This project was supported by the Berthold-Leibinger-Foundation, Ditzingen, Germany.

Intracoronary ACh testing has been approved by the local ethics committee and the protocol follows the guidelines of our institution for human research. A previous JoVE article covered a protocol showing preparation of the ACh solutions as well as preparation of the syringes for intracoronary injection of ACh15.
1. Preparation of the ACh solutions and preparation of the syringes for intracoronary injection of ACh
<ol>
	<li>Please refer to a previously published JoVE article15.</li>
</ol>
2. Preparation of adenosine solution for intracoronary injection
<ol>
	<li>Take 1 ampoule of 6 mg adenosine (with 2 mL solvent) into a syringe (this corresponds to a dose of 3 mg/mL).</li>
	<li>Add the 6 mg of adenosine to 100 mL of 0.9% sodium chloride solution and mix gently.</li>
	<li>Fill a 10 mL syringe with 3.5 mL of the adenosine solution (approximately 200 &#181;g of adenosine).</li>
	<li>Perform the last step 3 times for preparation of 3 injections.</li>
</ol>
3. Diagnostic coronary angiography
<ol>
	<li>Depending on arterial access route, inject local anaesthesia either in proximity to the right femoral artery (usually 15 mL of mepivacaine) or in proximity to the right radial artery (usually 2 mL of mepivacaine).</li>
	<li>To confirm the success of local anaesthesia, prick the anesthetized-skin with the needle and ask the patient if pain is still present.</li>
	<li>Puncture the artery according to the Seldinger technique and insert the sheath (usually 5F). If possible, omit radial spasm prophylaxis in patients undergoing planned IDP. Perform coronary angiography under sterile conditions.</li>
	<li>Introduce the diagnostic catheter over a J-tipped wire through the radial artery sheath to the ascending aorta and advance it to the aortic root.</li>
	<li>Give 5000 IU of heparin.</li>
	<li>Engage the diagnostic catheter into the ostium of the right (RCA) and subsequently of the left coronary artery (LCA). Inject 2 mL of contrast to confirm correct positioning of the catheter.</li>
	<li>Perform coronary angiography in different views using manual injections of approximately 10 mL of contrast agent under fluoroscopy to visualize the coronary arteries. 
	&#8203;NOTE: Usually LAO 40&#176; and RAO 35&#176; are used for the RCA and LAO 45&#176;/ CRAN 25&#176;, RAO 30&#176;/ CRAN 30&#176; and RAO 20&#176;/ CAUD 30&#176; are used for the LCA.</li>
</ol>
4. Preparations for the IDP
<ol>
	<li>As&#160;a prerequisite for the IDP, exclude any epicardial stenosis of &#62;50% on visual assessment. 
	NOTE: The default artery for the IDP is the LCA as it allows the examination of the two vessels (left anterior descending artery (LAD) and left circumflex artery (LCX)) at the same time.</li>
	<li>Place a guiding catheter suitable for the LCA into the left main (this can be 5F or 6F, choice of the catheter depends on the patient&#39;s anatomy).</li>
	<li>Give another 5000 IU of heparin.</li>
	<li>Advance the Doppler flow-/pressure-wire carefully through the guiding catheter into the left main artery.</li>
	<li>After flushing to avoid any contrast in the catheter, calibrate the Doppler flow-/pressure-wire with the fractional flow reserve (FFR) sensor (localized either tip-adjacent or 1.5 cm offset depending on the wire type) in the left main (press Norm on the software of the computer system).</li>
	<li>Place the tip of the wire&#160;in the proximal-mid portion of the vessel (usually LAD). Perform fluoroscopy&#160;to record wire position.</li>
	<li>Assess and optimize the Doppler and ECG signal quality, if needed. 
	NOTE: This can be done by turning or pulling the wire in order to optimize the wire position. There is also the possibility for fine tuning of the Doppler-signal within the system settings (e.g., optimal tracing and scaling of ECG- and Doppler-signals, wall filter adjustment, etc.).</li>
	<li>Once a good signal is obtained, press Record to record the signals on the system. The patient is now ready for the IDP.</li>
</ol>
5. Carrying out the IDP
<ol>
	<li>Inject 6 mL of the lowest ACh concentration (0.36 &#181;g/mL) into the LCA (~ 2 &#181;g of ACh) within 20 s. Flush with 3-4 mL of saline. Perform continuous 12-lead ECG monitoring and ask the patient for recognizable anginal symptoms (e.g., chest pain, dyspnoea). Observe the Doppler-signal curves and record the average peak velocity (APV) during ACh injection.</li>
	<li>Perform coronary angiography of the LCA after ACh injection by manual injection of approximately 10 mL of contrast agent through the catheter. After every ACh dose, record and print the 12-lead ECG. Ask the patient for recognizable anginal symptoms. Give a 1 min pause between every dose. 
	NOTE: Usually a RAO 20&#176;/ CAUD 30&#176; projection is the best projection for ACh testing.</li>
	<li>Inject 6 mL of the medium ACh concentration (3.6 &#181;g/mL) into the LCA (~ 20 &#181;g of ACh). Inject within 20 s with continuous monitoring of the 12-lead ECG and the patient&#39;s symptoms. Flush with 3-4 mL of saline. Observe the Doppler-signal curves and record the APV during ACh injection. Perform coronary angiography of the LCA after the 6 mL injection of ACh as mentioned above.</li>
	<li>Inject 5.5 mL of the high&#160;ACh concentration (18 &#181;g/mL) into the LCA (~ 100 &#181;g of ACh). Inject within 20 s with continuous monitoring of the ECG and the patient&#39;s symptoms. Flush with 3-4 mL of saline. Observe the Doppler-signal curves and record the APV during ACh injection. Repeat coronary angiography of the LCA as described above. 
	NOTE: In most patients with coronary spasm, symptom reproduction, ECG changes or epicardial vasoconstriction develop at this dose. If bradycardia occurs during ACh injection, this can be resolved by slowing down the speed of the manual ACh injection. A slower injection over a period of 3 min compared to the 20 s injection is also feasible.</li>
	<li>If no epicardial spasm (i.e., &#62; 90% vasoconstriction) occurs at the 100 &#181;g dose continue with the 200 &#181;g of ACh dose (11 mL of the high&#160;ACh concentration (18 &#181;g/mL). Inject within 20 s with continuous monitoring of the ECG and the patient&#39;s symptoms. Flush with 3-4 mL of saline. Observe the Doppler-signal curves and record the APV during ACh injection. Repeat coronary angiography of the LCA. 
	NOTE: Slow down the speed of the manual ACh injection if bradycardia occurs as mentioned above.</li>
	<li>Inject 200 &#181;g of nitroglycerine into the LCA at the end of the ACh test or when severe symptoms (i.e.,&#160;severe angina or dyspnoea), ischemic ECG shifts or epicardial spasm occurs. Perform coronary angiography of the LCA after approximately one minute to document reversion of spasm.</li>
	<li>After the APV returns to the baseline and ECG as well as patient&#39;s symptoms have normalized, perform the next step (i.e., CFR, HMR assessment).</li>
	<li>Press Base to capture baseline values of APV as well as distal (Pd) and aortic (Pa) pressure.</li>
	<li>Quickly inject a bolus of 3.5 mL of the adenosine solution into the LCA (~ 200 &#181;g of adenosine) followed by a brief saline flush (10 mL).&#160;Press the Peak search button 3 heart beats after the injection to initiate peak search (maximal APV and minimal Pd) in order to avoid influences of flushing. The system calculates and displays the values for FFR, CFR and HMR. 
	NOTE: The intracoronary injection of adenosine is well tolerated by the patients with only few side effects such as palpitations.</li>
	<li>Repeat the previous steps (5.8 &#38; 5.9) until 2 concurring measurements have been successfully done. Calculate the mean FFR/CFR/HMR from the values of the measurements.</li>
	<li>Pull back the Doppler flow-/pressure-wire into the left main to check for pressure drift.&#160;In case of a significant pressure drift, recalibrate the pressure sensor of the wire (step 4.5) and repeat the CFR/HMR measurement.</li>
	<li>Pull out the Doppler flow-/pressure-wire and take a final image of the LCA to document that no vessel injury has occurred.</li>
</ol>

<ol>
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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=North+American+Expert+Review+of+Rotational+Atherectomy.">North American Expert Review of Rotational Atherectomy.</a> Circulation. Cardiovascular Interventions. 12 (5), 007448 (2019).</li><li>Nickenig, G., 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=Transcatheter+edge-to-edge+repair+for+reduction+of+tricuspid+regurgitation:+6-month+outcomes+of+the+TRILUMINATE+single-arm+study.">Transcatheter edge-to-edge repair for reduction of tricuspid regurgitation: 6-month outcomes of the TRILUMINATE single-arm study.</a> The Lancet. 394 (10213), 2002-2011 (2019).</li><li>Vakil, 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=Safety+and+efficacy+of+the+MitraClip+system+for+severe+mitral+regurgitation:+a+systematic+review.">Safety and efficacy of the MitraClip system for severe mitral regurgitation: a systematic review.</a> Catheterization and Cardiovascular Interventions. 84 (1), 129-136 (2014).</li><li>Cahill, T. 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Cardiovascular interventions. 13 (16), 1847-1864 (2020).</li><li>Sechtem, U., 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=Coronary+microvascular+dysfunction+in+stable+ischaemic+heart+disease+(non-obstructive+coronary+artery+disease+and+obstructive+coronary+artery+disease).">Coronary microvascular dysfunction in stable ischaemic heart disease (non-obstructive coronary artery disease and obstructive coronary artery disease).</a> Cardiovascular Research. 116 (4), 771-786 (2020).</li><li>Beltrame, J. 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=International+standardization+of+diagnostic+criteria+for+vasospastic+angina.">International standardization of diagnostic criteria for vasospastic angina.</a> European Heart Journal. 38 (33), 2565-2568 (2017).</li><li>Ong, P., 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=International+standardization+of+diagnostic+criteria+for+microvascular+angina.">International standardization of diagnostic criteria for microvascular angina.</a> International journal of cardiology. 250, 16-20 (2018).</li><li>Knuuti, J., 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=2019+ESC+Guidelines+for+the+diagnosis+and+management+of+chronic+coronary+syndromes.">2019 ESC Guidelines for the diagnosis and management of chronic coronary syndromes.</a> European Heart Journal. 41 (3), 407-477 (2020).</li><li>Ford, T. J., 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=Ischemia+and+No+Obstructive+Coronary+Artery+Disease:+Prevalence+and+Correlates+of+Coronary+Vasomotion+Disorders.">Ischemia and No Obstructive Coronary Artery Disease: Prevalence and Correlates of Coronary Vasomotion Disorders.</a> Circulation. Cardiovascular Interventions. 12 (12), 008126 (2019).</li><li>Suda, 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=Coronary+Functional+Abnormalities+in+Patients+With+Angina+and+Nonobstructive+Coronary+Artery+Disease.">Coronary Functional Abnormalities in Patients With Angina and Nonobstructive Coronary Artery Disease.</a> Journal of the American College of Cardiology. 74 (19), 2350-2360 (2019).</li><li>Ford, T. J., 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=Stratified+Medical+Therapy+Using+Invasive+Coronary+Function+Testing+in+Angina:+The+CorMicA+Trial.">Stratified Medical Therapy Using Invasive Coronary Function Testing in Angina: The CorMicA Trial.</a> Journal of the American College of Cardiology. 72 (23), 2841-2855 (2018).</li><li>Ong, P., Athanasiadis, A., Sechtem, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracoronary+Acetylcholine+Provocation+Testing+for+Assessment+of+Coronary+Vasomotor+Disorders.">Intracoronary Acetylcholine Provocation Testing for Assessment of Coronary Vasomotor Disorders.</a> Journal of Visualized Experiments. (114), (2016).</li><li>Sara, J. D., 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=Prevalence+of+Coronary+Microvascular+Dysfunction+Among+Patients+With+Chest+Pain+and+Nonobstructive+Coronary+Artery+Disease.">Prevalence of Coronary Microvascular Dysfunction Among Patients With Chest Pain and Nonobstructive Coronary Artery Disease.</a> JACC. Cardiovascular Interventions. 8 (11), 1445-1453 (2015).</li><li>Kunadian, V., 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+EAPCI+Expert+Consensus+Document+on+Ischaemia+with+Non-Obstructive+Coronary+Arteries+in+Collaboration+with+European+Society+of+Cardiology+Working+Group+on+Coronary+Pathophysiology+&amp;+Microcirculation+Endorsed+by+Coronary+Vasomotor+Disorders+International+Study+Group.">An EAPCI Expert Consensus Document on Ischaemia with Non-Obstructive Coronary Arteries in Collaboration with European Society of Cardiology Working Group on Coronary Pathophysiology &amp; Microcirculation Endorsed by Coronary Vasomotor Disorders International Study Group.</a> European Heart Journal. 41 (37), 3504-3520 (2020).</li><li>Ong, P., 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=High+prevalence+of+a+pathological+response+to+acetylcholine+testing+in+patients+with+stable+angina+pectoris+and+unobstructed+coronary+arteries.+The+ACOVA+Study+(Abnormal+COronary+VAsomotion+in+patients+with+stable+angina+and+unobstructed+coronary+arteries.">High prevalence of a pathological response to acetylcholine testing in patients with stable angina pectoris and unobstructed coronary arteries. The ACOVA Study (Abnormal COronary VAsomotion in patients with stable angina and unobstructed coronary arteries.</a> Journal of the American College of Cardiology. 59 (7), 655-662 (2012).</li><li>Mohri, 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=Angina+pectoris+caused+by+coronary+microvascular+spasm.">Angina pectoris caused by coronary microvascular spasm.</a> The Lancet. 351 (9110), 1165-1169 (1998).</li><li>Sun, H., 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=Coronary+microvascular+spasm+causes+myocardial+ischemia+in+patients+with+vasospastic+angina.">Coronary microvascular spasm causes myocardial ischemia in patients with vasospastic angina.</a> Journal of the American College of Cardiology. 39 (5), 847-851 (2002).</li><li>Waxman, S., Moreno, R., Rowe, K. A., Verrier, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+primary+coronary+dilation+induced+by+transatrial+delivery+of+nitroglycerin+into+the+pericardial+space:+a+novel+approach+for+local+cardiac+drug+delivery.">Persistent primary coronary dilation induced by transatrial delivery of nitroglycerin into the pericardial space: a novel approach for local cardiac drug delivery.</a> Journal of the American College of Cardiology. 33 (7), 2073-2077 (1999).</li><li>Fearon, W. F., Kobayashi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasive+Assessment+of+the+Coronary+Microvasculature:+The+Index+of+Microcirculatory+Resistance.">Invasive Assessment of the Coronary Microvasculature: The Index of Microcirculatory Resistance.</a> Circulation. Cardiovascular Interventions. 10 (12), (2017).</li><li>Om, S. 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The authors declare that they have no conflict of interest.

Management of patients with angina and unobstructed coronary arteries is often demanding and sometimes frustrating. An important step during the work-up of these patients is that the underlying pathophysiological mechanism(s) for the patient&#39;s symptoms are adequately investigated. This is challenging as often not only one mechanism is responsible and various aetiologies including cardiac and non-cardiac as well as coronary and non-coronary need to be taken into account.
Frequently patients with chest pain of unknown origin are scheduled for invasive diagnostic coronary angiography in search of stenosing epicardial coronary disease. Several studies have shown that despite convincing symptoms and abnormal non-invasive stress tests such patients have unobstructed coronary arteries in more than 50% of cases12,18. Although it is correct that the yield of patients with relevant epicardial stenoses needs to be improved it should not be neglected that functional coronary disorders can be responsible for such a clinical presentation. We and others have shown that impaired coronary vasodilatation and/or coronary spasm may account for more than 60% of such cases12,18. Establishing a diagnosis in these often unsettled patients represents an important step in patient management. Thus, it is important to take the opportunity of the diagnostic coronary angiography for further testing. Although this may prolong catheter laboratory time for approximately 30 minutes, establishing a diagnosis may prevent patients from coming back for repeated diagnostic angiography in the future and allow the initiation of targeted pharmacological treatments.
In this context several protocols for an IDP have been developed over the last years. This involves assessment of vasoconstriction/spasm as well as vasodilatation and microvascular resistance. Some centres have added additional assessments to their protocol including measurements of lactate concentrations in coronary sinus blood samples during ACh testing (in search of microvascular spasm)19,20 or performing an ACh re-challenge after documentation of spasm and injection of nitroglycerine to assess the protective effect of nitroglycerine. The latter aspects will be covered in other contributions of this JoVE methods collection.
When discussing critical steps in the protocol presented here the first aspect is the vasodilatory effect of nitroglycerin. As coronary angiography is often performed via the radial artery some medication is usually given to prevent radial artery spasm (e. g., nitroglycerin/verapamil). This may have an impact on subsequent vasomotor testing as studies have shown that nitroglycerine may have an effect on epicardial tone for up to 15-20 minutes21. However, a study comparing the effects of any radial artery spasm prophylaxis on ACh testing has not been published so far. In this context it is also debatable when to perform ACh testing (i. e., before or after FFR/CFR/HMR testing). If ACh testing is performed after FFR/CFR/HMR testing,&#160;the vasodilatory effects of nitroglycerine may still be present and influence the results of ACh testing14. This is why it is recommended to perform ACh testing before FFR/CFR/HMR testing. However, there has been no direct comparison of these two protocols yet.
Another critical step in the protocol is the use and the positioning of the Doppler flow-/pressure-wire. To avoid any intravascular complications the wire should be placed with caution and ideally in the proximal-mid part of the vessel. For an application in patients with intermediate stenoses especially in the distal portion of the vessel placement with a microcatheter may be advisable. Although the Doppler flow-/pressure-wire has the advantage that a direct Doppler-signal can be heard and seen on the screen obtaining a good signal may sometimes be challenging. A combination of turning and pulling the wire as well as fine tuning with the remote control (e.g., adjustment of scale factor, curve detection and wall filter) solves the problem in most cases.
One important limitation of the method lies in the fact that only the LCA is tested with this protocol. The reason for testing the LCA as the default artery is that two vessels can be challenged at the same time. Nevertheless, in the rare cases in whom the IDP reveals no abnormality in the LCA the RCA should be assessed. Another limitation is that the assessment of microvascular resistance is a rather novel approach and, thus, optimal cut-off values in patients with unobstructed coronary arteries are still a matter of debate. Depending on the method used, either the index of microvascular resistance (IMR; thermodilution method) or the HMR (Doppler technique) is provided. Currently used cut-off values for the diagnosis of microvascular dysfunction are &#62; 25 for IMR22 and &#62; 1.917 or &#62; 2.47 for HMR.
The IDP as presented in this article represents one of the most comprehensive forms of coronary vasomotor testing. A major advantage in comparison to non-invasive testing protocols lies in the fact that non-invasive protocols usually are not able to assess coronary spasm. Although it has been suggested to be feasible in a recent publication from Korea23 there is still a lot of scepticism regarding patient safety as multivessel spasm during non-invasive ergonovine testing may not be adequately controlled. It can be expected that future randomized clinical trials continue to demonstrate the usefulness of the IDP in conjunction with stratified medical therapy. Moreover, the IDP represents the perfect platform for evaluation of new pharmacological agents for treatment of the different endotypes of coronary vasomotor disorders.

In recent years interventional cardiology has made substantial progress in various areas. This not only comprises interventional treatment of the heart valves using transcatheter aortic valve replacement and edge-to-edge repair of the mitral and tricuspid valve, but also coronary interventions1,2,3,4,5,6. Among the latter are advances in techniques for treatment of chronic total occlusions as well as calcified lesions using rotablation and shock wave therapy. In addition to these rather structural coronary interventional procedures invasive diagnostic procedures (IDP) have now been established in search of functional coronary disorders (i.e., coronary spasm and microvascular dysfunction)7. The latter comprise a heterogeneous group of conditions frequently but not exclusively occurring in patients with angina pectoris and unobstructed coronary arteries. The main mechanisms underlying these vasomotor disorders are impaired coronary vasodilatation, enhanced vasoconstriction/spasm as well as enhanced coronary microvascular resistance. The latter is often due to obstructive microvascular disease8. Anatomically, coronary vasomotor disorders may occur in the epicardial arteries, the coronary microcirculation or both. The Coronary Vasomotor Disorders International Study group (COVADIS) has published definitions for the diagnosis of these disorders9,10 and recent guidelines of the European Society of Cardiology (ESC) on the management of patients with chronic coronary syndrome have made recommendations for adequate patient assessment depending on the clinical condition11. Moreover, recent publications have delineated the various endotypes that can be derived from an IDP12,13. Such an approach has a benefit for the individual patient as randomized studies have shown better quality of life in patients undergoing an IDP followed by stratified medical therapy according to the test result compared to usual care by the general practitioner14. Currently, there is a debate about the most appropriate protocol for testing of such vasomotor disorders. The aim of this article is to describe a protocol where acetylcholine (ACh) provocation testing in search of coronary spasm is followed by Doppler wire-based assessment of coronary flow reserve (CFR) and hyperemic microvascular resistance (HMR) using adenosine (Figure 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cannula 0,95 x 50 mm (arterial punction)</td>
			<td>BBraun</td>
			<td>4206096</td>
			<td></td>
		</tr>
		<tr>
			<td>Cannula 23 G 0,6 x 25 mm (local anesthesia)</td>
			<td>BBraun</td>
			<td>4670025S-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Coronary angiography suite (AXIOM Artis MP eco)</td>
			<td>Siemens</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Contrast agent Imeron 350 with a 10 mL syringe for contrast injection</td>
			<td>Bracco Imaging</td>
			<td>30699.04.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Diagnostic catheter (various manufacturers)</td>
			<td>e.g. Medtronic</td>
			<td>DXT5JR40</td>
			<td></td>
		</tr>
		<tr>
			<td>Glidesheath Slender 6 Fr</td>
			<td>Terumo</td>
			<td>RM*RS6J10PQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin 5,000 IU (25,000 IU / 5 mL)</td>
			<td>BBraun</td>
			<td>1708.00.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Mepivacaine 10 mg/mL</td>
			<td>PUREN Pharma</td>
			<td>11356266</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride solution 0.9 % (1 x 100 mL)</td>
			<td>BBraun</td>
			<td>32000950</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 2 mL (1x) (local anesthesia)</td>
			<td>BBraun</td>
			<td>4606027V</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 10 mL (1x) (Heparin)</td>
			<td>BBraun</td>
			<td>4606108V</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetylcholine chloride (vial of 20 mg acetylcholine chloride powder and 1 ampoule of 2 mL diluent)</td>
			<td>Bausch &#38; Lomb</td>
			<td>NDC 240208-539-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Cannula 20 G 70 mm (2x)</td>
			<td>BBraun</td>
			<td>4665791</td>
			<td></td>
		</tr>
		<tr>
			<td>Glyceryle Trinitrate 1 mg/mL (5 mL)</td>
			<td>Pohl-Boskamp</td>
			<td>07242798</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride solution 0.9 % (3 x 100 mL)</td>
			<td>BBraun</td>
			<td>32000950</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 2 mL (1x)</td>
			<td>BBraun</td>
			<td>4606027V</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 5 mL (5x)</td>
			<td>BBraun</td>
			<td>4606051V</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 10 mL (1x)</td>
			<td>BBraun</td>
			<td>4606108V</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 50 mL (3x)</td>
			<td>BBraun</td>
			<td>4187903</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 6 mg/2 mL</td>
			<td>Sanofi-Aventis</td>
			<td>30124.00.00</td>
			<td></td>
		</tr>
		<tr>
			<td>ComboMap Pressure/Flow System</td>
			<td>Volcano</td>
			<td>Model No. 6800 (Powers Up)</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure/Flow Guide Wire</td>
			<td>Volcano</td>
			<td>9515</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride solution 0.9 % (1 x 100 mL)</td>
			<td>BBraun</td>
			<td>32000950</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 10 mL (3x)</td>
			<td>BBraun</td>
			<td>4606108V</td>
			<td></td>
		</tr>
	</tbody>
</table>

testing acetylcholine followed by adenosine for invasive diagnosis of coronary vasomotor disorders

More than 50% of patients with signs and symptoms of myocardial ischemia undergoing coronary angiography have unobstructed coronary arteries. Coronary vasomotor disorders (impaired vasodilatation and/or enhanced vasoconstriction/spasm) represent important functional causes for such a clinical presentation. Although impaired vasodilatation may be assessed with non-invasive techniques such as positron emission tomography or cardiac magnetic resonance imaging, there is currently no reliable non-invasive technique for the diagnosis of coronary spasm available. Thus, invasive diagnostic procedures (IDP) have been developed for the diagnosis of coronary vasomotor disorders including spasm testing as well as assessment of coronary vasodilatation. The identification of the underlying type of disorder (so called endotype) allows the initiation of targeted pharmacological treatments. Despite the fact that such an approach is recommended by the current European Society of Cardiology guidelines for the management of chronic coronary syndromes based on the CorMicA study, comparability of results as well as multicenter trials are currently hampered by major differences in institutional protocols for coronary functional testing. This article describes a comprehensive IDP protocol including intracoronary acetylcholine provocation testing for diagnosis of epicardial/microvascular spasm, followed by Doppler wire-based assessment of coronary flow reserve (CFR) and hyperemic microvascular resistance (HMR) in search of coronary vasodilatory impairment.

According to the diagnostic criteria suggested by COVADIS9, vasospastic angina can be diagnosed if the following criteria apply during ACh provocation testing: transient ECG changes indicating ischemia, reproduction of the patient&#180;s usual anginal symptoms and &#62; 90% vasoconstriction of an epicardial vessel&#160;as confirmed during coronary angiography (Figure 2).
Spasm of the coronary microvasculature can be diagnosed if the patient&#180;s symptoms and ischemic ECG alterations occur during provocation testing in the absence of epicardial vasospasm10 (Figure 3).
Impaired microvascular vasodilatation can be diagnosed by interpreting the CFR and HMR measurements following adenosine injections. Depending on the cut-off values applied, a reduced CFR is defined as &#60; 2.012,13 or &#8804; 2.516, respectively (Figure 4). For HMR, data on optimal cut-off values is scarce, but an increased microvascular resistance is currently defined as a HMR &#62; 1.917 or &#62; 2.47 (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62134/62134fig01.jpg" /> 
Figure 1: Flow chart of the Invasive Diagnostic Procedure. After exclusion of any epicardial stenosis during diagnostic angiography, the vasoconstrictive potential of the coronary arteries is tested by intracoronary injection of incremental doses of ACh. After spasm provocation testing, assessment of vasodilatation by intracoronary injection of adenosine is performed, followed by measurement of CFR and HMR. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62134/62134fig01large.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/62134/62134fig02.jpg" /> 
Figure 2: 58-year old female patient with diffuse epicardial spasm during ACh provocation testing. A) Baseline measurement before ACh injection showing neither stenosis nor ischemic ECG changes. B) Diffuse epicardial spasm of the LAD after intracoronary injection of 200 &#181;g ACh into the left main, accompanied by T-inversion in lead aVL and descending ST-depression in leads I and V2-V6 (red arrows)&#160;during reproduction of patient&#180;s symptoms. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62134/62134fig02large.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/62134/62134fig03.jpg" /> 
Figure 3: 61-year old female patient with microvascular spasm during ACh provocation testing. A) Baseline measurement before ACh injection showing neither stenosis nor ischemic ECG changes. B) Minor vasoconstriction of epicardial vessels after intracoronary injection of 100 &#181;g ACh into the left main. The patient experienced her usual symptoms, going along with ST-segment depression in leads II, V4-V6 (red arrows). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62134/62134fig03large.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/62134/62134fig04.jpg" /> 
Figure 4: Assessment of vasodilatation by measurement of CFR. After injection of adenosine, APV increased insufficiently from 36 cm/s at rest (A) by approx. 50% to 55 cm/s (B), leading to a pathological CFR of 1.5. Measurements to be performed until two concurring readings are obtained (additional measurements not shown); CFR equates to mean of measurements. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62134/62134fig04large.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/62134/62134fig05.jpg" /> 
Figure 5: Assessment of vasodilatation by measurement of HMR. For HMR calculation, average peak velocity (APV) and distal coronary artery pressure (Pd) are measured after injection of adenosine, leading to a pathological HMR of 2.3. Measurements to be performed until two concurring readings are obtained (additional measurements not shown); HMR equates to mean of measurements. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62134/62134fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Testing Acetylcholine Followed by Adenosine for Invasive Diagnosis of Coronary Vasomotor Disorders at JoVE.com

Testing Acetylcholine Followed by Adenosine for Invasive Diagnosis of Coronary Vasomotor Disorders

Coronary vasomotion disorders represent frequent functional causes of angina in patients with unobstructed coronaries. The underlying mechanism of angina (endotype) in these patients can be determined by a comprehensive invasive diagnostic procedure based on acetylcholine provocation testing followed by Doppler-derived assessment of the coronary flow reserve and microvascular resistance.

More than 50% of patients with signs and symptoms of myocardial ischemia undergoing coronary angiography have unobstructed coronary arteries. Coronary ...

testing-acetylcholine-followed-adenosine-for-invasive-diagnosis

Nanyang Technological University

Research

JoVE Journal

Medicine

3.2K Views.  Robert Bosch Hospital. Coronary vasomotion disorders represent frequent functional causes of angina in patients with unobstructed coronaries. The underlying mechanism of angina (endotype) in these patients can be determined by a comprehensive invasive diagnostic procedure based on acetylcholine provocation testing followed by Doppler-derived assessment of the coronary flow reserve and microvascular resistance.

Video: Testing Acetylcholine Followed by Adenosine for Invasive Diagnosis of Coronary Vasomotor Disorders

FISH for Pre-implantation Genetic Diagnosis

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a cohort generated by assisted reproduction technology (ART). This selection may be required because of familial monogenic disease (e.g. cystic fibrosis), or because one partner carries a chromosome rearrangement (e.g. a two-way reciprocal translocation). PGD is available for couples who have had previous affected children, and/or in the case of chromosome rearrangements, recurrent miscarriages, or infertility. Oocytes aspirated following ovarian stimulation are fertilized by in vitro immersion in semen (IVF) or by intracytoplasmic injection of an individual spermatozoon (ICSI). Pre-implantation cleavage-stage embryos are biopsied, usually by the removal of a single cell on day 3 post-fertilization, and the biopsied cell is tested to establish the genetic status of the embryo. Fluorescence in situ hybridization (FISH) on the fixed nuclei of biopsied cells with target-specific DNA probes is the technique of choice to detect chromosome imbalance associated with chromosome rearrangements, and to select female embryos in families with X-linked disease for which there is no mutation-specific test. FISH has also been used to screen embryos for spontaneous chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, the predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people's hands and it is not recommended for routine clinical use. We describe the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to PGD in a clinical setting.

This article describes the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to pre-implantation genetic diagnosis (PGD) in a clinical setting.

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

Intracoronary Acetylcholine Provocation Testing for Assessment of Coronary Vasomotor Disorders

Intracoronary acetylcholine provocation testing (ACH-test) is an established method for assessment of epicardial coronary artery spasm in the catheterization laboratory which was introduced more than 30 years ago. Due to the short half-life of acetylcholine it can only be applied directly into the coronary arteries. Several studies have demonstrated the safety and clinical usefulness of this test. However, acetylcholine testing is only rarely applied in the U.S. or Europe. Nevertheless, it has been shown that 62% of Caucasian patients with stable angina and unobstructed coronary arteries on coronary angiography suffer from coronary vasomotor disorders that can be diagnosed with acetylcholine testing. In recent years it has been appreciated that the ACH-test not only assesses the presence of epicardial spasm but that it can also be useful for the detection of coronary microvascular spam. In such cases no epicardial spasm is seen after injection of acetylcholine but ischemic ECG shifts are present together with a reproduction of the patient's symptoms during the test. This article describes the experience with the ACH-test and its implementation in daily clinical routine.

Intracoronary acetylcholine testing has been established for the assessment of epicardial coronary spasm more than 30 years ago. Recently, the focus has shifted towards the microcirculation and it has been shown that microvascular spasm can be detected using ACH-testing. This article describes the ACH-test and its implementation in daily routine.

Intracoronary acetylcholine provocation testing (ACH-test) is an established method for assessment of epicardial coronary artery spasm in the ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

Acetylcholine Re-Challenge After Intracoronary Nitroglycerine Administration

Coronary artery spasm (CAS) can be diagnosed in a large proportion of patients with recurrent angina with non-obstructive coronary artery disease (ANOCA) using acetylcholine (ACh) spasm provocation testing. CAS can further be divided into different subtypes (e.g., focal, diffuse epicardial, or microvascular spasm), each with different pathophysiological mechanisms that may require tailored drug treatment. The evidence behind the role of nitrates in the setting of each CAS subtype is lacking, and the effectivity can vary on a per-patient basis. In order to assess on a per-patient level whether nitroglycerine (NTG) can prevent inducible spasm, the vasospastic ACh dose can be readministered after NTG administration as part of the spasm provocation test. The preventive effect of NTG is assessed by evaluating improvements in the severity of induced symptoms, ischemic ECG changes, and by reassessing the site and mode of spasm on angiography. This technique can therefore be used to assess the nitrate responsiveness on a per-patient level and unmask co-existing microvascular spasm in patients with epicardial spasm that is prevented with NTG. The NTG rechallenge, therefore, allows to further guide targeted therapy for CAS and provide new insights into the pathophysiological mechanism behind vasospastic disorders.

This protocol presents the acetylcholine rechallenge after nitroglycerine as an add-on procedure to spasm provocation testing. The purpose of this technique is to unmask co-existing microvascular spasm&#160;in patients with epicardial spasm&#160;and to assess the protective efficacy of nitroglycerine on a per-patient level to guide medical therapy.

Coronary artery spasm (CAS) can be diagnosed in a large proportion of patients with recurrent angina with non-obstructive coronary artery disease (ANOCA) ...

Measurement of Myocardial Lactate Production for Diagnosis of Coronary Microvascular Spasm

In about a quarter of patients with angina and non-obstructive coronary arteries, no epicardial spasm is noted on coronary arteriography during an angina attack. Since the pressure-rate product is almost identical at rest and the onset of attack in those patients, the decrease in coronary blood flow rather than increased myocardial oxygen consumption is likely to explain myocardial ischemia, indicating a substantial involvement with coronary microvascular spasm (MVS). Myocardial lactate production, which could be defined as a negative myocardial lactate extraction ratio (ratio of the coronary arterial-venous difference in lactate concentration to arterial concentration), is considered indicative of objective evidence to support the emerging myocardial ischemia. Thus, monitoring of the myocardial lactate production and the emergence of chest pain and ischemic electrocardiographic changes during acetylcholine (ACh) provocation testing is of significant value for detecting the entity of MVS. Practically, 1 min after incremental doses of ACh (20, 50, and 100 &#956;g) are administered into the left coronary artery (LCA), paired samples of 1 mL of blood are collected from the LCA ostium and coronary sinus for measurement of lactate concentration by a calibrated automatic lactate analyzer. Then, the development of MVS could be confirmed by negative myocardial lactate extraction ratio despite the absence of angiographically demonstrable epicardial coronary spasm or before its occurrence throughout ACh provocation testing. In conclusion, assessment of myocardial lactate production is essential and valuable for the diagnosis of MVS.

Myocardial lactate production (coronary arterial-venous difference in serum lactate level) during coronary spasm provocation testing is considered as a highly sensitive marker that reflects acetylcholine-induced myocardial ischemia due to microvascular spasm. This article presents the procedures to assess myocardial lactate production for the diagnosis of coronary microvascular spasm.

In about a quarter of patients with angina and non-obstructive coronary arteries, no epicardial spasm is noted on coronary arteriography during an angina ...

Basophil Activation Test for Allergy Diagnosis

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and specific IgE (sIgE) determination in the evaluation of IgE-mediated allergic reactions to food, insect venom, drugs, as well as some forms of chronic urticaria. However, the role of this technique in the diagnostic algorithms is highly variable and not well determined.
BAT is based on the determination of basophil response to allergen/drug cross-linking IgE activation through the measurement of activation markers (such as CD63, CD203c) by flow cytometry. This test can be a useful and complementary tool to avoid controlled challenge tests&#160;to confirm allergy diagnosis, especially in subjects experiencing severe life-threatening reactions. In general, the performance of BAT should be considered if i) the allergen/drug produces false positive results in ST; ii) there is no allergen/drug source to use for ST or sIgE determination; iii) there is discordance between patient history and ST or sIgE determination; iv) symptoms suggest that ST may result in systemic response; v) before considering a CCT to confirm the culprit allergen/drug. The main limitations of the test are related to non-optimal sensitivity, especially in drug allergy, the need to perform the test no longer than 24 h after sample extraction, and the lack of standardization between laboratories in terms of procedures, concentrations, and cell markers.

The basophil activation test is a complementary in vitro diagnostic test for the evaluation of IgE-mediated allergic reactions based on the detection of basophil activation in the presence of a specific stimulus through the measure of activation markers by flow cytometry.

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and ...

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the three-dimensional (3D) architecture of the organ at the whole-organ level. Liver architecture is characterized by multiple branching lumenized structures, which can be filled with resin, including vascular and biliary trees, establishing a highly stereotyped pattern in the otherwise hepatocyte-rich parenchyma. This protocol describes the pipeline for performing double resin casting micro-computed tomography, or &#34;DUCT&#34;. DUCT entails injecting the portal vein and common bile duct with two different radiopaque synthetic resins, followed by tissue fixation. Quality control by clearing one lobe, or the entire liver, with an optical clearing agent, allows for pre-screening of suitably injected samples. In the second part of the DUCT pipeline, a lobe or the whole liver can be used for micro-computed tomography (microCT) scanning, (semi-)automated segmentation, and 3D rendering of the portal venous and biliary networks. MicroCT results in 3D coordinate data for the two resins allowing for qualitative as well as quantitative analysis of the two systems and their spatial relationship. DUCT can be applied to postnatal and adult mouse liver and can be further extended to other tubular networks, for example, vascular networks and airways in the lungs.

Double resin casting micro-computed tomography, or DUCT, enables visualization, digitalization, and segmentation of two tubular systems simultaneously to facilitate 3D analysis of organ architecture. DUCT combines ex vivo injection of two radiopaque resins followed by micro-computed tomography scanning and segmentation of the tomographic data.