Comparison of Anti-D Titration Testing by Automated Column Agglutination Technology on Glass-Beads and Standard Tube Method

— Introduction: Antibody titration is important in the follow-up of alloimmunised pregnant women and anti-D is still the most relevant specificity for hemolytic feto-neonatal disease risk. Column agglutination technology is an attractive alternative to traditional tube method for antibody titration because of its technical ease, operator independence, and automation possibility. This study's objective was to compare automated column agglutination technology on glass-beads and standard tube method in performing anti-D titrations. Methods: Serial plasma dilutions from 67 samples with detectable anti-D (IP, passive or AI, immune) were prepared with Ortho Vision platform and tested against R0r 3% reagent red cell using Anti-IgG Ortho BioVue cassettes. The reaction grades read by the system were reported and Titre Score (TS) was also calculated. Titration results were compared with those obtained by parallel serial dilution with the tube technique against the same singledose phenotype 3% red cells, following AABB standard method. Results: Anti-D titer range was 1-512 both in column and in tube. Column titer was identical to tube titer in 68.6% of samples, differed of 1 dilution in 22.5% and of 2 dilutions in only 9% of cases. The grade of agreement between the two methods was assessed and the bias in the mean differences was computed. In the IP antibody group (n=37) anti-D column titration results were 0.27 (95% CI = -0.73 to 1.27) additional dilutions greater than in tube. The median TS values in tube test were 3 (range 3- 23) in prophylactic category and 29.5 (range 3-90) in the immune group. In BioVue titration, the median TS values were 6 (range 3-33) and 40 (range 3-103), respectively. At a tube titer of 16, the sensitivity of BioVue titrations in immune patients was maximal (100%) at the same column titer of 16, whereas specificity was maximal (100%) at a column titer of 32. Conclusions: Our data show a good correlation in anti-D titration results against single-dose reagent cells between automated column agglutination technology on glass-beads and standard technique in tube. A maximum difference of 2 dilutions was observed with column data showing an overall higher sensitivity, as expected, but with a high percentage of concordance. The grade of agreement between the two methods was particularly good in patients with low titer anti-D with supposed passive nature. TS results were also evaluated and confirmed as a good tool to predict the nature, passive or immune, of detected anti-D. Further data are needed to compare titration results in immune anti-D and to determine clinically significant range for referral to a high-risk obstetrician.