Applications of automated simultaneous double fluorescence (SDF): I. Unbiased, quantitative HLA-A,B phenotyping

An automated HLA typing method has been developed and standardized. The procedure involves simultaneous excitation and reading of two fluorochromes, carboxy-fluorescein diacetate and propidium iodide, by a purpose-built Terasaki tray scanner known as the Astroscan 2100. Data obtained from 95 donors typed by this method and by the classical NIH dye exclusion method prove this system to be suitable for routine HLA-A,-B typing (similarity coefficient 0.9861). This comparison has highlighted significant observer bias, whereby the observer tends to downgrade very weakly positive reactions to negative and upgrade moderately positive reactions to strong positive. Elimination of this bias reduces the chances of any clinically disastrous consequence.