The authors are grateful to Nikki Sabourin-Gibbs, Rouen University Hospital, for her help in editing the manuscript. The authors would like to thank the communication department of Rouen University Hospital and the teams of the Medical Training Center for their participation in the video shoot.

1. Preparation of connectors and infusion lines
<ol>
	<li>Sterilely retrieve the connectors.</li>
	<li>On the 3-way extension stopcock, screw 1 connector into each socket of the stopcock and 1 connector into the end of the extension line. Take 0.9% NaCl with a 50 mL syringe to purge the extension and the lines. Purge each 3-way extension with 0.9% NaCl through the 2 connectors of the stopcock.</li>
	<li>Take the infusion manifold. Unscrew each single-use cap. Screw 1 connector into each socket of the manifold to replace the caps. Purge the infusion line through each connector by turning each stopcock sequentially. 
	NOTE: At the end of this step, there are 3 extensions with 3-way stopcock with purged connectors and an infusion manifold with purged connectors.</li>
</ol>
2. Placement of the catheter
<ol>
	<li>Place the central venous catheter sterilely according to usual practice in the unit or a previously described protocol9.</li>
	<li>Check the permeability of each line by aspiration of blood and then reinjection of 0.9% NaCl serum. Clamp the lines.</li>
</ol>
3. Installation of the connectors
<ol>
	<li>Screw the extension&#8217;s connector of each 3-way extension stopcock into each catheter line. These connectors, directly connected to the lines of the catheter, are the &#34;proximal connectors&#34;. Unclamp the lines.</li>
	<li>Connect the infusion manifold line to one of the connectors of the 3-way stopcock of the distal line. 
	NOTE: There is no need to place a vein guard on the manifold or extensions.</li>
</ol>
4. Use of connectors and infusion lines
<ol>
	<li>Before each infusion, disinfect the end of the connector for 3 s with a sterile compress soaked in 70% isopropyl alcohol. Connect the syringe or tubing directly to the connector by screwing and injecting. After injection or infusion, unscrew the device. Do not remove the connector.</li>
	<li>If needed, rinse an unused line with 3 mL of saline. There is no need to clamp or infuse an unused line with a vein guard. Leave the proximal valve in place even if the line is not perfused.</li>
</ol>
5. Replacement and maintenance of connectors and infusion lines
<ol>
	<li>Always change the connectors every 7 days except for the proximal connectors. Thus, every week, the nurse prepares the extensions with 3-way stopcock and the infusion manifold in sterile conditions (see step 1).</li>
	<li>Unscrew the used extensions at the proximal valve and screw the new sterile and purged extensions to the catheter on the proximal connector (see step 3).</li>
	<li>Only change a proximal connector if it is soiled (to maintain the catheter in a &#34;closed&#34;&#160;system).</li>
	<li>In case of macroscopic contamination, rinse the connector with 10 mL of 0.9% NaCl. If there is still contamination, replace the connector. In the event of transfusion, infusion of lipid solution (e.g. propofol) or parenteral nutrition, change the tubing and connectors of the line concerned every 24 hours.</li>
	<li>Change other infusion lines and tubing according to the practices and protocols of each unit.</li>
</ol>

<ol>
	<li>Worth, L. J., McLaws, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+it+possible+to+achieve+a+target+of+zero+central+line+associated+bloodstream+infections.">Is it possible to achieve a target of zero central line associated bloodstream infections.</a> Current Opinion in Infectious Diseases. 25 (6), 650-657 (2012).</li><li>Kelly, L. J., Jones, T., Kirkham, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Needle-free+devices:+keeping+the+system+closed.">Needle-free devices: keeping the system closed.</a> British Journal of Nursing (Mark Allen Publishing). 26 (2), 14-19 (2017).</li><li>Casey, A. L., Karpanen, T. J., Nightingale, P., Elliott, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+risk+of+microbial+contamination+associated+with+six+different+needle-free+connectors.">The risk of microbial contamination associated with six different needle-free connectors.</a> British Journal of Nursing (Mark Allen Publishing). 27 (2), 18-26 (2018).</li><li>Salgado, C. D., Chinnes, L., Paczesny, T. H., Cantey, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+rate+of+catheter-related+bloodstream+infection+associated+with+use+of+a+needleless+mechanical+valve+device+at+a+long-term+acute+care+hospital.">Increased rate of catheter-related bloodstream infection associated with use of a needleless mechanical valve device at a long-term acute care hospital.</a> Infection Control and Hospital Epidemiology. 28 (6), 684-688 (2007).</li><li>Jarvis, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Health+care-associated+bloodstream+infections+associated+with+negative-+or+positive-pressure+or+displacement+mechanical+valve+needleless+connectors.">Health care-associated bloodstream infections associated with negative- or positive-pressure or displacement mechanical valve needleless connectors.</a> Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America. 49 (12), 1821-1827 (2009).</li><li>Btaiche, I. F., Kovacevich, D. S., Khalidi, N., Papke, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+needleless+connectors+on+catheter-related+bloodstream+infections.">The effects of needleless connectors on catheter-related bloodstream infections.</a> American Journal of Infection Control. 39 (4), 277-283 (2011).</li><li>Chernecky, C., Waller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+five+needleless+intravenous+connectors.">Comparative evaluation of five needleless intravenous connectors.</a> Journal of Advanced Nursing. 67 (7), 1601-1613 (2011).</li><li>. Infusion Resource Library - BD Available from: <a target="_blank" href="https://www.bd.com/en-us/offerings/capabilities/infusion/infusion-resource-library?contenttype=22_productline=115">https://www.bd.com/en-us/offerings/capabilities/infusion/infusion-resource-library?contenttype=22_productline=115</a> (2019)</li><li>Kim, S. C., Klebach, C., Heinze, I., Hoeft, A., Baumgarten, G., Weber, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+supraclavicular+fossa+ultrasound+view+for+central+venous+catheter+placement+and+catheter+change+over+guidewire.">The supraclavicular fossa ultrasound view for central venous catheter placement and catheter change over guidewire.</a> Journal of Visualized Experiments. (94), 52160 (2014).</li><li>Clavier, 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=Impact+of+MaxZeroTM+needle-free+connector+on+the+incidence+of+central+venous+catheter-related+infections+in+surgical+intensive+care+unit.">Impact of MaxZeroTM needle-free connector on the incidence of central venous catheter-related infections in surgical intensive care unit.</a> Australian Critical Care: Official Journal of the Confederation of Australian Critical Care Nurses. , (2018).</li><li>Lehn, R. A., Gross, J. B., McIsaac, J. H., Gipson, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Needleless+connectors+substantially+reduce+flow+of+crystalloid+and+red+blood+cells+during+rapid+infusion.">Needleless connectors substantially reduce flow of crystalloid and red blood cells during rapid infusion.</a> Anesthesia and Analgesia. 120 (4), 801-804 (2015).</li><li>Ferroni, 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=Pulsative+flushing+as+a+strategy+to+prevent+bacterial+colonization+of+vascular+access+devices.">Pulsative flushing as a strategy to prevent bacterial colonization of vascular access devices.</a> Medical Devices (Auckland, N.Z). 7, 379-383 (2014).</li><li>Flynn, J. M., Keogh, S. J., Gavin, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sterile+v+aseptic+non-touch+technique+for+needle-less+connector+care+on+central+venous+access+devices+in+a+bone+marrow+transplant+population:+A+comparative+study.">Sterile v aseptic non-touch technique for needle-less connector care on central venous access devices in a bone marrow transplant population: A comparative study.</a> European Journal of Oncology Nursing: The Official Journal of European Oncology Nursing Society. 19 (6), 694-700 (2015).</li><li>Merrill, K. C., Sumner, S., Linford, L., Taylor, C., Macintosh, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+universal+disinfectant+cap+implementation+on+central+line-associated+bloodstream+infections.">Impact of universal disinfectant cap implementation on central line-associated bloodstream infections.</a> American Journal of Infection Control. 42 (12), 1274-1277 (2014).</li><li>Mermel, L. A., Bert, A., Chapin, K. C., LeBlanc, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraoperative+stopcock+and+manifold+colonization+of+newly+inserted+peripheral+intravenous+catheters.">Intraoperative stopcock and manifold colonization of newly inserted peripheral intravenous catheters.</a> Infection Control and Hospital Epidemiology. 35 (9), 1187-1189 (2014).</li><li>Rosenthal, V. D., Maki, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+study+of+the+impact+of+open+and+closed+infusion+systems+on+rates+of+central+venous+catheter-associated+bacteremia.">Prospective study of the impact of open and closed infusion systems on rates of central venous catheter-associated bacteremia.</a> American Journal of Infection Control. 32 (3), 135-141 (2004).</li><li>Bouza, E., 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+needleless+closed+system+device+(CLAVE)+protects+from+intravascular+catheter+tip+and+hub+colonization:+a+prospective+randomized+study.">A needleless closed system device (CLAVE) protects from intravascular catheter tip and hub colonization: a prospective randomized study.</a> The Journal of Hospital Infection. 54 (4), 279-287 (2003).</li><li>Tabak, Y. P., Jarvis, W. R., Sun, X., Crosby, C. T., Johannes, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meta-analysis+on+central+line-associated+bloodstream+infections+associated+with+a+needleless+intravenous+connector+with+a+new+engineering+design.">Meta-analysis on central line-associated bloodstream infections associated with a needleless intravenous connector with a new engineering design.</a> American Journal of Infection Control. 42 (12), 1278-1284 (2014).</li><li>Wallace, M. C., Macy, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+Central+Line-Associated+Bloodstream+Infection+Rates+in+Patients+in+the+Adult+Intensive+Care+Unit.">Reduction of Central Line-Associated Bloodstream Infection Rates in Patients in the Adult Intensive Care Unit.</a> Journal of Infusion Nursing: The Official Publication of the Infusion Nurses Society. 39 (1), 47-55 (2016).</li><li>Btaiche, I. F., Kovacevich, D. S., Khalidi, N., Papke, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+needleless+connectors+on+catheter-related+thrombotic+occlusions.">The effects of needleless connectors on catheter-related thrombotic occlusions.</a> Journal of Infusion Nursing: The Official Publication of the Infusion Nurses Society. 34 (2), 89-96 (2011).</li><li>Williams, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catheter+Occlusion+in+Home+Infusion:+The+Influence+of+Needleless+Connector+Design+on+Central+Catheter+Occlusion.">Catheter Occlusion in Home Infusion: The Influence of Needleless Connector Design on Central Catheter Occlusion.</a> Journal of Infusion Nursing: The Official Publication of the Infusion Nurses Society. 41 (1), 52-57 (2018).</li></ol>

TC, PG and BV previously conducted a study on PPNC valves. BD provided the connectors for this previous work but had no role in the trial initiation, study design, data collection, data analysis, data interpretation, writing of the report, or the decision to submit. The other authors have nothing to disclose. The drafting of this protocol was formalized in 2013 with validation by Carefusion consultants.

Setting up the connectors slightly increases the total time required to place the catheter. However, their use has several advantages: no need to maintain a continuous saline perfusion for non-infused lines, rapid stopping of infusion of the lines if necessary (in case of urgent transport of the patient for example), no need to use single-use caps several times a day to close the infusion lines. The use of connectors simplifies the daily use of catheter infusion lines without any particular complications. It should be noted that during our four year experience of these PPNC connectors there have been no complications with rapid fluid administration while this has been described with other types of connectors11. It is essential to maintain complete asepsis when installing the connectors in order to keep the infusion system closed. In addition, regular examination of the connectors for contamination is essential to be able to rinse or change the implicated connectors and avoid bacterial growth. A recent work reports the in vitro efficacy of a pulsative flushing technique to prevent bacterial colonization of vascular access devices but this technique has not been evaluated directly on needleless connectors12. Due to the technical aspect of this kind of connector, nurses must be trained before using these connectors in everyday practice. In this context, the use of Aseptic Non Touch Technique, which is a safe method for managing intravascular devices, is very relevant13. Thus, even after the connectors are set up, direct contact with the connectors should be minimized as much as possible to minimize the risk of contamination. In this context, the use of a disinfectant cap on needleless connectors can help to limit the contact with connectors and is effective in reducing central line-associated bloodstream infections14.
The inclusion of extension tubing, stopcocks and manifolds increase the complexity of the system and expose it to a risk of contamination of stopcocks and manifolds15. Our choice of extensions and infusion lines is the result of a reflection on the balance between patient safety and infectious risk. In the ICU, continuous uninterrupted infusion of certain drugs is essential (e.g., catecholamine, sedation). In our protocol, the use of a 3-way extension stopcock allows medication relays to be easily made without ever interrupting the infusion, which is a guarantee of safety for the patient. The use of a unique 4-port manifold with a perfusion line allows the infusion of several drugs simultaneously (e.g., antibiotics, analgesics, insulin) but the number of these manifolds should be limited to prevent the risk of infection.
Some studies have shown interest in a closed system to prevent colonization and catheter infections16,17. Additionally, several studies have reported a decrease in catheter infection after using PPNCs10,18,19. It is also possible that these connectors may decrease thrombotic catheter occlusions, but there is insufficient evidence to conclude a direct causal link between needleless connectors and catheter occlusions20,21. Moreover, data in the literature support the safe and prolonged use of these devices10,16,19. However, different department may sometimes use different methods to mount connectors on infusion lines and most studies do not describe the method used to install connectors on catheters. It could appear interesting that studies on connectors report their installation protocol to better assess potential differences of practice between teams.

Central venous catheter-related infections (CRI) are a severe complication of central venous catheters in intensive care unit (ICU). The decline in CRI remains an ever-present objective, with a final goal of &#34;zero catheter related infection&#34;1. Needle-free connectors were initially designed and promoted to avoid blood exposure for healthcare workers. There are two main designs of connectors: split septum (no internal moving parts) and closed valve systems (internal moving components) but both designs can be combined in one connector2. Needle-free connectors are categorized according to the type of fluid displacement that occurs after disconnection of a male Luer valve: negative (blood reflux into the catheter), neutral, and positive (with a push of blood out of the catheter lumen)2,3.
Some connectors have been described as a cause of catheter-related infections, in particular in the intensive care unit (ICU)4,5,6. A new generation of needle-free connectors with minimal internal complexity, a reduction or elimination of interstitial or dead space, a visible fluid path to help assess proper flushing technique, and a flat access surface, etc. has been designed to lower the risk of infection. In vitro, these connectors have shown low bacterial colonization7. There are global recommendations from the laboratory manufacturing these connectors; however, there is no practical description of how to install them on catheters8. Hence, it is possible that each team uses them differently. Therefore, we propose a formalized protocol for the installation of these connectors on central venous lines in the ICU.
We present the installation of a positive pressure needleless connector (PPNC) with an internal silicone piston in our ICU but this protocol is applicable with any positive displacement valve. This valve is a mechanical needle-free connector with positive displacement.

<table>
	<tbody>
		<tr>
			<td>BD MaxZero&#8482;&#160;needle-free connector</td>
			<td>Becton Dickinson</td>
			<td>MZ1000-07</td>
			<td>we present the installation of the connector with MaxZero but this protocol is applicable with any positive displacement valve.</td>
		</tr>
		<tr>
			<td>4-port mannifold with PE/PVC extension</td>
			<td>Cair-LGL</td>
			<td>RPB4310A</td>
			<td></td>
		</tr>
		<tr>
			<td>PE/PVC extension line with 3-way stopcock</td>
			<td>Cair-LGL</td>
			<td>PE3302M</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl 0.9% 250 ml</td>
			<td>Baxter</td>
			<td>2B1322</td>
			<td></td>
		</tr>
		<tr>
			<td>BD&#160;Plastipak&#8482; 50mL Luer-Lock Syringe</td>
			<td>BECTON DICKINSON MEDICAL</td>
			<td>613-3925</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol to set up needle-free connector with positive displacement on central venous catheter in intensive care unit

Needle-free connectors were initially designed and promoted to avoid blood exposure for healthcare workers. Some recent data suggest that the latest generation of connectors (with positive displacement) may be of interest for reducing central venous line infections. We have been using needle-free connectors for several years in our intensive care unit and here we present a protocol for installing these connectors on central venous catheters. After insertion of the catheter and control of the permeability of the lines, the connectors must be purged with 0.9% NaCl before being connected. The connectors replace all disposable caps used on infusion stopcocks and manifolds. All the connectors are changed every 7 days as recommended by the manufacturer (except when there is macroscopic contamination, which requires an immediate change of the connector). Before each injection, the connector must be disinfected for at least 3 seconds with 70% isopropyl alcohol. The connectors must not be disconnected (unless changed), as the injection is done through the device. Setting up the connectors slightly increases the total time required to place the catheter and there is no formal evidence that these connectors reduce the incidence of infectious or thrombotic complications. However, these devices simplify the management of central venous lines and prevent the catheter circuit from &#34;opening&#34; once it has been sterilely installed.

Once all the elements are in place, the catheter has connectors on almost all the junctions between two infusion lines (Figure 1). Thus, it has proximal connectors on each line and two connectors on the sockets of the 3-way extensions (Figure 2). Each infusion line has connectors at all its female sockets (Figure 3). Once the assembly is in place, any injection or infusion (continuous or discontinuous) must be made, after disinfection, through one of the connectors of the infusion lines or extensions (Figure 4).
Maintaining the connectors in place keeps the infusion system closed as well as sterile mounted and minimizes the risk of bacterial contamination. One of our previous works, the first study that prospectively analyzed these devices over several years, showed a significant decrease in CRI incidence during the six-year period framing the introduction of the connectors (Figure 5)10. In this work, the incidence of CRI before using connectors was 6.2 CRI/1000 catheter-day vs. 2.7 CRI/1000 catheter-days after using connectors10. Moreover, we did not find any increase of CRI after the beginning of connector use (Figure 5) and there was no significant difference concerning the kind of bacteria species involved in CRI10. We have not identified any specific complications associated with the use of these connectors during these years of use.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59801/59801fig01.jpg" /> 
Figure 1: Global view of the catheter once the connectors have been placed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59801/59801fig01large.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/59801/59801fig02.jpg" /> 
Figure 2: Proximal part of the catheter with proximal connectors (A) and connectors on the 3-way extension stopcock (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59801/59801fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59801/59801fig03.jpg" /> 
Figure 3: 4-port manifold with connectors. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59801/59801fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59801/59801fig04.jpg" /> 
Figure 4: Steps of injection through a connector.&#160;(A) Disinfect the surface of the connector for 3 seconds with 70% isopropyl alcohol. Wait until the connector is dry. (B) Insert the tip of the syringe or infuser into the connector. (C) Inject or infuse the medication. (D) Remove the syringe or infuser. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59801/59801fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59801/59801fig05.jpg" /> 
Figure 5: Evolution of catheter-related infection incidence before and after the use of connectors.&#160;Figure taken from Clavier et al.10 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59801/59801fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Protocol to Set Up Needle-Free Connector with Positive Displacement on Central Venous Catheter in Intensive Care Unit at JoVE.com

A Protocol to Set Up Needle-Free Connector with Positive Displacement on Central Venous Catheter in Intensive Care Unit

We present a protocol to show the installation of a needle-free connector with positive displacement on a central venous catheter.

Needle-free connectors were initially designed and promoted to avoid blood exposure for healthcare workers. Some recent data suggest that the latest ...

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11.9K Views.  Rouen University Hospital. We present a protocol to show the installation of a needle-free connector with positive displacement on a central venous catheter.

Video: A Protocol to Set Up Needle-Free Connector with Positive Displacement on Central Venous Catheter in Intensive Care Unit

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

The Supraclavicular Fossa Ultrasound View for Central Venous Catheter Placement and Catheter Change Over Guidewire

The supraclavicular fossa ultrasound view can be useful for central venous catheter (CVC) placement. Venipuncture of the internal jugular veins (IJV) or subclavian veins is performed with a micro-convex ultrasound probe, using a neonatal abdominal preset with a probe frequency of 10 Mhz at a depth of 10-12 cm. Following insertion of the guidewire into the vein, the probe is shifted to the right supraclavicular fossa to obtain a view of the superior vena cava (SVC), right pulmonary artery and ascending aorta. Under real-time ultrasound view, the guidewire and its J-tip is visualized and pushed forward to the lower SVC. Insertion depth is read from guidewire marks using central venous catheter. CVC is then inserted following skin and venous dilation. The supraclavicular fossa view is most suitable for right IJV CVC insertion. If other insertion sites are chosen the right supraclavicular fossa should be within the sterile field. Scanning of the IJVs, brachiocephalic veins and SVC can reveal significant thrombosis before venipuncture. Misplaced CVCs can be corrected with a change over guidewire technique under real-time ultrasound guidance. In conjunction with a diagnostic lung ultrasound scan, this technique has a potential to replace chest radiograph for confirmation of CVC tip position and exclusion of pneumothorax. Moreover, this view is of advantage in patients with a non-p-wave cardiac rhythm were an intra-cardiac electrocardiography (ECG) is not feasible for CVC tip position confirmation. Limitations of the method are lack of availability of a micro-convex probe and the need for training.

The overall goal is to enable clinicians to insert a central venous catheter under real-time ultrasound guidance via the right supraclavicular fossa view of the lower part of the superior vena cava. This view is useful for different catheter insertion sites, thrombosis detection before insertion and ultrasound guided correction of misplaced catheters.

The supraclavicular fossa ultrasound view can be useful for central venous catheter (CVC) placement. Venipuncture of the internal jugular veins (IJV) or ...

Setting Up a Stroke Team Algorithm and Conducting Simulation-based Training in the Emergency Department - A Practical Guide

Time is of the essence when caring for an acute stroke patient. The ultimate goal is to restore blood flow to the ischemic brain. This can be&#160;achieved by either&#160;thrombolysis with recombinant tissue-plasminogen activator (rt-PA), the standard therapy for stroke patients who present within the first hours of symptom onset without contraindications, or by an endovascular approach, if a proximal brain vessel occlusion is detected. As the efficacy of both therapies declines over time, every minute saved along the way will improve the patient&#39;s outcome.
This critical situation requires thorough work and precise communication with the patient, the family and colleagues from different professions to acquire all relevant information and reach the right decision while carefully monitoring the patient. This is a high fidelity situation. In nonmedical high-fidelity environments such as aviation, Crew Resource Management (CRM) is used to enhance safety and team efficiency.
This guide shows how a Stroke Team algorithm, which is transferable to other hospital settings, was established and how regular simulation-based trainings were performed. It requires determination and endurance to maintain these time-consuming simulation trainings on a regular basis over the course of time. However, the resulting improvement of team spirit and excellent door-to-needle times will benefit both the patients and the work environment in any hospital.
A dedicated Stroke Team of 7 persons who are notified 24/7 by a collective call via speed dial and run a binding algorithm that takes approximately 20 min, was established. To train everybody involved in this algorithm, a simulation-based team training for all new Stroke Team members was conceived and conducted at monthly intervals. This led to a relevant and sustained reduction of the mean door-to-needle time to 25 min, and enhanced the feeling of stroke readiness especially in junior doctors and nurses.

Every min counts in acute stroke care. This guide shows how to establish a Stroke Team algorithm and enhance its performance with regular simulation training. The principles of Crew Resource Management (CRM) facilitate a straight workflow, reduce door-to-needle times and increase staff satisfaction.

Time is of the essence when caring for an acute stroke patient. The ultimate goal is to restore blood flow to the ischemic brain. This can ...

Use of a Central Venous Line for Fluids, Drugs and Nutrient Administration in a Mouse Model of Critical Illness

This protocol describes a centrally catheterized mouse model of prolonged critical illness. We combine the cecal ligation and puncture method to induce sepsis with the use of a central venous line for fluids, drugs and nutrient administration to mimic the human clinical setting. Critically ill patients require intensive medical support in order to survive. While the majority of patients will recover within a few days, about a quarter of the patients need prolonged intensive care and are at high risk of dying from non-resolving multiple organ failure. Furthermore, the prolonged phase of critical illness is hallmarked by profound muscle weakness, and endocrine and metabolic changes, of which the pathogenesis is currently incompletely understood. The most widely used animal model in critical care research is the cecal ligation and puncture model to induce sepsis. This is a very reproducible model, with acute inflammatory and hemodynamic changes similar to human sepsis, which is designed to study the acute phase of critical illness. However, this model is hallmarked by a high lethality, which is different from the clinical human situation, and is not developed to study the prolonged phase of critical illness. Therefore, we adapted the technique by placing a central venous catheter in the jugular vein allowing us to administer clinically relevant supportive care, to better mimic the human clinical situation of critical illness. This mouse model requires an extensive surgical procedure and daily intensive care of the animals, but it results in a relevant model of the acute and prolonged phase of critical illness.

This protocol describes a centrally catheterized mouse model of prolonged critical illness. We combine the cecal ligation and puncture method to induce sepsis with the use of a central venous line for fluids, drugs and nutrient administration to mimic the human clinical setting.

This protocol describes a centrally catheterized mouse model of prolonged critical illness. We combine the cecal ligation and puncture method to induce ...

Observational Study Protocol for Repeated Clinical Examination and Critical Care Ultrasonography Within the Simple Intensive Care Studies

Longitudinal evaluations of critically ill patients by combinations of clinical examination, biochemical analysis and critical care ultrasonography (CCUS) may detect adverse events of interventions such as fluid overload at an early stage. The Simple Intensive Care Studies (SICS) is a research line that focuses on the prognostic and diagnostic value of combinations of clinical variables.
The SICS-I specifically focused on the use of clinical variables obtained within 24 h of acute admission for prediction of cardiac output (CO) and mortality. Its sequel, SICS-II, focuses on repeated evaluations during ICU admission. The first clinical examination by trained researchers is performed within 3 h after admission consisting of physical examination and educated guessing. The second clinical examination is performed within 24 h after admission and includes physical examination and educated guessing, biochemical analysis and CCUS assessments of heart, lungs, inferior vena cava (IVC) and kidney. This evaluation is repeated at days 3 and 5 after admission. CCUS images are validated by an independent expert, and all data is registered in an online secured database. Follow-up at 90 days includes registration of complications and survival status according to patient&#39;s medical charts and the municipal person registry. The primary focus of SICS-II is the association between venous congestion and organ dysfunction.
The purpose of publishing this protocol is to provide details on the structure and methods of this on-going prospective observational cohort study allowing answering multiple research questions. The design of the data collection of combined clinical examination and CCUS assessments in critically ill patients are explicated. The SICS-II is open for other centers to participate and is open for other research questions that can be answered with our data.

Structured protocols are necessary to provide answers on research questions in critically ill patients. The Simple Intensive Care Studies (SICS) provides an infrastructure for repeated measurements in critically ill patients including clinical examination, biochemical analysis and ultrasonography. SICS projects have specific focus but the structure is flexible to other investigations.

Longitudinal evaluations of critically ill patients by combinations of clinical examination, biochemical analysis and critical care ultrasonography (CCUS) ...

Protocol and Guidelines for Point-of-Care Lung Ultrasound in Diagnosing Neonatal Pulmonary Diseases Based on International Expert Consensus

Ultrasound is a safe bedside imaging tool that obviates the use of ionizing radiation diagnostic procedures. Due to its convenience, the lung ultrasound has received increasing attention from neonatal physicians. Nevertheless, clear reference standards and guideline limits are needed for accurate application of this diagnostic modality. This document aims to summarize expert opinions and to provide precise guidance to help facilitate the use of the lung ultrasound in the diagnosis of neonatal lung diseases.

Lung ultrasound is a noninvasive and valuable tool for bedside evaluation of neonatal lung diseases. However, a relative lack of reference standards, protocols and guidelines may limit its application. Here, we aim to develop a standardized neonatal lung ultrasound diagnostic protocol to be used in clinical decision-making.

Ultrasound is a safe bedside imaging tool that obviates the use of ionizing radiation diagnostic procedures. Due to its convenience, the lung ultrasound ...

Clinical Practice Protocol of Creative Music Therapy for Preterm Infants and Their Parents in the Neonatal Intensive Care Unit

Creative music therapy for preterm infants and their parents (CMT) has emerged as a promising family-integrated early intervention involving communicative musicality to improve infant development, parental well-being, and bonding. It aims at relaxing and nurturing the infant as well as promoting safety and social interaction for the parent-infant dyad. A music therapist specially trained in CMT hums or sings in an infant-directed, improvised, lullaby style continually adjusting to the individual needs, expressions, and breathing pattern of the preterm infant. Based on the principles of family-integrated care, the family is incorporated individually in the therapeutic process, namely by delivering CMT during kangaroo care (KC) and by motivating and facilitating parental vocal interaction with their infant to strengthen the parent-infant bonding. CMT aims at relaxing, stimulating, and coregulating premature infants at a time when many other interventions are still risky and can overwhelm the vulnerable patient group. CMT may be advantageous not by educating and teaching parents, but rather by uncovering the intuitive capacities of parenting that are often overshadowed by the traumatic experience of preterm birth. However, CMT can only be provided when the infants are clinically stable. CMT with parental integration is feasible when parents are available and receptive to participate. This paper presents a detailed protocol on how to use CMT to empower preterm infants and their families.

Creative music therapy for preterm infants and their parents has emerged as a promising family-integrated early intervention. We present a detailed protocol on how to use vocal interaction, humming, or singing to empower preterm infants and their families.

Creative music therapy for preterm infants and their parents (CMT) has emerged as a promising family-integrated early intervention involving communicative ...

Halogenated Agent Delivery in Porcine Model of Acute Respiratory Distress Syndrome via an Intensive Care Unit Type Device

Acute respiratory distress syndrome (ARDS) is a common cause of hypoxemic respiratory failure and death in critically ill patients, and there is an urgent need to find effective therapies. Preclinical studies have shown that inhaled halogenated agents may have beneficial effects in animal models of ARDS. The development of new devices to administer halogenated agents using modern intensive care unit (ICU) ventilators has significantly simplified the dispensing of halogenated agents to ICU patients. Because previous experimental and clinical research suggested potential benefits of halogenated volatiles, such as sevoflurane or isoflurane, for lung alveolar epithelial injury and inflammation, two pathophysiologic landmarks of diffuse alveolar damage during ARDS, we designed an animal model to understand the mechanisms of the effects of halogenated agents on lung injury and repair. After general anesthesia, tracheal intubation, and the initiation of mechanical ventilation, ARDS was induced in piglets via the intratracheal instillation of hydrochloric acid. Then, the piglets were sedated with inhaled sevoflurane or isoflurane using an ICU-type device, and the animals were ventilated with lung-protective mechanical ventilation during a 4 h period. During the study period, blood and alveolar samples were collected to evaluate arterial oxygenation, the permeability of the alveolar-capillary membrane, alveolar fluid clearance, and lung inflammation. Mechanical ventilation parameters were also collected throughout the experiment. Although this model induced a marked decrease in arterial oxygenation with altered alveolar-capillary permeability, it is reproducible and is characterized by a rapid onset, good stability over time, and no fatal complications.
We have developed a piglet model of acid aspiration that reproduces most of the physiological, biological, and pathological features of clinical ARDS, and it will be helpful to further our understanding of the potential lung-protective effects of halogenated agents delivered through devices used for inhaled ICU sedation.

We describe a model of hydrochloric acid-induced acute respiratory distress syndrome (ARDS) in piglets receiving sedation with halogenated agents, isoflurane and sevoflurane, through a device used for inhaled intensive care sedation. This model can be used to investigate the biological mechanisms of halogenated agents on lung injury and repair.

Acute respiratory distress syndrome (ARDS) is a common cause of hypoxemic respiratory failure and death in critically ill patients, and there is an urgent ...

A Reproducible Intensive Care Unit-Oriented Endotoxin Model in Rats

Sepsis and septic shock remain the leading cause of death in intensive care units. Despite significant improvements in sepsis management, mortality still ranges between 20 and 30%. Novel treatment approaches in order to reduce sepsis-related multiorgan failure and death are urgently needed. Robust animal models allow for one or multiple treatment approaches as well as for testing their effect on physiological and molecular parameters. In this article, a simple animal model is presented.
First, general anesthesia is induced in animals either with the use of volatile or by intraperitoneal anesthesia. After placement of an intravenous catheter (tail vein), tracheostomy, and insertion of an intraarterial catheter (tail artery), mechanical ventilation is started. Baseline values of mean arterial blood pressure, arterial blood oxygen saturation, and heart rate are recorded.
The injection of lipopolysaccharides (1 milligram/kilogram body weight) dissolved in phosphate-buffered saline induces a strong and reproducible inflammatory response via the toll-like receptor 4. Fluid corrections as well as the application of norepinephrine are performed based on well-established protocols.
The animal model presented in this article is easy to learn and strongly oriented towards clinical sepsis treatment in an intensive care unit with sedation, mechanical ventilation, continuous blood pressure monitoring, and repetitive blood sampling. Also, the model is reliable, allowing for reproducible data with a limited number of animals in accordance with the 3R (reduce, replace, refine) principles of animal research. While animal experiments in sepsis research cannot easily replaced, repetitive measurements allow for a reduction of animals and keeping septic animals anesthetized diminishes suffering.

Here, we present a reproducible intensive care unit-oriented endotoxin model in rats.

Sepsis and septic shock remain the leading cause of death in intensive care units. Despite significant improvements in sepsis management, mortality still ...

Lung Rapid Recovery Procurement Combined with Abdominal Normothermic Regional Perfusion in Controlled Donation after Circulatory Death

Controlled donation after circulatory death (cDCD) has contributed to increasing donor numbers all over the world. Experiences published in the last years confirm that the outcomes after lung transplantation from cDCD are similar to those from brain death donors; however, the utilization of lungs from asystole donors remains low. Several reasons may be involved: different legal frameworks among countries and centers with different premortem interventions, inadequate lung donor care before procurement, or even poor experience with cDCD procedures and protocols.
Initially, the rapid recovery technique was commonly employed for the procurement of thoracic and abdominal organs in cDCD, but, in the last decade, abdominal normothermic regional perfusion (ANRP) with extracorporeal membrane oxygenation devices has become a useful method to restore blood flow to abdominal organs, allowing their quality improvement and their functional assessment prior to transplantation. This makes the donation procedure more complex and generates doubts about injury to the grafts due to dual temperature.
The aim of this article is to describe a protocol based on a single center experience with Maastricht III donors combining lung cooling rapid recovery in the thorax and abdominal normothermic regional perfusion. Tips and tricks focused on premortem interventions and lung procurement procedure techniques are explained. This may help to minimize the reluctance among professionals to use this combined technique and encourage other donor centers to use it, despite the increased complexity of the procedure.

The protocol combines a lung cooling rapid recovery technique with abdominal normothermic regional perfusion for abdominal graft procurement in controlled asystole donors, which is a safe and useful method to expand the donor pool.

Controlled donation after circulatory death (cDCD) has contributed to increasing donor numbers all over the world. Experiences published in the last years ...