We thank Claudia Corazza for her invaluable English language support. This work was supported by Institute of Health &#39;&#39;Carlos III&#39;&#39; (ISCIII) of MINECO (grant cofunded by ERDF: &#34;Una manera de hacer Europa&#34;; Grants Nos. PI20/01715; PI18/00095; PI17/01410; PI17/01318; PI17/01237 and RETIC ARADYAL RD16/0006/0001; Andalusian Regional Ministry of Health (Grant Nos. PI-0127-2020, PIO-0176-2018; PE-0172-2018; PE-0039-2018; PC-0098-2017; PI-0075-2017; PI-0241-2016). ID is a Clinical Investigator (B-0001-2017) and AA holds a Senior Postdoctoral Contract (RH-0099-2020), both supported by Andalusian Regional Ministry of Health (cofunded by ESF: &#34;Andaluc&#237;a se mueve con Europa&#34;).

The protocol performance was conducted according to the Declaration of Helsinki principles and approved by the local Ethics Committee (Comit&#233; de &#201;tica para la Investigaci&#243;n Provincial de M&#225;laga, Spain). All subjects were informed orally about the research study and they signed the corresponding informed consent form.
NOTE: The present protocol details the BAT procedure that the authors use daily. However, this is not a standardized method and differences with procedures published by other authors exist. The main protocol modifications are related to the use of IL-3 in the stimulation buffer, incubation time with the stimulus, method for stopping basophil degranulation, and flow cytometry strategies. Moreover, different commercially available kits for BAT include specific protocols recommended by the manufacturer.
1. Sample preparation
<ol>
	<li>Collect peripheral blood in 9 mL heparinized tubes and maintain the sample at room temperature (RT) in a rotor until it is required for the experimental protocol.</li>
	<li>Label 5 mL cytometer tubes for negative controls (2 tubes), positive controls (2 tubes) and different concentrations of allergen/drug (1 tube per each allergen/drug concentration tested). Place the tubes in a rack where tubes fit perfectly without slipping.</li>
	<li>Prepare stimulation buffer in double distilled water containing 2% (v/v) HEPES, 78 mg/L NaCl, 3.7 mg/L KCl, 7.8 mg/L CaCl2, 3.3 mg/L MgCl2, 1 g/L HSA. Adjust pH to 7.4 and add IL-3 at 2 ng/mL. Usually prepare 100 mL and divide in 2.5 mL aliquots to be frozen at -20 &#176;C.</li>
	<li>Prepare positive controls in PBS-Tween-20 0.05% (v/v) (PBS-T): Positive control 1, N-Formylmethionyl-leucyl-phenylalanine (fMLP) (4 &#181;M), to confirm quality of basophils; Positive control 2, Anti-IgE (0.05 mg/mL) as a IgE-mediated positive control.</li>
	<li>Prepare the allergen/drug in PBS-T at 2x the desired final concentration. 
	&#8203;NOTE: Optimal allergen/drug concentrations to be used must be previously determined by using a wide range of concentrations, by dose-response curves, and by cytotoxicity studies following the same protocol steps139.</li>
</ol>
2. Staining mix preparation
<ol>
	<li>Add monoclonal antibodies labelled with fluorochrome to the stimulation buffer following manufacturer&#180;s recommended antibody concentration or by previous antibody titration. In this protocol we add 1 &#181;L of each antibody (CCR3-APC and CD203c-PE for basophil identification; CD63-FITC for basophil activation)140 per 20 &#181;L of stimulation buffer. 
	NOTE: Protect the staining mix preparation from the light.</li>
	<li>Add 23 &#181;L of staining mix to each tube.</li>
</ol>
3. Blood stimulation
<ol>
	<li>Add 100 &#181;L of PBS-T to tubes 1 and 2 (negative control), 100 &#181;L of fMLP to tube 3, 100 &#181;L of anti-IgE to tube 4, and 100 &#181;L of the different allergen/drug concentrations to the following tubes. Incubate for 10 min at 37 &#176;C in a thermostatic bath with medium agitation to prewarm reagents.</li>
	<li>Gently add 100 &#181;L of blood to each tube to avoid hemolysis. Gently vortex tubes and incubate for 25 min at 37 &#176;C in a thermostatic bath with medium agitation.</li>
	<li>Stop the degranulation, keeping tubes at 4 &#176;C for at least 5 min. 
	&#8203;NOTE: The protocol can be paused here at 4 &#176;C for 30-45 min if required141,142,143.</li>
</ol>
4. Erythrocytes lysing
<ol>
	<li>Add 2 mL of 1x lysing buffer to each tube to lyse erythrocytes. Vortex each tube and incubate for 5 min at RT. 
	NOTE: In this step, cells are fixed due to fixative agents (formaldehyde) contained in the buffer.</li>
	<li>Centrifuge at 300 x g at 4 &#176;C for 5 min. Decant the supernatant, overturning the rack into a sink. Cells remain at the bottom of the tubes.</li>
	<li>Add 3 mL of PBS-T to each tube to wash cells. Vortex each tube.</li>
	<li>Centrifuge at 300 x g at 4 &#176;C for 5 min. Decant the supernatant, overturning the rack into a sink. 
	&#8203;NOTE: Keep samples at 4 &#176;C, protected from the light until flow cytometer acquisition.</li>
</ol>
5. Flow cytometry acquisition
<ol>
	<li>Acquire samples by flow cytometer (e.g., BD FACSCalibur Flow Cytometer). Connect the flow cytometer to the computer software and wait for the cytometer to be ready. Load the template and instrument settings (Table 1).</li>
	<li>Start sample acquisition.</li>
	<li>Use the following cytometer strategies for selection of activated basophils139.
	<ol>
		<li>Gate the lymphocytes from the Side Scatter (SSC) - Forward Scatter (FSC) plot.</li>
		<li>Gate the basophils from the lymphocyte population as CCR3+CD203c+ cells. Acquire at least 500 basophils per tube.</li>
		<li>Show a CCR3 - CD63 plot to analyze activation using CD63 as activation marker. Set the CD63 negative threshold to approximately 2.5% using the negative control tubes.</li>
		<li>Acquire all the samples.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

BAT is a complementary in vitro diagnostic test for the evaluation of IgE-mediated allergic reactions that has shown to be useful in the diagnosis of reactions induced by different triggers such as drugs, food, or inhalants, as well as in some forms of chronic urticaria. In general, BAT performance should be considered if i) the allergen/drug produces false positive results in ST; ii) the allergen/drug is not available to be used for ST or sIgE quantification; iii) discordance between clinical history and ST or ...

IgE-mediated allergy diagnosis is based on clinical history, skin tests (STs), quantification of serum specific IgE (sIgE), and, if it is required and indicated, controlled challenge tests (CCTs)1,2,3,4,5,6. However, clinical history can be unreliable since there may be a lack of accurate information, and STs and CCTs are not risk-free procedures that can be contraindicated in subjects experiencing severe life-threatening reactions1,2,3,4,5,6. These issues, together with the fact that the determination of sIgE by validated and commercial fluoro-enzyme immunoassays is only available for few allergens and drugs, have highlighted the important role of other in vitro functional assays such as basophil activation test (BAT).
Basophils are key effector cells involved in IgE-mediated allergic reactions that are activated upon cross-linking of adjacent sIgE bound on high-affinity receptors (Fc&#949;RI) on cell surface after allergen/drug exposure. Basophil activation triggers cell degranulation and release of pre-formed and new synthesized inflammatory mediators contained in intracytoplasmatic secretion granules7,8,9. BAT is an in vitro method that tries to mimic the basophil activation in the presence of a stimulus (allergen or drug) and determines changes in the expression of basophil activation markers by flow cytometry7,10. There are different strategies to identify basophils (IgE+, CCR3+, CRTH2+, CD203c+) and to measure cell activation (mainly upregulation of CD63 and CD203c) using combinations of fluorochrome-labelled antibodies7,10. CD63, the best clinically validated activation marker11,12,13,14, is a membrane protein anchored at the secretory granules containing histamine that, after cell activation and fusion of the granules with the membrane, is expressed on basophil surface15,16,17,18,19,20,21. CD203c is a surface marker that is constitutively expressed on basophils and upregulated after Fc&#949;RI stimulation, which has also shown reliable results in BAT15,22,23,24,25. Besides, it appears to co-express with CD6326.
In the last decades, BAT has shown to be useful in the diagnosis of IgE-mediated allergic reactions induced by different triggers like drugs, food, or inhalants, as well as in some forms of chronic urticaria, as described below. However, the position of this technique in the diagnostic algorithms is highly variable and not well determined.
Drug hypersensitivity 
BAT has shown to be useful as a complementary test for selected drugs and patients, particularly for those who experience severe reactions due to the fact that diagnostic value of ST is not well established for most drugs, as they are validated and standardized for a limited number of drugs27,28,29,30. In addition, quantification of sIgE is available only for a limited number of drugs, with lower sensitivity than ST27,28,29,30,31,32. Therefore, the diagnosis of drug hypersensitivity usually relies upon drug provocation test, which can be contraindicated in subjects experiencing severe life-threatening reactions33.
Promising results have been reported for the use of BAT in selected patients reporting immediate hypersensitivity reactions to different drugs like betalactams (BLs)20,34,35,36,37,38,39, neuromuscular blocking agents (NMBAs)19,22,40,41,42,43,44,45, fluoroquinolones (FQs)46,47,48,49, pyrazolones50,51,52, radiocontrast media (RCM)53,54,55,56, and platinum compounds57,58,59. BAT has been reported to have a sensitivity and specificity between 51.7-66.9% and 89.2-97.8%, respectively; and positive and negative predictive values are described to range between 93.4% and 66.3%, respectively27,31. Moreover, BAT has been proposed as a predictive biomarker for breakthrough reactions during desensitization with platinum compounds, as CD203c expression is increased compared to CD63 in patients with high risk of adverse reactions during drug desensitization57.
It is of note that BAT is only useful in drug hypersensitivity when the reaction involves basophil degranulation; therefore, it is not useful in reactions resulting from enzymatic inhibition of cyclooxygenase 142.
Food allergy 
BAT has emerged as a potential diagnostic tool for food allergy because determination of serum sIgE to the whole allergen extract or single allergens is often ambiguous, requiring oral food challenge to confirm the diagnosis, which, similarly to drug hypersensitivity, is a costly and not risk-free procedure60. Several studies have shown relevant results with cow&#39;s milk61,62, egg61,63, wheat64,65,66,67,68, peanut63,69,70,71,72, hazelnut73,74,75,76,77, shellfish78, peach79,80,81, apple21, celery, and carrot82,83.
The main added value of BAT in the diagnosis of food allergy compared to STs and sIgE in serum is that it shows a higher specificity and similar sensitivity. Thus, BAT is a useful tool to differentiate clinically allergic patients from sensitized, but tolerant, subjects that have both high specificity (75-100%) and sensitivity (77-98%)63,69,84. Sensitivity and specificity values depend on the allergen and other factors as phenotypes (e.g., oral allergy syndrome versus anaphylaxis), age, and geography-related sensitization patterns63,85.
BAT using single allergen components can potentially improve diagnostic accuracy for some food allergens61,80. There are studies using seed storage proteins (e.g., Ara h 1, Ara h 2, Ara h 3 and Ara h 6 from peanut)86; lipid transfer proteins (e.g., Pru p 3 from peach and Ara h 9 from peanut)80,86; and Bet v 1 homologues (e.g., Ara h 8 from peanut)87. Other potential utilities are related to the identification of the culprit allergen in cases of pollen-food allergy syndrome21,87,88, allergy to red meat89, or food-dependent exercise-induced anaphylaxis66.
Interestingly, BAT can provide information about the severity and the threshold of allergic reactions since patients with more severe reactions show a greater proportion of activated basophils, as observed in studies of peanut and cow&#39;s milk-allergic patients84,90,91; and patients reacting to trace amounts of the allergen show a greater basophil sensitivity84,90,92. These data suggest that BAT may be useful to identify high-risk allergic patients who require closer follow-up and more intensified education93. Moreover, it has been reported that BAT can predict food challenge responses70,91,92,94 and thresholds of reactivity90,95 to help determine when food can be safely (re)introduced84. However, these findings are controversial in some studies63,96 and more research are required.
On the other hand, BAT has been used to monitor resolution of food allergy, either naturally or under immunomodulatory treatments, over time, which until now has been only assessed by oral food challenge, with the associated risks and costs84,97,98,99,100,101,102,103,104,105,106,107,108. Moreover, it has also been used to monitor the effect of omalizumab in food allergy as basophil activation decreases during treatment with omalizumab, but it increases after cessation of treatment109.
Inhalant allergy 
BAT is rarely beneficial in inhalant allergy as diagnosis can be routinely established by sIgE quantification and ST. However, in cases of local allergic rhinitis (undetectable levels of sIgE and negative STs with positive nasal provocation tests), BAT has allowed diagnosis in 50% of cases110. It has been further reported a correlation between basophil sensitivity and response to nasal/bronchial provocation tests, as well as between asthma severity and efficacy of treatment with omalizumab111,112.
BAT has also been used to monitor allergen immunotherapy for house dust mite and pollens, as basophil sensitivity decreases during immunotherapy, likely due to interference of blocking IgG antibodies113,114,115,116,117.
Hymenopteravenom allergy 
Diagnosis of hymenoptera venom allergy is routinely based on ST and serum sIgE. BAT has shown a high sensitivity (85-100%) and specificity (83-100%) and it has been reported to be useful in cases that yield equivocal results or in patients with a suggestive clinical history of venom allergy but undetectable sIgE and negative ST118,119. However, BAT appears not to be predictive of severity for these reactions120,121.
Up to 60% of patients exhibit sIgE to both wasp and bee venom, and the identification of dominant allergen is crucial for adequate immunotherapy treatment. In these cases, BAT has been reported to be useful in the identification of the dominant allergen119,122,123,124. Although sIgE to the major allergens of bee and wasp venoms may reduce the utility of BAT in patients with double positivity to both venoms, it provides useful information mainly in subjects with negative results in sIgE determinations123.
Some studies suggest that BAT may be useful as a predictive biomarker for side effects during the build-up phase of venom immunotherapy as this treatment option has been reported to decrease basophil sensitivity. However, reactivity does not decrease and this BAT utility is nowadays controversial13,120,125,126,127,128,129,130.
Urticaria and angioedema 
A subset of chronic urticaria patients have an autoinmune pathophysiology, due to IgE autoantibodies to autoallergens and IgG autoantibodies that target Fc&#949;RI or IgE-Fc&#949;RI complexes present on the mast cell surface131,132. In clinical practice, diagnosis of this type of chronic urticaria has relied on positive autologous serum ST, which has risk of accidental infection. BAT has been proposed as an in vitro test for diagnosing and monitoring patients with suspected chronic urticaria. Both CD63 and CD203c expression on the surface of basophils has been reported to be increased following stimulation with sera from chronic urticaria patients, showing the detection of active autoantibodies133,134,135,136,137. Recently, it has been reported that patients with positive BAT often experience the most active disease state, assessed by urticaria activity score, and require higher doses of antihistamines together with third-line treatments (cyclosporine A or omalizumab), compared to those with negative BAT138.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5 mL Round Bottom Polystyrene Test Tube, without Cap, Nonsterile</td>
			<td>Corning</td>
			<td>352008</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-human CD193 (CCR3) Antibody</td>
			<td>BioLegend</td>
			<td>310708</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSCalibur Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C1016</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-human CD63 Antibody</td>
			<td>BioLegend</td>
			<td>353006</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Thermo-Fisher</td>
			<td>15630106</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysing Solution 10x concentrated</td>
			<td>BD Biosciences</td>
			<td>349202</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Formyl-Met-Leu-Phe</td>
			<td>Sigma-Aldrich</td>
			<td>F3506</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-human CD203c (E-NPP3) Antibody</td>
			<td>BioLegend</td>
			<td>324606</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified Mouse Anti-Human IgE</td>
			<td>BD Biosciences</td>
			<td>555857</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IL-3</td>
			<td>R&#38;D Systems</td>
			<td>203-IL</td>
			<td></td>
		</tr>
		<tr>
			<td>Sheath Fluid</td>
			<td>BD Biosciences</td>
			<td>342003</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>TUBE 9 mL LH Lithium Heparin</td>
			<td>Greiner Bio-One</td>
			<td>455084</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

BAT performed with allergens or drugs allows investigation of IgE-dependent hypersensitivity reactions. Basophil reactivity should be measured in at least two optimal concentrations in order to obtain the best results34 and activation is visualized by the upregulation of CD63 on the cell surface. In the case of allergens, moreover, to confirm the basophil reactivity, the basophil sensitivity should be analyzed by measuring the reactivity at multiple decreasing allergen concentrations<sup class="xr...

Watch this Scientific Journal Video about Basophil Activation Test for Allergy Diagnosis at JoVE.com

Basophil Activation Test for Allergy Diagnosis

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 ...

basophil-activation-test-for-allergy-diagnosis

Research

JoVE Journal

Medicine

7.9K Views.  Hospital Regional Universitario de Málaga. 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. 

Video: Basophil Activation Test for Allergy Diagnosis

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 ...

The Trier Social Stress Test Protocol for Inducing Psychological Stress

This article demonstrates a psychological stress protocol for use in a laboratory setting. Protocols that allow researchers to study the biological pathways of the stress response in health and disease are fundamental to the progress of research in stress and anxiety.1 Although numerous protocols exist for inducing stress response in the laboratory, many neglect to provide a naturalistic context or to incorporate aspects of social and psychological stress. Of psychological stress protocols, meta-analysis suggests that the Trier Social Stress Test (TSST) is the most useful and appropriate standardized protocol for studies of stress hormone reactivity.2 In the original description of the TSST, researchers sought to design and evaluate a procedure capable of inducing a reliable stress response in the majority of healthy volunteers.3 These researchers found elevations in heart rate, blood pressure and several endocrine stress markers in response to the TSST (a psychological stressor) compared to a saline injection (a physical stressor).3 Although the TSST has been modified to meet the needs of various research groups, it generally consists of a waiting period upon arrival, anticipatory speech preparation, speech performance, and verbal arithmetic performance periods, followed by one or more recovery periods. The TSST requires participants to prepare and deliver a speech, and verbally respond to a challenging arithmetic problem in the presence of a socially evaluative audience.3 Social evaluation and uncontrollability have been identified as key components of stress induction by the TSST.4 In use for over a decade, the goal of the TSST is to systematically induce a stress response in order to measure differences in reactivity, anxiety and activation of the hypothalamic-pituitary-adrenal (HPA) or sympathetic-adrenal-medullary (SAM) axis during the task.1 Researchers generally assess changes in self-reported anxiety, physiological measures (e.g. heart rate), and/or neuroendocrine indices (e.g. the stress hormone cortisol) in response to the TSST. Many investigators have adopted salivary sampling for stress markers such as cortisol and alpha-amylase (a marker of autonomic nervous system activation) as an alternative to blood sampling to reduce the confounding stress of blood-collection techniques. In addition to changes experienced by an individual completing the TSST, researchers can compare changes between different treatment groups (e.g. clinical versus healthy control samples) or the effectiveness of stress-reducing interventions.1

This article describes a protocol for inducing psychological stress in participants, which enables researchers to measure psychological, physiological and neuroendocrine responses to stress within single participants or between groups.

This article demonstrates a psychological stress protocol for use in a laboratory setting.  Protocols that allow researchers to study the biological ...

Angiogenesis in the Ischemic Rat Lung

The adult lung is perfused by both the systemic bronchial artery and the entire venous return flowing through the pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that responds to a need for enhanced lung perfusion and shows robust neovascularization. Pulmonary vascular ischemia induced by pulmonary artery obstruction has been shown to result in rapid systemic arterial angiogenesis in man as well as in several animal models. Although the histologic assessment of the time course of bronchial artery proliferation in rats was carefully described by Weibel 1, mechanisms responsible for this organized growth of new vessels are not clear. We provide surgical details of inducing left pulmonary artery ischemia in the rat that leads to bronchial neovascularization. Quantification of the extent of angiogenesis presents an additional challenge due to the presence of the two vascular beds within the lung. Methods to determine functional angiogenesis based on labeled microsphere injections are provided.

The lung is perfused by both the systemic bronchial artery and pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that shows robust neovascularization. Cessation of pulmonary blood flow promotes brisk bronchial angiogenesis. We provide surgical details of inducing left pulmonary artery ischemia that promotes bronchial neovascularization.

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

Transabdominal Ultrasound for Pregnancy Diagnosis in Reeves' Muntjac Deer

Reeves' muntjac deer (Muntiacus reevesi) are a small cervid species native to southeast Asia, and are currently being investigated as a potential model of prion disease transmission and pathogenesis. Vertical transmission is an area of interest among researchers studying infectious diseases, including prion disease, and these investigations require efficient methods for evaluating the effects of maternal infection on reproductive performance. Ultrasonographic examination is a well-established tool for diagnosing pregnancy and assessing fetal health in many animal species1-7, including several species of farmed cervids8-19, however this technique has not been described in Reeves' muntjac deer. Here we describe the application of transabdominal ultrasound to detect pregnancy in muntjac does and to evaluate fetal growth and development throughout the gestational period. Using this procedure, pregnant animals were identified as early as 35 days following doe-buck pairing and this was an effective means to safely monitor the pregnancy at regular intervals. Future goals of this work will include establishing normal fetal measurement references for estimation of gestational age, determining sensitivity and specificity of the technique for diagnosing pregnancy at various stages of gestation, and identifying variations in fetal growth and development under different experimental conditions.

Transabdominal ultrasound is described as an effective, noninvasive means for assessing reproductive status in Reeves' muntjac deer. These methods can be used to achieve early pregnancy diagnosis and to evaluate fetal viability. Future applications of this technique include estimation of gestational age and effects of maternal disease on fetal development.

Reeves' muntjac deer (Muntiacus reevesi) are a small cervid species native to southeast Asia, and are currently being investigated as a potential model of ...

Diagnosis of Musculus Gastrocnemius Tightness - Key Factors for the Clinical Examination

Common foot and ankle pathologies have been linked to isolated Musculus gastrocnemius tightness (MGT). Various examination techniques have been described to assess MGT. Still, a standardized examination procedure is missing. Literature argues for weightbearing examination but the degree of knee flexion needed to eliminate the restraining effect of the M. gastrocnemius on ankle dorsiflexion (ADF) is unknown. This manuscript investigates the effect of knee flexion on ankle dorsiflexion and provides a detailed description of a standardized examination protocol. Examination on 20 healthy individuals revealed, that 20&#176; of knee flexion is sufficient to fully eliminate the influence of the M. gastrocnemius on ADF. This builds the prerequisite for a standardized examination for MGT. Non-weightbearing and weightbearing examination of ADF has to be conducted with the knee fully extended and at least 20&#176; flexed. Two investigators should conduct non-weightbearing testing with the subject in supine position. In order to obtain reliable results, the axis of the fibula should be marked. One examiner can conduct weightbearing examination with the subject in lunge stance. Isolated MGT is present if ADF is impaired with the knee fully extended and knee flexion results in a significant ADF increase. The herein presented standardized examination is the prerequisite for future studies aiming at establishing norm values.

Diagnosis of Musculus Gastrocnemius Tightness - Key Factors for the Clinical Examination

Isolated Musculus gastrocnemius tightness is a common cause for foot and ankle pathologies. Currently no standardized examination procedure exists. This manuscript demonstrates that 20 degree of knee flexion eliminates the restraining effect of the M. gastrocnemius on ankle dorsiflexion and presents a video description of a standardized examination protocol.

Common foot and ankle pathologies have been linked to isolated Musculus gastrocnemius tightness (MGT). Various examination techniques have been described ...

Hydra, a Computer-Based Platform for Aiding Clinicians in Cardiovascular Analysis and Diagnosis

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect of different cardiovascular risk factors (e.g., diabetes, raised blood pressure, unhealthy diet, tobacco use, stress, etc.) that commonly coexist and act multiplicatively. Most CVDs can be prevented by an early identification of the highest risk factors and an appropriate treatment. The stratification of cardiovascular risk factors involves a wide range of parameters and tests that specialists use in their clinical practice. In addition to cardiovascular (CV) risk stratification, ambulatory blood pressure monitoring (ABPM) also provides relevant information for diagnostic and treatment purposes. This work presents a list of protocols based on the Hydra platform, a web-based system for clinical decision support which incorporates a set of functionalities and services that are required for complete cardiovascular analysis, risk assessment, early diagnosis, treatment and monitoring of patients over time. The program includes tools for inputting and managing comprehensive patient data, organized into different checkups to track the evolution over time. It also has a risk stratification tool to compute a CV risk factor based upon several risk stratification tables of reference. Additionally, the program includes a tool that incorporates ABPM analysis and allows the extraction of valuable information by monitoring blood pressure over a specific period of time. Finally, the reporting service summarizes the most relevant information in a set of reports that aid clinicians in their clinical decision-making process.

This article presents a protocol based on Hydra &#8212; a web-based system for clinical decision support that integrates a full and detailed set of functionalities and services required by physicians for complete cardiovascular analysis, risk assessment, early diagnosis, treatment, and monitoring over time.

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect ...

International Expert Consensus and Recommendations for Neonatal Pneumothorax Ultrasound Diagnosis and Ultrasound-guided Thoracentesis Procedure

Pneumothorax (PTX) represents accumulation of the air in the pleural space. A large or tension pneumothorax can collapse the lung and cause hemodynamic compromise, a life-threatening disorder. Traditionally, neonatal pneumothorax diagnosis has been based on clinical images, auscultation, transillumination, and chest X-ray findings. This approach may potentially lead to a delay in both diagnosis and treatment. The use of lung US in diagnosis of PTX together with US-guided thoracentesis results in earlier and more precise management. The recommendations presented in this publication are aimed at improving the application of lung US in guiding neonatal PTX diagnosis and management.

Pneumothorax is a common emergency and critical disease in newborn infants that needs rapid, clear diagnosis and timely treatment. Diagnosis and treatment based on chest X-rays are associated with delayed management and radiation damage. Lung ultrasound (US) provides useful guidance for rapid, accurate diagnosis and the precise thoracentesis of pneumothorax.

Pneumothorax (PTX) represents accumulation of the air in the pleural space. A large or tension pneumothorax can collapse the lung and cause hemodynamic ...

Diagnosis and Surgical Treatment of Human Brucellar Spondylodiscitis

Brucellar spondylodiscitis (BS) is the most prevalent and significant osteoarticular presentation of human Brucellosis, which is commonly manifested in pastoral communities. It is difficult to differentially diagnose and usually leads to irreversible neurologic deficits and spinal deformities. The initial diagnosis of BS is based on clinical findings and radiographic assessments, and the confirmed diagnosis should be established by the isolation of Brucella species from the blood and/or the standard tube agglutination test. Differential diagnosis of multifocal BS from either degenerative disc diseases or tuberculosis is especially highlighted. The surgical approach, either endoscopic or open, is demonstrated in detail, accompanied by radiographic evidence of structural compression or severe instability. Further, the crucial surgical steps, including single-stage transforaminal decompression, debridement, interbody fusion, and internal fixation, are explained. Moreover, perioperative care and postoperative rehabilitation are also addressed. Taken together, this clinical algorithm presents a practical guide that has yielded substantially satisfactory outcomes in the past decades, which can also be introduced for large-scale application to manage human BS, especially in endemic regions.

We describe a clinical algorithm, based on decades of front-line experience of diagnosis and surgical treatment of human Brucellar spondylodiscitis in the largest medical center of the China's Xinjiang Pastoral Area.

Brucellar spondylodiscitis (BS) is the most prevalent and significant osteoarticular presentation of human Brucellosis, which is commonly manifested in ...

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.

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 ...

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

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Hydra, a Computer-Based Platform for Aiding Clinicians in Cardiovascular Analysis and Diagnosis

International Expert Consensus and Recommendations for Neonatal Pneumothorax Ultrasound Diagnosis and Ultrasound-guided Thoracentesis Procedure

Diagnosis and Surgical Treatment of Human Brucellar Spondylodiscitis

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

Measurement of Myocardial Lactate Production for Diagnosis of Coronary Microvascular Spasm