# D‐Alloantibody Titration Assessment Study: In Search of a Common Antibody Titration Platform—A BEST Collaborative Study

## TL;DR

This study compares different methods for measuring anti-D antibodies in pregnant women to improve accuracy and guide clinical decisions.

## Contribution

The study establishes a correlation between tube, gel, and CFA methods for anti-D titration.

## Key findings

- Gel-based methods produced higher titers than tube testing, especially at lower dilutions.
- CFA showed strong correlation with both tube and gel methods.
- A tube titer of 8–16 corresponds to a gel titer of 32–128 when using R2R2 cells.

## Abstract

Alloimmunization against D‐antigen can cause severe Hemolytic Disease of the Fetus and Newborn (HDFN). Traditionally, anti‐D‐titers are measured using a saline indirect antiglobulin test (tube testing). Anti‐D‐titers ≥8 during pregnancy trigger an escalation in maternal care. Tube testing is labor‐intensive and known for imprecision. Automated gel‐based titration is more sensitive and precise than tube titration for the detection of anti‐D. A gel titer correlated with potential fetal anemia has not been established, as studies comparing gel and tube titers provide widely variable results. This multicenter study tested anti‐D samples in parallel to characterize the difference in sensitivity between tube and automated gel assays.

Patients alloimmunized to RhD had samples tested using tube and automated gel titration methods. A total of 647 samples were tested in parallel. A subset of 141 samples also had anti‐D levels quantified using continuous flow analysis (CFA). Controlled lots of R2R2 red blood cells and standardized reagents were utilized.

Results demonstrated that gel‐based methods yielded mean titers 2.5–3 dilutions higher than tube; this difference diminished at tube titers >128. Notably, several samples previously considered negative by tube were positive by gel. Anti‐D levels quantified by CFA demonstrated a good correlation with tube and gel testing (R = 0.75–0.9 for tube; R = 0.85–0.89 for gel).

A tube titer of 8 to 16 correlates with an automated gel titer of 32–128 when R2R2 cells are used. Results using the CFA method correlate well with tube and gel analyses.