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.
Le MMA est une technique laborieuse qui exige une expertise à la fois dans la culture de tissus et la microscopie. Il y a plusieurs étapes essentielles pour assurer le succès: 1) la génération de la monocouche monocytes; 2) opsonisation des globules rouges, et 3) la quantification manuelle. La monocouche monocyte ne colle pas très fortement à la lame de la chambre, de sorte que le pH physiologique doit être maintenue tout au long de l'essai 11 et un nombre suffisant de PBMC doit être semée. pipetage Vigoureuse, ce qui pourrait perturber les cellules adhérentes, devrait être évitée. Une approche est de toujours supprimer et ajouter des solutions du même coin de la chambre et de veiller à ce que le mouvement est lent et régulier. De même, lors de la dernière étape de lavage pour éliminer l'excès de globules rouges, le mouvement devrait être lente et régulière. Cela garantit une perturbation minimale à la monocouche, tout en supprimant la majorité des hématies un-phagocyté. lavage inadéquat conduira à un fond élevé de globules rouges contaminants, ce qui rend qua manuelntification difficile. D' autre part, les groupes R 2 R 2 , les globules rouges doivent être suffisamment opsonisées afin d' obtenir un indice phagocytaire moyenne de 80 à 120 pour le contrôle de la phagocytose. Cette gamme de phagocytaire souhaité un équilibre entre la facilité de comptage (par exemple, monocytes avec plus de 5 phagocytées hématies sont difficiles à quantifier avec précision) et le maintien d' une quantité adéquate de la phagocytose pour l' analyse statistique. Le degré de opsonisation peut être confirmé par un IAT, et une lecture entre 3+ à 4+ est nécessaire. Le R 2 R 2 hématies doivent être jetés quand il y a excès de lyse pendant le lavage, lorsque le surnageant devient rouge foncé, ou quand une diminution significative de la phagocytose est observée dans des expériences en raison du vieillissement des cellules de stockage. Enfin, la quantification manuelle à l'aide du microscope peut être difficile, surtout lorsque l'on compare les chiffres entre le personnel de laboratoire et entre les expériences. En examinant le même domaine sur chaque puits, ou simplement en comptant plus de cellulesUn nombre plus uniforme peut être obtenue. formation Side-by-side avec un technicien expérimenté et l'utilisation d'un ensemble désigné de diapositives de formation est recommandé.
Une critique majeure de la MMA est la subjectivité de l'étape manuelle de quantification. Cependant, avec une formation adéquate, la cohérence peut être obtenue à travers différents compteurs. Une autre limitation est les différences donateurs à-donateurs intrinsèques monocytes capacités phagocytaires et en R 2 R 2 surface niveaux d'expression de l' antigène, ce qui est une source de variation de données lorsqu'ils traitent avec des échantillons humains.
D'autres techniques alternatives sont disponibles pour l'examen de la phagocytose FcyR médiée. La majorité des kits commerciaux utilisent la sortie fluorescente pour surveiller la phagocytose (par exemple, bioparticules, la protéine de fluorescence sensible au pH, ou des billes de latex fluorescentes IgG marqués). L'utilisation de la sortie fluorescente n'offre une quantification plus objective, mais on doit aussi conexaminer le disponibilité, le coût et la formation associée à l'utilisation d'un microscope à fluorescence ou d'un cytomètre de flux, ainsi que la dépendance qui s'ensuit sur des kits disponibles dans le commerce.
Enfin, cet essai peut être modifié en fonction de la question de recherche. Par exemple, lors du test de l' inhibition médicamenteuse de la phagocytose, les monocytes peuvent soit être pré-traitées ou co-incubées avec les médicaments et les globules rouges opsonisés ( par exemple, un test de compétition). La signalisation en aval des anticorps de sous-types différents, des anticorps chimères ou des constructions recombinantes peuvent également être testés. Avec les récentes percées dans le développement d'un antigène nul universel de sang 24, le MMA pourrait être utilisé dans les écrans initiaux de ces hématies antigène nul avec divers anticorps pour déterminer s'il est en effet une efficacité réduit dans le déclenchement de la phagocytose.
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) |