Advancing Detection and Functional Characterisation of D Variant Red Cells: Development of Enhanced FMH Assays and RhIg Interaction Models to Improve Transfusion and Pregnancy Management

The accurate detection and immunological characterisation of D weak/partial (D var) red cells remains a critical challenge in obstetric transfusion practice, particularly in the context of fetomaternal haemorrhage (FMH) estimation and the efficacy of RhIg anti-D prophylaxis (RhIg) administration. Conventional flow cytometric techniques often rely on antibody binding-induced fluorescence shift to detect D-positive fetal red cells. Weakened or partial expression of the D antigen, as seen in D variant phenotypes, can result in insufficient shift and a significant underestimation of FMH volume. This has clinical implications for the prevention of alloimmunisation in D-negative individuals.
This professional doctorate project aims to design, optimise, and evaluate novel flow cytometric and functional assays to improve the detection, typing, and immunological assessment of D var red cells. Central to this work is the use of rare D var cell stocks sourced from the International Blood Group Reference Laboratory (IBGRL) and RCI sample referrals to facilitate robust comparison across variant types.

Areas of research will be:

• An enhanced FMH quantification assay to amplify the signal shift of D var cells will be developed either through secondary antibodies directed against the BRAD3 anti-D monoclonal antibody, or through brighter conjugated fluorescent markers.
• Exploration of the feasibility of a combined anti-D and fetal haemoglobin (HbF) dual-labelling assay, wherein anti-D binding is followed by fixation and permeabilisation to allow development of an alternative testing profile to be used upon detection of either maternal or fetal D var cells during standard testing.
• To investigate the potential to classify D var type based on the ratio of positive shift compared a calibration standard of a known normal D type expression profile e.g. rr Vs R2R2
• To use the developed flow cytometric assays and antibody concentration ELISA to evaluate the in vitro interaction between RhIg and D var red cells to understand antibody uptake profiles over defined time periods in a model of simulated maternal circulation (D neg maternal Vs normal D; Dvar cells; D var maternal Vs normal D cells).
• Development and application of functional assays using donor-derived buffy coat preparations to model and assess the in vivo clearance of D-positive and D variant red cells following RhIg exposure.

Together these areas of research will address diagnostic and functional uncertainties surrounding D var phenotypes in transfusion medicine. By refining FMH estimation techniques and advancing our understanding of RhIg interactions with D variant antigens, this work contributes toward safer and more precise management of D var pregnancies. Further, it supports the broader integration of D var testing strategies in routine practice and will look to inform future UK transfusion guidelines.

Outputs

Not completed / published yet