Methods of Anti-ABO Titre Estimation
Primary Testing Methods
The tube test (conventional method) and column agglutination technology (CAT) are the two main methods for estimating anti-ABO titres, with flow cytometry emerging as an alternative technique. 1, 2
Tube Test (Conventional Method)
The tube test remains widely used and involves two primary techniques:
Immediate spin (IS) technique at room temperature (RT) - This method detects primarily IgM antibodies through direct agglutination and typically shows higher median titers for anti-A and anti-B in blood groups A and B individuals 2
Indirect antiglobulin test (IAT) - This technique detects both IgM and IgG antibodies, with the tube IAT method showing higher titers than gel card IAT for blood groups A and B, though gel-IAT shows the highest titer for anti-A antibody in blood group O 1, 2
Serial dilutions are performed (typically 1:2 to 1:1024), and the reciprocal of the highest dilution showing macroscopic agglutination is reported as the titer 3, 4
Column Agglutination Technology (CAT)
CAT, particularly automated systems like VISION Max, offers several advantages:
Automated CAT can be performed with or without dithiothreitol (DTT) - Testing without DTT measures total antibodies (IgM + IgG), while testing with DTT specifically measures IgG antibodies after IgM destruction 2, 4
CAT without DTT shows higher median titers than tube AHG method, especially for group O individuals, providing more sensitive results that include IgG data 2
Automated CAT demonstrates superior reproducibility with no significant inter-instrument or inter-laboratory variability (P ≥ 0.99), addressing a major limitation of manual tube testing 5
Greater than 90% of CAT titre values fall within one dilution of tube test results, demonstrating substantial agreement (k = 0.73) and high correlation (ρ ≥ 0.75) for most blood groups 5, 4
Flow Cytometry (FCM)
Flow cytometry represents a newer approach:
FCM with anti-IgM antibodies shows the highest titers compared to both tube and gel card tests with RT incubation, regardless of blood group tested 1
FCM with anti-IgG antibodies can be used to specifically detect IgG antibodies, though tube IAT typically shows higher titers than FCM with anti-IgG for blood groups A and B 1
Critical Technical Considerations
Method-Specific Differences
Gel titers are consistently one to two dilutions higher than tube titers and show sensitivity to reagent red cell lots, which must be considered when interpreting results 3
Each method demonstrates different detection capacity for each ABO antibody depending on the blood group tested, making direct comparison between methods challenging 1
The critical titer threshold varies by method - For gel testing, a critical direct agglutinin titer of 64 is considered too low and should be increased to 128 or higher to identify truly high-titer units 3
Standardization Requirements
Testing should be performed in duplicate (calibrators, controls, and samples) to account for the relatively high coefficient of variation compared to automated clinical chemistry assays 6
Each laboratory should determine its local cut-off value even when using commercial kits, testing at least 50-100 healthy normal individuals 6
Cut-off values should be calculated using the method of percentiles rather than adding standard deviations to the mean value, as the distribution of values is not Gaussian 6
Recommended Testing Algorithm
For Routine Clinical Use
Use automated CAT with and without DTT for titration of anti-A and anti-B, especially in group O individuals, to obtain both total antibody and IgG-specific data 2
Modified CAT (MCAT) approach - Perform initial testing at 1:32 dilution, then conduct additional testing from 1:64 to 1:1024 only if agglutination is present, which provides superior safety, time-efficiency, and cost-effectiveness 4
Quality Assurance Considerations
Automated CAT shows lower risk priority number (RPN) scores in failure mode and effect analysis (33,700 vs. 184,300 for tube test), indicating improved safety 4
Turnaround time and cost are comparable between tube test and CAT (approximately 14-15 hours and $1,300-1,400), though MCAT reduces both (13.5 hours and $900) 4
Common Pitfalls and Caveats
Caution must be exercised when interpreting results across different methods, as significant differences in titers occur depending on the detection method and blood group tested 1
The method used must always be specified in the test report, as results are not directly comparable between techniques 1, 2
Gel method sensitivity to reagent lots requires careful quality control and may necessitate adjustment of critical titer thresholds 3
Insurance coverage adjustment may be necessary considering the actual cost of reagents and personnel, particularly when implementing more comprehensive testing approaches like CAT with and without DTT 2