Algorithm to identify circulating tumor cell clusters using in vivo flow cytometer

Recent studies in oncology have addressed the importance of detecting circulating tumor cell clusters because circulating tumor cell clusters might survive and metastasize more easily than single circulating tumor cells.Signals with larger peak widths detected by in vivo flow cytometer(IVFC)have been used to identify cell clusters in previous studies.However,the accuracy of this criterion might be greatly degraded by variance in blood°ow and the rolling behaviors of circulating tumor cells.Here,we propose a criterion and algorithm to distinguish cell clusters from single cells.In this work,we first used area-based and volume-based models for single°uorescent cells.Simulating each model,we analyzed the corresponding morphology of IVFC signals from cell clusters.According to the Rayleigh criterion,the valley between two adjacent peak signals from two distinguishable cells should be lower than 73.5%of the peak values.A novel signal processing algorithm for IVFC was developed based on this criterion.The results showed that cell clusters can be reliably identied using our proposed algorithm.Intravital imaging was also performed to further support our algorithm.With enhanced accuracy,IVFC is a powerful tool to study circulating cell clusters.

Temporal change of plankton size structure preserved by Lugol's solution:a FlowCAM study

Plankton size structure is crucial for understanding marine ecosystem dynamics and the associated biogeochemical processes.A fixation step by acid Lugol’s solution has been commonly employed to preserve plankton samples in the field.However,the acid Lugol’s solution can bias the estimation of size structure and the preserved plankton size structure can vary with time.Here,we explore the impact of sample storage time on the size-structure of the plankton community preserved by Lugol’s solution.Two short-term experiments and one long-term experiment were conducted to explore the change of plankton community size structure with the storage time:covering from a week to a month,and to nearly seven months based on particle-size data obtained by continuous Flow Cytometer and Microscope(FlowCAM)measurements.We found a linear change of plankton size with the storage time in short-term periods(less than 3 months)with a decrease of the slope but an increase of the intercept for the normalized biomass size spectrum(NBS S).However,there were opposite trends for NBSS with increasing slope but decreasing intercept after3 months.The potential causes of the distinct patterns of the NBSS parameters are addressed in terms of the interplay between particle aggregation and fragmentation.We found large changes in plankton biovolume and abundance among different size classes,which may indicate a distinct effect of acid Lugol’s solution on various plankton size classes.The mechanism driving temporal change in the size-structure of the Lugolfixed plankton community was further discussed in terms of particle aggregation and fragmentation.Finally,we emphasize that the effect of storage time should be taken into account when interpreting or comparing data of plankton community acquired from samples with various storage durations.

STUDYING THE ROLE OF MACROPHAGES IN CIRCULATING PROSTATE CANCER CELLS BY IN VIVO FLOW CYTOMETRY

Metastasis is a very complicated multi-step process and accounts for the low survival rate of the cancerous patients.To metastasize,t he malignant cells must detach from the primary tumor and migrate to secondary sites in the body through either blood or lymph circulation.Macrophages appear to be directly involved in tumor progression and metastasis.However,the role of macrophages in affecting cancer metast asis has not been fully elucidated.Here,we have utilized an emerging technique,namely in vivo flow cytometry(IVFC)to study the depletion kinetics of circulating prostate cancer cells in mice and determine how depletion of macrophages by the liposome encapsulated clodronate affects the depletion kinetics.Our results show diferent depletion kinetics of PC-3 cells between the macrophagedeficient group and the control group.The number of circulating tumor cells(CTCs)in the macrophage-deficient group decreases in a slower manner compared to the control mice group.The differences in depletion kinetics indicate that the absence of macrophages facilitates the stay of prostate cancer cells in circulation.In addition,our imaging data suggest that macrophages might be able to arrest,phagocytose and digest PC-3 cells.Therefore,phagocy tosis may mainly contribute to the de-pletion kinetic diferences.The developed methods elaborated here would be useful to study the relationship between macr ophages and tumor metastasis in small animal cancer models.

Microchip imaging cytometer:making healthcare available,accessible,and affordable

The Microchip Imaging Cytometer(MIC)is a class of integrated point-of-care detection systems based on the combination of optical microscopy and flow cytometry.MIC devices have the attributes of portability,cost-effectiveness,and adaptability while providing quantitative measurements to meet the needs of laboratory testing in a variety of healthcare settings.Based on the use of microfluidic chips,MIC requires less sample and can complete sample preparation automatically.Therefore,they can provide quantitative testing results simply using a finger prick specimen.The decreased reagent consumption and reduced form factor also help improve the accessibility and affordability of healthcare services in remote and resource-limited settings.In this article,we review recent developments of the Microchip Imaging Cytometer from the following aspects:clinical applications,microfluidic chip integration,imaging optics,and image acquisition.Following,we provide an outlook of the field and remark on promising technologies that may enable significant progress in the near future.

The Role of Kappa and Lambda in Subclassification of B Cell Lymphoblastic Leukemia in Sudanese Patients Using Flow Cytometry

Background: B-cell Acute lymphoblastic leukemia (B-ALL) is a neoplasm of lymphoblasts which are of B-cell lineage typically composed of small to medium sized blast cells, moderately condensed to dispersed chromatin with scanty cytoplasm and inconspicuous nucleoli, involving the bone marrow and/or blood. Methods and materials: This is a prospective cross-sectional study in which 50 blood and/or bone marrow samples of newly diagnosed patients (B-ALL) were tested for immunophenotyping. All samples were prepared for surface and cytoplasmic markers including kappa and lambda human antibody for 10 minutes in dark place and then run by the Flow Cytometer. Results: 64% of the study populations were males and 36% were females. Cases were classified according to immunophenotype and the age into different subtypes and showed the following frequencies: Pro B-ALL (8%), early pre B-ALL (56%), common B-ALL (16%), Pre-B-ALL (14%) and Mature B-ALL (only 6%). Surface immunoglobulin was positive in 10% and negative in 90% of all patients, showing 100% positivity in mature B-ALL and totally negative in other subtypes. While cytoplasmic immunoglobulin was positive in 16% and negative in 84% of all patients and was positive in 100% of Pre-B-ALL and in 50% of mature B-ALL. Surface kappa was more expressed in mature B-ALL than lambda giving a ratio of 2:1, while cytoplasmic kappa:lambda was 6:1 in Pre-B-ALL. Conclusion: Kappa and lambda have important role in B-ALL classification which necessitates their presence in immunophenotyping of B-ALL.

Experimental Study on the Enhancement of Murine Splenic Lymphocyte Proliferation by Lycium Barbarum Glycopeptide

In order to investigate the immunoactivity of Lycium Barbarum glycopeptide (LBG), the routinely prepared murine splenic lymphocyte suspension was separately added into the samples with different concentrations (500, 100, 10, 1 μg/ml) of LBG as LBG groups. Blank control group in the absence of Lycium Barbarum glycopeptide or ConA and positive control group in the presence of 0.5 ml ConA but in the absence of LBG were created. 0.5 ml LBG samples with different concentrations in combination with 0.5 ml ConA (10 μg/ml) into each well to observe the synergic effects of LBG and ConA as LBG+ConA groups. After incubation for 72 h at 37 ℃, the samples were analyzed by CFSE-labeled cells combined with flow cytometry, and MTT. Flow cytometry revealed that both LBG could enhance the murine splenic lymphocyte proliferative reaction. Combined use of LBG and ConA had synergic effects. MTT demonstrated that sample A could obviously promote the murine splenic lymphocyte proliferative reaction as compared with control group (P<0.01), while sample B could also enhance the lymphocyte proliferation at a high dose. In combination with ConA, sample A had synergic effects at high dose, while sample B showed obviously synergic effects (P<0.05). It was concluded that both samples (A and B) had strong immunocompetence.

Expression of Costimulatory Molecules B7/CD28 in Systemic Lupus Erythematosus

Summary: The expression of the costimulatory molecules B7/CD28 in peripheral blood mononuclear cells (PBMC) of the patients with systemic lupus erythematosus (SLE) and its relation to the pathogenesis of SLE were studied. The expression of the costimulatory molecules in PBMC in 30 patients with active SLE and 20 cases of healthy controls was detected by using the techniques of immunofluorescence and flow cytometer. The result showed that the expression percentage of CD28+, CD4+CD28+ in T cells of PBMC from the patients with SLE decreased significantly as compared with that in healthy control group, while the expression percentage of CD80+, CD19+CD80+ in B cells was significantly increased than that in healthy control group (P<0.01). It suggested that the abnormal expression of costimulatory molecules B7/CD28 played a role in the pathogenesis of SLE.

Preliminary investigations on Arctic microalgae by jointapplication of fluorescent instruments

In vivo fluorescence has a wide application in analyzing microalgae, including assessing phytoplankton biomass, rates of primary production and physiological status. This study describes a preliminary investigation on the joint application of the three kinds of fluorescence analysis in the physiological study of microalgae. Flow cytometry and fluorescence spectrometry were used to obtain the in vivo static fluorescence information of pigments, and a Pulsed-Amplitude-Modulation chlorophyll fluorometer was used to detect the dynamic fluorescence of chlorophyll. The validity of the joint application was proved by analyzing two labora- tory cultured Arctic microalgae, Pseudo-nitzschia delicatissima （Bacillariophyceae） and Thalassiosira sp. The higher value of minimum fluorescence yield in dark-adapted state （Fo）, actual photochemical efficiency of PSll （ФPSII）, and electron transport rate （ETR） exhibited positive results in a higher cell abundance and chlorophyll a content of P. delicatissima; whereas higher fl-carotene content of Thalassiosira sp. played an important role in the protection of photosynthesis.

Increase of tumor necrosis factor receptor 1 expression in women with unexplained early spontaneous abortion

Objective: To investigate membrane tumor necrosis factor receptor 1 protein expression level in decidua and concentration of soluble tumor necrosis factor receptor 1 in serum in women with unexplained early spontaneous abortion, threatened abortion, and compare the levels with healthy pregnant women. Methods: Thirty-seven women with unexplained early spontaneous abortion, 27 women with threatened abortion, and 34 healthy pregnant women undergoing artificial abortion of pregnancy at 6 - 10 weeks of gestation were selected. Decidual samples were collected when women were undergoing artificial abortion, and blood samples were collected at the same time. The level of membrane tumor necrosis factor receptor 1 in decidua was detected by flow cytometer, and the concentration of soluble tumor necrosis factor receptor 1 in sera was measured with an enzyme-linked immunosorbent assay. Results: The percentages of membrane tumor necrosis factor receptor 1 positive decidual cells were 16.42 ± 7.10 Mean ± SD for women with unexplained early spontaneous abortion and 13. 14 ± 6.30 for healthy pregnant women ( P < 0.05). Serum concentration of soluble tumor necrosis factor receptor 1 was significantly higher in women with unexplained early spontaneous abortion than in healthy pregnant women and in women with threatened abortion, and no difference was found between healthy pregnant women and women with threatened abortion. Conclusion: Women with unexplained early spontaneous abortion present significantly higher expression of tumor necrosis factor receptor 1 than healthy pregnant women, suggesting that over-expression of tumor necrosis factor receptor 1 may contribute to the development of early spontaneous abortion.

Spherical Nucleic Acid-Amplified Digital Flow Cytometric Bead Assay for the Ultrasensitive Detection of Protein and Exosome

Comprehensive Summary,Most conventional digital bioassays rely on the use of fully-sealed microchambers as independent units to compartmentalize the target molecules and the signal generation reaction,which require specialized equipment or proprietary reagents/consumables.Herein,we report a microchamber-free and spherical nucleic acid(SNA)-amplified digital flow cytometric bead assay(dFBA)for ultrasensitive protein and exosome analysis with simple workflows,easily accessible instruments/reagents,and high discriminating ability towards the fluorescence-positive and fluorescence-negative beads.In this dFBA,microbeads are employed as independent carriers to anchor the single target molecule-initiated signal amplification reaction,avoiding the use of sealed droplets or microwell microchambers.Meanwhile,antibody-functionalized SNAs(FSNAs)with a high density of DNA probes act as a bridge for efficiently amplified target-to-DNA signal conversion,which allows the use of DNA-based rolling circle amplification(RCA)as the fluorescence signal amplification technique to quantify non-nucleic acid targets.Even a single target-induced on-bead RCA and fluorescence enriching are sufficient to make the target-loaded bead bright enough to be clearly discriminated from the negative ones just by use of a most common flow cytometer(FCM).This dFBA has successfully realized the digital analysis of ultralow levels of protein and exosome biomarkers,enlarging the toolbox of digital bioassays for clinical applications.

EFFECT OF ELECTROACUPUNCTURE ON RED BLOOD CELL IMMUNE AND T-CELL SUBGROUP IN THE RAT

In the present study, the effect of electroacupuncture (EA) on immune system was observed in the rat by using micro- whole blood direct immunofluorescence staining assay to detect changes of the peripheral blood T lymphocyte subgroup and employing red blood cell (RBC) C 3b receptor- yeast rosette test and red blood cell-IC rosette test to analyze erythrocytic immune function. Results showed that after EA of “Zusanli" (ST 36), CD+ 4, RBC-C 3bRR and RBC-ICR in the peripheral blood of the normal rats increased significantly while CD+ 8 had no any considerable changes and a positive correlation between CD+ 4 and RBC-C 3bRR was found. In immunosuppression model rats, the values of CD+ 4 and RBC-C 3bRR were obviously lower than those of the normal control group while CD+ 8 had no any striking changes; but after EA treatment, there were no evident differences between EA group and normal control group in the above-mentioned indexes. There were also no any significant differences between non-acupoint group and normal control group in those indexes. Results suggest that EA of “Zusanli" (ST 36) can raise T cell immune function and RBC adhesion function in both normal rats and immunosuppression model rats, both of which present a positive correlation.