The Unusual Pathobiology of Hemoglobin Constant Spring Red Blood Cells by S.L. Schrier, A. Bunyaratvej, A. Khuhapinant, S. Fucharoen, M. Aljurf, L.M. Snyder, C.R. Keifer, L. Ma, and N. Mohandas Blood Volume 89(5):1762-1769 March 1, 1997 ©1997 by American Society of Hematology

An analysis of patients with variant forms of α-thalassemia from Bangkok, Thailand. An analysis of patients with variant forms of α-thalassemia from Bangkok, Thailand. The top panel shows the Hb concentration. The subsequent four panels are parameters derived from the H*3 Bayer Hematology Analyzer indicating the mean cell volume (MCV), the mean cell Hb content (MCH), and the mean cell Hb concentration (MCHC), while the bottom panel indicates the percent of cells that are hypochromic (percent of RBC where the MCHC is less than 28 g/dL). The error bars indicate 1 standard deviation about the mean. Abbreviations: α-T, α-thalassemia-1 trait; CS-T, heterozygous HbCS; CS/CS, homozygous CS; HbH, α-thalassemia-1 trait/α-thalassemia-2 trait; HbH/CS, α thalassemia-1 trait/αCSα. The symbols indicate P values showing significant differences from the normal. The following symbols indicate the respective P values: NS, not significant; #, < .01; ⋄, < .0005; ▵, < .0001. The number of analyses done for the top four panels are indicated within the bars in the top panel. The number of analyses done to determine the extent of hypochromia (bottom panel) are indicated within or on the bars in that panel. S.L. Schrier et al. Blood 1997;89:1762-1769 ©1997 by American Society of Hematology

Parameters for the RBCs and reticulocytes from a normal patient, a patient with classical HbH, and a patient with classical HbH/CS. Parameters for the RBCs and reticulocytes from a normal patient, a patient with classical HbH, and a patient with classical HbH/CS. The distribution curves for the entire RBC population are shown in gray, whereas the reticulocyte population is displayed in red. S.L. Schrier et al. Blood 1997;89:1762-1769 ©1997 by American Society of Hematology

Relative membrane rigidity and membrane mechanical stability of variant α-thalassemia RBCs. HbCS/CS RBCs exhibited the greatest increase in membrane rigidity while HbH/CS, HbH, and CS trait RBCs exhibited intermediate increases in membrane rigidity (A). Relative membrane rigidity and membrane mechanical stability of variant α-thalassemia RBCs. HbCS/CS RBCs exhibited the greatest increase in membrane rigidity while HbH/CS, HbH, and CS trait RBCs exhibited intermediate increases in membrane rigidity (A). The α-thalassemia-1 trait RBC membranes exhibited normal membrane rigidity (A). Representative membrane mechanical stability data for the variant RBCs are shown (B). The rate of decline of deformability index of membranes from HbCS/CS was the slowest, indicating that these membranes exhibit the greatest increase in membrane mechanical stability. The rate of decline of deformability index of membranes of HbH/CS and HbH RBCs was intermediate between that of HbCS/CS and normal membranes, indicating somewhat less of an increase in membrane mechanical stability. The abbreviations are the same as those in Fig 1. S.L. Schrier et al. Blood 1997;89:1762-1769 ©1997 by American Society of Hematology

The left panel (Transfer Gel) identifies the SDS-PAGE analysis of solubilized membranes of normal controls, patients with HbH, HbH/CS, and patients with homozygous HbCS/CS. The left panel (Transfer Gel) identifies the SDS-PAGE analysis of solubilized membranes of normal controls, patients with HbH, HbH/CS, and patients with homozygous HbCS/CS. Splenectomized patients are indicated by splx. The globin band, running at approximately 15 kD, appears at the bottommost portion of the gel. The arrow indicates bands seen only in HbCS variants that run at a position consistent with 19 kD molecular weight. Western blotting of similar patients is shown on the two righthand panels with a monoclonal anti–α-globin in the center and anti–β-globin MoAb on the right. The anti–β-globin antibody shows accumulations of β-globin in HbH and HbH/CS, but the largest accumulations occurred in splenectomized patients with HbH and HbH/CS. Of importance is the fact that the anti–α-globin antibody reacted specifically with the 19-kD band. S.L. Schrier et al. Blood 1997;89:1762-1769 ©1997 by American Society of Hematology

Membrane skeletons, prepared as described, were subjected to SDS-PAGE analysis. Membrane skeletons, prepared as described, were subjected to SDS-PAGE analysis. Characteristic of membrane skeletons is the prominence of α- and β-spectrin, band 4.1, and actin. Relatively faint bands identified as αCS, because of their position at 19 kD, are seen only in the two patients with HbH/CS and the patient with HbCS/CS. S.L. Schrier et al. Blood 1997;89:1762-1769 ©1997 by American Society of Hematology

Thiol-disulfide exchange chromatography was performed on a normal control, a β-thalassemia intermedia patient who had been splenectomized, an HbH patient who had been splenectomized, and a patient with homozygous HbCS/CS. Thiol-disulfide exchange chromatography was performed on a normal control, a β-thalassemia intermedia patient who had been splenectomized, an HbH patient who had been splenectomized, and a patient with homozygous HbCS/CS. The unbound fraction was analyzed by SDS-PAGE and the αCS was identified at a position consistent with a molecular weight of 19 kD. Note the presence of globin bands at the bottom of the gel. These have previously been shown to be αA in β-thalassemia intermedia and βA in HbH.2 S.L. Schrier et al. Blood 1997;89:1762-1769 ©1997 by American Society of Hematology