The authors would like to thank Prof(s). Piccoli and Pastori of the Department of Medical and Surgical Sciences, University of Padova, Italy, for providing the BIVA software. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

The following protocol was approved (REF. 3057) and follows the guidelines of the human research ethics committee of Instituto Nacional de Ciencias M&#233;dicas y Nutrici&#243;n SZ. Furthermore, prior consent was obtained from the patients for this study.
NOTE: This procedure is to be used for measuring bioelectrical impedance analysis using tetrapolar multi-frequency equipment (see Table of Materials) and will provide accurate resistance and reactance values at a single frequency of 50 kHz, as well as the ratio between 200 kHz and 5 kHz impedance values (200/5 kHz).
1. Before testing
<ol>
	<li>Perform standardization of the person who will perform the measurements, as someone with a qualification in the area or who has extensive experience in making measurements.</li>
	<li>Ask the patient to refrain from eating for 4 to 5 h before testing.</li>
	<li>Periodically test the equipment to verify the impedance measurement are as accurate as possible, according to the guidelines provided by the manufacturer, using a test resistor with a known value of 500 &#937; (range 496-503 &#937;). Make sure the adhesive electrodes correspond to the manufacturer&#39;s recommendation.</li>
	<li>Clean the equipment using a chlorhexidine wipe, and then wash your hands. If the screen of the equipment shows the legend: change the battery, then replace the battery.</li>
	<li>If the patient is conscious, explain the procedure to them. Obtain the age and accurate measurement of the patient&#39;s height (in cm) and introduce these data into the equipment.</li>
	<li>Remove the shoe and sock from the right foot, as well as any metal objects, such as watches or bracelets worn by the patient. Place the patient in a supine position for 5 min with legs and arms spread out around 45&#176; before taking the measurements, verifying that they are not in contact with any other part of their body. In patients with obesity, in order to avoid contact between the thighs, place a sheet between their legs.</li>
</ol>
2. Measurement of BIA parameters
<ol>
	<li>Clean the surfaces where the electrodes will be placed with a 70% alcohol pad twice. Place two electrodes on the right hand dorsally, one behind the knuckle of the third metacarpophalangeal (middle finger) and the other on the wrist, next to the ulna head carpal joint. It might be helpful to draw an imaginary straight line between the protruding bones on the wrist, and then place each electrode in the center of that line.</li>
	<li>Place two electrodes on the right foot, one behind the third metatarsophalangeal joint and the tarsal joint on the ankle between the medial and lateral malleoli. To place the electrodes, follow the bones underneath. Ensure that the distance between the electrodes on the foot and hand is at least 5 to 10 cm, according to the size of the hand.</li>
	<li>Connect the lead wires to the equipment, with the red alligator clip nearest to the nails and the black clip nearest to the ankle or wrist; ensure that the wires do not cross between them.</li>
	<li>Ensure that the patient is not talking or moving during the measurements, as this will affect the results.</li>
	<li>The patient&#39;s ID will come up on the first screen. Scroll through and change the patient&#39;s parameters (sex, age, height, and weight). Ensure that the electrodes are stuck down correctly, and press Enter. It will show: measuring, on the screen. It takes about 6 to 10 s to measure, and a beep will sound when the measurement is complete.</li>
</ol>
3. Analysis of bioimpedance parameters
<ol>
	<li>The equipment will display the raw impedance values (Z) at four different frequencies: 5, 50, 100, and 200 kHz, as well as the resistance and reactance at 50 kHz, which are the values needed to classify a patient with fluid overload.</li>
	<li>Download the software named BIVA tolerance R-Xc graph13&#160;(see Table of Materials) and open it.</li>
	<li>Observe that the software is in a workbook in a spreadsheet program with seven worksheets: guide, reference populations, point graph, path, subjects, Z-score, Z-graph.</li>
	<li>Right click on the Reference Population sheet, choose the line of the selected reference population, and copy and paste it into the second row (yellow row).</li>
	<li>Right click on the Subjects sheet and, in the second row, insert the following data: the subject ID assigned to the patient. In the second column named Seq, always put the number 1; and optionally fill out the surname and name columns. In the sex column, enter F for female patient and M for male patient. In the next two columns, input resistance and reactance at 50 kHz each. Insert the height (in cm) and weight (in kg) in the next two columns.</li>
	<li>In the Popul Code column, insert the number that appears in the first column of the reference population sheet. In group code, randomly chose a number between 1 and 10 (this number will be required in the Point graph sheet), insert the patient&#39;s age in the next column.</li>
	<li>In the spreadsheet program menu, go to the Complements tab and right-click on the Calculate option to obtain the values of resistance and reactance adjusted by height and phase angle.</li>
	<li>Right-click on the Point Graph sheet, and observe that 50%, 75%, and 95% tolerance ellipses are drawn for the selected reference population (i.e., the population in the first yellow row at the top of the reference population sheet).</li>
	<li>In the dialog box, select groups, right-click on the number placed in the group code located in the subjects&#39; sheet, and right-click on OK. Next, the BIVA graph will appear, with the subject vector as a geometric figure (&#916;, &#8226;, &#9633;).</li>
	<li>Patients with vectors that fall outside the lower pole of the 75% tolerance ellipse will be classified as fluid overload (see Figure 1).</li>
	<li>Divide Z at 200 kHz by Z at 5 kHz-which reflects the total body water and extracellular water compartment, respectively-in order to obtain the impedance ratio (Imp-R). A value &#8805;0.85 indicates fluid overload. 
	NOTE: In new tetrapolar multi-frequency devices, the R-Xc graph is already included; however, it is important to ensure that the reference population is correct.</li>
</ol>

<ol>
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Clinical Nutrition ASPEN Association of hyperhydration evaluated by bioelectrical.</a> Clinical Nutrition ESPEN. 28, 12-20 (2018).</li><li>Kammar-Garc&#237;a, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Bioelectrical+Impedance+Analysis+parameters+for+the+detection+of+fluid+overload+in+the+prediction+of+mortality+in+patients+admitted+at+the+emergency+department.">Comparison of Bioelectrical Impedance Analysis parameters for the detection of fluid overload in the prediction of mortality in patients admitted at the emergency department.</a> Journal of Parenteral and Enteral Nutrition. 45 (2), 414-422 (2021).</li><li>Kammar-Garc&#237;a, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOFA+score+plus+impedance+ratio+predicts+mortality+in+critically+ill+patients+admitted+to+the+emergency+department:+Retrospective+observational+study.">SOFA score plus impedance ratio predicts mortality in critically ill patients admitted to the emergency department: Retrospective observational study.</a> Healthcare (Basel). 10 (5), 810 (2022).</li><li>Frank Peacock, W., Soto, K. 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D., Li, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioimpedance+illness+marker+compared+to+phase+angle+as+a+predictor+of+malnutrition+in+hospitalized+patients.">Bioimpedance illness marker compared to phase angle as a predictor of malnutrition in hospitalized patients.</a> Clinical Nutrition. 32, 85 (2013).</li><li>Castillo-Martinez, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioelectrical+impedance+and+strength+measurements+in+patients+with+heart+failure:+comparison+with+functional+class.">Bioelectrical impedance and strength measurements in patients with heart failure: comparison with functional class.</a> Nutrition. 23 (5), 412-418 (2007).</li><li>Earthman, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Body+composition+tools+for+assessment+of+adult+malnutrition+at+the+bedside:+A+tutorial+on+research+considerations+and+clinical+applications.">Body composition tools for assessment of adult malnutrition at the bedside: A tutorial on research considerations and clinical applications.</a> Journal of Parenteral and Enteral Nutrition. 39 (7), 787-822 (2015).</li><li>Piccoli, A., Pastori, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BIVA+software.">BIVA software.</a> Department of Medical and Surgical Sciences. , (2002).</li><li>Basso, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+management+in+the+intensive+care+unit:+bioelectrical+impedance+vector+analysis+as+a+tool+to+assess+hydration+status+and+optimal+fluid.">Fluid management in the intensive care unit: bioelectrical impedance vector analysis as a tool to assess hydration status and optimal fluid.</a> Blood Purification. 36 (3-4), 192-199 (2013).</li><li>Piccoli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioelectrical+impedance+measurement+for+fluid+status+assessment.">Bioelectrical impedance measurement for fluid status assessment.</a> Contributions to Nephrology. 164, 143-152 (2010).</li><li>National Institutes of Health Technology. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioelectrical+impedance+analysis+in+body+composition+measurement:+National+Institutes+of+Health+Technology+Assessment+Conference+Statement.">Bioelectrical impedance analysis in body composition measurement: National Institutes of Health Technology Assessment Conference Statement.</a> The American Journal of Clinical Nutrition. 64, 524-532 (1996).</li><li>Silva, A. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioelectrical+impedance+analysis+(BIA):+a+proposal+for+standardization+of+the+classical+method+in+adults.">Bioelectrical impedance analysis (BIA): a proposal for standardization of the classical method in adults.</a> Journal of Physics: Conference Series. 47, 407 (2012).</li><li>Di Somma, S., Gori, C. S., Grandi, T., Risicato, M. 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The authors declare no competing interests.

It is important to mention that different bioelectrical impedance analysis (BIA) approaches have been proposed in the published literature, including the use of multiple frequencies at 1-500 kHz (MF-BIA), phase-sensitive single frequency (SF-BIA) at 50 kHz, and spectroscopic BIA at 5 kHz to 2 MHz. Studies have provided inconsistent results, concerning the agreement regarding single- and multiple-frequency BIA equipment6, including source current, frequency, total impedance range over which the current is within a specified tolerance, resolution, and accuracy of the displayed impedance, as has been described in the National Institutes of Health (NIH) Technology Assessment Conference Statement21. Bioelectrical impedance spectroscopy (BIS) instruments present an important limitation: R and Xc of total body raw data cannot be obtained, which must be calculated or modeled from other segmental impedance parameters, and they seem to present an under-estimation when compared with phase-sensitive single- and multiple-frequency devices5,22. Therefore, we do not recommend the use of such technology.
On its own, MF-BIA is a phase-sensitive tetrapolar instrument that directly measures phase angle and impedance at different frequencies (5, 50, 100, 200, and 500 kHz), reporting a 0.5% deviation and an accuracy of 500 &#937; for each frequency, allowing differentiation between intracellular and extracellular water, based on the principle that: at lower frequencies, the current flows through extracellular water while, at higher frequencies, it flows through a total body of water. As this kind of device provides raw data, the IR can be calculated, as has been previously described6,23.
It is also important to consider that the type of electrode, and the specific anatomical location of electrodes, in addition to the position of the subject to be measured, can influence the raw bioelectrical values. Thus, one should avoid extrapolating results obtained with different equipment in patients who are decompensated (e.g., heart, renal, or hepatic failure), or who are suffering an acute event or another chronic disease6. It is essential to implement a protocol to standardize a method in order to determine fluid overload at admission. Therefore, obtaining a basal fluid distribution status allows for early and appropriate therapeutic approaches to be taken.
Clinical practice guidelines and manufacturers do not recommend BIA assessment to be performed in patients with cardiac implantable electronic devices (CIEDs), such as pacemakers and implantable cardioverter defibrillators, as it can cause electromagnetic interference due to the applied electric current. However, when the low magnitude of the electric current transferred to the body is inferior to the susceptibility limits of the CIED, and with the absence of alterations in its function, BIA is considered safe and can be performed in this group of patients24.
Another consideration to take into account is that BIA and BIVA cannot be performed in patients with any amputation or with abnormal physical structure21.
Some limitations of the measurement technique that may not be controlled in the context of patients at emergency admission include fasting time, alcohol consumption, previous physical exercise, and bladder voiding25.
When a fluid overload is detected-and based on the assumption that it is the result of fluid accumulation-the use of diuretics is frequent in clinical practice; however, it is possible that the main pathophysiological mechanisms could be related to fluid re-distribution, rather than accumulation, and high doses of furosemide may be detrimental to renal function. For example, in heart failure patients with diastolic dysfunction and pulmonary edema, high systolic blood pressure can be treated with vasodilators (nitrates), thus avoiding the use of diuretics26. Therefore, it is essential to interpret the BIVA results in the context of the patient&#39;s diagnosis, physical examination, and biomarkers (e.g., hemoglobin, albumin, sodium, and creatinine).
Finally, in order to illustrate how BIVA can be used, in a previous report, we found that the patients classified as having fluid overload, according to BIVA at admission to emergency department-even with an accumulated balance of 1212 mL fluids, a value that is considered normal-showed statistically significant higher disease severity with respect to SOFA, and presented higher mortality, compared to those with normal fluid status, thus demonstrating the usefulness of BIVA in critically ill patients27.

Fluid overload (FO), defined as an excess of total body fluid or a relative excess in one or more fluid compartments1, is frequently observed in critically ill patients and is associated with higher morbidity and mortality1,2,3. The range of alterations in hydration status is wide; can indicate renal, cardiac, or hepatic failure; and/or maybe the result of excessive oral intake or iatrogenic error4. Routine assessment of hydration status is challenging in emergency departments, as the gold standard of radioisotopic volume measurement requires specialized techniques, is costly and time-consuming, and may fail to identify early disturbances in hydration status. Hence, other less-accurate methods are generally used, including clinical examination and accumulated fluid balance (volume in mL in 24 h)5. Accurate and sensitive determination of fluid volume status is necessary to help clinicians in controlling body fluids, managing intravenous fluid administration, and maintaining hemodynamic stability, thus allowing patients to receive early treatment3,5,6. Errors in the volume assessment can lead to a lack of necessary treatment or to implementation of unnecessary therapy, such as excess fluid administration, both of which are related to increased hospitalization costs, complications, and mortality4.
Interest has recently increased in bioelectrical impedance analysis (BIA), which has been considered an alternative method for the classification of an individual&#39;s hydration status. BIA is a safe, non-invasive, portable, quick, bed-side, and easy-to-use method, designed for the estimation of body compartment composition. The analysis measures the opposition generated by soft tissues to the flow of an injected alternating electric current into the body (800 &#181;A), through four surface electrodes placed on the hands and feet. Total body water estimated by BIA has been shown to have a high correlation with that obtained by deuterium dilution (r = 0.93, p = 0.01)7.
Phase-sensitive BIA devices evaluate the direct measurement of phase angle and impedance (Z50), obtaining the resistance (R) and reactance (Xc) in single-frequency mode (50 kHz) or multi-frequency mode (5 kHz to 200 kHz)8. Dividing the R and Xc values by the subject&#39;s height (in m) squared-to control for inter-individual differences in conductor length-and plotting them in an R-Xc graph is the method used in bioelectrical impedance vector analysis (BIVA) to estimate the fluid status. BIVA is an alternative impedance approach, developed by Piccoli et al.9, which uses the spatial relationship between R (i.e., the opposition to the flow of an alternating current through intra- and extra-cellular ionic solutions) and Xc to assess soft tissue hydration, independent of the multiple-regression prediction equations generated in limited and specific samples10. Therefore, the classification of fluid status is more precise and accurate than the quantification of total body water. The R and Xc values of a subject produce a vector whose position can be evaluated relative to tolerance intervals in the R-Xc graph, which can be interpreted as indicating normal or abnormal hydration, based on the distance from the mean vector derived from a healthy reference population11,12,13.
In a previous study, we compared different bioelectrical impedance analysis parameters for the detection of fluid overload and prediction of mortality in patients admitted to an emergency department (ED) and demonstrated that BIVA (relative risk = 6.4; 95% confidence interval from 1.5 to 27.9; p = 0.01) and impedance ratio (relative risk = 2.7; 95% confidence interval from 1.1 to 7.1; p = 0.04) improved the estimation of the probability of 30-day mortality3.
Fluid overload can also be estimated using the impedance ratio (imp-R), which is the ratio between impedance measured at 200 kHz and impedance measured at 5 kHz obtained by the multi-frequency bioelectrical impedance equipment. Imp-R considers conduction in total body water (Z200) and in extracellular water fluid spaces (Z5). The penetration of a current into cells is frequency-dependent and, the 200/5 kHz ratio describes the ratio of greater to lesser current entry into cells3,8. If the difference between these two values decreases over time, it may indicate that the cells are becoming less healthy14.
Imp-R values &#8804;0.78 in males and &#8804;0.82 in females have been observed in healthy individuals15. Values nearer to 1.0 indicate that the two impedances are closer to each other, and the body cell is less healthy. In the case of critical illness, the resistance of the cell membrane at 5 kHz is reduced, and the difference between the impedance values at 5 and 200 kHz is markedly lower, indicating cellular worsening3. Values &#62; 1.0 suggest device error16,17. Thus, the objective of the present study is to demonstrate how to evaluate the presence of fluid overload through bioelectrical impedance vectorial analysis, as well as by using the impedance ratio, measured with tetrapolar multi-frequency equipment in patients admitted to the emergency department.

<table><tbody><tr><td>Alcohol 70% swabs</td><td>NA</td><td>NA</td><td>Any brand can be used</td></tr><tr><td>BIVA software 2002</td><td>NA</td><td>NA</td><td>Is a sofware created for academic use, can be download in http://www.renalgate.it/formule_calcolatori/bioimpedenza.htm in &quot;LE FORMULE DEL Prof. Piccoli&quot; section</td></tr><tr><td>Chlorhexidine Wipes</td><td>NA</td><td>NA</td><td>Any brand can be used</td></tr><tr><td>Examination table</td><td>NA</td><td>NA</td><td>Any brand can be used</td></tr><tr><td>Leadwires square socket</td><td>BodyStat</td><td>SQ-WIRES</td><td></td></tr><tr><td>Long Bodystat 0525 electrodes</td><td>BodyStat</td><td>BS-EL4000</td><td></td></tr><tr><td>Quadscan 4000 equipment</td><td>BodyStat</td><td>BS-4000</td><td>Impedance measuring range: 20 - 1300 &amp;Omega; ohms&lt;br/&gt; Test Current: 620 &amp;mu;A&lt;br/&gt; Frequency: 5, 50, 100, 200 kHz&lt;br/&gt; Accuracy: Impedance 5 kHz: +/- 2 &amp;Omega;&lt;br/&gt; Impedance 50 kHz: +/- 2 &amp;Omega;&lt;br/&gt; Impedance 100 kHz: +/- 3 &amp;Omega;&lt;br/&gt; Impedance 200 kHz: +/- 3 &amp;Omega;&lt;br/&gt; Resistance 50 kHz: +/- 2 &amp;Omega;&lt;br/&gt; Reactance 50 kHz: +/- 1 &amp;Omega;&lt;br/&gt; Phase Angle 50 kHz: +/- 0.2&amp;deg;&lt;br/&gt; Calibration: A resistor is supplied for independent verification from time to time. The impedance value should read between 496 and 503 &amp;Omega;.</td></tr></tbody></table>

evaluation of fluid overload by bioelectrical impedance vectorial analysis

Early detection and management of fluid overload are critically important in acute illness, as the impact of therapeutic intervention can result in decreased or increased mortality rates. Accurate fluid status assessment entails appropriate therapy. Unfortunately, as the gold standard method of radioisotopic fluid measurement is costly, time-consuming, and lacks sensitivity in the acute care clinical setting, other less-accurate methods are typically used, such as clinical examination or 24 h output. Bioelectrical impedance vectorial analysis (BIVA) is an alternative impedance-based approach, where the raw parameter resistance and reactance of a subject are plotted to produce a vector, the position of which can be evaluated relative to tolerance intervals in an R-Xc graph. The fluid status is then interpreted as normal or abnormal, based on the distance from the mean vector derived from a healthy reference population. The objective of the present study is to demonstrate how to evaluate the presence of fluid overload through bioelectrical impedance vectorial analysis and the impedance ratio measured with tetrapolar multi-frequency equipment in patients admitted to the emergency department.

As an example of the method presented above, we present the results for two women admitted to the emergency department. Bioelectrical impedance analysis was assessed at admission using a phase-sensitive multi-frequency device (see Table of Materials), and the obtained resistance (R) and reactance (Xc) values were used to calculate the BIVA graph. The results show that patients with overhydration had worse prognoses and clinical characteristics such as SOFA and Charlson index scores, which are related to fluid overload.
In Figure 2, the results plotted with &#916; denote a 77-year-old female (height = 155 cm) with normal fluid status and the following bioimpedance results: R = 586.7, Xc = 62.1. The data of clinical variables were as follows: sequential organ failure assessment score (SOFA) = 3; Charlson comorbidity index score = 5; the primary cause of hospital admission = hypotonic hyponatremia secondary to diuretic use and diarrhea; and length of hospital stay = 2 days.
Meanwhile, the results plotted with &#9633; denote a 62-year-old female (height = 149 cm) with fluid overload and bioimpedance results R = 332.6, Xc = 33.6. Data of clinical variables were as follows: SOFA = 16; Charlson comorbidity index = 4; primary cause of hospital admission = septic shock secondary to soft tissue infection; length of hospital stay = 3 days. This patient died due to the progression of refractory shock, with acute respiratory distress syndrome that progressively worsened.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64331/64331fig01.jpg" /> 
Figure 1: RXc-graph of the bioelectrical impedance vector analysis to classify the fluid status of a patient. Individual vectors below 75% (+2 standard deviation) can be classified as fluid overload &#916;. Abbreviations: R = resistance, Xc = reactance, H = height. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64331/64331fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64331/64331fig02.jpg" /> 
Figure 2: RXc-graph with the data of two female patients admitted to the emergency department. &#916; is patient in the 50% tolerance ellipse showing a normal fluid status. &#9633; is patient below the 75% ellipse classified with fluid overload. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64331/64331fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Evaluation of Fluid Overload by Bioelectrical Impedance Vectorial Analysis at JoVE.com

Evaluation of Fluid Overload by Bioelectrical Impedance Vectorial Analysis

In this study, we demonstrate how to evaluate the presence of fluid overload through bioelectrical impedance vectorial analysis (BIVA) and the impedance ratio measured using tetrapolar multi-frequency equipment in patients admitted to the emergency department. BIVA and impedance ratio are reliable and useful tools to predict poor outcomes.

Early detection and management of fluid overload are critically important in acute illness, as the impact of therapeutic intervention can result in ...

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2.4K Views.  Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán. In this study, we demonstrate how to evaluate the presence of fluid overload through bioelectrical impedance vectorial analysis (BIVA) and the impedance ratio measured using tetrapolar multi-frequency equipment in patients admitted to the emergency department. BIVA and impedance ratio are reliable and useful tools to predict poor outcomes.

Video: Evaluation of Fluid Overload by Bioelectrical Impedance Vectorial Analysis

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

Stroke is the third cause of mortality and the leading cause of disability in the World. Ischemic stroke accounts for approximately 80% of all strokes. However, the thrombolytic tissue plasminogen activator (tPA) is the only treatment of acute ischemic stroke that exists. This led researchers to develop several ischemic stroke models in a variety of species. Two major types of rodent models have been developed: models of global cerebral ischemia or focal cerebral ischemia. To mimic ischemic stroke in patients, in whom approximately 80% thrombotic or embolic strokes occur in the territory of the middle cerebral artery (MCA), the intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies. This model was first developed in rats by Koizumi et al. in 1986 1. Because of the ease of genetic manipulation in mice, these models have also been developed in this species 2-3.
	Herein, we present the transient MCA occlusion procedure in C57/Bl6 mice. Previous studies have reported that physical properties of the occluder such as tip diameter, length, shape, and flexibility are critical for the reproducibility of the infarct volume 4. Herein, a commercial silicon coated monofilaments (Doccol Corporation) have been used. Another great advantage is that this monofilament reduces the risk to induce subarachnoid hemorrhages. Using the Zeiss stereo-microscope Stemi 2000, the silicon coated monofilament was introduced into the internal carotid artery (ICA) via a cut in the external carotid artery (ECA) until the monofilament occludes the base of the MCA. Blood flow was restored 1 hour later by removal of the monofilament to mimic the restoration of blood flow after lysis of a thromboembolic clot in humans. The extent of cerebral infarct may be evaluated first by a neurologic score and by the measurement of the infarct volume. Ischemic mice were thus analyzed for their neurologic score at different post-reperfusion times. To evaluate the infarct volume, staining with 2,3,5-triphenyltetrazolium chloride (TTC) was usually performed. Herein, we used cresyl violet staining since it offers the opportunity to test many critical markers by immunohistochemistry. In this video, we report the MCAO procedure; neurological scores and the evaluation of the infarct volume by cresyl violet staining.

The intraluminal middle cerebral occlusion model in mice is herein presented. The extent of cerebral infarct is evaluated by a neurologic score and cresyl violet staining, an alternative staining to TTC, offering the great advantage to test in parallel many interest markers.

Stroke is the third  cause of mortality and the leading cause of disability in the World. Ischemic  stroke accounts for approximately 80% of all strokes. ...

Ascending Aortic Constriction in Rats for Creation of Pressure Overload Cardiac Hypertrophy Model

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart failure. Here, we describe a detailed surgical procedure for creating pressure overload and cardiac hypertrophy in rats by constriction of the ascending aorta using a small metallic clip. After anesthesia, the trachea is intubated by inserting a cannula through a half way incision made between two cartilage rings of trachea. Then a skin incision is made at the level of the second intercostal space on the left chest wall and muscle layers are cleared to locate the ascending portion of aorta. The ascending aorta is constricted to 50&#8211;60% of its original diameter by application of a small sized titanium clip. Following aortic constriction, the second and third ribs are approximated with prolene sutures. The tracheal cannula is removed once spontaneous breathing was re-established. The animal is allowed to recover on the heating pad by gradually lowering anesthesia. The intensity of pressure overload created by constriction of the ascending aorta is determined by recording the pressure gradient using trans-thoracic two dimensional Doppler-echocardiography. Overall this protocol is useful to study the remodeling events and contractile properties of the heart during the gradual onset and progression from compensated cardiac hypertrophy to heart failure stage.

We describe a stepwise procedure for creating pressure overload and left ventricular hypertrophy in Wistar rats by constriction of the ascending aorta using a small metallic clip. This model is extensively used for studying remodeling changes during cardiac hypertrophy and for identifying and evaluating strategies for regression of such changes.

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts

Spinal muscular atrophy (SMA), a lethal neurological disease caused by the loss of SMN1, presents a unique case in the field of antisense oligonucleotide (AON)-mediated therapy. While SMN1 mutations are responsible for the disease, AONs targeting intronic splice silencer (ISS) sites in SMN2, including FDA-approved nusinersen, have been shown to restore SMN expression and ameliorate the symptoms. Currently, many studies involving AON therapy for SMA focus on investigating novel AON chemistries targeting SMN2 that may be more effective and less toxic than nusinersen. Here, we describe a protocol for in vitro evaluation of exon inclusion using lipotransfection of AONs followed by reverse transcription polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), and Western blotting. This method can be employed for various types of AON chemistries. Using this method, we demonstrate that AONs composed of alternating locked nucleic acids (LNAs) and DNA nucleotides (LNA/DNA mixmers) lead to efficient SMN2 exon inclusion and restoration of SMN protein at a very low concentration, and therefore, LNA/DNA mixmer-based antisense oligonucleotides may be an attractive therapeutic strategy to treat splicing defects caused by genetic diseases. The in vitro evaluation method described here is fast, easy, and sensitive enough for the testing of various novel AONs.

Various antisense oligonucleotides (AONs) have been shown to induce exon inclusion (splice modulation) and rescue SMN expression for spinal muscular atrophy (SMA). Here, we describe a protocol for AON lipotransfection to induce exon inclusion in the SMN2 gene and the evaluation methods to determine the efficacy in SMA patient fibroblasts.

Spinal muscular atrophy (SMA), a lethal neurological disease caused by the loss of SMN1, presents a unique case in the field of antisense oligonucleotide ...

A Pulmonary Trunk Banding Model of Pressure Overload Induced Right Ventricular Hypertrophy and Failure

Right ventricular (RV) failure induced by sustained pressure overload is a major contributor to morbidity and mortality in several cardiopulmonary disorders. Reliable and reproducible animal models of RV failure are therefore warranted in order to investigate disease mechanisms and effects of potential therapeutic strategies. Banding of the pulmonary trunk is a common method to induce isolated RV hypertrophy but in general, previously described models have not succeeded in creating a stable model of RV hypertrophy and failure.
We present a rat model of pressure overload induced RV hypertrophy caused by pulmonary trunk banding (PTB) that enables different phenotypes of RV hypertrophy with and without RV failure. We use a modified ligating clip applier to compress a titanium clip around the pulmonary trunk to a pre-set inner diameter. We use different clip diameters to induce different stages of disease progression from mild RV hypertrophy to decompensated RV failure.
RV hypertrophy develops consistently in rats subjected to the PTB procedure and depending on the diameter of the applied banding clip, we can accurately reproduce different disease severities ranging from compensated hypertrophy to severe decompensated RV failure with extra-cardiac manifestations.
The presented PTB model is a valid and robust model of pressure overload induced RV hypertrophy and failure that has several advantages to other banding models including high reproducibility and the possibility of inducing severe and decompensated RV failure.

We present a surgical method to induce right ventricular hypertrophy and failure in rats.

Right ventricular (RV) failure induced by sustained pressure overload is a major contributor to morbidity and mortality in several cardiopulmonary ...

Continuous Venous-Arterial Doppler Ultrasound During a Preload Challenge

A preload challenge (PC) is a clinical maneuver that, first, increases the cardiac filling (i.e., preload) and, second, calculates the change in cardiac output. Fundamentally, a PC is a bedside approach for testing the Frank-Starling-Sarnoff (i.e., "cardiac function") curve. Normally, this curve has a steep slope such that a small change in the cardiac preload generates a large change in the stroke volume (SV) or cardiac output. However, in various disease states, the slope of this relationship flattens such that increasing the volume into the heart leads to little rise in the SV. In this pathological scenario, additional cardiac preload (e.g., intravenous fluid) is unlikely to be physiologically effective and could lead to harm if organ congestion evolves. Therefore, inferring both the cardiac preload and output is clinically useful as it may guide intravenous (IV) fluid resuscitation. Accordingly, the goal of this protocol is to describe a method for contemporaneously tracking the surrogates of cardiac preload and output using a novel, wireless, wearable ultrasound during a well-validated preload challenge.

The Frank-Starling-Sarnoff curve is clinically important and describes the relationship between cardiac preload and output. This report illustrates a novel method of simultaneous jugular venous and carotid arterial Doppler velocimetry as transient surrogates of cardiac preload and output, respectively; this approach is enabled by wireless, wearable Doppler ultrasound.

A preload challenge (PC) is a clinical maneuver that, first, increases the cardiac filling (i.e., preload) and, second, calculates the change in cardiac ...

Predicting Prognosis in Acute Heart Failure Using Phase Angle & BIVA Z-Scores

Clinical Application of Phase Angle and BIVA Z-Score Analyses in Patients Admitted to an Emergency Department with Acute Heart Failure

Acute heart failure is characterized by neurohormonal activation, which leads to sodium and water retention and causes alterations in body composition, such as increased body fluid congestion or systemic congestion. This condition is one of the most common reasons for hospital admission and has been associated with poor outcomes. The phase angle indirectly measures intracellular status, cellular integrity, vitality, and the distribution of spaces between intracellular and extracellular body water. This parameter has been found to be a predictor of health status and an indicator of survival and other clinical outcomes. In addition, phase angle values of &lt;4.8&#176; upon admission were associated with higher mortality in patients with acute heart failure. However, low phase angle values may be due to alterations-such as the shifting of fluids from an intracellular body water (ICW) compartment to an ECW (extracellular body water) compartment and a concurrent decrease in body-cell mass (which can reflect malnutrition)-that are present in heart failure. Thus, a low phase angle may be due to overhydration and/or malnutrition. BIVA provides additional information about the body-cell mass and congestion status with a graphical vector (R-Xc graph). In addition, a BIVA Z-score analysis (the number of standard deviations from the mean value of the reference group) that has the same pattern as that of the ellipses for the percentiles on the original R-Xc graph can be used to detect changes in soft-tissue mass or tissue hydration and can help researchers compare changes in different study populations. This protocol explains how to obtain and interpret phase angle values and BIVA Z-score analyses, their clinical applicability, and their usefulness as a predictive marker for the prognosis of a 90-day event in patients admitted to an emergency department with acute heart failure.

Author Spotlight: Unveiling Prognostic Indicators in Heart Failure - The Role of Phase Angle and Bioelectrical Impedance Analysis 

In this protocol, we explain how to obtain and interpret phase angle values and bioelectrical impedance vectorial analysis (BIVA) Z-score obtained by bioelectrical impedance in patients with acute heart failure admitted to the Emergency Department and their clinical applicability as a predictive marker for the prognosis of a 90-day event.

Acute heart failure is characterized by neurohormonal activation, which leads to sodium and water retention and causes alterations in body composition, ...

Developing an Exercise Program for Rheumatoid Arthritis Patients

Evaluation of Changes in Hydration and Body Cell Mass with Bioelectrical Impedance Analysis after Exercise Program for Rheumatoid Arthritis Patients

Rheumatoid arthritis (RA) is a debilitating disease that can result in complications such as rheumatoid cachexia. While physical exercise has shown benefits for RA patients, its impact on hydration and body cell mass remains uncertain. The presence of pain, inflammation, and joint changes often restrict activity and make traditional body composition assessments unreliable due to altered hydration levels. Bioelectrical impedance is a commonly used method for estimating body composition, but it has limitations since it was primarily developed for the general population and does not consider changes in body composition. On the other hand, bioelectrical impedance vectorial analysis (BIVA) offers a more comprehensive approach. BIVA involves graphically interpreting resistance (R) and reactance (Xc), adjusted for height, to provide valuable information about hydration status and the integrity of the cell mass.
Twelve women with RA were included in this study. At the beginning of the study, hydration and body cell mass measurements were obtained using the BIVA method. Subsequently, the patients participated in a six-month dynamic exercise program encompassing cardiovascular capacity, strength, and coordination training. To evaluate changes in hydration and body cell mass, the differences in the R and Xc parameters, adjusted for height, were compared using BIVA confidence software. The results showed notable changes: resistance decreased after the exercise program, while reactance increased. BIVA, as a classification method, can effectively categorize patients into dehydration, overhydration, normal, athlete, thin, cachectic, and obese categories. This makes it a valuable tool for assessing RA patients, as it provides information independent of body weight or prediction equations. Overall, the implementation of BIVA in this study shed light on the effects of the exercise program on hydration and body cell mass in RA patients. Its advantages lie in its ability to provide comprehensive information and overcome the limitations of traditional body composition assessment methods.

Author Spotlight: Implementation of BIVA for Analyzing Disease Risk Factors in Patients with Low Body Cell Mass 

This protocol assesses alterations in hydration and body cell mass status using bioelectrical impedance vectorial analysis following a dynamic exercise program designed for patients with rheumatoid arthritis. The dynamic exercise program itself is detailed, highlighting its components focused on cardiovascular capacity, strength, and coordination. The protocol details steps, instruments, and limitations.

Rheumatoid arthritis (RA) is a debilitating disease that can result in complications such as rheumatoid cachexia. While physical exercise has shown ...

Evaluation of Hydration Status by Bioelectrical Impedance Vector Analysis in Patients with Ischemic Heart Disease Undergoing Exercise Stress Test

Ischemic heart disease (IHD) represents a group of clinical syndromes characterized by myocardial ischemia, leading to an impairment in the myocardial blood supply and compromised perfusion. Several clinical variables assessed through a stress test, such as oxygen uptake (VO2) and heart rate oxygen pulse (HR/O2), have been attributed as cardiopulmonary prognostic factors in patients with IHD. However, other factors like hydration status (HS), potentially affecting the cardiopulmonary response, have been barely addressed. Unbalanced HS has a short-term effect on plasma volume and the sympathetic nervous system, which impacts blood volume, and lowers VO2 and HR/O2. Recently, bioelectrical impedance analysis (BIA), a method based on the opposition of body tissues (including fluid volume) to a low electrical current, has been widely used to assess HS by obtaining two components: resistance (R) and reactance (Xc) and using prediction formulas. However, several limitations as chronic illness or abnormal fluid status, may affect the results. In this sense, alternative BIA methods, such as bioelectrical impedance vector analysis (BIVA), have become relevant. R and Xc (adjusted by height) result in a vector plotted on the R/Xc graph, which allows interpreting the HS as normal or abnormal according to the distance of the mean vector. This study aims to describe how to determine HS by BIVA using a single-frequency device and compare the results with the cardiopulmonary response in patients with IHD.

Hydration status imbalances can have a short-term effect on direct and indirect determinants of oxygen uptake and pulse, and morbidity and mortality prognostic factors in ischemic heart disease. This protocol describes the technique for assessment of hydration status through bioelectrical impedance vector analysis and cardiopulmonary response during exercise stress test.

Ischemic heart disease (IHD) represents a group of clinical syndromes characterized by myocardial ischemia, leading to an impairment in the myocardial ...