Quantitation of Antibody Binding to Erythrocytes in LISS

Abstract

The amount of antibody bound to cells in a low ionic strength solution (LISS) has been quantitated for several antibodies including anti-D, anti-c, anti-Kell, anti-Fy^a, and anti-Jk^a. With the exception of the Kell antibodies there was an enhancement of the rate of antibody uptake in LISS. For Rh antibodies the amount bound after a 5-min LISS incubation is comparable to that bound after 45 min in saline. For Kell antibodies a smaller amount was bound in LISS than in saline. The effect of the ratio of serum to cells was also studied, and with several antibodies there was an increase in the amount of antibody bound with a higher serum to cell ratio irrespective of suspending medium. For Kell antibodies this ratio appears to be of greater importance than the ionic strength for antibody detection. A modification to the LISS method to increase the serum to cell ratio is, therefore, presented.

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

“…Thus, antibodies that are generally unreactive (undetectable) at low concentrations may be detectable with LISS. The amount of antibody bound per cell is also affected by the serum‐to‐packed‐cell ratio 7 . A 50‐percent increase in antibody bound can be obtained by increasing the ratio from 20:1 to 40:1.…”

“…Method 1 is based on our original method, Methods 2 and 3 are “in‐house” modifications to that method, and Methods 4 and 5 are based on those of Christensson et al 5 and Hilden et al, 6 respectively. The modifications included in Methods 2 and 3 were a 20‐minute sensitization based on the low‐ionic‐strength saline solution (LISS) method described by Merry et al 7 and the use of LISS throughout as a wash buffer to help minimize the loss of low‐affinity antibodies. The inclusion of normal‐ionic‐strength saline solution/bovine serum albumin (BSA) as the serum diluent in Method 3 was based on our experience in quantifying serum levels of monoclonal anti‐D by flow cytometry.…”