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GA Green and VK Kalra
USC School of Medicine, Biochemistry Department, Los Angeles 90033.
Previously we demonstrated that sickle erythrocytes sedimenting at high
densities after gradient centrifugation contain higher levels of surface
immunoglobulin bound in vivo in comparison to low-density erythrocytes from
the same patient. The present study examines the possibility that binding
of autologous IgG to sickle erythrocytes may be associated with the
sickling phenomenon. In the present study we subjected low-density
erythrocytes to prolonged sickling under nitrogen in the presence of
platelet-poor autologous plasma with added glucose for 24 hours (37 degrees
C). After reoxygenation IgG bound in vitro was quantified by a
nonequilibrium 125iodinated protein A-binding assay and by flow cytometry.
Results show that sickle erythrocytes incubated under nitrogen bound
significantly (P less than .001) more IgG, 439 +/- 41, molecules of IgG per
cell (mean +/- SD) compared with sickle cells incubated under oxygenation
(227 +/- 12 molecules of IgG per red cell) or compared with 196 +/- 26
molecules IgG per cell for untreated sickle cells. In contrast, normal
erythrocytes incubated in autologous plasma exhibited no detectable IgG
binding in vitro under either oxygenation or deoxygenation. Flow cytometry
shows that deoxygenation of sickle cells generated a two-to-sixfold
increase in the subpopulation of brightly fluorescent IgG-positive cells in
comparison to oxygenated sickle cells and a 13.5% +/- 3.1% (mean +/- SD)
increase in median fluorescence intensity for fluorescein
isothiocyanate-labeled deoxygenated sickled cells compared with labeled
oxygenated sickle cells. Our studies demonstrate that prolonged sickling
will induce in vitro binding of autologous IgG to sickle erythrocytes.
These findings indicate that sickle erythrocytes may be unique when
compared with erythrocytes from other nonimmunologic hemolytic anemias or
senescent red cells in that the primary events producing surface antigens
recognized by autoantibody may include the sickling process. These findings
also suggest that sickling in vivo may generate membrane alterations in
sickle erythrocytes that lead to cumulative binding of autoantibody in
vivo.
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| Copyright © 1988 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||