استخدام الوحيدات أحادي الطبقة الفحص لتقييم البلعمة بوساطة مستقبلات Fcγ
Summary
The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fcγ receptor (FcγR)-mediated phagocytosis.
Abstract
Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fcγ receptor (FcγR)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express FcγRs (e.g., FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIA) that are involved in immune responses. The MMA exploits the mechanism of FcγR-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate FcγRs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out FcγR-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo FcγR-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of FcγR-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.
Introduction
The monocyte monolayer assay (MMA) is an in vitro assay originally developed to better predict blood transfusion outcomes in patients with auto- or alloantibodies to red blood cells (RBCs)1-5. By assessing the effect of anti-RBC antibodies in mediating Fcγ receptor (FcγR)-mediated phagocytosis using this in vitro assay, it is possible to predict the clinical outcome in vivo. Indeed, the MMA has been used successfully to avoid immune destruction of antibody-bound RBCs, despite the transfusion of serologically incompatible blood5. The typical pre-transfusion procedure for compatibility testing, also termed crossmatching, involves serological methods that include typing the patient’s blood for ABO and Rh antigens and screening for the presence of anti-RBC antibodies in the patient6. Blood matched for ABO/Rh is selected, and if antibodies are present, an attempt to identify them is made so that blood for transfusion can be further selected to avoid these antigens. An ideal crossmatch result occurs when all donor blood is serologically compatible with the patient’s blood, which reduces the risk of post-transfusion hemolysis7. However, this system falls short for the small group of patients who have become alloimmunized upon repeated transfusion or pregnancy. These patients produce alloantibodies against specific RBC antigens. Some produce antibodies to antigens of very high frequency in the general population, and thus become progressively more difficult to crossmatch8,9. Adding to the complexity, not all alloantibodies are clinically significant; in other words, the binding of an alloantibody to RBCs detected by a serology test does not necessarily result in hemolysis when antigen-positive, incompatible blood is transfused. The MMA was originally developed to assess the potential clinical significance of serologically incompatible blood in a transfusion setting1-5.
Since extravascular hemolysis of antibody-bound RBCs is known to be mediated by the mononuclear phagocyte system, primary monocytes/macrophages are utilized in the development of diagnostic assays. The first assay to study the interaction of monocytes, RBCs, and antibodies was published in 1975, but the sub-optimal conditions used led only to rosette formation (the binding of RBCs to the periphery of the monocyte), and no phagocytosis was observed10. Significant modifications to the assay were made by several groups, leading to an assay for which the level of phagocytosis of alloantibody-bound RBCs could be correlated to the clinical outcome of hemolysis1-5. Recently, the optimal storage conditions of clinical samples and further optimization of assay conditions were examined to enhance the utility of a clinical MMA crossmatch using autologous patient samples11.
Three other diagnostic techniques have been employed in addition to the MMA in predicting transfusion outcomes: the 51Cr release test, the rosette test, and the chemiluminescence test (CLT). In the 51Cr release test, the patient is injected with 51Cr-labeled donor RBCs, and the half-life of the labeled RBCs is monitored and is predictive of post-transfusion survival or clearance12,13. As this method uses radioactive materials, it is rarely performed anymore. The rosette test involves mixing and incubating monocytes with RBCs and quantifying the level of rosette formation (with no phagocytosis)14. The clinical significance of antibodies in vivo involves active phagocytosis by macrophages found in the spleen and/or the liver; thus, this method does not provide a relevant readout of phagocytosis. The CLT uses luminol to monitor the oxidative burst during monocyte phagocytosis of RBC, since luminol fluoresces blue when oxidized in the phagosome15. This method is good, but contamination by neutrophils can confound the readout. Parallel comparisons have been made to evaluate the sensitivity, practicality, and reproducibility of the four available methods, and both the CLT and MMA were ranked superior16. However, the CLT has been mainly utilized in assessing hemolytic disease of the fetus and newborn (HDFN), and the assay’s optimal pH of 8.0 might compromise the level of phagocytosis11.
In addition to its diagnostic and clinical utility, the MMA has been modified for other research purposes. Indeed, the MMA can not only serve as a functional assay to address discrepancies between serology and biology, it has also been used to retrospectively investigate the cause of hemolysis after intravenous immunoglobulin (IVIG) therapy17. It has also been used to examine the structure-function of chemical inhibitors of FcγR-mediated phagocytosis18-20 and to study the downstream signaling of FcγR-mediated phagocytosis21. In our laboratory, in addition to using a human MMA, we are developing a murine MMA using primary mouse peripheral blood mononuclear cells (PBMCs) and autologous RBCs. The rationale is to screen antibodies that can induce FcγR-mediated phagocytosis as an intermediate to developing an in vivo autoimmune hemolytic anemia (AIHA) mouse model (unpublished data). The various modifications focus on different aspects of the IgG antibody and FcγR interaction that induce phagocytosis.
Protocol
Representative Results
Discussion
مجلس العمل المتحد هو أسلوب شاقة تتطلب الخبرة في كل من زراعة الأنسجة والفحص المجهري. هناك العديد من الخطوات الهامة لضمان النجاح: 1) جيل من أحادي الطبقة الوحيدات. 2) طهاية من كرات الدم الحمراء، و 3) الكمي اليدوي. لا تلتزم أحادي الطبقة الوحيدات بقوة إلى الشريحة الغرفة، لذلك يجب المحافظة على درجة الحموضة الفسيولوجية في جميع أنحاء فحص 11، وينبغي أن المصنف وجود عدد كاف من PBMCs. pipetting لقوي، والتي قد تعكر صفو خلايا الالتزام، ينبغي تجنبها. نهج واحد هو إزالة دائما وإضافة حلول من نفس زاوية الغرفة والتأكد من أن الحركة بطيئة وثابتة. وبالمثل، خلال خطوة الغسيل الأخيرة لإزالة الزائد كرات الدم الحمراء، وينبغي أن تكون الحركة بطيئة وثابتة. وهذا يضمن الحد الأدنى من اضطراب أحادي الطبقة في حين لا يزال إزالة الغالبية العظمى من كرات الدم الحمراء-المبتلعة الامم المتحدة. وغسل غير كافية يؤدي إلى خلفية عالية من كرات الدم الحمراء الملوثة، الأمر الذي يجعل منه اليدويntification صعوبة. ثانيا، كرات الدم الحمراء R 2 R 2 يجب opsonized بما فيه الكفاية للحصول على متوسط مؤشر أكلة من 80-120 لمراقبة البلعمة. هذا النطاق أكلة المطلوب توازنا بين سهولة العد (على سبيل المثال، المبتلعة حيدات مع أكثر من 5 كرات الدم الحمراء يصعب تحديدها بدقة) والحفاظ على كمية كافية من البلعمة للتحليل الإحصائي. درجة طهاية يمكن تأكيد من قبل معهد التكنولوجيا التطبيقية، وهناك حاجة إلى القراءة بين 3+ 4+ ل. وR 2 R يجب التخلص 2 كرات الدم الحمراء عندما يكون هناك تحلل الزائد أثناء الغسيل، عندما يتحول طاف الأحمر الداكن، أو عندما لوحظ انخفاض كبير في البلعمة في التجارب نظرا لشيخوخة الخلايا في التخزين. وأخيرا، وتحديد يدوي باستخدام المجهر ويمكن أن تكون خادعة، وخاصة عند المقارنة بين التهم بين أفراد المختبر وبين التجارب. من خلال دراسة نفس المجال على كل بئر، أو ببساطة عن طريق عد المزيد من الخلايا، يمكن الحصول على عدد أكثر اتساقا. ويوصى التدريب جنبا إلى جنب مع فني من ذوي الخبرة واستخدام مجموعة معينة من الشرائح التدريب.
نقد كبير من مجلس العمل المتحد هو ذاتية الخطوة الكمي اليدوية. ومع ذلك، مع التدريب المناسب، والاتساق ويمكن الحصول عبر عدادات مختلفة. الحد الآخر هو الجوهرية الخلافات المانحة إلى الجهات المانحة في قدرات أكلة الوحيدات وفي R 2 R 2 سطح مستويات التعبير مستضد، والتي هي مصدر التباين في البيانات عند التعامل مع العينات البشرية.
تتوفر لدراسة البلعمة بوساطة FcγR تقنيات بديلة أخرى. غالبية مجموعات تجارية تستخدم الناتج الفلورسنت لمراقبة البلعمة (على سبيل المثال، bioparticles وحساسة درجة الحموضة البروتين مضان، أو المسمى مفتش الخرز اللاتكس الفلورية). استخدام الناتج الفلورسنت لا تقدم الكمي أكثر موضوعية، ولكن يحتاج واحد أيضا لخداعسدر توافر والتكلفة والتدريب المرتبطة باستخدام المجهر الفلورسنت أو قياس التدفق الخلوي، فضلا عن الاعتماد التي تلت ذلك على مجموعات المتاحة تجاريا.
وأخيرا، وهذا الفحص يمكن تعديلها تبعا لسؤال البحث. على سبيل المثال، عند اختبار تثبيط المخدرات البلعمة، وحيدات يمكن أن تكون إما قبل المعالجة أو المشارك حضنت مع كل من المخدرات وكرات الدم الحمراء opsonized (أي فحص المنافسة). إشارات المصب من الأجسام المضادة من أنواع فرعية مختلفة، والأجسام المضادة خيالية، أو يبني المؤتلف ويمكن أيضا اختبار. مع اختراقات الأخيرة في تطوير مستضد باطل العالمي الدم 24، مجلس العمل المتحد يمكن استخدامها في الشاشات الأولية لهذه كرات الدم الحمراء مستضد لاغيا مع مختلف الأجسام المضادة لتقييم ما إذا كان هناك بالفعل فعالية خفضت في التسبب البلعمة.
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank the Canadian Blood Services for a Graduate Fellowship Program Award to T.N.T. This research received financial support from the Canadian Blood Services’ Centre for Innovation, funded by the federal government (Health Canada) and the provincial and territorial ministries of health. The views herein do not reflect the views of the federal, provincial, or territorial governments in Canada.
Materials
| Acid citrate dextrose (ACD) vacutainers | BD | REF364606 | |
| RPMI 1640 | Sigma | R8758 | |
| HEPES | Bioshop | HEP003.100 | |
| Fetal bovine serum | Multicell | 080150 | |
| Gentamicin | Gibco | 15710-64 | |
| Ficoll-Paque PLUS | GE | 17-1440-03 | https://www.gelifesciences.com/ |
| Phosphate buffered saline | Sigma | D8537 | |
| 8-chamber slides | Lab-Tek-ll | 154534 | |
| R2R2 (cDE/cDE) red blood cells | Canadian Blood Services | Commercially available (e.g. http://www.bio-rad.com/en-ca/product/reagent-red-blood-cells) | |
| Polyclonal anti-D from human serum | Gamma Biologics | DIN 02247724 | Can be substituted with commercially available monoclonal anti-D or with Rh immune globulin |
| 100% methanol | Caledon | 6700-1-42 | |
| Polyvinyl alcohol resin | Sigma | P8136 | Can be substituted with commercially available mount |
| UltraPure glycerine | Invitrogen | 15514-011 | |
| Cover slips | VWR | 48366 067 | |
| Novaclone anti-IgG | Immucorgamma | 5461023 | Optional for IAT (http://www.fda.gov/downloads/biologicsbloodvaccines/…/ucm081743.pdf) |
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