לפני הניתוח אימון <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> במהלך בדיקת CT בטן ניתן לראות בקלות את צינורות המרה (המופיע בתוך parenchyma הכבד עם צבע כהה יותר) מוגדל נרחב בצידו השמאלי של 0.3 הכבד D שחזור הפגינו נוכחות של עורק הכבד הימני שמקורם ישירות האאורטה ו אין הפרעות כלי דם נוספים. </li><li style=";text-align:right;direction:rtl"> הכבד ניתן לחלק תפקודית תוך 8 קטעים כפי שמוצג בסרטון ובהתחשב כי התרחבות הוגבל קטע 2 ו -3 (מגזרים משמאל), וכן sectoriectomy שמאל לפרוסקופי תוכנן. </li></ol> Intraoperative נוהל <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> החולה יוצב בעמדה פרקדן ואת לעמוד מנתח בין הרגליים של המטופל. 5 trocars משמשים והניח כפי שמוצג בסרטון </li><li style=";text-align:right;direction:rtl"> מהדק דם מושם על שילוש הפורטל על מנת להפחית את הדימום (manouvre של פרינגל) </li><li style=";text-align:right;direction:rtl"> אגף parenchyma הכבד מתבצעת באמצעות שילוב electrocautery או דו קוטבית הרמוני אזמל לאורך הרצועה falciform בין קטע III ו-IV </li><li style=";text-align:right;direction:rtl"> לאחר הקטע השלישי pedicle הוא גזור לגמרי זה יכול להיות מחולק באמצעות אנדו 45mm GIA עם מחסנית וסקולרית. </li><li style=";text-align:right;direction:rtl"> ההליך הוא מנהל עם חלוקה נוספת של parenchyma עד הראשי pedicle למגזר השנייה הוא הגיע, מבודדים ולבסוף מחולק באמצעות endoGIA 45 מ&quot;מ עם מחסנית וסקולרית </li><li style=";text-align:right;direction:rtl"> רצועה משולשים שמאל מחולק כיום באמצעות electrocautery ועל שמאל וריד הכבד מופרד מחולק באמצעות מהדק של כלי הדם </li><li style=";text-align:right;direction:rtl"> משטח כבד לוודא שהדימום; ארגון קרן מוחל על פני השטח הכבד ואת מהדק וסקולרית מוסר. בשלב זה דבק הפיברין מוחל על מנת להפחית את הסיכון שלאחר הניתוח דליפות מרה. הדגימה ממוקם endobag והוציאו </li></ol>

<ol>
	<li>Nguyen, K. T., Gamblin, T. C., Geller, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laparoscopic+liver+resection+for+cancer.">Laparoscopic liver resection for cancer.</a> Future Oncol. 4 (5), 661-670 (2008).</li><li>Gumbs, A. A., Gayet, B., Gagner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laparoscopic+liver+resection:+when+to+use+the+laparoscopic+stapler+device.">Laparoscopic liver resection: when to use the laparoscopic stapler device.</a> HPB (Oxford). 10 (4), 296-303 (2008).</li><li>Pulitan&#242;, C., Catena, M., Arru, M., Guzzetti, E., Comotti, L., Ferla, G., Aldrighetti, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laparoscopic+liver+resection+without+portal+clamping:+a+prospective+evaluation.">Laparoscopic liver resection without portal clamping: a prospective evaluation.</a> Surg Endosc. 22 (10), 2196-2200 (2008).</li><li>Yonem, O., Bayraktar, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+characteristics+of+Caroli's+disease.">Clinical characteristics of Caroli's disease.</a> World J Gastroenterol. 13 (13), 1934-1937 (2007).</li></ol>

ניתוח לפרוסקופי ניתן לבצע בבטחה גם כריתה הכבד הן מחלות שפירים וממאירים. הפחתת טראומה ניתוחית באמצעות גישה פולשנית מאפשרת למזער את שכיחות הסיבוכים שלאחר הניתוח שלאחר הניתוח ואת השהות בבית החולים.

laparoscopic left liver sectoriectomy of caroli's disease limited to segment ii and iii

מחלת קארולי מוגדר התרחבות חריגה של צינוריות המרה התוך hepatica: השכיחות שלה היא נמוכה מאוד (1 באוכלוסייה 1000000) וגם ברוב המקרים הכבד כולו הוא עניין השתלת כבד היא טיפול הבחירה. במקרה של התרחבות מוגבל באונה השמאלית או הימנית, כריתה כבד יכול להתבצע. במשך שנים רבות את הגישה הסטנדרטית עבור כריתה הכבד כבר laparotomy פורמלי באמצעות חתך גדול בבטן זה מאופיין בתחלואה שלאחר operatie משמעותי. לאחרונה, פולשנית, בגישה לפרוסקופית הוצע טכניקה כירורגית אפשרית כריתה הכבד הן מחלות שפירים וממאירים. היתרונות העיקריים של הגישה פולשנית מיוצג על ידי הפחתה משמעותית של הטראומה כירורגית המאפשרת התאוששות מהירה פחות סיבוכים שלאחר הניתוח. וידאו זה מציג מקרה של מחלה של קארולי התרחשו גבר בן 58 הודה במחלקת גסטרואנטרולוגיה על התפרצות פתאומית של כאבי בטן הקשורים חום (&gt; 38C °), בחילות רועד. אולטרסאונד בטן הפגינו הרחבה משמעותית של התוך הכבד צינוריות מרה שמאל מוצב ללא הוכחות של כיס המרה או צינור משותף אבני מרה. ממצאים אלה אוששו מחשב בטן טומוגרפיה ברזולוציה גבוהה. Sectoriectomy לפרוסקופי שמאל תוכנן. חמש trocars ו 30 ° אופטיים שימש, חקר חלל הבטן לא הראה שום הידבקויות או עדויות של מחלות אחרות. כדי לשלוט על זרימת הדם אל הכבד, מהדק כלי דם הונח על הכבד pedicle (פרינגל s manouvre), חלוקת Parenchymal מתבצעת תוך שימוש משולב של 5 מ&quot;מ מלקחיים דו קוטבית 5 מבתר קולי מ&quot;מ. צינור מורחבים קשות בכבד שמאל היה מבודד מחולק באמצעות מהדק 45mm וסקולרית אנדוסקופית. דיסקציה הכבד נמשכה עד הבידוד של הסניף הפורטל הראשי השמאלי שהיה מחולק אז עם מחסנית נוספת של כלי הדם מהדק 45 מ&quot;מ. בשלב שלו הכבד שמאל נשאר מחובר רק על ידי עורק הכבד משמאל: חלוקת רצועה המשולש בוצע באמצעות וו monopolar ואת וריד הכבד מבודדים מחולק באמצעות מהדק של כלי הדם. Haemostatis היה מעודן על ידי יישום של ארגון קרן קרישה ואף דימום התגלה גם לאחר הסרת מהדק כלי הדם (הזמן הכולל של פרינגל 27 דקות). כמובן לאחר הניתוח היה העלאת אירועים, מינימלי של בדיקות תפקודי כבד נרשם ביום שלאחר הניתוח 1 אבל חזרו למסלולם בבית שוחרר ביום שלאחר הניתוח 3.

Watch this Scientific Journal Video about Laparoscopic Left Liver Sectoriectomy of Caroli's Disease Limited to Segment II and III at JoVE.com

Laparoscopic Left Liver Sectoriectomy of Caroli&#039;s Disease Limited to Segment II and III

וידאו זה מציג מקרה של sectoriectomy לפרוסקופי שמאל perfomed הכבד בגבר בן 53 שנים סובל ממחלת מוגבל קטע II ו-III של הכבד של קרולי.

Sectoriectomy לפרוסקופי כבד שמאל של מחלת קארולי מוגבלת למגזר II ו-III

Caroli's disease is defined as a abnormal dilatation of the intra-hepatica bile ducts: Its  incidence is extremely low  (1 in 1,000,000 population) and in ...

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Laparoscopic Left Liver Sectoriectomy of Caroli's Disease Limited to Segment II and III

14.1K Views.  University of Insubria. וידאו זה מציג מקרה של sectoriectomy לפרוסקופי שמאל perfomed הכבד בגבר בן 53 שנים סובל ממחלת מוגבל קטע II ו-III של הכבד של קרולי.

Video: Laparoscopic Left Liver Sectoriectomy of Caroli's Disease Limited to Segment II and III

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

Matrigel מבוסס גיבוש Tube Assay להעריך את פעילות Vasculogenic של תאים סרטניים

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (&tilde;100 &mu;m) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2.
SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7.
With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11.
In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

ב אנדוציטוזה כבד vivo ואחריו טיהור בתאי הכבד על ידי זלוף כבד

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

הבידוד של הריבוזום פוליפפטידים המתהווה כרוכים<em&gt; במבחנה</em&gt; כדי לזהות אתרים השהה של תרגום לאורך ה-mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro and in vivo. Fluorescent probes are particularly useful in determining the bio-distribution of administered large molecules, where the addition of a small-molecule fluorescent label is unlikely to affect the kinetics or bio-distribution of the compound. A variety of methods exist to examine bio-distribution that vary significantly in the amount of effort required and whether the resulting measurements are fully quantitative, but using multiple methods in conjunction can provide a rapid and effective system for analyzing bio-distributions.
Ex vivo whole-organ imaging is a method that can be used to quickly compare the relative concentrations of fluorescent molecules within tissues and between multiple types of tissues or treatment groups. Using an imaging platform designed for live-animal or whole-organ imaging, fluorescence within intact tissues can be determined without further processing, saving time and labor while providing an accurate picture of the overall bio-distribution. This process is ideal in experiments attempting to determine the tissue specificity of a compound or for the comparison of multiple different compounds. Quantitative tissue histology on the other hand requires extensive further processing of tissues in order to create a quantitative measure of the labeled compounds. To accurately assess bio-distribution, all tissues of interest must be sliced, scanned, and analyzed relative to standard curves in order to make comparisons between tissues or groups. Quantitative tissue histology is the gold standard for determining absolute compound concentrations within tissues.
Here, we describe how both methods can be used together effectively to assess the ability of different administration methods and compound modifications to target and deliver fluorescently labeled molecules to the central nervous system1.

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Ex vivo whole-organ imaging is a rapid method for determining the relative concentrations of fluorescently labeled compounds within and between tissues or treatment groups. Conversely, quantitative fluorescence histology, while more labor intensive, allows for the quantification of the absolute tissue levels of labeled molecules.

השימוש של<em&gt; Ex Vivo</em&gt; הדמיה Whole-איברים היסטולוגיה רקמות כמותי כדי לקבוע את ביו-הפצה של מולקולות שכותרתו fluorescently

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro ...

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell motility, or cell signaling. The green algae Chlamydomonas reinhardtii has proven to be an insightful model in the study of centriole architecture, function, and protein composition. Despite great advances toward understanding centriolar architecture, one of the current challenges is to determine the precise localization of centriolar components within structural regions of the centriole in order to better understand their role in centriole biogenesis. A major limitation lies in the resolution of fluorescence microscopy, which complicates the interpretation of protein localization in this organelle with dimensions close to the diffraction limit. To tackle this question, we are providing a method to purify and image a large number of C. reinhardtii centrioles with different orientations using super-resolution microscopy. This technique allows further processing of data through fluorescent single-particle averaging (Fluo-SPA) owing to the large number of centrioles acquired. Fluo-SPA generates averages of stained C. reinhardtii centrioles in different orientations, thus facilitating the localization of distinct proteins in centriolar sub-regions. Importantly, this method can be applied to image centrioles from other species or other large macromolecular assemblies.

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

We have developed a strategy to purify and image a large number of centrioles in different orientations amenable for super-resolution microscopy and single-particle averaging.

בידוד והדמיה פלורסצנטיות עבור שחזור יחיד-חלקיקים של Chlamydomonas Centrioles

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell ...

Isolation of Human Primary Valve Cells for In vitro Disease Modeling

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve causes aortic stenosis and contributes to heart failure and stroke. Disease pathogenesis is multifactorial, and stresses such as inflammation, extracellular matrix remodeling, turbulent flow, and mechanical stress and strain contribute to the osteogenic differentiation of valve endothelial and valve interstitial cells. However, the precise initiating factors that drive the osteogenic transition of a healthy cell into a calcifying cell are not fully defined. Further, the only current therapy for CAVD-induced aortic stenosis is aortic valve replacement, whereby the native valve is removed (surgical aortic valve replacement, SAVR) or a fully collapsible replacement valve is inserted via a catheter (transcatheter aortic valve replacement, TAVR). These surgical procedures come at a high cost and with serious risks; thus, identifying novel therapeutic targets for drug discovery is imperative. To that end, the present study develops a workflow where surgically removed tissues from patients and donor cadaver tissues are used to create patient-specific primary lines of valvular cells for in vitro disease modeling. This protocol introduces the utilization of a cold storage solution, commonly utilized in organ transplant, to reduce the damage caused by the often-lengthy procurement time between tissue excision and laboratory processing with the benefit of greatly stabilizing cells of the excised tissue. The results of the present study demonstrate that isolated valve cells retain their proliferative capacity and endothelial and interstitial phenotypes in culture upwards of several days after valve removal from the donor. Using these materials allows for the collection of control and CAVD cells, from which both control and disease cell lines are established.

This protocol describes the collection of human aortic valves extracted during surgical aortic valve replacement procedures or from cadaveric tissue, and the subsequent isolation, expansion, and characterization of patient specific primary valve endothelial and interstitial cells. Included are important details regarding the processes needed to ensure cell viability and phenotype specificity.

בידוד תאי שסתום ראשוניים אנושיים למידול מחלות חוץ גופיות

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve ...

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is a growing global health issue, and the impact of NAFLD is compounded by the current lack of effective treatments. Considerable limiting factors hindering the timely and accurate diagnosis (including grading) and monitoring of NAFLD, as well as the development of potential therapies, are the current inadequacies in the characterization of the hepatic microenvironment structure and the scoring of the disease stage in a spatiotemporal and non-invasive manner. Using a diet-induced NAFLD mouse model, we investigated the use of in vivo micro-computed tomography (CT) imaging techniques as a non-invasive method to assess the progression stages of NAFLD, focusing predominantly on the hepatic vascular network due to its significant involvement in NAFLD-related hepatic dysregulation. This imaging methodology allows for longitudinal analysis of liver steatosis and functional tissue uptake, as well as the evaluation of the relative blood volume, portal vein diameter, and density of the vascular network. Understanding the adaptations of the hepatic vascular network during NAFLD progression and correlating this with other ways of characterizing the disease progression (steatosis, inflammation, fibrosis) using the proposed method can pave the way toward the establishment of new, more efficient, and reproducible approaches for NAFLD research in mice. This protocol is also expected to upgrade the value of preclinical animal models for investigating&#160;the development of novel therapies against disease progression.

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease

Using a diet-induced non-alcoholic fatty liver disease (NAFLD) mouse model, we describe the use of novel in vivo micro-computed tomography imaging techniques as a non-invasive method to assess the progression stages of NAFLD, focusing predominantly on the hepatic vascular network due to its significant involvement in NAFLD-related hepatic dysregulation.

רומן in vivo טכניקות הדמיה טומוגרפיה מיקרו-ממוחשבת להערכת ההתקדמות של מחלת כבד שומני לא אלכוהולי

Non-alcoholic fatty liver disease (NAFLD) is a growing global health issue, and the impact of NAFLD is compounded by the current lack of effective ...

איתור וכימות DNA של נגיף הפטיטיס B בסרום אנושי

Real-Time Polymerase Chain Reaction-Based Detection and Quantification of Hepatitis B Virus DNA

Hepatitis B virus (HBV) is a significant cause of liver disease worldwide. It can lead to acute or chronic infections, making individuals highly susceptible to fatal cirrhosis and liver cancer. Accurate detection and quantification of HBV DNA in the blood are essential for diagnosing and monitoring HBV infection. The most common method for detecting HBV DNA is real-time PCR, which can be used to detect the virus and assess the viral load to monitor the response to antiviral therapy. Here, we describe a detailed protocol for the detection and quantification of HBV DNA in human serum or plasma using an IVD-marked real-time PCR-based kit. The kit uses primers and probes that target the highly conserved core region of the HBV genome and can accurately quantify all HBV genotypes (A, B, C, D, E, F, G, H, I, and J). The kit also includes an endogenous internal control to monitor possible PCR inhibition. This assay runs for 40 cycles, and its cutoff is 38 Ct. For the quantification of HBV DNA in clinical samples, a set of 5 quantification standards is provided with the kit. The standards contain known concentrations of HBV-specific DNA that are calibrated against the 4th WHO International Standard for HBV DNA for the nucleic acid test (NIBSC code 10/266). The standards are used to validate the functionality of the HBV-specific DNA amplification and to generate a standard curve, allowing the quantification of HBV DNA in a sample. HBV DNA as low as 2.5 IU/mL was detected using the PCR kit. The high sensitivity and reproducibility of the kit make it a powerful tool in clinical laboratories, aiding healthcare professionals in effectively diagnosing and managing HBV infections.

מחבר זרקור: התקדמות ואתגרים בזיהוי וירוס הפטיטיס B 

Real-time polymerase chain reaction (PCR)-based detection and quantification of hepatitis B virus (HBV) DNA is a sensitive and accurate method for diagnosing and monitoring HBV infection. Here, we present a protocol for HBV DNA detection and the viral load measurement of a sample.