How the Crosslinker Amount Influences the Final Properties of Hydroxyethyl Methacrylate Cryogels.

The investigation of the mechanical, thermal, and adsorption properties of hydroxyethyl methacrylate (HEMA) cryogels as a function of a reactant ratio is herein reported to better address materials for specific applications. To this aim, cryogels have been synthesized using different monomer/crosslinker (N,N'-methylene-bisacrylamide-MBAA) ratios. The study of SEM images made it possible to identify the trend in the material's macroporosity. As would be expected, the average measured pore width decreased as the amount of MBAA increased while the number of pores grew. Swelling capacity ranges from 8.7 gW/ggel (grams of water per gram of gel) to 9.3 gW/ggel. These values are strictly connected with the pore's size and distribution, revealing that the water uptake for the most crosslinked sample is inferior to other samples. The equilibrium-adsorption capacity (Qe) towards the methylene violet (MV) was also assessed, revealing no remarkable differences after 24 h of a batch test. As expected, thermogravimetric analysis (TGA) also showed no significant changes in stability that ranged from a maximum weight loss temperature (T Max) of 420 °C to 425 °C, which increased as a function of crosslinker content. Conversely, compression strength measurements showed a notable difference of about 50% in modulus (Ec), moving from the higher to the lower HEMA/MBAA ratio. These new comparative results indicate how slight variations in the reactant's ratio can steadily improve the mechanical properties of the HEMA cryogel without affecting its adsorption efficiency. This can be helpful in the design of materials for water and energy purposes. Since swelling properties are needed in the case of biomedical applications, the HEMA/MBAA ratio should be tuned versus high values.

ZnO-pHEMA Nanocomposites: An Ecofriendly and Reusable Material for Water Remediation.

The design of new hybrid nanocomposites based on poly(2-hydroxyethylmethacrylate) (pHEMA) graphene oxide (GO) cryosponges, wherein ZnO nanolayers have been deposited to induce photocatalytic properties, is reported here. Atomic layer deposition at low temperature is specifically selected as the deposition technique to stably anchor ZnO molecules to the pendant polymer OH groups. Furthermore, to boost the pHEMA cryogel adsorption capability versus organic dyes, GO is added during the synthetic procedure. The morphology, the crystallinity, and the chemical composition of the samples are deeply investigated by scanning electron microscopy, transmission electron microscopy, X-ray diffraction analyses, Fourier transform infrared spectroscopy, and thermogravimetric analysis. Swelling properties, mechanical performance, and adsorption kinetics models of the hybrid materials are also evaluated. Finally, the adsorption and photocatalytic performance are tested and compared for all of the samples using methylene blue as a dye. Particularly, the adsorption efficiency of ZnO/pHEMA and ZnO/pHEMA-GO nanocomposites, as well as their in situ regeneration via photocatalysis, renders such devices very appealing for advanced wastewater treatment technology.

Validation rules for blood smear revision after automated hematological testing using Mindray CAL-8000.

BACKGROUND: This article was aimed to test the use of validation rules for blood smear review after automated hematological testing using Mindray CAL-8000 (two hematological analyzers and one autoslider). METHODS: This study was based on 1013 peripheral blood samples (PB) referred for routine hematological testing. Results of testing on CAL-8000 were analyzed using both locally derived and International Consensus Group for Hematology (ICGH) validation rules, and then compared with data obtained by optical microscopy (OM). A workflow analysis was also completed. RESULTS: The overall agreement with locally derived and ICGH criteria was 91% and 85%, but a higher sensitivity was observed for locally derived criteria (0.97 vs 0.95). The percentage of false negative and false positive samples was 2.1% and 7.1% using ICGH criteria, and was 1.4% and 14% using locally defined rules. The throughput of CAL-8000 system was 208 samples/h, with a percentage of OM analysis comprised between 14% and 17%, and sensitivity of 0.97. As regards personnel activity, we estimated 0.8 full-time equivalent (FTE) of technical staff and 0.7 FTE of personnel for clinical validation of data and blood smear review. CONCLUSION: These results show that customization of validation rules is necessary for enhancing the quality of hematological testing and optimizing workflow.

Clinical significance of cell population data (CPD) on Sysmex XN-9000 in septic patients with our without liver impairment.

BACKGROUND: This study evaluated the clinical significance of cell population data (CPD) parameters obtained on Sysmex XN-9000 in septic patients admitted to intensive care unit (ICU) and stratified according to liver function. METHODS: The study population consisted in 84 patients, 44 of whom did not develop sepsis (NS), whereas the remaining 40 developed sepsis (SE) (n=24) or septic shock (SS) (n=16). Two hundred ostensibly healthy blood donors [healthy subjects (HS)], undergoing routine blood testing before a regular blood donation, were studied. RESULTS: Except for neutrophils and lymphocytes cell size (NE-FCS and LY-Z), all other CPD values were significantly different in ICU patients compared to HS. Neutrophils and monocytes fluorescence intensity (NE-SFL and MO-X) values were significantly higher in SS compared to sepsis and not develop sepsis patients. The value of many parameters was also different according to liver function. Overall, MO-X and neutrophils fluorescence intensity (NE-SFL) exhibited the best performance for diagnosing sepsis in all patients (AUC, 0.75 and 0.72), as well as in those with (AUC, 0.95 and 0.89) or without (AUC, 0.72 for both) liver impairment. These parameters were also significantly correlated with Sequential Organ Failure Assessment (SOFA) score. CONCLUSIONS: This study suggested that some novel CPD parameters (namely NE-SFL and MO-X) may provide useful information for diagnosis and management of sepsis.

Optimization of Cellular analysis of Synovial Fluids by optical microscopy and automated count using the Sysmex XN Body Fluid Mode.

BACKGROUND: This study was planned to assess the impact of pre-treating synovial fluid (SF) samples with hyaluronidase (HY), defining the best procedure for optical microscopy (OM) analysis and evaluating the performance of Sysmex XN-9000 Body Fluid module (XN-BF). METHODS: The cell count by OM was carried out both with and without HY pre-treatment, and using 3 different types of staining reagents. The evaluation of XN-BF included data comparison with OM (100 SFs), carryover, Limit of Blank (LoB), Limit of Detection (LoD), Limit of Quantitation (LoQ) and linearity. RESULTS: Unlike cell count in Burker's chamber and staining with Stromatol, pre-treatment with HY and staining with Methylene Blue and Turk's promoted cell clustering. The SF samples pre-treated with HY displayed excellent morphological quality, contrary to samples without HY pre-treatment. Excellent correlation was found between total cells counting with both OM and XN-BF. Satisfactory agreement was also observed between polymorphonuclear neutrophils compared to XN-BF parameter, whereas mononuclear cell count on XN-BF had suboptimal agreement with OM. The carryover was negligible. The LoB, LoD, LoQ and linearity were excellent. CONCLUSION: XN-BF displays excellent performance, which makes it a reliable and practical alternative to OM for SF samples analysis in clinical laboratories.

Assessment of blood sample stability for complete blood count using the Sysmex XN-9000 and Mindray BC-6800 analyzers.

BACKGROUND: Different hematological analyzers have different analytical performances that are often reflected in the criteria for sample stability of the complete blood count. This study aimed to assess the stability of several hematological parameters using the XN-9000 Sysmex and BC-6800 Mindray analyzers. METHODS: The impact of storage at room temperature and 4°C was evaluated after 2, 4, 6, 8, 24, 36 and 48h using ten normal and 40 abnormal blood samples. The variation from the baseline measurement was evaluated by the Steel-Dwass-Critchlow-Fligner test and by Bland-Altman plots, using quality specifications and critical difference as the total allowable variation. RESULTS: Red blood cells and reticulocyte parameters (i.e. hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration, red blood cell distribution width, immature reticulocyte fractions, low-fluorescence reticulocytes, middle-fluorescence reticulocytes, high fluorescence mononuclear cells) showed less stability compared to leukocyte and platelet parameters (except for monocyte count and mean platelet volume). The bias for hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration and red blood cell distribution width coefficient of variation was higher than the critical difference after 8h using both analyzers. CONCLUSION: Blood samples measured with both analyzers do not show analytically significant changes in up to 2h of storage at room temperature and 4°C. However, the maximum time for analysis can be extended for up to 8h when the bias is compared to the critical difference.

Erythroblastaemia in natalizumab-treated patients with multiple sclerosis.

BACKGROUND: Natalizumab is a monoclonal antibody that significantly reduces the occurrence of relapses in relapse-remitting multiple sclerosis (RRMS) patients. Early papers on the clinical use of natalizumab in RRMS patients reported erythroblastemia as occasional and transient. OBJECTIVES: to determine the prevalence and absolute count of erythroblasts (nucleated red blood cells, NRBCs) in peripheral blood of RRMS patients in different treatment groups and healthy controls from the same geographic area using the same equipment for laboratory analysis. METHODS: We retrospectively evaluated the samples of 203 consecutive RRMS patients including 26 subjects on natalizumab, 17 on fingolimod, 72 on interferon, 41 on glatiramer acetate, 47 treatment-naïve and 240 healthy controls from the same geographic area. Blood samples were processed using an XN-9000-Hematology Analyzer and subsequent microscopic verification. In the natalizumab-treated patients we performed an additional analysis in order to detect the expression of CD34+ cells in peripheral blood, as confirmation of a bone marrow mobilization. RESULTS: The prevalence of patients with NRBCs positivity was significantly higher in natalizumab-treated patients (92%) compared with the other treatment groups and healthy controls (0%) (p<0.0005). The median absolute NRBCs count was significantly higher in natalizumab-treated patients (median 0.020, p<0.0005) than in the other treatment groups and healthy controls. Natalizumab-treated patients also had higher levels of white blood cells than all other groups and lower haemoglobin levels than healthy subjects (p<0.01), but no morphologic alterations were evident at a subsequent review of red blood cells, platelets and white blood cells. CD34+ cells levels were consistent with mobilization of haematopoietic stem cells from the bone marrow (median 8 cells/µL, IQR 5-12). CONCLUSIONS: We confirm erythroblastaemia as a frequent finding of natalizumab treatment in RRMS patients. More extended knowledge and adequate long-term observation of this phenomenon are essential to better understand any pathological implication.

Evaluation of Mindray BC-6800 body fluid mode for automated cerebrospinal fluid cell counting.

BACKGROUND: Cellular analysis in cerebrospinal fluid (CSF) provides important diagnostic information in various medical conditions. The aim of this study was to evaluate the application of Mindray BC-6800 body fluid (BF) mode in cytometric analysis of CSF compared to light microscopy (LM). METHODS: One hundred and twenty-nine consecutive CSF samples were analyzed by BC-6800-BF mode as well as by LM. The study also included limits of blank (LoB), limit of detection (LoD), limit of quantitation (LoQ), carryover and linearity. Results White blood cells LoQ was 4.0×106 cells/L. Linearity was good and carryover was negligible. As for the total and white blood cells, the BC-6800-BF parameters vs. LM showed both bias ranged from -10.28 to 0.06×106 cells/L. Polymorphonuclear and mononuclear cells ranged from 6.64 to 10.90%. For white blood cell the diagnostic agreement was 93% at the cut-off >5.0×106 cells/L, and for polymorphonuclear and mononuclear at the cut-off >50% was 91% and 92%, respectively. CONCLUSIONS: BC-6800-BF offers rapid and accurate counts in clinically relevant concentration ranges, replacing LM for most samples. However, in samples with abnormal cell counts or with abnormal white blood cell differential scattergrams the need to microscopic review for a correct clinical outcome remains.

Cell Population Data and reflex testing rules of cell analysis in pleural and ascitic fluids using body fluid mode on Sysmex XN-9000.

BACKGROUND: Although optical microscopy (OM) remains the reference technique for analysis of ascitic (AF) and pleural (PF) fluids, novel hematological analyzers are equipped with modules for body fluid (BF) analysis. This study was aimed to analyze the performance of XN-BF module in Sysmex XN-9000, and to develop validation rules for automated cell counts in BFs. METHODS: The evaluation of XN-BF module included assessment of carryover, Limit of Blank (LoB), Limit of Detection (LoD), Limit of Quantitation (LoQ), linearity, data comparison with OM, and development of rules for assisting the validation of automated analysis of BFs and activating reflex testing. RESULTS: The carryover was negligible. The LoB, LoD, LoQ and linearity were always excellent. The comparison with OM was characterized by Pearson's correlations ranging from r=0.50 to r=0.99 (p<0.001), modest bias and high diagnostic concordance (Area Under the Curve between 0.85 and 0.99). The use of instrument-specific cut-offs further increased diagnostic concordance. The implementation of reflex testing rules based on XN-BF data increased sensitivity and specificity of BFs classification to 0.98 and 0.95. CONCLUSIONS: Our results suggest that the XN-BF module on Sysmex-9000 may be a suitable alternative to OM for screening BF samples, especially when specific validation rules are used.

Reflex Testing Rules for Cell Count and Differentiation of Nucleated Elements in Pleural and Ascitic Fluids on Sysmex XE-5000.

Flow cytometry is widely used in many laboratories for automated nucleated cell counts and their differentiation in body fluids. The implementation of new reflex testing rules on these automated instruments could open new frontiers in laboratory workflow, improving characterization of body fluids and clinical diagnosis and decreasing costs. Ascitic (150) and pleural (33) fluids were collected and assessed by XE-5000 and optical microscopy. Cell counts performed with the methods showed a Pearson's correlation of 0.98 (p < 0.0001), Passing-Bablok regression y = 0.99x + 2.44, and bias of 32.3. In ascitic fluids, the best diagnostic performance was found for polymorphonuclear and neutrophil counts on XE-5000, which exhibited areas under the curve (AUCs) 0.98 (p < 0.0001) and 0.99 (p < 0.0001), respectively. In pleural fluids the best diagnostic performance was found for polymorphonuclear percent parameter, which displayed 0.97 (p < 0.0001). Specific reflex test rules based on these parameters were characterized by 92% diagnostic concordance, 1.00 sensitivity, and 0.84 specificity with optical microscopy. The application of a set of reflex testing rules may improve the diagnostic performance of XE-5000, increasing its reliability for routine automated cell count in body fluids. We acknowledge that further studies should be planned to validate our findings according to clinical data.

Assessment of blood sample stability for complete blood count using the Sysmex XN-9000 and Mindray BC-6800 analyzers

Background: Different hematological analyzers have different analytical performances that are often reflected in the criteria for sample stability of the complete blood count. This study aimed to assess the stability of several hematological parameters using the XN-9000 Sysmex and BC-6800 Mindray analyzers. Methods: The impact of storage at room temperature and 4 ◦C was evaluated after 2, 4, 6, 8, 24, 36 and 48h using ten normal and 40 abnormal blood samples. The variation from the baseline measurement was evaluated by the Steel­Dwass­Critchlow­Fligner test and by Bland­Altman plots, using quality specifications and critical difference as the total allowable variation. Results: Red blood cells and reticulocyte parameters (i.e. hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration, red blood cell distribution width, immature reticulocyte fractions, low-fluorescence reticulocytes, middle-fluorescence reticulocytes, high fluorescence mononuclear cells) showed less stability compared to leukocyte and platelet parameters (except for monocyte count and mean platelet volume). The bias for hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration and red blood cell distribution width coefficient of variation was higher than the critical difference after 8h using both analyzers. Conclusion: Blood samples measured with both analyzers do not show analytically signifi- cant changes in up to 2h of storage at room temperature and 4 ◦C. However, the maximum time for analysis can be extended for up to 8h when the bias is compared to the critical difference

Extended leukocyte differential count and C-reactive protein in septic patients with liver impairment: diagnostic approach to evaluate sepsis in intensive care unit.

BACKGROUND: Sepsis is still a major cause of death in intensive care units (ICUs) worldwide. Patients with liver impairment express an imbalanced cytokine response which alters common sepsis biphasic nature. Cytokines measurement is expensive, often unavailable, whereas leukocytes (WBC) evaluation performed through hematology analyzers can provide a practical strategy for monitoring inflammatory response. METHODS: A total of 200 healthy subjects (HS) and 84 patients (18 with, 66 without liver impairment) admitted to ICU, were assessed for International Sepsis Definitions, Sequential Organ Failure Assessment (SOFA) and Model for End-Stage Liver Disease (MELD) scores. We tested 1,022 peripheral blood samples using Sysmex XN-9000, estimating diagnostic accuracy of leukocyte differential count and nontraditional parameters through receiver operating characteristics (ROC) curves analysis compared to clinical classification. RESULTS: Median value of all-leukocyte parameters was different in ICU patients compared to HS. Leukocytes, neutrophils (NE) and immature granulocytes (IGs) in sepsis and septic shock (SS) were higher than no sepsis (NS), with an area under the curve: 0.81, 0.82 and 0.78 respectively. Lymphocytes (LY) and monocytes (MO) were significantly associated with liver impairment. CONCLUSIONS: Diagnostic accuracy of all-leukocyte parameters may provide valuable information for diagnosis and follow-up of sepsis in ICU patients, especially those with liver impairment.

Novel pH responsive calix[8]arene hydrogelators: self-organization processes at a nanometric scale.

Water soluble amphiphilic calix[8]arenes have been found to form pH-responsive and reversible supramolecular hydrogels at sub-millimolar concentration. Depending on their upper rim functionalization, pH variations trigger hydrogelation under basic or acidic conditions. Studies on self-organization processes by atomic force microscopy (AFM) measurements on differently charged surfaces are reported.

Polycationic calix[8]arenes able to recognize and neutralize heparin.

A mutual induced fit mechanism is responsible for the exceptional complexation performances exhibited by calix[8]arene polycations towards heparin. The recognition process was studied in comparison with two other heparin antagonists: protamine and polylysine. The arrangement of multiple functional groups on the flexible macrocyclic scaffold of calix[8]arene, with respect to the conformationally rigid protamine and low ordered polylysine, allowed a mutual adaptability between calixarene polycations and heparin, significantly enhancing the recognition performances. Fluorescence, NMR titration, and activated partial thromboplastin time (aPTT) experiments confirmed that these calixarene derivatives have a very high specificity and affinity towards heparin neutralization as in aqueous solution as in blood. Analogous results were obtained with low molecular weight heparin (LMWH) whose effect protamine is unable to completely reverse.

Designed calix[8]arene-based ligands for selective tryptase surface recognition.

Basic amino acid calix[8]arene receptors for tryptase surface recognition have been synthesized. The tetrameric arrangement and the negative charge distribution close to the active sites of the enzyme, have suggested the design of complementary multifunctional receptors that might bind to the active region of the protein blocking the approach of the substrate. Kinetic inhibition analysis on recombinant lung tryptase have showed a time-dependent competitive inhibition with both initial and steady-state rate constants in the nanomolar range.

A chiroptical molecular switch with distinct chiral and photochromic entities and its application in optical switching of a cholesteric liquid crystal.

Two new structurally related photoswitches are described, in which azobenzene photochromism is combined with the chirality of a 2,2'-dihydroxy-1,1'-binaphthyl unit. In system 1 the chiral binaphthyl moiety is bridged by a methylene tether, locking the biaryl chirality while in system 2 the biaryl core is unbridged and has considerable conformational flexibility. Both compound are capable of inducing cholesteric liquid crystalline phases and proved to be good photoswitches both in solution and in a liquid crystalline matrix. Compound 2 is capable of completely reversing the liquid crystalline chirality which is unique for a chiroptical molecular switch where the switching unit and the chiral moiety are separate entities.