all cases. We discuss the implications for flow cytometric diagnosis of BSS.
Introduction: Successful treatment of acute myeloid leukemia (AML) remains a clinical challenge due to the toxicity of current therapies and the relatively poor responses to the current standard ...therapy. Thus, treatments that address novel therapeutic targets are urgently needed. Endoglin, also known as CD105, is a receptor of the transforming growth factor-beta (TGF-β) superfamily, found to be expressed in functional long-term repopulating hematopoietic stem cells. CD105 is expressed in several cancers, including AML. However, because CD105 expression has been studied mostly in the context of solid tumors and angiogenesis, relatively little is known about CD105 expression and its function in hematopoietic malignancies. Ongoing studies from our group have revealed distinct expression of this receptor in the majority of the blast population in cases of AML and acute lymphoid leukemia (ALL). Importantly, utilizing a xenograft model we found leukemia-forming-activity to be enriched in the CD105+ fraction of primary AML blasts. Based on these encouraging initial findings, we investigated the therapeutic potential of inhibiting CD105 signaling in AML using TRC105, a monoclonal antibody to CD105 that is currently being investigated in clinical trials for the treatment of multiple solid tumors.
Materials and Methods: Sub-lethally irradiated NSG mice were intravenously injected with 5x105 human AML blast cells isolated from primary engrafted recipient mice. After infusion, mice were randomly divided into groups and treated with TRC105 (2mg/kg - IV. every 3 days) or IgG isotype control. Treatment started 2 or 30 days after the injection of AML blasts, and mice were monitored every 2 weeks for the presence of human CD45+ cells in the peripheral blood by FACS. Bone marrow and spleen were harvested at a predefined endpoint for analyses.
Results: Inhibition of CD105 signaling using TRC105 suppressed the ability of AML blasts to give rise to leukemia in vivo. In mice that were treated on D+2 of AML inoculation, untreated or IgG control treated mice displayed a clear sub-population of human blast cells in the peripheral blood (~ 20%) at week 4, which increased significantly by week 8 (~60%) and 12 (~ 90%). In contrast, TRC105-treated mice exhibited significantly lower levels of human leukemia by week 4 (~5%), which remained low by week 12 (~10%). Similar results were observed in the spleen. Remarkably, treatment with TRC105 starting at day 30, once human engraftment was detected, inhibited further development of leukemia and increased survival. The sub-population of human blasts in peripheral blood in this group remained low by week 12 (~15%), unlike their untreated controls (~ 90%).
Conclusion: Inhibition of CD105 using TRC105, a CD105 antibody in Phase 2 trials in solid cancer patients, inhibited the ability of injected human primary AML blasts to produce leukemia in NSG mice. Therefore TRC105 represents a potential novel therapy for AML. This is of particular relevance if we consider the population of AML patients that are either unable to receive intensive chemotherapy (i.e., elderly AML) or those who do not respond to standard chemotherapy.
Theuer:TRACON Pharmaceuticals: Employment. Perlingeiro:TRACON Pharmaceuticals: Research Funding.
Fanconi anemia is an autosomal recessive or X-linked genetic disorder characterized by bone marrow (BM) failure/aplasia. Failure of hematopoiesis results in depletion of the BM stem cell reservoir, ...which leads to severe anemia, neutropenia and thrombocytopenia, frequently requiring therapeutic interventions including hematopoietic stem cell transplantation (HSCT). Successful BMT requires reconstitution of normal immunity.
In the present study, we performed a detailed analysis of the distribution of peripheral blood subsets of T, B and NK lymphocytes in 23 patients with Fanconi anemia before and after BM transplantation (at days D+30, D+60 and D+100, D+180 D+270 and D+360). In parallel, we also compared the patients groups who received related and unrelated bone marrow, pre and one year after BMT.
After transplantation, we found different kinetics of recovery for the distinct major lymphocyte subsets. NK cells were the first to recover, followed by cytotoxic CD8+T-cells and B-cells, and finally also the CD4+ T-helper cells. Early recovery was at the expenses of memory cells which would therefore be potentially derived from the graft whereas, recent thymic emigrant (CD31+ CD45RA+) and naive CD4+ or CD8+T-cells, rose only 6 months after HSCT, in the presence of immunosuppressive graft-vs-host disease (GVHD) prophylactic agents. While only slight differences were observed in the early recovery of cytotoxic CD8+ T-cells among cases receiving a graft from related vs. unrelated donors, cases suffering from GVHD displayed more marked differences vsother patients.
Overall, our results support the utility of post-transplant monitoring of peripheral blood lymphoid subsets, for improved monitoring and follow-up of Fanconi anemia patients undergoing BMT.
No relevant conflicts of interest to declare.
Mature circulating endothelial cells (CEC) and circulating endothelial progenitor cells (EPC) have been described in several conditions associated with endothelial injury. Their role in deep vein ...thrombosis (DVT) has not been previously evaluated.
In this pilot study we evaluated the time course of CEC and EPC release after vena cava experimental DVT in mice, using the FeCl3 model. We also evaluated their presence in patients with DVT at different phases of the disease (acute and chronic phase). CEC and EPC were evaluated by Flow Cytometry.
In mice, both CEC and EPC were increased 24 hours after DVT induction, peaking 48 hours thereafter. After 72 hours, CEC counts decreased sharply, whereas EPC counts decreased less substantially. In DVT patients we observed a significant increase in CEC counts immediately after DVT compared to healthy individuals. Patients with chronic disease also presented a significant elevation of these cell count. In a subgroup of patients for whom serial samples were available, CEC counts decreased significantly after 9-15 months of the acute event.
Our results suggest the participation of these cells in the reparative processes that follows DVT, both at immediate and late time-points. The different kinetics of CEC and EPC release in experimental DVT suggests a heterogeneous role for these cells in the reparative events after DVT.
Abstract 1156▪▪This icon denotes a clinically relevant abstract
Bernard-Soulier syndrome (BSS) is a rare hereditary bleeding disorder, caused by mutations within the glycoprotein (GP) Ib alpha, GP Ib ...beta and GP IX genes that encode three of four subunits of the platelet GP Ib-V-IX adhesion receptor. In the present study we evaluated the mutations involved in the diagnosis of BSS from twenty-two unrelated patients. Patients and Methods: All patients were followed in two large bleeding disorder reference centers in Brazil. The diagnosis of BSS was established based on the presence of mucocutaneous bleeding and macrotrombocytopenia, and was confirmed by platelet ristocetin aggregation and flow cytometry for the platelet GP Ib alpha (CD 42b), GP Ib beta (CD 42c), and GP IX (CD 42a). Available first- and second-degree relatives were also contacted for clinical, laboratory and molecular evaluation. Genomic DNA from all index cases was used for sequence analysis of the three genes, GP1BA, GP1BB and GP9, and the results were confirmed in relatives when available. Results: Twenty-two unrelated patients with the confirmed diagnosis of BSS were enrolled in this study. Among twenty-two index cases, twenty-one had one or two mutations identified, including six novel mutations. We also identified two mutations that have been previously reported, GP1BA C209S (Simsek, et al., 1994), and GP9 A140T (Wang, et al., 2004). The six novel mutations correspond to conserved regions, and they consist of two mutations in the GP1BA (L51R, and L99P), three in GP1BB (M-25A, L72R, and L112P), and one in GP9 (P52Q). One of these novel mutations, the GP1BB L112P was observed in twelve of twenty-two unrelated cases. PCR-restriction fragment length analysis of genomic DNA from a hundred normal unrelated controls were performed to evaluate the presence of GP1BA L99P and GP1BB L112P mutations by using the AlwN I and Alu I restriction enzymes, respectively. All controls were negative for both mutations. Interestingly, when we analyzed the relatives of the index cases indentified with the mutations GP1BA L99P and GP1BB M-25A, we found evidence of mild macrotrombocytopenia in the heterozygote carriers of these mutations. The relatives heterozygous for GP1BB L112P showed normal platelet count and morphology. However, in three index cases with mild macrotrombocytopenia, GP1BB L112P in heterozygosity was the only mutation identified. Furthermore, site-directed mutagenesis was carried out to evaluate the expression of the novel mutations GP1BA L51R, and GP1BA L99P. Compared to the GP1BA wild-type, both mutations were only minimally expressed in CHO bIX cells, which stably express GP Ib beta and GP IX. Conclusions: We identified in this study eight distinct mutations among twenty-two unrelated SBS patients, including six novel mutations. The GP1BB L112P mutation was found in twelve of the twenty-two index cases, suggesting that this could be due to a founder effect. Identifying such a frequent mutation in this population of BSS patients will be helpful for genetic diagnosis of this condition in Brazil. Furthermore these mutations significantly add to the mutation database of BSS and will inevitably provide insights into the function of GP Ib-V-IX.
No relevant conflicts of interest to declare.