1.Effect of Shenxiong Huanglian Jiedu Decoction on Neuronal Damage and Aβ Clearance in Mice Model of Alzheimer's Disease
Jing LIU ; Kang CHEN ; Yushun ZHOU ; Zhezuo ZHANG ; Guran YU ; Hao LI
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(7):43-52
ObjectiveTo investigate the effects of Shenxiong Huanglian Jiedu decoction on the clearance of amyloid β-protein (Aβ) and neuronal damage in the mouse model of Alzheimer's disease (AD). MethodsA total of 36 SPF-grade 2-month-old C57BL/6J mice were used in this study, and the modeling was performed by bilateral hippocampal injection of Aβ oligomers in C57BL/6J mice. The experiment was conducted with a blank group, a sham operation group, a model group, low- and high-dose (3.27,6.54 g·kg-1, respectively) Shenxiong Huanglian Jiedu decoction groups, and a positive control (donepezil hydrochloride, 0.65 mg·kg-1) group. At the end of the drug intervention, the learning and memory abilities and the activities of mice were evaluated by the Morris water maze and open field tests. Brain histopathology was examined by hematoxylin-eosin and Nissl staining. Additionally, in vivo imaging was employed to measure the metabolism of fluorescent Aβ in the cerebrospinal fluid, and staining of ionized calcium-binding adapter molecule-1 (Iba-1) was employed to assess microglial activation in the hippocampal tissue. Additionally, neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) levels in the brain tissue and serum were determined by the immunofluorescence assay and enzyme-linked immunosorbent assay. Western blot was conducted to determine the expression of inflammation and pathway-related proteins in the hippocampal tissue. ResultsCompared with the blank group and the sham operation group, the escape latency of the mice in the model group was prolonged, the platform residence time was shortened, the hippocampal tissue showed pathological manifestations such as neuronal pyknosis, Nissl body dissolution, and microglia activation. The metabolic rate of fluorescent Aβ through cerebrospinal fluid was slowed down, and the expression levels of BDNF, NT-3, and interleukin-10 (IL-10) in the hippocampus were significantly decreased (P<0.01). The expression levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and phosphorylated nuclear transcription factor-κB (p-NF-κB p65) in hippocampus were significantly increased (P<0.05, P<0.01). Compared with the model group, the escape latency of mice in the low and high dose groups of Chinese medicine and donepezil group was shortened, and the platform residence time was prolonged. Neuronal karyopyknosis, Nissl body dissolution and microglia activation in hippocampus were improved. Fluorescence Aβ was metabolized faster by cerebrospinal fluid. The expression of BDNF and NT-3 in hippocampus was increased (P<0.01), and the expression of TLR4, MyD88 and p-NF-κB p65 was significantly decreased (P<0.05, P<0.01). The expression of TNF-α in the hippocampus of the high-dose group was significantly decreased (P<0.05), and the expression of IL-10 was significantly increased (P<0.05). The expression of TNF-α, IL-6 and IL-1β in the hippocampus of the donepezil group was significantly decreased (P<0.05, P<0.01). ConclusionShenxiong Huanglian Jiedu decoction may mitigate neuronal damage and enhance cerebrospinal fluid flow in the mouse model of AD, thereby promoting the clearance of Aβ and improving the learning and memory abilities. These beneficial effects are likely mediated through the inhibition of microglial activation, reduction of inflammation, and modulation of the TLR4/MyD88/NF-κB signaling pathway.
2.Effect of Shenxiong Huanglian Jiedu Decoction on Neuronal Damage and Aβ Clearance in Mice Model of Alzheimer's Disease
Jing LIU ; Kang CHEN ; Yushun ZHOU ; Zhezuo ZHANG ; Guran YU ; Hao LI
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(7):43-52
ObjectiveTo investigate the effects of Shenxiong Huanglian Jiedu decoction on the clearance of amyloid β-protein (Aβ) and neuronal damage in the mouse model of Alzheimer's disease (AD). MethodsA total of 36 SPF-grade 2-month-old C57BL/6J mice were used in this study, and the modeling was performed by bilateral hippocampal injection of Aβ oligomers in C57BL/6J mice. The experiment was conducted with a blank group, a sham operation group, a model group, low- and high-dose (3.27,6.54 g·kg-1, respectively) Shenxiong Huanglian Jiedu decoction groups, and a positive control (donepezil hydrochloride, 0.65 mg·kg-1) group. At the end of the drug intervention, the learning and memory abilities and the activities of mice were evaluated by the Morris water maze and open field tests. Brain histopathology was examined by hematoxylin-eosin and Nissl staining. Additionally, in vivo imaging was employed to measure the metabolism of fluorescent Aβ in the cerebrospinal fluid, and staining of ionized calcium-binding adapter molecule-1 (Iba-1) was employed to assess microglial activation in the hippocampal tissue. Additionally, neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) levels in the brain tissue and serum were determined by the immunofluorescence assay and enzyme-linked immunosorbent assay. Western blot was conducted to determine the expression of inflammation and pathway-related proteins in the hippocampal tissue. ResultsCompared with the blank group and the sham operation group, the escape latency of the mice in the model group was prolonged, the platform residence time was shortened, the hippocampal tissue showed pathological manifestations such as neuronal pyknosis, Nissl body dissolution, and microglia activation. The metabolic rate of fluorescent Aβ through cerebrospinal fluid was slowed down, and the expression levels of BDNF, NT-3, and interleukin-10 (IL-10) in the hippocampus were significantly decreased (P<0.01). The expression levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and phosphorylated nuclear transcription factor-κB (p-NF-κB p65) in hippocampus were significantly increased (P<0.05, P<0.01). Compared with the model group, the escape latency of mice in the low and high dose groups of Chinese medicine and donepezil group was shortened, and the platform residence time was prolonged. Neuronal karyopyknosis, Nissl body dissolution and microglia activation in hippocampus were improved. Fluorescence Aβ was metabolized faster by cerebrospinal fluid. The expression of BDNF and NT-3 in hippocampus was increased (P<0.01), and the expression of TLR4, MyD88 and p-NF-κB p65 was significantly decreased (P<0.05, P<0.01). The expression of TNF-α in the hippocampus of the high-dose group was significantly decreased (P<0.05), and the expression of IL-10 was significantly increased (P<0.05). The expression of TNF-α, IL-6 and IL-1β in the hippocampus of the donepezil group was significantly decreased (P<0.05, P<0.01). ConclusionShenxiong Huanglian Jiedu decoction may mitigate neuronal damage and enhance cerebrospinal fluid flow in the mouse model of AD, thereby promoting the clearance of Aβ and improving the learning and memory abilities. These beneficial effects are likely mediated through the inhibition of microglial activation, reduction of inflammation, and modulation of the TLR4/MyD88/NF-κB signaling pathway.
3.Principles, technical specifications, and clinical application of lung watershed topography map 2.0: A thoracic surgery expert consensus (2024 version)
Wenzhao ZHONG ; Fan YANG ; Jian HU ; Fengwei TAN ; Xuening YANG ; Qiang PU ; Wei JIANG ; Deping ZHAO ; Hecheng LI ; Xiaolong YAN ; Lijie TAN ; Junqiang FAN ; Guibin QIAO ; Qiang NIE ; Mingqiang KANG ; Weibing WU ; Hao ZHANG ; Zhigang LI ; Zihao CHEN ; Shugeng GAO ; Yilong WU
Chinese Journal of Clinical Thoracic and Cardiovascular Surgery 2025;32(02):141-152
With the widespread adoption of low-dose CT screening and the extensive application of high-resolution CT, the detection rate of sub-centimeter lung nodules has significantly increased. How to scientifically manage these nodules while avoiding overtreatment and diagnostic delays has become an important clinical issue. Among them, lung nodules with a consolidation tumor ratio less than 0.25, dominated by ground-glass shadows, are particularly worthy of attention. The therapeutic challenge for this group is how to achieve precise and complete resection of nodules during surgery while maximizing the preservation of the patient's lung function. The "watershed topography map" is a new technology based on big data and artificial intelligence algorithms. This method uses Dicom data from conventional dose CT scans, combined with microscopic (22-24 levels) capillary network anatomical watershed features, to generate high-precision simulated natural segmentation planes of lung sub-segments through specific textures and forms. This technology forms fluorescent watershed boundaries on the lung surface, which highly fit the actual lung anatomical structure. By analyzing the adjacent relationship between the nodule and the watershed boundary, real-time, visually accurate positioning of the nodule can be achieved. This innovative technology provides a new solution for the intraoperative positioning and resection of lung nodules. This consensus was led by four major domestic societies, jointly with expert teams in related fields, oriented to clinical practical needs, referring to domestic and foreign guidelines and consensus, and finally formed after multiple rounds of consultation, discussion, and voting. The main content covers the theoretical basis of the "watershed topography map" technology, indications, operation procedures, surgical planning details, and postoperative evaluation standards, aiming to provide scientific guidance and exploration directions for clinical peers who are currently or plan to carry out lung nodule resection using the fluorescent microscope watershed analysis method.
4.Study on the influence of different scanning positions based on chest phantom of CT scan on chest for image quality and radiation dose
Yan SUI ; Shihua TAO ; Kang LIU ; Xinghui GAI ; Zhiyuan GAO ; Zhaorui CHEN ; Hao GONG ; Dewu YANG
China Medical Equipment 2025;22(9):17-20
Objective:To explore the influence of different scanning positions based on chest phantom of computed tomography(CT)scan on chest on image quality and radiation dose.Methods:A thermoluminescent dosimeter(TLD)was placed at the breast area of simulating anthropoid chest phantom.GE Revolution evo CT was used to conduct scan on the conventional supine position(supine group)and prone position(prone group)for chest phantom.Different noise indexes(NI=10-23)were adjusted to control ration doses,and other parameters were fixed,and each group collected 12 sequence images.The average value(AV),standard deviation(SD)of the CT scan at region of interest(ROI)under different scanning positions were recorded to calculate the signal-to-noise ratio(SNR)and contrast-to-noise ratio(CNR)of the image.The radiation dose at the breast area was measured by TLD,and the volume CT dose index(CTDIvol)and dose-length product(DLP)were recorded.Results:Under different scanning positions,the radiation dose of breast organs in the prone group was lower than that in the supine group,there was a statistically significant difference between the two groups(t=6.57,P<0.05),while there were not statistically significant differences in CTDIvol and DLP between the two groups(P>0.05).There were not statistically significant differences in the CT values,SD,SNR,CNR of lung tissue,and the CT values of breast tissue between the two groups of images(P>0.05).The SD,SNR and CNR of breast tissue in the prone group were lower than those in the supine group,and the differences were statistically significant(t=-13.33,-10.59,6.70,P<0.05).There were no statistically significant differences in the subjective scores of the clarity of the edge of the tissue within lung,the layers of soft tissue of the breast,noise,and artifacts in the bone tissue between the two groups of images(P>0.05).Conclusion:When low-dose CT physical examination on chest is conducted in clinical practice,the scanning of prone position during undergoing CT scan on chest can obtain image quality that can meet the requirements in diagnosing lung,and reduce the radiation dose on the breast,and conform to the technical principle of optimal radiation protection.
5.Application and process optimization of automated magnetic bead sorting technology in T-SPOT.TB assay for tuberculosis infection
Tian ZHENG ; Xiao CHEN ; Hao LIU ; Meijuan KONG ; BeiPei KANG ; Jun XI ; Ke ZHOU ; Jiayun LIU
Chinese Journal of Preventive Medicine 2025;59(10):1779-1786
To evaluate the clinical application value of magnetic bead sorting technology (MBS) using the T-cell select kit combined with an automatic cell sorter for isolating peripheral blood mononuclear cells (PBMCs) to realize automated specimen pre-processing and process optimization in T-SPOT.TB assay. A cross-sectional study was conducted involving 300 patients recruited from the first affiliated hospital of Air Force Medical University from March 2023 to July 2024. Among them, there were 167 males and 133 females. The age range of the patients was 18 to 65 years, with a median age of 49.0 (33.3, 59.0) years and a mean age of (46.4±13.9) years. 4 ml of anticoagulated whole blood samples in duplicate were collected from each patient. One fresh sample (0-4 h) was processed immediately using density gradient centrifugation (DGC) for PBMC isolation, while the other was processed using MBS method at either 0-4 h or 34-54 h post-collection. The cycles for cell enrichment step of the automatic cell sorter were adjusted from the conventional 4 cycles to 2 cycles. Statistical analysis was performed using Cohen′s Kappa test to evaluate the concordance of T-SPOT.TB results across all processing groups. The results showed that for fresh samples (0-4 h), the T-SPOT.TB results for samples processed with 4-cycle and 2-cycle MBS enrichment steps demonstrated positive concordance rates of 91.7% and 93.1%, negative concordance rates of 100% for both, overall concordance rates of 98.0% and 97.9%, and Kappa values of 0.94 and 0.95, respectively, compared with the reference results for paired samples processed using DGC. Correspondingly, the results for samples processed with the 4-cycle and 2-cycle MBS methods at 34-54 h post-collection yielded positive concordance rates of 90.0% and 81.3%, negative concordance rates of 95.1% and 100%, overall concordance rates of 93.0% and 82.9%, and Kappa values of 0.86 and 0.43, respectively, compared with the reference results for fresh samples processed using DGC. In conclusion,for 0-4 h samples, the T-SPOT.TB results from both 4-cycle and 2-cycle MBS enrichment protocols were highly consistent with the reference results from conventional DGC methods. However, for 34-54 h stored samples, results from the 4-cycle MBS method rather than the 2-cycle protocol exhibited significantly superior concordance with the reference results. The MBS method using T-Cell Select kit achieves automated sample pre-processing for T-SPOT.TB assay, reduces hands-on time, and provides a viable alternative with reliable results for long-stored specimens.
6.Effects of Yiqi Juanbi Formula on chondrocyte pyroptosis in collagen-induced arthritic rats via NF-κB/NLRP3/Caspase-1 signaling pathway
Xin-yu CUI ; Hao-lin LI ; Wei-qing LI ; Hui-qin KANG ; Wei-gang CHENG ; Pei-xin HE ; Cai-hong YANG ; Ping CHEN ; Hai-dong WANG
Chinese Traditional Patent Medicine 2025;47(9):2880-2887
AIM To investigate the effects of Yiqi Juanbi Formula on chondrocyte pyroptosis in rat models of collagen-induced arthritis(CIA).METHODS Fifty rats were subcutaneously injected at the tail base with an emulsion containing equal volumes of bovine type Ⅱ collagen and incomplete Freund's adjuvant(IFA)to establish the CIA models.These rats were then randomly assigned to the model group,the methotrexate group(0.35 mg/kg),and the low-dose,medium-dose,and high-dose Yiqi Juanbi Formula groups(9.4,18.7,37.4 g/kg),in contrast to the ten intact rats serving in the normal control group.Following four weeks of intragastric administration,the rats had their general conditions observed;their joint swelling and arthritis indices measured;their ankle joint pathology assessed by HE staining;their serum levels of IL-1β,IL-18 and TNF-ɑ detected by ELISA;their mRNA expressions of NLRP3,Caspase-1,GSDMD,IL-1β,IL-18 and TNF-ɑ in ankle cartilage quantified by RT-qPCR;their protein expressions of NF-κB,NLRP3 and Caspase-1 in ankle cartilage analyzed by Western blot;and their NLRP3 and GSDMD positive expressions in ankle cartilage examined by immunohistochemistry.RESULTS Compared to the control group,the model group showed significantly increased joint swelling and arthritis indices(P<0.01);elevated serum levels of IL-1 β,IL-18 and TNF-ɑ(P<0.01);pathological changes including cartilage surface defects,reduced cell count,altered cellular morphology,irregular cell arrangement,and significant inflammatory cell infiltration in synovial tissue;upregulated mRNA expressions of NF-κB,NLRP3,Caspase-1,GSDMD,IL-1β,IL-18 and TNF-ɑ(P<0.01)and increased protein expressions of NF-κB,NLRP3 and Caspase-1(P<0.01)in ankle cartilage;enhanced positive expressions of NLRP3 and GSDMD in ankle cartilage(P<0.01).Compared to the model group,the groups intervened with methotrexate or medium-or high-dose Yiqi Juanbi Formula exhibited reduced joint swelling and arthritis indices(P<0.01);alleviated pathological damage in ankle joints;decreased serum levels of IL-1β,IL-18 and TNF-ɑ(P<0.01);downregulated mRNA expressions of NF-κB,NLRP3,Caspase-1,GSDMD,IL-1β,IL-18 and TNF-ɑ(P<0.05,P<0.01),and reduced protein expressions of NF-κB,NLRP3 and Caspase-1(P<0.05,P<0.01)in ankle cartilage;and diminished positive expressions of NLRP3 and GSDMD in ankle cartilage(P<0.01).CONCLUSION Yiqi Juanbi Formula alleviates inflammation in CIA rats,potentially by inhibiting the activation of the NF-κB/NLRP3/Caspase-1 signaling pathway,thereby suppressing chondrocyte pyroptosis.
7.Expression characteristics of OPG/RANKL/RANK and the relationship with fibrosis in myocardial tissues of rats with chronic heart failure
Xin YANG ; Xue-kun CAI ; Ze-long WU ; An-tao CHEN ; Zi-hao CHEN ; Xuan XIE ; Jia-kang OU ; Zhao-qi HUANG
The Chinese Journal of Clinical Pharmacology 2025;41(1):71-75
Objective To study the expression characteristics of osteoprotegerin(OPG)/receptor activator of nuclear factor-κB ligand(RANKL)/receptor activator of nuclear factor-κB(RANK)system and the relationship with fibrosis in myocardial tissues of rats with chronic heart failure.Methods SD rats were randomly divided into sham-operation group(12 rats)and model group.In sham-operation group,surgical thread was passed through the abdominal aorta without constricting it after laparotomy;in model group,establish the heart failure model by abdominal aorta coarctation.The successful model rats were randomly divided into model 1 week(12 rats),model 2 weeks(11 rats),model 4 weeks(11 rats),model 8 weeks(11 rats)and model 12 weeks groups(11 rats).The end point of the study is at week 12.The contents of hydroxyproline(HYP),total myocardial collagen and collagen volume fraction(CVF)were compaired in all proups.The expression levels of OPG,RANKL and RANK proteins in cardiomyocytes were determined by Western blot.Results The contents of HYP in sham-operation,model 1 week,model 2 weeks,model 4 weeks,model 8 weeks and model 12 weeks group were(0.25±0.04),(0.37±0.05),(0.45±0.04),(0.60±0.05),(0.82±0.10)and(1.03±0.07)μg·mg-1;the total myocardial collagen contents were(1.87±0.31),(2.73±0.38),(3.36±0.31),(4.47±0.37),(6.08±0.74)and(7.67±0.49)μg·mg-1;the CVF were(1.95±0.23)%,(2.40±0.25)%,(3.65±0.25)%,(5.43±0.29)%,(6.97±0.36)%and(9.38±0.49)%;the relative expression levels of OPG protein were 0.64±0.07,0.80±0.07,1.02±0.07,1.32±0.11,2.13±0.12 and 2.84±0.16;the relative expression levels of RANKL protein were 0.71±0.08,1.06±0.07,1.53±0.07,2.62±0.12,4.46±0.14 and 6.11±0.16;the relative expression levels of RANK protein were 0.30±0.05,0.45±0.05,0.63±0.06,0.98±0.07,1.43±0.10 and 1.63±0.10.With the extention of time,the above indexs of all model groups were significantly higher than those in the sham-operation group(all P<0.05).There were positive linear correlation between the relative expression levels of OPG,RANKL,RANK protein and the levels of CVF and total contents in cardiomyocytes of rats with chronic heart failure(allP<0.01).Conclusions In the process of chronic heart failure,the expression of OPG/RANKL/RANK axis is obviously enhanced,in which the up-regulation of RANKL level is most obvious.The expression level of OPG/RANKL/RANK is positively correlated with CVF and total myocardial collagen content.
8.Influence of body mass index on the analgesic effect of sufentanil in elderly patients after proximal femoral nail anti-rotation surgery
Ke GU ; Hao WANG ; Tie-sheng CHEN ; Ji-kang XU ; Zhen TIAN
Journal of Regional Anatomy and Operative Surgery 2025;34(6):540-543
Objective To investigate the influence of body mass index(BMI)on individualized analgesic effect,inflammatory factors and safety of sufentanil in elderly patients after proximal femoral nail anti-rotation(PFNA)surgery.Methods A total of 161 elderly patients who received PFNA surgery in Nanjing Drum Tower Hospital Group Suqian Hospital from January 2022 to December 2023 were selected as study subjects.Patients with BMI<18.5 kg/m2 were set as Group A,patients with BMI ranging from 18.5 kg/m2 to 23.9 kg/m2 were set as Group B,patients with BMI ranging from 24.0 kg/m2 to 35.0 kg/m2 were set as Group C.After operation,individualized analgesia was performed with an intravenous patient-controlled analgesia pump of 2 μg/kg sufentanil based on body weight,and the postoperative pain degree,inflammatory factors,analgesia condition and adverse reactions of patients in the three groups were compared.Results At 8 hours,12 hours,24 hours and 48 hours after surgery,the pain visual analogue scale(VAS)scores of patietns in group B and group C were significantly lower than those in group A(P<0.05).At 12 hours,24 hours and 48 hours after surgery,the pain VAS scores of patients in group C were significantly lower than those in group B(P<0.05).The levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and interleukin-6(IL-6)1 day after surgery of patients in group B and group C were significantly lower than those in group A(P<0.05).The pressing times of analgesia pump and the duration of obvious pain within 48 hours after surgery of patients in group A were significantly more/longer than those in group B and group C(P<0.05),and the analgesic satisfaction score was significantly lower than those in group B and group C(P<0.05).The total incidence of analgesic adverse reactions in group C was significantly higher than those in group A and group B(P<0.05).Conclusion BMI may affect the individualized analgesic effect,inflammatory factors and safety of sufentanil in elderly patients undergoing PFNA surgery.Underweight patients may have insufficient analgesia,while overweight or obese patients may have excessive analgesia,which may affect the analgesic safety.
9.Study on the influence of different scanning positions based on chest phantom of CT scan on chest for image quality and radiation dose
Yan SUI ; Shihua TAO ; Kang LIU ; Xinghui GAI ; Zhiyuan GAO ; Zhaorui CHEN ; Hao GONG ; Dewu YANG
China Medical Equipment 2025;22(9):17-20
Objective:To explore the influence of different scanning positions based on chest phantom of computed tomography(CT)scan on chest on image quality and radiation dose.Methods:A thermoluminescent dosimeter(TLD)was placed at the breast area of simulating anthropoid chest phantom.GE Revolution evo CT was used to conduct scan on the conventional supine position(supine group)and prone position(prone group)for chest phantom.Different noise indexes(NI=10-23)were adjusted to control ration doses,and other parameters were fixed,and each group collected 12 sequence images.The average value(AV),standard deviation(SD)of the CT scan at region of interest(ROI)under different scanning positions were recorded to calculate the signal-to-noise ratio(SNR)and contrast-to-noise ratio(CNR)of the image.The radiation dose at the breast area was measured by TLD,and the volume CT dose index(CTDIvol)and dose-length product(DLP)were recorded.Results:Under different scanning positions,the radiation dose of breast organs in the prone group was lower than that in the supine group,there was a statistically significant difference between the two groups(t=6.57,P<0.05),while there were not statistically significant differences in CTDIvol and DLP between the two groups(P>0.05).There were not statistically significant differences in the CT values,SD,SNR,CNR of lung tissue,and the CT values of breast tissue between the two groups of images(P>0.05).The SD,SNR and CNR of breast tissue in the prone group were lower than those in the supine group,and the differences were statistically significant(t=-13.33,-10.59,6.70,P<0.05).There were no statistically significant differences in the subjective scores of the clarity of the edge of the tissue within lung,the layers of soft tissue of the breast,noise,and artifacts in the bone tissue between the two groups of images(P>0.05).Conclusion:When low-dose CT physical examination on chest is conducted in clinical practice,the scanning of prone position during undergoing CT scan on chest can obtain image quality that can meet the requirements in diagnosing lung,and reduce the radiation dose on the breast,and conform to the technical principle of optimal radiation protection.
10.Application and process optimization of automated magnetic bead sorting technology in T-SPOT.TB assay for tuberculosis infection
Tian ZHENG ; Xiao CHEN ; Hao LIU ; Meijuan KONG ; BeiPei KANG ; Jun XI ; Ke ZHOU ; Jiayun LIU
Chinese Journal of Preventive Medicine 2025;59(10):1779-1786
To evaluate the clinical application value of magnetic bead sorting technology (MBS) using the T-cell select kit combined with an automatic cell sorter for isolating peripheral blood mononuclear cells (PBMCs) to realize automated specimen pre-processing and process optimization in T-SPOT.TB assay. A cross-sectional study was conducted involving 300 patients recruited from the first affiliated hospital of Air Force Medical University from March 2023 to July 2024. Among them, there were 167 males and 133 females. The age range of the patients was 18 to 65 years, with a median age of 49.0 (33.3, 59.0) years and a mean age of (46.4±13.9) years. 4 ml of anticoagulated whole blood samples in duplicate were collected from each patient. One fresh sample (0-4 h) was processed immediately using density gradient centrifugation (DGC) for PBMC isolation, while the other was processed using MBS method at either 0-4 h or 34-54 h post-collection. The cycles for cell enrichment step of the automatic cell sorter were adjusted from the conventional 4 cycles to 2 cycles. Statistical analysis was performed using Cohen′s Kappa test to evaluate the concordance of T-SPOT.TB results across all processing groups. The results showed that for fresh samples (0-4 h), the T-SPOT.TB results for samples processed with 4-cycle and 2-cycle MBS enrichment steps demonstrated positive concordance rates of 91.7% and 93.1%, negative concordance rates of 100% for both, overall concordance rates of 98.0% and 97.9%, and Kappa values of 0.94 and 0.95, respectively, compared with the reference results for paired samples processed using DGC. Correspondingly, the results for samples processed with the 4-cycle and 2-cycle MBS methods at 34-54 h post-collection yielded positive concordance rates of 90.0% and 81.3%, negative concordance rates of 95.1% and 100%, overall concordance rates of 93.0% and 82.9%, and Kappa values of 0.86 and 0.43, respectively, compared with the reference results for fresh samples processed using DGC. In conclusion,for 0-4 h samples, the T-SPOT.TB results from both 4-cycle and 2-cycle MBS enrichment protocols were highly consistent with the reference results from conventional DGC methods. However, for 34-54 h stored samples, results from the 4-cycle MBS method rather than the 2-cycle protocol exhibited significantly superior concordance with the reference results. The MBS method using T-Cell Select kit achieves automated sample pre-processing for T-SPOT.TB assay, reduces hands-on time, and provides a viable alternative with reliable results for long-stored specimens.

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