Razi Journal of Medical Sciences
مجله علوم پزشکی رازی
RJMS
Medical Sciences
http://rjms.iums.ac.ir
39
journal39
2228-7043
2228-7051
en
jalali
1400
10
1
gregorian
2022
1
1
28
11
online
1
fulltext
fa
طراحی و تهیه لام تشخیصی آنتیبادیهای ضد سیتوپلاسم نوتروفیل (ANCA) با روش ایمونوفلورسانس غیرمستقیم
Design and Preparation of Diagnostic Slides for Antineutrophil Cytoplasmic Antibodies (ANCA) by Indirect Immunofluorescence Method
ایمنیشناسی
Immunology
پژوهشي
Research
<div style="text-align: justify;"><span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="tab-stops:10.5pt"><span style="direction:rtl"><span style="unicode-bidi:embed"><span style="line-height:115%"><span style="font-family:Calibri,"sans-serif""><b><span lang="FA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:#0070c0">زمینه و هدف: </span></span></span></span></b><span lang="FA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black">آزمایش آنتیبادیها علیه سیتوپلاسم نوتروفیلها </span></span></span></span><span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black">(</span></span></span></span><span dir="LTR" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:black">ANCA</span></span></span></span><span lang="FA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black">) اولین درخواست در تشخیص اولیه و غربال بیماران مشکوک به بیماریهای خودایمن واسکولیت عروق کوچک و همچنین پیگیری روند درمان بیماران است که با استفاده از روش ایمونوفلورسانس غیرمستقیم (</span></span></span></span><span dir="LTR" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:black">IFA</span></span></span></span><span lang="FA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black">) حضور آنتیبادیها علیه آنتیژنها درون سیتوپلاسم نوتروفیلها را بررسی میشود. هدف از این مطالعه طراحی و تهیه لامهای تشخیص آنتیبادیها علیه سیتوپلاسم نوتروفیلها (</span></span></span></span><span dir="LTR" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:black">ANCA</span></span></span></span><span lang="FA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black">) به روش ایمونوفلورسانس غیرمستقیم است.</span></span></span></span></span></span></span></span></span></span></span></span><br>
<span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="tab-stops:10.5pt"><span style="direction:rtl"><span style="unicode-bidi:embed"><span style="line-height:115%"><span style="font-family:Calibri,"sans-serif""><b><span lang="FA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:#0070c0">روش کار: </span></span></span></span></b> <span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black">با استفاده از روش گرادیانت شیب غلظت و محلول دکستران</span></span></span></span><span lang="FA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black">،</span></span></span></span> <span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black">نوتروفیلها از خون محیطی افراد سالم جدا شدند. خلوص و بازده سلولهای استخراج شده توسط رنگ آمیزی رایت گیمسا، لام نئوبار و دستگاه شمارش گر اتوماتیک بررسی شد. با استفاده از فیکساتور اتانول، نوتروفیلهای جدا شده بروی اسلاید فیکس و رنگ آمیزی فلورسانت </span></span></span></span><span lang="FA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black">ش</span></span></span></span><span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black">دند. </span></span></span></span></span></span></span></span></span></span></span></span><br>
<span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="tab-stops:10.5pt"><span style="direction:rtl"><span style="unicode-bidi:embed"><span style="line-height:115%"><span style="font-family:Calibri,"sans-serif""><b><span lang="FA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:#0070c0">یافتهها: </span></span></span></span></b><span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black">خلوص و تعداد نوتروفیلهای جدا شده توسط دستگاه شمارشگر سلولی بررسی شد. بسته به نوتروفیل ورودی بطور میانگین (</span></span></span></span><span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:115%"><span style="color:black">±</span></span></span><span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black">انحراف استاندارد) تعداد نوتروفیلهای جدا شده برابر با </span></span></span></span><span dir="LTR" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:black">)</span></span></span></span><span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black">950</span></span></span></span><span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:115%"><span style="color:black">±</span></span></span><span dir="LTR" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:black">(</span></span></span></span><span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black">6000 سلول در هر میکرولیتر بود. میانگین خلوص نوتروفیلهای جدا شده برابر با 1/94 درصد بود. همچنین توسط رنگ آمیزی تریپان بلو درصد سلولهای زنده بیش از 85 درصد قبل از فیکساسیون تعیین گردید. آنالیز نتایج رنگآمیزی فلورسانس غیر مستقیم </span></span></span></span><span dir="LTR" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:black">ANCA</span></span></span></span><span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black"> بر روی لامهای تهیه شده در مقایسه با برند تجاری مرجع کاملا منطبق بود.</span></span></span></span></span></span></span></span></span></span></span></span><br>
<span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="tab-stops:10.5pt"><span style="direction:rtl"><span style="unicode-bidi:embed"><span style="line-height:115%"><span style="font-family:Calibri,"sans-serif""><b><span lang="FA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:#0070c0">نتیجهگیری: </span></span></span></span></b><span lang="FA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black">نتایج این مطالعه نشان میدهد که روش بکار برده شده میتواند برای تجاری سازی و تولید لامهای </span></span></span></span><span dir="LTR" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:black">ANCA</span></span></span></span><span style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black"> جهت اهداف تشخیصی بکار گرفته شود. در این زمینه علاوه بر بهینه نمودن مراحل جداسازی و فیکس نمودن سلولها نیاز است جهت تولید در تعداد انبوه و تجاری، پایداری لامهای تهیه شده در زمان انبارش و نگهداری نیز ارزیابی قرار گرفته و بهینه شود.</span></span></span></span><span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black"></span></span></span></span></span></span></span></span></span></span></span></span><br>
</div>
<div style="text-align: justify;"><span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="tab-stops:10.5pt"><span style="line-height:115%"><span style="font-family:Calibri,"sans-serif""><b><span style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:#0070c0">Background & Aims:</span></span></span></span></b> <span style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:black">One of the most important tests for the detection of antibodies against neutrophil cytoplasm in autoimmune vasculitis diseases is the Antineutrophil Cytoplasmic Antibodies (ANCA) test. The ANCA test is the first order in the initial diagnosis and screening of patients with suspected autoimmune diseases, including small-vessel vasculitis, as well as the follow-up of patients' treatment, which uses indirect immunofluorescence assay (IFA) to detect the presence of antibodies against antigens in the cytoplasm of neutrophils. In addition to vasculitis, the evaluation of this antibody is used in other autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and inflammatory bowel diseases (IBD). </span></span></span></span></span></span></span></span></span></span><br>
<span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="tab-stops:10.5pt"><span style="line-height:115%"><span style="font-family:Calibri,"sans-serif""><span style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:black">To prepare IFA slides for diagnostic purposes, neutrophils extracted from human peripheral blood are used as substrates. The IFA is a common and acceptable method in medical diagnostic laboratories for the detection of these autoantibodies. The first experience in the development of this technique back to the work of Koons and Kaplan, although today, this method has been widely developed in the field of laboratory diagnosis as well as research in basic medical sciences. A distinct advantage of the immunofluorescence assay is its applicability to fresh tissue and fixed cells. </span></span></span></span></span></span></span></span></span></span><br>
<span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="tab-stops:10.5pt"><span style="line-height:115%"><span style="font-family:Calibri,"sans-serif""><span style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:black">This method is based on the specific antigen binding to specific antibodies conjugated with fluorescent dyes and fluorescent light emission. There is two common types of immunofluorescence assay include direct and indirect immunofluorescence used for detection autoantibodies. In the direct immunofluorescence method, specific antibodies are conjugated to fluorescent dyes, and by binding to their specific antigen, the green fluorescent light is recognized under a fluorescent microscope. Although this procedure is faster, it is less often applied in medical diagnostic labs. In the IFA method, a specific antibody against the target antigen (called the primary antibody) binds to the target antigen recognized by a secondary antibody that is against the fixed region of the primary antibody and conjugated with fluorescent dyes, such as fluorescein isothiocyanate (FITC). In the indirect immunofluorescence assay, due to the binding of several secondary antibodies to the primary antibody, the generated fluorescence signals will be amplified, which in turn increases the fluorescence intensity and sensitivity. The aim of this study was to design and prepare slides for antibodies detection against antigens in cytoplasm neutrophils by the indirect immunofluorescence method. </span></span></span></span><span dir="RTL" lang="FA" style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"B Mitra""><span style="color:black"></span></span></span></span></span></span></span></span></span></span><br>
<span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="tab-stops:10.5pt"><span style="line-height:115%"><span style="font-family:Calibri,"sans-serif""><span style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:black">Furthermore, the next step by optimizing fluorescent patterns to detect different types of antibodies against neutrophil cytoplasm by indirect immunofluorescence method. Since 1980, the search for antibodies against the neutrophil cytoplasm has been recognized as one of the most effective diagnostic tools for diseases associated with small to medium-sized vasculitis. ANCA in the diagnosis and follow-up of the treatment of vasculitic diseases including granulomatosis with polyangiitis, formerly known as Wegener granulomatosis, microscopic polyangiitis, Eosinophilic granulomatosis with polyangiitis, formerly known as Churg – Strauss syndrome, is used. Most laboratories around the world use IFA for screening and early detection of ANCA.</span></span></span></span></span></span></span></span></span></span><br>
<span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="tab-stops:10.5pt"><span style="line-height:115%"><span style="font-family:Calibri,"sans-serif""><span style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:black">After the ANCA test is positive, the target antigens of the antibodies are evaluated by the ELISA method. The two most common targets of autoantibodies are myeloperoxidase (MPO) and proteinase-3 (PR-3) in patients with vasculitis. </span></span></span></span></span></span></span></span></span></span><br>
<span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="tab-stops:10.5pt"><span style="line-height:115%"><span style="font-family:Calibri,"sans-serif""><span style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:black">However, a clinical association of these vasculitis diseases with other antigens such as elastase, lactoferrin, azurocidin, lysozyme, H-Lamp-2, and other lesser-known antigens has also been observed.</span></span></span></span></span></span></span></span></span></span><br>
<span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="tab-stops:10.5pt"><span style="line-height:115%"><span style="font-family:Calibri,"sans-serif""><span style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:black">Antibodies against neutrophil cytoplasm have also been reported in other small vessel vasculitis diseases such as microscopic polyangiitis (MPA), eosinophilic granulomatosis with polyangiitis (EGPA), and necrotizing crescentic glomerulonephritis. Initially, the non-commercial ELISA method was used to diagnose PR3-ANCA and MPO-ANCA, but inconsistent results between laboratories showed the global need for standardization of this test . In 1998, fifteen laboratory centers around the world evaluated and standardized the standard method for measuring antibodies against neutrophil cytoplasm (ANCA) and its specific antigens, including PR3 and MPO, by indirect immunofluorescence and ELISA in patients with idiopathic systemic vasculitis. . The result of this standardization increased the detection value of ANCA by immunofluorescence and ELISA methods.</span></span></span></span></span></span></span></span></span></span><br>
<span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="tab-stops:10.5pt"><span style="line-height:115%"><span style="font-family:Calibri,"sans-serif""><span style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:black">One of the most important pillars of the commercialization of a product is the supply of raw materials with appropriate and sustainable quality. The first step in producing ANCA slides is the isolation of high purity and standard quality peripheral blood neutrophil cells. Peripheral blood neutrophils are short-lived, highly sensitive cells that cannot be stored for long periods of time, and it is recommended that these cells be isolated from whole blood immediately after blood sampling. To date, several methods have been proposed to isolate neutrophils from peripheral blood. In this study, it was shown that using the dextran method and subsequent separation by centrifugal gradient method with filament is one of the cost-effective methods with high purity to provide the ANCA test substrate.</span></span></span></span></span></span></span></span></span></span><br>
<span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="tab-stops:10.5pt"><span style="line-height:115%"><span style="font-family:Calibri,"sans-serif""><b><span style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:#0070c0">Methods:</span></span></span></span></b> <span style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:black">Using the Ficoll density gradient centrifugation and dextran solution, neutrophils were isolated from the peripheral blood of healthy individuals (without a history of any disease, especially autoimmune diseases in themselves and first-degree relatives). The purity and efficiency of the isolated cells were evaluated by Wright Giemsa staining, the neobar slide, and an automatic counting machine. Isolated neutrophils were fixed on slides using an ethanol fixative. At this stage, by selecting the best protocol, in terms of time and type of neutrophil fixation stage, they were fixed on the slide. In the next step, using positive and negative samples that have already been determined by ANCA with valid commercial kits (IVD certified), the slides containing fixed cells were fluorescent stained and compared with control samples.</span></span></span></span></span></span></span></span></span></span><br>
<span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="tab-stops:10.5pt"><span style="line-height:115%"><span style="font-family:Calibri,"sans-serif""><b><span style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:#0070c0">Results:</span></span></span></span></b><span style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:black"> The number and purity of neutrophils isolated were evaluated by a Sysmex automatic cell counter. Depending on the peripheral blood neutrophils, the mean(±standard deviation) of the number of isolated neutrophils was 6000(±950) cells per microliter. According to white blood cell differential count, the mean purity of isolated neutrophils was 94.1%. Also, by trypan blue staining, the percentage of viable cells was determined to be more than 85% before fixation. The results of ANCA indirect fluorescent staining on the prepared slides were completely consistent with the results of control samples. </span></span></span></span></span></span></span></span></span></span><br>
<span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="tab-stops:10.5pt"><span style="line-height:115%"><span style="font-family:Calibri,"sans-serif""><b><span style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:#0070c0">Conclusion:</span></span></span></span></b> <span style="font-size:9.0pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:black">The results of this study showed that the method used can be applied for the commercialization and production of ANCA slides. In this regard, in addition to optimizing the steps of separation and fixing the neutrophils, it is necessary to evaluate and optimize the stability of the prepared slides during storage and production in order to produce in large numbers.</span></span></span></span></span></span></span></span></span></span><br>
<span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="tab-stops:10.5pt"><span style="line-height:115%"><span style="font-family:Calibri,"sans-serif""><span style="font-size:9.5pt"><span style="line-height:115%"><span style="font-family:"Times New Roman","serif""><span style="color:black"></span></span></span></span></span></span></span></span></span></span><br>
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آنتی بادی علیه سیتوپلاسم نوتروفیل ها (ANCA), لام تشخیصی, ایمونوفلورسانت غیرمستقیم (IFA), نوتروفیل, واسکولیت
ANCA, Diagnosis slide, Indirect immunofluorescence assay, Neutrophil, Vasculitis
89
98
http://rjms.iums.ac.ir/browse.php?a_code=A-10-6176-2&slc_lang=fa&sid=1
Majid
Khoshmirsafa
مجید
خوش میرصفا
khoshmirsafa.m@iums.ac.ir
3900319475328460063336
3900319475328460063336
No
Immunology Research Center, Institute of Immunology and Infectious Diseases, Iran University of Medical Sciences, Tehran, Iran, & Department of Immunology, Iran University of Medical Sciences, Tehran, Iran
استادیار ایمونولوژی، مرکز تحقیقات ایمونولوژی، پژوهشکده ایمونولوژی و بیماریهای عفونی، دانشگاه علوم پزشکی ایران، تهران، ایران، و گروه ایمونولوژی، دانشگاه علوم پزشکی ایران، تهران، ایران
Mohammad-Ali
Assarehzadegan
محمدعلی
عصاره زادگان
assareh.ma@iums.ac.ir
3900319475328460063337
3900319475328460063337
Yes
Immunology Research Center, Institute of Immunology and Infectious Diseases, Iran University of Medical Sciences, Tehran, Iran, & Department of Immunology, Iran University of Medical Sciences, Tehran, Iran
دانشیار ایمونولوژی، : مرکز تحقیقات ایمونولوژی، پژوهشکده ایمونولوژی و بیماریهای عفونی، دانشگاه علوم پزشکی ایران، تهران، ایران، و گروه ایمونولوژی، دانشگاه علوم پزشکی ایران، تهران، ایران
Faezeh
Shahba
فائزه
شهبا
Shahba.f@iums.ac.ir
3900319475328460063338
3900319475328460063338
No
Immunology Research Center, Institute of Immunology and Infectious Diseases, Iran University of medical Sciences, Tehran, Iran, & Department of Immunology, Iran University of Medical Sciences, Tehran, Iran
دانشجوی کارشناسی ارشد ایمونولوژی، مرکز تحقیقات ایمونولوژی، پژوهشکده ایمونولوژی و بیماریهای عفونی، دانشگاه علوم پزشکی ایران، تهران، ایران، و گروه ایمونولوژی، دانشگاه علوم پزشکی ایران، تهران، ایران