scholarly journals A Flow Cytometry-Based Assay for the Measurement of Total Complement Activity in the Serum and Clinical Practice

2022 ◽  
Vol 2022 ◽  
pp. 1-12
Author(s):  
Xuewei Ding ◽  
Shijun Li ◽  
Hui Liu

Objective. To develop a novel sensitive and accurate assay suitable for high-volume testing of the total complement activity in the serum for clinical laboratories. Methods. The total complement activity (TCA) to be measured was quantified by detecting the number of fragments produced by erythrocyte lysis and the erythrocyte fragmentation index (EFI), indicating TCA. EFI = M × M 2 / M 1 + M 2 , where M is the number of erythrocyte fragments (removed from the background), M 1 is the number of unagglutinated red cells, M 2 is the number of agglutinated red cell groups, and M 2 / M 1 + M 2 is the agglutination coefficient indicating the degree of erythrocyte agglutination. Mild changes in hemolysin and erythrocyte concentrations were made to optimize the testing conditions. The same serum samples were tested for 10 consecutive days to determine the stability of the experimental results. Serum EFI was detected in both nephrotic syndrome patients and healthy subjects. Results. There was a linear relationship between hemolysin and erythrocyte agglutination ( r = 0.999 , P < 0.001 ). A good linear relationship existed between EFI and TCA ( r = 0.991 , P < 0.001 ). The results were not affected by slight fluctuations in the concentrations of hemolysin or erythrocytes. The interbatch CV = 8.6 % of the test results showed good stability. There was a significant difference in the EFI between nephrotic syndrome patients and healthy individuals, P < 0.001 , and EFI was reduced in nephrotic syndrome patients compared to healthy individuals. Conclusion. The flow cytometry-based assay for TCA was sensitive and accurate and had potential value for clinical application.

2021 ◽  
Author(s):  
Fen-fen Ni ◽  
Guang-lei Liu ◽  
shi-lei Jia ◽  
Ran-ran Chen ◽  
Li-bing Liu ◽  
...  

Abstract Background: The specific etiology and mechanism of idiopathic nephrotic syndrome (INS) in children remain unclear, so we investigated the pathogenesis of INS by measuring the effects two specific miRNAs on Th2 cells in children with this disease. Methods: Flow cytometry was used to measure the levels of Th2 cells and a cytometric bead array was used to measure the levels of IgE, interleukin (IL) -4, and IL-13. RT-PCR was used to measure the levels of miR-24 and miR-27 in CD4+TCD25− cells. PBMCs were isolated using Ficoll density gradient centrifugation, and transfected with different mimic or inhibitor miRNAs. RT-PCR was used to measure the expression of different RNAs, and flow cytometry was used to determine the percentages of Th2 cells. Results: Children with active INS had higher percentages of Th2 cells than healthy controls (P<0.05), but there was no significant difference for children in remission. The plasma levels of IgE, IL-4, and IL-13 were significantly increased in children with active INS (P<0.05). miR-24 and miR-27 were at lower levels in children with active non-atopic INS (P<0.05). Transfection experiments indicated that upregulation of each miRNA decreased the percentage of Th2 cells and the level of IL-4 (P<0.05), and down-regulation of each miRNA had the opposite effects (P<0.05). Conclusion: Children with active INS, with or without atopy, had higher levels of IgE, possibly related to their higher levels of IL-13 and IL-4 due to drift toward Th2 cells. miR-24 and miR-27 suppress the expression of Th2 cells and have a critical function in Th2 expression in INS.


2019 ◽  
Author(s):  
Abdelhakam G. Tamomh ◽  
Xiaojun Jin ◽  
Hui Liu

Abstract Background The goal of this paper is to compare and evaluate the differences in total activity and Heat Tolerance of complement distribution Between Black and Yellow populations.Methods Blood samples of Black and Yellow healthy individuals were randomly collected, and plasma was obtained. The test plasma was diluted in a five series concentration, following with standard reaction and experimental reaction measurements. The basic principle of heat tolerance temperature calculation is to consider the ratio value (ratio method) in which heat tolerance of TCA calculated and plotted according to the area of trapezoidal and triangle as follows: C x°C=OD1-OD5+(OD2-OD5+OD3-OD5+OD4-OD5)*2. Where Cx°C is the total complement activity of the specimen measured in the two reaction conditions.Results The total activity of complement in Black individuals was statistically significantly lower than Yellow individuals (P< 0.05). The heat tolerance of total complement activity was higher among blacks than yellow individuals with no differences between the two groups (P> 0.05). The frequency values of total activity of complement distributed in the yellow individuals was statistically higher than Blacks (P<0.05) and The heat tolerance of total complement activity was statistically higher significant among Blacks than Yellow individuals (P<0.05).Conclusions There is a high differences in total complement activity and heat Tolerance distribution between Black and Yellow populations, may predict health status and innate immunity level differences between African race and Asian race.


1986 ◽  
Vol 32 (2) ◽  
pp. 275-278 ◽  
Author(s):  
D W Bowden ◽  
M Rising ◽  
G Akots ◽  
A Myles ◽  
R J Broeze

Abstract This is a rapid, homogeneous, liposome-based assay for total complement activity in human serum. Liposome-encapsulated enzyme is unmasked by the action of complement on liposomes carrying surface-bound immune complexes. The amount of unmasked enzyme, proportional to the concentration of added complement, is quantified by measuring the absorbance of enzymically produced product at 410 nm. Complement activity in serum samples is extrapolated from a standard curve generated from dilutions of a guinea pig serum containing a known activity of complement. Interassay CVs were less than 7.0% and intra-assay CVs less than 2.8% for serum pools with complement activities spanning the normal range. Test results correlate as well with those of the hemolytic complement test (r = 0.80) as the latter correlates with itself (r = 0.82), and also correlate reasonably with measurements of complement components C3 (r = 0.62) and C4 (r = 0.74). Values for a normal population are reported. Advantages of this test include stability of reagents, speed, accuracy, simplicity, and avoidance of radioisotopes.


2021 ◽  
Vol 9 ◽  
Author(s):  
Fen-fen Ni ◽  
Guang-lei Liu ◽  
Shi-lei Jia ◽  
Ran-ran Chen ◽  
Li-bing Liu ◽  
...  

Purpose: We investigated the pathogenesis of idiopathic nephrotic syndrome (INS) by measuring the effects two specific miRNAs on Th2 cells in children with this disease.Methods: After informed consent, we enrolled 20 children with active INS before steroid initiation, 20 children with INS in remission after steroid therapy, and 20 age-matched healthy controls. Flow cytometry was used to measure the levels of Th2 cells and a cytometric bead array was used to measure the levels of IgE, interleukin (IL)−4, and IL-13. RT-PCR was used to measure the levels of miR-24 and miR-27 in CD4+TCD25− cells. PBMCs were isolated using Ficoll density gradient centrifugation, and transfected with different mimic or inhibitor miRNAs. RT-PCR was used to measure the expression of different RNAs, and flow cytometry was used to determine the percentage of Th2 cells.Results: Relative to healthy controls, children with active INS had higher percentages of Th2 cells (P &lt; 0.05), but there was no significant difference in controls and children in remission. The plasma levels of IgE, IL-4, and IL-13 were significantly increased in children with active INS (P &lt; 0.05). There were lower levels of miR-24 and miR-27 in children with active non-atopic INS (P &lt; 0.05). Transfection experiments indicated that upregulation of each miRNA decreased the percentage of Th2 cells and the level of IL-4 (P &lt; 0.05), and down-regulation of each miRNA had the opposite effects (P &lt; 0.05).Conclusion: Children with active INS, with or without atopy, had higher levels of IgE, possibly related to their higher levels of IL-13 and IL-4 due to a drift toward Th2 cells. miR-24 and miR-27 suppressed the expression of Th2 cells and have a critical function regulating Th2 cell expression in INS.


2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Andréa Tavares Dantas ◽  
Sayonara Maria Calado Gonçalves ◽  
Anderson Rodrigues de Almeida ◽  
Rafaela Silva Guimarães Gonçalves ◽  
Maria Clara Pinheiro Duarte Sampaio ◽  
...  

Objective. To determine active TGF-β1 (aTGF-β1) levels in serum, skin, and peripheral blood mononuclear cell (PBMC) culture supernatants and to understand their associations with clinical parameters in systemic sclerosis (SSc) patients.Methods. We evaluated serum samples from 56 SSc patients and 24 healthy controls (HC). In 20 SSc patients, we quantified spontaneous or anti-CD3/CD28 stimulated production of aTGF-β1 by PBMC. The aTGF-β1 levels were measured by ELISA. Skin biopsies were obtained from 13 SSc patients and six HC, and TGFB1 expression was analyzed by RT-PCR.Results. TGF-β1 serum levels were significantly higher in SSc patients than in HC (p< 0.0001). Patients with increased TGF-β1 serum levels were more likely to have diffuse subset (p= 0.02), digital ulcers (p= 0.02), lung fibrosis (p< 0.0001), positive antitopoisomerase I (p= 0.03), and higher modified Rodnan score (p= 0.046). Most of our culture supernatant samples had undetectable levels of TGF-β1. No significant difference in TGFB1 expression was observed in the SSc skin compared with HC skin.Conclusion. Raised active TGF-β1 serum levels and their association with clinical manifestations in scleroderma patients suggest that this cytokine could be a marker of fibrotic and vascular involvement in SSc.


2005 ◽  
Vol 76 (4) ◽  
pp. 526-533 ◽  
Author(s):  
Takehiro Mitsui ◽  
Yukie Tsukamoto ◽  
Shigeru Suzuki ◽  
Chikao Yamazaki ◽  
Kazuo Masuko ◽  
...  

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 414.2-415
Author(s):  
X. Huang ◽  
T. W. Li ◽  
J. Chen ◽  
Z. Huang ◽  
S. Chen ◽  
...  

Background:Ankylosing spondylitis (AS) is a type of common, chronic inflammatory disease that compromises the axial skeleton and sacroiliac joints, causing inflammatory low back pain and progressive spinal stiffness, over time some patients develop spinal immobility and ankylosis which can lead to a decrease in quality of life. The last few decades, evidence has clearly indicated that neutrophil also plays key roles in the progression of AS. However, the immunomodulatory roles and mechanisms of neutrophils in AS are poorly understood. T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) has been reported as an important regulatory molecule, expressed and regulated on different innate immune cells, plays a pivotal role in several autoimmunity diseases. Recent study indicates that Tim3 is also expressed on neutrophils. However, the frequency and roles of Tim3-expressing neutrophils in AS was not clear.Objectives:In this study, we investigated the expression of Tim3 on neutrophils in AS patients and explored the correlation between the level of Tim3-expressing neutrophils and the disease activity and severity of AS.Methods:Patients with AS were recruited from Guangdong Second Provincial General Hospital (n=62). Age/sex-matched volunteers as Healthy controls (HC) (n=39). The medical history, clinical manifestations, physical examination, laboratory measurements were recorded. The expression of costimulatory molecules including programmed death 1 (PD-1), Tim-3 on neutrophils were determined by flow cytometry. The mRNA expression of PD-1 and Tim-3 was determined by real-time PCR. The levels of Tim3-expressing neutrophils in AS patients were further analyzed for their correlation with the markers of inflammation such as ESR,CRP,WBC and neutrophil count(NE), as well as disease activity and severity of AS. The expression of Tim3 on neutrophils was monitored during the course of treatment (4 weeks).Results:The expression of Tim3 on neutrophils in patients with AS was increased compared to the HC (Figure 1A). However, significant difference was observed in the frequency of PD-1-expressing neutrophils between AS patients and HC (Figure 1B). The expression analysis of Tim-3 mRNA, but not PD-1, confirmed the results obtained from flow cytometry (Figure 1C). The level of Tim3-expressing neutrophils in patients with AS showed an positive correlation with ESR, CRP and ASAS-endorsed disease activity score (ASDAS) (Figure 1D). Moreover, the frequency of Tim3-expressing neutrophils in active patients(ASDAS≥1.3) was increased as compare with the inactive patients (ASDAS<1.3) (Figure 1E). As shown in Figure 1F, the frequency of Tim3-expressing neutrophils decreased after the treatment.Conclusion:Increased Tim-3 expression on neutrophils may be a novel indicator to assess disease activity and severity in AS, which may serves as a negative feedback mechanism preventing potential tissue damage caused by excessive inflammatory responses in AS patients.References:[1]Han, G., Chen, G., Shen, B. & Li, Y., Tim-3: an activation marker and activation limiter of innate immune cells. FRONT IMMUNOL 4 449 (2013).[2]Vega-Carrascal, I. et al., Galectin-9 signaling through TIM-3 is involved in neutrophil-mediated Gram-negative bacterial killing: an effect abrogated within the cystic fibrosis lung. J IMMUNOL 192 2418 (2014).Figure 1.(A,B)The expression of Tim3 and PD-1 on neutrophils in AS and HC were determined by flow cytometry.(C) The expression of Tim3 and PD-1 on neutrophils in AS and HC were determined by RT-PCR.(D)The correction between Tim3-expressing neutrophils and ESR,CRP,ASDAS.(E) The expression of Tim3 on neutrophils in active and inactive patients.(F) Influence of treatment on the frequency of Tim3-expressing neutrophils.Disclosure of Interests:None declared


2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 376.2-376
Author(s):  
E. Berglin ◽  
A. Esberg ◽  
J. Dahlqvist ◽  
J. Sjöwall ◽  
A. Lundquist ◽  
...  

Background:Etiology and pathogenesis of ANCA-associated vasculitis (AAV) is multifactorial and understanding of the processes leading from a healthy immune system to autoimmunity and on to debut of symptoms in AAV is rudimentary.Objectives:To identify inflammatory proteins related to the early processes preceding AAV development, and potential novel biomarkers, using large-scale protein analysesMethods:The Swedish National Patient Register of in-patient carevand the Swedish Cause of Death Register with discharge diagnosis from ICD-9 and-10 for AAV were co-analysed with the registers of 4 different blood biobanks to identify AAV individuals with available samples predating onset of symptom. Of the pre-AAV cases 86 (36 male, 50 female; mean age (SD); 51.9 (16.9) years) were identified with at least one plasma or serum sample (28 plasma, and 100 serum) pre-dating symptom onset (mean (SD); -4.3 (3.1) years), and 14 had 2-3 samples. Serum and plasma control samples matched for sex, age and sampling date were identified (n=198; 82 male, 116 female; mean age (SD); 51.9±15.9 years). The samples were analysed for levels of 92 proteins using proximity extension assay (OLINK inflammation panel, SciLifeLab, Uppsala, Sweden). Data were analysed using routine statistical methods, random forest and Partial Least square-discriminant analysis (PLS-DA).Results:As previously described for the assay significant difference between plasma and serum samples were observed both in pre-AAV individuals and controls. In pre-AAV plasma samples significantly increased concentrations of interleukin (IL)-2, chemokine ligand (CCL)-4, fibroblast growth factor (FGF)21, IL-4 and CCL20 were found closer to symptom onset, (<5 years) than later (> 5 years) and compared with controls. In serum tumor necrosis factor receptor superfamily member (TNFRSF)9, CXCL9, osteoprotegerin and vascular endothelial growth factor-A were significantly increased <5 years before onset vs. later (>5 years) and compared with controls. PLS-DA score scattered plot separated the pre-AAV individuals from healthy controls (R2=0.26), with significantly increased levels of CCL23, CXCL5, and matrix metalloproteinases-1 (MMP-1),transforming growth factor-ß, orosomucoid, en-rage (S100A12) and IL-7 and decreased FGF-19 level in serum. Binary logistic regression analyses comparing tertiles for these proteins confirmed significantly increased odds ratios for disease development of CCL23, CXCL5 and MMP-1. The findings were confirmed in random forest analysis where these factors were among the 20 most discriminatory factors between pre-symptomatic AAV and controls.Conclusion:In serum samples collected years before symptom onset of AAV, proteins involved in immune system activation were increased, suggesting that the inflammatory process is initiated long before clinical manifestations of the disease appear. These findings propose the elevated proteins as novel biomarkers for disease progression.References:[1]Watts et al. Ann Rheum Dis 2007;66:222-22Acknowledgments:Vasculitis Foundation, USADisclosure of Interests:Ewa Berglin: None declared, Anders Esberg: None declared, Johanna Dahlqvist: None declared, Johanna Sjöwall: None declared, Anders Lundquist: None declared, Kristina Lejon: None declared, Ingegerd Johansson: None declared, Aladdin J Mohammad Speakers bureau: lecture fees from Roche and Elli Lilly Sweden, PI (GiACTA study), Solbritt Rantapää Dahlqvist: None declared


Author(s):  
Michela Bottani ◽  
Aasne K. Aarsand ◽  
Giuseppe Banfi ◽  
Massimo Locatelli ◽  
Abdurrahman Coşkun ◽  
...  

Abstract Objectives Thyroid biomarkers are fundamental for the diagnosis of thyroid disorders and for the monitoring and treatment of patients with these diseases. The knowledge of biological variation (BV) is important to define analytical performance specifications (APS) and reference change values (RCV). The aim of this study was to deliver BV estimates for thyroid stimulating hormone (TSH), free thyroxine (FT4), free triiodothyronine (FT3), thyroglobulin (TG), and calcitonin (CT). Methods Analyses were performed on serum samples obtained from the European Biological Variation Study population (91 healthy individuals from six European laboratories; 21–69 years) on the Roche Cobas e801 at the San Raffaele Hospital (Milan, Italy). All samples from each individual were evaluated in duplicate within a single run. The BV estimates with 95% CIs were obtained by CV-ANOVA, after analysis of variance homogeneity and outliers. Results The within-subject (CV I ) BV estimates were for TSH 17.7%, FT3 5.0%, FT4 4.8%, TG 10.3, and CT 13.0%, all significantly lower than those reported in the literature. No significant differences were observed for BV estimates between men and women. Conclusions The availability of updated, in the case of CT not previously published, BV estimates for thyroid markers based on the large scale EuBIVAS study allows for refined APS and associated RCV applicable in the diagnosis and management of thyroid and related diseases.


Cancers ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2565
Author(s):  
Yixing Wu ◽  
Hongmei Zeng ◽  
Qing Yu ◽  
Huatian Huang ◽  
Beatrice Fervers ◽  
...  

Several exosome proteins, miRNAs and KRAS mutations have been investigated in the hope of carrying out the early detection of pancreatic cancer with high sensitivity and specificity, but they have proven to be insufficient. Exosome RNAs, however, have not been extensively evaluated in the diagnosis of pancreatic cancer. The purpose of this study was to investigate the potential of circulating exosome RNAs in pancreatic cancer detection. By retrieving RNA-seq data from publicly accessed databases, differential expression and random-effects meta-analyses were performed. The results showed that pancreatic cancer had a distinct circulating exosome RNA signature in healthy individuals, and that the top 10 candidate exosome RNAs could distinguish patients from healthy individuals with an area under the curve (AUC) of 1.0. Three (HIST2H2AA3, LUZP6 and HLA-DRA) of the 10 genes in exosomes had similar differential patterns to those in tumor tissues based on RNA-seq data. In the validation dataset, the levels of these three genes in exosomes displayed good performance in distinguishing cancer from both chronic pancreatitis (AUC = 0.815) and healthy controls (AUC = 0.8558), whereas a slight difference existed between chronic pancreatitis and healthy controls (AUC = 0.586). Of the three genes, the level of HIST2H2AA3 was positively associated with KRAS status. However, there was no significant difference in the levels of the three genes across the disease stages (stages I–IV). These findings indicate that circulating exosome RNAs have a potential early detection value in pancreatic cancer, and that a distinct exosome RNA signature exists in distinguishing pancreatic cancer from healthy individuals.


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