Monitoring of minimal residual disease (MRD) by flow cytometry (FCM) is a powerful prognostic tool for predicting outcomes in acute lymphoblastic leukemia (ALL). To apply FCM-MRD in large, collaborative trials, dedicated laboratory staff must be educated to concordantly high levels of expertise and their performance quality should be continuously monitored. We sought to install a unique and comprehensive training and quality control (QC) program involving a large number of reference laboratories within the international Berlin-Frankfurt-Münster (I-BFM) consortium, in order to complement the standardization of the methodology with an educational component and persistent quality control measures. Our QC and quality assurance (QA) program is based on four major cornerstones: (i) a twinning maturation program, (ii) obligatory participation in external QA programs (spiked sample send around, United Kingdom National External Quality Assessment Service (UK NEQAS)), (iii) regular participation in list-mode-data (LMD) file ring trials (FCM data file send arounds), and (iv) surveys of independent data derived from trial results. We demonstrate that the training of laboratories using experienced twinning partners, along with continuous educational feedback significantly improves the performance of laboratories in detecting and quantifying MRD in pediatric ALL patients. Overall, our extensive education and quality control program improved inter-laboratory concordance rates of FCM-MRD assessments and ultimately led to a very high conformity of risk estimates in independent patient cohorts.

Early clearance of leukemic cells during induction therapy of childhood acute lymphoblastic leukemia (ALL) is a basis for treatment optimization. Currently, the most widely used methods for the detection of minute residual malignant cells in the bone marrow and/or peripheral blood, minimal residual disease (MRD), are PCR and flow cytometry (FCM). Immunophenotypic modulation (IM) is a well known factor that can hamper the accurate FCM analysis.Aim: To report the IM detected by 8-color FCM during the BFM-type remission induction in 24 consecutive MRD-positive samples of children with B-cell precursor ALL and the possible implications for MRD detection.Patients and methods: Between 2010 and 2012 we prospectively followed up the MRD on days 15 and 33 of induction treatment in bone marrow (BM) samples and on day 8 in peripheral blood (PB). The IM was assessed by comparative analyses of the changes in the mean fluorescence intensity of 7 highly relevant antigens expressed by the leukemic cells and normal B-lymphocytes.Results: IM occurred, to different extents, in all analyzed day 15 BM and in most day 33 BM samples. Statistically significant changes in the MFI-levels of four CDs expressed by the leukemic blasts were observed: downmodulation of CD10, CD19 and CD34 and upmodulation of CD20. No changes in the expression of CD38, CD58 and CD45 were noticed.Conclusions: Measuring the MRD by standardized 8-color flow cytometry helps improve the monitoring of the disease, leading to better therapeutic results. However, the IM of the different antigens expressed by the leukemic blasts should be taken into consideration and cautiously analyzed.

Despite multimodal therapy, the prognosis of patients with metastatic Ewing sarcoma (ES) remains poor, with new treatments urgently needed. The disialoganglioside GD2, a well-established tumor-associated antigen, is expressed in 40% to 90% of ES cells, making it a suitable therapeutic target. Here we report 3 cases with newly diagnosed, metastatic, GD2-positive ES or Ewing-like sarcoma treated with the anti-GD2 antibody dinutuximab beta in addition to standard chemotherapeutic regimens. Treatment was well-tolerated, and all patients achieved complete remission, without evidence of relapse. First-line anti-GD2 immunotherapy in patients with metastatic, GD2-positive ES or Ewing-like sarcoma represents a promising therapeutic option that warrants further clinical evaluation.

Background: During the last four decades the prognosis of childhood acute myeloid leukemia (AML) has been substantially improved due to an increase in complete remission (CR) rates, event-free survival (EFS) and reduced early mortality. The relapsed AML still remains a therapeutic challenge. Aim: To report the AML treatment results of the Bulgarian pediatric oncohematological centers. Materials and methods: Retrospective analysis of the treatment results of children and adolescents (age from 0 to 20 years) with primary AML. Unified AML BFM- backbone type treatment protocol is used. Results: This study included 97 newly diagnosed patients (44 girls and 53 boys) with AML in Bulgaria between 2003 and 2016. The median age at diagnosis was 10.2 years. The most frequent FAB-morphologic subtype was M2 followed by M4. First complete remission (CR1) was achieved in 83 patients (85.6%). The 13-year EFS was 49%, while the overall survival (OS) was 54.6%. Twenty seven (27.8%) patients relapsed, with only 5 of them being still alive towards the end of the study period. Conclusion: The EFS and OS for the children with AML in Bulgaria are comparable with those reported by other European groups. The prognosis of relapsed AML remains still unfavorable for the past 13 years.

Minimal residual disease refers to a leukemia cell population that is resistant to chemotherapy or radiotherapy and leads to disease relapse. The assessment of MRD is crucial for making an accurate prognosis of the disease and for the choice of optimal treatment strategy. Here, we review the advantages and disadvantages of the available genetic and phenotypic methods and focus on the multiparametric flow cytometry as a promising method with greater sensitivity, speed, and standardization options. In addition, we discuss how the application of automated data analysis outweighs the use of complex combinations of windows and gates in classical analysis, thus eliminating subjective evaluation.

Background: Elevated serum IgE has been reported in severe COVID-19, suggesting that mast cell activation, allergic-like responses, and possible viral immune evasion occur. Objective: This study aimed to assess serum IgE, IgG, eosinophils, basophils, IL-10, and IL-33 in COVID-19 patients, and evaluate the infiltration of mast cells, basophils, and plasma cells in fatal cases. Methods: This retrospective study included 21 patients with severe COVID-19 or related respiratory conditions hospitalized in Plovdiv, Bulgaria (February 2020–May 2022). Serum immunoglobulins were quantified via immunoassays; IL-10 and IL-33 were also measured. Lung tissues from 30 autopsies were examined histologically and immunohistochemically using CD117 (mast cells) and CD138 (plasma cells). Results: Elevated IgE (>100 IU/mL) occurred in 10/21 patients, with two patients exhibiting levels exceeding 1000 IU/mL. High IgE correlated with reduced eosinophils and basophils, except in post-COVID lobar pneumonia. IL-10 was significantly increased, while IL-33 was reduced in acute and long COVID. Lung histology showed the accumulation of mast cells and plasma cells (5–20/field) during the diffuse alveolar damage and acute respiratory distress syndrome (ARDS) phases, but not in later fibrotic stages. Basophils are located near capillary basement membranes and the endothelium. Conclusions: SARS-CoV-2 may induce IgE-driven allergic-like mechanisms that contribute to severity. Monitoring IgE and mast cell activity may provide prognostic and therapeutic value, while elevated IgG4 could mitigate the effects of IgE.