Studies on abnormal hemoglobins. V. The distribution of type S,sickle cell,hemoglobin and type F,alkali resistant,hemoglobin within the red cell population in sickle cell anemia

1. By transfusing sickle cell anemia erythrocytes with a relatively high concentration of F hemoglobin into normal recipients, it was demonstrated that thedisappearance rates of the transfused cells and of their alkali resistant pigmentconsistently showed great discrepancies. These observations suggest an unequaldistribution of the F pigment within the erythrocyte population. A nonuniformdistribution of F hemoglobin could also be detected in vitro by exposing sicklecell anemia bloods to mechanical trauma for a longer period of time. The cellsmost resistant to trauma contained a higher percentage of F hemoglobin thanthe original blood specimen.

2. The red cell population of patients with sickle cell anemia seems to be composed of three main fractions: (1) cells containing S hemoglobin and no or littleF hemoglobin, (2) cells containing both pigments and (3) cells containing Fpigment with no or little S hemoglobin.

3. The erythrocytes carrying mostly S hemoglobin have the shortest life span,whereas the red cells containing mostly F hemoglobin have the longest survivaltime.

4. The significance of these findings in regard to clinical and genetic aspectsof sickle cell anemia is discussed. No direct correlation is demonstrable in anindividual patient between the absolute amounts of either type S or type Fhemoglobin and the severity of the anemia. The latter depends on the variablesize of the portion of red cells containing mostly S hemoglobin, and also on theability of the marrow to replace this particular fraction.

Submitted on August 5, 1952 Accepted on September 10, 1952     

Studies on abnormal hemoglobins. VIII. The gelling phenomenon of sickle cell hemoglobin: its biologic and diagnostic significance

1. When sufficiently concentrated sickle cell hemoglobin containing solutionsare exposed to a constant stream of CO₂ gas, the hemolysates gel. This gellingphenomenon is indicative of the presence of S hemoglobin and cannot be obtainedwith any other type of human hemoglobin in the absence of S pigment. Thelowest S hemoglobin concentration (Gm. per cent) of a hemolysate at which thegelling phenomenon can still be elicited is designated as its lowest gelling point.

2. A simple apparatus was developed to analyze the gelling phenomenon understandardized conditions. It could be shown that the lowest gelling points ofhemolysates prepared from erythrocytes of the sickle cell trait (containing A +S hemoglobins), of the "C variant" (containing C + S hemoglobins), and fromsickle cell anemia cells (containing S + F hemoglobins) differ distinctly. Furtherexperiments suggest that the presence of A hemoglobin decreases the minimalamount of S pigment required for gel formation, and that type C hemoglobinreduces this amount even further. F hemoglobin seems to exert no significantinfluence on the gelling phenomenon. Serum albumin is also capable of decreasingthe amount of S hemoglobin required for gelation.

3. A sickled erythrocyte is visualized as an S hemoglobin tactoid or gel, specifically influenced by the companion pigment which interacts with the S compound.Thus, in the sickle cell trait, a positive sickling test is not only caused by thepresence of S hemoglobin, but also by its interaction with A hemoglobin. Onlyin the sickle cell anemia cells does sickling seem to depend solely upon the interaction of the S hemoglobin molecules.

4. The readily demonstrable differences of the lowest gelling points of hemolysates prepared from the various types of sickling red cells form the basis of thediagnostic gelling test which distinguishes sharply between sickle cell anemia andsickle cell trait erythrocytes. By this procedure atypical cases of sickle cell disease,for example, those whose erythrocytes contain C hemoglobin, may also bedetected.

Submitted on April 21, 1953 Accepted on May 25, 1953     

Studies on abnormal hemoglobins. VI. Electrophoretic demonstration of type S (sickle cell) hemoglobin in erythrocytes incapable of showing the sickle cell phenomenon

1. A family is reported in which 2 children have unmistakable sickle cellanemia, but the mother’s red cells do not sickle; the father and a third childexhibit the typical sickle cell trait. Electrophoretic studies of the hemoglobinsolution prepared from the mother’s erythrocytes demonstrated, however, thepresence of a small amount (5 per cent) of type S hemoglobin. This abnormalcomponent was identified by its mobility and by addition experiments.

2. The finding that the mother’s erythrocytes contain small quantities of Shemoglobin, but are incapable of sickling, confirms the genetic concept thatsickle cell anemia will only develop when both parents transmit the gene for Shemoglobin.

3. The physico-chemical and possible medico-legal aspects of the fact that Shemoglobin is sometimes only detectable by means of electrophoresis, are discussed.

Submitted on August 5, 1952 Accepted on September 10, 1952     

Studies on abnormal hemoglobins. I. Their demonstration in sickle cell anemia and other hematologic disorders by means of alkali denaturation

1. By exposing hemoglobin solutions to an alkaline reagent at a pH of 12.7 itwas found that normal pigment is completely denatured within one minute.Thus even small amounts of more resistant hemoglobins, which may also bepresent in the solution, can be readily detected. Fetal hemoglobin, which isalkali resistant, may remain demonstrable until the end of the second year oflife.

2. Alkali resistant hemoglobins were regularly encountered in sickle cellanemia (but not in the trait), in the more fully developed Mediterranean syndromes, and in 1 out of 4 families with hereditary spherocytosis. In addition tothese hereditary hemolytic disorders, abnormally denaturing hemoglobin fractions were observed in 3 instances of chronic aregenerative anemia, and, irregularly, in patients with untreated pernicious anemia, acute and chronicleukemia and myelophthisic anemia. All other kinds of anemias were found tohave only normally denaturing pigments.

3. Three definite types of hemoglobin are identifiable at present by means ofelectrophoresis and denaturation. These have been designated as type N (normaladult), type F (fetal), and type S (sickle cell hemoglobin). The hypothesis isadvanced that the resistant fraction in the hereditary hemolytic syndromes mayrepresent a continued production of fetal pigment beyond the physiologic agelimit and the appearance of the abnormal hemoglobins in the "acquired" disorders may indicate a reactivation of such a mechanism. The implications ofsuch an assumption for the distribution of the various types of hemoglobin insickling erythrocytes are discussed.

4. The diagnostic significance of the denaturation test and its limitationsare outlined.

     

Studies on human myoglobin. I. Myoglobin in sickle cell disease

The problem whether patients with sickle cell anemia have not only a pathologic (type S) hemoglobin, but also an abnormal myoglobin, was investigated bymeans of spectroscopic and electrophoretic methods. Purified, hemoglobin-freemet-myoglobin preparations, obtained from normal adults and from individualswith sickle cell anemia, were compared. No differences could be detected witheither technic.

The electrophoretic studies revealed that adult met-myoglobin moves fasterin an acid than in an alkaline buffer. Regardless of the buffer used, adult met-myoglobin moves always slower than either met-hemoglobin A or S. The met-compounds of hemoglobin A and S move faster than their respective oxy-compounds in an acid medium, but slower in an alkaline medium.

Submitted on May 24, 1955 Accepted on June 12, 1955     

The effect of human plasma transfusion on the fecal urobilinogen excretion on sickle cell anemia

1. The results of transfusions of whole plasma and blood on the fecal urobilinogen excretion of 5 children with sickle cell anemia have been reported.

2. The phenomenon observed by Josephs—a reduction in urobilinogen outputfollowing plasma transfusions—has been confirmed.

3. It has been observed further that in certain patients the continued use ofplasma transfusions induces a reversal of this phenomenon, namely an increase inurobilinogen output after each transfusion.

4. The possible significance of these phenomena is discussed and attention iscalled to the limitations inherent in accepting urobilinogen excretion as a validindex of hemoglobin destruction in certain disorders in which a defect of hemoglobin metabolism may be present.

     

Hematologic observations on patients with sickle cell anemia sustained at normal hemoglobin levels by multiple transfusions

(1) Three sickle cell patients were sustained at normal hemoglobin levels for3-4 months by means of repeated transfusions of fresh blood.

(2) In response to transfusions, there was a decline in reticulocytes to normallevels during the first 5-7 days of observation. During periods in which the hemoglobin was maintained at high-normal or super-normal levels, the reticulocytevalues were depressed below the normal range. A distinct reticulocyte responsewas observed when the hemoglobin declined to approximately 11.0 Gm. per centfollowing cessation of transfusions.

(3) Employing anti-M differential agglutination, a simple exponential declinewas observed in the number of sickle cells in each patient’s circulation duringthe period of sustained normal hemoglobin concentration.

(4) The continued production of new sickle cells during the first week ofobservation complicated the interpretation of the differential agglutinationdata, but provided indirect evidence for the presence of an especially short-livedproportion of the patient’s cells. Support for this concept was derived from theradioactive iron utilization studies performed in Case 1.

Submitted on November 14, 1955 Accepted on March 9, 1956     

Hybrid erythrocytes for membrane studies in sickle cell disease.

A hybrid erythrocyte model for membrane studies in sickle cell disease has been developed. The model consists of normal red cell membranes containing hemoglobin S and sickle cell membranes containing hemoglobin A. In hybrids, complete hemoglobin exchange has been achieved together with restoration of low membrane permeability to potassium. Normal membranes containing HbS sickle upon deoxygenation and assume the characteristic appearance of irreversibly sickled cells (ISC) after prolonged anoxia. It is suggested that the hybrid model will be useful in defining further the process of ISC formation and in studying the influence of sickle hemoglobin upon the function of the surrounding membrane.     

Supersaturation in sickle cell hemoglobin solutions.

The kinetic inhibition of the gelation of hemoglobin S is compared to the change in hemoglobin S soulbility, when the solubility is altered by carbon monoxide, pH, or urea. By means of a new technique, the delay time and the extent of gelation are measured on the same sample. They delay time, td, is found to be proportional to a high power (30-40) of the hemoglobin S solubility. Togehter with the previously reported concentration dependence, this result demonstrates that the rate is proportional to a high power of the supersaturation, S, defined as the ratio of the total hemoglobin S concentration to the equilibrium solubility. The results obey the supersaturation equation td-1 = gammaSn, where gamma is an empirical constant (about 10(-7) sec-1) and n is about 35. The supersaturation equation can successfully account for observations on the kinetics of cell sickling and is therefore used to estimate the increase in the delay time for sickling necessary to produce significant clinical benefit to patients with sickle cell disease.     

Fetal hemoglobin in sickle cell anemia

Fetal hemoglobin (HbF) is the major genetic modulator of the hematologic and clinical features of sickle cell disease, an effect mediated by its exclusion from the sickle hemoglobin polymer. Fetal hemoglobin genes are genetically regulated, and the level of HbF and its distribution among sickle erythrocytes is highly variable. Some patients with sickle cell disease have exceptionally high levels of HbF that are associated with the Senegal and Saudi-Indian haplotype of the HBB-like gene cluster; some patients with different haplotypes can have similarly high HbF. In these patients, high HbF is associated with generally milder but not asymptomatic disease. Studying these persons might provide additional insights into HbF gene regulation. HbF appears to benefit some complications of disease more than others. This might be related to the premature destruction of erythrocytes that do not contain HbF, even though the total HbF concentration is high. Recent insights into HbF regulation have spurred new efforts to induce high HbF levels in sickle cell disease beyond those achievable with the current limited repertory of HbF inducers.     

Microchromatographic quantitation of fetal hemoglobin in patients with sickle cell disease.

A microchromatographic procedure (Isolab Fast Hb Test System) which was developed for the quantitation of Hb AI (10) has been found useful for the quantitation of Hb F in samples that contain Hb S and/or Hb C but no Hb A (% Fmicro). This method has been evaluated through analyses of known mixtures of Hb F and Hb S. The Hb Fmicro levels in patients with sickle cell anemia and related conditions were compared with results obtained by alkali denaturation (% FAD) and conventional DEAE-cellulose chromatography (% FDE). This microchromatographic technique is a fast, simple, and sensitive method for Hb F quantitation in patients with Sickle cell disease.     

Quantitative analysis of erythrocytes containing fetal hemoglobin (F cells) in children with sickle cell disease

Variation in the level of fetal hemoglobin (HbF) accounts for much of the clinical heterogeneity observed in patients with sickle cell disease (SCD). The HbF level has emerged as an important prognostic factor in both sickle cell pain and mortality, and a % HbF of 10–20% has been suggested as a threshold level for diminished clinical severity. The number of erythrocytes that contain HbF (termed F cells) may also be critically important, as F cells resist intravascular sickling and have preferential in vivo survival. Since F cells can be enumerated with high accuracy using flow cytometry methods, we prospectively studied a cohort of 242 children with SCD. Children with HbS and hereditary persistence of fetal hemoglobin (S/HPFH) had essentially 100% F cells. In contrast, children with homozygous sickle cell anemia (HbSS), HbS/β⁰ thalassemia, or HbS/β⁺ thalassemia had significantly lower mean % F cell values (55.9, 61.6, and 51.3%, respectively; P < 0.001), and children with HbSC had even fewer F cells (27.0%; P < 0.001). There was a highly significant correlation between the % F cells and the log (% HbF), which was observed for the total population of children (r = 0.95, P < 0.001), as well as for each of the individual subgroups of children with HbSS (r = 0.94, P < 0.001), HbSC (r = 0.89, P < 0.001), or HbS/β⁰ thalassemia and HbS/β⁺ thalassemia (r = 0.95, P <0.001). This logarithmic correlation between % F cells and % HbF has not been previously described and has important implications for the pharmacologic manipulation of HbF in patients with SCD. Am. J. Hematol. 54:40–46, 1997 © 1997 Wiley-Liss, Inc.     

A new form of hereditary persistence of fetal hemoglobin in blacks and its association with sickle cell trait.

A new form of hereditary persistence of fetal hemoglobin (HPFH) producing 3%-8% Hb F in heterozygotes and an elevation of F-cell counts as measured by both the Kleihauer test and an antibody fluorescent procedure was found during the study of a black family. Individuals with this anomaly also had sickle cell trait. A sickle cell homozygote who had apparently inherited the HPFH determinant had 20.3% Hb F. Both types of gamma-chains were present in equal proportions in the Hb F of these individuals. A population study revealed other AS individuals with increased Hb F synthesis, three of whom were sibs. The presence of this previously unrecognized form of HPFH might explain the mild clinical manifestations and the hemoglobin phenotypes of sickle cell homozygotes with unusual elevations of Hb F.     

Determination of deoxyhemoglobin S polymer in sickle erythrocytes upon deoxygenation.

We have used 13C/1H magnetic double-resonance spectroscopy to measure the amount of sickle hemoglobin polymer within sickle erythrocytes as a function of oxygen saturation. We previously showed that the methods of cross-polarization and scalar decoupling could be used to measure accurately the polymer fraction in deoxygenated sickle hemoglobin solutions [Noguchi, C.T., Torchia, D.A. & Schechter, A.N. (1979) Proc. Natl. Acad. Sci. USA 76, 4936-4940]. Our measurements show that the amount of intracellular deoxyhemoglobin S polymer increases monotonically with decreasing oxygen saturation. Polymer can be detected at oxygen saturation values above 90%. This result can be theoretically explained by the excluded volume effect of the oxyhemoglobin S in the cell. The very high total intracellular hemoglobin concentration (34 g/dl) reduces the amount of soluble deoxyhemoglobin S to about 3 g/dl at 90% oxygen saturation. The agreement between theory and experiment indicates that the equilibrium properties of intracellular polymerization can be described by the analyses resulting from studies of concentrated sickle hemoglobin solutions. The curve for polymer formation as a function of oxygen saturation is roughly hyperbolic whereas that for cell sickling is sigmoidal; the difference is most apparent for measurements at pH 7.65. Intracellular polymer formation may in general have a different relationship to oxygen saturation than cell sickling and may be a more meaningful parameter of the pathophysiological process in sickle cell anemia than cell morphology. In addition, measurements of intracellular polymer should be useful in evaluating potential therapeutic agents.     

Immunologic studies of hemoglobins. I. The production of anti-hemoglobin sera and their immunologic characteristics

1. Antisera to hemoglobin solutions obtained from normal adult, cord bloodand sickle cell anemia erythrocytes have been prepared by immunizing rabbitswith adjuvant mixtures. Furthermore, anti F hemoglobin sera have been similarly prepared by using purified fetal hemoglobin. A species specific anti guineapig hemoglobin serum was also developed.

2. Although anti F hemoglobin sera appear to be specific for fetal hemoglobin,no such immunologic specificity could be detected between anti A and antiSCA sera obtained from rabbits.

3. It is suggested that sera prepared by using hemoglobin solutions obtainedfrom normal and sickle cell anemia erythrocytes may contain anti F antibodies inaddition to anti A and anti S precipitins and that fetal hemoglobin may be present in many human adult blood specimens.

Submitted on November 19, 1952 Accepted on January 31, 1953     

Glycosylated hemoglobin levels in Sudanese sickle cell anemia patients

Glycosylated hemoglobin was measured, by a colorimetric method, in 49 patients with sickle cell anemia attending Khartoum Teaching Hospital. The level obtained (4.9%, SD 1.3) was significantly lower than the control value (5.6%, SD 0.2; p less than 0.0025). Expressed as percentage of the control value, the glycosylated hemoglobin level in these patients was 81%. This falls midway between the 90% reported in a benign form of sickle cell anemia in Saudi Arabia and the 58% reported in a severe form in an American Black group, which gives support to the observed heterogeneity of sickle cell anemia. Alternatively, glycosylated hemoglobin level in 27 subjects with sickle cell trait (5.6%, SD 0.2) was identical to that of the controls. The state of hemolysis in the sickle cell anemia patients, as indicated by bilirubin levels, did not correlate with the glycosylated hemoglobin values. Although glycosylated hemoglobin measurement is affected by red cell survival, it does not reflect the state of ongoing hemolysis.     

Fetal hemoglobin levels and beta s globin haplotypes in an Indian populations with sickle cell disease

To further explore the cause for variation in hemoglobin F (Hb F) levels in sickle cell disease, the beta globin restriction-fragment length polymorphism haplotypes were determined in a total of 303 (126 SS, 141 AS, 17 S beta degrees, 7 A beta, degrees and 12 AA) Indians from the state of Orissa. The beta s globin gene was found to be linked almost exclusively to a beta S haplotype ( -++-), which is also common in Saudi Arabian patients from the Eastern Province (referred to as the Asian beta s haplotype). By contrast, the majority of beta A and beta degree thalassemia globin genes are linked to haplotypes common in all European and Asian populations (+-----[+/-]; --++-++). Family studies showed that there is a genetic factor elevating Hb F levels dominantly in homozygotes (SS). This factor appears to be related to the Asian beta s globin haplotype, and a mechanism for its action is discussed. There is also a high prevalence of an independent Swiss type hereditary persistence of fetal hemoglobin (HPFH) determinant active in both the sickle cell trait and in sickle cell disease.     

Membrane-associated sickle hemoglobin: a major determinant of sickle erythrocyte rigidity

Micropipette aspiration tests on single erythrocytes have previously shown that the static rigidity (membrane shear modulus) of oxygenated sickle cells increased with increasing hemoglobin concentration, whereas the rigidity of normal cells was independent of hemoglobin concentration. Moreover, it was observed that after mechanical extension, sickle cells exhibited persistent deformation more frequently and to a greater extent than normal cells. To ascertain if differences in association of normal and sickle hemoglobin with the membrane could account for these observations, we measured rheologic properties of normal membranes reconstituted with sickle hemoglobin and sickle membranes reconstituted with normal hemoglobin. The static rigidity of normal ghosts reloaded with sickle hemoglobin was higher than those of either normal ghosts reloaded with normal hemoglobin or native normal cells. On the other hand, the increased rigidity of native sickle cells decreased to near-normal values following reconstitution with normal hemoglobin. Furthermore, we observed that normal ghosts reconstituted with sickle hemoglobin exhibited persistent bumps after mechanical extension, but no bumps formed on normal ghosts reconstituted with normal hemoglobin. Moreover residual bumps were not produced on sickle cells reloaded with normal hemoglobin. Since mechanical characteristics peculiar to sickle cells could be induced in normal cells by incorporation of sickle hemoglobin, and since normal characteristics could be restored to sickle cells by incorporation of normal hemoglobin, we suggest that the interaction of sickle hemoglobin with the cell membrane is responsible for augmented static rigidity of oxygenated sickle erythrocytes.     

The relationship between fetal hemoglobin level and glycosylation in sickle cell disease

Glycosylated hemoglobin (glyco Hb) was determined by affinity chromatography and Hb S1 and Hb F by Bio-Rex 70 chromatography in patients with sickle cell anemia, SC disease, S beta+-thalassemia, and S beta 0-thalassemia. SC and S beta-thalassemia patients had normal levels of glyco Hb whereas SS patients had significantly lower levels. Within each group of patients a direct correlation existed between Hb F and glyco Hb or Hb S1 levels. A similar relationship was noticed when glyco Hb and Hb F levels were compared in red cell populations of various densities (ages). Hb F seems to influence glycosylation through its effect on red cell survival.     

Catalysis of soluble hemoglobin oxidation by free iron on sickle red cell membranes

Abnormal deposition of hemichrome on the inner aspect of the sickle red cell membrane promotes premature cell demise. The steps proximate to hemichrome formation in these cells are poorly understood. To test the hypothesis that the pathologic deposits of free ferric iron located on the inner aspect of sickle cell membranes would be redox active and promote oxidation of soluble oxyhemoglobin, we incubated native versus iron-stripped sickle or normal ghost membranes with oxyhemoglobin S. We found that sickle membranes exerted an exaggerated effect on methemoglobin formation in solution, an effect completely accounted for by their abnormal content of free iron. This ability of sickle membranes to promote hemoglobin oxidation was not diminished by catalase or by presence of a high-affinity, iron-inactivating chelator that is unable to remove membrane iron. Examination of those membranes likewise revealed that their free iron content promoted deposition of additional heme-protein. These results establish that the potential redox couple formed by membrane-associated ferric iron and cytoplasmic oxyhemoglobin is promotive of hemoglobin oxidation and deposition of hemichrome on the membrane. This predicts that removal of pathologic membrane iron might help prevent the detrimental formation of methemoglobin and hemichrome in vivo, insofar as this is accelerated by transition metal.