Ultrasensitive and label free electrochemical immunosensor for detection of ROR1 as an oncofetal biomarker using gold nanoparticles assisted LDH/rGO nanocomposite.

In the present article, we developed a highly sensitive label-free electrochemical immunosensor based on NiFe-layered double hydroxides (LDH)/reduced graphene oxide (rGO)/gold nanoparticles modified glassy carbon electrode for the determination of receptor tyrosine kinase-like orphan receptor (ROR)-1. In this electrochemical immunoassay platform, NiFe-LDH/rGO was used due to great electron mobility, high specific surface area and flexible structures, while Au nanoparticles were prepared and coated on the modified electrodes to improve the detection sensitivity and ROR1 antibody immobilizing (ROR1Ab). The modification procedure was approved by using cyclic voltammetry and differential pulse voltammetry based on the response of peak current to the step by step modifications. Under optimum conditions, the experimental results showed that the immunosensor revealed a sensitive response to ROR1 in the range of 0.01-1 pg mL-1, and with a lower limit of quantification of 10 attogram/mL (10 ag mL-1). Furthermore, the designed immunosensor was applied for the analysis of ROR1 in several serum samples of chronic lymphocytic leukemia suffering patients with acceptable results, and it also exhibited good selectivity, reproducibility and stability.

Smartphone based on-chip fluorescence imaging and capillary flow velocity measurement for detecting ROR1+ cancer cells from buffy coat blood samples on dual-layer paper microfluidic chip.

Diagnosis of hematological cancer requires complete white blood cell count, followed by flow cytometry with multiple markers, and cytology. It requires substantial time and specialized training. A dual-layer paper microfluidic chip was developed as a quicker, low-cost, and field-deployable alternative to detect ROR1+ (receptor tyrosine-like orphan receptor one) cancer cells from the undiluted and untreated buffy coat blood samples. The first capture layer consisted of a GF/D glass fiber substrate, preloaded with cancer specific anti-ROR1 conjugated fluorescent particles to its center for cancer cell capture and direct smartphone fluorescence imaging. The second flow layer was comprised of a grade 1 cellulose chromatography paper with wax-printed four channels for wicking and capillary flow-based detection. The flow velocity was used as measure of antigen concentration in the buffy coat sample. In this manner, intact cells and their antigens were separated and independently analyzed by both imaging and flow velocity analyses. A custom-made smartphone-based fluorescence microscope and automated image processing and particle counter software were developed to enumerate particles on paper, with the limit of detection of 1 cell/µL. Flow velocity analysis showed even greater sensitivity, with the limit of detection of 0.1 cells/µL in the first 6 s of assay. Comparison with capillary flow model revealed great alignment with experimental data and greater correlation to viscosity than interfacial tension. Our proposed device is able to capture and on-chip image ROR1+ cancer cells within a complex sample matrix (buffy coat) while simultaneously quantifying cell concentration in a point-of-care manner.

Wnt5a/Ror2 pathway contributes to the regulation of cholesterol homeostasis and inflammatory response in atherosclerosis.

Atherosclerosis (AS) is characterized by lipids metabolism disorder and inflammatory response. Accumulating evidence has demonstrated that Wingless type 5a (Wnt5a) is implicated in cardiovascular diseases through non-canonical Wnt cascades. However, its precise role during the pathogenesis of AS is still unclear. Therefore, the present study aims to investigate the role and the underlying mechanism of Wnt5a/receptor tyrosine kinase-like orphan receptor 2 (Ror2) pathways in the promotion of AS process through affecting lipid accumulation and inflammation. In atherosclerotic clinical samples, Wnt5a levels were measured by using enzyme-linked immunosorbent assay (ELISA) assay. In vivo experiments were conducted by using apolipoprotein E knockout (apoE-/-) mice model. Vascular smooth muscle cells (VSMCs) were applied for in vitro studies. Wnt5a was highly expressed in both of atherosclerotic clinical samples and apoE-/- mice. The knockdown of Wnt5a significantly inhibited cholesterol accumulation and inflammatory response. Additionally, the lipopolysaccharide (LPS)-induced inflammation aggravated the cholesterol accumulation and decreased adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) expression in VSMCs. Depletion of intracellular cholesterol by ß-cyclodextrin (ß-CD) led to the upregulation of ABCA1 and the inhibition of inflammation. Conversely, the overexpression of Wnt5a inhibited ABCA1 expression, facilitated cholesterol accumulation, impared cholesterol efflux, promoted NF-κB nuclear translocation and the inflammatory cytokines secretion. Moreover, the knockdown of Ror2 increased ABCA1 expression and reduced Wnt5a-induced cholesterol accumulation and inflammatory responses. Furthermore, the knockdown of ABCA1 enhanced cholesterol accumulation and inflammatory response. Therefore, Wnt5a/Ror2 pathway was critical in regulating cholesterol homeostasis and inflammatory response, which might be a promising therapeutic target for AS therapy.

Which Markers Should the used for Diagnostic Chronic Lymphocytic Leukemia Immunophenotyping Scoring System by Flow Cytometry?

BACKGROUND: The scoring system used for chronic lymphocytic leukemia (CLL) cannot make an accurate diagnosis in some cases. Novel markers are available for the differential diagnosis of CLL, especially from MCL. However, these markers are still not incorporated into diagnostic algorithms. We investigated the role of CD43, CD81, CD200, and ROR1 in the differential diagnosis of CLL and their expression in non-CLL cases. METHODS: We investigated the role of CD43, CD81, CD20, and ROR1 in the differential diagnosis of CLL by incorporating them into the diagnostic panel after studying peripheral blood or bone marrow samples of 165 patients with 8-color flow cytometry. RESULTS: CD43 positivity was a sensitive marker but had a lower specificity for CLL. CD43 had high diagnostic value for CLL (sensitivity 100%, specificity 88.5%, AUC 98.0%). CD200 was a specific marker for CLL (sensitivity 98%, specificity 90%, AUC: 96%). CD81 expression was highest in the MCL cases, with a median expression rate of 68.5% (range: 54 - 82.5%). It was negative in all the CLL cases. For CLL, CD81 negativity had a sensitivity of 95%, a specificity of 82% and an AUC of 92%. ROR1 was positive in all CLL and MCL cases. CD79b, on the other hand, was a fairly sensitive and specific marker for MCL. CONCLUSIONS: CD43, CD81, CD200, and ROR1 should be incorporated into diagnostic algorithms for the differential diagnosis of CLL, especially from MCL.