Background/Case Studies: Prenatal antibody titration guides monitoring of alloimmunized pregnancies at risk for fetal anemia. The traditional method, conventional tube test (CTT), is a manual technique limited by procedural variation, unstable endpoints, and poor inter-laboratory reproducibility. An alternative method, gel column agglutination (GCA), is easier to standardize, has stable endpoints, and can be automated; however, GCA does not have established critical titer thresholds and lacks clinical correlation data, impeding broad implementation. This study compared CTT and GCA methods for measuring prenatal anti-c and anti-K antibody titers.
Study
Design/Methods: We conducted a method comparison study using stored, frozen plasma specimens from pregnant women (January 2019-December 2023) with reported anti-c or anti-K antibodies. Group O donor red blood cells expressing a single dose of the corresponding antigen were selected as indicator cells and prepared as 2% and 0.8% suspensions for CTT and GCA, respectively. Master serial twofold dilutions of patient plasma in saline were tested in parallel by CTT and GCA. Titer endpoint was defined as the reciprocal of the highest dilution yielding macroscopically visible agglutination (≥1+). Titer results were log-transformed and analyzed with Wilcoxon matched-pairs signed rank test.
Results/Findings: We performed paired titrations on 34 specimens from 13 patients with anti-c and 29 specimens from 19 patients with anti-K. Among these, 26% (9/34) with anti-c and 14% (4/29) with anti-K were nonreactive (titer=0) with neat plasma with both methods. The median anti-c titer was 1 [range 0-8] by CTT and 2 [range 0-32] by GCA. The median anti-K titer was 32 [range 0-1024] by CTT and 16 [range 0-512] by GCA. GCA titers were within one doubling dilution of CTT titers for 56% of specimens with anti-c and 97% of specimens with anti-K (Figure A). Among anti-c GCA titrations, 21% (7/34) were greater than 2 dilutions from the CTT result. For specimens with detectable titers, GCA titers were higher than CTT titers for anti-c (median difference 2 [95% confidence interval (CI) 1-2], p< 0.0001) but not significantly different for anti-K (median difference 0, p=0.06). Conclusions: Differences between GCA and CTT antibody titer results were antibody-dependent. For anti-c, GCA titers were generally 2 dilutions higher than the corresponding CTT titers. For anti-K, where the concept of a critical titer may be less clinically useful, results were more consistent between methods. Future studies correlating GCA titers with clinical outcomes will be valuable for establishing evidence-based critical titer thresholds specific to this methodology.
