Name: basophil activation test for allergy diagnosis
Uploaded: 2021-05-31T14:00:00
Description: Watch this Scientific Journal Video about Basophil Activation Test for Allergy Diagnosis at JoVE.com

Vi takker Claudia Corazza for hendes uvurderlige engelsksprogede støtte. Dette arbejde blev støttet af Institut for Sundhed »Carlos III« (ISCIII) under MINECO (tilskud medfinansieret af EFRU: »Una manera de hacer Europa«; Tilskud nr. PI20/01715; Pi18/00095; Pi17/01410; Pi17/01318; PI17/01237 og RETIC ARADYAL RD16/0006/0001; Det andalusiske regionale sundhedsministerium (tilskud nr. PI-0127-2020, PIO-0176-2018; PE-0172-2018; PE-0039-2018; Pc-0098-2017; Pi-0075-2017; PI-0241-2016). ID er en klinisk investigator (B-0001-2017), og AA har en seniorpostdoktorkontrakt (RH-0099-2020), begge støttet af det andalusiske regionale sundhedsministerium (medfinansieret af ESF: "Andalucía se mueve con Europa").

Protokollens udførelse blev udført i overensstemmelse med Helsingforserklæringens principper og godkendt af den lokale etiske komité (Comité de Ética para la Investigación Provincial de Málaga, Spanien). Alle forsøgspersoner blev informeret mundtligt om forskningsundersøgelsen, og de underskrev den tilsvarende informerede samtykkeformular.
BEMÆRK: Den nuværende protokol beskriver BAT-proceduren, som forfatterne bruger dagligt. Dette er dog ikke en standardiseret metode, og...

BAT er en supplerende in vitro-diagnostisk test til evaluering af IgE-medierede allergiske reaktioner, der har vist sig at være nyttige til diagnosticering af reaktioner induceret af forskellige udløsere såsom lægemidler, mad eller inhalanter samt i nogle former for kronisk urticaria. Generelt bør BAT-ydeevne overvejes, hvis i) allergenet / lægemidlet producerer falske positive resultater i ST; ii) allergenet /lægemidlet er ikke tilgængeligt til brug for ST- eller sIgE-kvantificering iii) der er uoverens...

IgE-medieret allergidiagnose er baseret på klinisk historie, hudtest (ST'er), kvantificering af serumspecifik IgE (sIgE) og, hvis det kræves og indikeres, kontrollerede udfordringstest (CCT'er)1,2,3,4,5,6. Klinisk historie kan dog være upålidelig, da der kan være mangel på nøjagtige oplysninger, og ST'er og CCT'er er ikke risikofrie procedurer, der kan kontraindiceres hos forsøgspersoner, der oplever alvorlige livstruende reaktioner 1,2,3,4,5,6 . Disse spørgsmål sammen med det faktum, at bestemmelsen af sIgE ved validerede og kommercielle fluor-enzymimmunoassays kun er tilgængelig for få allergener og lægemidler, har fremhævet den vigtige rolle, som andre in vitro funktionelle assays såsom basofilaktiveringstest (BAT) spiller.
Basofiler er nøgleeffektorceller involveret i IgE-medierede allergiske reaktioner, der aktiveres ved tværbinding af tilstødende sIgE bundet på højaffinitetsreceptorer (FcεRI) på celleoverfladen efter allergen / lægemiddeleksponering. Basofilaktivering udløser celledegranulering og frigivelse af præformede og nye syntetiserede inflammatoriske mediatorer indeholdt i intracytoplasmatiske sekretionsgranulater 7,8,9. BAT er en in vitro-metode, der forsøger at efterligne basofilaktiveringen i nærvær af en stimulus (allergen eller lægemiddel) og bestemmer ændringer i ekspressionen af basofile aktiveringsmarkører ved flowcytometri 7,10. Der er forskellige strategier til at identificere basofiler (IgE+, CCR3+, CRTH2+, CD203c+) og til at måle celleaktivering (hovedsageligt opregulering af CD63 og CD203c) ved hjælp af kombinationer af fluorokrommærkede antistoffer 7,10. CD63, den bedst klinisk validerede aktiveringsmarkør 11,12,13,14, er et membranprotein forankret i sekretorgranulatet indeholdende histamin, der efter celleaktivering og fusion af granulerne med membranen udtrykkes på basofiloverflade 15,16,17,18,19,20,21 . CD203c er en overflademarkør, der konstitutivt udtrykkes på basofiler og opreguleres efter FcεRI-stimulering, som også har vist pålidelige resultater i BAT 15,22,23,24,25. Desuden ser det ud til at co-udtrykke med CD6326.
I de sidste årtier har BAT vist sig at være nyttig til diagnosticering af IgE-medierede allergiske reaktioner induceret af forskellige udløsere som lægemidler, mad eller inhalanter samt i nogle former for kronisk urticaria, som beskrevet nedenfor. Placeringen af denne teknik i de diagnostiske algoritmer er imidlertid meget variabel og ikke godt bestemt.
Overfølsomhed over for lægemidler BAT har vist sig at være nyttig som en supplerende test for udvalgte lægemidler og patienter, især for dem, der oplever alvorlige reaktioner på grund af det faktum, at diagnostisk værdi af ST ikke er veletableret for de fleste lægemidler, da de er valideret og standardiseret for et begrænset antal lægemidler27,28,29,30. Derudover er kvantificering af sIgE kun tilgængelig for et begrænset antal lægemidler med lavere følsomhed end ST 27,28,29,30,31,32. Derfor er diagnosen lægemiddeloverfølsomhed normalt afhængig af lægemiddelprovokationstest, som kan kontraindiceres hos personer, der oplever alvorlige livstruende reaktioner33.
Der er rapporteret lovende resultater for brugen af BAT hos udvalgte patienter, der rapporterer øjeblikkelige overfølsomhedsreaktioner over for forskellige lægemidler som betalactamer (BL'er)20,34,35,36,37,38,39, neuromuskulære blokeringsmidler (NMBA'er)19,22,40,41,42, 43,44,45, fluorquinoloner (FQ'er)46,47,48,49, pyrazoloner 50,51,52, radiokontrastmedier (RCM)53,54,55,56 og platinforbindelser57,58,59 . BAT er rapporteret at have en følsomhed og specificitet mellem henholdsvis 51,7-66,9% og 89,2-97,8%; og positive og negative prædiktive værdier er beskrevet til at ligge mellem 93,4% og 66,3%, henholdsvis27,31. Desuden er BAT blevet foreslået som en prædiktiv biomarkør for gennembrudsreaktioner under desensibilisering med platinforbindelser, da CD203c-ekspression øges sammenlignet med CD63 hos patienter med høj risiko for bivirkninger under lægemiddeldesensibilisering57.
Det er værd at bemærke, at BAT kun er nyttig ved lægemiddeloverfølsomhed, når reaktionen involverer basofildegranulering; Derfor er det ikke nyttigt i reaktioner som følge af enzymatisk hæmning af cyclooxygenase 142.
Fødevareallergi BAT er opstået som et potentielt diagnostisk værktøj til fødevareallergi, fordi bestemmelse af serum-sIgE til hele allergenekstraktet eller enkelte allergener ofte er tvetydig, hvilket kræver oral fødevareudfordring for at bekræfte diagnosen, hvilket på samme måde som lægemiddeloverfølsomhed er en dyr og ikke risikofri procedure60. Flere undersøgelser har vist relevante resultater med komælk 61,62, æg61,63, hvede 64,65,66,67,68, jordnødde 63,69,70,71,72, hasselnød 73,74,75,76 ,77, skaldyr78, fersken 79,80,81, æble21, selleri og gulerod82,83.
Den største merværdi af BAT ved diagnosticering af fødevareallergi sammenlignet med ST'er og sIgE i serum er, at det viser en højere specificitet og lignende følsomhed. BAT er således et nyttigt værktøj til at differentiere klinisk allergiske patienter fra sensibiliserede, men tolerante forsøgspersoner, der har både høj specificitet (75-100%) og følsomhed (77-98%)63,69,84. Følsomhed og specificitetsværdier afhænger af allergenet og andre faktorer som fænotyper (fx oral allergisyndrom versus anafylaksi), alder og geografirelaterede sensibiliseringsmønstre63,85.
BAT ved hjælp af enkelte allergenkomponenter kan potentielt forbedre diagnostisk nøjagtighed for nogle fødevareallergener61,80. Der er undersøgelser, der bruger frølagringsproteiner (f.eks. Ara h 1, Ara h 2, Ara h 3 og Ara h 6 fra jordnødder)86; lipidoverførselsproteiner (f.eks. Pru p 3 fra fersken og Ara h 9 fra jordnødder)80,86; og Bet v 1 homologer (f.eks. Ara h 8 fra peanut)87. Andre potentielle værktøjer er relateret til identifikation af synderallergenet i tilfælde af pollen-fødevareallergisyndrom 21,87,88, allergi over for rødt kød 89 eller fødevareafhængig træningsinduceret anafylaksi66.
Interessant nok kan BAT give information om sværhedsgraden og tærsklen for allergiske reaktioner, da patienter med mere alvorlige reaktioner viser en større andel af aktiverede basofiler, som observeret i undersøgelser af jordnødde- og komælkallergiske patienter 84,90,91; og patienter, der reagerer på spormængder af allergenet, viser en større basofilfølsomhed 84,90,92. Disse data tyder på, at BAT kan være nyttigt til at identificere allergiske højrisikopatienter, der kræver tættere opfølgning og mere intensiveret uddannelse93. Desuden er det blevet rapporteret, at BAT kan forudsige fødevareudfordringsrespons 70,91,92,94 og tærskler for reaktivitet90,95 for at hjælpe med at bestemme, hvornår fødevarer sikkert kan (gen)indføres 84. Disse resultater er imidlertid kontroversielle i nogle undersøgelser63,96 og mere forskning er påkrævet.
På den anden side er BAT blevet brugt til at overvåge opløsningen af fødevareallergi, enten naturligt eller under immunmodulerende behandlinger, over tid, som indtil nu kun er blevet vurderet ved oral fødevareudfordring med de tilknyttede risici og omkostninger 84,97,98,99,100,101,102,103,104,105,106 ,107,108. Desuden er det også blevet brugt til at overvåge effekten af omalizumab i fødevareallergi, da basofilaktivering falder under behandling med omalizumab, men det stiger efter ophør af behandling109.
Inhalationsallergi BAT er sjældent gavnligt ved inhalationsallergi, da diagnosen rutinemæssigt kan etableres ved sIgE-kvantificering og ST. I tilfælde af lokal allergisk rhinitis (uopdagelige niveauer af sIgE og negative ST'er med positive nasale provokationstest) har BAT imidlertid tilladt diagnose i 50% af tilfældene110. Det er yderligere rapporteret en sammenhæng mellem basofilfølsomhed og respons på nasale / bronchiale provokationstest samt mellem astma sværhedsgrad og effekt af behandling med omalizumab111,112.
BAT er også blevet brugt til at overvåge allergenimmunterapi for husstøvmide og pollen, da basofilfølsomheden falder under immunterapi, sandsynligvis på grund af interferens med blokerende IgG-antistoffer 113,114,115,116,117.
Hymenopteragift allergi Diagnose af hymenoptera giftallergi er rutinemæssigt baseret på ST og serum sIgE. BAT har vist en høj følsomhed (85-100%) og specificitet (83-100%), og det er blevet rapporteret at være nyttigt i tilfælde, der giver tvetydige resultater eller hos patienter med en suggestiv klinisk historie med giftallergi, men uopdagelig sIgE og negativ ST118,119. BAT ser imidlertid ikke ud til at forudsige sværhedsgraden for disse reaktioner120.121.
Op til 60% af patienterne udviser sIgE til både hvepse- og bigift, og identifikation af dominerende allergen er afgørende for tilstrækkelig immunterapibehandling. I disse tilfælde er BAT rapporteret at være nyttig til identifikation af det dominerende allergen 119,122,123,124. Selvom sIgE til de vigtigste allergener af bi- og hvepsegifte kan reducere nytten af BAT hos patienter med dobbelt positivitet over for begge gifte, giver det nyttig information hovedsageligt hos forsøgspersoner med negative resultater i sIgE-bestemmelse123.
Nogle undersøgelser tyder på, at BAT kan være nyttig som en prædiktiv biomarkør for bivirkninger i opbygningsfasen af giftimmunterapi, da denne behandlingsmulighed er rapporteret at reducere basofilfølsomheden. Reaktiviteten falder dog ikke, og dette BAT-værktøj er i dag kontroversielt 13.120.125.126.127.128.129.130.
Urticaria og angioødem En delmængde af kroniske urticaria-patienter har en autoinmune patofysiologi på grund af IgE-autoantistoffer mod autoallergener og IgG-autoantistoffer, der er målrettet mod FcεRI- eller IgE-FcεRI-komplekser, der er til stede på mastcelleoverfladen131.132. I klinisk praksis har diagnosen af denne type kronisk urticaria været afhængig af positiv autolog serum ST, som har risiko for utilsigtet infektion. BAT er blevet foreslået som en in vitro-test til diagnosticering og overvågning af patienter med mistanke om kronisk urticaria. Både CD63- og CD203c-ekspression på overfladen af basofiler er rapporteret at være øget efter stimulering med sera fra kroniske urticaria-patienter, hvilket viser påvisning af aktive autoantistoffer 133,134,135,136,137. For nylig er det blevet rapporteret, at patienter med positiv BAT ofte oplever den mest aktive sygdomstilstand, vurderet ud fra urticaria-aktivitetsscore, og kræver højere doser antihistaminer sammen med tredjelinjebehandlinger (cyclosporin A eller omalizumab) sammenlignet med dem med negativ BAT138.

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			<td>APC anti-human CD193 (CCR3) Antibody</td>
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basophil activation test for allergy diagnosis

Basofilaktiveringstesten (BAT) er en supplerende in vitro-diagnostisk test, der kan bruges ud over klinisk historie, hudtest (ST) og specifik IgE (sIgE) bestemmelse ved evaluering af IgE-medierede allergiske reaktioner på mad, insektgift, lægemidler samt nogle former for kronisk urticaria. Imidlertid er denne tekniks rolle i de diagnostiske algoritmer meget variabel og ikke godt bestemt.
BAT er baseret på bestemmelse af basofilrespons på allergen/lægemiddel-tværbindende IgE-aktivering gennem måling af aktiveringsmarkører (såsom CD63, CD203c) ved flowcytometri. Denne test kan være et nyttigt og komplementært værktøj til at undgå kontrollerede udfordringstest for at bekræfte allergidiagnose, især hos personer, der oplever alvorlige livstruende reaktioner. Generelt bør udførelsen af BAT overvejes, hvis i) allergenet / lægemidlet producerer falske positive resultater i ST; ii) der er ingen allergen / lægemiddelkilde til brug for ST- eller sIgE-bestemmelse; iii) der er uoverensstemmelse mellem patienthistorie og ST- eller sIgE-bestemmelse iv) symptomer tyder på, at ST kan resultere i systemisk respons; v) før du overvejer en CCT for at bekræfte synderallergenet / lægemidlet. Testens vigtigste begrænsninger er relateret til ikke-optimal følsomhed, især i lægemiddelallergi, behovet for at udføre testen ikke længere end 24 timer efter prøveekstraktion og manglen på standardisering mellem laboratorier med hensyn til procedurer, koncentrationer og cellemarkører.

BAT udført med allergener eller lægemidler tillader undersøgelse af IgE-afhængige overfølsomhedsreaktioner. Basofilreaktivitet skal måles i mindst to optimale koncentrationer for at opnå de bedste resultater34 , og aktivering visualiseres ved opregulering af CD63 på celleoverfladen. I tilfælde af allergener skal basofilfølsomheden desuden analyseres ved at måle reaktiviteten ved flere faldende allergenkoncentrationer114 for at bekræfte basofilreaktiviteten. Denn...

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

Basophil Activation Test for Allergy Diagnosis

Basofilaktiveringstesten er en supplerende in vitro-diagnostisk test til evaluering af IgE-medierede allergiske reaktioner baseret på påvisning af basofilaktivering i nærvær af en specifik stimulus gennem måling af aktiveringsmarkører ved flowcytometri.

Basophil aktiveringstest til allergidiagnose

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 ...

Basofilaktiveringstesten er en gratis in vitro-diagnostisk test til evaluering af IgE-medierede allergiske reaktioner, der har vist sig at være nyttige til reaktioner induceret af lægemidler, mad eller inhalanter samt i nogle former for kronisk urticaria. Denne test er baseret på bestemmelse af basofilrespons på et allergen eller lægemiddel, der tværbinder IgE-aktivering gennem måling af aktiveringsmarkører ved flowcytometri. Basofilaktiveringstesten kan være et nyttigt og gratis værktøj til at undgå kontrollerede udfordringstest for at bekræfte allergidiagnosen, især hos personer, der oplever alvorlige livstruende reaktioner.Desuden har denne test den fordel, at den er i stand til at analysere responsen mod ethvert lægemiddel eller allergen sammenlignet med andre in vitro-metoder, der kun er tilgængelige for nogle få lægemidler og allergener. Testens vigtigste begrænsninger er relateret til ikke-optimal følsomhed, især i lægemiddelallergi. Behovet for at udføre testen ikke længere end 24 timer efter prøveekstraktion og manglen på standardisering mellem laboratorier og diagnostiske algoritmer.Begynd med at indsamle perifert blod i ni milliliter hepariniserede rør, og læg prøverne i en rotor for at opretholde dem ved stuetemperatur, indtil de er nødvendige for den eksperimentelle protokol. Mærk to fem milliliter cytometerrør hver for negativ kontrol og positiv kontrol. Mærk også et rør hver for de forskellige allergen- eller lægemiddelkoncentrationer, der testes.Placer derefter rørene i et stativ, så rørene passer perfekt uden at glide. For den første positive kontrol fremstilles en fire mikromolær opløsning af N-Formylmethionyl-leucyl-phenylalanin eller fMLP i 0,05% PBS-T For at bekræfte kvaliteten af basofiler. Til den anden positive kontrol fremstilles en 0,05 milligram pr. milliliter anti-IgE-opløsning ved hjælp af PBS-T.Forbered farvningsblandingen ved at tilføje en mikroliter hver af CCR3-APC-, CD203c-PE- og CD63-FITC-antistoffer pr. 20 mikroliter stimuleringsbuffer. Tilsæt derefter 23 mikroliter af denne farvningsblanding til hvert rør. Tilsæt 100 mikroliter PBS-T til de negative kontrolrør.Tilsæt 100 mikroliter fMLP til et af de positive kontrolrør og tilsæt 100 mikroliter anti-IgE til det andet til resten af rørene. Tilsæt 100 mikroliter af de forskellige allergen- eller lægemiddelkoncentrationer og inkuber ved 37 grader Celsius i 10 minutter. Efter inkubation tilsættes 100 mikroliter blod forsigtigt til hvert rør for at undgå hæmolyse.Derefter hvirvles forsigtigt rørene og inkuberes dem i 25 minutter ved 37 grader Celsius i det termostatiske bad med medium omrøring. Hold rørene ved fire grader Celsius i mindst fem minutter for at stoppe degranuleringen. Til hvert rør tilsættes to milliliter lysbuffer for at lyse erythrocytterne.Hvirvel hvert rør og inkuber derefter rørene i fem minutter ved stuetemperatur. Centrifuge rørene ved 300 g ved fire grader Celsius i fem minutter. Vend derefter stativet i vasken for at dekantere supernatanten, mens cellerne forbliver i bunden af rørene.Tilsæt tre milliliter PBS-T til hvert rør for at vaske cellerne og hvirvle rørene. Udfør en anden runde centrifugering ved hjælp af det samme sæt parametre, og dekanter supernatanten ved at vælte stativet i vasken. Hold derefter prøverne ved fire grader Celsius beskyttet mod lys indtil flowcytometri.Tilslut flowcytometeret til computersoftwaren, og vent, indtil cytometeret er klar. Indlæs skabelon- og instrumentindstillingerne som beskrevet i tekstmanuskriptet. Start processen med prøveopkøb.For at vælge aktiverede basofiler skal du først gate lymfocytterne fra Side Scatter-Forward Scatter-plottet. Derefter gate basofilerne fra lymfocytpopulationen som CCR3 + CD203c + celler og erhverve mindst 500 basofiler pr. Rør. Indstil CD63-tærsklen til ca. 2,5% ved hjælp af de negative kontrolrør og analyser prøverne.IgE-afhængige overfølsomhedsreaktioner blev undersøgt ved at udføre en basofilaktiveringstest eller BAT ved hjælp af allergener og lægemidler. Basofilfølsomhed blev analyseret ved at måle reaktiviteten ved flere faldende allergenkoncentrationer, der hjælper med at bestemme den allergiske koncentration, der inducerer responsen fra 50% basofiler eller EC50. For det første blev lymfocytter gated fra siden scatter-forward scatter plot.Derefter blev basofiler indhegnet fra lymfocytpopulationen som CCR3 + CD203c + celler. Derefter blev et CCR3-CD63-plot brugt til at analysere basofilaktivering ved hjælp af CD63 som en aktiveringsmarkør for to lægemidler og forskellige koncentrationer af et allergen. Testen udføres med frisk fuldblod og må udføres højst 24 timer efter blodekstraktion.Den stimulus, der anvendes i testen, bør ikke omfatte hjælpestoffer, og lægemidlets kemiske egenskaber skal overvejes. Andre yderligere in vitro-metoder til diagnosticering af IgE-medierede allergiske reaktioner eller bestemmelse af sIgE eller histaminfrigivelsestesten.

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Medicine

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 ...

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 for Pregnancy Diagnosis in Reeves&#039; Muntjac Deer

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.

Transabdominal Ultralyd til graviditetsdiagnose i 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 ...

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.

diagnose af<em&gt; Musculus Gastrocnemius</em&gt; Tæthed - nøglefaktorer for den kliniske undersøgelse

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.

Hydra, en Computer-baseret Platform til medvirken klinikere i hjerte-kar-analyse og diagnose

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.

International ekspert konsensus og anbefalinger til neonatal Pneumothorax ultralyddiagnose og ultralyd-guidet 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 ...

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.

Diagnose og kirurgisk behandling af human Brucellar Spondylodiscitis

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.

Test acetylcholin efterfulgt af adenosin for invasiv diagnose af koronar vasomotoriske lidelser

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.

Måling af myokardielaktatproduktion til diagnose af koronar mikrovaskulær spasme

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

High-speed Video Microscopy Analysis for First-line Diagnosis of Primary Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD) is a congenital disorder predominantly inherited in an autosomal recessive trait. The disorder causes disturbance in the motion of cilia, leading to severe impairment of mucociliary clearance (MCC). If undiagnosed or diagnosed too late, the condition leads to the development of bronchiectasis and serious damage to the lungs in later life. Most of the methods for diagnosing PCD are time-consuming and demand extensive economic resources to establish them. High-speed video microscopy analysis (HSVMA) is the only diagnostic tool to visualize and analyze living respiratory cells with beating cilia in vitro. It is fast, cost-effective, and, in experienced hands, very reliable as a diagnostic tool for PCD. In addition, classical diagnostic measures such as transmission electron microscopy (TEM) are not applicable for some mutations as morphological changes are absent. 
This paper describes the process of collecting respiratory epithelial cells, the further preparation of the specimen, and the process of HSVMA. We also describe how brushed cells can be successfully kept unharmed and beating by keeping them in a nourishing medium for storage and transport to the investigation site in cases where a clinic does not possess the equipment to perform HSVMA. Also shown are videos with pathologic beating patterns from patients with a mutation in the dynein arm heavy chain 11 gene (DNAH11), which cannot be diagnosed with TEM; the result of an inconclusive HSVMA due to infection of the upper airways, as well as an unsuccessful brushing with superimposition of red blood cells. With this article, we would like to encourage every unit dealing with pulmonology patients and rare lung diseases to perform HSVMA as part of their daily routine diagnostics for PCD or send the specimens over to a center specializing in performing HSVMA.

High-speed video microscopy analysis is a relatively easy-to-perform, fast, cost-effective, and, in experienced hands, a considerably reliable tool for first-line diagnostics of primary ciliary dyskinesia, which should be available in every center involved in diagnostics and the treatment of severe lung diseases.

Højhastigheds videomikroskopianalyse til førstelinjediagnose af primær ciliær dyskinesi

Primary ciliary dyskinesia (PCD) is a congenital disorder predominantly inherited in an autosomal recessive trait. The disorder causes disturbance in the ...