Background/Case Studies: Antibody titration is a semi-quantitative method used to estimate clinically significant antibody levels and assess HDFN risk. Despite its clinical value, the process is prone to variability due to the manual serial dilution process and subjective grading. Automation may reduce these limitations, but results must be validated against the tube method. This study compares traditional tube testing with automated gel testing on two IH-500 NEXT systems at separate LabCorp sites and assesses the platform’s intra-assay precision.
Study
Design/Methods: Alloantibody-positive samples were titrated using the tube method per AABB guidelines, carrying out two-fold dilutions using saline, 37°C incubation, saline washes, anti-IgG AHG addition, centrifugation, and result interpretation. Site 1 used the CAP method (weakest positive as endpoint), while Site 2 used the last 1+ reaction. Within seven days, parallel titration was performed on two IH-500 NEXT systems using anti-IgG IH-Cards and IH-Titration Solution. Double-dose antigen-positive red cells were used, except for anti-K, which used single-dose cells.
Results/Findings: At Site 1, a total of 16 samples were analyzed with the following alloantibody specificities: anti-c (2), -E (3), -K (3), -S, -Fya (4), -Jka, -s, and anti-N. At Site 2, 13 samples were tested, comprising anti-D (3), -c (2), -E (3), -K (2), -Jka, -s, and anti-M. At Site 1, 94% of the samples exhibited higher titers when tested with the automated gel system compared to the conventional tube method. Notably, 81% of these titers exceeded the tube method by at least two dilutions. The average increase in titer was 2.3 dilutions (SD: 0.9). At Site 2, all samples (100%) showed higher titers with the automated gel system, with 62% exceeding the tube method by two or more dilutions. The mean increase in titer was 2.2 dilutions (SD: 1.1). To assess the intra-assay precision of the IH-500 NEXT system, 12 samples containing anti-D (2), -K (4), -Jka, -s, -c (2), -M, and -E were tested in duplicate. Nine samples (75%) produced identical titers upon repeat testing. The remaining three samples (25%) showed a variation of no more than ±1 dilution, which is within the acceptable range of assay variability.
Conclusions: Automated antibody titration has demonstrated high accuracy and a strong potential for standardization. This study emphasizes the importance of correlating results between the traditional tube method and the automated gel technique to define appropriate new cutoff values prior to clinical implementation. Although interpretation of tube titers varied between sites, both observed an average increase of 2.2 to 2.3 dilutions with the automated gel method. This consistent rise suggests that cutoff thresholds may need to be adjusted when transitioning to the automated platform.
