In Situ Self-Assembled Formation of Nitrogen-Rich Ag@Ti3C2 Film for Sensitive Detection and Spatial Imaging of Pesticides with Laser Desorption/Ionization Mass Spectrometry (LDI-MS).

Pesticide residues are hazardous to human health; thus, developing a rapid and sensitive method for pesticide detection is an urgent need. Herein, novel nitrogen-rich Ag@Ti3C2 (Ag@N-Ti3C2) was synthesized via an ecofriendly, ultraviolet-assisted strategy, followed by in situ formation of a highly homogeneous film on target carriers via a facile water evaporation-induced self-assembly process. Ag@N-Ti3C2 shows greater surface area, electrical conductivity, and thermal conductivity than Ti3C2. This Ag@N-Ti3C2 film overcomes the limitations of conventional matrixes and allows laser desorption/ionization mass spectrometry (LDI-MS) to provide fast and high-throughput analysis of pesticides (e.g., carbendazim, thiamethoxam, propoxur, dimethoate, malathion, and cypermethrin) with ultrahigh sensitivity (detection limits of 0.5-200 ng/L), enhanced reproducibility, extremely low background, and good salt tolerance. Furthermore, the levels of pesticides were quantified with a linear range of 0-4 µg/L (R2 > 0.99). This Ag@N-Ti3C2 film was used for high-throughput analysis of pesticides spiked in traditional Chinese herbs and soft drink samples. Meanwhile, high-resolution Ag@N-Ti3C2 film-assisted LDI-MS imaging (LDI MSI) was used to successfully explore spatial distributions of xenobiotic pesticides and other endogenous small molecules (e.g., amino acids, saccharides, hormones, and saponin) in the roots of plants. This study presents the new Ag@N-Ti3C2 self-assembled film equably deposits on the ITO slides and provides a dual platform for pesticide monitoring and has the advantages of high conductivity, accuracy, simplicity, rapid analysis, minimal sample volume requirement, and an imaging function.

Validation of the single-platform ISHAGE protocol for enumeration of CD34+ hematopoietic stem cells in umbilical cord blood in a Brazilian center.

BACKGROUND: This study aims to validate the single-platform method for enumeration of CD34+ cells, by comparing the performance of two different commercial kits, as well as to evaluate the efficiency of the AccuriTM C6 cytometer in providing direct counts of absolute cell numbers. METHOD: We evaluated 20 samples from umbilical cord blood (UCB), comparing the two different methodologies for enumeration of CD34+ cells: single and dual-platform. For the assessment of the single-platform, Procount and SCE kits were used, both of which use fluorescent beads as a counting reference to obtain absolute CD34+ cells numbers. Moreover, after the acquisition of samples in flow cytometer AccuriTM C6, following the protocol established for each kit, the number of CD34+ cells was recalculated, considering the cell count provided by the AccuriTM C6. MAIN RESULTS: In our analysis, the results showed a strong correlation between the number of CD34+ cells/µL (r2=0.77) when comparing the SCE kit and the current dual-platform method. On the other hand, the comparison between Procount kit and dual-platform results showed a moderate correlation for the number of CD34+/µL cells (r2=0.64). CONCLUSION: Our results showed that the AccuriTM C6 flow cytometer can be used safely, applying both the dual and single platform analysis strategy. Considering the ISHAGE protocol-based single-platform approach, as the most appropriate methodology for CD34+ cells enumeration, our results demonstrated that the SCE kit has great potential for national standardization of UCB samples analysis methodology.

Validation of the single-platform ISHAGE protocol for enumeration of CD34+ hematopoietic stem cells in umbilical cord blood in a Brazilian center

Abstract Background This study aims to validate the single-platform method for enumeration of CD34+ cells, by comparing the performance of two different commercial kits, as well as to evaluate the efficiency of the AccuriTM C6 cytometer in providing direct counts of absolute cell numbers. Method We evaluated 20 samples from umbilical cord blood (UCB), comparing the two different methodologies for enumeration of CD34+ cells: single and dual-platform. For the assessment of the single-platform, Procount and SCE kits were used, both of which use fluorescent beads as a counting reference to obtain absolute CD34+ cells numbers. Moreover, after the acquisition of samples in flow cytometer AccuriTM C6, following the protocol established for each kit, the number of CD34+ cells was recalculated, considering the cell count provided by the AccuriTM C6. Main Results In our analysis, the results showed a strong correlation between the number of CD34+ cells/μL (r2 = 0.77) when comparing the SCE kit and the current dual-platform method. On the other hand, the comparison between Procount kit and dual-platform results showed a moderate correlation for the number of CD34+/μL cells (r2 = 0.64). Conclusion Our results showed that the AccuriTM C6 flow cytometer can be used safely, applying both the dual and single platform analysis strategy. Considering the ISHAGE protocol-based single-platform approach, as the most appropriate methodology for CD34+ cells enumeration, our results demonstrated that the SCE kit has great potential for national standardization of UCB samples analysis methodology.

Time and temperature stability of T-cell subsets evaluated by a dual-platform method.

INTRODUCTION: T-cell subset enumeration in HIV patients is routinely performed for monitoring infection stage and response to antiretroviral therapy. Studies have examined the effect of specimen refrigeration and age for single-platform (SP) methods, but there is limited data for time and temperature requirements of dual-platform (DP) methods. METHODS: Using a DP method, we analyzed peripheral blood (PB) from 52 HIV patients at room temperature (RT) at 24, 72, and 96 hours. PBs from 34 HIV patients had baseline RT analysis within 24 hours, and then were refrigerated and analyzed at 24, 48, and 72 hours. The coefficient of variation (CV) and residuals (changes in lymphocyte subsets) were recorded at each time point and compared to assess the precision and bias under the various conditions. Testing performance under different conditions was compared by linear regression. RESULTS: Mean CV was ≤7.3% and median residuals were <30/µl for absolute CD4 and CD8 determinations. There was good correlation between baseline analysis data at RT and at various time points, both at RT and 4°C. CONCLUSIONS: Our results are similar to those published for SP methods for aging or refrigerated specimens. The high level of agreement between measurements supports the robustness of this DP methodology.

CD45-assisted PanLeucogating for accurate, cost-effective dual-platform CD4+ T-cell enumeration

Background: North American and European guidelines for dual-platform (DP) flow cytometry recommendabsolute CD4 T-cell counts to be calculated from two parameters: the absolute lymphocyte counts obtainedon a hematology analyzer and the percentages of CD4 cells among lymphocytes (CD4%/lympho) obtainedby flow cytometry. Nevertheless, the identification of lymphocytes is error-prone: a poor match betweenthese common denominators in the two systems is the main source of inaccuracy. In contrast, total leucocytecounts (white cell counts [WCC]) and CD4% among the gated CD45 leucocytes (CD4%/leuco) can bedetermined with greater accuracy. Methods: We introduced "PanLeucogating," i.e., we used total leucocytesas the common denominator for improving the precision of DP absolute CD4 counting. Correlations andBland-Altman tests were used for statistical analysis. Results: First, 22 stabilized blood product sampleswere provided by U.K. National External Quality Assessment Scheme (NEQAS) and a higher accuracy andprecision of CD4 counts were documented using PanLeucogating compared with lymphocyte gating. Next,183 fresh and 112 fixed (TransFix) whole blood samples were used to compare DP methods and single-platform (SP) methodology, including both volumetric and bead-based techniques. A particularly highcorrelation and comparable precision of absolute CD4 counts were observed between the SP volumetricmethod and DP PanLeucogating (R2 0.990; bias 6 SD 17%). The SP volumetric method showed lowerlevels of agreement with the DP lymphocyte gating (R2 0.758; bias 14 SD 51%) and with the SPbead-based method (R2 0.923; bias 4 SD 31%). Conclusions: These observations show that DPleucocyte counts (WCC) should replace lymphocyte counts as the "common denominator" although CD4%/lymphovalues can, as an extra step, be also provided readily if requested. When coupled with quality controlfor WCC on hematology analyzers, the DP method with CD45 PanLeucogating represents a robust CD4 T-cellassay that is as accurate as the SP volumetric technique. This DP method uses only two, CD45 and CD4,antibody reagents and can be run on any pair of hematological analyzer plus flow cytometer.Background: North American and European guidelines for dual-platform (DP) flow cytometry recommendabsolute CD4 T-cell counts to be calculated from two parameters: the absolute lymphocyte counts obtainedon a hematology analyzer and the percentages of CD4 cells among lymphocytes (CD4%/lympho) obtainedby flow cytometry. Nevertheless, the identification of lymphocytes is error-prone: a poor match betweenthese common denominators in the two systems is the main source of inaccuracy. In contrast, total leucocytecounts (white cell counts [WCC]) and CD4% among the gated CD45 leucocytes (CD4%/leuco) can bedetermined with greater accuracy. Methods: We introduced "PanLeucogating," i.e., we used total leucocytesas the common denominator for improving the precision of DP absolute CD4 counting. Correlations andBland-Altman tests were used for statistical analysis. Results: First, 22 stabilized blood product sampleswere provided by U.K. National External Quality Assessment Scheme (NEQAS) and a higher accuracy andprecision of CD4 counts were documented using PanLeucogating compared with lymphocyte gating. Next,183 fresh and 112 fixed (TransFix) whole blood samples were used to compare DP methods and single-platform (SP) methodology, including both volumetric and bead-based techniques. A particularly highcorrelation and comparable precision of absolute CD4 counts were observed between the SP volumetricmethod and DP PanLeucogating (R2 0.990; bias 6 SD 17%). The SP volumetric method showed lowerlevels of agreement with the DP lymphocyte gating (R2 0.758; bias 14 SD 51%) and with the SPbead-based method (R2 0.923; bias 4 SD 31%). Conclusions: These observations show that DPleucocyte counts (WCC) should replace lymphocyte counts as the "common denominator" although CD4%/lymphovalues can, as an extra step, be also provided readily if requested. When coupled with quality controlfor WCC on hematology analyzers, the DP method with CD45 PanLeucogating represents a robust CD4 T-cellassay that is as accurate as the SP volumetric technique. This DP method uses only two, CD45 and CD4,antibody reagents and can be run on any pair of hematological analyzer plus flow cytometer.