Objective:Explore the clinical utility of TRBC1/TRBC2 dual staining by flow cytometry in determining clonality in T-cell lymphomas.Methods:This is a retrospective case-control study. A total of 40 patients with T-cell lymphoma involving bone marrow from the First Affiliated Hospital of Zhengzhou University were enrolled between December 10, 2024 and March 5, 2025. This cohort included 30 cases of mature T-cell lymphoma, 16 males and 14 females, age 62 (54, 71)years, and 10 cases of T-lymphoblastic leukemia/lymphoma[age 11(7, 33)years ]. Additionally, 30 control subjects without T-cell lymphoproliferative disorders were included (17 males and 13 females[age 55(47, 67)years]. Multiparameter flow cytometry (FCM) was performed to analyze TRBC1 and TRBC2 expression patterns in different αβ-T cell subsets within bone marrow samples. Neoplastic T lymphocytes were identified and gated based on immunophenotypic markers (CD3, CD2, CD5, CD7, etc.), followed by characterization of their TRBC1 and TRBC2 expression profiles. Statistical comparisons among multiple groups were conducted using the Kruskal-Wallis test, while the Wilcoxon rank-sum test was employed for pairwise comparisons.Results:In the mature T-cell lymphoma group, 66.7% (21/30) of cases demonstrated monotypic expression of either TRBC1 or TRBC2. Despite the rest 33.3% (9/30) of cases with surface CD3 negativity showed complete loss of both surface and intracellular TRBC1 and TRBC2 (dual-negative), TCR gene rearrangement positivity confirmed their biologically monoclonal proliferation. In contrast, control group αβ-T cells and their subsets exhibited polytypic TRBC1 and TRBC2 expression. Compared to control αβ-T cells, mature T-cell lymphomas showed statistically significant differences in TRBC1 ( U=270.00, P<0.05) and TRBC2 ( U=300.00, P<0.05) distribution. Within control group T-cell subsets, using a threshold of>85% TRBC1-or TRBC2-positive cells, T-cell clones of uncertain significance (T-CUS) were detected in 23% (7/30) of controls, in which 12 clonal proliferations were totally found. These T-CUS clones were significantly associated with CD57+T cells, suggesting a possible link to immunosenescence or chronic antigen stimulation. Among T-ALL/LBL patients, 2 cases showed intracellular TRBC monotypic expression, while 8 cases exhibited dual-negative intracellular TRBC expression, which aids in differentiating T-ALL/LBL from normal thymocytes (which usually display polytypic TRBC expression). Conclusion:Multiparameter flow cytometry (FCM) combined with TRBC1/TRBC2 dual staining can effectively distinguish neoplastic T cells from normal T lymphocyte populations. This approach serves as a crucial determinant for assessing T-cell clonality and can definitively identify TRBC subtypes (TRBC1 or TRBC2).

Objective:To investigate the characteristics of lactate dehydrogenase A (LDHA) gene expression in bone marrow mononuclear cells of children with acute myeloid leukemia (AML), and its association with clinical efficacy and prognosis, and the clinical significances.Methods:A retrospective case series study was conducted. Forty newly-treated AML (non-acute promyelocytic leukemia) children confirmed by morphology, immunology, cytogenetics, and molecular biology at the Children's Hospital Affiliated to Zhengzhou University from February 2021 to March 2022 were included in the AML group, and the pediatric patients were given induction therapy, consolidation therapy and maintenance therapy according to the Chinese Children's Leukemia Group AML-2019 protocol. Eighteen children undergoing bone marrow examination for non-hematologic diseases (iron-deficiency anemia, idiopathic thrombocytopenic purpura, etc.) during the same period served as the control group. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect LDHA mRNA expression levels in bone marrow mononuclear cells before treatment. Patients were categorized into high and low LDHA expression groups based on the median relative expression of LDHA mRNA. LDHA mRNA levels were compared between the AML and control groups, as well as among AML patients with different treatment responses. The distribution of LDHA high or low expression patients was analyzed across clinical characteristic subgroups. Kaplan-Meier method was used for overall survival analysis of children with high and low expression of LDHA, and log-rank test was used for inter group comparison.Results:There were 26 males (65.0%) and 14 females (35.0%) in the AML group, with an age of (4.9±3.3) years; there were 9 males (50.0%) and 9 females (50.0%) in the control group, with an age of (4.2±2.8) years; there was no statistically significant difference in age and gender between the two groups (both P > 0.05). The relative expression level of LDHA mRNA before treatment in bone marrow mononuclear cells of AML patients was higher than that of the control group (4.32±1.21 vs. 1.05±0.38), and the relative expression level of LDHA mRNA before treatment in AML patients who did not improve after induction therapy (8 cases) was higher than that in AML patients who achieved complete remission (CR) after induction therapy (32 cases) (5.89±1.45 vs. 3.76±1.02), and the differences were statistically significant (both P < 0.05). The median relative expression level of LDHA mRNA in AML patients was 2.90, and 20 patients with high LDHA expression and 20 patients with low LDHA expression were classified according to this value. The proportion of AML patients with LDHA high expression in the CR after induction therapy group was lower than that in the NR group [37.5% (12/32) vs. 8/8], and the difference was statistically significant ( P = 0.003). However, there was no statistically significant difference in the proportion of patients with high LDHA expression among the subgroups of gender, age ≥ 10 years, France-America-Britain classification, white blood cell count ≥ 50×10 9, and risk stratification (all P > 0.05). Forty AML children were followed up for a median of 23 months (ranging from 6 to 36 months); 9 cases (22.5%) died, including 2 cases with LDHA low expression and 7 cases with LDHA high expression, accounting for 10.0% and 35.0% of the two groups, respectively. Kaplan-Meier survival analysis showed that the overall survival of the LDHA low expression group was better than that of the LDHA high expression group, and the difference was statistically significant ( P = 0.036). Conclusions:LDHA is highly expressed in bone marrow mononuclear cells of children with AML, and its expression level may be related to the efficacy and overall survival, which may be a potential biomarker for prognostic evaluation.

Copper is an indispensable trace element that participates in numerous metabolic and signaling processes in both its oxidized and reduced states.It is intimately associated with several facets of tumor development,and alterations in copper homeostasis can substantially influence processes such as tumor cell growth,metastasis,modulation of the tumor microenvironment,oxidative stress,cell signaling,and the evasion of tumor cells from immune surveillance.Copper metabolism in tumor cells predominantly promotes immune escape by regulating the expression of programmed death ligand 1.In view of its crucial role in tumor immunity,modulating copper metabolism has emerged as a prospective therapeutic approach.This paper reviews the regulatory mechanisms of copper within the human body and investigates how disruptions to copper metabolism impact tumorigenesis and progression,along with the immune and tumor microenvironments.We also discuss the research value of copper as a target for tumor immunotherapy,thus providing a theoretical basis for future research and clinical applications.

Copper is an indispensable trace element that participates in numerous metabolic and signaling processes in both its oxidized and reduced states.It is intimately associated with several facets of tumor development,and alterations in copper homeostasis can substantially influence processes such as tumor cell growth,metastasis,modulation of the tumor microenvironment,oxidative stress,cell signaling,and the evasion of tumor cells from immune surveillance.Copper metabolism in tumor cells predominantly promotes immune escape by regulating the expression of programmed death ligand 1.In view of its crucial role in tumor immunity,modulating copper metabolism has emerged as a prospective therapeutic approach.This paper reviews the regulatory mechanisms of copper within the human body and investigates how disruptions to copper metabolism impact tumorigenesis and progression,along with the immune and tumor microenvironments.We also discuss the research value of copper as a target for tumor immunotherapy,thus providing a theoretical basis for future research and clinical applications.

Objective:Explore the clinical utility of TRBC1/TRBC2 dual staining by flow cytometry in determining clonality in T-cell lymphomas.Methods:This is a retrospective case-control study. A total of 40 patients with T-cell lymphoma involving bone marrow from the First Affiliated Hospital of Zhengzhou University were enrolled between December 10, 2024 and March 5, 2025. This cohort included 30 cases of mature T-cell lymphoma, 16 males and 14 females, age 62 (54, 71)years, and 10 cases of T-lymphoblastic leukemia/lymphoma[age 11(7, 33)years ]. Additionally, 30 control subjects without T-cell lymphoproliferative disorders were included (17 males and 13 females[age 55(47, 67)years]. Multiparameter flow cytometry (FCM) was performed to analyze TRBC1 and TRBC2 expression patterns in different αβ-T cell subsets within bone marrow samples. Neoplastic T lymphocytes were identified and gated based on immunophenotypic markers (CD3, CD2, CD5, CD7, etc.), followed by characterization of their TRBC1 and TRBC2 expression profiles. Statistical comparisons among multiple groups were conducted using the Kruskal-Wallis test, while the Wilcoxon rank-sum test was employed for pairwise comparisons.Results:In the mature T-cell lymphoma group, 66.7% (21/30) of cases demonstrated monotypic expression of either TRBC1 or TRBC2. Despite the rest 33.3% (9/30) of cases with surface CD3 negativity showed complete loss of both surface and intracellular TRBC1 and TRBC2 (dual-negative), TCR gene rearrangement positivity confirmed their biologically monoclonal proliferation. In contrast, control group αβ-T cells and their subsets exhibited polytypic TRBC1 and TRBC2 expression. Compared to control αβ-T cells, mature T-cell lymphomas showed statistically significant differences in TRBC1 ( U=270.00, P<0.05) and TRBC2 ( U=300.00, P<0.05) distribution. Within control group T-cell subsets, using a threshold of>85% TRBC1-or TRBC2-positive cells, T-cell clones of uncertain significance (T-CUS) were detected in 23% (7/30) of controls, in which 12 clonal proliferations were totally found. These T-CUS clones were significantly associated with CD57+T cells, suggesting a possible link to immunosenescence or chronic antigen stimulation. Among T-ALL/LBL patients, 2 cases showed intracellular TRBC monotypic expression, while 8 cases exhibited dual-negative intracellular TRBC expression, which aids in differentiating T-ALL/LBL from normal thymocytes (which usually display polytypic TRBC expression). Conclusion:Multiparameter flow cytometry (FCM) combined with TRBC1/TRBC2 dual staining can effectively distinguish neoplastic T cells from normal T lymphocyte populations. This approach serves as a crucial determinant for assessing T-cell clonality and can definitively identify TRBC subtypes (TRBC1 or TRBC2).

Objective:To summarize the morphological characteristics of diffuse large B-cell lymphoma (DLBCL) cells and investigate the prognosis value of the characteristics and the number of DLBCL cells in bone marrow.Methods:Retrospective study. We collected 79 cases newly diagnosed with DLBCL in the First Affiliated Hospital of Zhengzhou University from January 2020 to August 2022. 30 cases newly diagnosed without bone marrow involvement were selected as controls, whose mean age 58 years (30-82 years). The DLBCL cells were evaluated by the bone marrow smear, biopsy and flow cytometry separetely.The detection rate of DLBCL cells in the bone marrow was compared, to analyse the relationship between the morphological characteristics of DLBCL in the smear, clinical characteristics and flow cytometry parameters, and the prognostic value of DLBCL detected in the bone marrow smear and its quantity was analyzed. Logistic regression was used to analyze the correlation between the detection of DLBCL cells in bone marrow smears and the age, clinical stage, and the number of extraderules involved organs. Multivariate Cox regression was used to analyze the influence of DLBCL cells detection and its number on the prognosis of patients.Results:(1) The positive rates of DLBCL cells in bone marrow biopsy, bone marrow smear and flow cytometry were 4.86%, 5.14% and 9.27% respectively. (2) The morphological characteristics of 79 cases in bone marrow smears were described: more than 2 times the volume of the cell body of the lymphocyte, the shape was different, round or quasi-round or irregular shape, can be seen pseudopodia or protrusion; The volume of cytoplasm was moderate, vacuoles were visible, and a few perinuclear areas were visible. The nucleus were different in shape, round or quasi-round or irregularly shaped, with a majority of them having multiple nuclei and a few of them having delicate and loose chromatin. Most nucleoli were medium or large obviously, with a majority of them having 1-2 nucleoli and a few having more than 3.Sergiosomes and hemophagocytosis were observed in some DLBCL cases, tumor cell aggregation phenomenon was observed in a few DLBCL cases, occasionally pathological mitosis.(3) DLBCL cells in bone marrow smear was positively related to the age of patients, clinical stage and the number of extranodal organs involved(regression coefficient were 2.012, 2.754, 2.028, P<0.05);The volume of DLBCL cells in bone marrow smear was positively correlated with the ratio of CD4 and CD8(regression coefficient is 2.545, P<0.05);The vacuoles in cytoplasm and the pseudopod of tumor were both negative relationship with the quantity of CD38 expressed on DLBCL cells(regression coefficient was -2.465, -3.045, P<0.05); (4) DLBCL cells in bone marrow smear was an independent risk factor for PFS and OS( RR=7.059, P<0.05); RR=5.409, P<0.05). Conclusion:The appearace of DLBCL cells in bone marrow smear with prognosis, and could be used for clinical staging.

Objective:To investigate the correlation between the number of circulating DLBCL cell and the marrow tumor cell burden and the prognostic indicators in patients with DLBCL, and to evaluate the feasibility of circulating DLBCL cell reflecting the marrow tumor burden and disease progression. Optimization of FCM for screening circulating DLBCL cell was done to monitor MRD and recurrence.Methods:We conducted a retrospective study in 75 diagnosed DLBCL patients in the First Affiliated Hospital of Zhengzhou University from June 2020 to February 2021, including 43 males and 32 females aged 61 (37-85) years. According to the diagnosis and treatment criteria, the patients were divided into initial and recurrence group ( n = 53), partial response(PR)group ( n=14) and complete response(CR)group ( n=8). According to the positive criteria of circulating DLBCL cells, 48 cases were divided into circulating DLBCL positive group and 27 cases were negative group. 30 anemia patients with non-B-cell tumor-related diseases were selected as the control group, including 16 males and 14 females, aged 52 (30-79) years. 70 healthy subjects, including 36 males and 34 females, aged 39 (25-57), were selected for methodology optimization. FCM was used to detect the ratio of marrow and circulating DLBCL cells in each group, and analyze the connection between circulating DLBCL cells and clinical indicators. Statistical analysis was performed using t test, χ 2 test, Kruskal-Wallis H test, Spearman rank correlation, and Logistic regression. Results:(1) Bone marrow and circulating DLBCL cells were not detected in CR group and control group; The positive rate of circulating DLBCL cells in the initial/recurrent group and PR groups was 75.47% and 57.14%, respectively. The proportion of bone marrow and circulating DLBCL cells was positively correlated in the two groups ( P value was <0.001 and 0.020, respectively). (2) The proportion of bone marrow and circulating DLBCL cells in the initial and recurrent groups, PR group, CR group and control group decreased successively ( P<0.05). The proportion of DLBCL cells was 27.72% (initial and recurrent bone marrow group), 26.92% (initial and recurrent circulating group), 3.23% (bone marrow PR group) and 1.67% (circulating PR group), respectively. (3) Compared with the negative group, the circulating DLBCL cell positive group had increased LDH, β 2-MG, and CMYC expression(≥80%), with decreased LYM, HGB<100 g/L, B symptoms, PD-L1 expression, and age ≥60 years, showing higher ECOG, aaIPI/IPI scores and Ann staging ( P<0.05). Age ≥60, B symptoms, and PD-L1 expression were independent risk factors for circulating DLBCL cells ( P<0.05). Conclusions:The detectable rate of circulating DLBCL cell could be improved by optimizing the preoperative treatment conditions of FCM. Circulating DLBCL cells can reflect the tumor burden and disease progression. Detecting circulating DLBCL cells may improve patients′ compliance.

Idiopathic thrombocytopenic purpura (ITP) is a common bloody disease with a high incidence in children, but its diagnostic method is exclusive diagnosis, and the existing detection techniques are mostly invasive, which may cause secondary injury to patients and also may increase the risk of disease. In order to make up for the lack of the detection method, this study made a preliminary exploration on the diagnosis of children's ITP from the perspective of infrared thermography. In this study, a total of 11 healthy children and 22 ITP children's frontal infrared thermal images were collected, and the pattern characteristic (PFD), average temperature (Troi) and maximum temperature (MAX) characteristics of 7 target areas were extracted. The weighted PFD parameters were correlated with the platelet count commonly used in clinical diagnosis, and the sensitivity and specificity of the weighted PFD parameters for children's ITP were calculated through the receiver operating characteristic curve (ROC). The final results showed that the difference of the weighted PFD parameters between healthy children and ITP children was statistically significant, and the parameters negatively correlated with platelet count. Under the ROC curve, the area under the curve (AUC) of this parameter is as high as 92.1%. Based on the research results of this paper, infrared thermography can clearly show the difference between ITP children and healthy children. It is hoped that the methods proposed in this paper can non-invasively and objectively describe the characteristics of ITP infrared thermal imaging of children, and provide a new ideas for ITP diagnosis.
Area Under Curve ; Child ; Humans ; Platelet Count ; Purpura, Thrombocytopenic, Idiopathic ; Thermography

Objective To setup a measurement of human bone marrow micromegakaryocyte which based on CD41a and PI double‐labeled flow cytometric analysis ,and study the significance in the diagnosis of MDS .Methods In 42 cases of MDS patients , their bone marrow megakaryocytes were obtained by Percoll density gradient separation medium .The megakaryocyte glycoproteinⅡb/Ⅲa(CD41a)were marked with fluorescein isothiocyanate through its corresponding monoclonal antibody ,and their DNA were marked with PI .Then the megakaryocyte ploidy was analyzed by flow cytometry(FCM ) .Results The method for micromegakaryo‐cyte identification and analysis was established .In 42 patients with MDS ,the detection rate of micromegakaryocyte was 90 .5 per‐cent by FCM analysis ,but only 54 .8 percent by Wright‐Giemsa staining test and 64 .3 percent by immunohistochemistry ,the differ‐ence among them was statistically significant(χ2 = 13 .640 ,P= 0 .001) .The 42 patients with MDS were divided into two groups (low‐risk group and high‐risk group) .The detection rates of micromegakaryocyte were 81 .8 percent in low‐risk group and 100 per‐cent in high‐risk group separately by FCM analysis ,the difference was statistically significant(χ2 =4 .019 ,P=0 .045) .Conclusion The detection rate of micromegakaryocyte by FCM with CD41a and PI double marker is higher than that by cytochemical staining . The detection rate of micromegakaryocyte in the high‐risk group is higher than that of the low‐risk group ,which shows that the de‐tection of micromegakaryocyte is of great significance for MDS prognosis assessment .