The Monocyte Monolayer Assay (MMA)

Most patients have negative results in pretransfusion testing when their serum or plasma is tested for unexpected antibodies to red cells. For most patients with positive red cell antibody screens, the antibody–for example, anti-K or anti-E–is easily identified and antigen-negative blood can be obtained if needed.  But some patients have multiple antibodies or antibodies to high-prevalence antigens that make obtaining compatible blood a challenge.

The outcome of an incompatible red cell transfusion is hard to predict. Antibodies reacting at room temperature (IgM) rarely cause transfused red cell destruction, but antibodies reactive at 37°C and/or the antiglobulin test phase often do. The monocyte monolayer assay (MMA) is a test that can be useful in predicting the survival of incompatible red cells if a transfusion is required and compatible red cells are not available. The most frequent use of the MMA is in a patient with an antibody to a high-prevalence antigen, such as anti-Lub  or anti-Yta, that is known to vary in clinical significance in vivo. Antigen-negative units are usually found by the American Rare Donor Program (ARDP). If an MMA is negative, rare blood is not required for that specific transfusion and is preserved for another patient in need. The MMA is neither used for IgM antibodies nor to predict hemolytic disease of the fetus or newborn. Interferences with the MMA include autoantibodies and high-titer, low-avidity antibodies. If an antigen-unknown (presumed antigen-positive) unit is transfused based on a negative MMA, then to predict survival of a subsequent transfusion, an MMA must be performed on a pretransfusion sample drawn as close to the time of the next transfusion as possible. A consultation with your immunohematology reference laboratory is helpful in determining if an MMA would be useful.

The MMA is performed by very few US laboratories. It is technically demanding and requires almost a full day to complete, similar to the time requirement of allogeneic adsorption procedures. Mononuclear cells are separated from freshly drawn blood using a density gradient and incubated on a solid platform to isolate the monocytes (monocytes adhere; lymphocytes do not). Antigen-positive and -negative reagent red cells are incubated in test tubes, in most cases with and without a source of fresh complement, along with positive and negative controls. Supernatant is removed from the monolayer of monocytes, and the test red cells are added. After incubation, the platform is rinsed, fixed, and stained for evaluation. Figures 1 and 2 show reactive and nonreactive monocytes respectively.

There are several MMA methods published. It is important that the laboratory performing the test has validated the assay by having clinical correlates with the MMA interpretation.

The MMA has been used for nearly 30 years by the American Red Cross National Reference Laboratory for Blood Group Serology (NRLBGS) in Philadelphia, Pennsylvania, and for over 35 years by the American Red Cross Immunohematology Reference Laboratory in Pomona, California.1  Experience with the MMA has been published by multiple laboratories and reviewed recently.2 The MMA was also reviewed in a recent podcast.3

The MMA is used conservatively but is tremendously useful in those occasions when antigen-negative blood is available in insufficient quantities or not at all.

References

1. Nance SJ, Arndt P, Garratty G. Predicting the clinical significance of red cell alloantibodies using a monocyte monolayer assay. Transfusion1987;27:449-452.

2. Nance SJT. The monocyte monolayer assay, an in vitro method for prediction of in vivo survival of transfused incompatible red blood cells: a review. Immunohematology 2023;39:61-69.

3. Chaffin J. Blood Bank Guy [Internet]. [place unknown]: Joe Chaffin, MD; 1998-2023. [Podcast], 093CE: The Mighty MMA! with Sandy Nance; 2021 Oct 13 [cited 2023 Nov 17]; [55 min, 52 sec]. Available from: https://www.bbguy.org/2021/10/13/093/

Figure 1. Reactive monocyte with adherent and phagocytosed red cells in the MMA. Photograph from MMA performed at NRLBGS, Philadelphia

Figure 2. Nonreactive monocyte in the MMA. Photograph from MMA performed at NRLBGS, Philadelphia

Authors

  • Paul M. Mansfield MLS(ASCP)SBBCM is the Director of the Immunohematology Reference Laboratory and the National Reference Laboratory for Blood Group Serology at the American Red Cross in Philadelphia. Paul received his BS in Medical Laboratory Science from Northeastern University in Boston in 1997. He started his career as a Medical Laboratory Scientist in Blood Banking at Brigham and Women’s Hospital in Boston in 1997. He moved to Philadelphia in 2001 and began as a technologist in the Immunohematology Reference Laboratory. He was promoted to several positions of increasing responsibility and ultimately became Director of the Laboratory in 2021. He attained his SBB from the ASCP in 2008. He is very active with the AABB in support of the IRLs as a member of the IRL Accreditation Committee and the IRL Standards Committee. He is also a member of the Invitational Conference of Investigational Immunohematologists.

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  • Sandy Nance is the emeritus Senior Director for National Laboratories for the American Red Cross and has provided leadership to multiple programs at the Red Cross, including the American Rare Donor Program and the national American Red Cross SBB Program. She is also an emeritus Adjunct Assistant Professor at the University of Pennsylvania, Division of Transfusion Medicine & Therapeutic Pathology Department of Pathology and Laboratory Medicine. She has a master’s degree in Pathology from the University of Maryland and received her SBB from the Johns Hopkins Medical Institutions. Sandy developed of the use of polyethylene glycol (PEG) for serologic testing as well as the monocyte monolayer assay (MMA).

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