This work was performed on behalf of the International Federation of Clinical Chemistry Working Group on CSF Proteins, which has the following composition: Kaj Blennow (Chair) and Henrik Zetterberg, Gothenburg University, Sweden; Les Shaw and Magdalena Korecka, University of Philadelphia, PA, USA; Ingrid Zegers, Institute for Reference Materials and Measurements, Geel, Belgium; Piotr Lewczuk, Universit&#228;tsklinikum Erlangen, Germany; and Rand Jenkins, PPD Bioanalytical Laboratory, Richmond, VA, USA; Randall Bateman, Washington University, MO, USA; and H Vanderstichele, Innogenetics NV, Ghent, Belgium. This work was also part of the Global Consortium for Biomarker Standardization CSF Reference Methods Working Group, which is led by Maria C. Carrillo, Ph.D., Senior Director, Medical &amp; Scientific Relations, Alzheimer's Association. The study was supported by The Swedish Research Council (grants #14002, #521-2011-4709, and #2013-2546); the Knut and Alice Wallenberg Foundation; Demensf&#246;rbundet; and Emil and Wera Cornell's, Aina Wallstr&#246;m and Mary-Ann Sj&#246;blom's, Gun and Bertil Stohne's, Magnus Bergvall's, and Gamla Tj&#228;narinnor's Foundations.

NOTA: Questo protocollo richiede aliquote di almeno 50 ml, con una concentrazione di 50 mg / ml per ciascun peptide Ap, come materiale di partenza. I peptidi Ap vanno dissolte in 20% acetonitrile (ACN) e il 4% di soluzione di ammoniaca concentrata in acqua deionizzata (v / v) e conservati a 80 ° C. Attenzione: Vedi Tabella 1 per le informazioni di sicurezza. 1. preparazione delle soluzioni <ol><li> Preparare 100 ml di 20% ACN e 4% ammoniaca concentrata in acqua deionizzata (v / v) diluendo 20 mL di ACN e 4 ml di ammoniaca concentrata (~ 25%) in acqua deionizzata. Regolare il volume finale a 100 ml con acqua deionizzata. Fare fresco tutti i giorni. </li><li> Preparare 50 mL di 5 M guanidina cloridrato sciogliendo 26,08 g di guanidina cloridrato in acqua deionizzata fino ad un volume finale di 50 ml. Conservare a 20 ° C e fare mensile fresco. </li><li> Preparare 200 ml di acido fosforico 4% in acqua deionizzata (v /v) diluendo 9,4 mL di acido fosforico concentrato (~ 85%) in acqua deionizzata. Regolare il volume finale a 200 ml con acqua deionizzata. Conservare in frigorifero e fare settimanale fresco. </li><li> Preparare 50 mL di 75% ACN e ammoniaca concentrata 10% (v / v) in acqua deionizzata diluendo 37,5 mL di ACN e 5 ml di ammoniaca concentrata (~ 25%) in acqua deionizzata. Regolare il volume finale di 50 ml con acqua deionizzata. Fare fresco tutti i giorni. </li><li> Scongelare almeno 2,5 ml di CSF umano per i calibratori, ottenuti da campioni avanzi de-identificato da analisi cliniche di routine. </li><li> Preparare CSF artificiale contenente 150 mM Na, 3,0 mM K, 1.4 mM Ca, Mg 0,8 mM, 1,0 mM P, e 155 mM Cl in acqua deionizzata e aggiungere sieroalbumina bovina ad una concentrazione finale di 4 mg / mL. Solo 1 ml è necessaria per l&#39;analisi, ma preparare un grande volume, un&#39;aliquota di esso, e conservare per uso futuro. </li></ol> 2. Preparazione dei calibratori <ol><li> Preparare 0,5 ml di 4 mg / ml 15 NAβ 1-42 peptide aggiungendo 40 ml di 50 mg / mL 15 NAβ 142 al 0.46 mL di 20% ACN e ammoniaca concentrata 4% in una provetta da 0,5 ml. Miscelare con un miscelatore vortex per 1 minuto. </li><li> Preparare 2 mL di 100 ng / mL 15 NAβ 1-42 peptide aggiungendo 50 ml di 4 mg / mL 15 NAβ 142 al 1.95 mL di 20% ACN e ammoniaca concentrata 4% in una provetta 2 mL. Miscelare con un miscelatore vortex per 1 minuto. </li><li> Preparare sei soluzioni calibratori (AF) mescolando i volumi di ogni soluzione indicata nella tabella 2. Utilizzare 0.5, 1.5, e 2 tubi ml microcentrifuga. Miscelare con un miscelatore vortex per 1 minuto. </li><li> Preparare i calibratori finali (in duplicato) in 0,5 ml microprovette con l&#39;aggiunta delle soluzioni di taratura corrispondenti e CSF umano secondo la Tabella 3. Mix su un vortex per 1 minuto. </li></ol> 3. Preparazione dello standard interno <ol><li> Preparare 2 ml di 0,8 mg / mL 13 CAβ 1-42 peptide aggiungendo 32 ml di 50 mg / mL 13 CAβ 142 al 1.968 ml di 20% ACN e ammoniaca concentrata 4% in una provetta 2 mL. Miscelare con un miscelatore vortex per 1 minuto. </li><li> Preparare 5 ml di 16 ng / mL 13 CAβ 1-42 peptide aggiungendo 0,1 ml di 0,8 mg / ml a 4,9 ml di 20% ACN e ammoniaca concentrata 4% in una provetta da microcentrifuga 5. Miscelare con un miscelatore vortex per 1 minuto. </li></ol> 4. Preparazione del campione Fattore di risposta NOTA: Il fattore di risposta (RF) determinazione è eseguita per determinare la concentrazione del peptide marcato usato per la calibrazione (15 NAβ 1-42). Ciò richiede che la concentrazione del nativo Ap 1-42 peptide è stata determinata tramite analisi amminoacidica (AAA). Così, il volume e la concentrazione di nativi aliquote peptide Ap 1-42necessità di soddisfare i requisiti del AAA. <ol><li> Preparare 0,5 ml di 4 mg / ml nativo (senza etichetta) Ap 1-42 con l&#39;aggiunta di 40 ml di 50 mg / ml nativo Ap 1-42 a 0,46 ml del 20% ACN e ammoniaca concentrata 4% in una provetta da 0,5 ml. Miscelare con un miscelatore vortex per 1 minuto. </li><li> Preparare un 2-ml 40 ng mL mix di nativi e 15 NAβ 1-42 / aggiungendo 20 ml di 4 mg / ml nativo Ap 1-42 e 20 ml di 4μg / mL 15 NAβ 1-42 a 1,96 ml del 20% ACN e 4% concentrate di ammoniaca in una provetta da microcentrifuga 2. Miscelare con un miscelatore vortex per 1 minuto. </li><li> Aggiungere 20 ml di 40 ng / mL mix di 0,38 ml di liquido cerebrospinale artificiale in una provetta 0,5 microcentrifuga. Preparare i duplicati e mescolare su un vortex per 1 minuto. </li></ol> Preparazione del campione 5. NOTA: Scongelare i campioni da misurare a temperatura ambiente su un rullo stanza. <ol><li>Aggiungere 0,18 ml di ogni calibratore, fattore di risposta, e campione non noto (compreso il controllo di qualità [QC] campioni, se utilizzato) ad una piastra a 96 profondo bene 1 proteina mL, secondo la figura 1 (assumendo un piatto pieno è utilizzato). Assicurarsi di aggiungere i campioni in, o in prossimità, il fondo dei pozzetti. </li><li> Aggiungere 20 ml di standard interno ad ogni pozzetto (ad esempio, calibratori, i fattori di risposta, QC, e le incognite); è fondamentale per rilasciare la goccia sul lato del pozzo vicino alla superficie del campione senza immergendo la punta della pipetta. </li><li> Aggiungere 0,2 ml di 5 M guanidina-cloridrato in ciascun pozzetto. </li><li> Posizionare il piatto del campione, un vortex e miscelare i campioni per 45 min a 1100 rpm. La frequenza ottimale potrebbe differire a seconda della strumentazione. Impostare la frequenza e l&#39;ampiezza del mixer in modo che le soluzioni sono accuratamente miscelati e non gocce di standard interno o CSF ​​sono lasciati non miscelato sul lato dei pozzetti. </li><li> Aggiungere 0,2 ml di acido fosforico 4%in ciascun pozzetto. Vortex mescolare brevemente. </li></ol> 6. estrazione in fase solida NOTA: In tutte lavaggio, carico, e passi di eluizione, applicare il vuoto più basso possibile, dopo l&#39;aggiunta della soluzione e aumentare gradualmente come necessario per caricare o eluire la soluzione. Disabilitare il vuoto tra ogni carico e passo eluizione. <ol><li> Mettere un vassoio di serbatoio per i rifiuti sotto una modalità mista, scambio cationico, 96 pozzetti estrazione in fase solida (SPE) placca nella camera di collettore piatto di estrazione. </li><li> Condizionare la assorbente SPE con l&#39;aggiunta di 0,2 ml di metanolo ad ogni pozzetto. </li><li> Equilibrare il sorbente aggiungendo 0,2 mL di acido fosforico 4% a ciascun pozzetto. </li><li> Trasferire tutti i campioni (circa 0,62 ml in ogni pozzetto) dalla piastra profondo bene alla piastra SPE. Utilizzare una pipetta a otto canali per il trasferimento dei campioni dalla piastra profondo bene alla piastra SPE; non è cruciale per la trasmissione completa o volumi uguali di tutti i campioni, poiché i campioni contengono un internoal standard che compensare le variazioni. </li><li> Lavare il sorbente dopo i campioni hanno attraversato aggiungendo 0,2 mL di acido fosforico 4% a ciascun pozzetto. </li><li> Dopo che il solvente di lavaggio è eluito dalla sorbente, sostituire il vassoio serbatoio con una piastra di raccolta o tubi. </li><li> Eluire il campione dalla sorbente di due volte aggiungendo 50 ml di 75% ACN / 10% ammoniaca concentrata e notare che questa soluzione richiede molto basso vuoto per passare attraverso l&#39;assorbente. Ricordarsi di disattivare il vuoto tra ogni aggiunta. <ol><li> OPTIONAL: Sigillare la piastra di raccolta o tubi e congelarli a -80 ° C. Rimuovere il sigillo dalla piastra raccolta o tubi prima di procedere al punto 6.8.2. </li><li> Asciugare le eluati utilizzando centrifugazione a vuoto (senza applicazione di calore); questo può richiedere da una a diverse ore, a seconda della centrifuga a vuoto. </li><li> Sigillare i contenitori e congelarli a -80 ° C. </li></ol></li></ol> 7. chromato Liquidgrafia <ol><li> Preparare fase mobile A (5% ACN e ammoniaca concentrata 0,3% in acqua deionizzata [v / v]), B (acqua deionizzata 4% e ammoniaca concentrata 0,1% in ACN [v / v]), e lavaggio dell&#39;ago (50% ACN e 4% concentrato ammoniaca in acqua deionizzata [v / v]). <ol><li> Per 500 ml di fase mobile A, aggiungere 25 ml di ACN e 1,5 ml di ammoniaca concentrata di acqua deionizzata. Regolare il volume finale a 500 ml con acqua deionizzata. </li><li> Per 500 ml di mobili fase B, aggiungere 500 ml di ammoniaca concentrata e 25 ml di acqua deionizzata ad ACS. Regolare il volume finale di 500 ml con ACN. </li><li> Preparare 250 ml di lavaggio dell&#39;ago aggiungendo 120 mL di ACN e 10 ml di ammoniaca concentrata di acqua deionizzata. Regolare il volume finale a 250 ml con acqua deionizzata. </li><li> Mettere il fasi mobili A e B e lavare l&#39;ago bottiglie aperte in un bagno di ultrasuoni per 20 minuti prima di utilizzarlo con il sistema LC </li></ol></li><li> Sciogliere ogni campione con 25 ml di 20% ACN e il 4% Concentrasoluzione di ammoniaca Ted e metterli su un agitatore per 20 min. Centrifuga verso il basso i campioni e metterli nel campionatore automatico (tenere a 7 ° C). </li><li> Iniettare 20 ml di campione su un 1 x 250 mm 2 polistirene-divinilbenzene (fase inversa) colonna monolitica mantenuta a 50 ° C. <ol><li> Utilizzare il gradiente LC mostrato nella Tabella 4 con una portata di 0,3 mL / min. Deviazione i primi due e gli ultimi cinque minuti sprecare (post-colonna) mediante una valvola di deviazione per ridurre la contaminazione dello spettrometro di massa. </li></ol></li></ol> Analisi 8. Messa spettrometrica NOTA: Questi parametri sono stati utilizzati per uno spettrometro di massa ibrido quadrupolo-Orbitrap dotato di aheated sorgente di ionizzazione elettrospray. <ol><li> Impostare i parametri per la sorgente di ioni secondo la tabella 5. </li><li> Impostare lo strumento MS per isolare i 4+ carica stati di natale Ap 1-42 (1,129.48 di massaa-charge ratio [m / z]), 15 NAβ 1-42 (1,143.00 m / z), e 13 CAβ 1-42 (1,179.50 m / z) nell&#39;analizzatore di massa a quadrupolo con una larghezza isolamento di 2,5 m / z. </li><li> Frammentare i peptidi isolati nella cella di collisione con un&#39;energia di collisione normalizzata (NCE) di 17,0. Questo potrebbe essere necessario sintonizzati per ogni strumento, anche dello stesso tipo (e soprattutto se si utilizzano altri tipi di strumenti, come un triplo quadrupolo MS). </li><li> Registrare il spettri frammento con una risoluzione di 17.500, con un obiettivo di controllo automatico del guadagno di 2 x 10 5 cariche e un tempo massimo di iniezione di 250 ms. </li></ol> 9. Elaborazione dati <ol><li> Utilizzare la somma degli ioni prodotto (con una tolleranza massa di ± 250 unità di massa milli [MMU]) nella Tabella 6, per calcolare le aree cromatografiche per ciascun peptide. Si noti che i tipi di ioni ed i numeri sono indicati solo per Ap nativo <sub&gt; ioni 1-42 di prodotto, dal momento che sono gli stessi per entrambi 15 NAβ 1-42 e 13 CAβ 1-42. </li><li> Determinare il fattore di risposta media dei due campioni fattore risposta dividendo l&#39;area sotto la curva (picco cromatografico) del 15 NAβ 1-42 con l&#39;area sotto la curva del nativo Ap 1-42. </li><li> Regolare la concentrazione del 15 NAβ 1-42 utilizzata per la calibrazione, moltiplicandolo per il fattore di risposta calcolato nel passo 9.2. </li><li> Costruire una curva di taratura riportando i rapporti di superficie di 15 NAβ 1-42 al 13 CAβ 1-42 da due serie di calibratori contro la concentrazione. </li><li> Calcolare la pendenza e intercetta della curva di calibrazione utilizzando la regressione lineare. </li><li> Calcolare il rapporto di nativo Ap 1-42 area a standard interno (13 CAβ 1-42) per uncampioni noti. </li><li> Estrapolare la concentrazione dei campioni sconosciuti dalla curva di calibrazione utilizzando la pendenza e l&#39;intercetta ottenuto nel passaggio 9.5. </li></ol>

<ol>
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JP and EP reports no disclosures. HZ has served on the advisory boards of Roche Diagnostics, Eli Lilly, and Pharmasum Therapeutics. KB has served as a consultant or on the advisory boards for Fujirebio Europe, IBL International, and Roche Diagnostics. HZ and KB are co-founders of Brain Biomarker Solutions in Gothenburg AB, a GU Venture-based platform company at the University of Gothenburg.

Per il metodo descritto, invece di utilizzare una matrice surrogata, abbiamo usato il metodo surrogata analita 13, 14, 15, 16, che consente la calibrazione in CSF umano. L&#39;approccio analita surrogato coinvolge due diversi standard marcati con isotopi. Uno (15 NAβ 142) è utilizzato per generare la curva di calibrazione in CSF umano, mentre un altro (13 CA?...

La malattia di Alzheimer (AD) è la forma più comune di demenza e colpisce circa 35 milioni di persone in tutto il mondo 1. Le caratteristiche neuropatologici della malattia ampiamente creduto di essere al centro della patogenesi sono grovigli neurofibrillari intracellulari di hyperphosphorylated proteina tau 2 e placche extracellulari costituito da (Ap) peptidi 3 aggregati beta-amiloide. In linea con questo, la valutazione della patologia placca in vivo da biomarcatori è stato recentemente inserito nei criteri diagnostici di ricerca per AD 4. Per misurazioni CSF di Ap 1-42, vari test immunologici sono disponibili e sono utilizzati in molti laboratori clinici 5. La concentrazione di Ap 1-42 in CSF è inferiore di circa il 50% in pazienti con AD rispetto al cognitivo normale anziani, riflettendo la deposizione del peptide in placche nel brain 6, 7. Questi biomarcatori sono principalmente analizzati utilizzando saggi immunologici (ad esempio, le tecniche a base di anticorpi), ma questi test possono essere influenzati da effetti di matrice 8. L&#39;uso di saggi immunologici su piattaforme tecnologiche diverse e la mancanza di test di standardizzazione 9, 10 rende l&#39;introduzione di concentrazioni di cut-off globali difficili 11, 12. Un RMP analiticamente convalidato avrebbe permesso la calibrazione uniforme delle piattaforme di analisi diverse, idealmente con conseguente migliore comparabilità tra le piattaforme di analisi e in un migliore controllo dei fattori che contribuiscono alla variabilità complessiva di misura. La quantificazione assoluta di Ap 1-42 con il metodo LC-MS / MS sviluppata supera molti dei problemi connessi con tecn a base di anticorpiiques. Il metodo, indicato come un RMP dal comitato misto per la tracciabilità in Medicina di Laboratorio (JCTLM archivio di identificazione numero C11RMP9), sarà utilizzato per determinare la concentrazione assoluta di Ap 1-42 in un materiale di riferimento certificato (CRM) per armonizzare CSF Ap 1 misurazioni -42 attraverso tecniche e piattaforme analitiche. Il flusso di lavoro descritto deve essere rilevante per lo sviluppo di metodi di riferimento candidato per peptidi e proteine ​​all&#39;interno di altre aree della medicina.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Eppendorf Protein LoBind microcentrifuge tubes, 0.5 mL</td>
			<td>Eppendorf</td>
			<td>022431081</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Protein LoBind microcentrifuge tubes, 1.5 mL</td>
			<td>Eppendorf</td>
			<td>022431081</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Protein LoBind microcentrifuge tubes, 2 mL</td>
			<td>Eppendorf</td>
			<td>022431102</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Protein LoBind microcentrifuge tubes, 5 mL</td>
			<td>Eppendorf</td>
			<td>0030 108.310</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Protein LoBind Deepwell Plates 96</td>
			<td>Eppendorf</td>
			<td>951032905</td>
			<td></td>
		</tr>
		<tr>
			<td>Micronic 0.75 mL polypropylene tubes, V-bottom</td>
			<td>Micronic</td>
			<td>MPW32071BC3</td>
			<td>Tubes used for collecting SPE eluate, but other collection devices can be used such as 96-well plates.</td>
		</tr>
		<tr>
			<td>Micronic Split TPE Capcluster for 96 ind. tubes</td>
			<td>Micronic</td>
			<td>MP53026</td>
			<td>Caps for Micronic tubes.</td>
		</tr>
		<tr>
			<td>Micronic Loborack-96 white w high cover bar coded</td>
			<td>Micronic</td>
			<td>MPW51015BC3</td>
			<td>Holder for Micronic tubes.</td>
		</tr>
		<tr>
			<td>Biohit Optifit tips 1.2 mL</td>
			<td>Sartorius</td>
			<td>791210</td>
			<td>Used to reach all the way to the bottom of the LoBind deepwell plates (96-well). Other narrow tips might work as well.</td>
		</tr>
		<tr>
			<td>Waters Oasis MCX 96-well &#181;Elution Plate</td>
			<td>Waters</td>
			<td>186001830BA&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Waters Plate manifold reservoir tray</td>
			<td>Waters</td>
			<td>WAT058942</td>
			<td></td>
		</tr>
		<tr>
			<td>Waters Extraction Plate Manifold for Oasis 96-Well Plates</td>
			<td>Waters</td>
			<td>186001831</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide solution, puriss. p.a., reag. ISO, reag. Ph. Eur., ~25% NH3 basis</td>
			<td>Sigma-Aldrich</td>
			<td>30501-1L-D</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile, Far UV HPLC Gradient grade, 2.5 L</td>
			<td>Fisher Scientific</td>
			<td>A/0627/17X</td>
			<td></td>
		</tr>
		<tr>
			<td>ortho-Phosphoric acid 85%, ACS,ISO,Reag. Ph Eur</td>
			<td>Merck Millipore</td>
			<td>1005731000</td>
			<td></td>
		</tr>
		<tr>
			<td>Guanidine Hydrochloride, 500 g</td>
			<td>Thermo Scientific</td>
			<td>24110</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Q-exactive Hybrid Quadrupole Orbitrap Mass Spectrometer</td>
			<td>Thermo Scientific</td>
			<td>IQLAAEGAAPFALGMAZR</td>
			<td>With bundled Dionex Ultimate 3000 LC &#38; autosampler.</td>
		</tr>
		<tr>
			<td>Dionex ProSwift RP-4H Monolith Column (1.0 x 250mm)</td>
			<td>Dionex</td>
			<td>066640</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin, lyophilized powder, suitable for (for molecular biology), Non-acetylated</td>
			<td>Sigma-Aldrich</td>
			<td>B6917</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta-Amyloid (1-42), Ultra Pure, TFA</td>
			<td>rPeptide</td>
			<td>A-1002-2</td>
			<td></td>
		</tr>
		<tr>
			<td>15N Beta-Amyloid (1-42), Uniformly labeled</td>
			<td>rPeptide</td>
			<td>A-1102-2</td>
			<td></td>
		</tr>
		<tr>
			<td>13C Beta-Amyloid (1-42), Uniformly labeled</td>
			<td>rPeptide</td>
			<td>A-1106-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate shakers, TiMix 2</td>
			<td>Edmund B&#252;hler</td>
			<td>6110 000</td>
			<td></td>
		</tr>
	</tbody>
</table>

absolute quantification of a&#946;1-42 in csf using a mass spectrometric reference measurement procedure

Alzheimer's disease (AD) is the most common neurodegenerative disease among the elderly and accounts for 60-80% of all cases of dementia. Currently, the diagnosis of AD is based on cognitive tests and mental state exams, but the peptide amyloid-beta (A&#946;) in cerebrospinal fluid (CSF) is increasingly used in clinical trials and settings. As for most protein and peptide biomarkers, quantification is performed using antibody-based techniques, such as enzyme-linked immunosorbent assay (ELISA). However, intra- and inter-laboratory variability in these assays hamper its use as a diagnostic marker in clinical routine.
An antibody-independent Reference Measurement Procedure (RMP) was developed based on solid-phase extraction (SPE) and liquid chromatography (LC)-tandem mass spectrometry (MS/MS), where stable, isotope-labeled A&#946; peptides were used as internal standards, enabling absolute quantification. A high-resolution quadrupole-orbitrap hybrid instrument was used for the measurements. The method allows for the quantification of CSF A&#946;1-42 between 150-4,000 pg/mL.

Il setup piatto in Figura 1 è utilizzato per un piatto pieno di campioni. Se un minor numero di campioni sconosciuti sono da analizzare, il secondo calibratore, set RF, e QC deve essere posto dopo la prima metà dei campioni sconosciuti. Come si vede nella figura 2, i calibratori sono vicini alla linea di regressione, con basse deviazioni standard. Questo metodo ha un livello inferiore di qua...

Watch this Scientific Journal Video about Absolute Quantification of AÃŽÂ²1-42 in CSF Using a Mass Spectrometric Reference Measurement Procedure at JoVE.com

Absolute Quantification of A&#946;1-42 in CSF Using a Mass Spectrometric Reference Measurement Procedure

A reference measurement procedure for the absolute quantification of A&#946;1-42 in human CSF based on solid-phase extraction and liquid chromatography tandem mass spectrometry is described.

Quantificazione assoluta di A?<sub&gt; 1-42</sub&gt; In CSF Utilizzando un riferimento alla procedura di misurazione di massa spettrometrica

Alzheimer's disease (AD) is the most common neurodegenerative disease among the elderly and accounts for 60-80% of all cases of dementia. Currently, the ...

absolute-quantification-a1-42-csf-using-mass-spectrometric-reference

Research

JoVE Journal

Medicine

7.5K Views.  The Sahlgrenska Academy at University of Gothenburg, Sahlgrenska University Hospital. A reference measurement procedure for the absolute quantification of Aβ1-42 in human CSF based on solid-phase extraction and liquid chromatography tandem mass spectrometry is described.

Video: Absolute Quantification of Aβ1-42 in CSF Using a Mass Spectrometric Reference Measurement Procedure

Magnetic Resonance Imaging Quantification of Pulmonary Perfusion using Calibrated Arterial Spin Labeling

This demonstrates a MR imaging method to measure the spatial distribution of pulmonary blood flow in healthy subjects 
during normoxia (inspired O2, fraction (FIO2) = 0.21) hypoxia (FIO2 = 0.125), and hyperoxia 
(FIO2 = 1.00). In addition, the physiological responses of the subject are monitored in the MR scan environment. MR images 
were obtained on a 1.5 T GE MRI scanner during a breath hold from a sagittal slice in the right lung at functional residual capacity. An arterial 
spin labeling sequence (ASL-FAIRER) was used to measure the spatial distribution of pulmonary blood flow 1,2 and a multi-echo fast 
gradient echo (mGRE) sequence 3 was used to quantify the regional proton (i.e. H2O) density, allowing the quantification 
of density-normalized perfusion for each voxel (milliliters blood per minute per gram lung tissue). 
With a pneumatic switching valve and facemask equipped with a 2-way non-rebreathing valve, different oxygen concentrations 
were introduced to the subject in the MR scanner through the inspired gas tubing. A metabolic cart collected expiratory gas via expiratory tubing. Mixed expiratory O2 and CO2 concentrations, oxygen consumption, carbon dioxide production, respiratory exchange ratio, 
respiratory frequency and tidal volume were measured. Heart rate and oxygen saturation were monitored using pulse-oximetry. 
Data obtained from a normal subject showed that, as expected, heart rate was higher in hypoxia (60 bpm) than during normoxia (51) or hyperoxia (50) and the arterial oxygen saturation (SpO2) was reduced during hypoxia to 86%. Mean ventilation was 8.31 L/min BTPS during hypoxia, 7.04 L/min during normoxia, and 6.64 L/min during hyperoxia. Tidal volume was 0.76 L during hypoxia, 0.69 L during normoxia, and 0.67 L during hyperoxia. 
Representative quantified ASL data showed that the mean density normalized perfusion was 8.86 ml/min/g during hypoxia, 8.26 ml/min/g during normoxia and 8.46 ml/min/g during hyperoxia, respectively. In this subject, the relative dispersion4, an index of global heterogeneity, was increased in hypoxia (1.07 during hypoxia, 0.85 during normoxia, and 0.87 during hyperoxia) while the fractal dimension (Ds), another index of heterogeneity reflecting vascular branching structure, was unchanged (1.24 during hypoxia, 1.26 during normoxia, and 1.26 during hyperoxia). 
Overview. This protocol will demonstrate the acquisition of data to measure the distribution of pulmonary perfusion noninvasively under conditions of normoxia, hypoxia, and hyperoxia using a magnetic resonance imaging technique known as arterial spin labeling (ASL). 
Rationale: Measurement of pulmonary blood flow and lung proton density using MR technique offers high spatial resolution images which can be quantified and the ability to perform repeated measurements under several different physiological conditions. In human studies, PET, SPECT, and CT are commonly used as the alternative techniques. However, these techniques involve exposure to ionizing radiation, and thus are not suitable for repeated measurements in human subjects.

A MR imaging method to study the distribution of pulmonary blood flow under a variety of physiological conditions, in this case exposure to three different inspired oxygen concentrations: hypoxia, normoxia, and hyperoxia, is described. This technique utilizes human pulmonary physiology research techniques in an MR scanning environment.

Magnetic Resonance Imaging Quantificazione di perfusione polmonare mediante calibrati Etichettatura arteriosa Spin

This demonstrates a MR imaging method to measure the spatial distribution of pulmonary blood flow in healthy subjects 
during normoxia (inspired O2, ...

Ricerca

Medicina

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

Segmentazione e misurazione dei volumi FAT nei modelli murini di obesità Utilizzo di X-ray Tomografia Computerizzata

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

Epidurale Misurazione della pressione intracranica in ratto utilizzando una fibra ottica trasduttore di pressione

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

Quantificazione del cancro al seno Cellula Invasività Utilizzando una tridimensionale (3D) Modello

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United States. Tacrolimus is a narrow therapeutic index drug and as such requires therapeutic drug monitoring and dose adjustment based on its whole blood trough concentrations. To facilitate home therapeutic drug and adherence monitoring, the collection of dried blood spots is an attractive concept. After a finger stick, the patient collects a blood drop on filter paper at home. After the blood is dried, it is mailed to the analytical laboratory where tacrolimus is quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in combination with a simple manual protein precipitation step and online column extraction.
For tacrolimus analysis, a 6-mm disc is punched from the saturated center of the blood spot. The blood spot is homogenized using a bullet blender and then proteins are precipitated with methanol/0.2 M ZnSO4 containing the internal standard D2,13C-tacrolimus. After vortexing and centrifugation, 100 &#181;l of supernatant is injected into an online extraction column and washed with 5 ml/min of 0.1 formic acid/acetonitrile (7:3, v:v) for 1 min. Hereafter, the switching valve is activated and the analytes are back-flushed onto the analytical column (and separated using a 0.1% formic acid/acetonitrile gradient). Tacrolimus is quantified in the positive multi reaction mode (MRM) using a tandem mass spectrometer.
The assay is linear from 1 to 50 ng/ml. Inter-assay variability (3.6%-6.1%) and accuracy (91.7%-101.6%) as assessed over 20 days meet acceptance criteria. Average extraction recovery is 95.5%. There are no relevant carry-over, matrix interferences and matrix effects. Tacrolimus is stable in dried blood spots at RT and at +4 &#176;C for 1 week. Extracted samples in the autosampler are stable at +4 &#176;C for at least 72 hr.

Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.

Quantificazione del immunosoppressore Tacrolimus su macchie di sangue secco Uso LC-MS / MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

Mass quantitativa spettrometrica Profiling di proteomi cancro-cellulari derivate da liquido e tumori solidi

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control seizures. However, a significant minority of patients continues to exhibit post-operative seizures, even in those cases in which the suspected source of seizures has been correctly localized and resected. The protocol presented here combines a clinical procedure routinely employed during the pre-operative evaluation of temporal lobe epilepsy (TLE) patients with a novel technique for network analysis. The method allows for the evaluation of the temporal evolution of mesial network parameters. The bilateral insertion of foramen ovale electrodes (FOE) into the ambient cistern simultaneously records electrocortical activity at several mesial areas in the temporal lobe. Furthermore, network methodology applied to the recorded time series tracks the temporal evolution of the mesial networks both interictally and during the seizures. In this way, the presented protocol offers a unique way to visualize and quantify measures that considers the relationships between several mesial areas instead of a single area.

This protocol describes a procedure to track the evolution of mesial network measures in temporal lobe epilepsy (TLE) patients. It is based on the combination of intracranial recordings with a novel numerical technique for data analysis. Specifically, we present a protocol for network analyses of foramen ovale recordings.

Network Analysis di forame ovale elettrodi Recordings in farmaco-resistenti epilessia del lobo temporale pazienti

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control ...

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel therapeutics are urgently required. The ability of dosed TB drugs to reach and sterilize bacteria within poorly-vascularized necrotic regions (caseum) of pulmonary granulomas is crucial for successful therapeutic intervention. Effective therapeutic regimens must therefore contain drugs with favorable caseum penetration properties. Current LC/MS methods for quantifying drug levels in biological tissues have limited spatial resolution capabilities, making it difficult to accurately determine absolute drug concentrations within small tissue compartments such as those found within necrotic granulomas. Here we present a protocol combining laser capture microdissection (LCM) of pathologically-distinct tissue regions with LC/MS quantification. This technique provides absolute quantification of drugs within granuloma caseum, surrounding cellular lesion and uninvolved lung tissue and, therefore, accurately determines whether bactericidal concentrations are being achieved. In addition to tuberculosis research, the technique has many potential applications for spatially-resolved quantification of drugs in diseased tissues.

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS

Here, we describe a protocol using laser capture microdissection coupled with LC/MS analysis to spatially-quantify drug distributions within pulmonary tuberculosis granulomas. The approach has broad applicability to quantifying drug concentrations within tissues at high spatial detail.

Spaziali quantificazione delle droghe nelle lesioni di tubercolosi polmonare di Laser Capture Microdissection liquido cromatografia spettrometria di massa (LCM-LC/MS)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel ...

Measurement of Carotenoids in Perifovea using the Macular Pigment Reflectometer

The macular pigment reflectometer (MPR) objectively measures the overall macular pigment optical density (MPOD) and further provides the lutein optical density (L-OD) and zeaxanthin optical density (Z-OD) in the central 1 degree of the fovea. A modification of the technique was developed to evaluate in vivo carotenoid density eccentric to the fovea. An adjustable track system with red LED lights was placed 6.1 m away from the participant to facilitate ocular fixation. Lights were spaced appropriately to create increments of 1 degree retinal disparity during the reflectometry measurements. All reflectometry measurements were obtained with pupillary dilation. The mean MPR-MPOD value for the central measurement was 0.593 (SD 0.161) with an L-OD to Z-OD ratio of 1:2.61. The MPR-MPOD value at 1 degree was 0.248 and the mean MPR-MPOD value at 2 degrees in the parafoveal region was 0.143. The L-OD to Z-OD ratio at 1 degree and 2 degrees off center was 1.38:1.0 and 2.08:1.0, respectively. The results demonstrate that MPOD measurements obtained using the MPR decrease as a function of retinal eccentricity and that there is a higher concentration of zeaxanthin centrally compared to lutein. The L-OD to Z-OD ratio changes with foveal eccentricity, with twice more lutein than zeaxanthin at 2 degrees off center. Our technique successfully provides a quick in vivo method for the measurement of macular pigment optical density at various foveal eccentricities. The results agree with prior published in vivo and in vitro xanthophyll carotenoid density distribution measurements.

We present a protocol to determine the levels of overall macular pigment, lutein, and zeaxanthin optical density in the central and parafoveal regions of the retina. The protocol includes a novel adjustable track system used to measure macular pigment optical density in the foveal eccentricity.

Misurazione dei carotenoidi in Perifovea utilizzando il Riflessometro pigmento maculare

The macular pigment reflectometer (MPR) objectively measures the overall macular pigment optical density (MPOD) and further provides the lutein optical ...

Using an Automated Hirschberg Test App to Evaluate Ocular Alignment

A smartphone app has been developed to perform the automated photographic Hirschberg test for objective measurement of ocular misalignment. By computing the difference in corneal reflection generated by the phone camera flash relative to the iris center based on high resolution images, the app can measure misalignment with a much higher precision than the naked eye performing the Hirschberg test. It has been validated in a previous clinical evaluation study by comparing to the clinical gold standard-prism and alternate cover test. The goal of this article is to describe the testing techniques regarding how to use the app to measure ocular alignment for different fixation distances, without or with cover to break fusion, as well as angle kappa, so that users can use the app to perform equivalent tests typically done in clinic using prisms.

We present a protocol of using a smartphone app to perform Hirschberg test for measuring manifest and intermittent ocular misalignment (strabismus) under near and far fixation conditions.

Utilizzo di un'app di test Hirschberg automatizzata per valutare l'allineamento oculare

A smartphone app has been developed to perform the automated photographic Hirschberg test for objective measurement of ocular misalignment. By computing ...