Development and application of amplified luminescent proximity homogeneous assay for quantitation of heparin-binding protein.

A fast and highly sensitive amplified luminescent proximity homogeneous assay (AlphaLISA) method was developed for quantitation of plasma heparin-binding protein levels. In this study, a method directly coupling donor and acceptor beads modified with aldehyde groups to anti-HBP antibodies was proposed, which can effectively simplify the steps and shorten the reaction time to achieve faster detection. Therefore, the developed method required only 15 min of reaction time to generate results. Compared with the approved commercial kit, the developed method had a wider linear range (2.78-500 ng/mL). The excellent linear range means that the method can better exploit the value of HBP in clinical applications. Meanwhile, results of amplified luminescent proximity homogeneous assay and fluorescence dry quantitative immunoassay had good correlation and consistency (ρ = 0.9181). Moreover, the plasma HBP concentrations of patients with bacterial infection were significantly higher than those of healthy individuals (P < 0.0001), indicating the potential applicability of the proposed method for predicting the incidence of bacterial infections. Importantly, the newly developed method is expected to serve as an alternative to the traditional assay method and provides a completely new platform for other biomarkers that require rapid detection.

Differential expression of circular RNAs in plasma exosomes from patients with ankylosing spondylitis.

Exosomes are being extensively studied as a source of valuable new biomarkers. The underlying mechanism of ankylosing spondylitis (AS) may include changes in the circular RNAs (circRNAs) of exosomes. However, there is a lack of reports on the role of exosomal cirRNAs in the plasma of patients with AS. We isolated exosomes from the plasma of patients with AS and healthy individuals (controls). Subsequently, we investigated the circRNA profiles of the exosomes via high-throughput RNA sequencing and identified 56 differentially expressed circRNAs in the exosomes of patients with AS compared with those of the healthy controls. Gene Ontology demonstrated that the differentially expressed circRNAs were mainly involved in the negative regulation of the activity of the transcription factor nuclear factor-κB and bone remodelling that is potentially related to AS. Kyoto Encyclopedia Genes and Genome demonstrated that the most highly AS-correlated pathways that were identified were 'notch signalling pathway' and those primarily involved with 'cholinergic synapse'. Finally, we validated five differentially expressed circRNAs using quantitative real-time polymerase chain reaction and predicted their potential functions through the circRNA-miRNA-mRNA network. Our study is the first to report changes in exosomal circRNAs from plasma samples of patients with AS, and provide novel targets for further investigation of molecular mechanisms and potential intervention therapy targets of AS.

Establishment of heparin-binding protein time-resolved immunoassay and some potential clinical applications.

AIM: To establish a highly sensitive time-resolved fluorescence immunoassay of heparin-binding protein (HBP-TRFIA) and evaluate its application value for bacterial or fungal infections in tumor patients. METHODS: Two types of HBP monoclonal specific antibodies against different epitopes of the antigen molecule were used as coating antibodies and Eu3+-labeled antibodies, respectively. The double-antibody sandwich method was used in establishing HBP-TRFIA, and the methodology was evaluated. The established HBP-TRFIA was used in detecting HBP concentration in the plasma samples of healthy individuals, patients with bacterial or fungal infections, and infected or uninfected patients with various types of tumors. RESULTS: The linear range of HBP-TRFIA was (0.11-530 ng/mL). Plasma HBP concentrations detected through HBP-TRFIA were consistent with the results of fluorescence quantitative immunochromatography (ρ = 0.964). The plasma HBP concentrations of infected tumor patients were significantly higher than those of uninfected tumor patients (P < 0.01). CONCLUSION: This study successfully established a highly sensitive HBP-TRFIA, which was highly comparable to commercially available fluorescent quantitative immunochromatographic kits and was able to facilitate the timely diagnosis of bacterial or fungal infections in patients with tumor.

[Pathogenesis study of inherited dysfibrinogenemia].

OBJECTIVE: To explore the pathogenesis of a family with inherited dysfibrinogenemia. METHODS: Coagulation parameters of peripheral venous blood of a family with inherited dysfibrinogenemia from November 2012 were measured. And platelet and fibrinogen functions were examined by thromboelastogram. The antigen concentration of fibrinogen was detected by immune nephelometry. All exons and exon-intron boundaries of FGA, FGB and FGG were amplified and subjected to mutation screening by direct/reverse sequencing. And the influences of mutant fibrinogen structure and function were analyzed and predicated by a molecular structure model. RESULTS: The values of activated partial thromboplastin time (APTT), D-dimer and fibrinogen antigen of the propositus and his mother (I-2), younger brother (II-3), younger sister (II-2) and daughter (III-1) were all in normal reference value ranges.However thrombin time (TT) was significantly prolonged and the activity of fibrinogen was much lower compared to its antigenicity. Thromboelastogram indicated normal function of platelet and impaired function of fibrinogen of I-2, II-2 and III-1.However the fibrinogen functions of proband and II-3 became much more impaired. Mutation screening demonstrated the homozygous mutation of proband and II-3 while I-2, II-2 and III-1 showed heterozygous mutation of FGG c.1001 A>C (p. Asn308Thr). No mutation was detected among other family members and reducing SDS-PAGE immunoblot showed no variants. Asn308, located at the interface of fibrinogen dimmer, participated in the fibrous structure assembling from the structure model. And mutation at this position will affect the stability of fiber structure. CONCLUSION: FGG c.1001 A>C mutation may account for dominant genetic dysfibrinogenemia in these family members.

Biological variations of hematologic parameters determined by UniCel DxH 800 hematology analyzer.

CONTEXT: The Coulter DxH 800 hematology analyzer can determine conventional hematologic parameters. It also provides many new hematologic parameters, some of which show potential clinical utility. OBJECTIVES: To study, for the first time, the biological variations of new hematologic parameters and reinvestigate the biological variations of conventional hematologic parameters using the newest Coulter hematology analyzer. DESIGN: Forty adult volunteers (21 women and 19 men) were included. All participants maintained their normal lifestyles. Blood samples were drawn in duplicate by a single experienced phlebotomist and analyzed within 2 hours using a single analyzer. Before each batch analysis, the instrument quality controls were performed using the same lots of reagents. RESULTS: Within-subject and between-subject biological variations for the conventional hematologic parameters were compatible with published data. The analytic variation of the DxH 800 for these parameters appeared smaller. Index of individuality (ratio of within-subject to between-subject biological variation) for all parameters was low. In addition, intraday and interday biological variations of most parameters studied are fairly constant among the population examined. CONCLUSIONS: These observations are clinically valuable. Data on within-subject biological variation and analytic precision may be used to generate objective delta-check values for use in quality management. Comparing within-subject and between-subject biological variation on new parameters may allow us to decide the utility of traditional population-based reference ranges. Furthermore, documentation of biological variations of new parameters is an essential prerequisite in the development of any clinical application in the future.

Biological variations of leukocyte numerical and morphologic parameters determined by UniCel DxH 800 hematology analyzer.

CONTEXT: The Coulter DxH 800 hematology analyzer can determine leukocyte numerical parameters (total leukocyte counts and differentials). It also measures intrinsic biophysical properties of these cells in their near-native state. These morphologic measurements are known as cell population data (CPD). OBJECTIVE: To study, for the first time, the biological variations of morphologic parameters or CPD and reinvestigate numerical parameters using the newest Coulter hematology analyzer. Design.-Forty adult volunteers (21 women, 19 men) were included. All participants maintained their normal lifestyles. Blood samples were drawn in duplicate by a single experienced phlebotomist and analyzed within 2 hours using a single analyzer. Before each batch analysis, the instrument quality controls were performed using the same lots of reagents. RESULTS: Within-subject (CV(I)) and between-subjects (CV(G)) biological variations for numerical parameters are smaller than previously reported. Cell population data have much smaller overall CV(I) and CV(G) compared to numerical parameters, suggesting that these parameters are less variable around the homeostatic set point intraindividually and interindividually. Index of individuality (ratio of CV(I)/CV(G)) for CPD was low. In addition, intraday and interday biological variations of all parameters are fairly constant. CONCLUSIONS: These observations are clinically valuable. Data on CV(I) and analytical precision may be used to generate objective delta-check values for use in quality management. Comparing CV(I) and CV(G) on CPD may allow us to decide the utility of traditional population-based reference ranges. Documentation of CPD on biological variations is an essential prerequisite in the development of any new application clinically.

[Effect of total flavonoids from Chrysanthemun morifolium on learning and memory in aging mice].

OBJECTIVE: To investigate the effect of total fiavonoids from Chrysanthemun morifolium (TFCM) on learning and memory, and cholinergic system function in aging mice. METHODS: The aging mice model was established by subcutaneous injection of D-galactose. ICR mice were divided into five groups (n=10): contrA group, model group, and TFCM groups. Mice in TFCM groups were given TFCM (50,100 or 150 mg/kg) by gastric irrigation once a day. Learning and memory ability were evaluated by Morris water maze test. The MDA content, SOD and Ach E activity were also measured. RESULTS: Compared with control group, learning and memory ability declined in the D-galactose-induced aging mice; meanwhile MDA content and AchE activity increased, SOD activity decreased. Treatment with TFCM (100, 150 mg/kg) ameliorated the decrease in learning and memory ability of aging mice. Compared with model group, TFCM (100, 150 mg/kg) could also decrease MDA content and Ach E activity, and increase SOD activity in aging mice. CONCLUSION: TFCM may improve the learning and memory ability of aging mice. The mechanism is involved in its antioxidative characteristic and improvement of central cholinergic system function.