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Use of Four-Color Flow Cytometric Assay for Discrimination of Hematogone from Lymphoblast: Critical Issue for MRD Assessment in B-ALL Patients
Dr Mehdi Allahbakhshian Farsani, Dr Esmaeil Shahabi Satlsar , Dr Alireza Mohseni, Dr Mohammad Mosleh, Dr Mahdieh Mehrpouri , Dr Mahnaz Agaeipour, Dr Mohammad Hossein Mohammadi , Dr Roohollah Gholampour, Dr Farzaneh Jadali,
Volume 10, Issue 1 (1-2020)
Abstract
Background: Hematogones are normal B-cell precursor which can be seen in different physiological and pathological conditions. Due to variation in B-cell acute lymphoblastic leukemia (B-ALL) blasts immunophenotyping and interference of hematogones in minimal residual disease (MRD) assessment, precise discrimination of hematogones is very crucial.  The purpose of this study was to evaluate the expression pattern of surface markers in hematogones and compare them with lymphoblasts.
Material and Methods: In this applied study, flow cytometric analysis was performed using Coulter FC-500 and MXP software in 4-color combination and 6 different tubes. In this study, 85 patients diagnosed with acute lymphoblastic leukemia were evaluated. Out of these patients, 45 were boys and 40 were girls. Patients aged from 1 to 15 years old. In addition, 27 bone marrow samples from other patients aged 4 to 18 years were included in this investigation. These samples had been obtained for other diagnostic purposes, such as immune thrombocytopenic purpura and juvenile idiopathic arthritis.
Results: During flow cytometric analysis, hematogones showed expressions of CD19, CD20, CD22, CD10, CD45, CD81, CD123, CD9, CD34 (partial expression), and tdt (partial expression). In these patients, hematgones were negative for CD66c expression. Lymphoblastic cells were positive for CD19, CD20 (in some cases), CD22, CD10, CD45, CD81, CD123, CD58, CD9, CD66c, CD34 (in most cases), and TDT. CD81 mean fluorescence intensity (MFI) in hematogones was higher than that in lymphoblasts. (112.5 (30-251) vs. 17.5 (5-30); P<0.0001)
Conclusion: According to findings of this study, it seems that the use of CD81, CD58, CD123, CD66c, CD9, and CD81 MFI in combination with B-Cells associated markers can be very effective in differentiating hematogone from lymphoblast.

The Role of Autophagy-Associated Genes in the Pathogenesis of Patients with Acute Lymphoblastic Leukemia
Dr Mahdieh Mehrpouri, Dr Esmail Shahabi Satlsar, Dr Hamed Mohammadi,
Volume 15, Issue 2 (3-2025)
Abstract
Background: Expanding the knowledge of the underlying molecular mechanisms in acute lymphoblastic leukemia (ALL) is of great importance to improving treatment outcomes. Autophagy, a critical and evolutionarily conserved pathway, plays an important role in maintaining cellular homeostasis under stressful conditions. This pathway consists of several sequential steps. The present study aimed to evaluate the expression levels of autophagy-related protein 3 (ATG3), autophagy-related protein 5 (ATG5), autophagy-related protein 7 (ATG7), autophagy-related protein 14 (ATG14), and urothelial cancer-associated 1 (UCA1) genes in B-ALL patients in order to better comprehend the autophagy pathway in B-ALL.
Materials and Methods: This research is a case-control study. The bone marrow of 50 newly diagnosed patients with B-ALL (mean age = 12.3 years) and 15 healthy controls (mean age = 13.4 years) was evaluated by real-time PCR to analyze the expression of the aforementioned genes. Additionally, morphological, immunophenotypic, and molecular analyses were conducted to examine the phenotypes, genotypes, and percentage of lymphoblasts, respectively.
Results: The findings revealed that B-ALL patients exhibited significantly higher expression of ATG3, ATG5, ATG7, and ATG14 genes compared to the healthy volunteers (P < 0.001). However, there was no significant difference in UCA1 levels between the two groups (P > 0.05). Interestingly, ATG3, ATG5, ATG7, ATG14, and UCA1 had similar mRNA expression levels in the patients with different types of chromosome abnormalities and immunophenotypes.
Conclusion: Based on these results, the substantial increase in the expression of ATG3, ATG5, ATG7, and ATG14 genes suggests that the autophagy pathway is activated in B-ALL patients. This activation may contribute to tumor growth. Furthermore, the detection of autophagy gene expression could serve as a novel marker to monitor the response of B-ALL patients to treatment.

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