ALBERT

Description: Red cell lysis in isotonic solutions containing NH4Cl, NH4HCO3, and a carbonic anhydrase enzyme inhibitor (acetazolamide) is a function of erythrocyte enzyme activity and permeability of cells to the inhibitor. Erythrocyte carbonic anhydrase activity is at least fivefold greater and acetazolamide permeability about tenfold less for adults than for newborns. In this setting, greater than 99.9% of red cells from adults can be hemolyzed at a time when greater than 25% of those from newborns remain intact. This easily applied method may be useful when antenatal diagnosis of hemoglobinopathies is otherwise precluded by contaimination with maternal erythrocytes. The feasibility of differential hemolysis via NH4Cl--HCO3-mediated, acetazolamide- modulated reactions is shown by the successful isolation of the few fetal-origin erythrocytes present in grossly nonbloody amniotic fluids and, in one instance, by approximately 3300-fold enrichment of apparently authentic fetal-origin red cells from the arm blood of a woman in her 18th wk of pregnancy.

Description: Levels of fetal hemoglobin (HbF) bearing reticulocytes (F reticulocytes) range from 2% to 50% in patients with sickle cell (SS) anemia. To learn whether any portion of such variation in F cell production is regulated by loci genetically separable from the beta- globin gene cluster, percentages of F reticulocytes were compared in 59 sib pairs composed solely of SS members, including 40 pairs from Jamaica and 19 from the United States. We reasoned that differences in F reticulocyte levels might arise (1) from any of several kinds of artifact, (2) via half-sib status, or (3) because one or more genes regulating F cell production segregate separately from beta S. We minimized the role of artifact by assay of fresh samples from 84 SS individuals, including both members of 38 sib pairs. In 78 of the 84 subjects, serial values for percent F reticulocytes fell within 99.9% confidence limits or were alike by t test (P greater than or equal to .05). This left 32 sib pairs for which F reticulocyte levels in each member were reproducible. When sib-sib comparisons were limited to these 32 pairs, percentages of F reticulocytes were grossly dissimilar within 12 Jamaican and 3 American sibships. Within them, the probability that sibs were alike was always less than or equal to .005 and usually less than or equal to 10(-4). We next minimized the contribution of half-sibs among Jamaicans by a combination of paternity testing and sib-sib comparison of beta-globin region DNA restriction fragment length polymorphisms, especially among discordant pairs. We thereafter concluded that at least seven to eight Jamaican pairs were composed of reproducibly discordant full sibs. There is thus little doubt that there are genes regulating between-patient differences in F cell production that are separate from the beta-globin gene cluster. Still unanswered is (1) whether or not these genes are actually linked to beta S, (2) why F reticulocyte levels in Americans tend to be lower than in Jamaicans, and (3) whether or not differences in F cell production among SS patients are regulated by several major loci or by only one.

Description: Levels of fetal hemoglobin (HbF) bearing reticulocytes (F reticulocytes) range from 2% to 50% in patients with sickle cell (SS) anemia. To learn whether any portion of such variation in F cell production is regulated by loci genetically separable from the beta- globin gene cluster, percentages of F reticulocytes were compared in 59 sib pairs composed solely of SS members, including 40 pairs from Jamaica and 19 from the United States. We reasoned that differences in F reticulocyte levels might arise (1) from any of several kinds of artifact, (2) via half-sib status, or (3) because one or more genes regulating F cell production segregate separately from beta S. We minimized the role of artifact by assay of fresh samples from 84 SS individuals, including both members of 38 sib pairs. In 78 of the 84 subjects, serial values for percent F reticulocytes fell within 99.9% confidence limits or were alike by t test (P greater than or equal to .05). This left 32 sib pairs for which F reticulocyte levels in each member were reproducible. When sib-sib comparisons were limited to these 32 pairs, percentages of F reticulocytes were grossly dissimilar within 12 Jamaican and 3 American sibships. Within them, the probability that sibs were alike was always less than or equal to .005 and usually less than or equal to 10(-4). We next minimized the contribution of half-sibs among Jamaicans by a combination of paternity testing and sib-sib comparison of beta-globin region DNA restriction fragment length polymorphisms, especially among discordant pairs. We thereafter concluded that at least seven to eight Jamaican pairs were composed of reproducibly discordant full sibs. There is thus little doubt that there are genes regulating between-patient differences in F cell production that are separate from the beta-globin gene cluster. Still unanswered is (1) whether or not these genes are actually linked to beta S, (2) why F reticulocyte levels in Americans tend to be lower than in Jamaicans, and (3) whether or not differences in F cell production among SS patients are regulated by several major loci or by only one.

Description: Red cell lysis in isotonic solutions containing NH4Cl, NH4HCO3, and a carbonic anhydrase enzyme inhibitor (acetazolamide) is a function of erythrocyte enzyme activity and permeability of cells to the inhibitor. Erythrocyte carbonic anhydrase activity is at least fivefold greater and acetazolamide permeability about tenfold less for adults than for newborns. In this setting, greater than 99.9% of red cells from adults can be hemolyzed at a time when greater than 25% of those from newborns remain intact. This easily applied method may be useful when antenatal diagnosis of hemoglobinopathies is otherwise precluded by contaimination with maternal erythrocytes. The feasibility of differential hemolysis via NH4Cl--HCO3-mediated, acetazolamide- modulated reactions is shown by the successful isolation of the few fetal-origin erythrocytes present in grossly nonbloody amniotic fluids and, in one instance, by approximately 3300-fold enrichment of apparently authentic fetal-origin red cells from the arm blood of a woman in her 18th wk of pregnancy.