Cell Volume Measurements Research Articles

An analytical observational study for diagnostic accuracy of volume, conductivity & scatter (VCS) indices of neutrophils for diagnosis of sepsis in an emergency hospital setting.

Background & objectives The newer technique using an innovative volume conductivity scatter (VCS) technology is emerging as a surrogate for sepsis diagnosis. The VCS technology offers a more objective method to measure cell volume (V), characterize conductivity (C) and light scatter (S) directly from more than 8,000 white blood cells (WBCs). However, diagnostic performance of VCS parameters in sepsis has not been extensively tested in routine hospital emergency settings. Therefore, the present study aimed to investigate the diagnostic and prognostic performance of VCS markers of neutrophils in our local hospital emergency setting. Methods It was an observational analytical study with 41 cases of sepsis and 43 healthy controls aged above 18 yr. Individuals with acute coronary syndrome and individuals with already diagnosed Human Immunodeficiency Virus (HIV) infection were excluded from the study. Results The mean neutrophil volume (MNV) values were not significantly different between cases and controls (P 0.138) whereas mean neutrophil conductance (MNC) and mean neutrophil scatter (MNS) measurements were significantly higher among cases as compared to controls (both P-values <0.001). According to Receiver Operating Characteristics (ROCs) curve analysis, MNV in our study failed to show statistically significant discriminatory ability in sepsis (AUC 0.54) whereas MNC (AUC 0.98) and MNS (AUC 0.95) showed marked discriminatory ability in diagnosing sepsis in this study cohort. Interpretation & conclusions Among VCS parameters, MNV failed as a standalone biomarker of sepsis in routine emergency setting whereas MNC and MNS had statistically significant diagnostic and discriminatory accuracies among hospitalized affected individuals with sepsis.

Prevention of Cerebrospinal Fluid Leakage in the Anterior Transpetrosal Approach.

Background: The anterior transpetrosal approach (ATPA) is effective for reaching petroclival lesions, and it allows for complications such as impaired venous return and neuropathy to be resolved. However, there is still room for improvement regarding cerebrospinal fluid (CSF) leakage. Here, we aim to focus on describing specific preoperative, intraoperative, and postoperative countermeasures for preventing CSF leakage when using the ATPA. Methods: Eleven patients treated using the ATPA, who were treated at our hospital from June 2019 to February 2023, were included in this descriptive study. Preoperatively, we performed a 3D simulation of the opened air cells. Then, we classified patterns of dural closure into three types based on intradural manipulation and whether it involved opened air cells or not. Intraoperatively, we performed a dural closure that included the use of more-watertight sutures (DuraGen®) and an endoscope. Furthermore, temporal bone air cell volume measurements were performed to confirm the correlation between the volume and factors related to CSF leakage. Results: No postoperative CSF leakage was observed in any patient. The temporal bone air cell volumes significantly corelated with the air cells of the petrous apex, the high-risk tract in the petrous apex, and postoperative fluid collection in mastoid air cells. Conclusions: We have described countermeasures for preventing CSF leakage when using the ATPA. Preoperative simulations and the use of multiple-layered dural reconstructions with endoscopes could be considered more reliable methods for preventing CSF leakage when using the ATPA.

Temperature Dependence of Membrane Permeability Parameters for Five Cell Types Using Nonideal Thermodynamic Assumptions to Mathematically Model Cryopreservation Protocols.

Cryopreservation is the process of preserving biological matter at subzero temperatures for long-term storage. During cryopreservation, cells are susceptible to various injuries that can be mitigated by controlling the cooling and warming profiles and cryoprotective agent (CPA) addition and removal procedures. Mathematical modeling of the changing cell volume at different temperatures can greatly reduce the experiments needed to optimize cryopreservation protocols. Such mathematical modeling requires as inputs the cell membrane permeabilities to water and CPA and the osmotically inactive fraction of the cell. Since the intra- and extracellular solutions are generally thermodynamically nonideal, our group has been incorporating the osmotic virial equation to model the solution thermodynamics that underlie the cell volume change equations, adding the second and third osmotic virial coefficients of the grouped intracellular solute to the cell osmotic parameters that must be measured. In our previous work, we reported methods to obtain cell osmotic parameters at room temperature by fitting experimental cell volume kinetic data with equations that incorporated nonideal solution thermodynamics assumptions. Since the relevant cell volume excursions occur at different temperatures, the temperature dependence of the osmotic parameters plays an important role. In this work, we present a new two-part fitting method to obtain five cell-type-specific parameters (water permeability, dimethyl sulfoxide permeability, osmotically inactive fraction, and the second and third osmotic virial coefficients of the intracellular solution) from experimental measurements of equilibrium cell volume and cell volume as a function of time at room temperature and 0 °C for five cell types, namely, human umbilical vein endothelial cells (HUVECs), H9c2 rat myoblasts, porcine corneal endothelial cells (PCECs), the Jurkat T-lymphocyte cell line, and human cerebral microvascular endothelial cells (hCMECs/D3 cell line). The fitting method in this work is based on both equilibrium and kinetic cell volume data, enabling us to solve some technical challenges and expand our previously reported measurement technique to 0 °C. Finally, we use the measured parameters to model the cell volume changes for a HUVEC cryopreservation protocol to demonstrate the impact of the nonideal thermodynamic assumptions on predicting the changing cell volume during freezing and thawing.

Measurement of red cell, plasma, and blood volume: A perspective.

Measurement of red cell, plasma, and blood volume: A perspective.

Dialyzer reprocessing: Considerations and pitfalls for effective and safe hemodialysis.

Dialyzer reprocessing for dialyzer reuse in the same patient has been developed since the early time in hemodialysis history to save cost and time related to reassembling the new dialyzer during that time. The procedure can reduce the first-use and allergic reactions from using incompatible cellulosic dialyzer membrane by altering some manufacturing chemicals. All of established literatures regarding recent dialyzer reprocessing methods and considerations were extensively reviewed and summarized. Dialyzer reprocessing can be performed by multiple protocols but involves common steps including bedside rinsing after use, cleaning, dialyzer testing to prevent excessive drop in dialyzer clearance and membrane integrity, high-level disinfection or sterilization either by chemicals or heat, storage, and preparation for subsequent dialysis session by adequate rinsing to reduce the residual reprocessing chemical to the safe level. Compared with the single-use strategy, evidence is conflicting for the mortality advantages or disadvantages of dialyzer reuse, with some showing increased mortality in patients receiving peracetic acid sterilization. Keys for the effective and safe dialyzer reuse involve strict adherence to specific manufacturer's protocol, adequate dialysis water quality complied with the Association for the Advancement of Medical Instrumentation standard, measurement of the total cell volume to prevent inadequate hemodialysis, and infectious control consideration. In the present era, single-use strategy is increasingly adopted due to the decreased cost for dialyzer manufacturing. Environmental concerns of higher solid waste from dialyzer disposal in single-use dialysis should be compared with the liquid waste from reprocessing chemicals along with plastic waste and cardboard in reuse dialysis. Dialyzer reprocessing with adequate regulation is considered as an acceptable option for cost-effective hemodialysis, compared with the single-use strategy.

Prevalence of Bovine Babesiosis in Selected District of East Wollega Zone, Western Ethiopia

Introduction. Bovine babesiosis is a hemoparasitic disease that severely impacts cattle, leading to significant morbidity and mortality. This study aimed to determine the prevalence of bovine babesiosis and identify associated risk factors. Methods. A cross-sectional study was conducted in two selected districts of East Wollega, Oromia region, Ethiopia. Blood samples were collected from a purposively selected group of 384 animals (268 females and 116 males), and thin and thick smears were prepared to identify Babesia parasites. Anemia was assessed using packed cell volume measurements. Results. Bovine babesiosis prevalence was 5.2%. Babesia bovis infected 3.91%, and Bovis bigemina infected 1.30% of animals. Significant correlations (p&lt;0.05) were found between husbandry practices and previous anti-Babesia drug treatment. No significant associations (p&gt;0.05) were observed with age, sex, breed, or body condition. Males (6.0%) had a higher prevalence, highest in animals of more than seven years (6.1%) and mature animals (5.2%). The lowest prevalence (4.2%) occurred in young animals. Low body condition cattle (7.1%) had higher infection rates than medium (5.0%) and high (1.2%) scores. Conclusion. Bovine babesiosis was prevalent in the study area and poses a significant threat to cattle production overall.

Aquaporin-4 and GPRC5B: old and new players in controlling brain oedema.

Brain oedema is a life-threatening complication of various neurological conditions. Understanding molecular mechanisms of brain volume regulation is critical for therapy development. Unique insight comes from monogenic diseases characterized by chronic brain oedema, of which megalencephalic leukoencephalopathy with subcortical cysts (MLC) is the prototype. Variants in MLC1 or GLIALCAM, encoding proteins involved in astrocyte volume regulation, are the main causes of MLC. In some patients, the genetic cause remains unknown. We performed genetic studies to identify novel gene variants in MLC patients, diagnosed by clinical and MRI features, without MLC1 or GLIALCAM variants. We determined subcellular localization of the related novel proteins in cells and in human brain tissue. We investigated functional consequences of the newly identified variants on volume regulation pathways using cell volume measurements, biochemical analysis and electrophysiology. We identified a novel homozygous variant in AQP4, encoding the water channel aquaporin-4, in two siblings, and two de novo heterozygous variants in GPRC5B, encoding the orphan G protein-coupled receptor GPRC5B, in three unrelated patients. The AQP4 variant disrupts membrane localization and thereby channel function. GPRC5B, like MLC1, GlialCAM and aquaporin-4, is expressed in astrocyte endfeet in human brain. Cell volume regulation is disrupted in GPRC5B patient-derived lymphoblasts. GPRC5B functionally interacts with ion channels involved in astrocyte volume regulation. In conclusion, we identify aquaporin-4 and GPRC5B as old and new players in genetic brain oedema. Our findings shed light on the protein complex involved in astrocyte volume regulation and identify GPRC5B as novel potentially druggable target for treating brain oedema.

The Ca2+ channel TRPV4 is dispensable for Ca2+ influx and cell volume regulation during hypotonic stress response in human keratinocyte cell lines

Cell swelling as a result of hypotonic stress is counteracted in mammalian cells by a process called regulatory volume decrease (RVD). We have recently discovered that RVD of human keratinocytes requires the LRRC8 volume-regulated anion channel (VRAC) and that Ca2+ exerts a modulatory function on RVD. However, the ion channel that is responsible for Ca2+ influx remains unknown. We investigated in this study whether the Ca2+-permeable TRPV4 ion channel, which functions as cell volume sensor in many cell types, may be involved in cell volume regulation during hypotonic stress response of human keratinocytes. We interfered with TRPV4 function in two human keratinocyte cell lines (HaCaT and NHEK-E6/E7) by using two TRPV4-specific inhibitors (RN1734 and GSK2193874), and by creating a CRISPR/Cas9-mediated genetic TRPV4−/− knockout in HaCaT cells. We employed electrophysiological patch clamp analysis, fluorescence-based Ca2+ imaging and cell volume measurements to determine the functional importance of TRPV4. We could show that both hypotonic stress and direct activation of TRPV4 by the specific agonist GSK1016790A triggered intracellular Ca2+ response. Strikingly, the Ca2+ increase upon hypotonic stress was neither affected by genetic knockout of TRPV4 in HaCaT cells nor by pharmacological inhibition of TRPV4 in both keratinocyte cell lines. Accordingly, hypotonicity-induced cell swelling, downstream activation of VRAC currents as well as subsequent RVD were unaffected both in TRPV4 inhibitor-treated keratinocytes and in HaCaT-TRPV4−/− cells. In summary, our study shows that keratinocytes do not require TRPV4 for coping with hypotonic stress, which implies the involvement of other, yet unidentified Ca2+ channels.

P20 Novel phenotypic, impedance-based fast antibiotic susceptibility test (AST) can distinguish between MRSA and MSSA in 2 h

Abstract Background Antimicrobial resistance (AMR) is a global problem and is forecast that by 2050 it will cause 10 million deaths a year. Current antimicrobial susceptibility tests (ASTs) used clinically, can take 24–48 h or longer to report results, requiring that initial treatment uses broad spectrum antibiotics. The novel impedance-based fast AST (iFAST) method can report results within 2 h of exposure to an antibiotic. The impedance cytometer measures changes in the electrical charge flow between electrodes when bacterial cells flow individually through a microfluidic channel by an AC current of multiple frequencies. This is interpreted as a read-out of electrical radius (a surrogate measure of cell volume) and opacity (a measure of the resistance properties of the bacterial membranes). Exposure to antibiotics can change the electrical characteristics of the bacterial cell in size and opacity compared with the control sample. The number of exposed cells within a contour defined by a control sample, is used to assess how the cells following exposure. Objectives To demonstrate concordance of this novel rapid method with the current gold standard AST, as performed in a diagnostic laboratory. To distinguish between MRSA and MSSA isolates. Cefoxitin was used as a surrogate for methicillin in laboratory testing as outlined in EUCAST guidelines. Sequentially collected bacterial isolates were used in the clinical microbiology laboratory of University Hospital Southampton from May–July 2022. Methods 60 blinded concurrent clinical isolates of S. aureus were taken from the clinical workflow and tested on the iFAST. S. aureus isolates were streaked onto blood plates and incubated at 37°C for 2 h. The bacteria were diluted into 3 mL of saline solution. The concentration of the bacteria was adjusted to 5 × 105 cfu/mL, in line with EUCAST guidelines, exposed to the beta lactam antibiotic cefoxitin for 2 h at the EUCAST breakpoint concentration of 8 mg/L. Following exposure, the samples were diluted 1:20 in saline solution and measured on the iFAST. Cefoxitin-exposed cells were compared with a control sample, which has not been challenged with antibiotic. The iFAST results were compared with gold standard disk diffusion assay results, generated in the clinical lab as part of normal clinical service. Results The iFAST results showed 100% concordance with disk diffusion susceptibility testing for cefoxitin carried out in parallel by the clinical laboratory. The data showed different electrical impedance changes for both resistant and susceptible isolates. Susceptible strains showed a decrease in electrical radius, suggesting that the cells are smaller and a significant reduction in overall cell count was observed. Resistant strains showed a distinctive increase in electrical radius, suggesting that the cells are larger in size when exposed to cefoxitin. However, this and other factors that may contribute to an increase in electrical radius are currently being investigated. An electrical MIC carried out for selected strains, was used to confirm that the transition from one response to the other corresponded to the MIC measured by microbroth dilution. An analysis of the data with hospital laboratory staff, suggested that the iFAST can provide an MRSA confirmation to clinicians 16 h before the current AST's, which could benefit patient management and treatment. Conclusions The iFAST can rapidly differentiate between MRSA and MSSA isolates in concordance with current susceptibility testing in the clinical setting. The results help to show how the iFAST could reduce the time taken to provide critical and accurate antibiotic treatment to patients.

Artificial confocal microscopy for deep label-free imaging.

Widefield microscopy of optically thick specimens typically features reduced contrast due to "spatial crosstalk", in which the signal at each point in the field of view is the result of a superposition from neighbouring points that are simultaneously illuminated. In 1955, Marvin Minsky proposed confocal microscopy as a solution to this problem. Today, laser scanning confocal fluorescence microscopy is broadly used due to its high depth resolution and sensitivity, but comes at the price of photobleaching, chemical, and photo-toxicity. Here, we present artificial confocal microscopy (ACM) to achieve confocal-level depth sectioning, sensitivity, and chemical specificity, on unlabeled specimens, nondestructively. We equipped a commercial laser scanning confocal instrument with a quantitative phase imaging module, which provides optical path-length maps of the specimen in the same field of view as the fluorescence channel. Using pairs of phase and fluorescence images, we trained a convolution neural network to translate the former into the latter. The training to infer a new tag is very practical as the input and ground truth data are intrinsically registered, and the data acquisition is automated. The ACM images present significantly stronger depth sectioning than the input (phase) images, enabling us to recover confocal-like tomographic volumes of microspheres, hippocampal neurons in culture, and 3D liver cancer spheroids. By training on nucleus-specific tags, ACM allows for segmenting individual nuclei within dense spheroids for both cell counting and volume measurements. In summary, ACM can provide quantitative, dynamic data, nondestructively from thick samples, while chemical specificity is recovered computationally.

The Synergistic Effect of Theranekron and Cisplatin on the Neuroblastoma (SH-SY5Y) Cell Line.

Neuroblastoma (NB), a pediatric tumor with high prevalence and mortality rates, was the focus of this investigation. This study aimed to assess the combined antitumor potential of theranekron (Trn) and cisplatin in this cell line. The interaction between theranekron (10-100 µM) and cisplatin (40 µM) was examined. The cytotoxicity of theranekron was evaluated using MTT assays, cell volume analyses, apoptosis assessments, and measurements of mitochondrial membrane potential. These evaluations were conducted over a 24-hour incubation period. The coadministration of 100 µM theranekron with cisplatin exhibited a cytotoxic effect of approximately 60%. Furthermore, it led to a reduction of up to 38% in cell volume. Notably, SH-SY5Y cells demonstrated an early apoptosis rate of 34.4%, accompanied by an eightfold decrease in mitochondrial membrane potential compared to the control group. Theranekron demonstrated synergism and significantly enhanced the efficacy of cisplatin (P 0.001). Nevertheless, further in-depth investigations are necessary to elucidate the intricacies of these synergistic effects.

Predictors of Incomplete Adherence and Immuno-virologic Failure among HIV Infected Patients in Osun State, Nigeria

BACKGROUND: To identify and address modifiable baseline clinical and non- clinical predictors related to negative outcomes, and identify high risk patients who need priority attention in order to prevent future failure of treatment in HIV patients. METHODS: The study was a longitudinal clinical based cohort study of One hundred and forty (140) HIV infected adults who were initially ART naive and commenced ART in June- July 2013 were followed up to June 2014. Three out of the participants were lost to follow up, one participant died and 1 participant was transferred to another treatment centre, while a total of one hundred and thirty-five (135) patients were finally included in the immuno-virologic analysis. Adherence was assessed using both self-report questionnaire and measurement of Mean Cell Volume (MCV) which was obtained using automated haematology analyzer. CD4 counts were analyzed using flow cytometry method and HIV-RNA levels were measured by using the RT-Polymerase Chain Reaction technique. RESULTS: Incomplete adherence was recorded in 28 (20.7%) of the study participants, immunologic failure (decline in CD4 count to or below baseline or CD4 count change &lt; 50 cells/ul at 12 months) was observed in 39 (28.8%) of the participants. Virologic failure rate (viral load &gt; 400 copies/ml) was 27.4% and immuno-virologic failure rate was 31.9%. Using logistic regression model, immunologic failure was associated with male gender (OR=1.29; p=0.008), non-disclosure of status (OR=1.24; p=0.01), baseline anaemia (OR=1.15; p=0.009) and incomplete adherence by self-report (OR=3.28; p=0.001). Virologic failure was associated with no formal education (OR=2.29; p=0.01), non-disclosure of status (OR=1.04; p = &lt; 0.01), non-adherence (OR=2.74; p=0.015), being unemployed (OR=0.57; p=0.04). CONCLUSIONS: The study observed that immunologic recovery and virologic suppression rate within the first year of treatment was significant, although the rate of incomplete adherence obtained still needs improvement. Effort to promote social coping particularly to patients who were unemployed, re-strategizing on improving patient education and counseling especially to patients with no formal education, focusing on campaigns against stigmatization will help in addressing predictors of negative immuno-virologic outcomes. Baseline HIV-RNA level should also be considered in addition to CD4 testing in order to identify virological failure and thus preventing the emergence of drug resistance in HIV/AIDS management.

Determination of the Pre-breakdown Electric Field near a Cathode Streamer in Water

The electrical strength of liquids under the influence of an impulse voltage is determined by the streamer initiation and propagation processes. For a streamer to grow and develop, a strong field must be present near its head. The field strength near a cathode streamer developing in water is estimated. Computer simulation of electro-optical studies of the pre-breakdown stage in water in the submicrosecond range that have already been carried out is performed, and the relevant processes are analyzed. During the simulation, all geometric, optical, and electrophysical conditions of the experiment were fully reproduced. A cathode streamer developing in a system of spherical electrodes in water is considered. The mathematical simulation was carried out using the finite element method inside the measuring cell volume. The experimental kerrogram intensity distribution field was numerically reproduced using the proposed model. The obtained 2D matrix was visualized, and the calculated kerrogram was compared with the experimental one. Calculations with a step of 2.5–5 µm allowed the fragments of interest to be analyzed in detail. Two streamer model versions were considered: a simplified one and a modified one, with the latter being closer to the real streamer kind. Computer simulation of the field near the streamer has shown the following. If the permittivity is assumed to be independent of the field strength, its value at the cathode streamer head does not exceed 1.9 MV/cm. At the same time, if the permittivity non-linearity is taken into account, the field strength estimated using both the streamer model was found to be around 2.4–3.1 MV/cm. It is shown that, despite the fact that the field strength somewhat exceeds the threshold value equal to 3 MV/cm, at which the dipole saturation state is possible, this does not lead to a noticeable change in the result. Thus, it has been confirmed that the field strength near the cathode streamer is essentially lower compared with that near the anode streamer.

Identification of the chloride channel, leucine-rich repeat-containing protein 8, subfamily a (LRRC8A), in mouse cholangiocytes.

Chloride (Cl- ) channels in the apical membrane of biliary epithelial cells (BECs), also known as cholangiocytes, provide the driving force for biliary secretion. Although two Cl- channels have been identified on a molecular basis, the Cystic Fibrosis Transmembrane Conductance Regulator and Transmembrane Member 16A, a third Cl- channel with unique biophysical properties has been described. Leucine-Rich Repeat-Containing Protein 8, subfamily A (LRRC8A) is a newly identified protein capable of transporting Cl- in other epithelium in response to cell swelling. The aim of the present study was to determine if LRRC8A represents the volume-regulated anion channel in mouse BECs. Studies were performed in mouse small (MSC) and large (MLC) cholangiocytes. Membrane Cl- currents were measured by whole-cell patch-clamp techniques and cell volume measurements were performed by calcein-AM fluorescence. Exposure of either MSC or MLC to hypotonicity (190 mOsm) rapidly increased cell volume and activated Cl- currents. Currents exhibited outward rectification, time-dependent inactivation at positive membrane potentials, and reversal potential at 0 mV (ECl ). Removal of extracellular Cl- or specific pharmacological inhibition of LRRC8A abolished currents. LRRC8A was detected in both MSC and MLC by reverse transcription polymerase chain reaction and confirmed by western blot. Transfection with LRRC8A small interfering RNA decreased protein levels by >70% and abolished volume-stimulated Cl- currents. These results demonstrate that LRRC8A is functionally present in mouse BECs, contributes to volume-activated Cl- secretion, and, therefore, may be a target to modulate bile formation in the treatment of cholestatic liver disorders.

Patterns of Cell Death Induced by Thiohydantoins in Human MCF-7 Breast Cancer Cells.

Conventional therapies for breast cancer are still a challenge due to cytotoxic drugs not being highly effective with significant adverse effects. Thiohydantoins are biologically active heterocyclic compounds reported for several biological activities, including anticarcinogenic properties, etc. This work aims to assess the use of thiohydantoin as a potential antitumor agent against MCF-7 breast cancer cells. MTT and neutral red assays were used to assess the possible cytotoxic activity of compounds against MCF-7 cells. Cell volume measurement and analysis were performed by flow cytometry. Fluorescence analysis was carried out to determine patterns of cell death induced by thiohydantoins. The treatment with micromolar doses of thiohydantoins promoted a decrease in the viability of MCF-7 breast tumor cells. An increase in the ROS and NO production, reduction in cell volume, loss of membrane integrity, mitochondrial depolarization, and increased fluorescence for annexin-V and caspase-3 were also observed. These findings indicate cell death by apoptosis and increased autophagic vacuoles, stopping the cell cycle in the G1/ G0 phase. Our results indicate that thiohydantoins are cytotoxic to breast tumor cells, and this effect is linked to the increase in ROS production. This phenomenon changes tumorigenic pathways, which halt the cell cycle in G1/G0. This is an essential checkpoint for DNA errors, which may have altered how cells produce energy, causing a decrease in mitochondrial viability and thus leading to the apoptotic process. Furthermore, the results indicate increased autophagy, a vital process linked to a decrease in lysosomal viability and thus considered a cell death and tumor suppression mechanism.

Value Of Blood Volume Analysis In Patients With Left Ventricular Assist Devices.

Introduction Implantable left ventricular assist devices (LVAD) have dramatically changed the face of mechanical circulatory support of patients with advanced heart failure. With increased durability, reliability and safety these devices have extended the horizon of functional improvement, life quality and survival. Beyond crucial pre-operative and intra-operative issues, several device-host interface issues are important for successful outcomes. Among these are clear understanding of intravascular volume status, red blood cell volume status. Blood volume analysis (BVA) is a blood test that measures these 3 metrics using the indicator dilution technique, with rapid personalized and actionable results. We used BVA to evaluate 10 consecutive stable ambulatory patients supported with LVAD. Results The patients were seven men and three woman aged 32-70 years. Duration of LVAD support at the time of outpatient BVA (Daxor, Oak Ridge, TN) ranged from 46-1460 days. Two out of eight patients had LVAD (HM3(5), HW(4), HM2(1)implanted as bridge to transplant, all others as destination therapy. Eight out of ten patients were obese with deviations from normal body weight of 22-117% and average BMI of 32.6. All patients were felt to be improved and stable compared to pre-LVAD (INTERMACS 1-3) and none had evidence of device malfunction or drive line infection. All patients had variations in total blood volumes, plasma volume and red cell volume (Table1). Only two out of ten patients had all three BVA parameters within normal limits. Half of the patients had significant hypervolemia, which was not suspected on physical exam. Two out of ten patients had significant hypervolemia with polycythemia, which was not clinically suspected. Conclusions In summary, BVA and its individualized, unique metrics is a valuable test when used in patients with LVAD's. Despite improved functional status and adequate device performance wide variations are still present in patients being supported with LVAD's. Additionally, measurement of red blood cell volume is helpful in determined variations (anemia or polycythemia) that might not be noted clinically and might presage future hemolytic and/or thrombotic events despite adequate anticoagulation. Implantable left ventricular assist devices (LVAD) have dramatically changed the face of mechanical circulatory support of patients with advanced heart failure. With increased durability, reliability and safety these devices have extended the horizon of functional improvement, life quality and survival. Beyond crucial pre-operative and intra-operative issues, several device-host interface issues are important for successful outcomes. Among these are clear understanding of intravascular volume status, red blood cell volume status. Blood volume analysis (BVA) is a blood test that measures these 3 metrics using the indicator dilution technique, with rapid personalized and actionable results. We used BVA to evaluate 10 consecutive stable ambulatory patients supported with LVAD. The patients were seven men and three woman aged 32-70 years. Duration of LVAD support at the time of outpatient BVA (Daxor, Oak Ridge, TN) ranged from 46-1460 days. Two out of eight patients had LVAD (HM3(5), HW(4), HM2(1)implanted as bridge to transplant, all others as destination therapy. Eight out of ten patients were obese with deviations from normal body weight of 22-117% and average BMI of 32.6. All patients were felt to be improved and stable compared to pre-LVAD (INTERMACS 1-3) and none had evidence of device malfunction or drive line infection. All patients had variations in total blood volumes, plasma volume and red cell volume (Table1). Only two out of ten patients had all three BVA parameters within normal limits. Half of the patients had significant hypervolemia, which was not suspected on physical exam. Two out of ten patients had significant hypervolemia with polycythemia, which was not clinically suspected. In summary, BVA and its individualized, unique metrics is a valuable test when used in patients with LVAD's. Despite improved functional status and adequate device performance wide variations are still present in patients being supported with LVAD's. Additionally, measurement of red blood cell volume is helpful in determined variations (anemia or polycythemia) that might not be noted clinically and might presage future hemolytic and/or thrombotic events despite adequate anticoagulation.

Intravascular Volume Derangement and Value of Blood Volume Analysis in Stable Ambulatory Patients with Advanced Heart Failure Supported with Left Ventricular Assist Device

<h3>Purpose</h3> Heart failure is associated with elevated filling pressures and significant volume expansion. Durable left ventricular assist devices (LVAD) have been shown to improve survival and quality of life in patients with advanced heart failure. There is strong evidence suggesting structural, neurohormonal and hemodynamic improvement after LVAD implantation. The data on normalization of plasma volume is lacking. We sought to describe blood volume parameters in stable outpatient LVAD recipients. <h3>Methods</h3> We performed blood volume analysis (BVA) on 33 stable consecutive patients supported with LVADs in the outpatient setting. All BVAs were obtained as part of routine outpatient follow up. Results of the test were provided to clinicians for ongoing patient management. BVA is clinically approved and utilizes the Iodine131-tagged albumin tracer to provide quantitative measurement of total blood volume (TBV), plasma volume (PV) and red blood cell volume (RBC). The BVA-report provides absolute values (ml) and deviation from ideal TBV, PV, and RBC. A TBV deviation ≥±8% was considered to be either an excess or deficit of volume. <h3>Results</h3> Only 9% of stable ambulatory LVAD patients had normal blood volume results. 18/33 (54%) had significant anemia, 4/33 (12%) had polycythemia. Polycythemia in all of the patients was masked on complete blood count analysis, due to significant hypervolemia. One of the patients with masked polycythemia had fatal pump thrombosis. Plasma volume abnormality was found in 75% of patients (39% were hypervolemic, 36% were hypovolemic). 6 patients had repeat BVA after medical interventions and all of them were unsuccessful in normalizing plasma volume. <h3>Conclusion</h3> BVA is useful tool in outpatient management of patients with LVADs. As with advanced heart failure, correction of clinically unsuspected and undetected derangements in intravascular volume and red blood cell volumes likely have an impact on device and patient outcomes. These deviations may be recalcitrant to correction and further investigation is ongoing. Underdiagnosed polycythemia in LVAD patients may further contribute to hemo-compatibility issues and thrombosis. Measurement and treatment of derangements of intravascular volumes likely will improve performance and survival of patients and devices.

Impaired Regulatory Volume Decrease and Characterization of Underlying Volume-Activated Currents in Cystic Fibrosis Human Cholangiocyte Cell Line.

The volume-activated chloride channel (VACC) serves vital cellular functions in secretion and cell volume regulation via regulatory volume decrease (RVD) in various epithelia. Previously, we have shown that RVD in primary CF mouse cholangiocytes is impaired. Thus, the effect of CFTR defect on VACC and RVD in CF human immortalized cholangiocyte cell (HBDC) was examined in comparison with those in normal HBDC by using cell volume measurement and whole-cell patch clamp techniques, respectively. The CF HBDC had an impaired RVD, which was not further inhibited by removing the extracellular calcium or administering BAPTA-AM, NPPB, or DIDS. When exposed to a hypotonic solution, CF HBDC exhibited large, outwardly rectified currents with time-dependent inactivation at a positive potential. The amplitude of the outward currents was about three times that of the inward currents. The amplitude and reversal potential of VACC was dependent on chloride concentration. The VACC was significantly inhibited by replacing chloride with gluconate, glutamate, sucrose, or acetate in the hypotonic solution as well as by an administration of NPPB or tamoxifen, classical VACC inhibitors. Surprisingly, the VACC amplitude is greater in CF HBDC than in normal HBDC, suggesting that the channel density or open probability of VACC is increased, thus CFTR may have inhibitory effects on VACC. On the contrary, the amplitude of the volume-activated potassium current is lower in CF HBDC, suggesting the potassium channel density or open probability is decreased in CF cholangiocytes and/or CFTR may have regulatory effects on volume-activated potassium current. In conclusion, RVD is impaired in CF human cholangiocytes. The VACC of CF human cholangiocytes has similar electrophysiological characteristics as that of normal cholangiocytes but its activity is augmented in CF cholangiocytes, while volume-activated potassium current is decreased in CF human cholangiocytes, providing a fundamental underlying pathophysiologic mechanism for the impaired RVD in CF cholangiocytes.

Measuring the size and growth of single cells.

The size and growth of a cell can be described by three related physical parameters: volume, density and mass. All the three are coupled to numerous biochemical reactions and biophysical properties of a cell. It is therefore not surprising that cell size and growth pattern are tightly regulated across all kingdoms of life. Indeed, deregulation of cell size and growth has been found to be associated with diseases. Yet, how cells regulate their size and how cell size connects to cell function remain poorly understood, partly due to the difficulties to precisely measure the size and growth of single cells. In this review, we summarize methods of measuring cell volume, density, and mass, and discuss how the new technologies may advance our understanding of cell size control.

Temporal declines in Wadden Sea phytoplankton cell volumes observed within and across species

AbstractCell size is a master trait in the functional ecology of phytoplankton correlating with numerous morphological, physiological, and life‐cycle characteristics of species that constrain their nutrient use, growth, and edibility. In contrast to well‐known spatial patterns in cell size at macroecological scales or temporal changes in experimental contexts, few data sets allow testing temporal changes in cell sizes within ecosystems. To analyze the temporal changes of intraspecific and community‐wide cell size, we use the phytoplankton data derived from the Lower Saxony Wadden Sea monitoring program, which comprises sample‐ and species‐specific measurements of cell volume from 1710 samples collected over 14 yr. We find significant reductions in both the cell volume of most species and the weighted mean cell size of communities. Mainly diatoms showed this decline, whereas the size of dinoflagellates seemed to be less responsive. The magnitude of the trend indicates that cell volumes are about 30% smaller now than a decade ago. This interannual trend is overlayed by seasonal cycles with smaller cells typically observed in summer. In the subset of samples including environmental conditions, small community cell size was strongly related to high temperatures and low total phosphorus concentration. We conclude that cell size captures ongoing changes in phytoplankton communities beyond the changes in species composition. In addition, based on the changes in species biovolumes revealed by our analysis, we warn that using standard cell size values in phytoplankton assessment will not only miss temporal changes in size, but also lead to systematic errors in biomass estimates over time.