Poster Presentation The Australasian Society for Immunology 2017 Annual Scientific Meeting

Optimisation of monocyte monolayer assay (mma) for determining the clinical significance of red cell antibodies (#264)

Ji Yu 1 2 , Robert L Flower 1 3 , Tanya Powley 1 , YuanTong Gu 2 , Suvash Saha 2 , Emilie Sauret 2 , Melinda M Dean 1 3
  1. Clinical Services and Research, Australian Red Cross Blood Service, Kelvin Grove, QLD, Australia
  2. Faculty of Science and Engineering, Queensland University of Technology, Brisbane, QLD, Australia
  3. Faculty of Health, Queensland University of Technology, Brisbane, QLD, Australia

BACKGROUND:

One risk of transfusion is the development of alloantibodies against red blood cell (RBC) antigens not found on the patient’s RBC.  The monocyte monolayer assay (MMA) is an in vitro model which has been used to determine the clinical significance of alloantibodies and predict post-transfusion RBC survival. As the traditional MMA is labour intensive (1.5 days) we aimed to optimise the procedure and reduce turnaround time for use in Australia.   

METHODS:

Ficoll isolated PBMC followed by CD14 selection, or monocyte adherence, are typically used as the cell sources for MMA. More rapid whole blood monocyte isolation techniques are available (STEMCELL Technologies).  Monocyte purity, yield and platelet contamination were compared for these cell separation techniques (n=6). The reactivity of monocytes from the different cell isolations were then assessed using MMA. Briefly,  D+  RBC were sensitised with plasma containing anti-D (37°C, 1hr) and subsequently incubated with monocytes prepared on a Lab Tek 8-well plate (37°C, 1 hr, CO2).  Giemsa & Wright’s stains were used for visualisation and proportion of monocytes with adherent or phagocytosed RBC determined via microscopy.

RESULTS:

Monocytes separated directly from whole blood had both the highest purity (96.1% direct cf. 89.5% CD14+ selection, p=0.02) and the least PLT contamination (undetectable direct cf. 41.5 x 109/L PBMC isolation, p=0.001). The direct whole blood method also had comparative yield of monocytes/mL of starting whole blood. Monocytes isolated using this method were also comparable in the capacity to bind and/or phagocytose anti-D opsonised RBC, demonstrating their suitability for detecting clinically significant antibodies in vitro.

CONCLUSIONS:  

Using a direct monocyte isolation method, we successfully developed a MMA with reduced turnaround time (one day) to detect the clinical significance of RBC antibodies. This assay will improve blood transfusion safety and facilitate better patient management by reducing the risk of incompatible blood transfusion.