Objective:To investigate the possible effects of Tim-3/Galectin-9 signaling pathway and MDSC on acute graft-ver-sus-host disease(aGVHD)development in patients after Haplo-HDPSCT.Methods:A total of 42 patients underwent Haplo-HDPSCT and 20 healthy controls were enrolled in this study.Peripheral blood specimens were collected from all study subjects,the number of Tim-3+CD8+T,Granzyme B+CD8+T and MDSC were detected by FCM,and level of Galectin-9 was detected by ELISA.Apoptosis of CD8+T cells from peripheral blood of aGVHD patients were detected by FCM.Results:According to the Seattle International Diagnostic Criteria,there were 16 patients occurred aGVHD after transplantation,while 26 patients did not.①The number of Tim-3+CD8+T and Granzyme B+CD8+T cells in aGVHD patients were significantly higher than patients without aGVHD and healthy controls,while MDSC and Galectin-9 in aGVHD patients were lower than patients without aGVHD and healthy controls(P<0.05);②There were statistically significant differences in the number of Tim-3+CD8+T and Granzyme B+CD8+T cells and level of Galectin-9 between patients with mild(grade Ⅰ～Ⅱ)and severe(grade Ⅲ～Ⅳ)aGVHD groups(P<0.05),while there was no statistically significant difference in the number of MDSC(P=0.689);③Spearman analysis showed that level of Galectin-9 was positively correlated with the number of MDSC in patients without aGVHD(r=0.684,P<0.05);④Isolating CD8+T cells from peripheral blood of aGVHD patients,and found that addition of Galectin-9 increased rate of CD8+T cells apoptosis.Conclusion:Galectin-9 inhibits immune response of CD8+T cells through the Tim-3/Galectin-9 signaling pathway after Haplo-HDPSCT,and this can avoid or slow down the incidence of aGVHD;Galectin-9 is positively correlated with the number of MDSC,it may be associated with the occurrence of aGVHD.

Objective:To investigate the possible effects of Tim-3/Galectin-9 signaling pathway and MDSC on acute graft-ver-sus-host disease(aGVHD)development in patients after Haplo-HDPSCT.Methods:A total of 42 patients underwent Haplo-HDPSCT and 20 healthy controls were enrolled in this study.Peripheral blood specimens were collected from all study subjects,the number of Tim-3+CD8+T,Granzyme B+CD8+T and MDSC were detected by FCM,and level of Galectin-9 was detected by ELISA.Apoptosis of CD8+T cells from peripheral blood of aGVHD patients were detected by FCM.Results:According to the Seattle International Diagnostic Criteria,there were 16 patients occurred aGVHD after transplantation,while 26 patients did not.①The number of Tim-3+CD8+T and Granzyme B+CD8+T cells in aGVHD patients were significantly higher than patients without aGVHD and healthy controls,while MDSC and Galectin-9 in aGVHD patients were lower than patients without aGVHD and healthy controls(P<0.05);②There were statistically significant differences in the number of Tim-3+CD8+T and Granzyme B+CD8+T cells and level of Galectin-9 between patients with mild(grade Ⅰ～Ⅱ)and severe(grade Ⅲ～Ⅳ)aGVHD groups(P<0.05),while there was no statistically significant difference in the number of MDSC(P=0.689);③Spearman analysis showed that level of Galectin-9 was positively correlated with the number of MDSC in patients without aGVHD(r=0.684,P<0.05);④Isolating CD8+T cells from peripheral blood of aGVHD patients,and found that addition of Galectin-9 increased rate of CD8+T cells apoptosis.Conclusion:Galectin-9 inhibits immune response of CD8+T cells through the Tim-3/Galectin-9 signaling pathway after Haplo-HDPSCT,and this can avoid or slow down the incidence of aGVHD;Galectin-9 is positively correlated with the number of MDSC,it may be associated with the occurrence of aGVHD.

Objective:The aim of this study is to establish the reference interval of refined immune cell subsets by multi-parameter flow cytometry.Methods:In this cross sectional study, a total of 326 healthy participants were included and divided into two groups based on age: 18-40 years old group and 41-60 years old group. Peripheral venous blood was collected in a fasting status. Flow cytometry tests were performed according to previous consensus article. The analysis of reference interval was conducted according to the documents of Clinical and Laboratory Standards Institute (CLSI) EP28-A3c and Health Industry Standards of the People′s Republic of China WS/T 402-2024.Results:The T,B,NK,DC and monocyte refined immune cell subsets applicable to the reference range of the general population mainly include: CD3 +(56.4%-83.3%),CD4 +TEMRRA (0.2%-11.6%), CD4 +TEM (14.9%-52.8%), CD4 +CD28 +(76.3%-99.9%), CD19 +CD5 +(9.7%-45.8%), CD19 +CD27 -(45.7%-90.0%), CD19 +CD27 +(9.8%-54.0%), CD3 +CD16 +CD56 +(1.3%-20.2%), CD3 -CD19 -CD20 -CD14 -CD56 -HLA-DR +(0.4%-2.0%), Monocyte Mo1 subset CD14 +CD16 -(46.3%-94.6%), monocyte Mo2 subset CD14 +CD16 +(2.8%-49.7%), etc. When the Z-value between different age groups was higher than Z* (3.50 cut-off value), the reference intervals of these subsets should be established independently according to age. Conclusions:In this study, the reference intervals of refined subsets of immune cells by multi-parameter flow cytometry has been preliminarily established. For those subgroups that meet the grouping criteria, age should be fully considered in clinical applications and laboratory validation.

Immunesenescence is an important component of human aging and is closely associated with age-related diseases such as cancer, cardiovascular disease, and increased risk of infection. Detection of immune cell senescence markers by flow cytometry provides a means to assess the senescence status of various immune cells. Current studies on immunesenescence are mostly focused on specific immune cell subpopulations with limited reports describing the senescence markers of different types of immune cells in the panorama. Given variety of immune cells, the aging markers differed across cell types. This paper provides a comprehensive analysis of the aging-related flow cytometry biomarkers of different types of immune cells such as neutrophils, macrophages and dendritic cells, congenital lymphocytes, acquired lymphocytes and unconventional T lymphocytes. The detection of these markers by flow cytometry is advantageous in depicting the immune senescence status of the human body and may also be useful in the future for evaluating interventions and therapies against age related diseases. However, challenges such as such as inconsistent detection protocols for refined subpopulations of immune cells and conflicting research results need further investigation to promote the clinical application of flow cytometry technology in the assessment of immune senescence.

Objective:The aim of this study is to establish the reference interval of refined immune cell subsets by multi-parameter flow cytometry.Methods:In this cross sectional study, a total of 326 healthy participants were included and divided into two groups based on age: 18-40 years old group and 41-60 years old group. Peripheral venous blood was collected in a fasting status. Flow cytometry tests were performed according to previous consensus article. The analysis of reference interval was conducted according to the documents of Clinical and Laboratory Standards Institute (CLSI) EP28-A3c and Health Industry Standards of the People′s Republic of China WS/T 402-2024.Results:The T,B,NK,DC and monocyte refined immune cell subsets applicable to the reference range of the general population mainly include: CD3 +(56.4%-83.3%),CD4 +TEMRRA (0.2%-11.6%), CD4 +TEM (14.9%-52.8%), CD4 +CD28 +(76.3%-99.9%), CD19 +CD5 +(9.7%-45.8%), CD19 +CD27 -(45.7%-90.0%), CD19 +CD27 +(9.8%-54.0%), CD3 +CD16 +CD56 +(1.3%-20.2%), CD3 -CD19 -CD20 -CD14 -CD56 -HLA-DR +(0.4%-2.0%), Monocyte Mo1 subset CD14 +CD16 -(46.3%-94.6%), monocyte Mo2 subset CD14 +CD16 +(2.8%-49.7%), etc. When the Z-value between different age groups was higher than Z* (3.50 cut-off value), the reference intervals of these subsets should be established independently according to age. Conclusions:In this study, the reference intervals of refined subsets of immune cells by multi-parameter flow cytometry has been preliminarily established. For those subgroups that meet the grouping criteria, age should be fully considered in clinical applications and laboratory validation.

Immunesenescence is an important component of human aging and is closely associated with age-related diseases such as cancer, cardiovascular disease, and increased risk of infection. Detection of immune cell senescence markers by flow cytometry provides a means to assess the senescence status of various immune cells. Current studies on immunesenescence are mostly focused on specific immune cell subpopulations with limited reports describing the senescence markers of different types of immune cells in the panorama. Given variety of immune cells, the aging markers differed across cell types. This paper provides a comprehensive analysis of the aging-related flow cytometry biomarkers of different types of immune cells such as neutrophils, macrophages and dendritic cells, congenital lymphocytes, acquired lymphocytes and unconventional T lymphocytes. The detection of these markers by flow cytometry is advantageous in depicting the immune senescence status of the human body and may also be useful in the future for evaluating interventions and therapies against age related diseases. However, challenges such as such as inconsistent detection protocols for refined subpopulations of immune cells and conflicting research results need further investigation to promote the clinical application of flow cytometry technology in the assessment of immune senescence.

Objective:To explore the expression levels, clinical significances and prognostic evaluation value of T-cell immunoglobulin mucin-3 (Tim-3) and galectin-9 (Gal-9) in bone marrow cells of patients with newly diagnosed acute lymphocytic leukemia (ALL).Methods:Bone marrow samples from 30 newly diagnosed ALL patients admitted to First Affiliated Hospital of Xinjiang Medical University from September 2016 to September 2018 were selected, and peripheral blood samples from 20 healthy volunteers during the same period in the First Affiliated Hospital of Xinjiang Medical University were treated as the controls. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect mRNA relative expression levels of Tim-3 and Gal-9. Differences in mRNA expression of Tim-3 and Gal-9 among ALL patients with varied clinicopathological characteristics were compared. Overall survival (OS) analysis was performed by using the Kaplan-Meier method, Cox proportional hazards model was used to make univariate and multivariate survival analysis.Results:mRNA relative expression levels of Tim-3 and Gal-9 in 30 newly diagnosed ALL patients were higher than those in the healthy control group (2.86±0.47 vs. 0.45±0.05, t = 21.65, P<0.05; 9.79±0.58 vs. 0.96±0.23, t = 63.24, P<0.05). mRNA relative expression level of Tim-3 had statistically significant differences in patients with different ages, France-America-Britain (FAB) Cooperative Group classification, hazard grades and central nervous system invasion (all P<0.01). There were statistically significant differences in mRNA relative expression level of Gal-9 for patients with different ages, FAB Cooperative Group classification, white blood cell count (WBC), central nervous system invasion and NOTCH1 mutation (all P<0.01). All patients were grouped by mRNA relative expression levels of Tim-3 and Gal-9, and patients in high Tim-3 expression group (≥2.86) had worse overall survival (OS) compared with that for patients in low Tim-3 expression group (<2.86) ( P = 0.048). Patients in high Gal-9 expression group (≥9.79) had worse OS compared with that for patients in low Gal-9 expression group (<9.79) ( P = 0.031). Moreover, the OS in Tim-3 and Gal-9 both high expression group was worse than that in Tim-3 and Gal-9 both low expression group and in the low expression group of either of them (all P<0.05). There was no statistically significant difference in OS between the high Tim-3 expression with low Gal-9 expression group and the high Gal-9 expression with low Tim-3 expression group ( P > 0.05). Multivariate Cox regression analysis revealed that peripheral blood WBC≥11.4×10 9/L, BCR-ABL gene mutation, central nervous system invasion, and high expression of Tim-3 and Gal-9 were independent risk prognostic factors of OS for newly diagnosed ALL patients (all P<0.05) . There was a positive correlation between the expression levels of Tim-3 and Gal-9 ( r = 0.788, P<0.01). Conclusions:The high expression of Tim-3 and its ligand Gal-9 are independent effecting factors of poor prognosis in newly diagnosed ALL patients. The expression levels of Tim-3 and Gal-9 can be served as a potential prognostic indicator for ALL patients.

Objective To analyze the experience in diagnosis and surgical treatment for solid pseudopapillary neoplasm(SPN) of pancreas in children.Methods A retrospective study was performed in 12 pediatric patients with SPN who had been admitted to Affiliated Cancer Hospital of Zhengzhou University during January 2004 to December 2016,and their general data,demographic data,types of operations,postoperative complications and follow-up were analyzed.Results Among the 12 patients,3 cases were male and 8 cases were female,with average age 14.3 years old (11-17 years old).The main clinical manifestations included abdominal pain(4/12 cases,33.3％),abdominal mass (2/12 cases,16.7％) and trauma(2/12 cases,16.7％).In those 12 patients,33.3％ (4/12 cases) SPN was located at the head of the pancreas,and 66.7％ (8/12 cases) at the body and tail of it.The tumors were usually large,the largest diameter ranged from 4.0 to 15.3 cm(average largest diameter,8.2 cm).The color uhrasonography indicated heterogeneous echogenic mass and clear boundary.CT scanning indicated that the tumor was a low-density cystic mass with a clear boundary,with enhanced tumor real component and irregular reinforcement.No calcification was found in the patients.Dynamic enhanced magnetic resonance imaging scan revealed gradual strengthening solid components in tumor.All the patients received surgical resection,with distal pancreatectomy in 4 patients,pancreaticoduodenectomy in 4 patients,spleen-preserving distal pancreatectomy in 2 patients,Enucleation in 1 patient,and distal pancreatectomy and self-splenic slices transplantation in 1 patient.Lymphadenectomy was performed in 4 patients,and all the 21 removed lymph nodes were all negative.Pathological diagnosis confirmed the SPN in all the patients,among them 3 cases were malignant SPN,and one of them with tumor rupture and hemorrhage.The mean follow-up duration was 57.7 months(19-156 months) and no recurrence was found.Conclusion SPN is a rare neoplasm in children who go to see doctors because of clinical symptoms.Surgical resection,especially organs-preserving resection,may improve the long-term results.

Objective: To observe the expression levels of PD-1/PD-L1 costimulatory molecules and explore the clinical significance in patients with chronic lymphocytic leukemia (CLL) . Methods: The expression of PD-1/PD-L1 in peripheral blood CD8⁺ T cells, CD4⁺T cells, CD19⁺B, and dendrites cells (DC) was detected by flow cytometry in 57 CLL patients and 20 healthy controls. The correlations of PD-1/PD-L1 expression with disease stage, CD38 expression, ZAP-70 expression, chromosome karyotype abnormality and β₂-MG expression were analyzed. Results: ①Compared with control, CLL patients, including 39 males and 18 females with the median age of (63.7±10.7) years, had no statistically significant difference in age and gender (P>0.05) . CLL patients had the higher PD-1/PD-L1 expression than healthy controls (P<0.05) . ②In Rai staging, the later the stage, the higher expression of PD-1/PD-L1 (P<0.05) . ③PD-1 expression in CD8⁺CD38⁺ group (11 cases) was higher than that in CD8⁺CD38^- group (46 cases) (P=0.004) , and CD8⁺ poor prognosis chromosome group (14 cases) also had significant higher PD-1 expression than CD8⁺good prognosis chromosome group (28 cases) (P=0.004) . ④The expression of PD-L1 was higher in CD38⁺group, ZAP-70⁺group, and poor prognosis group, as compared to that in CD38^-group (P=0.002) , in ZAP-70^-group (P<0.001) , in good prognosis group (P=0.023) . There was no correlation between the expression of PD-1/PD-L1 and β₂-MG (P>0.05) . Conclusion This data reveals that PD-1/PD-L1 was highly expressed in CLL patients. Its expression levels were correlated with Rai stage, CD38, ZAP-70, chromosome karyotype, but not with β₂-MG. PD-1/PD-L1 may be a prognostic factor in patients with CLL.

OBJECTIVETo develop a reversed-phase HPLC method for simultaneous determination of gastrodin, adenosin, 4-hydroxybenzyl alcohol and 4-hydroxybenzaldehyde in Rhizoma Gastrodia.

METHODA Kromasil C18 column (4.6 mm x 250 mm, 5 microm) was used with a methanol-water-0.1% acetic acid gradient elution system. The eluates were detected at 270 nm, the flow rate was 1.0 mL x min(-1) and the column temperature was 35 degrees C.

RESULTThe linear range of gastrodin, adenosin, 4-hydroxybenzyl alcohol and 4-hydroxybenzaldehyde were 19.1-383 (r = 0.999 9), 0.620-12.4 (r = 0.999 9), 2.45-49.0 (r = 0.999 9), 0.280-5.63 mg x L(-1) (r = 0.999 6), respectively. The average recoveries (n = 9) of the four components were 96.7% -97.7%, RSD < 1.6%.

CONCLUSIONThe method is accurate, sensitive and reliable for determination of gastrodin, adenosin, 4-hydroxybenzyl alcohol and 4-hydroxybenzaldehyde in Rhizoma Gastrodia.