Early diagnosis of hemoglobinopathies
Antibodies specific for human hemoglobins F, A, S, and C were isolated by affinity chromatography from antisera produced in rabbits and goats. These reagents were shown to possess adequate sensitivity for identifying and quantifying hemoglobins A, S, and C in cord blood by radial immunodiffusion (RID). Furthermore, single immunodiffusion plates combining up to three of the antibodies at differing concentrations produced up to three nonoverlapping immunoprecipitin rings, retaining the capability for identifying multiple hemoglobins by a single application of hemolyzed whole blood to the RID plate. Isolated antibodies were conjugated with fluorescein isothiocy- anate and incubated with red blood cells which were obtained from placental cord blood or from amniotic fluid obtained by amniocentesis and maternal venous blood at 16-20 weeks of gestation, and suspended in a thin layer of agar. Using antibodies against hemoglobins F, A, and S this method proved to possess sufficient sensitivity for detecting each of the respective hemoglobins in individual cells in all of the above classes of specimens. Since the red blood cells obtained by amniocentesis are potentially a mixture of fetal and maternal cells the proportion of F cells to A cells in amniotic fluid and in maternal venous blood were estimated. In 33 such sequential specimen pairs, 29 contained a higher F cell to A cell ratio in amniotic fluid than in maternal blood. Red blood cells from maternal blood and from amniotic fluid were incubated in vitro with L-[14C]serine. Incorporation of the serine into cells was identified by autoradiography. Since a substantial proportion of fetal red blood cells are synthetically active this provides a method for identifying hemoglobin phenotype in fetal-origin cells in a fetal-maternal mixture. Initial experience with a short series of specimens examined in this manner indicates that in amniotic fluid the proportion of cells which contain both hemoglobin and L-[14C]serine is typically higher than in maternal venous blood. Diagnostic accuracy for hemoglobin phenotypes of the fetus by the combination of autoradiography and fluorescent cytoimmunodiffusion is being tested currently in a substantial unselected sample of amniotic fluid specimens. Criteria for definitive diagnosis of SS phenotype in the fetus are fluorescent anti-S pattern in synthetically active cells, absence or rare occurrence of fluorescent anti-A pattern in such active cells, and a higher relative frequency of synthetically active F cells in amniotic fluid than in maternal blood. © 1978 International Pediatric Research Foundation, Inc.
Headings, Verle E.; Anyaibe, Stephen I.O.; Bhattacharya, Syama P.; and Hopkins, Ernest L., "Early diagnosis of hemoglobinopathies" (1978). The Center For Sickle Cell Disease Faculty Publications. 332.