Name: standardized measurement of nasal membrane transepithelial potential difference (npd)
Uploaded: 2018-09-13T12:30:00
Duration: 9 min 47 s
Description: Watch this Scientific Journal Video about Standardized Measurement of Nasal Membrane Transepithelial Potential Difference NPD at JoVE.com

This research was supported by the Working Group for CFTR Function of the Standardization Committee (Clinical Trials Network, European Cystic Fibrosis Society) and The National Resources Center Working Group (Therapeutics Development Network, Cystic Fibrosis Foundation). Additional support was provided by the CF Foundation (Clancy FY09 to GMS) and NIH (DK072482 to SMR and GMS).

The protocol involving human subjects was approved by all participating Institutes&#39; Research Committee. Each center implementing the technique is responsible for the application to its institutional human research ethics committee for approval.
1. Solution Preparation
<ol>
	<li>Prepare Solutions #1, #2 and #3, which are base solutions, in 1 L batches prior to the procedure and stored on-site (Table 1). 
	NOTE: Amiloride is light sensitive and must be stored in the dark (see Table 1 for solution composition) (see SOP for detailed solution preparation11).
	<ol>
		<li>Buffer all solutions at pH 7.4 and filter with a 0.22 &#181;m bottle-top filter.</li>
		<li>For solution #3, add the phosphate-containing salts first, allow them to ionize to prevent crystallization (See Table 2 for solution composition). 
		NOTE: The sequence of mixing is critical for solution #3.</li>
		<li>Store these solutions at 4 &#176;C (stable for 3 months) or at -20 &#176;C (stable for 6 months).</li>
		<li>Prepare solutions #4 and #5 by adding agents on the day of the NPD test. Isoproterenol is light and oxidation sensitive and it loses its activity at room temperature (demonstrating 4% decay over 4 h at 4&#8211;8 &#176;C). Store at 4 &#176;C. 
		NOTE: ATP is light and oxidation sensitive (see Table 3).</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Compound</td>
			<td>Molecular Weight</td>
			<td>Concentration (mM)</td>
			<td>Composition (g/L)</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>58</td>
			<td>148</td>
			<td>8.58</td>
		</tr>
		<tr>
			<td>CaCl2 2H2O</td>
			<td>147</td>
			<td>2.25</td>
			<td>0.33</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>75</td>
			<td>4.05</td>
			<td>0.3</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>174</td>
			<td>2.4</td>
			<td>0.42</td>
		</tr>
		<tr>
			<td>KH2 PO4</td>
			<td>136</td>
			<td>0.4</td>
			<td>0.05</td>
		</tr>
		<tr>
			<td>MgCl2 6H2O</td>
			<td>203</td>
			<td>1.2</td>
			<td>0.24</td>
		</tr>
	</tbody>
</table>
Table 1: Solution Composition.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Compound</td>
			<td>Molecular Weight</td>
			<td>Concentration (mM)</td>
			<td>Composition (g/L)&#160;</td>
		</tr>
		<tr>
			<td>Na Gluconate</td>
			<td>218</td>
			<td>148</td>
			<td>33.26</td>
		</tr>
		<tr>
			<td>Ca Gluconate</td>
			<td>430</td>
			<td>2.25</td>
			<td>0.97</td>
		</tr>
		<tr>
			<td>K Gluconate</td>
			<td>234</td>
			<td>4.05</td>
			<td>0.95</td>
		</tr>
		<tr>
			<td>K2 HPO4</td>
			<td>174</td>
			<td>2.4</td>
			<td>0.42</td>
		</tr>
		<tr>
			<td>KH2 PO4</td>
			<td>136</td>
			<td>0.4</td>
			<td>0.05</td>
		</tr>
		<tr>
			<td>MgSO4 7H2O</td>
			<td>246</td>
			<td>1.2</td>
			<td>0.24</td>
		</tr>
	</tbody>
</table>
Table 2: Solution Composition.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution</td>
			<td>Solution Number</td>
			<td>Contents</td>
			<td>EDC Mark</td>
		</tr>
		<tr>
			<td>Ringers injection</td>
			<td>Solution #1/A</td>
			<td>Buffered ringers for injection</td>
			<td>RINGERS</td>
		</tr>
		<tr>
			<td>Ringers + amiloride</td>
			<td>Solution #2/B</td>
			<td>Buffered ringers + 100 &#956;M amiloride</td>
			<td>AMIL</td>
		</tr>
		<tr>
			<td>Zero Cl- + amiloride</td>
			<td>Solution #3/C</td>
			<td>Buffered zero Cl- + 100 &#956;M amiloride</td>
			<td>OCL</td>
		</tr>
		<tr>
			<td>Zero Cl- + amiloride + isoproterenol</td>
			<td>Solution #4/D</td>
			<td>Buffered zero Cl- + 100 &#956;M amiloride + 10 &#956;M isoproterenol</td>
			<td>ISO</td>
		</tr>
		<tr>
			<td>Zero Cl-+ amiloride + isoproterenol + ATP</td>
			<td>Solution #5/E</td>
			<td>Buffered zero Cl- + 100 &#956;M amiloride + 10 &#956;M isoproterenol + 100 &#956;M ATP</td>
			<td>ATP</td>
		</tr>
	</tbody>
</table>
Table 3: Solution List.
2. Catheter
<ol>
	<li>Use a PVC, sterile, single use, 2 lumens (0.7 mm inner &#216;) catheter with a round and smooth extremity (2.5 mm outer &#216;), which is specifically designed for NPD.
	<ol>
		<li>Make contacts with the mucosa by a side hole, 2 mm distant to the tip with a hole at the tip for perfusion (see step 10.1).</li>
		<li>Connect one of the two Luer-lock connections of the catheter to the measuring electrode and the other one to the perfusion pump. Use the channel stained in blue as the measuring lumen. Tag the catheter at each 0.5 cm interval for 10 cm. 
		NOTE: The dead space is 0.3 mL. It is preferable to use the above procedure for the preparation of the catheter if this is not possible follow the next two steps.</li>
		<li>Cut equal (~76 cm) lengths of PE50 and PE90 catheter tubing.</li>
		<li>Affix these together in a 1 cm piece of silicon rubber tubing. Insert a 25 G blunt tip needle snuggly into the opposite end of the PE-90 tubing. Insert a 25 G butterfly needle snuggly into the opposite end of the PE50 tubing making sure not to puncture the tubing as it is placed.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57006/57006fig4.jpg" /> 
Figure 4: Catheter used for NPD measurements. Inset box demonstrates the catheter tip with measuring hole.
3. Preparation of Agar Skin Bridge (Butterfly Needle) and Catheter
NOTE: The manipulation of the melted agar may cause burns, and this should be done with caution.
<ol>
	<li>Prepare 3% agar by mixing 3 g of agar with 100 mL of solution #1 in a wide-mouth bottle. Melt the agar in the microwave until soluble (transparent).
	<ol>
		<li>Fill the 10 mL syringe with warm agar.</li>
		<li>Connect the syringe subsequently to the butterfly needle (23 G) and to the marked lumen of the catheter. Inject the agar until it appears at the tip.</li>
		<li>Allow it to cool for at least 10 min.</li>
		<li>Ensure that the skin bridge and the catheter are fully filled and visualized to be free of air bubbles.</li>
		<li>Store the bulk skin bridges in solution #1 at 4 &#176;C and do not use after 1 week.</li>
	</ol>
	</li>
</ol>
4. If Using ECG cream
<ol>
	<li>Dilute the ECG cream with solution #1 (1:1, v/v Ringer). Let it rest until free of air bubbles.
	<ol>
		<li>Fill the 10 mL syringe with diluted ECG cream.</li>
		<li>Connect the syringe to the marked lumen of the catheter and slowly inject the ECG cream until it appears at the lower side hole.</li>
		<li>Ensure that the catheter is fully filled and free of air bubbles.</li>
	</ol>
	</li>
</ol>
5. Data Acquisition System
NOTE: The general setup of the data acquisition system is shown in Figure 5.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57006/57006fig5.jpg" /> 
Figure 5: Set up of the data acquisition system. Demonstrating connections of the bioamplifier and headstage to the computer interface as well as the electrode connections to the headstage11. <a href="https://www.jove.com/files/ftp_upload/57006/57006fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Connections
	<ol>
		<li>Connect the computer to the data acquisition system (Table of Materials) with a USB cable.</li>
		<li>To connect the data acquisition system to the bioamplifier, connect the BNC cable from the channel 1 input on the data acquisition system (front) to the output on the bioamplifier (back).</li>
		<li>Connect the bioamplifier to the headstage with a custom cable pre-connected to the headstage screws into the input portion in the front of the bioamplifier.</li>
		<li>Connect the headstage to the electrodes and the ground electrode. Use standard 2 mm female-female connectors to connect the front of headstage to the electrodes. 
		NOTE: The port is recessed and only fits one direction of the 2 mm cable: Red-measuring electrode to the patient nose (via nasal catheter); Black-Reference electrode to patient skin bridge; White to ECG electrode grounded to subject&#39;s skin.</li>
		<li>Set the bioamplifier as described: Offset: pull to activate, turn to adjust, leave in pulled position after adjustment; Voltage: DC; 1 mV cal: neutral position; Power: On to collect data, off to charge battery; Gain: set to 10; Band pass: LoFreq (Outer knob): DC; Band pass: HighFreq (Inner knob): 1kHz; Volume: Off.</li>
	</ol>
	</li>
</ol>
6. Adjusting the Head-stage Offset
<ol>
	<li>Connect the laptop and the amplifier in the sequence as indicated in the SOP11. Switch on the data acquisition system and then the laptop (the sequence is important for the software to recognize the apparatus used for data acquisition).
	<ol>
		<li>Adjust the Head-Stage Offset as per the SOP sequence.</li>
	</ol>
	</li>
</ol>
7. Offsets
NOTE: There are several offsets to be tested to ensure the stability of the electrical measuring system. (see Figure 6)
<ol>
	<li>For the Electrode Offset, place the reference (negative) electrode and measuring (positive) electrode together in the diluted ECG cream or the 3 M KCl. Ensure that the potential difference reading between the electrodes is near zero on the headstage.
	<ol>
		<li>For setting the catheter and/or skin bridge offset, place the Luer-lock end of the nasal catheter or the Luer-lock end of the skin bridge into the bath with the measuring (positive) electrode. Place the other end of the catheter in the ECG cream bath or 3 M KCL containing the reference (negative) electrode ensuring that the potential difference is near zero on the headstage.</li>
		<li>To set up a closed loop offset, ensure that the circuit is closed when replacing the nasal catheter in the bath of the electrodes. Check that the closed loop offset reads near 0 mV (=&#39;offset&#39;; &#177;2.5 mV). Adjust the headstage offset knob to bring the offset to 0 mV. 
		NOTE: This confirms that all connections within the circuit are intact. If this is not the case, the nasal catheter may not be intact (air bubbles in the agar or the ECG cream). Change the agar bridge or push the ECG cream in the setup. The electrode offset must be performed first, followed by the closed system (closed loop offset) with the electrode and the bridge (Figure 6).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57006/57006fig6.jpg" /> 
Figure 6: Setup of (A) Electrode offset, (B) Catheter (or bridge) offset, (C) Closed loop offset.
8. Syringe Set-up
NOTE: The following is the recommended set-up.
<ol>
	<li>Thaw solutions #1, #2 and #3 approximately 1 h before the measurement.
	<ol>
		<li>Connect the extension line to the stopcock closest to the catheter.</li>
		<li>Switch on all pumps and flush the catheter with solution #1 to completely flush off the catheter until stopcocks are clear of bubbles.</li>
	</ol>
	</li>
</ol>
9. Placement of the Reference and Measurement Electrode
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57006/57006fig7.jpg" /> 
Figure 7: Subject with measuring electrode and subcutaneous bridge ready for measurements.
<ol>
	<li>Have the study subject take a seated position facing the NPD operator. For more comfort, place feet on the optional anti-static mat and head on the orthoptic chin rest.
	<ol>
		<li>Connect the ground lead electrode to the ECG pad placed on the subject&#39;s arm (Figure 7).</li>
		<li>Either insert the subcutaneous needle into the dorsal forearm (agar system) or apply the reference electrode to the ECG cream on a previously minimally abraded area on the forearm (see points 7 through to 10 below).</li>
		<li>Check the connection to the subcutaneous space by measuring the potential difference with the skin (Finger PD) and asking the subject to close the &#34;measuring hole&#34; by pinching the catheter between the tip of his/her thumb and index finger.</li>
		<li>If the Finger PD is not -30 mV or more negative, check the insertion of the butterfly needle. Repeat the abrasion (for ECG cream set-up) and check bridges.</li>
		<li>Start the solution #1 syringe pump at 80 mL/h. Start with the right nostril.</li>
		<li>Measure the Finger PD as a steady negative voltage (typical range -40 to -80 mV).</li>
		<li>If using the ECG Cream System: dilute the ECG cream 1:1 and fill the catheter after a complete flush off out of the probe hole as previously seen for the Agar.</li>
		<li>Connect the catheter to a 50 mL syringe half-filled with the ECG cream to bathe the electrodes, allowing a check of the electrode offset and the bridge off set.</li>
		<li>Connect the reference Ag/Cl electrode to the subcutaneous space by slight previous minimal abrasion of the skin, the skin will appear &#8216;pink and shiny&#8217; when the level of the dermis is reached.</li>
		<li>Position the measuring electrode, covered with ECG cream, on the abraded skin. Check the finger PD as previously shown for the Agar system.</li>
	</ol>
	</li>
</ol>
10. Measurement of Basal PD
<ol>
	<li>Insert the nasal catheter into the right nostril using an illuminating rhinoscope (or equivalent) to visualize the inferior turbinate. Using the anterior tip as a landmark, advance the catheter targeting the inferior site of the inferior turbinate on the respiratory mucosa. Alternatively, if the placement is difficult, the probe hole can be placed in contact with the floor of the nostril. 
	NOTE: The catheter is rigid enough to be guided into the nostril by the operator. To facilitate the placement, one channel of the catheter is colored in blue and contains the probe-side hole in contact with the inferior turbinate. This prevents catheter rotation. The marks indicated on the catheter from 1 to 10 cm provide easy reference points.
	<ol>
		<li>Measure the PD at the inferior turbinate. For this purpose, ensure that the measuring hole of the catheter is closed by its placement against the mucosa of the inferior turbinate (mark Right Basal AT).</li>
		<li>Measure the PD at 3.0, 2.0, 1.5, 1.0, and 0.5 cm (distance within the inferior meatus from the inferior turbinate): mark Right Basal PDs.</li>
		<li>Maintain each measurement at the specified distance for approximately 5 s each to ensure a steady reading (&#177; 1 mV) and to facilitate accurate interpretation of basal PD values.</li>
		<li>Repeat the steps above in the left nostril, using the function key to mark the Left Basal PDs (3 cm, 2 cm, etc.) and the Left Basal AT.</li>
		<li>Using basal PD measures as a guide, insert the probe of the nasal catheter to the site of most negative signal (up to 3 cm from the anterior tip of the inferior nasal turbinate), and secure with a small piece of tape on the tip of the nose (or equivalent).</li>
	</ol>
	</li>
</ol>
11. NPD Tracing Sequential Perfusions
<ol>
	<li>For the Right Nostril
	<ol>
		<li>Verify that the solution is dripping from the patient&#39;s nose. Have the subject assume a comfortable position with their head down (often helped by having the subject rest their head on their hand or use a chinrest or other immobilizing device). Remind the subject to minimize the movement and avoid touching nose or the tubing, and to avoid talking.</li>
		<li>Turn solution #1 (Ringers) pump on (5 mL/min, or 300 mL/h). Record until a stable value is obtained (&#60;1 mV change/30 s). 
		NOTE: This takes approximately 3 min to achieve stability.</li>
		<li>Turn off the perfusion with solution #1.</li>
		<li>Start the perfusion with solution #2 (Amiloride). Record NPD for a minimum of 3 min (if the plateau voltage is in doubt, continue the recording for up to 5 min total).</li>
		<li>Start the perfusion with solution #3 (Zero Chloride). Record NPD for a minimum of 3 min (if the plateau voltage is not stable, continue the recording for up to 5 min total).</li>
		<li>Start the perfusion with solution #4 (Isoproterenol). Record NPD for a minimum of 3 min [if the plateau voltage is not stable (a steady voltage tracing for at least 30 s of &#60; 1 mV drift), continue the recording for up to 5 min total].</li>
		<li>Start the perfusion with solution #5 (ATP). Record NPD for a minimum of 1 min, until a peak hyperpolarizing response is obtained.</li>
		<li>Turn on the perfusion with the solution #1 (Ringers) and allow 30 s to flush the catheter.</li>
		<li>Turn off the solution #1 perfusion.</li>
		<li>Repeat the procedure for the left nostril.</li>
	</ol>
	</li>
</ol>
12. End of the Test
<ol>
	<li>Recheck and record stable Finger PD (&#34;Post Finger&#34;) for 5 s.
	<ol>
		<li>Remove the subject&#39;s cutaneous bridge and the bandage from the insertion site on the skin. For the AgCl/ECG Cream System, remove the electrode from the arm.</li>
		<li>Record the &#34;Final Closed Loop Offset&#34; voltage as described for measuring the initial closed loop offset (see step 7.1.3).</li>
		<li>Mark final offset with the Function key.</li>
		<li>Stop data acquisition (Press &#34;Start&#34;). 
		NOTE: The current SOP recommends the use of 100 &#181;M ATP to activate the purinergic calcium dependent Cl- secretion, to serve as a positive control for the test; however, this is an optional test.</li>
	</ol>
	</li>
</ol>

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M., 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=Diagnosis+of+Cystic+Fibrosis:+Consensus+Guidelines+from+the+Cystic+Fibrosis+Foundation.">Diagnosis of Cystic Fibrosis: Consensus Guidelines from the Cystic Fibrosis Foundation.</a> The Journal of Pediatrics. 181, S4-S15 (2017).</li><li>Mesbahi, M., 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=Changes+of+CFTR+functional+measurements+and+clinical+improvements+in+cystic+fibrosis+patients+with+non-p.Gly551Asp+gating+mutations+treated+with+ivacaftor.">Changes of CFTR functional measurements and clinical improvements in cystic fibrosis patients with non-p.Gly551Asp gating mutations treated with ivacaftor.</a> Journal of Cystic Fibrosis. 16, 45-48 (2017).</li><li>Accurso, 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=Sweat+chloride+as+a+biomarker+of+CFTR+activity:+proof+of+concept+and+ivacaftor+clinical+trial+data.">Sweat chloride as a biomarker of CFTR activity: proof of concept and ivacaftor clinical trial data.</a> Journal of Cystic Fibrosis. 13 (2), 139-147 (2014).</li><li>Accurso, 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=Effect+of+VX-770+in+Persons+with+Cystic+Fibrosis+and+the+G551D-CFTR+Mutation.">Effect of VX-770 in Persons with Cystic Fibrosis and the G551D-CFTR Mutation.</a> New England Journal of Medicine. 363, 1991-2003 (2010).</li><li>Sermet, I., 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=Measurement+of+nasal+potential+difference+in+young+children+with+an+equivocal+sweat+test+following+newborn+screening+for+cystic+fibrosis.">Measurement of nasal potential difference in young children with an equivocal sweat test following newborn screening for cystic fibrosis.</a> Thorax. 65 (6), 539-544 (2010).</li><li>Wilschanski, M., 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=Mutations+in+the+Cystic+Fibrosis+Transmembrane+Regulator+Gene+and+In+vivo+Transepithelial+Potentials.">Mutations in the Cystic Fibrosis Transmembrane Regulator Gene and In vivo Transepithelial Potentials.</a> American Journal of Respiratory and Critical Care Medicine. 174 (7), 787794 (2006).</li><li>Sermet-Gaudelus, I., 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=Clinical+Phenotype+and+Genotype+of+Children+with+Borderline+Sweat+Test+and+Abnormal+Nasal+Epithelial+Chloride+Transport.">Clinical Phenotype and Genotype of Children with Borderline Sweat Test and Abnormal Nasal Epithelial Chloride Transport.</a> American Journal of Respiratory and Critical Care Medicine. 182 (7), 929-936 (2010).</li><li>De Boeck, K., 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=CFTR+biomarkers:+time+for+promotion+to+surrogate+endpoint?.">CFTR biomarkers: time for promotion to surrogate endpoint?.</a> European Respiratory Journal. 14, 38 (2013).</li><li>US CFF-TDN (Cystic Fibrosis Foundation-Therapeutics Development Network) and the ECFS-CTN (European Cystic Fibrosis Society- Clinical Trials Network). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+Operating+Procedure+528.01.">Standard Operating Procedure 528.01.</a> Standardized Measurement of Nasal Membrane Transepithelial Potential Difference (NPD). , (2014).</li><li>Knowles, M., 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=Increased+bioelectric+potential+difference+across+respiratory+epithelia+in+CF.">Increased bioelectric potential difference across respiratory epithelia in CF.</a> New England Journal of Medicine. 305 (25), 1489-1493 (1981).</li><li>Solomon, G. M., 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=An+international+Randomised+Multicentre+Comparison+of+NPD+Techniques.">An international Randomised Multicentre Comparison of NPD Techniques.</a> Chest. 138, 919-928 (2010).</li><li>Sermet, I., 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=Chloride+Transport+in+Nasal+Ciliated+Cells+of+Cystic+Fibrosis+Heterozygotes.">Chloride Transport in Nasal Ciliated Cells of Cystic Fibrosis Heterozygotes.</a> American Journal of Respiratory and Critical Care Medicine. 171, 1026-1031 (2005).</li><li>Naehrlich, 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=Nasal+potential+difference+measurements+in+diagnosis+of+cystic+fibrosis:+an+international+survey.">Nasal potential difference measurements in diagnosis of cystic fibrosis: an international survey.</a> Journal of Cystic Fibrosis. 13 (1), 24-28 (2014).</li><li>Vermeulen, 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=Nasal+potential+measurements+on+the+nasal+floor+and+under+the+inferior+turbinate:+Does+it+matter?.">Nasal potential measurements on the nasal floor and under the inferior turbinate: Does it matter?.</a> Pediatric Pulmonology. 46 (2), 145-152 (2011).</li><li>Bronsveld, I., 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=Influence+of+perfusate+temperature+on+nasal+potential+difference.">Influence of perfusate temperature on nasal potential difference.</a> European Respiratory Journal. 42, 389-393 (2013).</li><li>Boyle, M., 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=A+multi-center+study+on+the+effect+of+solution+temperature+on+nasal+potential+difference+measurements.">A multi-center study on the effect of solution temperature on nasal potential difference measurements.</a> Chest. 124 (2), 482-489 (2003).</li><li>Solomon, G. M., 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=A+Multiple+Reader+Scoring+System+for+Nasal+Potential+Difference+Parameters.">A Multiple Reader Scoring System for Nasal Potential Difference Parameters.</a> Journal of Cystic Fibrosis. 16 (5), 573-578 (2017).</li><li>Beekman, J. M., 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=CFTR+functional+measurements+in+human+models+for+diagnosis,+prognosis+and+personalized+therapy:+Report+on+the+pre-conference+meeting+to+the+11th+ECFS+Basic+Science+Conference,+Malta,+26-29+March+2014.">CFTR functional measurements in human models for diagnosis, prognosis and personalized therapy: Report on the pre-conference meeting to the 11th ECFS Basic Science Conference, Malta, 26-29 March 2014.</a> Journal of Cystic Fibrosis. 13, 363-372 (2014).</li><li>Accurso, 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=Effect+of+VX-770+in+persons+with+cystic+fibrosis+and+the+G551D-CFTR+mutation.">Effect of VX-770 in persons with cystic fibrosis and the G551D-CFTR mutation.</a> New England Journal of Medicine. 363, 1991-2003 (2010).</li><li>Wilschanski, M., 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=Chronic+ataluren+(PTC124)+treatment+of+nonsense+mutation+cystic+fibrosis.">Chronic ataluren (PTC124) treatment of nonsense mutation cystic fibrosis.</a> European Respiratory Journal. 38, 59-69 (2011).</li><li>Accurso, 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=Sweat+Chloride+as+a+biomarker+of+CFTR+activity:+proof+of+concept+and+Ivacaftor+clinical+trial+data.">Sweat Chloride as a biomarker of CFTR activity: proof of concept and Ivacaftor clinical trial data.</a> Journal of Cystic Fibrosis. 13 (2), 139-147 (2014).</li><li>Rowe, S., 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=Clinical+Mechanism+of+the+Cystic+Fibrosis+Transmembrane+Conductance+Regulator+Potentiator+Ivacaftor+in+G551D-mediated+Cystic+Fibrosis.">Clinical Mechanism of the Cystic Fibrosis Transmembrane Conductance Regulator Potentiator Ivacaftor in G551D-mediated Cystic Fibrosis.</a> American Journal of Respiratory and Critical Care Medicine. 190 (2), 175-184 (2014).</li><li>Boyle, M., 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=A+CFTR+corrector+lumacaftor+and+a+CFTR+potentiator+(ivacaftor)+for+treatment+of+patients+with+cystic+fibrosis+who+have+a+phe508del+CFTR+mutation:+a+phase+2+randomised+controlled+trial.">A CFTR corrector lumacaftor and a CFTR potentiator (ivacaftor) for treatment of patients with cystic fibrosis who have a phe508del CFTR mutation: a phase 2 randomised controlled trial.</a> The Lancet Respiratory Medicine. 2 (7), 527-538 (2014).</li><li>Schulz, A., Tummler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-allergic+asthma+as+a+CFTR-related+disorder.">Non-allergic asthma as a CFTR-related disorder.</a> Journal of Cystic Fibrosis. 15 (5), 641-644 (2016).</li><li>Sloane, P. 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=A+pharmacologic+approach+to+acquired+cystic+fibrosis+transmembrane+conductance+regulator+dysfunction+in+smoking+related+lung+disease.">A pharmacologic approach to acquired cystic fibrosis transmembrane conductance regulator dysfunction in smoking related lung disease.</a> PLoS One. 7 (6), e39809 (2012).</li><li>Bienvenu, T., 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=Cystic+Fibrosis+Transmembrane+Conductance+Regulator+Channel+Dysfunction+in+Non-Cystic+Fibrosis+Bronchiectasis.">Cystic Fibrosis Transmembrane Conductance Regulator Channel Dysfunction in Non-Cystic Fibrosis Bronchiectasis.</a> American Journal of Respiratory and Critical Care Medicine. 181 (10), 1078-1083 (2010).</li><li>Werlin, S., 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=Genetic+and+electrophysiological+characteristics+of+recurrent+acute+pancreatitis.">Genetic and electrophysiological characteristics of recurrent acute pancreatitis.</a> Journal of Pediatric Gastroenterology and Nutrition. 60 (5), 675-679 (2015).</li><li>Dransfield, M. T., 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=Acquired+Cystic+Fibrosis+Transmembrane+Conductance+Regulator+Dysfunction+in+the+Lower+Airways+in+COPD.">Acquired Cystic Fibrosis Transmembrane Conductance Regulator Dysfunction in the Lower Airways in COPD.</a> Chest. 144 (2), 498-506 (2013).</li></ol>

The authors declare that they have no competing financial interests.

In vivo, NPD provides a unique measurement that can be performed repeatedly on a longitudinal basis and demonstrates that with repeated measurements, similar longitudinal results are observed on a group-wise and individual basis14,15. There is strong evidence that NPD has excellent discrimination validity for distinguishing CF from non-CF. 25 studies consistently demonstrated a statistically significant difference in Cl- and Na+ conductance between patients with CF and healthy controls10. While several previously developed indices demonstrate this capacity, we anticipate that new updates are necessary given recent standardizations of methodology7,8.
Modifications and Troubleshooting
This test requires several key steps to assure accurate measurements. This includes the electrodes and catheters closed loop offset to ensure that the system is performing to recommended standards. Patients must remain still and refrain from talking as this minimizes artifacts and catheter dislodgement. This makes the test difficult in non-cooperative patients and the technique has only been reported in one study in children below 6 years of age7.
Pre-inspection of the nasal epithelium is necessary to ensure that there are no crusts or mucus on the epithelium, which can affect measurements.
Very importantly, it must be pointed out that the location of the placement of the catheter is the subject of debate. The SOP presented here utilizes measurement under the inferior turbinate (IT). The placement of the catheter under the IT has been standardized and conducted in multicenter trials and, therefore, this is the recommended technique. Measurement under the IT is performed with the side-hole catheter, which may be difficult to maintain in firm contact with the nasal mucosa, whilst being in contact with the solutions. Other groups may measure the PD on the nasal floor, which is technically easier. Importantly, Vermeulen (2011) demonstrated that the 2 methods are comparable16.
The warming of the solutions remains a matter of debate between European and US-centers17,18. It has been advocated that using solutions at 37 &#176;C instead of 22 &#176;C increases the observed total chloride response by approximately 25% and the isoproterenol-dependent chloride response by approximately 95%18. However, warming increases variability, as assessed by a larger standard deviation of the total chloride response17. Therefore, as warming the solutions is an additional factor of variability, it is advised not to warm the solutions unless required on a study basis.
We have previously compared both of the electrode techniques and found that both the AgCl and Calomel electrode systems operated similarly in basal and stimulated currents in normal subjects13.
Limitations of the technique
This test is subject to significant within-subject variability. The variability of scoring is especially prevalent in patients with indeterminate tracings and this should be accounted for in diagnostic application19. Factors of variability include acute upper respiratory tract infection, extensive nasal polyps, prior sinus surgery and CF-related inflammation, which decrease&#160;its specificity and sensitivity20,10. Additionally, interpretation of tracings may be different between the readers, although expert readers demonstrate excellent agreement of quantitative scoring and interpretability in CF and non-CF tracings, contrasting with a significant variability in the confidence of the tracing19.
Intrinsic variability versus significant thresholds
Very importantly, the physiological variability of the measurement is considerable, as illustrated in different studies10, such as the CFTR gene therapy trials that demonstrated considerable variability in changes in chloride total transport and amiloride range21,22. Cross-sectional evaluation suggests that zero Cl- plus isoproterenol response above the threshold of -5 to -7 mV is the cut-off between CF and non-CF subjects10.
We nevertheless lack clear knowledge about the magnitude of change of this parameter representing an effective CFTR correction in phase-II trials with disease modifying therapies. To assess individual response, repeated tests monitoring the response to an intervention may be required to distinguish significant changes from intrinsic variability. Very importantly, future long-term studies with disease modifying drugs need to demonstrate that improvement in CFTR function correlates with improvement in clinically relevant outcomes or surrogate outcomes (such as improvement in FEV1) of CF disease. Indeed, a recent phase II Ivacaftor study demonstrated marked clinical benefit despite a small improvement in the chloride secretion23.
Such studies will help to establish if a cut-off value of improvement in trans-epithelial Cl-conductance might be a surrogate parameter for clinical benefit. This would be an important parameter for guiding the development of CFTR modifying therapies.
Significance with respect to existing methods: Sweat Test and Intestinal Current Measurements (ICM)
In patients with &#39;&#39;questionable&#39;&#39; cystic fibrosis, as assessed by an intermediate sweat Cl- concentration between 30 and 60 mM, NPD composite scores provided a highly sensitive tool to diagnose patients as &#39;&#39;CF-likely&#39;&#39; and &#39;&#39;CF-unlikely&#39;&#39;10. Intestinal current measurement (ICM), which provides an ex vivo measurement of the net Cl- fluxes across the rectal epithelium, also allows determination of residual CFTR function with a high sensitivity because CFTR is highly expressed in this epithelium.
Considering modification of CFTR function by CFTR modulators, the relation between these different CFTR biomarker changes is at present unclear. Although recent work based on Ivacaftor determined that NPD and sweat test are correlated4, it has not yet been established if a measurement in the respiratory tract is a better predictor of respiratory outcome than, for example, the sweat test24,25 or change in ICM. Furthermore, modifier drugs may also differ in their organ specific efficacies. In regard to NPD, it is important to note that changes in basal PD and amiloride response express Na+ transport, whilst changes in 0 Cl- and isoproterenol response express Cl- transport. It is yet to be established which of these is more important for disease amelioration.
Future Application of this Technique
The use of this technique is expected outside of the CF field. Since this technique is uniquely suited to demonstrate Na+ and Cl- ion channel, it can be applied to demonstrate dysfunction in airways diseases including asthma26, chronic bronchitis27, non-CF bronchiectasis28&#160;and recurrent pancreatitis29. In addition, modifications of this technique have been used in the lower airways (LAPD) to demonstrate lower airways-focused CFTR dysfunction in chronic obstructive pulmonary disease (COPD) patients with chronic bronchitis30.
NPD provides a sensitive in vivo biomarker of CFTR function, which can be used for both the diagnosis and, also, for proof-of-concept studies aiming to correct CFTR and ENaC channel activity in translational research. This allows longitudinal assessment of trans-epithelial function and holds promise as a strategy for personalized medicine to tailor the most efficient corrector for each patient with CF.

The overall goal of this method is to measure the nasal potential difference (NPD) which aims to investigate trans-epithelial ion transport in vivo1. This technique allows the measurement of sodium (Na+) and chloride (Cl-) transport. NPD has been used as a research tool since the late 1980s and was accepted in 1998 as a diagnostic procedure by the Cystic Fibrosis Foundation (CFF) consensus statement2 and in 2017 in the Cystic Fibrosis Foundation (CFF) Consensus Diagnostic Guidelines3. Indeed, biological CFTR dysfunction, which is the cause of CF, is evidenced by an increased Na+ absorption at the apical membrane and a defect in Cl- secretion. This functional test provides the advantage of an additional diagnosis tool when genetics is non-conclusive in patients with indeterminate intermediate sweat test results3. Although this information may also be obtained by intestinal current measurement biopsies (ICM), ICM is, however, only available in a few centers globally and needs further standardization. NPD is more available in approximately 60 global centers and, moreover, targets the respiratory epithelium that is the main location of the disease.
Given the information it provides on CFTR activity, it is also used in proof-of-concept studies aiming to assess functional restoration of CFTR protein by modulator therapies4,5,6,7,8. Indeed, data from studies with CFTR mRNA/gene editing, CFTR potentiator, and corrector therapies, highlight significant changes in Cl- and Na+ transport with therapy6,9 and confirms that NPD can be a responsive endpoint in clinical trials. As we lack sensitive clinical endpoints able to detect a subtle change in clinical status of the patient in the short term, this preclinical biomarker might be highly informative. The field of CFTR modulator therapies broadens quickly and we urgently need tests in vivo that are able to quickly decipher active compounds before going to large phase 3 trials10.
The physiological rationale of the technique is based on the measurement of the potential difference between the airway epithelium in the nostril and the subcutaneous compartment. Ion channel activities are explored by measuring the stable maximal baseline potential difference (PD), its changes after blocking the ENaC related Na+ absorption and driving Cl- secretion via different apical Cl- transporters including CFTR. CFTR dysfunction is shown by a minimal change in potential difference upon stimulation of Cl- secretion through a cAMP dependent pathway and an increased ENaC mediated Na+ absorption as detected by a more negative baseline potential difference and an enhanced response to amiloride. The mechanistic basis for CF versus normal PD is summarized in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57006/57006fig1.jpg" /> 
Figure 1: Summary Figure of Ion Channel Activity. (A) Ion activity in the respiratory epithelium demonstrating balanced activity of ENaC and CFTR in normal subjects and (B) loss of CFTR activity resulting in increased ENaC mediated sodium transport and reduced CFTR dependent chloride transport. ENaC: epithelial sodium channel, Na+: sodium, CFTR: cystic fibrosis transmembrane regulator, CL-: chloride, mV: millivolts, PD: potential difference, min: minute/s <a href="https://www.jove.com/files/ftp_upload/57006/57006fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
However, this test demonstrates some degree of variability both at repeated measurements within the same patient and among patients with the same genotype. This is of utmost importance to facilitate the interpretation of the changes after modulator treatment. Moreover, we still lack validated thresholds discriminating between CF and healthy subjects. This may be partially due to differences between the availability of clinical facilities and the techniques employed. Therefore, a considerable international effort aimed at standardization of the test is ongoing. Both the US CFF-TDN (Cystic Fibrosis Foundation-Therapeutics Development Network) and the ECFS-CTN (European Cystic Fibrosis Society-Clinical Trials Network) created a&#160;NPD Standard Operating Procedure (SOP) for the use in the multicenter and research trials. This recent collaborative work by the CTN and TDN has resulted in a combined, international SOP, bringing together the expertise of the CTN and TDN (2014)11. This paper presents the protocol and test techniques to employ NPD for CF diagnosis or for investigator-initiated proof-of-concept trials. Each center implementing the technique is responsible for the application to its institutional human research ethics committee for approval.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57006/57006fig2.jpg" /> 
Figure 2: Schematic of entire recommended NPD setup. Note that the recommended setup is shown, including sequential perfusion pumps, and the 4-stop-cock series setup. Specific connections and examples of components are shown in the SOP. (Diagram modified with permission from Solomon, G.M. Chest, 201013) <a href="https://www.jove.com/files/ftp_upload/57006/57006fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The general experimental flow is outlined in Figure 2, whereby NPD is measured between the exploring bridge positioned on the epithelium surface and reference bridge placed in the subcutaneous space, both connected to electrodes and a high-impedance voltmeter.
This is ensured by 2 different systems: there are 2 acceptable reference electrode setups: (i) balanced Ag/AgCl electrodes and an electrocardiogram (ECG) cream-filled bridge connected to the subcutaneous space by slight abrasion or (ii) saturated calomel half-cells and an agar filled 22- to 24-gauge needle introduced subcutaneously. The contact to the nasal mucosa is enabled by a double lumen catheter. One lumen is filled with agar or ECG cream and connected to the measuring electrode, the other allows perfusion onto the nasal mucosa of the different solutions.
The tip of the exploring tubing is placed onto the respiratory mucosa under the inferior nasal turbinate (Figure 3).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57006/57006fig3.jpg" /> 
Figure 3: Placement of exploring tubing onto respiratory mucosa. (A) External view showing placement. (B) Rhinoscopic view demonstrating placement. (C) Diagram indicating the anatomic location for catheter placement. PD: potential difference
To study the response of PD to several drugs, the superfusion solutions are applied via the second lumen of the catheter. There are several key steps regarding preparation and conduct of NPD measurements, which are detailed below in the protocol, from initial preparation through to data analysis.
After preparation of solutions and electrodes, adequate quality testing of the electrodes and catheters allows for the basic conduct of the test. Basal measurements are made along the inferior turbinate, which allows selection of the best place for measurement, usually that with the most negative measurement. Then sequential perfusions determine Na+ (ENaC) and Cl- (CFTR-dependent) ion flux via a change in the voltage across the nasal epithelium.

<table>
	<tbody>
		<tr>
			<td>KD Scientific infusion pump (or equivalent &#8211; such as programmable infusion pumps provided by the institution/hospital)</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Powerlab 4/30</td>
			<td>AD Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BMA-200 AC/DC portable bioamplifier</td>
			<td>AD Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IS0-Z isolation headstage for BMA-200</td>
			<td>AD Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Windows compatible PC - Minimum requirements of Windows XP or higher</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AD Instruments software: GLP Client V6 (Windows) or higher</td>
			<td>AD Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ECG electrode (ground for study subject)</td>
			<td>Hospital standard</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2 mini calomel reference electrodes</td>
			<td>Fisher Scientific</td>
			<td>13-620-79</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride KCl, Granular &#8211; USP, formula weight 76, qty: 500 gm</td>
			<td>Spectrum</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile container (such as specimen collection container , or similar) to be used for KCl calomel bath, with holes cut in lid to hold electrodes in place. (If not provided by electrode manufacturer.)</td>
			<td>Hospital standard</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2 electrodes: Ag/AgCl 8 mm TP electrode</td>
			<td>BIOPAC Systems</td>
			<td>UNSHLD-EL258</td>
			<td></td>
		</tr>
		<tr>
			<td>2 Ag/AgCl electrodes, B0194, plug 4 mm</td>
			<td>SLE Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Signacreme&#174; Conductive Electrode Cream</td>
			<td>Fisher Scientific</td>
			<td>Parker Labs ref # 17-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Skin abrasion device</td>
			<td>PROMED Feeling</td>
			<td>Ref 374901</td>
			<td></td>
		</tr>
		<tr>
			<td>Hi Di 541 M, Diamond tipped dental burrs</td>
			<td>Ash Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Becton Dickinson PE 50 tubing</td>
			<td>Fisher Scientific</td>
			<td>427411</td>
			<td></td>
		</tr>
		<tr>
			<td>Becton Dickinson PE 90 tubing</td>
			<td>Fisher Scientific</td>
			<td>427421</td>
			<td></td>
		</tr>
		<tr>
			<td>Silastic tubing, 0.062&#8221; ID, 0.095&#8221; OD</td>
			<td>Fisher Scientific</td>
			<td>508-007</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropore Surgical Tape Paper (25 mm x 9.1 m)</td>
			<td>3M</td>
			<td>1530-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Marquat double lumen catheter Length: 80 cm; Outer diameter: 2.5 mm; Internal diameter of the channels: 0.8 mm; Distance of the side-holes to the tip: 2 mm. EU label Agreement for NPD: I0202US</td>
			<td>Marquat</td>
			<td>I0202US</td>
			<td></td>
		</tr>
		<tr>
			<td>1&#34; X 10 yards silk tape</td>
			<td>3M Durapore</td>
			<td>1538-1</td>
			<td></td>
		</tr>
		<tr>
			<td>IV extension tubing (30&#34;, 50/box)</td>
			<td>International Limited</td>
			<td>IMN30</td>
			<td></td>
		</tr>
		<tr>
			<td>Three-way stopcock (50/box)</td>
			<td>Medex</td>
			<td>MX5311L</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile syringe filters (ANOTOP 25 sterile 50pk; 0.22-micron or smaller filters; or equivalent)</td>
			<td>Fisher Scientific</td>
			<td>09-926-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Becton Dickinson Intramedic Luer stub adapter (20G, for connection to PE90 if using nasal catheter produced at study site)</td>
			<td>Fisher Scientific</td>
			<td>427564</td>
			<td></td>
		</tr>
		<tr>
			<td>Becton Dickinson 23G, 0.75&#8221; Vacutainer (&#8220;butterfly&#8221;) needles (0.6 x 19 mm; 50U/box) (for connection to PE50) if using nasal catheter produced at study site)</td>
			<td>Fisher Scientific</td>
			<td>367283</td>
			<td></td>
		</tr>
		<tr>
			<td>Becton Dickinson Syringe 60 ml without needle Luer-Lok tip (40/Box)</td>
			<td>Fisher Scientific</td>
			<td>309653</td>
			<td></td>
		</tr>
		<tr>
			<td>Becton Dickinson Syringe 10 ml without needle Luer-Lok tip (100/Box</td>
			<td>Fisher Scientific</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>Single use sterile wipes (per institutional availability)</td>
			<td>Hospital standard</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70% EtOH (1 pint), Aaper Alcohol and Chemical Co. catalog number NC9274019 (or equivalent)</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Corning single use sterile bottle-top filters, 0.22 &#956;m pore size (0.15 &#8211; 1.0 litre volumes acceptable)</td>
			<td>Fisher Scientific</td>
			<td>430624</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer Cert Ph 10.00 (1L Sn04332) &#8211; for pH meter calibration</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer Cert Ph 4.00 (1L Sn04327) &#8211; for pH meter calibration</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer Cert Ph 7.00 (500 ml Sn04328) &#8211; for pH meter calibration</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable underpads (Blue Pads; 23&#34;X36&#34; 150/Box; or equivalent per hospital standard)</td>
			<td>SureCare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>23G, 0.75&#8221; Vacutainer &#8220;butterfly&#8221; needles (0.6x19 mm; 50U/box)</td>
			<td>Becton Dickinson</td>
			<td>367283</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Laboratories Agar (Noble 100g 0142-15-2; or equivalent)</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Welch Allyn Rhinoscope 71000-C (or equivalent)</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Welch Allyn Convertible Handle Battery 72300 (or equivalent) OR Otoscope with battery</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Head and chin rest (or equivalent; optional)</td>
			<td>Richmond Products, Inc</td>
			<td>629R</td>
			<td></td>
		</tr>
		<tr>
			<td>Static Dissipative Anti-Fatigue Matting&#160; (or equivalent)</td>
			<td>Fisher Scientific</td>
			<td>No. 791</td>
			<td></td>
		</tr>
		<tr>
			<td>REAGENTS FOR SOLUTIONS MIXED ON SITE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride, Granular &#8211; USP NaCl</td>
			<td>Spectrum</td>
			<td></td>
			<td>Formula Weight: 58; Size: 500 gm</td>
		</tr>
		<tr>
			<td>Calcium Chloride CaCl2&#8226;2H2O &#8211; USP</td>
			<td>Spectrum</td>
			<td></td>
			<td>Formula Weight: 147; Size: 500 gm</td>
		</tr>
		<tr>
			<td>Magnesium Chloride Hexahydrate Crystal, MgCl2&#8226;6H2O &#8211; USP</td>
			<td>Spectrum</td>
			<td></td>
			<td>Formula Weight: 203; Size: 500 gm</td>
		</tr>
		<tr>
			<td>Potassium Phosphate Dibasic, Anhydrous, Granular, K2HPO4 &#8211; USP</td>
			<td>Spectrum</td>
			<td></td>
			<td>Formula Weight: 174; Size: 500 gm</td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic Crystals &#8211; NF (KH2PO4)</td>
			<td>Spectrum</td>
			<td></td>
			<td>Formula Weight: 136; Size: 500 gm</td>
		</tr>
		<tr>
			<td>Sodium Gluconate- USP (monosodium salt)</td>
			<td>Spectrum</td>
			<td></td>
			<td>Formula Weight: 218; Size: 500 gm</td>
		</tr>
		<tr>
			<td>Calcium Gluconate &#8211; USP (Anhydrous Powder)</td>
			<td>Spectrum</td>
			<td></td>
			<td>Formula Weight: 430; Size: 500 gm</td>
		</tr>
		<tr>
			<td>Potassium Gluconate- USP (Anhydrous)</td>
			<td>Spectrum</td>
			<td></td>
			<td>Formula Weight: 234; Size: 500 gm</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate Heptahydrate &#8211; USP MgSO4&#8226;7H2O</td>
			<td>Spectrum</td>
			<td></td>
			<td>Formula Weight: 246; Size: 500 gm</td>
		</tr>
		<tr>
			<td>Amiloride HCl &#8211; USP</td>
			<td>Spectrum</td>
			<td></td>
			<td>Formula Weight: 302; Size: 5gm</td>
		</tr>
		<tr>
			<td>Adenosine 5&#8217;-Triphosphate (ATP) (Disodium salt)</td>
			<td>Spectrum</td>
			<td></td>
			<td>Formula Weight: 551; Size: 5gm</td>
		</tr>
		<tr>
			<td>Magnesium Chloride, Hexahydrate, Crystal &#8211; USP MgCl2&#8226;6H2O</td>
			<td>Spectrum</td>
			<td></td>
			<td>Formula Weight: 203; Size: 500 gm</td>
		</tr>
		<tr>
			<td>Double-distilled water (ddH2O)</td>
			<td>Hospital Pharmacy</td>
			<td></td>
			<td>Formula Weight: NA; Size: 1 L</td>
		</tr>
		<tr>
			<td>Isoproterenol HCL Injection - USP 1 mg/5 ml ampule</td>
			<td>Hospital Pharmacy</td>
			<td></td>
			<td>Formula Weight: 248; Size: single use</td>
		</tr>
		<tr>
			<td>Ringers Injection, USP or Ringers Irrigation</td>
			<td>Hospital Pharmacy</td>
			<td></td>
			<td>Formula Weight: NA; Size: 5 L</td>
		</tr>
	</tbody>
</table>

standardized measurement of nasal membrane transepithelial potential difference (npd)

We describe a standardized measurement of nasal potential difference (NPD). In this technique, cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial sodium channel (ENaC) function are monitored by the change in voltage across the nasal epithelium after the superfusion of solutions that modify ion channel activity. This is enabled by the measurement of the potential difference between the subcutaneous compartment and the airway epithelium in the nostril, utilizing a catheter in contact with the inferior nasal turbinate.
The test allows the measurement of the stable baseline voltage and the successive net voltage changes after perfusion of 100 &#181;M amiloride, an inhibitor of Na+ reabsorption in Ringer&#39;s solution; a chloride-free solution containing amiloride to drive chloride secretion and 10 &#181;M isoproterenol in a chloride-free solution with amiloride to stimulate the cyclic adenosine monophosphate (cAMP)-dependent chloride conductance related to CFTR.
This technique has the advantage of demonstrating the electrophysiological properties of two key components establishing the hydration of the airway surface liquid of the respiratory epithelium, ENaC, and CFTR. Therefore, it is a useful research tool for phase 2 and proof of concept trials of agents that target CFTR and ENaC activity for the treatment of cystic fibrosis (CF) lung disease. It is also a key follow-up procedure to establish CFTR dysfunction when genetic testing and sweat testing are equivocal. Unlike sweat chloride, the test is relatively more time consuming and costly. It also requires operator training and expertise to conduct the test effectively. Inter- and intra-subject variability has been reported in this technique especially in young or uncooperative subjects. To assist with this concern, interpretation has been improved through a recently validated algorithm.

In normal airway epithelia, Na+ absorption is the primary ion transport activity. This results in a negative airway surface potential difference with regard to the interstitium. Perfusion of the ENaC channel blocker amiloride leads to a less negative potential difference. Then, superfusion of Cl--free solution creates a chemical gradient for Cl-, which creates a more negative potential difference and activates all the Cl- transporters, including CFTR. Isoproterenol, which increases intracellular cAMP, further increases Cl- secretion by specifically activating CFTR and increases the potential difference.
By contrast, in CF subjects, absent or dysfunctional CFTR results in an increased ENaC mediated Na+ absorption12. As a result, the baseline potential difference is more negative. The depolarization observed with application of amiloride is larger, whereas there is minimal or no change in the potential difference upon stimulation of Cl- secretion through CFTR dependent pathways. This can be seen in the representative tracings in Figure 8, showing &#39;healthy&#39; vs &#39;CF&#39; tracings.
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/57006/57006fig8.jpg" /> 
Figure 8: Representative tracings of the &#39;healthy&#39; subject and the subject with CF. PD: potential difference, &#916;Amiloride: delta amiloride, 0 Cl-/Iso-: low chloride: the change in PD between completion of solution #2 and solution #4 perfusion, S1-S4: stages 1-4, Green line on graphs A and B indicate the NPD tracing and Black arrows indicate the difference in potential difference

Watch this Scientific Journal Video about Standardized Measurement of Nasal Membrane Transepithelial Potential Difference NPD at JoVE.com

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference NPD

Here, we present a standardized protocol to measure the nasal potential difference (NPD). Cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) function are evaluated by the change in the voltage across the nasal epithelium after superfusion of solutions that modify ion channel activity, providing an outcome measure.

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference (NPD)

We describe a standardized measurement of nasal potential difference (NPD). In this technique, cystic fibrosis transmembrane conductance regulator (CFTR) ...

This method can help answer key questions in the CF and ion transport fields about whether CF patients can mount a meaningful response to activation or restoration of CFTR function.The main advantage of this technique is that it measures the coordinated activity of the CFTR and epithelial sodium channel in the airway epithelium.Although this method can provide insight into cystic fibrosis, it can also be applied to other disease systems, such as genetic disorders of hyperactive epithelial sodium channel function.Demonstrating the procedure will be Thao Nguyen, biologist, and Aur

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Research

JoVE Journal

Medicine

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the human respiratory tract. NA is a non-invasive technique which absorbs fluid from the inferior turbinate, and causes minimal discomfort. NA has yielded reproducible results with the ability to frequently repeat sampling of the upper airway. 
By comparison, alternative methods of sampling the respiratory mucosa, such as nasopharyngeal aspiration (NPA) and conventional swabbing, are more invasive and may result in greater data variability. Other methods have limitations, for instance, biopsies and bronchial procedures are invasive, sputum contains many dead and dying cells and requires liquefaction, exhaled breath condensate (EBC) contains water and saliva, and lavage samples are dilute and variable.
BA can be performed through the working channel of a bronchoscope in clinic. Sampling is well tolerated and can be conducted at multiple sites in the airway. BA results in MLF samples being less dilute than bronchoalveolar lavage (BAL) samples.
This article demonstrates the techniques of NA and BA, as well as the laboratory processing of the resulting samples, which can be tailored to the desired downstream biomarker being measured. These absorption techniques are useful alternatives to the conventional sampling techniques used in clinical respiratory research.

This manuscript describes the use of nasal and bronchial absorption techniques, specifically using synthetic absorptive matrices (SAM) to sample the mucosal lining fluid (MLF) of the upper and lower airway. These methods provide better standardization and tolerability than existing respiratory sampling techniques.

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the ...

Standardized Technique of Aortic Valve Re-implantation for Valve-sparing Aortic Root Replacement

Despite the obvious advantages of the preservation of a normal aortic valve during aortic root replacement, the complexity of valve sparing procedures prevents a number of cardiac surgeons from incorporating them into their practice. The aim of this protocol is to describe a simplified and user-friendly technique of an aortic valve-sparing root replacement (VSRR) procedure by re-implantation of the aortic valve. Proper selection of patients and limitations of the technique are discussed.
In 54 consecutive patients, normal appearing aortic valves were re-implanted in a commercially available polyester prosthesis with pre-shaped sinuses by a simplified and standardized technique. Placement of the first row of the proximal suture line, choice of the prosthesis size, and adjustment of the height of the commissures of the patient to the fixed height of the sinus portion of the prosthesis were slightly modified from the reference techniques with the aim of increasing its feasibility for use by other cardiac surgeons. Early mortality and morbidity as well as 5-year survival, freedom from aortic valve reoperation, and freedom from recurrent moderate regurgitation were collected in all patients.
Thirty-day mortality, re-sternotomy for bleeding, re-sternotomy for mediastinitis, and the incidence of stroke were very low, 1.8% for each (1 of 54). No patient required permanent pace-maker implantation. At 5 years, survival, freedom from aortic valve reoperation, and freedom from recurrent moderate regurgitation were 97.5%, 95.2%, and 91.6%, respectively.
Mid-term results of our standardized technique of re-implantation of the aortic valve for valve-sparing aortic root replacement are very good and compare with more complex techniques reported by experienced surgeons. By following the present protocol of the standardized re-implantation technique, a greater number of cardiac surgeons can perform this procedure with comparable good results.

Valve-sparing aortic root replacement has the advantage of preserving the patient&#39;s own aortic valve. The complexity of the reported techniques to date restricts their use to a limited number of cardiac surgeons. This protocol describes step-by-step a standardized technique reproducible by a greater number of cardiac surgeons.

Despite the obvious advantages of the preservation of a normal aortic valve during aortic root replacement, the complexity of valve sparing procedures ...

Generation of Human Nasal Epithelial Cell Spheroids for Individualized Cystic Fibrosis Transmembrane Conductance Regulator Study

While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the genotype-directed therapy model currently in use has several limitations. First, rare or understudied mutation groups are excluded from definitive clinical trials. Moreover, as additional modulator drugs enter the market, it will become difficult to optimize the modulator choices for an individual subject. Both of these issues are addressed with the use of patient-derived, individualized preclinical model systems of CFTR function and modulation. Human nasal epithelial cells (HNEs) are an easily accessible source of respiratory tissue for such a model. Herein, we describe the generation of a three-dimensional spheroid model of CFTR function and modulation using primary HNEs. HNEs are isolated from subjects in a minimally invasive fashion, expanded in conditional reprogramming conditions, and seeded into the spheroid culture. Within 2 weeks of seeding, spheroid cultures generate HNE spheroids that can be stimulated with 3',5'-cyclic adenosine monophosphate (cAMP)-generating agonists to activate CFTR function. Spheroid swelling is then quantified as a proxy of CFTR activity. HNE spheroids capitalize on the minimally invasive, yet respiratory origin of nasal cells to generate an accessible, personalized model relevant to an epithelium reflecting disease morbidity and mortality. Compared to the air-liquid interface HNE cultures, spheroids are relatively quick to mature, which reduces the overall contamination rate. In its current form, the model is limited by low throughput, though this is offset by the relative ease of tissue acquisition. HNE spheroids can be used to reliably quantify and characterize CFTR activity at the individual level. An ongoing study to tie this quantification to in vivo drug response will determine if HNE spheroids are a true preclinical predictor of patient response to CFTR modulation.

Here we describe a method to generate three-dimensional spheroid cultures of human nasal epithelial cells. Spheroids are then stimulated to drive Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-dependent ion and fluid secretion, quantifying the change in the spheroid luminal size as a proxy for CFTR function.

While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the ...

A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment elevated myocardial infarction. However, reperfusion per se can result in further cardiomyocyte death, a phenomenon known as reperfusion injury. The opening of the mitochondrial permeability transition pore (mPTP), with the decrease of the mitochondrial membrane potential (MMP), or mitochondrial depolarization, is universally recognized as the final step of reperfusion injury and is responsible for mitochondrial and cardiomyocyte death. JC-1 is a lipophilic cationic dye that accumulates in mitochondria depending on the value of MMP. The higher the MMP is, the more JC-1 accumulates in the mitochondria. The increasing amounts of JC-1 in mitochondria can be reflected by a fluorescence emission shift from green (~530 nm) to red (~590 nm). Therefore, the reduction of the red/green fluorescence intensity ratio can indicate the depolarization of mitochondria. Here, we take advantage of JC-1 to measure MMP, or the opening of mPTP in human cardiac myocytes after hypoxia/reoxygenation, detected by flow cytometry.

Here, we present a protocol that uses JC-1 dye to assess the mitochondrial membrane potential of cells after being exposed to hypoxia/reoxygenation with or without a protective agent.

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment ...

Microelectrode Impalement Method to Record Membrane Potential from a Cannulated Middle Cerebral Artery

Membrane potential (Vm) of vascular smooth muscle cells determines vessel tone and thus blood flow to an organ. Changes in the expression and function of ion channels and electrogenic pumps that regulate Vm in disease conditions could potentially alter Vm, vascular tone, and blood flow. Thus, a basic understanding of electrophysiology and the methods necessary to accurately record Vm in healthy and diseased states are essential. This method will allow modulating Vm using different pharmacological agents to restore Vm. Although there are several methods, each with its advantages and disadvantages, this article provides protocols to record Vm from cannulated resistance vessels such as the middle cerebral artery using the microelectrode impalement method. Middle cerebral arteries are allowed to gain myogenic tone in a myograph chamber, and the vessel wall is impaled using high resistance microelectrodes. The Vm signal is collected through an electrometer, digitized, and analyzed. This method provides an accurate reading of the Vm of a vessel wall without damaging the cells and without changing the membrane resistance.

The primary goal of this article is to provide details of how to record membrane potential (Vm) from the middle cerebral artery using the microelectrode impalement method. The cannulated middle cerebral artery is equilibrated to gain myogenic tone, and the vessel wall is impaled using high resistance microelectrodes.

Membrane potential (Vm) of vascular smooth muscle cells determines vessel tone and thus blood flow to an organ. Changes in the expression and function of ...

DIPLOMA Approach for Standardized Pathology Assessment of Distal Pancreatectomy Specimens

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignant cancers. A minority (20%) of PDACs are found in the pancreatic body and tail. Accurate pathology assessment of the pancreatic specimen is essential for providing prognostic information and it may guide further treatment strategies. The recent 8th edition of the American Joint Committee on Cancer/Union for International Cancer Control (AJCC/UICC) staging system for pancreatic tumors has incorporated significant changes to tumor (pT) stage, which is predominantly based on tumor size. This change emphasizes the importance of careful block selection. Owing to the greater prevalence of tumors in the head of the pancreas, efforts are made to standardize the assessment of pancreatoduodenectomy specimens. However, consensus regarding the macroscopic assessment of distal (i.e., left) pancreatectomy specimens is lacking. The DIPLOMA approach includes the standardized measurement of pancreas and other resected organs, inking of relevant surgical margins and anatomical surfaces without removing covering layers of fat, measurement of tumor size (for T-stage), together with assessment of splenic vessel involvement (and other organs if present). All relevant margins are assessed, and relevant blocks are selected to confirm these parameters microscopically. The current protocol describes a standardized approach to the macroscopic assessment of distal pancreatectomy specimens. This approach was developed during several meetings with pathologists and surgeons during the preparation phase for an international multicenter trial (DIPLOMA, ISRCTN44897265), which focuses on radicality of distal pancreatectomy for pancreatic ductal adenocarcinoma. This standardized approach can be instrumental in the design of studies and will uniform reporting on the outcomes of distal pancreatectomy. The described technique is used in the DIPLOMA trial for pancreatic ductal adenocarcinoma but may also be useful for other indications.

The current study highlights a standardized approach to the macroscopic assessment of distal pancreatectomy specimens for pancreatic ductal adenocarcinoma, with special emphasis on the measurement of pancreatic dimensions and those of other organs, inking of margins, measurement of tumor size and proximity to margins, lymph node sampling and block selection.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignant cancers. A minority (20%) of PDACs are found in the pancreatic body and tail. ...

Standardized Model of Ventricular Fibrillation and Advanced Cardiac Life Support in Swine

Cardiopulmonary resuscitation after cardiac arrest, independent of its origin, is a regularly encountered medical emergency in hospitals as well as preclinical settings. Prospective randomized trials in human subjects are difficult to design and ethically ambiguous, which results in a lack of evidence-based therapies. The model presented in this report represents one of the most common causes of cardiac arrests, ventricular fibrillation, in a standardized setting in a large animal model. This allows for reproducible observations and various therapeutic interventions under clinically accurate conditions, hence facilitating the generation of better evidence and eventually the potential for improved medical treatment.

Cardiopulmonary resuscitation and defibrillation are the only effective therapeutic options during cardiac arrest caused by ventricular fibrillation. This model presents a standardized regimen to induce, assess, and treat this physiological state in a porcine model, thus providing a clinical approach with various opportunities for data collection and analysis.

Cardiopulmonary resuscitation after cardiac arrest, independent of its origin, is a regularly encountered medical emergency in hospitals as well as ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...

Culture and Imaging of Human Nasal Epithelial Organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis ...

Collection, Expansion, and Differentiation of Primary Human Nasal Epithelial Cell Models for Quantification of Cilia Beat Frequency

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary dyskinesia. However, the wider application of these techniques is limited by the extreme susceptibility of ciliary function to changes in environmental factors e.g., temperature, humidity, and pH. In the airway of patients with Cystic Fibrosis (CF), mucus accumulation impedes cilia beating. Cilia function has been investigated in primary airway cell models as an indicator of CF Transmembrane conductance Regulator (CFTR) channel activity. However, considerable patient-to-patient variability in cilia beating frequency has been found in response to CFTR-modulating drugs, even for patients with the same CFTR mutations. Furthermore, the impact of dysfunctional CFTR-regulated chloride secretion on ciliary function is poorly understood. There is currently no comprehensive protocol demonstrating sample preparation of in vitro airway models, image acquisition, and analysis of Cilia Beat Frequency (CBF). Standardized culture conditions and image acquisition performed in an environmentally controlled condition would enable consistent, reproducible quantification of CBF between individuals and in response to CFTR-modulating drugs. This protocol describes the quantification of CBF in three different airway epithelial cell model systems: 1) native epithelial sheets, 2) air-liquid interface models imaged on permeable support inserts, and 3) extracellular matrix-embedded three-dimensional organoids. The latter two replicate in vivo lung physiology, with beating cilia and production of mucus. The ciliary function is captured using a high-speed video camera in an environment-controlled chamber. Custom-built scripts are used for the analysis of CBF. Translation of CBF measurements to the clinic is envisioned to be an important clinical tool for predicting response to CFTR-modulating drugs on a per-patient basis.

This protocol describes nasal epithelial cell collection, expansion, and differentiation to organotypic airway epithelial cell models and quantification of cilia beat frequency via live-cell imaging and custom-built scripts.

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary ...