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.
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.
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.
MMA doku kültürü ve mikroskopi hem de uzmanlık gerektiren zahmetli bir tekniktir. başarı sağlamak için birkaç kritik adımlar vardır: monosit tek tabaka 1) nesil; RBCs 2) opsonizasyon, ve 3) manuel miktar. Monosit tek tabakalı odası slayt çok güçlü bir şekilde bağlı değildir, bu yüzden, fizyolojik pH tahlilinde 11 boyunca muhafaza edilmesi ve PBMC yeterli sayıda tohumlanır edilmelidir. yapıştırılır hücreleri bozacağı Dinç pipetleme, kaçınılmalıdır. Bir yaklaşım her zaman kaldırmak ve odanın aynı köşesinden çözümler ekleyebilir ve hareket yavaş ve istikrarlı olmasını sağlamak için olmaktır. Benzer şekilde, fazla RBC çıkarmak için son yıkama aşaması sırasında, hareket yavaş ve sabit olmalıdır. hala un-fagosite eritrosit çoğunluğunu kaldırılırken Bu tek tabaka minimal rahatsızlık sağlar. Yetersiz yıkama manuel nereye yapar kirlenmesine eritrosit yüksek arka plan yol açacaktırntification zor. İkinci olarak, R2 R2 eritrosit yeterli fagositoz kontrolü için 80-120 arasında bir ortalama fagositik endeksi elde etmek için opsonize gerekmektedir. Bu istenen fagositik aralığı sayma kolaylığı arasında bir denge ve istatistiksel analiz için fagositoz yeterli miktarda muhafaza (örneğin, 5'ten fazla olan monositler eritrositler doğru ölçmek zordur fagosite). opsonızasyonu derecesi bir IAT tarafından teyit edilebilir ve 4+ için 3+ arasında bir okuma gereklidir. 2 eritrosit var yüzer koyu kırmızı olduğunda, yıkama sırasında aşırı liziz, ya da atılmalıdır R 2R fagositoz önemli bir azalma nedeniyle depolama hücrelerinin yaşlanmaya deneylerde zaman. laboratuar personeli arasındaki ve deneyler arasındaki sayıları karşılaştırarak özellikle Son olarak, mikroskop kullanarak manuel miktar yanıltıcı olabilir. her bir aynı alan inceleyerek, ya da sadece daha fazla hücre sayılarak, Daha tutarlı bir sayısı elde edilebilir. deneyimli bir teknisyen ve eğitim slaytları belirli bir dizi kullanımı ile yan-yana eğitim tavsiye edilir.
MMA önemli bir eleştiri manuel miktar adım öznellik olduğunu. Ancak, yeterli eğitim, tutarlılık farklı sayaçlar üzerinden elde edilebilir. Başka bir sınırlama insan örnekleri ile ilgili veri varyasyon kaynağıdır 2 R 2 yüzey antijeni ifade seviyeleri, monosit fagositik yeteneklerine ve Ar içsel donör için-donör farklılıklarıdır.
Diğer alternatif teknikler FcyR aracılı fagositozu incelenmesi için kullanılabilir. Ticari kitler çoğunluğu fagositoz izlemek için flüoresan çıkış kullanır (örneğin, bioparticles, pH'a duyarlı floresan proteini veya IgG-etiketli floresan lateks boncuk). Floresan çıkış kullanımı daha objektif ölçümü teklif yok, ama bir de con gerekiyorbir flüoresan mikroskop ya da bir akış sitometresinde yanı sıra ticari olarak temin edilebilen kitler üzerine takip eden bağımlı kullanımı ile ortaya çıkan durumu, maliyet ve eğitim sider.
Son olarak, bu deney, bir araştırma söz bağlı olarak değiştirilebilir. Fagositoz ilaç engellenmesini test ederken, örneğin, monositler ön tedavi edilebilir ya da ilaç ve opsonize edilmiş RBCs (yani, bir rekabet deneyi) her ikisi ile ko-inkübe edilmiştir. farklı alt kimerik antikorlar veya yeniden birleştirici yapıları antikorların aşağı doğru sinyal de test edilebilir. Evrensel bir antijen boş kanın 24 gelişiminde son buluşlarla, MMA fagositoz tetikleyen bir alçaltılmış etkinliği gerçekten var olup olmadığını değerlendirmek için çeşitli antikorlar ile bu antijen boş RBCs ilk ekranlarında yararlanılabilir.
The authors have nothing to disclose.
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.
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) |