Anti‐D quantification by flow cytometry:a comparison of five methods

Abstract

Anti-D quantification by flow cytometry is influenced by the serum antibody characteristics and the method used. The differences between the flow cytometric and AutoAnalyzer techniques indicate that further validation of the flow cytometric method is required before routine use.

Quantification of anti‐D and fetomaternal hemorrhageby flow cytometry

“…This instrument may prove to be suitable for the quantification of anti‐D in antenatal samples. The article by Austin and McIntosh 6 in this issue of TRANSFUSION presents a timely comparison of several flow cytometric methods with AutoAnalyzer quantification of anti‐D in sera. Quite large differences—up to 100 percent—in anti‐D concentrations were obtained with the various flow cytometric methods, which were not fully explained by procedural variations such as serum:cell ratio, temperature of incubation with anti‐IgG, or the use of low‐ionic‐strength saline.…”

“…In contrast, assays using anti‐IgG reagents resulted in greater quantification with the IgG3 anti‐D than the IgG1 anti‐D; antiglobulin titers (from 20 μg/mL) were 32,000 (BRAD‐3) and 4,000 (BRAD‐5), and, in FC (using an Fab fragment of anti‐IgG conjugated with fluorescein isothiocyanate [FITC]), cells coated with IgG3 anti‐D gave at least 10‐percent greater fluorescence throughout the titration range than cells coated with IgG1 anti‐D. At lower concentrations of anti‐D, in the range of the standards used by Austin and McIntosh, 6 BRAD‐3 gave three times the fluorescence of BRAD‐5.…”

“…The composition of the anti‐D standards used in the AutoAnalyzer and the FC methods by Austin and McIntosh 6 are likely to be similar to those of most batches of prophylactic anti‐D made in the UK, as both are high‐titer pooled‐plasma preparations, containing on average 10 to 15 percent IgG3 9,10 . It is anticipated that prophylactic anti‐D may therefore be accurately quantified by both methods using these standards, although no comparison has yet been performed.…”

“…Austin and McIntosh 6 state that it may be inappropriate to compare anti‐D concentrations derived by FC and the AutoAnalyzer. Before either FC or ELISA can become routinely used for quantification of anti‐D, a comparison of results with clinical outcome (and, ideally, with titration and the AutoAnalyzer results) must be made with a large number of samples so that threshold levels can be set for obstetric intervention.…”

Applications of flow cytofluorometry to red blood cell immunology

“…In the USA, prenatal titrations of anti-D (using the antiglobulin test) are used to evaluate whether a woman should have invasive tests such as amniocentesis to assess the status of a fetus possibly affected by hemolytic disease of the newborn. Europeans have, for some years, quantitated antibodies in micrograms or IU, using a standard curve utilizing a standard anti-D and an AutoAnalyzer; more recently, FC has been applied (70)(71)(72)(73)(74).…”